HUE029962T2 - Egyláncú fehérjék kétláncú formájúvá történõ intracelluláris konverziójának módszerei - Google Patents

Egyláncú fehérjék kétláncú formájúvá történõ intracelluláris konverziójának módszerei Download PDF

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HUE029962T2
HUE029962T2 HUE13188050A HUE13188050A HUE029962T2 HU E029962 T2 HUE029962 T2 HU E029962T2 HU E13188050 A HUE13188050 A HU E13188050A HU E13188050 A HUE13188050 A HU E13188050A HU E029962 T2 HUE029962 T2 HU E029962T2
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Sanjiv Ghanshani
Linh Q Le
Yi Liu
Lance E Steward
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Allergan Inc
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Description

Description [0001] The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic applications, see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxins commercially available as pharmaceutical compositions include, BoNT/A preparations, such as, e.g., BOTOX® (Allergan, Inc., Irvine, CA), DYS-PORT®/RELOXIN®, (Beaufour Ipsén, Porton Down, England), NEURONOX® (Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A(Lanzhou Institute Biological Products, China) and XEOMIN® (Merz Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/B preparations, such as, e.g., MYOBLOC™/NEUROBLOC™ (Elan Pharmaceuticals, San Francisco, CA). As an example, BOTOX® is currently approved in one or more countries for the following indications: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and VII nerve disorder.
[0002] The therapeutic utility of Clostrdial toxins has been expanded beyond their current myo-relaxant applications to treat sensory nerve-based ailments, such as, e.g., various kinds of chronic pain, neurogenic inflammation and uro-gentital disorders, as well as non-neuronal-based disorders, such as, e.g., pancreatitis. One approach that is currently being exploited to expand Clostridial toxin-based therapies involves modifying a Clostridial toxin so that the modified toxin has an altered cell targeting capability for a non-Clostridial toxin target cell. This re-targeted capability is achieved by replacing a naturally-occurring targeting domain of a Clostridial toxin with a targeting domain showing a selective binding activity for a non-Clostridial toxin receptor present In a non-Clostridial toxin target cell. Such modifications to a targeting domain result in a modified toxin that is able to selectively bind to a non-Clostridial toxin receptor (target receptor) present on a non-Clostridial toxin target cell (re-targeted). A retargeted Clostridial toxin with a targeting activity for a non-Clostridial toxin target cell can bind to a receptor present on the non-Clostridial toxin target cell, translocate into the cytoplasm, and exert its proteolytic effect on the SNARE complex of the non-Clostridial toxin target cell.
[0003] Non-limiting examples of re-targeted Clostridial toxins with a targeting activity for a non-Clostridial toxin target cell are described in, e.g., Keith A. Foster et al., Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent Functions, U.S. Patent 5,989,545; Clifford C. Shone et ai., Recombinant Toxin Fragments, U.S. Patent 6,461,617; Stephan Donovan, Clostridial Toxin Derivativesand Methods ForTreating Pain, U.S. Patent6,500,436; Conrad P. Quinn et al., Methods and Compounds for the Treatment of Mucus Hypersecretion, U.S. Patent 6,632,440; Lance E. Steward et al., Methods And Compositions For The Treatment Of Pancreatitis, U.S. Patent 6,843,998; J. Oliver Dolly et al., Activatable Recombinant Neurotoxins, U.S. Patent 7,419,676; Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. Patent 7,514,088; Keith A. Foster et al., Inhibition of Secretion from Nonneural Cells, U .S. Patent Publication 2003/0180289; and Keith A. Fosteret al., Retargeted Toxin Conjugates, International Patent Publication WO 2005/023309. The ability to re-target the therapeutic effects associated with Clostridial toxins has greatly extended the number of medicinal applications able to use a Clostridial toxin therapy. As a non-limiting example, modified Clostridial toxins retargeted to sensory neurons are useful in treating various kinds of chronic pain, such as, e.g., hyperalgesia and allodynia, neuropathic pain and inflammatory pain, see, e.g,. Foster, supra, (1999); and Donovan, supra, (2002); and Stephan Donovan, Method For Treating Neurogenic Inflammation Pain with Botulinum Toxin and Substance P Components, U.S. Patent 7,022,329. As another non-limiting example, modified Clostridial toxins retargeted to pancreatic cells are useful in treating pancreatitis, see, e.g., Steward, supra, (2005).
[0004] Clostridial toxins, whether naturally occurring or modified, are processed into a di-chain form in order to achieve full activity. Naturally-occurring Clostridial toxins are each translated as a single-chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease (FIG. 1). This cleavage occurs within the discrete di-chain loop region created between two cysteine residues that form a disulfide bridge. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC), comprising the enzymatic domain, and an approximately 100 kDa heavy chain (HC), comprising the translocation and cell binding domains, the LC and HC being held together by the single disulfide bond and non-covalent interactions (FIG. 1). Recombinantly-produced Clostridial toxins generally substitute the naturally-occurring di-chain loop protease cleavage site with an exogenous protease cleavage site (FIG. 2). See e.g., Dolly, J.O. et al., Activatable Clostridial Toxins, U.S. Patent 7,419,676. Although re-targeted Clostridial toxins vary in their overall molecular weight because of the size of the targeting moiety, the activation process and its reliance on exogenous cleavage sites is essentially the same as that for recombinantly-produced Clostridial toxins. See e.g., Steward, L.E. et al., Activatable Clostridial Toxins, U.S. Patent Publication 2009/0005313; Steward, L.E. etal., Modified Clostridia Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S. Patent Application 111776,075; Steward, L.E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity for Clostridial Toxin Target Cells, U.S. Patent Publication 2008/0241881.
[0005] To date, the conversion of the single-chain form of a recombinantly produced Clostridial toxin or modified
Clostridial toxin into its di-chain form required an in vitro activation process. First, the bacterial cells used to recombinantly produce these toxins lack the naturally-occurring protease present in the Clostridial strains that produce the native toxins. Second, there has been no great need for bacteria! cells to produce activated toxins recombinantly because of safety concerns raised in handling activated toxins. See e.g., Dolly, U.S. 7,419,676, supra, (2008). However, if these concerns could be overcome, the production of recombinantly produced activated toxins would streamline the manufacturing process of recombinantly produced Clostridial toxins or modified Clostridial toxins. For example, currently the manufacture of recombinantly produced Clostridial toxins or modified Clostridial toxins involves the following purification steps: 1) immobilized metal affinity chromatography, 2) buffer exchange dialysis, 3) protease cleavage reaction , 4), ion exchange chromatography and and 5) addition of PEG and flash freezing for storage at -80 °C. The use of a bacterial cell that can protealytically cleave the recombinant Clostridial toxin intracellularly while it is still expressing the toxin can reduce the number of purification steps to the following: 1) immobilized metal affinity chromatography, 2) buffer exchange dialysis, 3) ion exchange chromatography, and 4) addition of PEG and flash freezing for storage at-80 °C. US 2004/018589 discloses an intracellular method of converting a single-chain Clostridial toxin comprising a di-chain loop region into a di-chain form comprising the step of growing an E. coli cell comprising a construct with an ORF encoding a single-chain Clostridial toxin which has inter alia a Factor Xa protease cleavage site, together with a construct comprising an ORF encoding a Factor Xa protease.
[0006] The present specification discloses a method of converting a single-chain protein comprising a di-chain loop region into its di-chain form that does not rely on an in vitro process for converting the single-chain form of the toxin into its di-chain form. This is accomplished by the use of cells that express both the protein and the protease necessary to convert it to active di-chain.
[0007] Thus, aspects of the present specification provide, a dual expression construct that includes an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be , e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. Polynucleotides, as well as the Clostridial toxins comprising a di-chain loop region comprising an exogenous protease cleavage site that they encode, are described in, e.g., Dolly, J.O. et al., Activatable Clostridia Toxins, U.S. Patent 7,132,259; Dolly, J.O. et al., Activatable Clostridia Toxins, U.S. Patent 7,419,676. Polynucleotides, as well as the proteins comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site that they encode, are described in, e.g., Steward, L.E. et al., Multivalent Clostridial Toxins, U.S. Patent Publication 2009/0048431 ; Steward, L.E. et al., Activatable Clostridia Toxins, U.S. Patent Publication 2009/0069238; Steward, L.E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S. Patent Application 11/776,075: Steward, L. E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity for Clostridial Toxin Target Cells, U.S. Patent Publication 20D8I0241881 ; Foster, K.A. et al., Fusion Proteins, U.S. Patent Publication 2009/0035822; Foster, K.A. et al., Non-Cytotoxic Protein Conjugates, U.S. Patent Publication 2009/0162341; Steward, L.E. etal., Activatable Clostridial Toxins, U.S. Patent Publication 2008/0032931 ; Foster, K.A. et al., Non-Cytofoxic Protein Conjugates, U.S. Patent Publication 2008/0187960; Steward, L.E. et al., Degradable Clostridial Toxins, U.S. Patent Publication 2008/0213830: Steward, L.E. et al., Modified Clostridial Toxins With Enhanced Translocation Capabilities and Altered Targeting Activity For Clostridial Toxin Target Cells, U.S. Patent Publication 2008/0241881; Dolly, J.O. et al., Activatable Clostridial Toxins, U.S. Patent 7,419,676; and a companion patent application Ghanshani, et al., Modified Clostridial Toxins Comprising an Integrated Protease Cleavage Site-Binding Domain, Attorney Docket No. 18468 PROV (BOT).
[0008] Other aspects of the present specification provide a cell comprising a dual expression construct that includes an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region. In further aspects, the single-chain protein comprising a dichain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0009] Yet other aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at a first temperature for a certain period of time in order to achieve maximal cell density, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; and ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its dichain form.
[0010] Still other aspects of the present specification provide an intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of: a) growing a cell comprising a dual expression construct at 37 °C for about 2 to about 3.5 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; and ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at 22 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
[0011] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37 °C for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a singlechain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 12 to about 16 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0012] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37 °C for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a singlechain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 20 to about 24 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0013] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37 °C for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 12 to about 16 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0014] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37 °C for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 20 to about 24 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0015] Other aspects of the present specification provide, an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0016] Other aspects of the present specification provide, an expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site.
[0017] Other aspects of the present specification provide a cell comprising an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0018] Yet other aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and ii) another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the expression constructs; and wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the singlechain protein into its di-chain form.
[0019] Still other aspects of the present specification provide an intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of: a) growing at 37 °C for about 2 to about 3.5 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site and ii) another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; b) growing the cell at 22 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the singlechain Clostridial toxin and the protease from the expression constructs; and wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
[0020] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37 °C for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 12 to about 16 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0021] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37 °C for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 20 to about 24 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0022] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37 °C for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 12 to about 16 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0023] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37 °C for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 20 to about 24 °C for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the domain organization of naturally-occurring Clostridial toxins. The single chain form depicts the amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, and a Hc binding domain. The di-chain loop region located between the translocation and enzymatic domains is depicted by the double SS bracket. This region comprises an endogenous di-chain loop protease cleavage site that upon proteolytic cleavage with a naturally-occurring protease, such as, e.g., an endogenous Clostridial toxin protease or a naturally-occurring protease produced in the environment, converts the single chain form of the toxin into the di-chain form.
[0025] FIG. 2 shows a schematic of the current paradigm of neurotransmitter release and Clostridial toxin intoxication in a central and peripheral neuron. FIG. 2A shows a schematic for the neurotransmitter release mechanism of a central and peripheral neuron. The release process can be described as comprising two steps: 1) vesicle docking, where the vesicle-bound SNARE protein of a vesicle containing neurotransmitter molecules associates with the membrane-bound SNARE proteins located at the plasma membrane; and 2) neurotransmitter release, where the vesicle fuses with the plasma membrane and the neurotransmitter molecules are exocytosed. FIG. 2B shows a schematic of the intoxication mechanism for tetanus and botulinum toxin activity in a central and peripheral neuron. This intoxication process can be described as comprising four steps: 1) receptor binding, where a Clostridial toxin binds to a Clostridial receptor system and initiates the intoxication process; 2) complex internalization, where after toxin binding, a vesicle containing the toxin/receptor system complex is endocytosed into the cell; 3) light chain translocation, where multiple events are thought to occur, including, e.g., changes in the internal pH of the vesicle, formation of a channel pore comprising the HN domain of the Clostridial toxin heavy chain, separation of the Clostridial toxin light chain from the heavy chain, and release of the active light chain and 4) enzymatic target modification, where the activate light chain of Clostridial toxin proteolytically cleaves its target SNARE substrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking and neurotransmitter release.
[0026] Clostridia toxins produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii and Clostridium butyricum are the most widely used in therapeutic and cosmetic treatments of humans and other mammals. Strains of C. Botulinum produce seven antigenically-distinct types of Botulinum toxins (BoNTs), which have been identified by investigating botulism outbreaks in man(BoNT/A,/B,/Eand/F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35% amino acid identity with each other and share the same functional domain organization and overall structural architecture. It is recognized by those of skill in the art that within each type of Clostridial toxin there can be subtypes that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently four BoNT/A subtypes, B0NT/AI, BoNT/A2, BoNT/A3 and BoNT/A4, with specific subtypes showing approximately 89% amino acid identity when compared to another BoNT/A subtype. While all seven BoNT serotypes have similar structure and pharmacological properties, each also displays heterogeneous bacteriological characteristics. In contrast, tetanus toxin (TeNT) is produced by a uniform group of C. tetani. Two other Clostridia species, C. baratii and C. butyricum, produce toxins, BaNT and BuNT, which are similar to BoNT/F and BoNT/E, respectively.
[0027] Each mature di-chain molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets core components of the neurotransmitter release apparatus; 2) a translocation, domain (HN) contained within the amino-terminal half of the HC that facilitates release of the LC from intracellular vesicles into the cytoplasm of the target cell; and 3) a binding domain (Hc) found within the carboxyl-terminal half of the HC that determines the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell. The Hc domain comprises two distinct structural features of roughly equal size that indicate function and are designated the HCN and Hcc subdomains. Table 1 gives approximate boundary regions for each domain found in exemplary Clostridial toxins.
Table 1. Clostridial Toxin Reference Sequences and Regions
[0028] The binding, translocation, and enzymatic activity of these three functional domains are all necessary for toxicity. While all details of this process are not yet precisely known, the overall cellular intoxication mechanism whereby Clostridial toxins enter a neuron and inhibit neurotransmitter release is similar, regardless of serotype or subtype. Although the applicants have no wish to be limited by the following description, the intoxication mechanism can be described as comprising at least four steps: 1 ) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) enzymatic target modification (FIG. 3). The process is initiated when the Hc domain of a Clostridial toxin binds to a toxin-specific receptor system located on the plasma membrane surface of a target cell. The binding specificity of a receptor complex is thought to be achieved, in part, by specific combinations of gangliosides and protein receptors that appear to distinctly comprise each Clostridial toxin receptor complex. Once bound, the toxin/receptor complexes are internalized by endo-cytosis and the internalized vesicles are sorted to specific intracellular routes. The translocation step appears to be triggered by the acidification of the vesicle compartment. This process seems to initiate two important pH-dependent structural rearrangements that increase hydrophobicity and promote formation of the di-chain form of the toxin. Once activated, light chain endopeptidase of the toxin is released from the intracellular vesicle into the cytosol where it appears to specifically target one of three known core components of the neurotransmitter release apparatus. These core proteins, vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking and fusion at the nerve terminal and constitute members of the soluble A/-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminal region, releasing a nine or twenty-six amino acid segment, respectively, and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conserved central portion of VAMP, and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the cytosolic membrane surface. The selective proteolysis of synaptic SNAREs accounts for the block of neurotransmitter release caused by Clostridial toxins in vivo. The SNARE protein targets of Clostridial toxins are common to exocytosis in a variety of non-neuronal types; in these cells, as in neurons, light chain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al., Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic Utility, 27(11) Trends Biochem. Sei. 552-558. (2002); Gio-vanna Lalii et al., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).
[0029] In an aspect of the specification, a modified Clostridial toxin comprises, in part, a single-chain modified Clostridial toxin and a di-chain modified Clostridial toxin. As discussed above, a Clostridial toxin, whether naturally-occurring or non-naturally-occurring, are initially synthesized as a single-chain polypeptide. This single-chain form is subsequently cleaved at a protease cleavage site located within a discrete di-chain loop region created between two cysteine residues that form a disulfide bridge by a protease. This posttranslational processing yields a di-chain molecule comprising a light chain (LC) and a heavy chain. As used herein, the term "di-chain loop region" refers to loop region of a naturally-occurring or non-naturally-occurring Clostridial toxin formed by a disulfide bridge located between the LC domain and the HC domain. As used herein, the term "single-chain modified Clostridial toxin" refers to any modified Clostridial toxin disclosed in the present specification that is in its single-chain form, i.e., the toxin has not been cleaved at the protease cleavage site located within the di-chain loop region by its cognate protease. As used herein, the term "di-chain modified Clostridial toxin" refers to any modified Clostridial toxin disclosed in the present specification that is in its di-chain form, i.e., the toxin has been cleaved at the protease cleavage site located within the di-chain loop region by its cognate protease.
[0030] Aspects of the present specification provide, in part, polynucleotide molecules. As used herein, the term "polynucleotide molecule" is synonymous with "nucleic acid molecule" and means a polymeric form of nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides.ofany length. Useful polynucleotide molecules, include, without limitation, naturally-occurring and non-naturally-occurring DNA molecules and naturally-occurring and non-naturally-occurring RNA molecules. Non-limiting examples of naturally-occurring and non-naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA.
[0031] Well-established molecular biology techniques that may be necessary to make a polynucleotide molecule encoding a modified Clostridial toxin disclosed in the present specification include, but not limited to, procedures involving polymerase chain reaction (PCR) amplification, restriction enzyme reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial transformation, nucleic acid purification, nucleic acid sequencing and recombination-based techniques are routine and well within the scope of one skilled in the art and from the teaching herein. Non-limiting examples of specific protocols necessary to make a polynucleotide molecule encoding a modified Clostridial toxin are described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a variety of commercially available products useful for making a polynucleotide molecule encoding a modified Clostridial toxin are widely available. These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
[0032] The methods disclosed in the present specification include, in part, an open reading frame. As used herein, the term "open reading frame" is synonymous with "ORF" and means any polynucleotide molecule that encodes a protein, or a portion of a protein. An open reading frame usually begins with a start codon (represented as, e.g. AUG for an RNA molecule and ATG in a DNA molecule in the standard code) and is read in codon-triplets until the frame ends with a STOP codon (represented as, e.g. UAA, UGA or UAG for an RNA molecule and TAA, TGA or TAG in a DNA molecule in the standard code). As used herein, the term "codon" means a sequence of three nucleotides in a polynucleotide molecule that specifies a particular amino acid during protein synthesis; also called a triplet or codon-triplet.
[0033] The methods disclosed in the present specification include, in part, an expression construct. An expression construct comprises a polynucleotide molecule including an open reading frame disclosed in the present specification operably-linked to an expression vector useful for expressing the polynucleotide molecule in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a polynucleotide molecule disclosed in the present specification, including, without limitation, a viral expression vector; a prokaryotic expression vector; eukaryotic expression vectors, such as, e.g., a yeast expression vector, an insect expression vector and a mammalian expression vector; and a cell-free (in vitro) expression vector. It is further understood that expression vectors useful to practice aspects of these methods may include those which express the polynucleotide molecule under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, CA; BD Biosciences Pharmingen, San Diego, CA; Invitrogen, Inc, Carlsbad, CA; EMD Biosciences-Novagen, Madison, Wl; QIAGEN, Inc., Valencia, CA; and Stratagene, La Jolla, CA. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.
[0034] The expression constructs disclosed in the present specification can comprise an open reading frame encoding a protein including a di-chain loop region comprising an exogenous protease cleavage site, wherein cleavage of the exogenous protease cleavage site converts the single-chain protein into its di-chain form. In aspects of this embodiment, a viral expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; a prokaryotic expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; a yeast expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; an insect expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; and a mammalian expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop. In other aspects of this embodiment, an expression construct - suitable for expressing a polynucleotide molecule disclosed in the present specification can be expressed using a cell-free extract. In an aspect of this embodiment, a cell-free expression vector is operably linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop.
[0035] In an embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, the single-chain Clostridial toxin comprises a linear amino-to-carboxyl order of 1) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial translocation domain and the Clostridial binding domain; 2) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial binding domain and the Clostridial translocation domain; 3) the Clostridial binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin enzymatic domain; 4) the Clostridial binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin enzymatic domain and the Clostridial binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0036] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a BoNT/A toxin enzymatic domain, a BoNT/A translocation domain, a BoNT/A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a BoNT/B enzymatic domain, a BoNT/B translocation domain, a BoNT/B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain, a BoNT/C1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a BoNT/D enzymatic domain, a BoNT/D translocation domain, a BoNT/D binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a BoNT/E enzymatic domain, a BoNT/E translocation domain, a BoNT/E binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a BoNT/F enzymatic domain, a BoNT/F translocation domain, a BoNT/F binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a BoNT/G enzymatic domain, a BoNT/G translocation domain, a BoNT/G binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a TeNT enzymatic domain, a TeNT translocation domain, a TeNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 9) a BaNT enzymatic domain, a BaNT translocation domain, a BaNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 10) a BuNT enzymatic domain, a BuNT translocation domain, a BuNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0037] In further other aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a BoNT/A toxin enzymatic domain, a BoNT/A translocation domain, a BoNT/A binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 2) a BoNT/B enzymatic domain, a BoNT/B translocation domain, a BoNT/B binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 3) a BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain, a BoNT/C1 binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 4) a BoNT/D enzymatic domain, a BoNT/D translocation domain, a BoNT/D binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 5) a BoNT/E enzymatic domain, a BoNT/E translocation domain, a BoNT/E binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 6) a BoNT/F enzymatic domain, a BoNT/F translocation domain, a BoNT/F binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 7) a BoNT/G enzymatic domain, a BoNT/G translocation domain, a BoNT/G binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 8) a TeNT enzymatic domain, a TeNT translocation domain, a TeNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 9) a BaNT enzymatic domain, a BaNT translocation domain, a BaNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 10) a BuNT enzymatic domain, a BuNT translocation domain, a BuNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site.
[0038] Examples of such Clostridial toxins comprising a di-chain loop region comprising an exogenous protease cleavage sit are described in, e.g., J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Patent 7,132,529; J. Oliver Dolly, et al., Activatable Recombinanf Neurotoxins, U.S. Patent 7,419,676; Lance Steward, et al., Leucine-Based Motifs and Clostridial Neurotoxins, U.S. Patent 6,903,187; Lance Steward, et al., Leucine-Based Motifs and Clostridial Neurotoxins, U.S. Patent 7,393,925; Wei-Jen Lin, et al., Neurotoxins with Enhanced Target Specificity, U.S. Patent 7,273,722; Lance Steward, et al., Modified Botulinum Neurotoxins, U.S. Patent 7,491,799; Lance E. Steward, et al., Optimized Expression of Active Botulinum Toxin Type E, U.S. Patent Publication 200810138893; Ester Fernandez-Salas, et al., Optimized Expression of Active Botulinum Toxin Type A, U.S. Patent Publication 2008/0057575.
[0039] In another embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, the single-chain protein comprises a linear amino-to-carboxyl order of 1) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial translocation domain and the non-Clostridial binding domain; 2) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the non-Clostridial binding domain and the Clostridial translocation domain; 3) the non-Clostridial binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin enzymatic domain; 4) the non-Clostridial binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin enzymatic domain and the non-Clostridial binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the non-Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0040] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an opioid binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an enkephalin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a bovine adrenomedullary-22 (BAM22) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an endomorphin binding domain, and a dichain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an endorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a dynorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a nociceptin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a hemorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a rimorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0041] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a melanocortin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an melanocyte stimulating hormone binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an adrenocorticotropin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a lipotropin binding domain, and a dichain loop region comprising an exogenous protease cleavage site.
[0042] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin peptide binding domain, and a dichain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin message-associated peptide (GMAP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0043] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a granin peptide binding domain, and a dichain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin C binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0044] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a tachykinin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an exprès- sión construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Substance P binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide K binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide gamma binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurokinin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a hemokinin binding domain, and a dichain loop region comprising an exogenous protease cleavage site; or 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a endokinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0045] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Neuropeptide Y related peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide Y (NPY) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Peptide YY (PYY) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pancreatic peptide (PP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pancreatic icosapeptide (PIP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0046] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurohormone peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a corticotropin-releasing hormone (CCRH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a parathyroid hormone (PTH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a thyrotropin-releasing hormone (TRH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a somatostatin binding domain, and a dichain loop region comprising an exogenous protease cleavage site.
[0047] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cytokine peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a ciliary neurotrophic factor (CNTF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a glycophorin-A (GPA) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a leukemia inhibitory factor (LIF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an interleukin (IL) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an onostatin M binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cardiotrophin-1 (CT-1) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cardiotrophin-like cytokine (CLC) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuroleukin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0048] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a kinin peptide binding domain, and a dichain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a kallidin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a desArg9 bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a desArglO bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0049] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Fibroblast growth factor (FGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-9 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-17 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation, domain, a FGF-18 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0050] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurotrophin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a nerve growth factor (NGF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a brain derived neurotrophic factor (BDNF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation, domain, a neurotrophin-3 (NT-3) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurotrophin-4/5 (NT-4/5) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a head activator peptide (HA) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0051] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a tumor necrosis factor (TNF)peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0052] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Glial derived growth factor (GDNF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1 ) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurturin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a persephrin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an artemin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0053] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Transformation growth factor ß (TGFß) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFßl binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFß2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFß3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFß4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0054] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Bone morphogenetic protein ß (BMP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP5 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP6 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP7 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP10 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0055] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth differentiation factor ß (GDF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain, a GDF3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF5 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF6 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain, a GDF7 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF10 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 9) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF11 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 10) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF15 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0056] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin peptide binding domain, and a dichain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin C binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin E binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an inhibin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0057] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Vascular endothelial growth factor (VEGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0058] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain, an insulin growth factor (IGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an IGF-1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an IGF-2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0059] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an Epidermal growth factor (EGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0060] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Glucagon like hormone peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a secretin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a glucagonlike peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0061] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pituitary adenylate cyclase activating peptide (PACAP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0062] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth hormone-releasing hormone (GH-RH) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0063] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth hormone-releasing hormone (GH-RH) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0064] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Vasoactive intestinal peptide (VIP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression constructcomprisesanopen reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a VIP1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a VIP2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0065] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Gastric inhibitory polypeptide (GIP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0066] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Calcitonin-related peptidesvisceral gut peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a gastrin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a gastrin-releasing peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cholecystokinin (CCK) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0067] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a protease activated receptor (PAR) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression constructcomprisesanopen readingframeencoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0068] Examples, of such proteins comprising a di-chain loop region comprising an exogenous protease cleavage site are described in, e.g., J. Oliver Dolly, etal., Activatable Recombinant Neurotoxins, U.S. Patent 7,132,529; J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Patent 7,419,676; Lance E. Steward et al., Multivalent Clostridial Toxin Derivativesand Methods of Their Use, U.S. Patent 7,514,088; Keith A. Foster etal., Re-targeted Toxin Conjugates, International Patent Publication WO 2005/023309: Lance E. Steward, et al., Activatable Recombinant Neurofoxins, U.S. Patent Publication 2008/0032930; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 200810032931: Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0161226: Lance E, Steward, etal., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0221012; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0004224: Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0005313: Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 200910018081; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0069238; and Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. Patent Publication 2009/0048431.
[0069] In another embodiment, an expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain. In aspects of this embodiment, the single-chain protein comprises a linear amino-to-carboxyl order of 1) an integrated protease cleavage site-binding domain, a Clostridial toxin translocation domain and a Clostridial toxin enzymatic domain; 2) an integrated protease cleavage site-binding domain, a Clostridial toxin enzymatic domain, and a Clostridial toxin translocation domain; 3) a Clostridial toxin enzymatic domain, an integrated protease cleavage sitebinding domain, and a Clostridial toxin translocation domain; 4) a Clostridial toxin translocation domain, an integrated protease cleavage site-binding domain, and a Clostridial toxin enzymatic domain; 5) a Clostridial toxin translocation domain, a Clostridial toxin enzymatic domain, and an integrated protease cleavage site-binding domain; and 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain.
[0070] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-opioid binding domain. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-enkephafin binding domain; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-bovine adrenomedullary-22 (BAM22) binding domain; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-endomorphin binding domain; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-endorphin binding domain; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-dynorphin binding domain; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-nociceptin binding domain; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-hemorphin binding domain; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-rimorphin binding domain [0071] Examples, of such proteins comprising integrated protease cleavage site-binding domain are described in, e.g., companion patent application Sanjiv Ghanshani, et al., Modified Clostridial Toxins Comprising an Integrated Protease Cleavage Site-Binding Domain.
[0072] The expression constructs disclosed in the present specification can comprise an open reading frame encoding a protease. In aspects of this embodiment, a viral expression vector is operably-linked to a polynucleotide molecule encoding a protease; a prokaryotic expression vector is operably-linked to a polynucleotide molecule encoding a protease; a yeast expression vector is operably-linked to a polynucleotide molecule encoding a protease; an insect expression vector is operably-linked to a polynucleotide molecule encoding a protease; and a mammalian expression vector is operably-linked to a polynucleotide molecule encoding a protease. In other aspects of this embodiment, an expression construct is suitable for expressing a polynucleotide molecule disclosed in the present specification can be expressed using a cell-free extract. In an aspect of this embodiment, a cell-free extract expression vector is operably linked to a polynucleotide molecule encoding a protease.
[0073] In aspect of this embodiment, an expression construct comprising an open reading frame encodes an enter-okinase, a human rhinovirus 3C protease, a human enterovirus 3C protease, a tobacco etch virus (TEV) protease, a Tobacco Vein Mottling Virus (TVMV) protease, a subtilisin protease, or a Caspase 3 protease. Examples of Enterokinase proteases and the polynucleotide molecules that encode them are described in, e.g., Edward R. LaValiie, Cloning of Enterokinase and Method of Use, U.S. Patent 5,665,566; Edward R. LaValiie, Cloning of Enterokinase and Method of Use, U.S. Patent 6,746,859. Examples of subtilisin proteases and the polynucleotide molecules that encode them are described in, e.g., Donn N. Rubingh, et al., Subtillisin Protease Variants having Amino Acid Deletions and Substitutions in Defined Epitope Regions, U.S. Patent 6,586,224.
[0074] In another aspect of this embodiment, an enterokinase is SEQ ID NO: 11. In another aspect of this embodiment, an enterokinase comprises amino acids 239-1035 of SEQ ID NO: 11. In yet another aspect of this embodiment, an enterokinase is a naturally occurring enterokinase variant, such as, e.g., an enterokinase isoform. In still another aspect of this embodiment, an enterokinase is a non-naturally occurring enterokinase variant, such as, e.g., a conservative enterokinase variant, a non-conservative enterokinase variant, an enterokinase chimeric, an active enterokinase fragment, or any combination thereof. In another aspect of this embodiment, an Enterokinase is one disclosed in U.S. Patent 5,665,566 or U.S. Patent 6,746,859. In another aspect of this embodiment, an enterokinase, a naturally occurring enterokinase variant, or a non-naturally occurring enterokinase variant is obtained from a species of mammal such as, e.g., a human, a cow, or a rodent.
[0075] In other aspects of this embodiment, an enterokinase comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 11 ; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 11. In yet other aspects of this embodiment, an enterokinase comprises a polypeptide having, e.g., at least 1,2, 3. 4, 5. 6, 7, 8, 9, 10, 20. 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11. In still other aspects of this embodiment, an enterokinase comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11.
[0076] In another aspect of this embodiment, a human rhinovirus3C protease is SEQ ID NO: 12. In yet another aspect of this embodiment, a human rhinovirus 3C protease is a naturally occurring human rhinovirus 3C protease variant, such as, e.g., a human rhinovirus 3C protease isoform. In still another aspect of this embodiment, a human rhinovirus 3C protease is a non-naturally occurring human rhinovirus 3C protease variant, such as, e.g., a conservative human rhinovirus 3C protease variant, a non-conservative human rhinovirus 3C protease variant, a human rhinovirus 3C protease chimeric, an active human rhinovirus 3C protease fragment, or any combination thereof. In another aspect of this embodiment, a human rhinovirus 3C protease, a naturally occurring human rhinovirus 3C protease variant, or a non-naturally occurring human rhinovirus 3C protease variant is obtained from a species of Rhinovirus.
[0077] In other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 12. In yet other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12. In still other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12.
[0078] In another aspect of this embodiment, a human enterovirus 3C protease is SEQ ID NO: 13. In yet another aspect of this embodiment, a human enterovirus 3C protease is a naturally occurring human enterovirus 3C protease variant, such as, e.g., a human enterovirus 3C protease isoform. In still another aspect of this embodiment, a human enterovirus 3C protease is a non-naturally occurring human enterovirus 3C protease variant, such as, e.g., a conservative human enterovirus 3C protease variant, a non-conservative human enterovirus 3C protease variant, a human enterovirus 3C protease chimeric, an active human enterovirus 3C protease fragment, or any combination thereof. In another aspect of this embodiment, a human enterovirus 3C protease, a naturally occurring human enterovirus 3C protease variant, or a non-naturally occurring human enterovirus 3C protease variant is obtained from a species of Enterovirus.
[0079] In other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 13; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 13. In yet other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13. In still other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13; or at most 1,2,3,4,5,6,7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13.
[0080] In another aspect of this embodiment, a TEV protease is SEQ ID NO: 14. In another aspect of this embodiment, a TEV protease comprises amino acids 2038-2270 of SEQ IS NO: 14. In another aspect of this embodiment, a TEV protease comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another aspect of this embodiment, a TEV protease is a naturally occurring TEV protease variant, such as, e.g., a TEV protease isoform. In still another aspect of this embodiment, a TEV protease is a non-naturally occurring TEV protease variant, such as, e.g., a conservative TEV protease variant, a non-conservative TEV protease variant, a TEV protease chimeric, an active TEV protease fragment, or any combination thereof. In another aspect of this embodiment, a TEV protease, a naturally occurring TEV protease variant, or a non-naturally occurring TEV protease variant is obtained from a species of Potyvirus.
[0081] In other aspects of this embodiment, a TEV protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet other aspects of this embodiment, a TEV protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 1,2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still other aspects of this embodiment, a TEV protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
[0082] In another aspect of this embodiment, a TVMV protease is SEQ ID NO: 24. In another aspect of this embodiment, a TEV protease comprises amino acids 2002-2236 of SEQ IS NO: 24. In yet another aspect of this embodiment, a TVMV protease is a naturally occurring TVMV protease variant, such as, e.g., a TVMV protease isoform. In still another aspect of this embodiment, a TVMV protease is a non-naturally occurring TVMV protease variant, such as, e.g., a conservative TVMV protease variant, a non-conservative TVMV protease variant, a TVMV protease chimeric, an active TVMV protease fragment, or any combination thereof. In another aspect of this embodiment, a TVMV protease, a naturally occurring TVMV protease variant, or a non-naturally occurring TVMV protease variant is obtained from a species of Potyvirus.
[0083] In other aspects of this embodiment, a TVMV protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24. In yet other aspects of this embodiment, a TVMV protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24. In still other aspects of this embodiment, a TVMV protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 1,2,3,4,5,6,7,8,9,10,20,30,40,50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24.
[0084] In another aspect of this embodiment, a subtilisin protease is SEQ ID NO: 25. In another aspect of this embodiment, a subtilisin protease comprises amino acids 107-365 of SEQ IS NO: 25. In yet another aspect of this embodiment, a subtilisin protease is a naturally occurring subtilisin protease variant, such as, e.g., a subtilisin protease isoform. In still another aspect of this embodiment, a subtilisin protease is a non-naturally occurring subtilisin protease variant, such as, e.g., a conservative subtilisin protease variant, a non-conservative subtilisin protease variant, a subtilisin protease chimeric, an active subtilisin protease fragment, or any combination thereof. In another aspect of this embodiment, a subtilisin protease, a naturally occurring subtilisin protease variant, ora non-naturally occurring subtilisin protease variant is obtained from a species of Bacillus.
[0085] In other aspects of this embodiment, a subtilisin protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25. In yet other aspects of this embodiment, a subtilisin protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25. In still other aspects of this embodiment, a subtilisin protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25.
[0086] In another aspect of this embodiment, a Caspase 3 protease is SEQ ID NO: 26. In yet another aspect of this embodiment, a Caspase 3 protease is a naturally occurring Caspase 3 protease variant, such as, e.g., a Caspase 3 protease isoform. In still another aspect of this embodiment, a Caspase 3 protease is a non-naturally occurring Caspase 3 protease variant, such as, e.g., a conservative Caspase 3 protease variant, a non-conservative Caspase 3 protease variant, a Caspase 3 protease chimeric, an active Caspase 3 protease fragment, or any combination thereof. In another aspect of this embodiment, a Caspase 3 protease, a naturally occurring Caspase 3 protease variant, or a non-naturally occurring Caspase 3 protease variant is obtained from a species of mammal such as, e.g., a human, a cow, or a rodent.
[0087] In other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 26; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 26. In yet other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26. In still other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26.
[0088] The methods disclosed in the present specification include, in part, a dual expression construct. A dual expression construct comprises two polynucleotide molecules, each including an open reading frame disclosed in the present specification operably-linked to an expression vector useful for expressing both polynucleotide molecules in a cell or cell-free extract. A wide variety of dual expression vectors can be employed for expressing a polynucleotide molecule disclosed in the present specification, including, without limitation, a viral dual expression vector; a prokaryotic dual expression vector; an eukaryotic dual expression vector, such as, e.g., a yeast dual expression vector, an insect dual expression vector and a mammalian dual expression vector; and a cell-free extract dual expression vector. It is further understood that dual expression vectors useful to practice aspects of these methods may include those which express the polynucleotide molecules under the control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of dual expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, EMD Biosciences-Novagen, Madison, Wl. The selection, making and use of an appropriate dual expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.
[0089] The dual expression constructs disclosed in the present specification can comprise an open reading frame encoding a protein including a di-chain loop region comprising an exogenous protease cleavage site and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop, thereby converting the single-chain protein into its di-chain form.
[0090] Thus, in an embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop as disclosed in the present specification.
[0091] In an aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, a di-chain loop region, and a TEV protease cleavage site, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0092] In an aspect of this embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop region. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, a di-chain loop region, and a TEV protease cleavage site, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0093] In an aspect of this embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-binding domain and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0094] The location of one of the open reading frames contained within the dual expression construct can be in any order relative to the location of the other open reading frame, with the proviso that transcription from both open reading frames can still occur. When a dual expression construct is made, transcriptional initiation from the first promoter region typically transcribes both open reading frames, whereas, transcriptional initiation from the second promoter region typically transcribes only one of the open reading frames. Thus, depending on the location of the open reading frame relative to the first and second promoter regions, twice as many transcripts can be made from one of the open reading frames.
[0095] Thus, in one embodiment, the open reading frame encoding a protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site is under the control of both the first promoter and second promoter regions. In an aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a Clostridial toxin comprising a TEV protease cleavage site located within the di-chain loop region is under the control of both the first promoter and second promoter regions. In another aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site is under the control of both the first promoter and second promoter regions. In yet another aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain is under the control of both the first promoter and second promoter regions.
[0096] In another embodiment, the open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a protease is under the control of both the first promoter and second promoter regions. In an aspect of this embodiment, the open reading frame encoding a Clostridial toxin comprising a di-chain loop region comprising a TEV protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions. In another aspect of this embodiment, the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions. In yet another aspect of this embodiment, the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions.
[0097] The 5’-3’ orientation of one of the open reading frames contained within the dual expression construct can be in any direction relative to the 5’-3’ orientation of the other open reading frame, with the proviso that transcription from both open reading frames can still occur. In one embodiment, the 5’-3’ orientation of one of the open reading frames is in the same direction as the 5’-3’ orientation of the other open reading frame. In another embodiment, the 5’-3’ orientation of one of the open reading frames is in the opposite direction as the 5’-3’ orientation of the other open reading frame. In an aspect of this embodiment, the 5’-3’ orientation of one of the open reading frames is convergent relative to the 5’-3’ orientation of the other open reading frame. In another aspect of this embodiment, the 5’-3’ orientation of one of the open reading frames is divergent relative to the 5’-3’ orientation of the other open reading frame.
[0098] The methods disclosed in the present specification include, in part, a protein comprising a di-chain loop region comprising an exogenous protease cleavage site. As used herein, the term "di-chain loop region" means the amino acid sequence of a Clostridial toxin containing a protease cleavage site used to convert the single-chain form of a Clostridial toxin into the di-chain form. Non-limiting examples of a Clostridial toxin di-chain loop region, include, a di-chain loop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1 ; a di-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; a di-chain loop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8; a di-chain loop region of BaNT comprising amino acids 421-435 of SEQ ID NO: 9; and a di-chain loop region of BuNT comprising amino acids 412-426 of SEQ ID NO: 10 (Table 2).
Table 2. Di-chain Loop Region of Clostridial Toxins
[0099] As mentioned above, Clostridial toxins are translated as a single-chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease. This post-translational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulphide bond and noncovalent interactions. While the identity of the protease is currently unknown, the di-chain loop protease cleavage site for many Clostridial toxins has been determined. In BoNTs, cleavage at K448-A449 converts the single polypeptide form of BoNT/A into the di-chain form; cleavage at K441-A442 converts the single polypeptide form of BoNT/B into the di-chain form; cleavage at K449-T450 converts the single polypeptide form of BoNT/C1 into the di-chain form; cleavage at R445-D446 converts the single polypeptide form of BoNT/D into the di-chain form; cleavage at R422-K423 converts the single polypeptide form of BoNT/E into the di-chain form; cleavage at K439-A440 converts the single polypeptide form of BoNT/F into the di-chain form; and cleavage at K446-S447 converts the single polypeptide form of BoNT/G into the di-chain form. Proteolytic cleavage of the single polypeptide form of TeNT at A457-S458 results in the di-chain form. Proteolytic cleavage of the single polypeptide form of BaNT at K431-N432 results in the di-chain form. Proteolytic cleavage of the single polypeptide form of BuNT at R422-K423 results in the di-chain form. Such a di-chain loop protease cleavage site is operably-linked in-frame to a modified Clostridial toxin as a fusion protein. However, it should also be noted that additional cleavage sites within the di-chain loop also appear to be cleaved resulting in the generation of a small peptide fragment being lost. As a non-limiting example, cleavage of a BoNT/A single-chain polypeptide ultimately results in the loss of a ten amino acid fragment within the di-chain loop.
[0100] It is envisioned that any molecule that comprises a di-chain loop region can be modified to include an exogenous protease cleavage site useful forthe disclosed methods. Examples of molecules that can have the di-chain loop modified to include an exogenous protease cleavage site useful for the disclosed methods include, e.g., Keith A. Foster et al., Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent Functions, U.S. Patent 5,989,545; Clifford C. Shone et al., Recombinant Toxin Fragments, U.S. Patent 6,461,617; Conrad P. Quinn et al., Methods and Compounds forthe Treatment of Mucus Hypersecretion, U.S. Patent 6,632,440; Lance E. Steward et al., Methods And Compositions For The Treatment Of Pancreatitis, U.S. Patent 6,843,998; Stephan Donovan, Clostridial Toxin Derivativesand Methods For Treading Pain, U.S. Patent 7,244,437; Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Patent7,413,742; Stephan Donovan, Clostridial Toxin Derivativesand Methods ForTreating Pain, U.S. Patent 7,425,338.
[0101] A di-chain loop region is modified by the addition of an exogenous protease cleavage site. As used herein, the term "exogenous protease cleavage site" is synonymous with a "non-naturally occurring protease cleavage site’ or "non- native protease cleavage site" and refers to a protease cleavage site that is not normally present in a di-chain loop region from a naturally occurring Clostridial toxin. It is envisioned that any and all exogenous protease cleavage sites that can be used to convert the single-chain polypeptide form of a Clostridial toxin into the di-chain form are useful to practice aspects of the present invention. Non-limiting examples of exogenous protease cleavage sites include, e.g., an enter-okinase protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a tobacco etch virus (TEV) protease cleavage site, a Tobacco Vein Mottling Virus (TVMV) protease cleavage site, a subtilisin protease cleavage site, or a Caspase 3 protease cleavage site.
[0102] It is envisioned that an exogenous protease cleavage site of any and all lengths can be useful in aspects of the present invention with the proviso that the exogenous protease cleavage site is capable of being cleaved by its respective protease. Thus, in aspects of this embodiment, an exogenous protease cleavage site can have a length of, e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 amino acids; or at most 6, 7. 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 amino acids.
[0103] In an embodiment, a di-chain loop region comprises an exogenous protease cleavage site. In aspects of this embodiment, a di-chain loop region is modified to comprise, e.g., an enterokinase protease cleavage site, a Tobacco Etch Virus protease cleavage site, a Tobacco Vein Mottling Virus protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a subtilisin cleavage site, and a Caspase 3 cleavage site. In other aspects of this embodiment, an exogenous protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, an exogenous protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0104] In an aspect of this embodiment, a di-chain loop region comprises a Tobacco Etch Virus protease cleavage site having the consensus sequence E-P5-P4-Y-P2-CT-G (SEQ ID NO: 27) or E-P5-P4-Y-P2-CT-S (SEQ ID NO: 28), where P2, P4 and P5 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a Tobacco Etch Virus protease cleavage site comprising SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38. In still other aspects of this embodiment, a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0105] In another aspect of this embodiment, a di-chain loop region comprises a Tobacco Vein Mottling Virus protease cleavage site having the consensus sequence P6-P5-V-R-F-Q*-G (SEQ ID NO: 39) or P6-P5-V-R-F-Q*-S (SEQ ID NO: 40), where P5 and P6 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a Tobacco Vein Mottling Virus protease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. In still other aspects of this embodiment, a Tobacco Vein Mottling Virus protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, ora BuNT. In other aspects of this embodiment, a Tobacco Vein Mottling Virus protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0106] In yet another aspect of this embodiment, a di-chain loop region comprises a human rhinovirus 3C protease cleavage site having the consensus sequence P5-P4-L-F-Q*-G-P (SEQ ID NO: 45), where P4 is G, A, V, L, I, M, S or T and P5 can any amino acid, with D or E preferred. In other aspects of the embodiment, a di-chain loop region comprises a human rhinovirus 3C protease cleavage site comprising SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In still other aspects of this embodiment, a human rhinovirus 3C protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a human rhinovirus 3C protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0107] In still another aspect of this embodiment, a di-chain loop region comprises a subtilisin protease cleavage site having the consensus sequence P6-P5-P4-P3-H*-Y (SEQ ID NO: 52) or P6-P5-P4-P3-Y-H* (SEQ ID NO: 53), where P3, P4 and P5 and P6 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a subtilisin protease cleavage site comprising SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. In still other aspects of this embodiment, a subtilisin protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, ora BuNT. In other aspects of this embodiment, a subtilisin protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0108] In a further aspect of this embodiment, a di-chain loop region comprises a Caspase 3 protease cleavage site having the consensus sequence D-P3-P2-D*P1’ (SEQ ID NO: 57), where P3 can be any amino acid, with E preferred, P2 can be any amino acid and P1 ’ can any amino acid, with G or S preferred. In other aspects of the embodiment, a dichain loop region comprises a Caspase 3 protease cleavage site comprising SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In still other aspects of this embodiment, a Caspase 3 protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, ora BuNT. In other aspects of this embodiment, a Caspase 3 protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0109] In yet another aspect of this embodiment, a di-chain loop region comprises an enterokinase protease cleavage site having the consensus sequence DDDDK (SEQ ID NO: 64). In other aspects of this embodiment, an enterokinase protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In yet other aspects of this embodiment, an enterokinase protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Patent 5,989,545; U.S. Patent 6,461,617; U.S. Patent 6,632,440; U.S. Patent 6,843,998; U.S. Patent 7,244,437; U.S. Patent 7,413,742; and U.S. Patent 7,425,338.
[0110] Ad i-chain loop region is modified to replace a naturally-occurring di-chain loop protease cleavage site for an exogenous protease cleavage site. In this modification, the naturally-occurring di-chain loop protease cleavage site is made inoperable and thus can not be cleaved by its protease. Only the exogenous protease cleavage site can be cleaved by its corresponding exogenous protease. In this type of modification, the exogenous protease site is operably-linked in-frame to a modified Clostridial toxin as a fusion protein and the site can be cleaved by its respective exogenous protease. Replacement of an endogenous di-chain loop protease cleavage site with an exogenous protease cleavage site can be a substitution of the sites where the exogenous site is engineered at the position approximating the cleavage site location of the endogenous site. Replacement of an endogenous di-chain loop protease cleavage site with an exogenous protease cleavage site can be the addition of an exogenous site where the exogenous site is engineered at a position different from the cleavage site location of the endogenous site, the endogenous site being engineered to be inoperable.
[0111] A naturally-occurring protease cleavage site contained within the di-chain loop region can be made inoperable by altering at least the two amino acids flanking the peptide bond cleaved by the naturally-occurring di-chain loop protease. More extensive alterations can be made, with the proviso that the two cysteine residues of the di-chain loop region remain intact and the region can still form a disulfide bridge. Non-limiting examples of an amino acid alteration include deletion of an amino acid or replacement of the original amino acid with a different amino acid. Thus, in one embodiment, a naturally-occurring protease cleavage site contained within the di-chain loop region is made inoperable by altering the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease. In other aspects of this embodiment, a naturally-occurring protease cleavage site contained within the di-chain loop region is made inoperable by altering, e.g., at least three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at least 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.
[0112] In still other aspects of this embodiment, a naturally-occurring di-chain protease cleavage site contained within the di-chain loop region is made inoperable by altering, e.g., at most three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at most 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.
[0113] The methods disclosed in the present specification include, in part, a cell. It is envisioned that any and all cells can be used. Thus, aspects of this embodiment include, without limitation, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroídes fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fiuorescens and Salmonella typhimurium; and eukaryotic cells including, without limitation, yeast strains, such as, e.g., those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and mammalian cells and cell lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human. Cell lines may be obtained from the American Type Culture Collection, European Collection of Cell Cultures and the German Collection of Microorganisms and Cell Cultures. Non-limiting examples of specific protocols for selecting, making and using an appropriate cell line are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer Academic Publishers, 1996) ; Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997) ; CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons, 1998) ; R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4th ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press, 3rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2nd ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.
[0114] The methods disclosed in the present specification include, in part, introducing into a cell an expression construct or dual expression construct as disclosed in the present specification. An expression construct or dual expression construct introduced into a cell can be transiently or stably maintained by that cell. Stably-maintained expression constructs or dual expression constructs may be extra-chromosomal and replicate autonomously, or they may be integrated into the chromosomal material of the cell and replicate non-autonomously. It is envisioned that any and all methods for introducing an expression construct or a dual expression construct disclosed in the present specification into a cell can be used. Methods useful for introducing an expression constructor a dual expression construct into a cell include, without limitation, chemical-mediated transfection such as, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE) dex-tran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated tranfection, such as, e.g., biolistic particle delivery, microinjection, protoplast fusion and electroporation; and viral-mediated transfection, such as, e.g., retroviral-mediated transfection, see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3rd ed. 2001). One skilled in the art understands that selection of a specific method to introduce an expression construct or a dual expression construct into a cell will depend, in part, on whether the cell will transiently contain the expression construct or dual expression construct, or whether the cell will stably contain the expression construct or dual expression construct. These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.
[0115] In an aspect of this embodiment, a chemical-mediated method, termed transfection, is used to introduce an expression construct ora dual expression constructdisclosed in the presentspecification into a cell. In chemical-mediated methods of transfection the chemical reagentforms a complex with the expression construct or dual expression construct that facilitates its uptake into the cells. Such chemical reagents include, without limitation, calcium phosphate-mediated, see, e.g., Martin Jordan & Florian Worm, Transfection of adherent and suspended cells by calcium phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationic polymer-mediated like polyethyleneimine (PEI)-mediated and polylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine strategies for plasmid delivery to brain-derived cells, 33(2) Methods 144-150 (2004). Such chemical-mediated delivery systems can be prepared by standard methods and are commercially available, see, e.g., CellPhect Transfection Kit(Amersham Biosciences, Piscataway, NJ); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc., La Jolla, CA); Lipofectamine™ Transfection Reagent (Invitrogen, Inc., Carlsbad, CA); ExGen 500 Transfection kit (Fermentas, Inc., Hanover, MD), and SuperFect and Effectene Transfection Kits (Qiagen, Inc., Valencia, CA).
[0116] In another aspect of this embodiment, a physically-mediated method is used to introduce an expression construct or a dual expression construct disclosed in the present specification into a cell. Physical techniques include, without limitation, electroporation, biolistic and microinjection. Biolistics and microinjection techniques perforate the cell wall in order to introduce the expression construct or dual expression construct into the cell, see, e.g., Jeike E. Biewenga et al., Plasmid-mediated gene transfer in neurons using the biolistics technique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O’Brien & Sarah C. R. Lummis, Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells, 33(2) Methods 121-125 (2004). Electroporation, also termed electropermeabiliza-tion, uses brief, high-voltage, electrical pulses to create transient pores in the membrane through which the polynucleotide molecules enter and can be used effectively for stable and transient transfections of all cell types, see, e.g., M. Golzio etal., In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression, 33(2) Methods 126-135 (2004); and Oliver Greschetal., Newnon-viral method for gene transfer into primary cells, 33(2) Methods 151-163 (2004).
[0117] In another aspect of this embodiment, a viral-mediated method, termed transduction, is used to introduce an expression construct or a dual expression construct disclosed in the present specification into a cell. In viral-mediated methods of transient transduction, the process by which viral particles infect and replicate in a host cell has been manipulated in order to use this mechanism to introduce the expression construct or dual expression construct into the cell. Viral-mediated methods have been developed from a wide variety of viruses including, without limitation, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); and Maurizio Federico, From lentiviruses to lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M. Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); Karim Benihoud et al, Adenovirus vectors for gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler, Adeno-associated viral vectors for gene transfer and gene therapy, 380(6) Biol. Chem. 613-622(1999); ChooiM. Lai etal., Adenovirus and adeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002); Edward A. Burton et al., Gene delivery using herpes simplex virus vectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al., Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); Ilya Frolov et al., Alphavirus-based expression vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sei. U. S. A. 11371-11377 (1996); Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1) Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4) Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann, Bacufovirus vectors: novel mammalian cell gene-delivery vehicles and their applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003), [0118] Adenoviruses, which are non-enveloped, double-stranded DNA viruses, are often selected for mammalian cell transduction because adenoviruses handle relatively large polynucleotide molecules of about 36 kb, are produced at high titer, and can efficiently infect a wide variety of both dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermens et al., Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci. Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches for generating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction using adenoviral-based system do not support prolonged protein expression because the nucleic acid molecule is carried by an episome in the cell nucleus, rather than being integrated into the host cell chromosome. Adenoviral vector systems and specific protocols for how to use such vectors are disclosed in, e.g., VIRAPOWER™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad, CA) and VIRAPOWER™ Adenoviral Expression System Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and ADEASY™ Adenoviral Vector System (Stratagene, Inc., La Jolla, CA) and ADEASY™ Adenoviral Vector System Instruction Manual 064004Í, Stratagene, Inc..
[0119] Introduction of an expression construct or dual expression construct disclosed in the present specification into a cell can also be achieved using single-stranded RNA retroviruses, such as, e.g., oncoretroviruses and lentiviruses. Retroviral-mediated transduction often produce transduction efficiencies close to 100%, can easily control the proviral copy number by varying the multiplicity of infection (MOI), and can be used to either transiently or stably transduce cells, see, e.g., Tiziana Tonini et al., Transient production of retroviral- and lentiviral-based vectors for the transduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004); Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); Félix Recillas-Targa, Gene transfer and expression in mammalian cell lines and transgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and Roland Wolkowicz et al., Lentiviral vectors for the delivery of DNA into mammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviral particles consist of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. The retrovirus infects a host cell by injecting its RNA into the cytoplasm along with the reverse transcriptase enzyme. The RNA template is then reverse transcribed into a linear, double stranded cDNAthat replicates itself by integrating into the host cell genome. Viral particles are spread both vertically (from parent cell to daughter cells via the provirus) as well as horizontally (from cell to cell via virions). This replication strategy enables long-term persistent expression since the nucleic acid molecules of interest are stably integrated into a chromosome of the host cell, thereby enabling long-term expression of the protein. For instance, animal studies have shown that lentiviral vectors injected into a variety of tissues produced sustained protein expression for more than 1 year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector, 272(5259) Science 263-267 (1996). The Oncoretroviruses-derived vector systems, such as, e.g., Moloney murine leukemia virus (MoMLV), are widely used and infect many different non-dividing cells. Lentiviruses can also infect many different cell types, including dividing and non-dividing cells and possess complex envelope proteins, which allows for highly specific cellular targeting.
[0120] Retroviral vectors and specific protocols for how to use such vectors are disclosed in, e.g., Manfred Gossen &
Hermann Bujard, Tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters, U.S. Patent 5,464,758, Hermann Bujard & Manfred Gossen, Methods for regulating gene expression, U.S. Patent 5,814,618, David S. Hogness, Polynucleotides encoding insect steroid hormone receptor polypeptides and cells transformed with same, U.S. Patent 5,514,578, and David S. Hogness, Polynucleotide encoding insectecdysonereceptor, U.S. Patent6,245,531 ; Elisabetta Vegeto et al., Progesterone receptor having C. terminal hormone binding domain truncations, U.S. Patent 5,364,791" Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Patent 5,874,534, and Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Patent 5,935,934. Furthermore, such viral delivery systems can be prepared by standard methods and are commercially available, see, e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto, CA) and BD™ Tet-Off and Tet-On Gene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003), GENESWITCH™ System (Invitrogen, Inc., Carlsbad, CA) and GENESWITCH™ System A Mifepristone-Regulated Expression System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad, CA) and VIRAPOWER™ Lentiviral Expression System Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System (Stratagene, La Jolla, CA) and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System Instruction Manual, 064005e.
[0121] The methods disclosed in the present specification include, in part, expressing an expression construct or dual expression construct disclosed in the present specification. It is envisioned that any of a variety of expression systems may be useful for expressing an expression construct or a dual expression construct disclosed in the present specification, including, without limitation, cell-based systems, and cell-free expression systems. Cell-based systems include, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Cell-free systems include, without limitation, wheat germ extracts, rabbit reticulocyte extracts and E. coli extracts and generally are equivalent to the method disclosed herein. Expression of an expression construct or dual expression construct using an expression system can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, viral-mediated expression, stably-integrated expression, and transient expression. Expression systems that include well-characterized vectors, reagents, conditions and cells are well-established and are readily available from commercial vendors that include, without limitation, Ambion, Inc. Austin, TX; BD Biosciences-Clontech, Palo Alto, CA; BD Biosciences Pharmingen, San Diego, CA; Invitrogen, Inc, Carlsbad, CA; QIAGEN, Inc., Valencia, CA; Roche Applied Science, Indianapolis, IN; and Stratagene, La Jolla, CA Non-limiting examples on the selection and use of appropriate heterologous expression systems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Harnes eds., Oxford University Press, 1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh, Expression Systemsfor Production of Heterologous Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001 ). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
[0122] A variety of cell-based expression procedures are useful for expressing an expression construct or a dual expression construct disclosed in the present specification. Examples included, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Viral expression systems include, without limitation, the VIRAPOWER™ Lentiviral (Invitrogen, Inc., Carlsbad, CA), the Adenoviral Expression Systems (Invitrogen, Inc., Carlsbad, CA), the ADEASY™ XL Adenoviral Vector System (Stratagene, La Jolla, CA) and the VIRAPORT® Retroviral Gene Expression System (Stratagene, La Jolla, CA). Non-limiting examples of prokaryotic expression systems include the CHAMPION™ pET Expression System (EMD Biosciences-Novagen, Madison, Wl), the TRIEX™ Bacterial Expression System (EMD Biosciences-Novagen, Madison, Wl), the QIAEXPRESS® Expression System (QIAGEN, Inc.), and the AFFINITY® Protein Expression and Purification System (Stratagene, La Jolla, CA). Yeast expression systems include, without limitation, the EASYSELECT™ Pichia Expression Kit (Invitrogen, Inc., Carlsbad, CA), the YES-ECHO™ Expression Vector Kits (Invitrogen, Inc., Carlsbad, CA) and the SPECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad, CA). Non-limiting examples of baculoviral expression systems include the BACULODIRECT™ (Invitrogen, Inc., Carlsbad, CA), the BAC-TO-BAC® (Invitrogen, Inc., Carlsbad, CA), and the BD BACULOGOLD™ (BD Biosciences-Pharmigen, San Diego, CA). Insect expression systems include, without limitation, the Drosophila Expression System (DES®) (Invitrogen, Inc., Carlsbad, CA), INSECTSELECT™ System (Invitrogen, Inc., Carlsbad, CA) and INSECTDIRECT™ System (EMD Biosciences-Novagen, Madison, Wl). Non-limiting examples of mammalian expression systems include the T-REX™ (Tetracycline-Regulated Expression) System (Invitrogen, Inc., Carlsbad, CA), the FLP-IN™ T-REX™ System (Invitrogen, Inc., Carlsbad, CA), the pcDNA™ system (Invitrogen, Inc., Carlsbad, CA), the pSecTag2 system (Invitrogen, Inc., Carlsbad, CA), the EXCHANGER® System, INTERPLAY™ Mammalian TAP System (Stratagene, La Jolla, CA), COMPLETE CONTROL® Inducible Mammalian Expression System (Stratagene, La Jolla, CA) and LACSWITCH® II Inducible Mammalian Expression System (Stratagene, La Jolla, CA).
[0123] Another procedure of expressing an expression construct or a dual expression construct disclosed in the present specification employs a cell-free expression system such as, without limitation, prokaryotic extracts and eukaryotic extracts. Non-limiting examples of prokaryotic cell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science, Indianapolis, IN), the ACTIVEPRO™ In Vitro Translation Kit (Ambion, Inc., Austin, TX), the ECOPRO™ System (EMD Biosciences-Novagen, Madison, Wl) and the Expressway™ Plus Expression System (Invitrogen, Inc., Carlsbad, CA). Eukaryotic cell extract include, without limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science, Indianapolis, IN), the TNT® Coupled Wheat Germ Extract Systems (Promega Corp., Madison, Wl), the Wheat Germ IVT™ Kit (Ambion, Inc., Austin, TX), the Retie Lysate IVT™ Kit (Ambion, Inc., Austin, TX), the PROTEINSCRIPT® II System (Ambion, Inc., Austin, TX) and the TNT® Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wl).
[0124] The methods disclosed in the present specification include, in part, growing a cell at a first temperature for a certain period of time and then growing the cell at a second temperature for a certain period of time. The first and second temperatures and the periods of time the cells are grown at the first and second temperatures are determined based on the desired amount of protein to be expressed by the cell, and the desired cleavage efficiency at the exogenous protease cleavage site located within the di-chain loop region to convert the single-chain protein into its di-chain form.
[0125] In one embodiment, a cell is grown at a first temperature for a certain period of time in order to achieve maximum cell density. In aspects of this embodiment, a cell is grown at about 37 °C for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, about 5.0 hours, about 6.0 hours, about 7.0 hours, about 8.0 hours, about 9.0 hours or about 10 hours. In other aspects of this embodiment, a cell is grown at about 42 °C for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, about 5.0 hours. In aspects of this embodiment, a cell is grown at about 30 °C for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, or about 5.0 hours. In yet other aspects, of this embodiment, a cell is grown at about 12 °C for about 2 hours to about 8 hours, at about 16 °C for about 2 hours to about 8 hours, at about 20 °C for about 2 hours to about 8 hours, or at about 24 °C for about 2 hours to about 8 hours. In still other aspects, of this embodiment, a cell is grown at about 12 °C to about 16 °C for about 2 hours to about 8 hours, or at about 20 °C to about 24 °C for about 2 hours to about 8 hours.
[0126] In another embodiment, a cell is grown at a second temperature for a certain period of time in order to achieve maximum induction of protein expression. In aspects of this embodiment, a cell is grown at about 37 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In other aspects of this embodiment, a cell is grown at about 30 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In yet other aspects of this embodiment, a cell is grown at about 25 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In still other aspects of this embodiment, a cell is grown at about 22 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In further aspects of this embodiment, a cell is grown at about 16 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours, In yet further aspects of this embodiment, a cell is grown at about 12 °C for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours.
[0127] Aspects of the present invention are as described in the claims.
EXAMPLES
Example 1 TEV Protease Variants [0128] The following example illustrates how to make and use TEV protease variants that have increased stability and/or solubility. A. Construction of pET29ITEV expression constructs.
[0129] In order to produce a TEV protease recombinantly, an open reading frame encoding the desired TEV protease was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA). Complementary oligonucleotides of 20 to 50 bases in length, spanning the entire open reading frame, were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHBI carrier vector at the Smal site to generate pUCBHB1/TEV plasmids. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATORT™ Chemistry 3.1 (Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied Biosystems, Foster City, CA).
[0130] The open reading frame encoding the TEV variants were codon-optimized forE, coli expression and all encode an approximately 250 amino acid proteolytic fragment of approximately 27.5 kDa, corresponding to residues 2038-2279 of the full-length TEV polyprotein fused to either an N- or C-terminal poly-histidine affinity purification tag. Recombinant expression of wild-type TEV protease results in a protein that has a propensity to cleave itself at Serine 219 to generate a truncated protease with greatly diminished proteolytic activity. Thus, to largely eliminate autoproteolysis and subsequent generation of this truncated product, TEV variants were synthesized where Serine 219 was changed to either Asparagine (S219N) or Valine (S219V). In addition, it is well documented that although recombinant wild-type TEV protease is expressed at very high levels in E, coli, it is almost entirely insoluble (Kapust et al., 2001). Thus, to improve solubility of the expressed TEV, several amino acid variants were made and tested to determine whether the changes resulted in increased protein solubility. The TEV variants synthesized are shown in Table 3. Variant 1 represented a codon-optimized TEV construct engineered with a C-terminal His-tag and the S219N mutation. Variant 11 was a construct with native DNA sequence of TEV protease engineered with an N-terminal tag and the S219N mutation.
[0131] To construct pET29/TEV variant expression constructs, a pUCBHB1/TEV constructwasdigested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding the TEV; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wl). Using a T4 DNA ligase procedure this insert was directionally ligated into a pET29 vector digested with the same restriction endonucleases in the multiple cloning site. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSys-tems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubatorforovernightgrowth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the TEV gene insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding TEV variants operably-linked to either a carboxyl terminal or amino-terminal polyhistidine affinity purification peptide. B. Analysis of TEV expression under differerit induction conditions.
[0132] To determine the best growth and protein induction conditions to use, pET29/TEV variants 9 and 10 (Table 3) were grown and induced in an IPTG induced media and an auto-inducing media. In addition, the length of induction was examined.
[0133] To induce expression with IPTG, cells harboring the TEV expression construct were first grown overnight to produce a starter culture. Fresh LB media was inoculated at 1:1000 with the overnight culture and allowed to grow, with shaking, at 37°C until OD600 reached 0.7, at which time IPTG was added to a final concentration of 0.6 mM. Cells were harvested 4 hrs. following induction and total cell lysates evaluated to detect target expression.
[0134] To express constructs under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kan-amycin was inoculated with a single colony of BL21 (DE3) cells harboring the appropriate expression construct and grown at 37 °C with shaking overnight. 1.0 μί of this starter culture was used to inoculate 1.0 mL of ZYP-5052 auto-induction media containing 50 μg/mL kanamycin. Cells were grown at 37 °C with shaking and aliquots removed at 5, 8, 12, 20, and 28 hours.
[0135] To determine total TEV protease expression, 40 μί of the induced cell culture from each time-point was mixed with an equal volume of 2x Laemmi Sample Buffer and incubated at 95 °C for 10 minutes. 2 μί of 1 unit^L Benzonase in 1 M MgS04 was added to this mixture and incubated at 95 °C for 5 minutes. A15 μί aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (In-vitrogen, Inc, Carlsbad, CA) under denaturing, reducing conditions. The gel was washed and fixed in Fix Solution comprising 10% methanol, 7% acetic acid for 30 minutes. Afterfixing, the Fix Solution was removed and the gel incubated with SYPRO Ruby Protein Gel Stain at room temperature for 3 hours. The gel was then destained in Destain Solution comprising 10% methanol, 7% acetic acid at room temperature for 3 hours. The image was visualized with a Typhoon 9410 Variable Mode Imager and Imager Analysis software (GE Healthcare, Amersham Biosciences, Piscataway, NJ).
[0136] To determine soluble TEV protease expression, 1.0 mL of the induced cell culture was lysed by adding 100 μί of a Cell Lysis Solution comprising 1 x FASTBREAK™ Cell Lysis reagent (Promega Corp., Madison, Wl), 500 mM NaCI, 250 units/mL benzonase nuclease (EMD Biosciences-Novagen, Madison, Wl), and 1 x Protease Inhibitor Cocktail III (EMD Biosciences-Calbiochem, Gibbstown, NJ) and incubated at room temperature for 25 minutes with constant vortexing. The lysate was centrifuged at 4300 rpm for 15 minutes to pellet debris. 800 μί of the supernatant was transferred to a clean tube, to which 30 μί of MagneHis magnetic beads were added and the mixture incubated for 5 minutes with constant rotation. After incubation, the magnetic beads were sequestered on a magnetic stand, the solution was removed, and the beads washed three times with 150 μί wash buffer comprising 500 mM NaCI. The protein was eluted with 80 μί of elution buffer, an equal volume of 2x Laemmli Sample Buffer was added, and the mixture incubated at 95 °C for 10 minutes. A 15 μί aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, CA) under denaturing, reducing conditions.
[0137] Results of the induction experiments indicated that auto-induction conditions resulted in 5-10-fold more expressed TEV protease relative to IPTG-induction. Comparison of total and soluble TEV protease expression in the autoinduction media revealed that although longer induction times resulted in more total protein, the amount of recoverable soluble TEV protease decreased. In fact, about 8 hours of expression at 37°C yielded the largest amount of soluble protein. Lastly, although both the TEV S219N and TEV S219V variants exhibited significantly less autoproteolysis, the TEV S219V variant showed more truncated product at prolonged induction times suggesting that the TEV S219V variant was more prone to autoproteolysis.
[0138] Once the growth and induction conditions were optimized using pET29/TEV variants 9 and 10, expression of all eleven pET29/TEV variants was examined in parallel under these conditions. The results indicated that the order of increasing yield of soluble TEV protease, from greatest to least of the five highest expressers, was from pET29/TEV variants 5, 10, 7, 3, and 6. In comparison, the TEV variant 11 was expressed at the lowest level of all. C. Large-scale expression and purification.
[0139] To rigorously compare TEV protease expression levels from the top five pET29/TEV variants, along with variant 11 as a control, under large-scale conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL Kanamycin was inoculated with a single colony of BL21 (DE3) cells harboring the appropriate expression construct and grown at 37°C with shaking overnight. 250 μί of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37°C with shaking for 8 hours. The cells were pelleted by centrifugation.
[0140] To lyse cells, the cell pellet was resuspended in a 5.0 mL/gram cell pellet of Lysis Solution comprising BUG-BUSTER™ Protein Extraction Reagent (EMD Biosciences-Novagen, Madison, Wl), 1 x protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, Gibbstown, NJ), 25 units/mL Benzonase nuclease, and 1 Kunit/mL rLysozyme (EMD Biosciences-Novagen, Madison, Wl). The cell suspension was incubated at room temperature on a platform rocker for 20 minutes, followed by incubation on ice for 15 minutes. The suspension was centrifuged at 4 °C for 30 minutes at 30,350 ref to pellet debris and the supernatant was transferred to a clean tube. To prepare the insoluble cell extract pellet for SDS-PAGE analysis, the pellet was resuspended to the original volume with 1x BUGBUSTER™ Protein Extraction Reagent.
[0141] To purify a TEV protease variant by IMAC purification, the clarified lysate was mixed with TALON™ SuperFlow Metal Affinity Cobalt Resin equilibrated with IMAC Wash Solution comprising 25 mM Sodium phosphate, pH 7.0, 500 mM NaCI, 10% glycerol and 35 mM imidazole. The lysate-resin mixture was incubated on a platform rocker at 4 °C for 1 hour and then transferred to a 20 mL disposable column support attached to a vacuum manifold. The column was washed twice with five column volumes of IMAC Wash Solution. The TEV protease was eluted from the resin with two column volumes of IMAC Elution Solution, comprising 25 mM sodium phosphate, pH 7.8, 500 mM NaCI, 10% glycerol and 500 mM imidazole, and collected in 1.0 mL fractions. Each fraction containing protein was identified by mixing 10 μι aliquot with 200 μι of QUICKSTART™ Bradford Dye reagent. Peak elution fractions were pooled and dialyzed for secondary ion exchange chromatography purification.
[0142] To dialyze an IMAC-purified TEV protease variant, the pooled sample comprising the peak elution fraction was dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4°C in 1 L of a Desalting Buffer with constant stirring overnight. For cation exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCI, pH 8.0.
[0143] To purify a TEV protease variant by cation exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-S1 cation exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCI gradient with Buffer B comprising 25 mM sodium phosphate, pH 7.0, 1 M NaCI at a flow rate of 1.0 mL/min as follows: 5% Buffer B for 3 mL, 20% Buffer B for 10 mL, 20% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at -80 °C. TEV variant 7 had the highest yield of soluble protease (ca. 35 mg/L) followed by variant 3 (ca. 24 mg/L) and variant 10 (ca. 23 mg/L). The remaining two variants, 5 and 6, had yields of 18 and 8 mg/L, respectively. Yield of the TEV variant 11 was ca. 0.6 mg/L. As such, all of the top five TEV variants containing a solubility enhancing amino acid change resulted in at least a 10-fold increase in soluble TEV protease purified relative to the TEV variant 11 that only comprised the autoproteolysis eliminating amino acid change (S219N). When comparing the rank order of yield of TEV protease from small- and large-scale expression studies, variant 5 exhibited the highest yield in small-scale expressions (Example 1C). However, it was variant 7 that had the highest yield in large-scale expressions. Repeat comparison of yields from large-scale batches consistently revealed variant 7 to be the highest expressing variant. As a result, variant 7 represented the lead TEV protease construct and was used for all subsequent studies described here.
[0144] To determine the proteolytic activity of TEV protease variants, a TEV protease variant, or AcTEV protease as a positive control, was added to 30 μι of a Reaction Solution comprising 50 mM Tris-HCI, pH 8.0, 1 mM DTT, and 2.5 μg of a TEV substrate and incubated at 30 °Cfor 30 minutes, 60 minutes, and 120 minutes. The reactions were quenched by adding 2 x Laemmi Sample Buffer and incubating the sample at 95 °C for 10 minutes. A 15 μί aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, CA) under denaturing, reducing conditions. The gel was washed and fixed in Fix Solution comprising 10% methanol, 7% acetic acid for 30 minutes. After fixing, the Fix Solution was removed and the gel incubated with SYPRO Ruby Protein Gel Stain at room temperature for 3 hours. The gel was then destained in Destain Solution comprising 10% methanol, 7% acetic acid at room temperature for 3 hours. The image was visualized with a Typhoon 9410 Variable Mode Imagerand analyzed with ImageQuantTL Image Analysis software (GE Healthcare, Amersham Biosciences, Piscataway, NJ). The ratio of intensities of uncleaved substrate and cleaved product was used to calculate percentage of cleaved TEV substrate. The results of the TEV protease activity assay are given in Table 4.
(continued)
Example 2
Intracellular activation of a Clostridial toxin with a TEV protease cleavage site using two different expression constructs [0145] The following example illustrates a procedure useful for expressing in a cell a Clostridial toxin comprising a dichain loop region comprising an exogenous protease cleavage site as disclosed in the present specification. A. Construction of pET29IBoNTIA-TEV expression construct.
[0146] In order to produce a BoNT/A comprising a TEV protease cleavage site located within the di-chain loop region, an open reading frame (SEQ ID NO: 87) encoding the desired BoNT/A-TEV (SEQ ID NO: 88) was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA). Complementary oligonucleotides of 20 to 50 bases in length, spanning the entire open reading frame of BoNT/A-TEV were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHBI carrier vector at the Smal site to generate the pUCBHB1/BoNT/A-TEV constructs. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR™ Chemistry 3.1 (Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied Biosystems, Foster City, CA).
[0147] To generate the pET29/BoNT/A-TEV expression construct, pUCBHB1/BoNT/A-TEV was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding BoNT/A-TEV; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wl). This insert was subcloned using a T4 DNA ligase procedure into a pET29 vector digested with the analogous restriction endonucleases to yield the appropriate pET29/BoNT/A-TEV expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding BoNT/A-TEV operably-linked to a carboxyl-terminal polyhistidine affinity purification peptide. B. Construction of pET22ITEV expression constructs.
[0148] To generate a pET22/TEV variant expression construct, a pET29/TEV variant 7 expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 77) encoding the TEV protease (SEQ ID NO: 78); and 2) enable this insert to be operably-linked to a pET22 vector (EMD Biosciences-Novagen, Madison, Wl). This insert was subcloned using a T4 DNA ligase procedure into a pET22 vector digested with the analogous restriction endonucleases to yield the appropriate pET22/TEV expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37 °C incubatorfor overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET22 expression construct comprising the polynucleotide molecule encoding TEV variant 7 operably-linked to an amino-terminal polyhistidine affinity purification peptide. C. Construction of cells comprising pET29IBoNTIA-TEV and pET22ITEV expression constructs.
[0149] To make a cell comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs, a pET29/BoNT/A-TEV expression construct was transformed into electro-competent E. coli BL21(DE3) cells harboring pET22/TEV variant 7 expression construct using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs. D. In situ activation of BoNTIA-TEV.
[0150] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0mLofPA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pET22/TEV expression constructs and grown at 37 °C with shaking overnight. About 1.0 μί of this starter culture was used to inoculate a 1.0 mL of ZYP-5052 containing 50 μg/mL kanamycin and 50 μg/mL ampicillin and grown at 37 °C with shaking for 3.5 hours and then at 22 °C with shaking for 18.5 hours. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0151] Following growth and induction, the cells were lysed and IMAC purified essentially as described in Example 1B. The IMAC purified samples were analyzed by SDS-PAGE and the gels stained essentially as described in Example 1 B.
[0152] The results indicate that when pET29/BoNT/A-TEV is expressed alone, an approximately 150 kDa band corresponding to the single-chain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. Thus, coexpression of BoNT/A-TEV and TEV protease in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV.
[0153] To confirm these results, a large scale expression of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pET22/TEV expression constructs was done. 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs and grown at 37 °C with shaking overnight. About 250 μί of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 50 μg/mL ampicillin and grown at 37 °C with shaking for 3.5 hours and then at 22 °C with shaking for 18.5 hours. The cells were pelleted by centrifugation. The cells were lysed and IMAC purified as described in Example 1C.
[0154] To dialyze the IMAC-purified BoNT/A-TEV for secondary ion exchange chromatography, the pooled sample comprising the peak elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4 °C in 1 L of a Desalting Buffer with constant stirring overnight. For anion exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCI, pH 8.0.
[0155] To purify BoNT/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCI gradient with Buffer B comprising 50 mM Tris-HCI, pH 8.0, 1 M NaCI at a flow rate of 0.5 mL/min as follows: 3% Buffer B for 3 mL, 7% Buffer B for 10 mL, 7% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at-80°C. Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. The results confirm the initial small scale experiments and indicate that the single-chain BoNT/A-TEV is converted to its di-chain form with near 100% efficiency.
[0156] To assess the activity of the BoNT/A-TEV di-chains, these toxins were evaulated in a cell-based assay and animal-based assay.
[0157] To test the activity of BoNT/A-TEV di-chains using a cell-based assay, an immuno-based BoNT/A activity assay using multiplex ECL sandwich ELISA was performed essentially as described in patent application Fernandez-Salas, et al., Immuno-Based BONTIA Activity Assays, Attorney Docket No. 18383 (BOT).
[0158] To obtain a BoNT/A-TEV treated cell lysate for analysis, approximately 50,000 cells from a stock culture of a
SiMa cell line were seeded into a poly-D-lysine 96-well plate containing a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX™ I with Earle’s salts, 1 xB27 supplement, 1 x N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 μg/mL of GTb1. These cells were incubated in a 37 °C incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM, 3.33 nM and 10.0 nMof a BoNT/A-TEV. After a 24 hr treatment, the cells were washed, incubated for an additional two days without toxin. To harvest the cells, the medium was aspirated, washed with 1 x PBS, and lysed by adding 30 μΙ of Lysis Buffer comprising 50 mM HEPES, 150 mM NaCI, 1.5 mM MgCI2, 1 mM EGTA, 1% Triton X-100 to each well, and the plate incubated on a shaker rotating at 500 rpm for 30 minutes at 4 °C. The plate was centrifuged at 4000 rpm for 20 minutes at 4 °C to pellet cell debris and the supernatant was transferred to a capture antibody coated 96-well plate to perform the detection step.
[0159] To prepare the a-SNAP-25 capture antibody solution, the a-SNAP-25 monoclonal antibody contained in the ascites from hybridoma cell line 2E2A6 was purified using a standard Protein A purification protocol To prepare the a-SNAP-25 detection antibody solution, a-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis, MO) was conjugated to Ruthenium(ll)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer’s instructions (Meso Scale Discovery, Gaithersburg, MD). To prepare the solid phase support comprising the capture antibody that was specific for a SNAP-25 cleaved product, approximately 5 μί of a-SNAP-25 monoclonal antibody 2E2A6 solution (20 μg/mL in 1 x PBS) was added to each well of a 96-well MSD High Bind plate and the solution was allowed to air dry in a biological safety cabinet for 2-3 hours in order to liquid evaporate the solution. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity.
[0160] To detect the presence of a cleaved SNAP-25 product by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated, 25 μί of a lysate from cells treated with BoNT/A was added to each well and the plates were incubated at 4 °C for 2 hrs. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 μι 1 x PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 μΙ of 5 μg/mL a-SNAP-25 detection antibody solution comprising 2% Amersham Blocking Reagent in 1 x PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After a-SNAP-25 detection antibody incubation, the wells were washed three times with 200 μι 1 x PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). The raw data obtained from the ECL imager was then transferred to SigmaPlot v. 9.0 and a 4-parameter logistics fit was used to define the dose-response curves. There were no constraints used for the 4-parameter logistic function when plotting the data. Graphical reports were generated using the following analysis: R2 (correlation coefficient), a (Maxtor data set), b (hillslope), and X0 ± SE (EC50 value ± standard error). The results from two independent runs indicate that the activity of both di-chains was nearly identical and within 2-fold of the native di-chain.
[0161] To test the activity of BoNT/A-TEV di-chains using an animal-based assay, an in vivo Digit Abduction Score (DAS) assay was performed. CD-1 Fe mice were weighed and placed into subsets of 10 animals for each discrete DAS assay. Mice were included into a particular subset based on the following criteria: 1) good health; 2) robust baseline DAS response of 0; 3) inclusion in a median weight range of X ± 2 g established for the selected subset and 4) weight greater than 17.0 g.
[0162] Each mouse was injected with 5 μί of one of seven different doses of BoNT/A-TEV (0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM, 3.33 nM and 10.0 nM) with a 30-gauge needle in the gastrocnemius muscle of the right hind limb. As a control, the gastrocnemius muscle of the left hind limb was injected with 5 μί of a solution not containing any BoNT/A-TEV. Mice were observed for the DAS response consecutively for the first 4 days. The DAS was read by lifting each mouse by the tail and precisely observing the injected hind limbs. The abduction or no abduction of the hind digits reveals the effect of paralysis due to the test toxin injected in the muscle. The digit abduction of the injected hind limb was compared with that of the non-injected hind limb and scored accordingly. DAS data was analyzed by calculating the ED50 dose based on peak mean DAS score and AUC (area under the curve) in terms of u/Kg and/or ng/Kg. This was accomplished as follows: 1) the mean peak DAS score for each dose was calculated in each study; 2) any dose that elicited more than five deaths in any study was eliminated from consideration; 3) the highest dose used in a given individual study was the lowest dose which elicited an average peak of 4.0; 4) the lowest dose used in a given individual study was the highest dose which elicited an average peak of 0; 5) curves were constructed for each individual study of average peak DAS vs. log (dose); 6) an AUC value was calculated for each group of 10 mice of the multiple groups in some studies; 7) curves were constructed for each individual study of average AUC vs. log (dose); 8) an x, y replicate response curve was constructed for each set of multiple identical studies; for each test toxin; 9) dose-response data were analyzed by non-linear regression (non-weighted) using a three-parameter logistic equation (Sigma Plot v 8.0; SPSS Science, Chicago, Illinois) using the following equation: y = a/(1 + (x/xO)b) where y is the response, a is the asymptotic ymax, b is the slope, x is the dose, and 0 is the ED50 dose, For peak ED50 determinations, Ymax was set to 4 (maximum DAS reading on scale). Mean (peak and/or AUC) ED50 values were computed for each eight-dose study performed.
[0163] The results from two independent runs indicate that the level of activity of both di-chains was nearly identical and within 2-fold of the native di-chain. Taken together, the cell-based assay and DAS assay data indicate that the process of intracellular activation yields di-chain rBoNT/A which was not only structurally comparable to the in-vitro nicked material but also functionally indistinguishable.
Example 3
Intracellular activation of a Clostridial toxin with a TEV protease cleavage site using two different expression constructs under control of independent promoters [0164] The following example illustrates a procedure useful for expressing in a cell a Clostridial toxin comprising a dichain loop region comprising an exogenous protease cleavage site as disclosed in the present specification. In this case, the formation of the di-chain form of the toxin is regulated by TEV protease under control of an independent promoter. A. Construction of pBADITEV expression construct.
[0165] In order to produce a TEV protease recombinantly, the expression of which was under control of an arabinose promoter (Pbad)· the °Pen reading frame encoding the TEV protease variant 7 (Table 3 [130]), minus an N-terminal His tag, was cloned into the expression vector pBAD/Myc-HisA to construct pBAD/TEV. To construct pBAD/TEV, an open reading frame encoding the TEV protease variant 7 (SEQ ID NO: 106), minus an N-terminal poly-histidine tag, was synthesized using standard procedures (BlueHeron Biotechnology, Carlsbad, CA). The synthetic fragment was also flanked by restriction sites to enable this insert to be operably-linked to a pBAD/Afyc-HisA vector (Life Technologies, Madison, Wl). Using a T4DNA ligase procedure this insert was directionally ligated into a pBAD/Afyc-HisA vector digested with the same restriction endonucleases in the multiple cloning site. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the TEV gene insert. This cloning strategy yielded a pBAD/TEV expression construct comprising the polynucleotide molecule encoding TEV variant 7 free of a polyhistidine affinity purification peptide. B. Construction of cells comprising pET29IBoNTIA-TEV and pBADITEV expression constructs.
[0166] To make a cell comprising pET29/BoNT/A-TEV and pBAD/TEV expression constructs, a pET29/BoNT/A-TEV expression construct (described in Example 2A) was transformed into electro-competent E. coli BL21(DE3) cells harboring pBAD/TEV variant 7 expression construct using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/BoNT/A-TEV and pBAD/TEV expression constructs. C. In situ activation of BoNTIA-TEV.
[0167] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pBAD/TEV expression constructs and grown at 37 °C with shaking overnight. 250 μί of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 100 μg/mL ampicillin and grown at 37 °C with shaking for 8 hours and then at 22 °C with shaking for 14 hours. At this point, TEV expression was induced with 0.2% L-arabinose and the culture was grown for an additional 4 hours at 22°C. As a control, BL21 (DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0168] Following growth and induction, the cells were lysed and IMAC purified essentially as described in Example 1C. To dialyze the IMAC-purified BoNT/A-TEV for secondary ion exchange chromatography, the pooled sample comprising the peak elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4 °C in 1 L of a Desalting Buffer with constant stirring overnight. For anion exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCI, pH 8.0.
[0169] To purify BoNT/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCI gradient with Buffer B comprising 50 mM Tris-HCI, pH 8.0, 1 M NaCI at a flow rate of 0.5 mL/min as follows: 3% Buffer B for 3 mL, 7% Buffer B for 10 mL, 7% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined.
[0170] Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. The results indicate that when pET29/BoNT/A-TEV is expressed alone, an approximately 150 kDa band corresponding to the single-chain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease under control of the PBAD promoter and induced with arabinose, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. Thus, co-expression of BoNT/A-TEV and TEV protease in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV. The results indicate that between 90-95% of the singlechain BoNT/A-TEV is converted to its di-chain form.
Example 4
Intracellular activation of a Clostridial toxin with a TEV protease cleavage site using a dual expression construct [0171] The following example illustrates methods useful for purifying and quantifying a Clostridial toxin comprising an exogenous protease cleavage site as disclosed in the present specification. A. Construction of pET29IBoNTIA-TEVI2xTEV dual expression construct.
[0172] To construct pET29/BoNT/A-TEV/2xTEV dual expression construct, a synthetic fragment (SEQ ID NO: 89) encoding the last 37 amino acids of BoNT/A-TEV as well as transcription (T7 promoter, lac operator site) and translation (RBS) elements necessary for E. coli expression and the entire coding region of TEV variant 7 was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA). Complementary oligonucleotides of 20 to 50 bases in length, were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHBI carrier vector at the Smal site to generate the pUCBHB1/BoNT/A-TEV_C-term/T7Prom/TEV plasmid. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR™ Chemistry 3.1 (Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied Biosystems, Foster City, CA.
[0173] To generate the pET29/BoNT/A-TEV/2xTEV expression construct, pUCBHB1/BoNT/A-TEV_C-term/T7Prom/TEV was digested with restriction endonucleases that 1) excise the insert comprising the C-terminus of BoNT/A-TEV, transcription and translation motifs necessary for E. coli expression of a second open reading frame, and the entire coding region of TEV variant 7; and 2) enable this insert to be operably-linked behind the BoNT/A gene in pET29/BoNT/A-TEV vector from Example 1A. This insert was subcloned using a T4 DNA ligase procedure into the pET29/BoNT/A-TEV vector digested with the analogous restriction endonucleases to yield the appropriate pET29/BoNT/A-TEV/2xTEV dual expression construct comprising the BoNT/A-TEV and TEV protease variant 7 open reading frames with the intervening transcription and translation elements of SEQ ID NO: 89. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubatorforovernightgrowth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET29 dual expression construct comprising the polynucleotide molecule encoding a BoNT/A-TEV variant operably-linked to a carboxyl terminal polyhistidine affinity purification tag and a TEV protease. The open reading frame organization was such that transcription initiation from the first T7 promoter yields an mRNA with the open reading frame encoding BoNT/A-TEV and the open reading frame encoding TEV protease. In addition, transcription initiation from the second T7 promoter yields mRNA with the open reading frame encoding only TEV protease. Thus, there would be twice as many transcripts encoding TEV protease compared to BoNT/A-TEV. B. In situ activation of BoNTIA-TEV from pET29IBoNTIA-TEVI2xTEV.
[0174] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0 mLof PA-0.5G media containing 50 μg/mL Kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pET29/BoNT/A-TEV/TEV dual expression construct and grown at 37 °C with shaking overnight. About 250 μΐ of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37 °C with shaking for 3.5 hours and then at 22 °C with shaking for 18.5 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21 (DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0175] The results indicate that when expressed alone, an approximately 150 kDa band corresponding to the singlechain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. The results also indicated that the single-chain BoNT/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of BoNT/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV. C. Construction of pRSFduetlTEVI2xBoNTIA-TEV dual expression constructs.
[0176] To determine if reversing the organization of the open reading frames encoding BoNT/A-TEV and the TEV protease would affect yield and cleavage efficiency of BoNT/A-TEV, a dual expression construct was made where transcription initiation from the first T7 promoter yields an mRNA with the open reading frames encoding TEV and BoNT/A-TEV and transcription initiation from the second T7 promoter yields mRNA with the open reading frame encoding only BoNT/A-TEV. Thus, there would be twice as many mRNA’s encoding BoNT/A-TEV compared to TEV protease.
[0177] To construct pRSFduet/TEV/2xBoNT/A-TEV dual expression construct, two sequential cloning reactions were performed. First, the open reading frame (SEQ ID NO: 91) encoding TEV variant 7 (SEQ ID NO: 22) was amplified by PCR from the pET29/TEV variant 7 expression construct. The 5’-end of the open reading frame encoding the polyhistidine affinity tag was excluded from the amplification to encode a tag-less protease. Following amplification, the PCR product was digested at the unique restriction sites, incorporated at the ends of the PCR product by means of the PCR primers, and cloned into the corresponding sites in MCSI (multiple cloning site) of the dual expression plasmid pRSFduet-1 (EMD Biosciences-Novagen, Madison, Wl) using a T4 DNA ligase procedure. This intermediate construct was designated pRSduet/TEV. Next, a pET29/BoNT-A/TEV expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 87) encoding the BoNT/A-TEV (SEQ ID NO: 88); and 2) enable this insert to be operably-linked to the MCS2 in pRSFduet/TEV- The BoNT/A-TEV insert was subcloned into the MCS2 of the pRSFduet vector using a T4 DNA ligase procedure to yield the appropriate pRSFdu-et/TEV/2xBoNT/A-TEV dual expression construct. This cloning strategy yielded a pRSFduet dual expression construct where transcription from the first T7 promoter would produce mRNA’s encoding TEV and BoNT/A-TEV and transcription from the second T7 promoter would produce mRNA’s encoding only BoNT/A-TEV.
[0178] This cloning strategy will yield a pRSFduet dual expression construct where the first T7 promoter will transcribe the open reading frame encoding BoNT/A-TEV and the second T7 promoter will transcribe the open reading encoding TEV protease. D. Construction of pET29IBoNTIA-TEVITEV dual expression construct.
[0179] To determine BoNT/A-TEV yields and efficiency of conversion to di-chain from a transcription unit configuration where BoNT/A-TEV and TEV could only be produced from their own independent mRNA’s, pET29/BoNT/A-TEV/TEV was constructed. To generate the pET29/BoNT/A-TEV/TEV dual expression construct, a short synthetic DNA fragment was used to incorporate a T7 terminator site (SEQ ID NO: 92) in the intervening sequence between the open reading frames of BoNT/A-TEV and TEV in the dual expression construct pET29/BoNT/A-TEV/2xTEV (Example 3A above). Using a T4 DNA ligase procedure, this was essentially accomplished by swapping the intervening region in pET29/BoNT/A-TEV/2xTEV which lacked a T7 terminator site with a synthetic DNA fragment harboring the intervening transcription and translation elements along with a T7 termination site of SEQ ID NO: 93. The resulting dual expression construct, designated pET29/BoNT/A-TEV/TEV, comprises the polynucleotide molecule encoding a BoNT/A-TEV variant operably-linked to a carboxyl terminal polyhistidine affinity tag and TEV protease, transcribed from the first and second T7 promoters, respectively. E. In situ activation of BoNTIA-TEV.
[0180] The growth and induction of di-chain forms of BoNT/A-TEV under auto-induction conditions was done essentially as described in Example 2D, except the BL21(DE3) cells comprising a pET29/BoNT/A-TEV/2xTEV dual expression construct, a pRSF/TEV/2xBoNT/A-TEV dual expression construct, or a pET29/BoNT/A-TEV/TEV dual expression construct were used and single colonies from each of these cell lines were used to inoculate four 1.0 mL cultures in parallel. After growth and induction, the four 1.0 mL replicates were pooled together for processing. The cells were lysed and IMAC purified, and analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent. The results indicate that BoNT/A-TEV was expressed at very comparable levels from cells containing any one of the three dual expression constructs; however, the extent of conversion to di-chain varied. Single-chain BoNT/A-TEV was converted to its di-chain form with ca. 96% efficiency when the proteins were expressed from pET29/BoNT/A-TEV/2xTEV, with ca. 81% efficiency when the proteins were expressed from pET29/BoNT/A-TEV/TEV, and with greater than 99% efficiency when the proteins were expressed from pRSFdu-et/TEV/2xBoNT/A-TEV.
Example 5
Intracellular activation of a protein comprising an integrated TEV protease cleavage site-opioid binding domain using a dual expression construct [0181] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an exogenous protease cleavage site disclosed in the present specification. A. Construction of pRSFduetlTEVI2xNociLHNIA-TEV dual expression construct.
[0182] To construct pRSFduet/TEV/2xNociLHN/A-TEV dual expression construct, a pET29/NociLHN/A-TEV expression construct was digested with restriction endonucleases that 1 ) excise the insert comprising the open reading frame (SEQ ID NO: 94) encoding the NociLHN/A-TEV (SEQ ID NO: 95); and 2) enable this insert to be operably-linked to the MCS2 of pRSFduet/TEV, a pRSFduet-1 vector harboring TEV variant 7 in MCSI (Described in Example 3C). The NociLHN/A-TEV insert was subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the appropriate pRSFduet/TEV/2xNociLHN/A-TEV dual expression construct. The ligation mixture was transformed into electro-competent E. coli BL21 (DE3) Acéllá cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pRSFduetdual expression construct where transcription from the first T7 promoter would produce mRNA’s encoding TEV and NociLHN/A-TEV and transcription from the second T7 promoter would produce mRNA’s encoding only NociLHN/A-TEV. B. In situ activation of NociLHNIA-TEV.
[0183] To produce di-chain forms of NociLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μς/ηιί kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pRSFdu-et/TEV/2xNociLHN/A-TEV dual expression construct and grown at 37 °C with shaking overnight. 250 μΙ. of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37 °C with shaking for 8 hours and then at 16 °C with shaking for 18 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21(DE3) cells harboring NociLHN/A-TEV alone were grown and induced as described above.
[0184] The results indicate that when expressed alone, an approximately 102 kDa band corresponding to the singlechain of NociLHN/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when NociLHN/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50.8 kDa and the other of approximately 51.3 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50.8 kDa and approximately 51.3 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 50.8 kDa and approximately 51.3 kDa bands seen under reducing conditions respectively correspond to the Clostridial toxin enzymatic domain and the Clostridial toxin translocation domain with the nociceptin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of NociLHN/A-TEV and that the single-chain NociLHN/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of NociLHN/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of NociLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-opioid binding domain and the subsequent formation of the di-chain form of NociLHN/A-TEV. C. Construction of pRSFduetlTEVI2xDynLHNIA-TEV dual expression construct.
[0185] pRSFduet/TEV/2xDynLHN/A-TEV dual expression construct was generated almost exactly as pRSFdu-et/TEV/2xNociLHN/A-TEV. A pET29/DynLHN/A-TEV expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 96) encoding the DynLHN/A-TEV (SEQ ID NO: 97); and 2) enable this insert to be operably-linked to the MCS2 of of pRSFduet/TEV (Described in Example 3C). The DynLH N/A-TEV insert was subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the appropriate pRSFduet/TEV/2xDynLHN/A-TEV dual expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pRSFduetdual expression construct where transcription from the first T7 promoter would produce mRNA’s encoding TEV and DynLHN/A-TEV and transcription from the second T7 promoter would produce mRNA’s encoding only DynLHN/A-TEV. D. In situ activation of DynLHNIA-TEV.
[0186] To produce di-chain forms of NociLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pRSFdu-et/TEV/2xDynLHN/A-TEV dual expression construct and grown at 37 °C with shaking overnight. 250 μί of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37 °C with shaking for 8 hours and then at 16 °C with shaking for 18 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21(DE3) cells harboring DynLHN/A-TEV alone were grown and induced as described above.
[0187] The results indicate that when expressed alone, an approximately 102 kDa band corresponding to the singlechain for of DynLH N/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when DynLHN/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50.8 kDa and the other of approximately 52 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50.8 kDa and approximately 52 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 50.8 kDa band corresponds to the Clostridial toxin enzymatic domain and an approximately 52 kDa band corresponds to the Clostridial toxin translocation domain with the dynorphin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of DynLHN/A-TEV and also that the single-chain DynLHN/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of DynLHN/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of DynLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-opioid binding domain and the subsequent formation of the di-chain form of DynLHN/A-TEV.
Example 6
Intracellular activation of a protein comprising an Integrated TEV protease cleavage site-Galanln binding domain using two different expression constructs [0188] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid binding domain disclosed in the present specification where the target protein and the protease are expressed from separate plasmids and under control of different promoters. A. Construction of pET29IGalLHNIA-TEV expression construct.
[0189] To construct the pET29/GalLHN/A-TEV expression construct, a pET29/DynLHN/A-TEV expression construct was first digested with restriction endonucleases to excise a DNA segment encoding the di-chain loop comprising an integrated TEV protease cleavage site- dynorphin binding domain. The resulting pET29/LHn/A framework fragment was ligated with a synthetic DNA fragment bracketed with the compatible restriction sites (SEQ ID NO: 98), comprising the di-chain loop comprising an integrated TEV protease cleavage site-galanin binding domain (SEQ ID NO: 99). The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded the pET29/GalLHN/A-TEV expression construct comprising the open reading frame (SEQ ID NO: 100) encoding the GalLHN/A-TEV (SEQ ID NO: 101) in which expression of GalLHN/A-TEV is under control of the T7 promoter. B. Construction of pColdIVITEV expression construct.
[0190] To generate an expression construct in which TEV is under control of the cold-shock promoter (csp), the open reading frame (SEQ ID NO: 91) encoding TEV variant 7 (SEQ ID NO: 22) was amplified by PCR from the pET29/TEV variant 7 expression construct. The 5’-end of the open reading frame encoding the poly-histidine affinity tag was excluded from the amplification to encode a tag-less protease. Following amplification, the PCR product was digested at the unique restriction sites, incorporated at the ends of the PCR product by means of the PCR primers, and cloned into the corresponding sites in the multiple cloning site of the expression plasmid pColdIV (Clontech Laboratories, Inc. Madison, Wl) using a T4 DNA ligase procedure. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded the pColdIV/TEV expression construct comprising the polynucleotide molecule encoding TEV variant 7 under control of the cold-shock promoter. C. Construction of cells comprising pET29IGalLHNIA-TEV and pColdIVITEV expression constructs.
[0191] To make a cell comprising pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs, the pET29/GalL-HN/A-TEV expression construct was transformed into electro-competent E. coli BL21(DE3) cells harboring pColdIV/TEV using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs. D. In situ activation of pET29IGalLHNIA.
[0192] To produce di-chain forms of GalLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μς/ιτιΙ- kanamycin and 100 μς/ιτιΙ- ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs and grown at 37 °C with shaking overnight. About 250 μι. of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 100 μg/mL ampicillin and grown at 37 °C with shaking for 8 hours and then at 15 °C with shaking for 18 hours. The cells were lysed and IMAC purified using Magne-His resin.
[0193] To purify di-chain GalLHN/A-TEV by Magne-His purification, induced cells from 250 mL expression cultures were resuspended in 16 mL of cold (4-6 °C) IMAC Wash Buffer consisting of 100 mM HEPES, pH 7.5,10 % v/v glycerol, 10 mM imidazole, 1 M NaCI. The cell suspension was transferred to a sealed-atmosphere treatment chamber (#101-021-006, Branson Ultrasonics Corporation) and sonicated by 15 pulses (10 sec, 30% amplitude, 0.5-inch disruptor horn) with 1 minute in between pulses (Sonifier® Digital 450, Branson Ultrasonics Corporation). During sonication the sealed-atmosphere treatment chamber was cooled by passing chilled water from a circulating water bath (3.5 °C) through the outer jacket of the chamber. Sonicated material was transferred from the treatment chamber to a clean Oakridge tube and centrifuged at 30,500 RCF for 30 min (SL-50T Rotor, Sorvall; FIBERLite® F21S-8X50 Rotor, Piramoon Technologies Inc.) at 4 °C to remove insoluble cellular debris. The clarified lysate was aspirated by syringe and passed first through a 0.8 μίτι and then a 0.45 μίτι syringe filter (Sartorius) in series into a clean 50 mL conical tube. Magne-His™ Protein Purification Resin (Promega Corp., Madison, Wl) was vortexed to a uniform suspension and 4 mL of the suspension transferred to the clarified lysate. The tube was sealed and inverted several times to mix the particles well. The mixture was incubated for 30 min with gentle rocking to bind the target protein at 16 °C. The tube was transferred to a MagneSil Magnetic Separation Unit (Promega Corp., Madison, Wl) and ~2 min were allowed for capture of the resin particles. The supernatant solution was removed and the tube removed from the separation unit. The resin was then resuspended in 10 mL IMAC Wash Buffer, captured on the magnetic separation unit, and the wash bufFer removed. The wash step was repeated two more times. To elute the target protein, the resin was resuspended in 5 mL of the Magne-His™ Elution Buffer (100 mM HEPES, pH 7.5, 500mM Imidazole) incubated at room temperature for 2 min, the resin captured on the magnetic separation unit and the supernatant solution transferred to a new tube. The elution step was repeated once.
[0194] To dialyze the IMAC-purified GalLHN/A-TEV for secondary ion exchange chromatography, the pooled elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4 °C in 1 L of a Desalting Buffer (Buffer A: 50 mM Tris-HCI, pH 8.0) with constant stirring overnight.
[0195] To purify di-chain GalLHN/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 1 mL/min. Bound protein was eluted by NaCI gradient with Buffer B comprising 50 mM Tris-HCI, pH 8.0, 1 M NaCI at a flow rate of 1 mL/min as follows: 7% Buffer B for 3 mL, 15% Buffer B for 7 mL, 10% to 50% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at-80 °C. Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B.
[0196] The results indicate that when GalLHN/A-TEV was co-expressed with TEV protease, two nearly superimposing bands were observed under reducing conditions, one of approximately 51.1 kDa and another of approximately 52.1 kDa. Moreover, when the same samples were run under non-reducing conditions, the two approximately 51.1 kDa and 52.1 kDa bands disappeared and a new band of approximately 103 kDa was observed. Taken together, these observations indicate that the approximately 51.1 kDa band corresponds to the Clostridial toxin enzymatic domain and the approximately 52.1 kDa band corresponds to the Clostridial toxin translocation domain with the galanin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of GalLHN/A-TEV and also that the single-chain GalLHN/A-TEV was converted to its di-chain form with approximately 90% efficiency. Thus, coexpression of GalLHN/A-TEV and TEV protease in these cells from independent plasmids results in cleavage of GalL-HN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-galanin binding domain and the subsequent formation of the di-chain form of GalLHN/A-TEV.
Example 7: Prophetic
Intracellular activation of a protein comprising an integrated TEV protease cleavage site-Galanin binding domain using a dual expression construct [0197] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid bidning domain disclosed in the present specification where the target protein and the protease are expressed from a dual expression plasmid. A. Construction of pRSFduetlTEVI2xGalLHNIA-TEV dual expression construct.
[0198] To construct pRSFduet/TEV/2xGalLHN/A-TEV dual expression construct similar to pRSFdu-etlTEVI2xNociLHNIA-TEVandpRSFduetlTEVI2xDynLHNIA-TEVconstructed before (See Example 4), a pET29/GalL-HN/A-TEV expression construct will be digested with restriction endonucleases to 1) excise the insert comprising the open reading frame (SEQ ID NO: 100) encoding the GalLHN/A-TEV (SEQ ID NO: 101); and 2) enable this insert to be operably-linked to the MCS2 of pRSFduet/TEV, a pRSFduet-1 vector harboring TEV variant 7 in MCSI (Described in Example 3C). The GalLHN/A-TEV insert will be subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the pRSFduet/TEV/2xGalLHN/A-TEV dual expression construct. The ligation mixture will be transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression constructs will be identified as kanamycin resistant colonies and candidate constructs confirmed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy will yield a pRSFduet dual expression construct where transcription from the first T7 promoter will produce mRNA’s encoding TEV and GalLHN/A-TEV and transcription from the second T7 promoter will produce mRNA’s encoding only GalLHN/A-TEV. B. In situ activation of GalLHNIA-TEV.
[0199] To produce di-chain forms of GalLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin will be inoculated with a single colony of BL21(DE3) cells comprising pRSFdu-et/TEV/2xGalLHN/A-TEV dual expression construct and grown at 37 °C with shaking overnight. 250 μΙ_ of this starter culture will be used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37 °C with shaking for 8 hours and then at 16 °C with shaking for 18 hours. The cells will be pelleted by centrifugation, lysed, IMAC purified, desalted, and purified by anion exchange chromatography as described in Example 5D. Purified target protein will be analyzed by SDS-PAGE under both reducing and non-reducing conditons, and the gels stained essentially as described in Example 1B to assess expression levels and the extent to which GalLHN/A-TEV produced from the pRSFdu-et/TEV/2xGalLHN/A-TEV dual expression construct is converted to its di-chain form.
Example 8
Intracellular activation of a protein comprising an integrated TEV protease cleavage site-Dynorphin binding domain using a dual expression construct in BEVS
[0200] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid binding domain disclosed in the present specification where the target protein and the protease are co-expressed in a dual expression construct and under control of two independent promoters in the baculovirus expression vector system (BEVS). A. Construction of pBAC-6ITEVIDynLHNIA-TEVdual expression construct.
[0201] To construct the pBAC-6/TEV/DynLHN/A-TEV dual expression construct, a synthetic fragment (SEQ ID NO: 107) encoding recombinant TEV variant7 downstream of the p10 promoter sequence and DynLHn/A-TEV downstream of the polH promoter sequence in the opposite orientation was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA). Complementary oligonucleotides of 20 to 50 bases in length were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHBI carrier vector at the Smal site to generate the pUCBHB1/p10-TEV/polH-DynLHN/A-TEV plasmid. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR™ Chemistry 3.1 (Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied Biosystems, Foster City, CA.
[0202] To generate the pBAC-6/TEV/DynLHN/A-TEV dual expression construct, pUCBHB1/p10-TEV/polH-DynL-HN/A-TEV was digested with restriction endonucleases that 1) excise the insert comprising the entire coding region of TEV variant 7 under control of the p10 promoter and DynLHN/A-TEV in the opposite direction under control of the polH promoter; and 2) enable this insert to be operably-linked to a pBAC-6 transfer vector (EMD Biosciences-Novagen, Madison, Wl). This insert was subcloned using a T4 DNA ligase procedure into the pBAC-6 transfer vector digested with the analogous restriction endonucleases to yield the engineered pBAC-6 dual expression construct comprising TEV protease variant 7 open reading frame downstream of the p10 promoter and a second open reading frame of DynLHN/A- TEV downstream of the polH promoter. The ligation mixture was transformed into electro-competent £. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, MD) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of ampicillin, and placed in a 37 °C incubator for overnight growth. Bacteria containing expression construct were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pBAC-6 dual expression construct comprising the polynucleotide molecule encoding a DynLH/A-TEV operably-linked to a carboxyl terminal polyhistidine affinity purification tag and TEV protease. B. Generation of high titer TEVIDynLHNIA-TEV recombinant baculovirus stock.
[0203] Before di-chain forms of DynLHN/A-TEV could be produced, high titre recombinant baculovirus stock comprising TEV/DynLHN/A-TEV were generated. Approximately 2x106 Sf9 insect cells were seeded in 35 mm dishes in a 2 mi-volume of insect cell culture medium ESF921. A transfection solution was prepared by mixing Solution A (comprising 2 μg of pBAC-6/TEV/DynLHN/A-TEV, 0.5 μg of linearized flashBAC baculovirus DNA (Oxford Expression Technologies, Oxford, UK), and 100 μΙ. of Transfection Medium) with solution B (comprising 6 μί of TRANSLT®-2020 transfection reagent and 100 μΙ. of Transfection Medium) and incubating at room temperature for 30 minutes. An additional 800 μΙ-of Transfection Medium was next added to the Solution A/B mixture, mixed gently, and added dropwise onto the cells. Cells were incubated at 28°C for 5 hours, at the end which 3 mL of ESF 921 was added to bring the final volume up to 4 mL in each well. The incubation was continued at 28°C for 4-5 days for the production of P0 recombinant virus. To generate higher titer P1 recombinant baculovirus seed stocks, virus isolated from P0 supernatant was titered using bacu/oQUANT (Oxford Expression Technologies, Oxford, UK) and further amplified in shake flasks. About 100-200 mL of Sf9 cells at a density of 2x106 cells/mL were infected with P0 virus at an MOI (multiplicity of infection) < 1 pfu/cell and incubated with shaking for 4 - 5 days. Following quantification, the high titer P1 stock was used to infect Tni cells for high-level protein expression. C. In situ activation of DynLHNIA-TEV.
[0204] To produce di-chain forms of DynHN/A-TEV, 50 mL Tni cells at a concentration of 1 x 106/mL were infected at an MOI of 5 with recombinant P1 virus stock comprising TEV/DynLHN/A-TEV and harvested 3 days post-infection (pi). The cells were lysed and IMAC purified using Magne-His resin.
[0205] To purify di-chain DynLHN/A-TEV by Magne-His purification, the cell pellet was resuspended in 20 mL of PBS w/o Ca2+ or Mg2+ in the presence of 100 μί Insect PopCulture Reagent and 20 μί (10U) Benzonase Nuclease, mixed gently and incubated for 15 minutes at room temperature. After clarifying the cell lysate by centrifugation at 16,000 rpm for 15 minutes at 4 °C, the supernatant was mixed with 4 mL of uniformly suspended Magne-His™ Protein Purification Resin (Promega Corp., Madison, Wl). The mixture was incubated for 20 min at room temperature with gentle rocking to bind the target protein. The tube was transferred to a MagneSil magnetic separation unit for about 2 min to allow capture of the resin particles. After removing the supernatant, the tube was removed from the separation unit and the resin resuspended in 10 mL of IMAC wash buffer. Again, the resin was captured on the magnetic separation unit and the wash buffer removed. The wash step was repeated two more times. To elute the target protein, the resin was resuspended in 2.5 mL of the Magne-His™ Elution Buffer (100 mM HEPES, pH 7.5, 500mM Imidazole), incubated at room temperature for 2 min, the resin captured on the magnetic separation unit, and the supernatant solution transferred to a new tube. The elution step was repeated again to maximize target recovery from the magnetic resin.
[0206] To dialyze the IMAC-purified DynLHN/A-TEV for secondary ion exchange chromatography, the pooled elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4 °C in 1 L of a Desalting Buffer (Buffer A: 50 mM Tris-HCI, pH 8.0) with constant stirring overnight.
[0207] To purify di-chain DynLHN/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 1 mL/min. Bound protein was eluted by NaCI gradient with Buffer B comprising 50 mM Tris-HCI, pH 8.0, 1 M NaCI at a flow rate of 1 mL/min as follows: 7% Buffer B for 3 mL, 15% Buffer B for 7 mL, 10% to 50% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were stored at -20 °C. Purified DynLHN/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B.
[0208] The results indicate that when DynLHN/A-TEV was co-expressed with TEV protease in insect cells and purified to near homogeneity, two nearly superimposing bands were observed under reducing conditions, one of approximately 51 kDa and another of approximately 52 kDa. Moreover, when the same samples were run under non-reducing conditions, the two approximately 50 kDa and 52 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 51 kDa band corresponds to the Clostridial toxin enzymatic domain and the approximately 52 kDa band corresponds to the Clostridial toxin translocation domain with the dynorphin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of dichain formation of DynLHN/A-TEV and also that the single-chain DynLHN/A-TEV was converted to its di-chain form with 80-90% efficiency. Thus, co-expression of DynLHN/A-TEV and TEV protease in insect cells infected with TEV/DynLHN/A-TEV recombinant baculovirus generated from pBAC-6/TEV/DynLHN/A-TEV dual expression construct results in cleavage of DynLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-dynorphin binding domain and the subsequent formation of the di-chain form of DynLHN/A-TEV.
[0209] Although aspects of the present invention have been described with reference to the disclosed embodiments, one skilled in the art will readily appreciate that the specific examples disclosed are only illustrative of these aspects and in no way limit the present invention.
SEQUENCE LISTING
[0210] <110> Ghanshani, Sanjiv Le, Linh Q. Liu, Li Steward, Lance E. <120> Methods of Intracellular Conversion of Single-Chain Proteins into their Di-chain Form <130 18469 (BOT) <150 US 61/286,963 <151 > 2010-01-25 <160> 107 <170> FastSEQ for Windows Version 4.0
<210> 1 <211 > 1296 <212> PRT <213> Clostridium botulinum Serotype A <400> 1
Met Pro Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15
Val Asp lie Ala Tyr lie Lys lie Pro Asn Ala Gly Gin Met Gin Pro 20 25 30
Val Lys Ala Phe Lys lie His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160
Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190
Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380
Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
Phe Asn Gly Gin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445
Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480
Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495
Asp Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510
Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin Leu 515 520 525
Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540
Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu 545 550 555 560
His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575
Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590
Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605
Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620
Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 640
Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655
Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685
Val Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys 705 710 715 720
Val Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735
Glu Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr Asn 740 745 750
Gin Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765
Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780
Asn Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800
Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815
Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830
Gin Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845
Ile Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg Leu Leu Ser 850 855 860
Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875 880
Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895
Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910
Gin Ile Gin Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925
Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940
Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn 945 950 955 960
Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975
Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gin Asp Thr Gin Glu 980 985 990
Ile Lys Gin Arg Val Val Phe Lys Tyr Ser Gin Met Ile Asn Ile Ser 995 1000 1005
Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020
Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gin Lys Pro 1025 1030 1035 1040
Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055
Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070
Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085
Asp Asn Gin Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100
Leu Gin Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 1110 1115 1120
Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135
Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150
Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165
Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180
Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala 1185 1190 1195 1200
Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215
Leu Ser Gin Val Val Val Met Lys Ser Lys Asn Asp Gin Gly Ile Thr 1220 1225 1230
Asn Lys Cys Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245
Phe Ile Gly Phe His Gin Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260
Asn Trp Tyr Asn Arg Gin Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280
Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295
<210 2 <211> 1291 <212> PRT <213> Clostridium botulinum Serotype B <400 2
Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 15 10 15
Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30
Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45
Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80
Thr Asn Asp Lys Lys Asn Ile Phe Leu Gin Thr Met Ile Lys Leu Phe 85 90 95
Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110
Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125
Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140
Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160
Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175
Ile Gin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gin 180 185 190
Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gin Glu 195 200 205
Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220
Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240
Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255
Phe Met Gin Ser Thr Asp Ala Ile Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
Gly Gly Gin Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285
Tyr Asp Lys Val Leu Gin Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300
Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320
Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335
Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350
Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365
Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380
Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400
Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gin Asn Lys Ala Ile 405 410 415
Asn Lys Gin Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430
Lys Ile Gin Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445
Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460
Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gin Ser Asn 465 470 475 480
Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495
Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510
Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gin Pro Ala Ile Lys 515 520 525
Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gin Tyr Leu Tyr Ser Gin 530 535 540
Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560
Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575
Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590
Trp Val Lys Gin Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605
Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620
Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640
Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655
Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670
Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685
Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gin Trp 690 695 700
Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720
Lys Ala Leu Asn Tyr Gin Ala Gin Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735
Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750
Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gin Ala Ile 755 760 765
Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780
Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800
Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815
Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830
Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845
Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860
Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880
Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895
Asn Gin Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910
Gin Asn Gin Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925
Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gin Asn 930 935 940
Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960
Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975
Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990
Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005
Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020
Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile 1025 1030 1035 1040
Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gin Phe Ile Trp Met 1045 1050 1055
Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gin Ser Asn Ile Glu 1060 1065 1070
Glu Arg Tyr Lys Ile Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085
Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100
Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu 1105 1110 1115 1120
Ile Leu Thr Arg Ser Lys Tyr Asn Gin Asn Ser Lys Tyr Ile Asn Tyr 1125 1130 1135
Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150
Ser Gin Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165
Leu Asp Phe Phe Asn Leu Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180
Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp 1185 1190 1195 1200
Ser Asp Glu Phe Tyr Asn Thr Ile Gin Ile Lys Glu Tyr Asp Glu Gin 1205 1210 1215
Pro Thr Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230
Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245
Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260
Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280
Gin Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
<210>3 <211> 1291 <212> PRT <213> Clostridium botulinum Serotype C1 <400>3
Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn 15 10 15
Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30
Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45
Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60
Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80
Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95
Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110
Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125
Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gin Gly Asn 130 135 140
Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160
Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175
Asn Asn Thr Phe Ala Ala Gin Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190
Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205
Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220
Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240
Ile Ala Ile Pro Asn Asp Gin Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255
Phe Tyr Ser Gin Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270
Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285
Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300
Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320
Glu Tyr Lys Gin Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335
Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350
Glu Leu Thr Gin Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365
Val Gin Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380
Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gin Asn Gly Phe Asn 385 390 395 400
Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gin Asn Leu Ser 405 410 415
Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430
Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445
Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460
Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470 475 480
Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495
Val Asp Gin Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gin Leu 500 505 510
Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525
Glu Asn Gin Val Phe Tyr Asp Asn Arg Thr Gin Asn Val Asp Tyr Leu 530 535 540
Asn Ser Tyr Tyr Tyr Leu Glu Ser Gin Lys Leu Ser Asp Asn Val Glu 545 550 555 560
Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575
Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590
Val Gin Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605
Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys lie Ser Asp 610 615 620
Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn lie Ser Asn 625 630 635 640
Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655
Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670
Ala Phe Val Ile Tyr Ser Lys Val Gin Glu Arg Asn Glu Ile Ile Lys 675 680 685
Thr Ile Asp Asn Cys Leu Glu Gin Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700
Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gin Phe 705 710 715 720
Asn Asn Ile Ser Tyr Gin Met Tyr Asp Ser Leu Asn Tyr Gin Ala Gly 725 730 735
Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750
Asp Lys Glu Asn Ile Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu 755 760 765
Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780
Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800
Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815
Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830
Lys Ala Lys Val Asn Asn Ser Phe Gin Asn Thr Ile Pro Phe Asn Ile 835 840 845
Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860
Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gin Asn Arg Lys 865 870 875 880
Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895
Gly Asp Val Gin Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910
Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gin Asn Glu Asn 915 920 925
Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940
Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960
Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975
Leu Val Phe Thr Leu Lys Gin Asn Glu Asp Ser Glu Gin Ser Ile Asn 980 985 990
Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005
Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 1010 1015 1020
Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile 1025 1030 1035 1040
Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr 1045 1050 1055
Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070
Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085
Phe Asn Ser Leu Gin Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 1095 1100
Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu 1105 1110 1115 1120
Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gin Ile Val Phe Asn Thr Arg 1125 1130 1135
Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg 1140 1145 1150
Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165
Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180
Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 1185 1190 1195 1200
Ile Gly Leu Arg Glu Gin Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215
Gin Ile Gin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gin Ile Phe 1220 1225 1230
Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245
Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 1255 1260
Val Pro Thr Val Lys Gin Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 1265 1270 1275 1280
Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285 1290
<210>4 <211 > 1276 <212> PRT <213> Clostridium botulinum Serotype D <400>4
Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 15 10 15
Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gin Asn Lys Leu lie Thr Thr 20 25 30
Pro Val Lys Ala Phe Met lie Thr Gin Asn lie Trp Val lie Pro Glu 35 40 45
Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60
Thr Ser Lys Tyr Gin Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80
Glu Gin Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95
Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110
Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125
Phe Thr Arg His Thr Thr Asn lie Ala Val Glu Lys Phe Glu Asn Gly 130 135 140
Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu lie Phe Gly 145 150 155 160
Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gin Gly 165 170 175
Gin Gin Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190
Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205
Gin Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220
Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gin Leu Tyr Gly 225 230 235 240
Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gin Val Ser Glu Gly 245 250 255
Phe Phe Ser Gin Asp Gly Pro Asn Val Gin Phe Glu Glu Leu Tyr Thr 260 265 270
Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gin Ile Glu Arg Ser Gin 275 280 285
Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300
Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320
Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335
Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350
Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gin Tyr Asn 355 360 365
Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380
Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395 400
Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gin Asn Ile Glu 405 410 415
Arg Asn Pro Ala Leu Gin Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430
Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445
Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460
Asp Ser Ile Ser Gin Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480
Thr Asn Val Gin Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485 490 495
Leu Asp Gly Gin Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510
Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525
Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540
Tyr Leu Glu Ser Gin Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560
Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575
Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gin Ala Gly 580 585 590
Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605
Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620
Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640
Gly Asn Phe Asn Gin Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655
Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670
Tyr Ser Ser Ile Gin Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685
Cys Leu Glu Gin Arg Val Lys Arg Trp Lys Asp Ser Tyr Gin Trp Met 690 695 700
Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gin Phe Asn His Ile Asn 705 710 715 720
Tyr Gin Met Tyr Asp Ser Leu Ser Tyr Gin Ala Asp Ala Ile Lys Ala 725 730 735
Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750
Ile Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765
Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780
Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800
Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 H±s Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830
Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845
Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860
Asn Asp Ser Lys Ile Leu Ser Leu Gin Asn Lys Lys Asn Ala Leu Val 865 870 875 880
Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gin 885 890 895
Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910
Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925
Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940
Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gin Asn Ser 945 950 955 960
Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gin 965 970 975
Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990
Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005
Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys 1010 1015 1020
Gin Ser Gin Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040
Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gin Met Leu Trp 1045 1050 1055
Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile 1060 1065 1070
Asn Ile Val Tyr Glu Gly Gin Ile Leu Arg Asn Val Ile Lys Asp Tyr 1075 1080 1085
Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr Ile Ile Asn Asp 1090 1095 1100
Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val Leu Val Leu 1105 1110 1115 1120
Val Gin Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135
Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140 1145 1150
Asp Asn Ile Ile Leu His Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165
Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gin Gly Gly Glu Cys Ser 1170 1175 1180
Gin Asn Cys Val Tyr Ala Leu Lys Leu Gin Ser Asn Leu Gly Asn Tyr 1185 1190 1195 1200
Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205 1210 1215
Cys Ser Gin Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala 1220 1225 1230
Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245
Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260
Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275
<210>5 <211 > 1252 <212> PRT <213> Clostridium botulinum Serotype E <400>5
Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 15 10 15
Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gin Glu Phe Tyr Lys Ser 20 25 30
Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45
Gly Thr Thr Pro Gin Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Glu Glu Lys 65 70 75 80
Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95
Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110
Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gin Phe His Ile Gly Asp 115 120 125
Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gin Asp Ile Leu 130 135 140
Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190
Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gin Asp Pro Ala Leu 195 200 205
Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gin Lys Gin Asn Pro Leu 225 230 235 240
Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255
Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gin Ser Asn Asp Ile Tyr 260 265 270
Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285
Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300
Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320
Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr Ile 340 345 350
Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365
Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380
Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400
Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415
Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445
Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460
Glu Asn Asp Leu Asp Gin Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480
Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gin Asn Asp Ala 485 490 495
Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gin His 500 505 510
Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540
Leu Leu Glu Gin Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560
Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Ser Trp lie 565 570 575
Gin Gin Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr 580 585 590
Val Asp Lys lie Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605
Ala Leu Asn Ile Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640
Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655
Ser Asp Asn Lys Asn Lys Val lie Lys Ala lie Asn Asn Ala Leu Lys 660 665 670
Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe lie Val Ser Asn 675 680 685
Trp Met Thr Lys Ile Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700
Tyr Gin Ala Leu Gin Asn Gin val Asn Ala lie Lys Thr lie lie Glu 705 710 715 720
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735
Lys Tyr Asp Ile Lys Gin Ile Glu Asn Glu Leu Asn Gin Lys Val Ser 740 745 750
Ile Ala Met Asn Asn lie Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765
Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780
Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gin His 785 790 795 800
Gly Ser Ile Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val Thr 805 810 815
Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830
Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860
Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880
Tyr Pro Thr Asn Lys Asn Gin Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895
Glu Val Asn Ile Ser Gin Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910
Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925
Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960
Trp Thr Leu Gin Asp Asn Ala Gly Ile Asn Gin Lys Leu Ala Phe Asn 965 970 975
Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990
Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005
Gly Asn Leu Ile Asp Gin Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020
Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040
Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055
Thr Glu Ile Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 1060 1065 1070
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100
Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120
Leu Tyr Ser Gly Ile Lys Val Lys Ile Gin Arg Val Asn Asn Ser Ser 1125 1130 1135
Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr Ile Asn Phe 1140 1145 1150
Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180
Asn Gin Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200
Phe Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215
Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230
Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245
Trp Gin Glu Lys 1250
<210>6 <211 > 1274 <212> PRT <213> Clostridium botulinum Serotype F <400 6
Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 15 10 15
Asp Thr Ile Leu Tyr Met Gin Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30
Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45
Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60
Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80
Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95
Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gin Glu Ile Ser 100 105 110
Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115 120 125
Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140
Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160
Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175
Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190
Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205
Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 220
Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240
Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gin Ala Pro Leu Met 245 250 255
Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270
Gin Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285
Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300
Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320
Gin Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335
Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350
Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365
Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380
Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400
Asn Arg Gly Gin Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415
Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430
Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445
Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460
Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn 465 470 475 480
Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495
Gin Thr Ile Pro Gin Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gin 500 505 510
Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525
Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540
Gin Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560
Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575
Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590
Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gin Lys 595 600 605
Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620
Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640
Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655
Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670
Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680 685
Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700
Ser Asn Trp Leu Thr Arg Ile Asn Thr Gin Phe Asn Lys Arg Lys Glu 705 710 715 720
Gin Met Tyr Gin Ala Leu Gin Asn Gin Val Asp Ala Ile Lys Thr Ala 725 730 735
Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750
Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765
Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780
Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu 785 790 795 800
Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810 815
Asp His Arg Ser Ile Leu Gly Glu Gin Thr Asn Glu Leu Ser Asp Leu 820 825 830
Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845
Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860
Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880
Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895
Tyr Ile Tyr Ser Thr Asn Arg Asn Gin Phe Gly Ile Tyr Asn Ser Arg 900 905 910
Leu Ser Glu Val Asn Ile Ala Gin Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925
Arg Tyr Gin Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930 935 940
Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960
Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975
Cys Glu Ile Ile Trp Thr Leu Gin Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990
Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005
Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020
Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu 1025 1030 1035 1040
Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 1045 1050 1055
Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn Thr 1060 1065 1070
Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro Asp 1075 1080 1085
Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100
Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr Leu 1105 1110 1115 1120
Asn Ser Gly Ile Leu Asn Ile Asn Gin Gin Arg Gly Val Thr Glu Gly 1125 1130 1135
Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile 1140 1145 1150
Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg 1155 1160 1165
Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr 1170 1175 1180
Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr 1185 1190 1195 1200
Ser Asn Leu Asn Asp Ser Leu Gly Gin Ile Ile Val Met Asp Ser Ile 1205 1210 1215
Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230
Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245
Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260
Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 1265 1270
<210>7 <211 > 1297 <212> PRT <213> Clostridium botulinum Serotype G <400>7
Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 15 10 15
Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30
Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45
Arg Phe Thr Tyr Gly Phe Gin Pro Asp Gin Phe Asn Alá Ser Thr Gly 50 55 60
Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80
Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95
Asn Arg Ile Asn Ser Lys Pro Ser Gly Gin Arg Leu Leu Asp Met Ile 100 105 110
Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125
Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gin 130 135 140
Pro Gly Ala Glu Asp Gin Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145 150 155 160
Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175
Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190
Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gin Glu 195 200 205
Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220
Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240
Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255
Phe Met Gin His Ser Asp Pro Val Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn lie 275 280 285
Tyr Asn Lys Ala Leu Gin Asn Phe Gin Asp Ile Ala Asn Arg Leu Asn 290 295 300
Ile Val Ser Ser Ala Gin Gly Ser Gly lie Asp Ile Ser Leu Tyr Lys 305 310 315 320
Gin Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335
Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350
Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly lie Lys Thr 355 360 365
Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro lie Lys Thr Glu Lys 370 375 380
Leu Leu Asp Asn Thr lie Tyr Thr Gin Asn Glu Gly Phe Asn lie Ala 385 390 395 400
Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gin Asn Lys Ala Val Asn 405 410 415
Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430
Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445
Gin Cys lie lie Val Asn Asn Glu Asp Leu Phe Phe lie Ala Asn Lys 450 455 460
Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr lie Ala Tyr Asn 465 470 475 480
Thr Gin Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gin Leu lie 485 490 495
Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510
Glu Pro Phe Thr Asn Phe Asp Asp lie Asp lie Pro Val Tyr lie Lys 515 520 525
Gin Ser Ala Leu Lys Lys lie Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540
Tyr Leu His Ala Gin Thr Phe Pro Ser Asn Ile Glu Asn Leu Gin Leu 545 550 555 560
Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575
Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590
Ser Leu Phe Val Asn Trp Val Lys Gly Val lie Asp Asp Phe Thr Ser 595 600 605
Glu Ser Thr Gin Lys Ser Thr lie Asp Lys Val Ser Asp Val Ser Ile 610 615 620
Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640
Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650 655
Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670
Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685
Asn Ala Leu Lys Lys Arg Asp Gin Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700
Ile Val Ser Gin Trp Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr Ile 705 710 715 720
Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gin Ser Gin Ala Ile Glu 725 730 735
Lys Ile Ile Glu Asp Gin Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750
Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gin Ser 755 760 765
Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe lie Asn Gin Cys Ser 770 775 780
Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu 785 790 795 800
Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815
Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830
Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845
Thr Lys Asp Thr Ile Leu Ile Gin Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860
Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880
Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895
Ile Phe Asn Asp Ile Gly Asn Gly Gin Phe Lys Leu Asn Asn Ser Glu 900 905 910
Asn Ser Asn Ile Thr Ala His Gin Ser Lys Phe Val Val Tyr Asp Ser 915 920 925
Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940
Asn Asn Asn Asp Ile Gin Thr Tyr Leu Gin Asn Glu Tyr Thr Ile Ile 945 950 955 960
Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975
Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990
Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005
Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile 1010 1015 1020
Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp 1025 1030 1035 1040
Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055
Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060 1065 1070
Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gin Ser Ser 1075 1080 1085
Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095 1100
Gin Tyr Tyr Leu Phe Asn Gin Gly Met Gin Asn Ile Tyr Ile Lys Tyr 1105 1110 1115 1120
Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135
Asn Ala Ala Ile Asn Tyr Gin Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140 1145 1150
Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn Ile Val 1155 1160 1165
Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu 1170 1175 1180
Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile Gin Thr Gin 1185 1190 1195 1200
Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215
Gin Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gin Ile Leu 1220 1225 1230
Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245
Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260
Ile Ser Gin Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu 1265 1270 1275 1280
Arg Leu Gly Cys Asn Trp Gin Phe Ile Pro Val Asp Glu Gly Trp Thr 1285 1290 1295
Glu
<210>8 <211> 1315 <212> PRT <213> Clostridium tetani <400>8
Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 15 10 15
Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30
Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45
Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60
Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80
Asp Ser Asp Lys Asp Arg Phe Leu Gin Thr Met Val Lys Leu Phe Asn 85 90 95
Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110
Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125
Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gin Asp Pro 130 135 140
Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160
Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175
Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190
Ile Met Gin Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205
Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220
Gin Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His 225 230 235 240
Gly Leu Tyr Gly Met Gin Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255
Gin Glu Ile Tyr Met Gin His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270
Phe Thr Phe Gly Gly Gin Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285
Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300
Lys Leu Ser Gin Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320
Ser Tyr Lys Gin Ile Tyr Gin Gin Lys Tyr Gin Phe Asp Lys Asp Ser 325 330 335
Asn Gly Gin Tyr Ile Val Asn Glu Asp Lys Phe Gin Ile Leu Tyr Asn 340 345 350
Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365
Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380
Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400
Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gin Asn Met 405 410 415
Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430
Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445
Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460
Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480
Lys Asn Ser Phe Ser Glu Glu Pro Phe Gin Asp Glu Ile Val Ser Tyr 485 490 495
Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510
Ile Val Asp Tyr Asn Leu Gin Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525
Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540
Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560
Tyr Gin Tyr Leu Tyr Ala Gin Lys Ser Pro Thr Thr Leu Gin Arg Ile 565 570 575
Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590
Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gin Gly Ala Gin 595 600 605
Gly Ile Leu Phe Leu Gin Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620
Asn Glu Ser Ser Gin Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640
Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gin Gly 645 650 655
Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670
Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685
Ser Ile Ala Glu Ser Ser Thr Gin Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700
Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720
Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gin Phe Gin Lys 725 730 735
Arg Ser Tyr Gin Met Tyr Arg Ser Leu Glu Tyr Gin Val Asp Ala Ile 740 745 750
Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765
Glu Gin Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780
Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800
Ser Arg Ser Phe Leu Val Asn Gin Met Ile Asn Glu Ala Lys Lys Gin 805 810 815
Leu Leu Glu Phe Asp Thr Gin Ser Lys Asn Ile Leu Met Gin Tyr Ile 820 825 830
Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845
Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860
Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880
Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895
Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910
Gin Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925
Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940
Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960
Val Ser Ala Ser His Leu Glu Gin Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975
Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990
Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005
Gly Glu Val Arg Gin Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020
Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg 1025 1030 1035 1040
Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala 1045 1050 1055
Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 1060 1065 1070
Lys Leu Asp Arg Cys Asn Asn Asn Asn Gin Tyr Val Ser Ile Asp Lys 1075 1080 1085
Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu 1090 1095 1100
Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn 1105 1110 1115 1120
Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser 1125 1130 1135
Ser Lys Asp Val Gin Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 1140 1145 1150
Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 1155 1160 1165
Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn 1170 1175 1180
Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val 1185 1190 1195 1200
Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn 1205 1210 1215
Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro 1220 1225 1230
Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245
Lys Thr Tyr Ser Val Gin Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260
Leu Gly Leu Val Gly Thr His Asn Gly Gin Ile Gly Asn Asp Pro Asn 1265 1270 1275 1280
Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp 1285 1290 1295
Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 1300 1305 1310
Thr Asn Asp 1315 <210> 9 <211> 1268
<212> PRT <213> Clostridium baratii <400>9
Met Pro Val Asn Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 15 10 15
Thr Thr Ile Leu Tyr Met Lys Met Pro Tyr Tyr Glu Asp Ser Asn Lys 20 25 30
Tyr Tyr Lys Ala Phe Glu Ile Met Asp Asn Val Trp Ile Ile Pro Glu 35 40 45
Arg Asn Ile Ile Gly Lys Lys Pro Ser Asp Phe Tyr Pro Pro Ile Ser 50 55 60
Leu Asp Ser Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80
Asp Ala Glu Lys Asp Arg Phe Leu Lys Thr Val Ile Lys Leu Phe Asn 85 90 95
Arg Ile Asn Ser Asn Pro Ala Gly Gin Val Leu Leu Glu Glu Ile Lys 100 105 110
Asn Gly Lys Pro Tyr Leu Gly Asn Asp His Thr Ala Val Asn Glu Phe 115 120 125
Cys Ala Asn Asn Arg Ser Thr Ser Val Glu Ile Lys Glu Ser Asn Gly 130 135 140
Thr Thr Asp Ser Met Leu Leu Asn Leu Val Ile Leu Gly Pro Gly Pro 145 150 155 160
Asn Ile Leu Glu Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn 165 170 175
Ile Ala Tyr Asp Pro Ser Glu Lys Gly Phe Gly Ser Ile Gin Leu Met 180 185 190
Ser Phe Ser Thr Glu Tyr Glu Tyr Ala Phe Asn Asp Asn Thr Asp Leu 195 200 205
Phe Ile Ala Asp Pro Ala Ile Ser Leu Ala His Glu Leu Ile His Val 210 215 220
Leu His Gly Leu Tyr Gly Ala Lys Gly Val Thr Asn Lys Lys Val Ile 225 230 235 240
Glu Val Asp Gin Gly Ala Leu Met Ala Ala Glu Lys Asp Ile Lys Ile 245 250 255
Glu Glu Phe Ile Thr Phe Gly Gly Gin Asp Leu Asn Ile Ile Thr Asn 260 265 270
Ser Thr Asn Gin Lys Ile Tyr Val Ile Leu Leu Ser Asn Tyr Thr Ala 275 280 285
Ile Ala Ser Arg Leu Ser Gin Val Asn Arg Asn Asn Ser Ala Leu Asn 290 295 300
Thr Thr Tyr Tyr Lys Asn Phe Phe Gin Trp Lys Tyr Gly Leu Asp Gin 305 310 315 320
Asp Ser Asn Gly Asn Tyr Thr Val Asn Ile Ser Lys Phe Asn Ala Ile 325 330 335
Tyr Lys Lys Leu Phe Ser Phe Thr Glu Cys Asp Leu Ala Gin Lys Phe 340 345 350
Gin Val Lys Asn Arg Ser Asn Tyr Leu Phe His Phe Lys Pro Phe Arg 355 360 365
Leu Leu Asp Leu Leu Asp Asp Asn Ile Tyr Ser Ile Ser Glu Gly Phe 370 375 380
Asn Ile Gly Ser Leu Arg Val Asn Asn Asn Gly Gin Asn Ile Asn Leu 385 390 395 400
Asn Ser Arg Ile Val Gly Pro Ile Pro Asp Asn Gly Leu Val Glu Arg 405 410 415
Phe Val Gly Leu Cys Lys Ser Ile Val Ser Lys Lys Gly Thr Lys Asn 420 425 430
Ser Leu Cys Ile Lys Val Asn Asn Arg Asp Leu Phe Phe Val Ala Ser 435 440 445
Glu Ser Ser Tyr Asn Glu Asn Gly Ile Asn Ser Pro Lys Glu Ile Asp 450 455 460
Asp Thr Thr Ile Thr Asn Asn Asn Tyr Lys Lys Asn Leu Asp Glu Val 465 470 475 480
Ile Leu Asp Tyr Asn Ser Asp Ala Ile Pro Asn Leu Ser Ser Arg Leu 485 490 495
Leu Asn Thr Thr Ala Gin Asn Asp Ser Tyr Val Pro Lys Tyr Asp Ser 500 505 510
Asn Gly Thr Ser Glu Ile Lys Glu Tyr Thr Val Asp Lys Leu Asn Val 515 520 525
Phe Phe Tyr Leu Tyr Ala Gin Lys Ala Pro Glu Gly Glu Ser Ala Ile 530 535 540
Ser Leu Thr Ser Ser Val Asn Thr Ala Leu Leu Asp Ala Ser Lys Val 545 550 555 560
Tyr Thr Phe Phe Ser Ser Asp Phe Ile Asn Thr Val Asn Lys Pro Val 565 570 575
Gin Ala Ala Leu Phe Ile Ser Trp Ile Gin Gin Val Ile Asn Asp Phe 580 585 590
Thr Thr Glu Ala Thr Gin Lys Ser Thr Ile Asp Lys Ile Ala Asp Ile 595 600 605
Ser Leu Ile Val Pro Tyr Val Gly Leu Ala Leu Asn Ile Gly Asn Glu 610 615 620
Val Gin Lys Gly Asn Phe Lys Glu Ala Ile Glu Leu Leu Gly Ala Gly 625 630 635 640
Ile Leu Leu Glu Phe Val Pro Glu Leu Leu Ile Pro Thr Ile Leu Val 645 650 655
Phe Thr Ile Lys Ser Phe Ile Asn Ser Asp Asp Ser Lys Asn Lys Ile 660 665 670
Ile Lys Ala Ile Asn Asn Ala Leu Arg Glu Arg Glu Leu Lys Trp Lys 675 680 685
Glu Val Tyr Ser Trp Ile Val Ser Asn Trp Leu Thr Arg Ile Asn Thr 690 695 700
Gin Phe Asn Lys Arg Lys Glu Gin Met Tyr Gin Ala Leu Gin Asn Gin 705 710 715 720
Val Asp Gly Ile Lys Lys Ile Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr 725 730 735
Leu Asp Glu Lys Asn Arg Leu Arg Ala Glu Tyr Asn Ile Tyr Ser Ile 740 745 750
Lys Glu Glu Leu Asn Lys Lys Val Ser Leu Ala Met Gin Asn Ile Asp 755 760 765
Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr Leu Met Lys Leu Ile Asn 770 775 780
Glu Ala Lys Ile Asn Lys Leu Ser Glu Tyr Asp Lys Arg Val Asn Gin 785 790 795 800
Tyr Leu Leu Asn Tyr Ile Leu Glu Asn Ser Ser Thr Leu Gly Thr Ser 805 810 815
Ser Val Pro Glu Leu Asn Asn Leu Val Ser Asn Thr Leu Asn Asn Ser 820 825 830
Ile Pro Phe Glu Leu Ser Glu Tyr Thr Asn Asp Lys Ile Leu Ile His 835 840 845
Ile Leu Ile Arg Phe Tyr Lys Arg Ile Ile Asp Ser Ser Ile Leu Asn 850 855 860
Met Lys Tyr Glu Asn Asn Arg Phe Ile Asp Ser Ser Gly Tyr Gly Ser 865 870 875 880
Asn Ile Ser Ile Asn Gly Asp Ile Tyr Ile Tyr Ser Thr Asn Arg Asn 885 890 895
Gin Phe Gly Ile Tyr Ser Ser Arg Leu Ser Glu Val Asn Ile Thr Gin 900 905 910
Asn Asn Thr Ile Ile Tyr Asn Ser Arg Tyr Gin Asn Phe Ser Val Ser 915 920 925
Phe Trp Val Arg Ile Pro Lys Tyr Asn Asn Leu Lys Asn Leu Asn Asn 930 935 940
Glu Tyr Thr Ile Ile Asn Cys Met Arg Asn Asn Asn Ser Gly Trp Lys 945 950 955 960
Ile Ser Leu Asn Tyr Asn Asn Ile Ile Trp Thr Leu Gin Asp Thr Thr 965 970 975
Gly Asn Asn Gin Lys Leu Val Phe Asn Tyr Thr Gin Met Ile Asp Ile 980 985 990
Ser Asp Tyr Ile Asn Lys Trp Thr Phe Val Thr Ile Thr Asn Asn Arg 995 1000 1005
Leu Gly His Ser Lys Leu Tyr Ile Asn Gly Asn Leu Thr Asp Gin Lys 1010 1015 1020
Ser Ile Leu Asn Leu Gly Asn Ile His Val Asp Asp Asn Ile Leu Phe 1025 1030 1035 1040
Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe 1045 1050 1055
Lys Ile Phe Asn Met Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr 1060 1065 1070
His Ser Glu Pro Asp Ser Thr Ile Leu Lys Asp Phe Trp Gly Asn Tyr 1075 1080 1085
Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Leu Asn Leu Leu Lys Pro Asn 1090 1095 1100
Met Ser Val Thr Lys Asn Ser Asp Ile Leu Asn Ile Asn Arg Gin Arg 1105 1110 1115 1120
Gly Ile Tyr Ser Lys Thr Asn Ile Phe Ser Asn Ala Arg Leu Tyr Thr 1125 1130 1135
Gly Val Glu Val Ile Ile Arg Lys Val Gly Ser Thr Asp Thr Ser Asn 1140 1145 1150
Thr Asp Asn Phe Val Arg Lys Asn Asp Thr Val Tyr Ile Asn Val Val 1155 1160 1165
Asp Gly Asn Ser Glu Tyr Gin Leu Tyr Ala Asp Val Ser Thr Ser Ala 1170 1175 1180
Val Glu Lys Thr Ile Lys Leu Arg Arg Ile Ser Asn Ser Asn Tyr Asn 1185 1190 1195 1200
Ser Asn Gin Met Ile Ile Met Asp Ser Ile Gly Asp Asn Cys Thr Met 1205 1210 1215
Asn Phe Lys Thr Asn Asn Gly Asn Asp Ile Gly Leu Leu Gly Phe His 1220 1225 1230
Leu Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Lys Asn Ile Arg Asn 1235 1240 1245
Asn Thr Arg Asn Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys Glu His 1250 1255 1260
Gly Trp Gin Glu 1265
<210> 10 <211> 1251 <212> PRT <213> Clostridium butyricum <400> 10
Met Pro Thr lie Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg 15 10 15
Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gin Gin Phe Tyr Lys Ser 20 25 30
Phe Asn lie Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val lie 35 40 45
Gly Thr lie Pro Gin Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Gin Glu Lys 65 70 75 80
Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asp 85 90 95
Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110
Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe lie lie Asn Asp 115 120 125
Ala Ser Ala Val Pro Ile Gin Phe Ser Asn Gly Ser Gin Ser Ile Leu 130 135 140
Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190
Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gin Asp Pro Ala Leu 195 200 205
Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gin Lys Gin Asn Pro Leu 225 230 235 240
Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255
Gly Thr Asp Leu Asn lie lie Thr Ser Ala Gin Ser Asn Asp lie Tyr 260 265 270
Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285
Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300
Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320
Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr Ile 340 345 350
Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365
Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380
Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400
Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415
Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445
Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460
Glu Asn Asp Leu Asp Gin Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480
Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gin Asn Asp Ala 485 490 495
Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gin His 500 505 510
Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540
Leu Leu Glu Gin Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560
Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Gly Trp Ile 565 570 575
Gin Gin Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr 580 585 590
Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605
Ala Leu Asn Ile Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640
Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655
Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670
Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685
Trp Met Thr Lys Ile Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700
Tyr Gin Ala Leu Gin Asn Gin Val Asn Ala Leu Lys Ala Ile Ile Glu 705 710 715 720
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735
Lys Tyr Asp Ile Glu Gin Ile Glu Asn Glu Leu Asn Gin Lys Val Ser 740 745 750
Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765
Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780
Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His 785 790 795 800
Gly Ser Ile Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val Ile 805 810 815
Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830
Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860
Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880
Tyr Pro Thr Asn Lys Asn Gin Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895
Glu Val Asn Ile Ser Gin Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910
Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925
Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960
Trp Thr Leu Gin Asp Asn Ser Gly Ile Asn Gin Lys Leu Ala Phe Asn 965 970 975
Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990
Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005
Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020
Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040
Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055
Thr Glu Ile Gin Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 1060 1065 1070
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100
Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120
Leu Tyr Ser Gly Ile Lys Val Lys Ile Gin Arg Val Asn Asn Ser Ser 1125 1130 1135
Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr Ile Asn Phe 1140 1145 1150
Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180
Asn Gin Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe 1185 1190 1195 1200
Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp 1205 1210 1215
Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr 1220 1225 1230
Asn Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp 1235 1240 1245
Gin Glu Lys 1250 <210> 11 <211> 1035 <212> PRT <213> Bos taurus <400> 11
Met Gly Ser Lys Arg Ser Val Pro Ser Arg His Arg Ser Leu Thr Thr 15 10 15
Tyr Glu Val Met Phe Ala Val Leu Phe Val Ile Leu Val Ala Leu Cys 20 25 30
Ala Gly Leu Ile Ala Val Ser Trp Leu Ser Ile Gin Gly Ser Val Lys 35 40 45
Asp Ala Ala Phe Gly Lys Ser His Glu Ala Arg Gly Thr Leu Lys Ile 50 55 60
Ile Ser Gly Ala Thr Tyr Asn Pro His Leu Gin Asp Lys Leu Ser Val 65 70 75 80
Asp Phe Lys Val Leu Ala Phe Asp Ile Gin Gin Met Ile Asp Asp Ile 85 90 95
Phe Gin Ser Ser Asn Leu Lys Asn Glu Tyr Lys Asn Ser Arg Val Leu 100 105 110
Gin Phe Glu Asn Gly Ser Ile Ile Val Ile Phe Asp Leu Leu Phe Asp 115 120 125
Gin Trp Val Ser Asp Lys Asn Val Lys Glu Glu Leu Ile Gin Gly Ile 130 135 140
Glu Ala Asn Lys Ser Ser Gin Leu Val Thr Phe His Ile Asp Leu Asn 145 150 155 160
Ser Ile Asp Ile Thr Ala Ser Leu Glu Asn Phe Ser Thr Ile Ser Pro 165 170 175
Ala Thr Thr Ser Glu Lys Leu Thr Thr Ser Ile Pro Leu Ala Thr Pro 180 185 190
Gly Asn Val Ser Ile Glu Cys Pro Pro Asp Ser Arg Leu Cys Ala Asp 195 200 205
Ala Leu Lys Cys Ile Ala Ile Asp Leu Phe Cys Asp Gly Glu Leu Asn 210 215 220
Cys Pro Asp Gly Ser Asp Glu Asp Asn Lys Thr Cys Ala Thr Ala Cys 225 230 235 240
Asp Gly Arg Phe Leu Leu Thr Gly Ser Ser Gly Ser Phe Glu Ala Leu 245 250 255
His Tyr Pro Lys Pro Ser Asn Asn Thr Ser Ala Val Cys Arg Trp Ile 260 265 270
Ile Arg Val Asn Gin Gly Leu Ser Ile Gin Leu Asn Phe Asp Tyr Phe 275 280 285
Asn Thr Tyr Tyr Ala Asp Val Leu Asn Ile Tyr Glu Gly Met Gly Ser 290 295 300
Ser Lys Ile Leu Arg Ala Ser Leu Trp Ser Asn Asn Pro Gly Ile Ile 305 310 315 320
Arg Ile Phe Ser Asn Gin Val Thr Ala Thr Phe Leu Ile Gin Ser Asp 325 330 335
Glu Ser Asp Tyr Ile Gly Phe Lys Val Thr Tyr Thr Ala Phe Asn Ser 340 345 350
Lys Glu Leu Asn Asn Tyr Glu Lys Ile Asn Cys Asn Phe Glu Asp Gly 355 360 365
Phe Cys Phe Trp He Gin Asp Leu Asn Asp Asp Asn Glu Trp Glu Arg 370 375 380
Thr Gin Gly Ser Thr Phe Pro Pro Ser Thr Gly Pro Thr Phe Asp His 385 390 395 400
Thr Phe Gly Asn Glu Ser Gly Phe Tyr He Ser Thr Pro Thr Gly Pro 405 410 415
Gly Gly Arg Arg Glu Arg Val Gly Leu Leu Thr Leu Pro Leu Asp Pro 420 425 430
Thr Pro Glu Gin Ala Cys Leu Ser Phe Trp Tyr Tyr Met Tyr Gly Glu 435 440 445
Asn Val Tyr Lys Leu Ser He Asn He Ser Ser Asp Gin Asn Met Glu 450 455 460
Lys Thr He Phe Gin Lys Glu Gly Asn Tyr Gly Gin Asn Trp Asn Tyr 465 470 475 480
Gly Gin Val Thr Leu Asn Glu Thr Val Glu Phe Lys Val Ser Phe Tyr 485 490 495
Gly Phe Lys Asn Gin He Leu Ser Asp He Ala Leu Asp Asp He Ser 500 505 510
Leu Thr Tyr Gly He Cys Asn Val Ser Val Tyr Pro Glu Pro Thr Leu 515 520 525
Val Pro Thr Pro Pro Pro Glu Leu Pro Thr Asp Cys Gly Gly Pro His 530 535 540
Asp Leu Trp Glu Pro Asn Thr Thr Phe Thr Ser He Asn Phe Pro Asn 545 550 555 560
Ser Tyr Pro Asn Gin Ala Phe Cys He Trp Asn Leu Asn Ala Gin Lys 565 570 575
Gly Lys Asn He Gin Leu His Phe Gin Glu Phe Asp Leu Glu Asn He 580 585 590
Ala Asp Val Val Glu He Arg Asp Gly Glu Gly Asp Asp Ser Leu Phe 595 600 605
Leu Ala Val Tyr Thr Gly Pro Gly Pro Val Asn Asp Val Phe Ser Thr 610 615 620
Thr Asn Arg Met Thr Val Leu Phe He Thr Asp Asn Met Leu Ala Lys 625 630 635 640
Gin Gly Phe Lys Ala Asn Phe Thr Thr Gly Tyr Gly Leu Gly He Pro 645 650 655
Glu Pro Cys Lys Glu Asp Asn Phe Gin Cys Lys Asp Gly Glu Cys He 660 665 670
Pro Leu Val Asn Leu Cys Asp Gly Phe Pro His Cys Lys Asp Gly Ser 675 680 685
Asp Glu Ala His Cys Val Arg Leu Phe Asn Gly Thr Thr Asp Ser Ser 690 695 700
Gly Leu Val Gin Phe Arg He Gin Ser He Trp His Val Ala Cys Ala 705 710 715 720
Glu Asn Trp Thr Thr Gin He Ser Asp Asp Val Cys Gin Leu Leu Gly 725 730 735
Leu Gly Thr Gly Asn Ser Ser Val Pro Thr Phe Ser Thr Gly Gly Gly 740 745 750
Pro Tyr Val Asn Leu Asn Thr Ala Pro Asn Gly Ser Leu He Leu Thr 755 760 765
Pro Ser Gin Gin Cys Leu Glu Asp Ser Leu He Leu Leu Gin Cys Asn 770 775 780
Tyr Lys Ser Cys Gly Lys Lys Leu Val Thr Gin Glu Val Ser Pro Lys 785 790 795 800
He Val Gly Gly Ser Asp Ser Arg Glu Gly Ala Trp Pro Trp Val Val 805 810 815
Ala Leu Tyr Phe Asp Asp Gin Gin Val Cys Gly Ala Ser Leu Val Ser 820 825 830
Arg Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Met 835 840 845
Glu Pro Ser Lys Trp Lys Ala Val Leu Gly Leu His Met Ala Ser Asn 850 855 860
Leu Thr Ser Pro Gin He Glu Thr Arg Leu He Asp Gin He Val He 865 870 875 880
Asn Pro His Tyr Asn Lys Arg Arg Lys Asn Asn Asp Ile Ala Met Met 885 890 895
His Leu Glu Met Lys Val Asn Tyr Thr Asp Tyr Ile Gin Pro Ile Cys 900 905 910
Leu Pro Glu Glu Asn Gin Val Phe Pro Pro Gly Arg Ile Cys Ser Ile 915 920 925
Ala Gly Trp Gly Ala Leu Ile Tyr Gin Gly Ser Thr Ala Asp Val Leu 930 935 940
Gin Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Lys Cys Gin Gin Gin 945 950 955 960
Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Val Cys Ala Gly Tyr Glu 965 970 975
Ala Gly Gly Val Asp Ser Cys Gin Gly Asp Ser Gly Gly Pro Leu Met 980 985 990
Cys Gin Glu Asn Asn Arg Trp Leu Leu Ala Gly Val Thr Ser Phe Gly 995 1000 1005
Tyr Gin Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Pro 1010 1015 1020
Arg Phe Thr Glu Trp Ile Gin Ser Phe Leu His 1025 1030 1035
<210> 12 <211> 183 <212> PRT <213> Human rhinovirus C <400> 12
Gly Pro Glu His Glu Phe Leu Asn Ala Leu Ile Arg Arg Asn Cys His 15 10 15
Ile Ile Thr Thr Asp Lys Gly Glu Phe Asn Leu Leu Gly Ile Tyr Ser 20 25 30
Asn Cys Ala Val Val Pro Thr His Ala Glu Pro Gly Asp Val Val Asp 35 40 45
Ile Asp Gly Arg Leu Val Arg Val Leu Lys Gin Gin Val Leu Thr Asp 50 55 60
Met Asn Asp Val Asp Thr Glu Val Thr Val Leu Trp Leu Asp Gin Asn 65 70 75 80
Glu Lys Phe Arg Asp Ile Arg Arg Phe Ile Pro Glu His Gin Gin Asp 85 90 95
Trp His Asn Ile His Leu Ala Thr Asn Val Thr Lys Phe Pro Met Leu 100 105 110
Asn Val Glu Val Gly His Thr Val Pro Tyr Gly Glu Ile Asn Leu Ser 115 120 125
Gly Asn Ala Thr Cys Arg Leu Tyr Lys Tyr Asp Tyr Pro Thr Gin Pro 130 135 140
Gly Gin Cys Gly Ala Val Leu Ala Asn Thr Gly Asn Ile Ile Gly Ile 145 150 155 160
His Val Gly Gly Asn Gly Arg Val Gly Tyr Ala Ala Ala Leu Leu Arg 165 170 175
Lys Tyr Phe Ala Glu Glu Gin 180
<210> 13 <211 > 183 <212> PRT <213> Human enterovirus 71 <220> <221 > mise feature <222> 124 <223> Xaa is unknown or other <400> 13
Gly Pro Ser Leu Asp Phe Ala Leu Ser Leu Leu Arg Arg Asn Val Arg 15 10 15
Gin Val Gin Thr Asp Gin Gly His Phe Thr Met Leu Gly Val Arg Asp 20 25 30
Arg Leu Ala Val Leu Pro Arg His Ser Gin Pro Gly Lys Thr lie Trp 35 40 45
Ile Glu His Lys Leu Val Asn Val Leu Asp Ala Val Glu Leu Val Asp 50 55 60
Glu Gin Gly Val Asn Leu Glu Leu Thr Leu Ile Thr Leu Asp Thr Asn 65 70 75 80
Glu Lys Phe Arg Asp lie Thr Lys Phe lie Pro Glu Asn lie Ser Thr 85 90 95
Ala Ser Asp Ala Thr Leu Val lie Asn Thr Glu His Met Pro Ser Met 100 105 110
Phe Val Pro Val Gly Asp Val Val Gin Tyr Gly Xaa Leu Asn Leu Ser 115 120 125
Gly Lys Pro Thr His Arg Thr Met Met Tyr Asn Phe Pro Thr Lys Ala 130 135 140
Gly Gin Cys Gly Gly Val Val Thr Ser Val Gly Lys Val lie Gly lie 145 150 155 160
His Ile Gly Gly Asn Gly Arg Gin Gly Phe Cys Ala Gly Leu Lys Arg 165 170 175
Ser Tyr Phe Ala Ser Glu Gin 180 <210> 14 <211> 3054 <212> PRT <213> Potyvirus <400> 14
Met Ala Leu Ile Phe Gly Thr Val Asn Ala Asn Ile Leu Lys Glu Val 15 10 15
Phe Gly Gly Ala Arg Met Ala Cys Val Thr Ser Ala His Met Ala Gly 20 25 30
Ala Asn Gly Ser Ile Leu Lys Lys Ala Glu Glu Thr Ser Arg Ala Ile 35 40 45
Met His Lys Pro Val Ile Phe Gly Glu Asp Tyr Ile Thr Glu Ala Asp 50 55 60
Leu Pro Tyr Thr Pro Leu His Leu Glu Val Asp Ala Glu Met Glu Arg 65 70 75 80
Met Tyr Tyr Leu Gly Arg Arg Ala Leu Thr His Gly Lys Arg Arg Lys 85 90 95
Val Ser Val Asn Asn Lys Arg Asn Arg Arg Arg Lys Val Ala Lys Thr 100 105 110
Tyr Val Gly Arg Asp Ser Ile Val Glu Lys Ile Val Val Pro His Thr 115 120 125
Glu Arg Lys Val Asp Thr Thr Ala Ala Val Glu Asp Ile Cys Asn Glu 130 135 140
Ala Thr Thr Gin Leu Val His Asn Ser Met Pro Lys Arg Lys Lys Gin 145 150 155 160
Lys Asn Phe Leu Pro Ala Thr Ser Leu Ser Asn Val Tyr Ala Gin Thr 165 170 175
Trp Ser Ile Val Arg Lys Arg His Met Gin Val Glu Ile Ile Ser Lys 180 185 190
Lys Ser Val Arg Ala Arg Val Lys Arg Phe Glu Gly Ser Val Gin Leu 195 200 205
Phe Ala Ser Val Arg His Met Tyr Gly Glu Arg Lys Arg Val Asp Leu 210 215 220
Arg Ile Asp Asn Trp Gin Gin Glu Thr Leu Leu Asp Leu Ala Lys Arg 225 230 235 240
Phe Lys Asn Glu Arg Val Asp Gin Ser Lys Leu Thr Phe Gly Ser Ser 245 250 255
Gly Leu Val Leu Arg Gin Gly Ser Tyr Gly Pro Ala His Trp Tyr Arg 260 265 270
His Gly Met Phe Ile Val Arg Gly Arg Ser Asp Gly Met Leu Val Asp 275 280 285
Ala Arg Ala Lys Val Thr Phe Ala Val Cys His Ser Met Thr His Tyr 290 295 300
Ser Asp Lys Ser Ile Ser Glu Ala Phe Phe Ile Pro Tyr Ser Lys Lys 305 310 315 320
Phe Leu Glu Leu Arg Pro Asp Gly Ile Ser His Glu Cys Thr Arg Gly 325 330 335
Val Ser Val Glu Arg Cys Gly Glu Val Ala Ala Ile Leu Thr Gin Ala 340 345 350
Leu Ser Pro Cys Gly Lys Ile Thr Cys Lys Arg Cys Met Val Glu Thr 355 360 365
Pro Asp Ile Val Glu Gly Glu Ser Gly Glu Ser Val Thr Asn Gin Gly 370 375 380
Lys Leu Leu Ala Met Leu Lys Glu Gin Tyr Pro Asp Phe Pro Met Ala 385 390 395 400
Glu Lys Leu Leu Thr Arg Phe Leu Gin Gin Lys Ser Leu Val Asn Thr 405 410 415
Asn Leu Thr Ala Cys Val Ser Val Lys Gin Leu Ile Gly Asp Arg Lys 420 425 430
Gin Ala Pro Phe Thr His Val Leu Ala Val Ser Glu Ile Leu Phe Lys 435 440 445
Gly Asn Lys Leu Thr Gly Ala Asp Leu Glu Glu Ala Ser Thr His Met 450 455 460
Leu Glu Ile Ala Arg Phe Leu Asn Asn Arg Thr Glu Asn Met Arg Ile 465 470 475 480
Gly His Leu Gly Ser Phe Arg Asn Lys Ile Ser Ser Lys Ala His Val 485 490 495
Asn Asn Ala Leu Met Cys Asp Asn Gin Leu Asp Gin Asn Gly Asn Phe 500 505 510
Ile Trp Gly Leu Arg Gly Ala His Ala Lys Arg Phe Leu Lys Gly Phe 515 520 525
Phe Thr Glu Ile Asp Pro Asn Glu Gly Tyr Asp Lys Tyr Val Ile Arg 530 535 540
Lys His Ile Arg Gly Ser Arg Lys Leu Ala Ile Gly Asn Leu Ile Met 545 550 555 560
Ser Thr Asp Phe Gin Thr Leu Arg Gin Gin Ile Gin Gly Glu Thr Ile 565 570 575
Glu Arg Lys Glu Ile Gly Asn His Cys Ile Ser Met Arg Asn Gly Asn 580 585 590
Tyr Val Tyr Pro Cys Cys Cys Val Thr Leu Glu Asp Gly Lys Ala Gin 595 600 605
Tyr Ser Asp Leu Lys His Pro Thr Lys Arg His Leu Val Ile Gly Asn 610 615 620
Ser Gly Asp Ser Lys Tyr Leu Asp Leu Pro Val Leu Asn Glu Glu Lys 625 630 635 640
Met Tyr Ile Ala Asn Glu Gly Tyr Cys Tyr Met Asn Ile Phe Phe Ala 645 650 655
Leu Leu Val Asn Val Lys Glu Glu Asp Ala Lys Asp Phe Thr Lys Phe 660 665 670
Ile Arg Asp Thr Ile Val Pro Lys Leu Gly Ala Trp Pro Thr Met Gin 675 680 685
Asp Val Ala Thr Ala Cys Tyr Leu Leu Ser Ile Leu Tyr Pro Asp Val 690 695 700
Leu Arg Ala Glu Leu Pro Arg Ile Leu Val Asp His Asp Asn Lys Thr 705 710 715 720
Met His Val Leu Asp Ser Tyr Gly Ser Arg Thr Thr Gly Tyr His Met 725 730 735
Leu Lys Met Asn Thr Thr Ser Gin Leu Ile Glu Phe Val His Ser Gly 740 745 750
Leu Glu Ser Glu Met Lys Thr Tyr Asn Val Gly Gly Met Asn Arg Asp 755 760 765
Val Val Thr Gin Gly Ala Ile Glu Met Leu Ile Lys Ser Ile Tyr Lys 770 775 780
Pro His Leu Met Lys Gin Leu Leu Glu Glu Glu Pro Tyr Ile Ile Val 785 790 795 800
Leu Ala Ile Val Ser Pro Ser Ile Leu Ile Ala Met Tyr Asn Ser Gly 805 810 815
Thr Phe Glu Gin Ala Leu Gin Met Trp Leu Pro Asn Thr Met Arg Leu 820 825 830
Ala Asn Leu Ala Ala Ile Leu Ser Ala Leu Ala Gin Lys Leu Thr Leu 835 840 845
Ala Asp Leu Phe Val Gin Gin Arg Asn Leu Ile Asn Glu Tyr Ala Gin 850 855 860
Val Ile Leu Asp Asn Leu Ile Asp Gly Val Arg Val Asn His Ser Leu 865 870 875 880
Ser Leu Ala Met Glu Ile Val Thr Ile Lys Leu Ala Thr Gin Glu Met 885 890 895
Asp Met Ala Leu Arg Glu Gly Gly Tyr Ala Val Thr Ser Glu Lys Val 900 905 910
His Glu Met Leu Glu Lys Asn Tyr Val Lys Ala Leu Lys Asp Ala Trp 915 920 925
Asp Glu Leu Thr Trp Leu Glu Lys Phe Ser Ala Ile Arg His Ser Arg 930 935 940
Lys Leu Leu Lys Phe Gly Arg Lys Pro Leu Ile Met Lys Asn Thr Val 945 950 955 960
Asp Cys Gly Gly His Ile Asp Leu Ser Val Lys Ser Leu Phe Lys Phe 965 970 975
His Leu Glu Leu Leu Lys Gly Thr Ile Ser Arg Ala Val Asn Gly Gly 980 985 990
Ala Arg Lys Val Arg Val Ala Lys Asn Ala Met Thr Lys Gly Val Phe 995 1000 1005
Leu Lys Ile Tyr Ser Met Leu Pro Asp Val Tyr Lys Phe Ile Thr Val 1010 1015 1020
Ser Ser Val Leu Ser Leu Leu Leu Thr Phe Leu Phe Gin Ile Asp Cys 1025 1030 1035 1040
Met Ile Arg Ala His Arg Glu Ala Lys Val Ala Ala Gin Leu Gin Lys 1045 1050 1055
Glu Ser Glu Trp Asp Asn Ile Ile Asn Arg Thr Phe Gin Tyr Ser Lys 1060 1065 1070
Leu Glu Asn Pro Ile Gly Tyr Arg Ser Thr Ala Glu Glu Arg Leu Gin 1075 1080 1085
Ser Glu His Pro Glu Ala Phe Glu Tyr Tyr Lys Phe Cys Ile Gly Lys 1090 1095 1100
Glu Asp Leu Val Glu Gin Ala Lys Gin Pro Glu Ile Ala Tyr Phe Glu 1105 1110 1115 1120
Lys Ile Ile Ala Phe Ile Thr Leu Val Leu Met Ala Phe Asp Ala Glu 1125 1130 1135
Arg Ser Asp Gly Val Phe Lys Ile Leu Asn Lys Phe Lys Gly Ile Leu 1140 1145 1150
Ser Ser Thr Glu Arg Glu Ile Ile Tyr Thr Gin Ser Leu Asp Asp Tyr 1155 1160 1165
Val Thr Thr Phe Asp Asp Asn Met Thr Ile Asn Leu Glu Leu Asn Met 1170 1175 1180
Asp Glu Leu His Lys Thr Ser Leu Pro Gly Val Thr Phe Lys Gin Trp 1185 1190 1195 1200
Trp Asn Asn Gin Ile Ser Arg Gly Asn Val Lys Pro His Tyr Arg Thr 1205 1210 1215
Glu Gly His Phe Met Glu Phe Thr Arg Asp Thr Ala Ala Ser Val Ala 1220 1225 1230
Ser Glu Ile Ser His Ser Pro Ala Arg Asp Phe Leu Val Arg Gly Ala 1235 1240 1245
Val Gly Ser Gly Lys Ser Thr Gly Leu Pro Tyr His Leu Ser Lys Arg 1250 1255 1260
Gly Arg Val Leu Met Leu Glu Pro Thr Arg Pro Leu Thr Asp Asn Met 1265 1270 1275 1280
His Lys Gin Leu Arg Ser Glu Pro Phe Asn Cys Phe Pro Thr Leu Arg 1285 1290 1295
Met Arg Gly Lys Ser Thr Phe Gly Ser Ser Pro Ile Thr Val Met Thr 1300 1305 1310
Ser Gly Phe Ala Leu His His Phe Ala Arg Asn Ile Ala Glu Val Lys 1315 1320 1325
Thr Tyr Asp Phe Val Ile Ile Asp Glu Cys His Val Asn Asp Ala Ser 1330 1335 1340
Ala Ile Ala Phe Arg Asn Leu Leu Phe Glu His Glu Phe Glu Gly Lys 1345 1350 1355 1360
Val Leu Lys Val Ser Ala Thr Pro Pro Gly Arg Glu Val Glu Phe Thr 1365 1370 1375
Thr Gin Phe Pro Val Lys Leu Lys Ile Glu Glu Ala Leu Ser Phe Gin 1380 1385 1390
Glu Phe Val Ser Leu Gin Gly Thr Gly Ala Asn Ala Asp Val Ile Ser 1395 1400 1405
Cys Gly Asp Asn Ile Leu Val Tyr Val Ala Ser Tyr Asn Asp Val Asp 1410 1415 1420
Ser Leu Gly Lys Leu Leu Val Gin Lys Gly Tyr Lys Val Ser Lys Ile 1425 1430 1435 1440
Asp Gly Arg Thr Met Lys Ser Gly Gly Thr Glu Ile Ile Thr Glu Gly 1445 1450 1455
Thr Ser Val Lys Lys His Phe Ile Val Ala Thr Asn Ile Ile Glu Asn 1460 1465 1470
Gly Val Thr Ile Asp Ile Asp Val Val Val Asp Phe Gly Thr Lys Val 1475 1480 1485
Val Pro Val Leu Asp Val Asp Asn Arg Ala Val Gin Tyr Asn Lys Thr 1490 1495 1500
Val Val Ser Tyr Gly Glu Arg Ile Gin Lys Leu Gly Arg Val Gly Arg 1505 1510 1515 1520
His Lys Glu Gly Val Ala Leu Arg Ile Gly Gin Thr Asn Lys Thr Leu 1525 1530 1535
Val Glu Ile Pro Glu Met Val Ala Thr Glu Ala Ala Phe Leu Cys Phe 1540 1545 1550
Met Tyr Asn Leu Pro Val Thr Thr Gin Ser Val Ser Thr Thr Leu Leu 1555 1560 1565
Glu Asn Ala Thr Leu Leu Gin Ala Arg Thr Met Ala Gin Phe Glu Leu 1570 1575 1580
Ser Tyr Phe Tyr Thr Ile Asn Phe Val Arg Phe Asp Gly Ser Met His 1585 1590 1595 1600
Pro Val Ile His Asp Lys Leu Lys Arg Phe Lys Leu His Thr Cys Glu 1605 1610 1615
Thr Phe Leu Asn Lys Leu Ala Ile Pro Asn Lys Gly Leu Ser Ser Trp 1620 1625 1630
Leu Thr Ser Gly Glu Tyr Lys Arg Leu Gly Tyr Ile Ala Glu Asp Ala 1635 1640 1645
Gly Ile Arg Ile Pro Phe Val Cys Lys Glu Ile Pro Asp Ser Leu His 1650 1655 1660
Glu Glu Ile Trp His Ile Val Val Ala His Lys Gly Asp Ser Gly Ile 1665 1670 1675 1680
Gly Arg Leu Thr Ser Val Gin Ala Ala Lys Val Val Tyr Thr Leu Gin 1685 1690 1695
Thr Asp Val His Ser Ile Ala Arg Thr Leu Ala Cys Ile Asn Arg Arg 1700 1705 1710
Ile Ala Asp Glu Gin Met Lys Gin Ser His Phe Glu Ala Ala Thr Gly 1715 1720 1725
Arg Ala Phe Ser Phe Thr Asn Tyr Ser Ile Gin Ser Ile Phe Asp Thr 1730 1735 1740
Leu Lys Ala Asn Tyr Ala Thr Lys His Thr Lys Glu Asn Ile Ala Val 1745 1750 1755 1760
Leu Gin Gin Ala Lys Asp Gin Leu Leu Glu Phe Ser Asn Leu Ala Lys 1765 1770 1775
Asp Gin Asp Val Thr Gly Ile Ile Gin Asp Phe Asn His Leu Glu Thr 1780 1785 1790
Ile Tyr Leu Gin Ser Asp Ser Glu Val Ala Lys His Leu Lys Leu Lys 1795 1800 1805
Ser His Trp Asn Lys Ser Gin Ile Thr Arg Asp Ile Ile Ile Ala Leu 1810 1815 1820
Ser Val Leu Ile Gly Gly Gly Trp Met Leu Ala Thr Tyr Phe Lys Asp 1825 1830 1835 1840
Lys Phe Asn Glu Pro Val Tyr Phe Gin Gly Lys Lys Asn Gin Lys His 1845 1850 1855
Lys Leu Lys Met Arg Glu Ala Arg Gly Ala Arg Gly Gin Tyr Glu Val 1860 1865 1870
Ala Ala Glu Pro Glu Ala Leu Glu His Tyr Phe Gly Ser Ala Tyr Asn 1875 1880 1885
Asn Lys Gly Lys Arg Lys Gly Thr Thr Arg Gly Met Gly Ala Lys Ser 1890 1895 1900
Arg Lys Phe Ile Asn Met Tyr Gly Phe Asp Pro Thr Asp Phe Ser Tyr 1905 1910 1915 1920
Ile Arg Phe Val Asp Pro Leu Thr Gly His Thr Ile Asp Glu Ser Thr 1925 1930 1935
Asn Ala Pro Ile Asp Leu Val Gin His Glu Phe Gly Lys Val Arg Thr 1940 1945 1950
Arg Met Leu Ile Asp Asp Glu Ile Glu Pro Gin Ser Leu Ser Thr His 1955 1960 1965
Thr Thr Ile His Ala Tyr Leu Val Asn Ser Gly Thr Lys Lys Val Leu 1970 1975 1980
Lys Val Asp Leu Thr Pro His Ser Ser Leu Arg Ala Ser Glu Lys Ser 1985 1990 1995 2000
Thr Ala Ile Met Gly Phe Pro Glu Arg Glu Asn Glu Leu Arg Gin Thr 2005 2010 2015
Gly Met Ala Val Pro Val Ala Tyr Asp Gin Leu Pro Pro Lys Asn Glu 2020 2025 2030
Asp Leu Thr Phe Glu Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr 2035 2040 2045
Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly 2050 2055 2060
His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr 2065 2070 2075 2080
Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser 2085 2090 2095
Leu His Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His 2100 2105 2110
Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe 2115 2120 2125
Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu 2130 2135 2140
Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser 2145 2150 2155 2160
Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe 2165 2170 2175
Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu 2180 2185 2190
Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn 2195 2200 2205
Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met 2210 2215 2220
Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg 2225 2230 2235 2240
Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe Met Ser 2245 2250 2255
Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met 2260 2265 2270
Asn Glu Leu Val Tyr Ser Gin Gly Glu Lys Arg Lys Trp Val Val Glu 2275 2280 2285
Ala Leu Ser Gly Asn Leu Arg Pro Val Ala Glu Cys Pro Ser Gin Leu 2290 2295 2300
Val Thr Lys His Val Val Lys Gly Lys Cys Pro Leu Phe Glu Leu Tyr 2305 2310 2315 2320
Leu Gin Leu Asn Pro Glu Lys Glu Ala Tyr Phe Lys Pro Met Met Gly 2325 2330 2335
Ala Tyr Lys Pro Ser Arg Leu Asn Arg Glu Ala Phe Leu Lys Asp Ile 2340 2345 2350
Leu Lys Tyr Ala Ser Glu Ile Glu Ile Gly Asn Val Asp Cys Asp Leu 2355 2360 2365
Leu Glu Leu Ala Ile Ser Met Leu Val Thr Lys Leu Lys Ala Leu Gly 2370 2375 2380
Phe Pro Thr Val Asn Tyr Ile Thr Asp Pro Glu Glu Ile Phe Ser Ala 2385 2390 2395 2400
Leu Asn Met Lys Ala Ala Met Gly Ala Leu Tyr Lys Gly Lys Lys Lys 2405 2410 2415
Glu Ala Leu Ser Glu Leu Thr Leu Asp Glu Gin Glu Ala Met Leu Lys 2420 2425 2430
Ala Ser Cys Leu Arg Leu Tyr Thr Gly Lys Leu Gly Ile Trp Asn Gly 2435 2440 2445
Ser Leu Lys Ala Glu Leu Arg Pro Ile Glu Lys Val Glu Asn Asn Lys 2450 2455 2460
Thr Arg Thr Phe Thr Ala Ala Pro Ile Asp Thr Leu Leu Ala Gly Lys 2465 2470 2475 2480
Val Cys Val Asp Asp Phe Asn Asn Gin Phe Tyr Asp Leu Asn Ile Lys 2485 2490 2495
Ala Pro Trp Thr Val Gly Met Thr Lys Phe Tyr Gin Gly Trp Asn Glu 2500 2505 2510
Leu Met Glu Ala Leu Pro Ser Gly Trp Val Tyr Cys Asp Ala Asp Gly 2515 2520 2525
Ser Gin Phe Asp Ser Ser Leu Thr Pro Phe Leu Ile Asn Ala Val Leu 2530 2535 2540
Lys Val Arg Leu Ala Phe Met Glu Glu Trp Asp Ile Gly Glu Gin Met 2545 2550 2555 2560
Leu Arg Asn Leu Tyr Thr Glu Ile Val Tyr Thr Pro Ile Leu Thr Pro 2565 2570 2575
Asp Gly Thr Ile Ile Lys Lys His Lys Gly Asn Asn Ser Gly Gin Pro 2580 2585 2590
Ser Thr Val Val Asp Asn Thr Leu Met Val Ile Ile Ala Met Leu Tyr 2595 2600 2605
Thr Cys Glu Lys Cys Gly Ile Asn Lys Glu Glu Ile Val Tyr Tyr Val 2610 2615 2620
Asn Gly Asp Asp Leu Leu Ile Ala Ile His Pro Asp Lys Ala Glu Arg 2625 2630 2635 2640
Leu Ser Arg Phe Lys Glu Ser Phe Gly Glu Leu Gly Leu Lys Tyr Glu 2645 2650 2655
Phe Asp Cys Thr Thr Arg Asp Lys Thr Gin Leu Trp Phe Met Ser His 2660 2665 2670
Arg Ala Leu Glu Arg Asp Gly Met Tyr Ile Pro Lys Leu Glu Glu Glu 2675 2680 2685
Arg Ile Val Ser Ile Leu Glu Trp Asp Arg Ser Lys Glu Pro Ser His 2690 2695 2700
Arg Leu Glu Ala Ile Cys Ala Ser Met Ile Glu Ala Trp Gly Tyr Asp 2705 2710 2715 2720
Lys Leu Val Glu Glu Ile Arg Asn Phe Tyr Ala Trp Val Leu Glu Gin 2725 2730 2735
Ala Pro Tyr Ser Gin Leu Ala Glu Glu Gly Lys Ala Pro Tyr Leu Ala 2740 2745 2750
Glu Thr Ala Leu Lys Phe Leu Tyr Thr Ser Gin His Gly Thr Asn Ser 2755 2760 2765
Glu Ile Glu Glu Tyr Leu Lys Val Leu Tyr Asp Tyr Asp Ile Pro Thr 2770 2775 2780
Thr Glu Asn Leu Tyr Phe Gin Ser Gly Thr Val Asp Ala Gly Ala Asp 2785 2790 2795 2800
Ala Gly Lys Lys Lys Asp Gin Lys Asp Asp Lys Val Ala Glu Gin Ala 2805 2810 2815
Ser Lys Asp Arg Asp Val Asn Ala Gly Thr Ser Gly Thr Phe Ser Val 2820 2825 2830
Pro Arg Ile Asn Ala Met Ala Thr Lys Leu Gin Tyr Pro Arg Met Arg 2835 2840 2845
Gly Glu Val Val Val Asn Leu Asn His Leu Leu Gly Tyr Lys Pro Gin 2850 2855 2860
Gin Ile Asp Leu Ser Asn Ala Arg Ala Thr His Glu Gin Phe Ala Ala 2865 2870 2875 2880
Trp His Gin Ala Val Met Thr Ala Tyr Gly Val Asn Glu Glu Gin Met 2885 2890 2895
Lys Ile Leu Leu Asn Gly Phe Met Val Trp Cys Ile Glu Asn Gly Thr 2900 2905 2910
Ser Pro Asn Leu Asn Gly Thr Trp Val Met Met Asp Gly Glu Asp Gin 2915 2920 2925
Val Ser Tyr Pro Leu Lys Pro Met Val Glu Asn Ala Gin Pro Thr Leu 2930 2935 2940
Arg Gin Ile Met Thr His Phe Ser Asp Leu Ala Glu Ala Tyr Ile Glu 2945 2950 2955 2960
Met Arg Asn Arg Glu Arg Pro Tyr Met Pro Arg Tyr Gly Leu Gin Arg 2965 2970 2975
Asn Ile Thr Asp Met Ser Leu Ser Arg Tyr Ala Phe Asp Phe Tyr Glu 2980 2985 2990
Leu Thr Ser Lys Thr Pro Val Arg Ala Arg Glu Ala His Met Gin Met 2995 3000 3005
Lys Ala Ala Ala Val Arg Asn Ser Gly Thr Arg Leu Phe Gly Leu Asp 3010 3015 3020
Gly Asn Val Gly Thr Ala Glu Glu Asp Thr Glu Arg His Thr Ala His 3025 3030 3035 3040
Asp Val Asn Arg Asn Met His Thr Leu Leu Gly Val Arg Gin 3045 3050 <210> 15 <211> 242 <212> PRT <213> Potyvirus <400> 15
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Ser Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 16 <211 >242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 216 <223> S219N <400> 16
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 17 <211> 242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 56 <223> L56V
<221 > VARIANT <222> 135 <223> S135G
<221 > VARIANT <222> 219 <223> S219N <400> 17
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 18 <211> 242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 17 <223> T17V
<221 > VARIANT <222> 68 <223> N68D
<221 > VARIANT <222> 77 <223> I77V
<221 > VARIANT <222> 219 <223> S219N <400> 18
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Val Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210 19 <211> 242 <212> PRT <213> Potyvirus <220
<221 > VARIANT <222> 44 <223> N44V
<221 > VARIANT <222> 56 <223> L56V
<221 > VARIANT <222> 135 <223> S135G
<221 > VARIANT <222> 219 <223> S219N <400> 19
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Val Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 20 <211> 242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 56 <223> L56V
<221 > VARIANT <222> 68 <223> N68D
<221 > VARIANT <222> 135 <223> S135G
<221 > VARIANT <222> 219 <223> S219N <400> 20
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 21 <211> 242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 17 <223> T17S
<221 > VARIANT <222> 56 <223> L56V
<221 > VARIANT <222> 68 <223> N68D
<221 > VARIANT <222> 77 <223> I77V
<221 > VARIANT <222> 219 <223> S219N <400> 21
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Val Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 22 <211> 242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 17 <223> T17S
<221 > VARIANT <222> 68 <223> N68D
<221 > VARIANT <222> 77 <223> I77V
<221 > VARIANT <222> 135 <223> S135G
<221 > VARIANT <222> 219 <223> S219N <400 22
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 15 10 15
Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Val Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 23 <211 >242 <212> PRT <213> Potyvirus <220>
<221 > VARIANT <222> 17 <223> T17S
<221 > VARIANT <222> 44 <223> N44V
<221 > VARIANT <222> 56 <223> L56V
<221 > VARIANT <222> 68 <223> N68D
<221 > VARIANT
<222> 77 <223> 177V
<221 > VARIANT <222> 135 <223> S135G <221 > VARIANT <222> (219) ... (219)
<223> S219N <400 23
Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro lie Ser Ser 15 10 15
Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Val Lys His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val Phe 50 55 60
Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Val Asp Gly Arg 65 70 75 80
Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin 85 90 95
Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu Val 100 105 110
Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140
Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160
Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175
Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190
Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu Pro 210 215 220
Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr 225 230 235 240
Ser Gin <210> 24 <211> 3023 <212> PRT <213> Potyvirus <400> 24
Met Ala Ala Thr Met lie Phe Gly Ser Phe Thr His Asp Leu Leu Gly 15 10 15
Lys Ala Met Ser Thr lie His Ser Ala Val Thr Ala Glu Lys Asp lie 20 25 30
Phe Ser Ser Ile Lys Glu Arg Leu Glu Arg Lys Arg His Gly Lys lie 35 40 45
Cys Arg Met Lys Asn Gly Ser Ile Tyr Ile Lys Ala Ala Ser Ser Thr 50 55 60
Lys Val Glu Lys Ile Asn Ala Ala Ala Lys Lys Leu Ala Asp Asp Lys 65 70 75 80
Ala Ala Phe Leu Lys Ala Gin Pro Thr Ile Val Asp Lys Ile Ile Val 85 90 95
Asn Glu Lys Ile Gin Val Val Glu Ala Glu Glu Val His Lys Arg Glu 100 105 110
Asp Val Gin Thr Val Phe Phe Lys Lys Thr Lys Lys Arg Ala Pro Lys 115 120 125
Leu Arg Ala Thr Cys Ser Ser Ser Gly Leu Asp Asn Leu Tyr Asn Ala 130 135 140
Val Ala Asn Ile Ala Lys Ala Ser Ser Leu Arg Val Glu Val Ile His 145 150 155 160
Lys Lys Arg Val Cys Gly Glu Phe Lys Gin Thr Arg Phe Gly Arg Ala 165 170 175
Leu Phe Ile Asp Val Ala His Ala Lys Gly His Arg Arg Arg Ile Asp 180 185 190
Cys Arg Met His Arg Arg Glu Gin Arg Thr Met His Met Phe Met Arg 195 200 205
Lys Thr Thr Lys Thr Glu Val Arg Ser Lys His Leu Arg Lys Gly Asp 210 215 220
Ser Gly Ile Val Leu Leu Thr Gin Lys Ile Lys Gly His Leu Ser Gly 225 230 235 240
Val Arg Asp Glu Phe Phe Ile Val Arg Gly Thr Cys Asp Asp Ser Leu 245 250 255
Leu Glu Ala Arg Ala Arg Phe Ser Gin Ser Ile Thr Leu Arg Ala Thr 260 265 270
His Phe Ser Thr Gly Asp Ile Phe Trp Lys Gly Phe Asn Ala Ser Phe 275 280 285
Gin Glu Gin Lys Ala Ile Gly Leu Asp His Thr Cys Thr Ser Asp Leu 290 295 300
Pro Val Glu Ala Cys Gly His Val Ala Ala Leu Met Cys Gin Ser Leu 305 310 315 320
Phe Pro Cys Gly Lys Ile Thr Cys Lys Arg Cys Ile Ala Asn Leu Ser 325 330 335
Asn Leu Asp Phe Asp Thr Phe Ser Glu Leu Gin Gly Asp Arg Ala Met 340 345 350
Arg Ile Leu Asp Val Met Arg Ala Arg Phe Pro Ser Phe Thr His Thr 355 360 365
Ile Arg Phe Leu His Asp Leu Phe Thr Gin Arg Arg Val Thr Asn Pro 370 375 380
Asn Thr Ala Ala Phe Arg Glu Ile Leu Arg Leu Ile Gly Asp Arg Asn 385 390 395 400
Glu Ala Pro Phe Ala His Val Asn Arg Leu Asn Glu Ile Leu Leu Leu 405 410 415
Gly Ser Lys Ala Asn Pro Asp Ser Leu Ala Lys Ala Ser Asp Ser Leu 420 425 430
Leu Glu Leu Ala Arg Tyr Leu Asn Asn Arg Thr Glu Asn Ile Arg Asn 435 440 445
Gly Ser Leu Lys His Phe Arg Asn Lys Ile Ser Ser Lys Ala His Ser 450 455 460
Asn Leu Ala Leu Ser Cys Asp Asn Gin Leu Asp Gin Asn Gly Asn Phe 465 470 475 480
Leu Trp Gly Leu Ala Gly Ile Ala Ala Lys Arg Phe Leu Asn Asn Tyr 485 490 495
Phe Glu Thr Ile Asp Pro Glu Gin Gly Tyr Asp Lys Tyr Val Ile Arg 500 505 510
Lys Asn Pro Asn Gly Glu Arg Lys Leu Ala Ile Gly Asn Phe Ile Ile 515 520 525
Ser Thr Asn Leu Glu Lys Leu Arg Asp Gin Leu Glu Gly Glu Ser Ile 530 535 540
Ala Arg Val Gly Ile Thr Glu Glu Cys Val Ser Arg Lys Asp Gly Asn 545 550 555 560
Tyr Arg Tyr Pro Cys Cys Cys Val Thr Leu Glu Asp Gly Ser Pro Met 565 570 575
Tyr Ser Glu Leu Lys Met Pro Thr Lys Asn His Leu Val Ile Gly Asn 580 585 590
Ser Gly Asp Pro Lys Tyr Leu Asp Leu Pro Gly Glu Ile Ser Asn Leu 595 600 605
Met Tyr Ile Ala Lys Glu Gly Tyr Cys Tyr Ile Asn Ile Phe Leu Ala 610 615 620
Met Leu Val Asn Val Asp Glu Ala Asn Ala Lys Asp Phe Thr Lys Arg 625 630 635 640
Val Arg Asp Glu Ser Val Gin Lys Leu Gly Lys Trp Pro Ser Leu Ile 645 650 655
Asp Val Ala Thr Glu Cys Ala Leu Leu Ser Thr Tyr Tyr Pro Ala Ala 660 665 670
Ala Ser Ala Glu Leu Pro Arg Leu Leu Val Asp His Ala Gin Lys Thr 675 680 685
Ile His Val Val Asp Ser Tyr Gly Ser Leu Asn Thr Gly Tyr His Ile 690 695 700
Leu Lys Ala Asn Thr Val Ser Gin Leu Glu Lys Phe Ala Ser Asn Thr 705 710 715 720
Leu Glu Ser Pro Met Ala Gin Tyr Lys Val Gly Gly Leu Val Tyr Ser 725 730 735
Glu Asn Asn Asp Ala Ser Ala Val Lys Ala Leu Thr Gin Ala Ile Phe 740 745 750
Arg Pro Asp Val Leu Ser Glu Leu Ile Glu Lys Glu Pro Tyr Leu Met 755 760 765
Val Phe Ala Leu Val Ser Pro Gly Ile Leu Met Ala Met Ser Asn Ser 770 775 780
Gly Ala Leu Glu Phe Gly Ile Ser Lys Trp Ile Ser Ser Asp His Ser 785 790 795 800
Leu Val Arg Met Ala Ser Ile Leu Lys Thr Leu Ala Ser Lys Val Ser 805 810 815
Val Ala Asp Thr Leu Ala Leu Gin Lys His Ile Met Arg Gin Asn Ala 820 825 830
Asn Phe Leu Cys Gly Glu Leu Ile Asn Gly Phe Gin Lys Lys Lys Ser 835 840 845
Tyr Thr His Ala Thr Arg Phe Leu Leu Met Ile Ser Glu Glu Asn Glu 850 855 860
Met Asp Asp Pro Val Leu Asn Ala Gly Tyr Arg Val Leu Glu Ala Ser 865 870 875 880
Ser His Glu Ile Met Glu Lys Thr Tyr Leu Ala Leu Leu Glu Thr Ser 885 890 895
Trp Ser Asp Leu Ser Leu Tyr Gly Lys Phe Lys Ser Ile Trp Phe Thr 900 905 910
Arg Lys His Phe Gly Arg Tyr Lys Ala Glu Leu Phe Pro Lys Glu Gin 915 920 925
Thr Asp Leu Gin Gly Arg Tyr Ser Asn Ser Leu Arg Phe His Tyr Gin 930 935 940
Ser Thr Leu Lys Arg Leu Arg Asn Lys Gly Ser Leu Cys Arg Glu Arg 945 950 955 960
Phe Leu Glu Ser Ile Ser Ser Ala Arg Arg Arg Thr Thr Cys Ala Val 965 970 975
Phe Ser Leu Leu His Lys Ala Phe Pro Asp Val Leu Lys Phe Ile Asn 980 985 990
Thr Leu Val Ile Val Ser Leu Ser Met Gin Ile Tyr Tyr Met Leu Val 995 1000 1005
Ala Ile Ile His Glu His Arg Ala Ala Lys Ile Lys Ser Ala Gin Leu 1010 1015 1020
Glu Glu Arg Val Leu Glu Asp Lys Thr Met Leu Leu Tyr Asp Asp Phe 1025 1030 1035 1040
Lys Ala Lys Leu Pro Glu Gly Ser Phe Glu Glu Phe Leu Glu Tyr Thr 1045 1050 1055
Arg Gin Arg Asp Lys Glu Val Tyr Glu Tyr Leu Met Met Glu Thr Thr 1060 1065 1070
Glu Ile Val Glu Phe Gin Ala Lys Asn Thr Gly Gin Ala Ser Leu Glu 1075 1080 1085
Arg Ile Ile Ala Phe Val Ser Leu Thr Leu Met Leu Phe Asp Asn Glu 1090 1095 1100
Arg Ser Asp Cys Val Tyr Lys Ile Leu Thr Lys Phe Lys Gly Ile Leu 1105 1110 1115 1120
Gly Ser Val Glu Asn Asn Val Arg Phe Gin Ser Leu Asp Thr Ile Val 1125 1130 1135
Pro Thr Gin Glu Glu Lys Asn Met Val Ile Asp Phe Glu Leu Asp Ser 1140 1145 1150
Asp Thr Ala His Thr Pro Gin Met Gin Glu Gin Thr Phe Ser Asp Trp 1155 1160 1165
Trp Ser Asn Gin Ile Ala Asn Asn Arg Val Val Pro His Tyr Arg Thr 1170 1175 1180
Glu Gly Tyr Phe Met Gin Phe Thr Arg Asn Thr Ala Ser Ala Val Ser 1185 1190 1195 1200
His Gin Ile Ala His Asn Glu His Lys Asp Ile Ile Leu Met Gly Ala 1205 1210 1215
Val Gly Ser Gly Lys Ser Thr Gly Leu Pro Thr Asn Leu Cys Lys Phe 1220 1225 1230
Gly Gly Val Leu Leu Leu Glu Pro Thr Arg Pro Leu Ala Glu Asn Val 1235 1240 1245
Thr Lys Gin Met Arg Gly Ser Pro Phe Phe Ala Ser Pro Thr Leu Arg 1250 1255 1260
Met Arg Asn Leu Ser Thr Phe Gly Ser Ser Pro Ile Thr Val Met Thr 1265 1270 1275 1280
Thr Gly Phe Ala Leu His Phe Phe Ala Asn Asn Val Lys Glu Phe Asp 1285 1290 1295
Arg Tyr Gin Phe Ile Ile Phe Asp Glu Phe His Val Leu Asp Ser Asn 1300 1305 1310
Ala Ile Ala Phe Arg Asn Leu Cys His Glu Tyr Ser Tyr Asn Gly Lys 1315 1320 1325
Ile Ile Lys Val Ser Ala Thr Pro Pro Gly Arg Glu Cys Asp Leu Thr 1330 1335 1340
Thr Gin Tyr Pro Val Glu Leu Leu Ile Glu Glu Gin Leu Ser Leu Arg 1345 1350 1355 1360
Asp Phe Val Asp Ala Gin Gly Thr Asp Ala His Ala Asp Val Val Lys 1365 1370 1375
Lys Gly Asp Asn Ile Leu Val Tyr Val Ala Ser Tyr Asn Glu Val Asp 1380 1385 1390
Gin Leu Ser Lys Met Leu Asn Glu Arg Gly Phe Leu Val Thr Lys Val 1395 1400 1405
Asp Gly Arg Thr Met Lys Leu Gly Gly Val Glu Ile Ile Thr Lys Gly 1410 1415 1420
Ser Ser Ile Lys Lys His Phe Ile Val Ala Thr Asn Ile Ile Glu Asn 1425 1430 1435 1440
Gly Val Thr Leu Asp Val Asp Val Val Val Asp Phe Gly Leu Lys Val 1445 1450 1455
Val Pro Asn Leu Asp Ser Asp Asn Arg Leu Val Ser Tyr Cys Lys Ile 1460 1465 1470
Pro Ile Ser Leu Gly Glu Arg Ile Gin Arg Phe Gly Arg Val Gly Arg 1475 1480 1485
Asn Lys Pro Gly Val Ala Leu Arg Ile Gly Glu Thr Ile Lys Gly Leu 1490 1495 1500
Val Glu Ile Pro Ser Met Ile Ala Thr Glu Ala Ala Phe Leu Cys Phe 1505 1510 1515 1520
Val Tyr Gly Leu Pro Val Thr Thr Gin Asn Val Ser Thr Ser Ile Leu 1525 1530 1535
Ser Gin Val Ser Val Arg Gin Ala Arg Val Met Cys Gin Phe Glu Leu 1540 1545 1550
Pro Ile Phe Tyr Thr Ala His Leu Val Arg Tyr Asp Gly Ala Met His 1555 1560 1565
Pro Ala Ile His Asn Ala Leu Lys Arg Phe Lys Leu Arg Asp Ser Glu 1570 1575 1580
Ile Asn Leu Asn Thr Leu Ala Ile Pro Thr Ser Ser Ser Lys Thr Trp 1585 1590 1595 1600
Tyr Thr Gly Lys Cys Tyr Lys Gin Leu Val Gly Arg Leu Asp Ile Pro 1605 1610 1615
Asp Glu Ile Lys Ile Pro Phe Tyr Thr Lys Glu Val Pro Glu Lys Val 1620 1625 1630
Pro Glu Gin Ile Trp Asp Val Met Val Lys Phe Ser Ser Asp Ala Gly 1635 1640 1645
Phe Gly Arg Met Thr Ser Ala Ala Ala Cys Lys Val Ala Tyr Thr Leu 1650 1655 1660
Gin Thr Asp Ile His Ser Ile Gin Arg Thr Val Gin Ile Ile Asp Arg 1665 1670 1675 1680 lieu Leu Glu Asn Glu Met Lys Lys Arg Asn Hi s Phe Asn Leu Val Val 1685 1690 1695
Asn Gin Ser Cys Ser Ser His Phe Met Ser Leu Ser Ser Ile Met Ala 1700 1705 1710
Ser Leu Arg Ala His Tyr Ala Lys Asn His Thr Gly Gin Asn Ile Glu 1715 1720 1725
Ile Leu Gin Lys Ala Lys Ala Gin Leu Leu Glu Phe Ser Asn Leu Ala 1730 1735 1740
Ile Asp Pro Ser Thr Thr Glu Ala Leu Arg Asp Phe Gly Tyr Leu Glu 1745 1750 1755 1760
Ala Val Arg Phe Gin Ser Glu Ser Glu Met Ala Arg Gly Leu Lys Leu 1765 1770 1775
Ser Gly His Trp Lys Trp Ser Leu Ile Ser Arg Asp Leu Ile Val Val 1780 1785 1790
Ser Gly Val Gly Ile Gly Leu Gly Cys Met Leu Trp Gin Phe Phe Lys 1795 1800 1805
Glu Lys Met His Glu Pro Val Lys Phe Gin Gly Lys Ser Arg Arg Arg 1810 1815 1820
Leu Gin Phe Arg Lys Ala Arg Asp Asp Lys Met Gly Tyr Ile Met His 1825 1830 1835 1840
Gly Glu Gly Asp Thr Ile Glu His Phe Phe Gly Ala Ala Tyr Thr Lys 1845 1850 1855
Lys Gly Lys Ser Lys Gly Lys Thr His Gly Ala Gly Thr Lys Ala His 1860 1865 1870
Lys Phe Val Asn Met Tyr Gly Val Ser Pro Asp Glu Tyr Ser Tyr Val 1875 1880 1885
Arg Tyr Leu Asp Pro Val Thr Gly Ala Thr Leu Asp Glu Ser Pro Met 1890 1895 1900
Thr Asp Leu Asn Ile Val Gin Glu His Phe Gly Glu Ile Arg Arg Glu 1905 1910 1915 1920
Ala Ile Leu Ala Asp Ala Met Ser Pro Gin Gin Arg Asn Lys Gly Ile 1925 1930 1935
Gin Ala Tyr Phe Val Arg Asn Ser Thr Met Pro Ile Leu Lys Val Asp 1940 1945 1950
Leu Thr Pro His Ile Pro Leu Lys Val Cys Glu Ser Asn Asn Ile Ala 1955 1960 1965
Gly Phe Pro Glu Arg Glu Gly Glu Leu Arg Arg Thr Gly Pro Thr Glu 1970 1975 1980
Thr Leu Pro Phe Asp Ala Leu Pro Pro Glu Lys Gin Glu Val Ala Phe 1985 1990 1995 2000
Glu Ser Lys Ala Leu Leu Lys Gly Val Arg Asp Phe Asn Pro Ile Ser 2005 2010 2015
Ala Cys Val Trp Leu Leu Glu Asn Ser Ser Asp Gly His Ser Glu Arg 2020 2025 2030
Leu Phe Gly Ile Gly Phe Gly Pro Tyr Ile Ile Ala Asn Gin His Leu 2035 2040 2045
Phe Arg Arg Asn Asn Gly Glu Leu Thr Ile Lys Thr Met His Gly Glu 2050 2055 2060
Phe Lys Val Lys Asn Ser Thr Gin Leu Gin Met Lys Pro Val Glu Gly 2065 2070 2075 2080
Arg Asp Ile Ile Val Ile Lys Met Ala Lys Asp Phe Pro Pro Phe Pro 2085 2090 2095
Gin Lys Leu Lys Phe Arg Gin Pro Thr Ile Lys Asp Arg Val Cys Met 2100 2105 2110
Val Ser Thr Asn Phe Gin Gin Lys Ser Val Ser Ser Leu Val Ser Glu 2115 2120 2125
Ser Ser His Ile Val His Lys Glu Asp Thr Ser Phe Trp Gin His Trp 2130 2135 2140
Ile Thr Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Ile Ile 2145 2150 2155 2160
Asp Gly Asn Ile Leu Gly Ile His Ser Leu Thr His Thr Thr Asn Gly 2165 2170 2175
Ser Asn Tyr Phe Val Glu Phe Pro Glu Lys Phe Val Ala Thr Tyr Leu 2180 2185 2190
Asp Ala Ala Asp Gly Trp Cys Lys Asn Trp Lys Phe Asn Ala Asp Lys 2195 2200 2205
Ile Ser Trp Gly Ser Phe Thr Leu Val Glu Asp Ala Pro Glu Asp Asp 2210 2215 2220
Phe Met Ala Lys Lys Thr Val Ala Ala Ile Met Asp Asp Leu Val Arg 2225 2230 2235 2240
Thr Gin Gly Glu Lys Arg Lys Trp Met Leu Glu Ala Ala His Thr Asn 2245 2250 2255
Ile Gin Pro Val Ala His Leu Gin Ser Gin Leu Val Thr Lys His Ile 2260 2265 2270
Val Lys Gly Arg Cys Lys Met Phe Ala Leu Tyr Leu Gin Glu Asn Ala 2275 2280 2285
Asp Ala Arg Asp Phe Phe Lys Ser Phe Met Gly Ala Tyr Gly Pro Ser 2290 2295 2300
His Leu Asn Lys Glu Ala Tyr Ile Lys Asp Ile Met Lys Tyr Ser Lys 2305 2310 2315 2320
Gin Ile Val Val Gly Ser Val Asp Cys Asp Thr Phe Glu Ser Ser Leu 2325 2330 2335
Lys Val Leu Ser Arg Lys Met Lys Glu Trp Gly Phe Glu Asn Leu Glu 2340 2345 2350
Tyr Val Thr Asp Glu Gin Thr Ile Lys Asn Ala Leu Asn Met Asp Ala 2355 2360 2365
Ala Val Gly Ala Leu Tyr Ser Gly Lys Lys Lys Gin Tyr Phe Glu Asp 2370 2375 2380
Leu Ser Asp Asp Ala Val Ala Asn Leu Val Gin Lys Ser Cys Leu Arg 2385 2390 2395 2400
Leu Phe Lys Asn Lys Leu Gly Val Trp Asn Gly Ser Leu Lys Ala Glu 2405 2410 2415
Leu Arg Pro Phe Glu Lys Leu Ile Glu Asn Lys Thr Arg Thr Phe Thr 2420 2425 2430
Ala Ala Pro Ile Glu Thr Leu Leu Gly Gly Lys Val Cys Val Asp Asp 2435 2440 2445
Phe Asn Asn His Phe Tyr Ser Lys His Ile Gin Cys Pro Trp Ser Val 2450 2455 2460
Gly Met Thr Lys Phe Tyr Gly Gly Trp Asn Glu Leu Leu Gly Lys Leu 2465 2470 2475 2480
Pro Asp Gly Trp Val Tyr Cys Asp Ala Asp Gly Ser Gin Phe Asp Ser 2485 2490 2495
Ser Leu Ser Pro Tyr Leu Ile Asn Ala Val Leu Arg Leu Arg Leu Ser 2500 2505 2510
Ser Met Glu Glu Trp Asp Val Gly Gin Lys Met Leu Gin Asn Leu Tyr 2515 2520 2525
Thr Glu Ile Val Tyr Thr Pro Ile Ser Thr Pro Asp Gly Thr Ile Val 2530 2535 2540
Lys Lys Phe Lys Gly Asn Asn Ser Gly Gin Pro Ser Thr Val Val Asp 2545 2550 2555 2560
Asn Thr Leu Met Val Val Leu Ala Met Tyr Tyr Ala Leu Ser Lys Leu 2565 2570 2575
Gly Val Asp Ile Asn Ser Gin Glu Asp Val Cys Lys Phe Phe Ala Asn 2580 2585 2590
Gly Asp Asp Leu Ile Ile Ala Ile Ser Pro Glu Leu Glu His Val Leu 2595 2600 2605
Asp Gly Phe Gin Gin His Phe Ser Asp Leu Gly Leu Asn Tyr Asp Phe 2610 2615 2620
Ser Ser Arg Thr Arg Asp Lys Lys Glu Leu Trp Phe Met Ser His Arg 2625 2630 2635 2640
Ala Leu Ser Lys Asp Gly Ile Leu Ile Pro Lys Leu Glu Pro Glu Arg 2645 2650 2655
Ile Val Ser Ile Leu Glu Trp Asp Arg Ser Ala Glu Pro His His Arg 2660 2665 2670
Leu Glu Ala Ile Cys Ala Ser Met Ile Glu Ala Trp Gly Tyr Thr Asp 2675 2680 2685
Leu Leu Gin Asn Ile Arg Arg Phe Tyr Lys Trp Thr Ile Glu Gin Glu 2690 2695 2700
Pro Tyr Arg Ser Leu Ala Glu Gin Gly Leu Ala Pro Tyr Leu Ser Glu 2705 2710 2715 2720
Val Ala Leu Arg Arg Leu Tyr Thr Ser Gin Ile Ala Thr Asp Asn Glu 2725 2730 2735
Leu Thr Asp Tyr Tyr Lys Glu Ile Leu Ala Asn Asn Glu Phe Leu Arg 2740 2745 2750
Glu Thr Val Arg Phe Gin Ser Asp Thr Val Asp Ala Gly Lys Asp Lys 2755 2760 2765
Ala Arg Asp Gin Lys Leu Ala Asp Lys Pro Thr Leu Ala Ile Asp Arg 2770 2775 2780
Thr Lys Asp Lys Asp Val Asn Thr Gly Thr Ser Gly Thr Phe Ser Ile 2785 2790 2795 2800
Pro Arg Leu Lys Lys Ala Ala Met Asn Met Lys Leu Pro Lys Val Gly 2805 2810 2815
Gly Ser Ser Val Val Asn Leu Asp His Leu Leu Thr Tyr Lys Pro Ala 2820 2825 2830
Gin Glu Phe Val Val Asn Thr Arg Ala Thr His Ser Gin Phe Lys Ala 2835 2840 2845
Trp His Thr Asn Val Met Ala Glu Leu Glu Leu Asn Glu Glu Gin Met 2850 2855 2860
Lys Ile Val Leu Asn Gly Phe Met Ile Trp Cys Ile Glu Asn Gly Thr 2865 2870 2875 2880
Ser Pro Asn Ile Ser Gly Val Trp Thr Met Met Asp Gly Asp Glu Gin 2885 2890 2895
Val Glu Tyr Pro Ile Glu Pro Met Val Lys His Ala Asn Pro Ser Leu 2900 2905 2910
Arg Gin Ile Met Lys His Phe Ser Asn Leu Ala Glu Ala Tyr Ile Arg 2915 2920 2925
Met Arg Asn Ser Glu Gin Val Tyr Ile Pro Arg Tyr Gly Leu Gin Arg 2930 2935 2940
Gly Leu Val Asp Arg Asn Leu Ala Pro Phe Ala Phe Asp Phe Phe Glu 2945 2950 2955 2960
Val Asn Gly Ala Thr Pro Val Arg Ala Arg Glu Ala His Ala Gin Met 2965 2970 2975
Lys Ala Ala Ala Leu Arg Asn Ser Gin Gin Arg Met Phe Cys Leu Asp 2980 2985 2990
Gly Ser Val Ser Gly Gin Glu Glu Asn Thr Glu Arg His Thr Val Asp 2995 3000 3005
Asp Val Asn Ala Gin Met His His Leu Leu Gly Val Lys Gly Val 3010 3015 3020 <210> 25 <211> 381 <212> PRT <213> Bacillus subtilis <400> 25
Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 15 10 15
Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Gly Lys 20 25 30
Ser Ser Thr Glu Lys Lys Tyr Ile Val Gly Phe Lys Gin Thr Met Ser 35 40 45
Ala Met Ser Ser Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gly 50 55 60
Lys Val Gin Lys Gin Phe Lys Tyr Val Asn Ala Ala Thr Ala Thr Leu 65 70 75 80
Asp Glu Lys Ala Val Lys Glu Leu Lys Gin Asp Pro Ser Val Ala Tyr 85 90 95
Val Glu Glu Asp His Ile Ala His Glu Tyr Ala Gin Ser Val Pro Tyr 100 105 110
Gly Ile Ser Gin Ile Lys Ala Pro Ala Leu His Ser Gin Gly Tyr Thr 115 120 125
Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser Ser 130 135 140
His Pro Asp Leu Asn Val Lys Gly Gly Ala Ser Phe Val Pro Ser Glu 145 150 155 160
Thr Asn Pro Tyr Gin Asp Gly Ser Ser His Gly Thr His Val Ala Gly 165 170 175
Thr Ile Ala Ala Leu Asn Asn Thr Ile Gly Val Leu Gly Val Ala Pro 180 185 190
Asn Ala Ser Leu Tyr Ala Val Lys Val Leu Asp Ser Thr Gly Ser Gly 195 200 205
Gin Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ser Asn Asn 210 215 220
Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala 225 230 235 240
Leu Lys Thr Val Val Asp Lys Ala Val Ser Ser Gly Ile Val Val Ala 245 250 255
Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly Ser Thr Ser Thr Val Gly 260 265 270
Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala Val Gly Ala Val Asn Ser 275 280 285
Ser Asn Gin Arg Ala Ser Phe Ser Ser Ala Gly Ser Glu Leu Asp Val 290 295 300
Met Ala Pro Gly Val Ser Ile Gin Ser Thr Leu Pro Gly Gly Thr Tyr 305 310 315 320
Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 325 330 335
Ala Ala Leu Ile Leu Ser Lys His Pro Thr Trp Thr Asn Ala Gin Val 340 345 350
Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr 355 360 365
Tyr Gly Lys Gly Leu Ile Asn Val Gin Ala Ala Ala Gin 370 375 380
<210> 26 <211> 277 <212> PRT <213> Rattus norvegicus <400 26
Met Asp Asn Asn Glu Thr Ser Val Asp Ser Lys Ser Ile Asn Asn Phe 15 10 15
Glu Thr Lys Thr Ile His Gly Ser Lys Ser Met Asp Ser Gly Ile Tyr 20 25 30
Leu Asp Ser Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45
Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Ser Ala Arg 50 55 60
Asn Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Met Ala 65 70 75 80
Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95
Met Glu Leu Met Asp Ser Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110
Ser Phe Val Cys Val Ile Leu Ser His Gly Asp Glu Gly Val Ile Phe 115 120 125
Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Leu Thr Ser Phe Phe Arg 130 135 140
Gly Asp Tyr Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160
Gin Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175
Gly Thr Asp Asp Asp Met Ala Cys Gin Lys Ile Pro Val Glu Ala Asp 180 185 190
Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205
Ser Arg Asp Gly Ser Trp Phe Ile Gin Ser Leu Cys Ala Met Leu Lys 210 215 220
Leu Tyr Ala His Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240
Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Leu Asp Ala Thr Phe 245 250 255
His Ala Lys Lys Gin Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270
Leu Tyr Phe Tyr His 275
<210> 27 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site consensus sequence <221 > VARIANT <222> 2, 3, 5 <223> Xaa can be any amino acid <400> 27
Glu Xaa Xaa Tyr Xaa Gin Gly 1 5
<210> 28 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site consensus sequence <221 > VARIANT <222> 2, 3, 5 <223> Xaa can be any amino acid <400> 28
Glu Xaa Xaa Tyr Xaa Gin Ser 1 5
<210> 29 <211>7 <212> PRT <213> Artificial Sequence <220 <223> Tobacco Etch Virus protease cleavage site <400 29
Glu Asn Leu Tyr Phe Gin Gly 1 5
<210> 30 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 30
Glu Asn Leu Tyr Phe Gin Ser 1 5
<210> 31 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 31
Glu Asn lie Tyr Thr Gin Gly 1 5
<210> 32 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400 32
Glu Asn Ile Tyr Thr Gin Ser 1 5
<210> 33 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 33
Glu Asn Ile Tyr Leu Gin Gly 1 5
<210> 34 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 34
Glu Asn Ile Tyr Leu Gin Ser 1 5
<210> 35 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 35
Glu Asn Val Tyr Phe Gin Gly 1 5
<210 36 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400 36
Glu Asn Val Tyr Ser Gin Ser 1 5
<210> 37 <211>7 <212> PRT <213> Artificial Sequence <220 <223> Tobacco Etch Virus protease cleavage site <400 37
Glu Asn Val Tyr Ser Gin Gly 1 5
<210> 38 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Etch Virus protease cleavage site <400> 38
Glu Asn Val Tyr Ser Gin Ser 1 5
<210> 39 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Vein Mottling Virus protease cleavage site consensus sequence <221 > VARIANT <222> 1, 2 <223> Xaa can be any amino acid <400> 39
Xaa Xaa Val Arg Phe Gin Gly 1 5
<210> 40 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Vein Mottling Virus protease cleavage site consensus sequence <221 > VARIANT <222> 1, 2 <223> Xaa can be any amino acid <400> 40
Xaa Xaa Val Arg Phe Gin Ser 1 5
<210> 41 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Vein Mottling Virus protease cleavage site <400> 41
Glu Thr Val Arg Phe Gin Gly 1 5
<210> 42 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Vein Mottling Virus protease cleavage site <400> 42
Glu Thr Val Arg Phe Gin Ser 1 5
<210> 43 <211>7 <212> PRT <213> Artificial Sequence <220 <223> Tobacco Vein Mottling Virus protease cleavage site <400 43
Asn Asn Val Arg Phe Gin Gly 1 5
<210> 44 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Tobacco Vein Mottling Virus protease cleavage site <400> 44
Asn Asn Val Arg Phe Gin Ser 1 5
<210> 45 <211>7 <212> PRT <213> Artificial Sequence <220 <223> Human Rhinovirus 3C protease cleavage site consensus sequence <221 > VARIANT <222> 1 <223> Xaa can be amino acid, with D or E preferred <221 > VARIANT <222> 2 <223> Xaa can be G, A, V, L, I, M, S or T <400 45
Xaa Xaa Leu Phe Gin Gly Pro 1 5
<210> 46 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Human Rhinovirus 3C protease cleavage site <400> 46
Glu Ala Leu Phe Gin Gly Pro 1 5
<210> 47 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Human Rhinovirus 3C protease cleavage site <400> 47
Glu Val Leu Phe Gin Gly Pro 1 5
<210> 48 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Human Rhinovirus 3C protease cleavage site <400> 48
Glu Leu Leu Phe Gin Gly Pro 1 5 <210> 49
<211>7 <212> PRT <213> Artificial Sequence <220 <223> Human Rhinovirus 3C protease cleavage site <400 49
Asp Ala Leu Phe Gin Gly Pro 1 5
<210> 50 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Human Rhinovirus 3C protease cleavage site <400> 50
Asp Val Leu Phe Gin Gly Pro 1 5
<210> 51 <211>7 <212> PRT <213> Artificial Sequence <220> <223> Human Rhinovirus 3C protease cleavage site <400> 51
Asp Leu Leu Phe Gin Gly Pro 1 5
<210> 52 <211>6 <212> PRT <213> Artificial Sequence <220> <223> Subtilisin protease cleavage site consensus sequence <221 > VARIANT <222> 1,2, 3,4 <223> Xaa can be any amino acid <400> 52
Xaa Xaa Xaa Xaa His Tyr 1 5
<210> 53 <211>6 <212> PRT <213> Artificial Sequence <220 <223> Subtilisin protease cleavage site consensus sequence <221 > VARIANT <222> 1,2, 3, 4 <223> Xaa can be any amino acid <400 53
Xaa Xaa Xaa Xaa His Tyr 1 5
<210> 54 <211>2 <212> PRT <213> Artificial Sequence <220> <223> Subtilisin protease cleavage site <400> 54
His Tyr 1
<210> 55 <211>2 <212> PRT <213> Artificial Sequence <220> <223> Subtilisin protease cleavage site <400> 55
Tyr His 1
<210> 55 <211>6 <212> PRT <213> Artificial Sequence <220> <223> Subtilisin protease cleavage site
I <400> 56
Pro Gly Ala Ala His Tyr 1 5
<210> 57 <211>5 <212> PRT <213> Artificial Sequence <220 <223> Caspase 3 protease cleavage site consensus sequence <221 > VARIANT <222> 2 <223> Xaa can be any amino acid with E preferred <221 > VARIANT <222> 3 <223> Xaa can be any amino acid <221 > VARIANT <222> 5 <223> Xaa can be any amino acid with G or S preferred <400 57
Asp Xaa Xaa Asp Xaa 1 5
<210> 58 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Caspase 3 protease cleavage site <400> 58
Asp Glu Val Asp Gly 1 5
<210> 59 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Caspase 3 protease cleavage site <400> 59
Asp Glu Val Asp Ser 1 5
<210> 60 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Caspase 3 protease cleavage site <400> 60
Asp Glu Pro Asp Gly 1 5
<210 61 <211>5 <212> PRT <213> Artificial Sequence <220 <223> Caspase 3 protease cleavage site <400 61
Asp Glu Pro Asp Ser 1 5
<210> 62 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Caspase 3 protease cleavage site <400> 62
Asp Glu Leu Asp Gly 1 5
<210> 63 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Caspase 3 protease cleavage site <400> 63
Asp Glu Leu Asp Ser 1 5
<210> 64 <211>5 <212> PRT <213> Artificial Sequence <220> <223> Enterokinase protease cleavage site consensus sequence <400> 64
Asp Asp Asp Asp Lys 1 5 <210> 65 <211> 753
<212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 65 atgggcgaat ctctgttcaa gggtccgcgt gattataacc cgatatcttc tactatttgt 60 catctgacta acgaaagcga cggccacacg acttctctgt acggtatcgg tttcggtccg 120 ttcatcatta ccaacaagca tctgttccgc cgtaacaacg gtaccctgct ggttcaatct 180 ctgcacggcg tcttcaaggt aaaaaatacc actacgctgc agcagcacct gattgacggc 240 cgtgacatga teatcatccg catgccgaaa gattttccgc cgttcccgca aaaactgaag 300 tttcgtgaac cgcaacgcga agaaegtatt tgcctggtta ccaccaactt tcagaccaaa 360 ageatgtett ctatggtttc cgatacctct tgcaccttcc caagctctga cggtattttc 420 tggaaacatt ggatccagac caaagatggt cagtgcggct ctccgctggt gtctacgcgt 480 gacggtttca tcgttggtat ccattctgct tetaaettea ctaacactaa caactacttt 540 acttccgttc cgaaaaactt catggagctg ctgactaacc aagaggccca gcagtgggtg 600 tccggttggc gcctgaacgc agattctgta ctgtggggtg gtcataaggt attcatgaac 660 aaaeeggagg agccgttcca geeggtcaaa gaggcgaccc agctgatgaa cgaactggtt 720 tactctcagg gtcaccacca tcaccaccat taa 753
<210> 66 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (S219N) with amino-terminus polyhistidine affinity tag <400> 66
Met Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser 15 10 15
Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser 20 25 30
Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu 35 40 45
Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gin Ser Leu His Gly Val 50 55 60
Phe Lys Val Lys Asn Thr Thr Thr Leu Gin Gin His Leu Ile Asp Gly 65 70 75 80
Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro 85 90 95
Gin Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu 100 105 110
Val Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp 115 120 125
Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp 130 135 140
Ile Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg 145 150 155 160
Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr 165 170 175
Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr 180 185 190
Asn Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp 195 200 205
Ser Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu 210 215 220
Pro Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val 225 230 235 240
Tyr Ser Gin Gly His His His His His His 245 250
<210> 67 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 67 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt ctactatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaaaatac cactacgctg 240 cagcagcacc tgattgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 68 <211 >250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (L56V, S135G, S219N) with amino-terminus polyhistidine affinity tag <400> 68
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly lie Gly Phe Gly Pro Phe 35 40 45 lie lie Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu lie Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 69 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 69 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac 180 ggtaccctgc tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagctctg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 70 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, N68D, I77V, S219N) with amino-terminus polyhistidine affinity tag <400> 70
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250 <210> 71
<211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 71 atgggtcacc accatcaeca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt ctactatttg tcatctgact aacgaaagcg acggccacac gaettetetg 120 taeggtateg gtttcggtcc gttcatcatt accgtgaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaaaatac cactacgctg 240 cagcagcacc tgattgaegg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaaegtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg teagtgegge 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttetaaette 540 actaacacta acaactactt tacttccgtt ccgaaaaact teatggaget gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggteataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 72 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (N44V, L56V, S135G, S219N) with amino-terminus polyhistidine affinity tag <400> 72
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly lie Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Val Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu lie Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 73 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 73 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt ctactatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tgattgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 74 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (L56V, N68D, S135G, S219N) with amino-terminus polyhistidine affinity tag <400> 74
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly lie Gly Phe Gly Pro Phe 35 40 45 lie lie Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu lie Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 75 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 75 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagctctg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210 76 <211 >250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, L56V, N68D, I77V, S219N) with amino-terminus polyhistidine affinity tag <400> 76
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 77 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 77 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac 180 ggtaccctgc tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 78 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, N68D, I77V, S135G, S219N) with amino-terminus polyhistidine affinity tag <400> 78
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250 <210> 79
<211> 753 <212> DNA <213> Artificial Sequence <220 <223> Codon-optimized TEV open reading frame <400 79 atgggcgaat ctctgttcaa gggtccgcgt gattataacc cgatatcttc ttctatttgt 60 catctgacta acgaaagcga cggccacacg acttctctgt acggtatcgg tttcggtccg 120 ttcatcatta ccgtgaagca tctgttccgc cgtaacaacg gtaccctggt ggttcaatct 180 ctgcacggcg tcttcaaggt aaaagacacc actacgctgc agcagcacct ggtcgacggc 240 cgtgacatga tcatcatccg catgccgaaa gattttccgc cgttcccgca aaaactgaag 300 tttcgtgaac cgcaacgcga agaacgtatt tgcctggtta ccaccaactt tcagaccaaa 360 agcatgtctt ctatggtttc cgatacctct tgcaccttcc caagcggtga cggtattttc 420 tggaaacatt ggatccagac caaagatggt cagtgcggct ctccgctggt gtctacgcgt 480 gacggtttca tcgttggtat ccattctgct tctaacttca ctaacactaa caactacttt 540 acttccgttc cgaaaaactt catggagctg ctgactaacc aagaggccca gcagtgggtg 600 tccggttggc gcctgaacgc agattctgta ctgtggggtg gtcataaggt attcatgaac 660 aaaccggagg agccgttcca gccggtcaaa gaggcgaccc agctgatgaa cgaactggtt 720 tactctcagg gtcaccacca tcaccaccat taa 753
<210> 80 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, N44V, L56V, N68D, I77V, S135G, S219N) with carboxyl-terminus polyhistidine affinity tag <400> 80
Met Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro lie Ser 15 10 15
Ser Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser 20 25 30
Leu Tyr Gly lie Gly Phe Gly Pro Phe Ile Ile Thr Val Lys His Leu 35 40 45
Phe Arg Arg Asn Asn Gly Thr Leu Val Val Gin Ser Leu His Gly Val 50 55 60
Phe Lys Val Lys Asp Thr Thr Thr Leu Gin Gin His Leu Val Asp Gly 65 70 75 80
Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro 85 90 95
Gin Lys Leu Lys Phe Arg Glu Pro Gin Arg Glu Glu Arg Ile Cys Leu 100 105 110
Val Thr Thr Asn Phe Gin Thr Lys Ser Met Ser Ser Met Val Ser Asp 115 120 125
Thr Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp 130 135 140
Ile Gin Thr Lys Asp Gly Gin Cys Gly Ser Pro Leu Val Ser Thr Arg 145 150 155 160
Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr 165 170 175
Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr 180 185 190
Asn Gin Glu Ala Gin Gin Trp Val Ser Gly Trp Arg Leu Asn Ala Asp 195 200 205
Ser Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu 210 215 220
Pro Phe Gin Pro Val Lys Glu Ala Thr Gin Leu Met Asn Glu Leu Val 225 230 235 240
Tyr Ser Gin Gly His His His His His His 245 250
<210> 81 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 81 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accgtgaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatggt gaaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 82 <211 >250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, N44V, L56V, N68D, I77V, S135G, S219V) with amino-terminus polyhistidine affinity tag <400> 82
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Val Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Val Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 83 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized TEV open reading frame <400> 83 atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac 60 ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg 120 tacggtatcg gtttcggtcc gttcatcatt accgtgaagc atctgttccg ccgtaacaac 180 ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg 240 cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300 ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360 accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420 ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480 tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc 540 actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac 600 caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt 660 ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc 720 cagctgatga acgaactggt ttactctcag taa 753
<210> 84 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (T17S, N44V, L56V, N68D, I77V, S135G, S219N) with amino-terminus polyhistidine affinity tag <400> 84
Met Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro 15 10 15
Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Val Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55 60
Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu 65 70 75 80
Gin Gin His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser 115 120 125
Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly 145 150 155 160
Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165 170 175
Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180 185 190
Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser 195 200 205
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr 225 230 235 240
Gin Leu Met Asn Glu Leu Val Tyr Ser Gin 245 250
<210> 85 <211> 750 <212> DNA <213> Artificial Sequence <220 <223> Native TEV open reading frame <400 85 atgcatcacc atcaccacca tggagaaagc ttgtttaagg gaccacgtga ttacaacccg 60 atatcgagca ccatttgtca tttgacgaat gaatctgatg ggcacacaac atcgttgtat 120 ggtattggat ttggtccctt catcattaca aacaagcact tgtttcgccg taataatgga 180 acactgttgg tccaatcact acatggtgta ttcaaggtca agaacaccac gactttgcaa 240 caacacctca ttgatgggag ggacatgata attattcgca tgcctaagga tttcccacca 300 tttcctcaaa agctgaaatt tagagagcca caaagggaag agcgcatctg tcttgtgaca 360 accaacttcc aaactaagag catgtctagc atggtgtcag acactagttg cacattccct 420 tcatctgatg gcatattctg gaagcattgg atccaaacca aggatgggca gtgtggcagt 480 ccattagtat caactagaga tgggttcatt gttggtatac actcagcatc gaatttcacc 540 aacacaaaca attatttcac aagcgtgccg aaaaacttca tggaattgtt gacaaatcag 600 gaggcgcagc agtgggttag tggttggcga ttaaatgctg actcagtatt gtgggggggc 660 cataaagttt tcatgaacaa acctgaagag ccttttcagc cagttaagga agcgactcaa 720 ctcatgaatg aattggtgta ctcgcaataa 750
<210> 86 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> TEV protease (S219N) with amino-terminus polyhistidine affinity tag <400> 86
Met His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro Arg 15 10 15
Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser 20 25 30
Asp Gly His Thr Thr Ser Leu Tyr Gly lie Gly Phe Gly Pro Phe lie 35 40 45 lie Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val 50 55 60
Gin Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu Gin 65 70 75 80
Gin His Leu lie Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys 85 90 95
Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg Glu Pro Gin Arg 100 105 110
Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gin Thr Lys Ser Met 115 120 125
Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly 130 135 140
Ile Phe Trp Lys His Trp Ile Gin Thr Lys Asp Gly Gin Cys Gly Ser 145 150 155 160
Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala 165 170 175
Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn 180 185 190
Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin Trp Val Ser Gly 195 200 205
Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe 210 215 220
Met Asn Lys Pro Glu Glu Pro Phe Gin Pro Val Lys Glu Ala Thr Gin 225 230 235 240
Leu Met Asn Glu Leu Val Tyr Ser Gin 245
<210> 87 <211> 3984 <212> DNA <213> Artificial Sequence <220> <223> Open reading frame encoding BoNT/A-TEV <400> 87 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag caaaaccaaa 1320 tccttgggcg gtggtggcga aaacctgtac ttccagggcg gtggcggtgg tgataagggc 1380 tataacaagg ccttaaatga tttatgcatc aaggtgaaca actgggactt gtttttctct 1440 ccatctgaag ataattttac taacgacttg aacaaaggag aggaaattac ttccgatacc 1500 aacatcgaag cagcggaaga gaatattagc ctggatctta ttcaacaata ttacctgacc 1560 tttaattttg ataacgagcc tgagaacatt tccattgaga atctcagctc tgacatcatc 1620 ggccagctgg aactgatgcc gaatatcgaa cgctttccta atggaaagaa atatgaattg 1680 gacaaataca ccatgttcca ctatctccgc gcgcaggagt ttgagcacgg caagtctcgt 1740 attgctctga ccaattcggt aaacgaagcc cttttaaatc cttcgcgtgt gtacaccttt 1800 ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg aagcggcgat gtttttggga 1860 tgggtggaac aactggtata tgactttacg gatgaaactt ctgaagtctc gaccaccgac 1920 aaaattgccg atattaccat tatcattccc tatattggcc ctgcactgaa cattggtaac 1980 atgctgtata aagatgattt tgtgggcgcc ctgatctttt caggcgctgt tatcctgctg 2040 gaatttatcc cggaaatcgc cattccagta ctcggtacct ttgcgctggt gtcctatatc 2100 gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc tcagtaaacg taacgaaaaa 2160 tgggatgagg tgtataagta tattgttacc aactggctcg ctaaagtaaa cacccagatt 2220 gacctgattc gcaagaagat gaaagaagcg ctggaaaacc aagcagaagc gaccaaagct 2280 attatcaact atcaatataa ccagtacaca gaggaagaaa agaataacat caacttcaac 2340 atcgacgact tatcttcaaa gctgaatgaa tctattaaca aagcgatgat taatattaac 2400 aagttcttga accaatgtag tgtcagctat ctgatgaact cgatgatccc ttacggtgtg 2460 aaacgtctgg aagacttcga tgcaagcctt aaagatgccc ttctgaagta tatttacgat 2520 aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag ataaagtcaa caacacgctg 2580 agtacagaca tcccttttca gctgtctaaa tatgtggaca atcagcgcct gctgtccacg 2640 tttacggaat acatcaaaaa catcatcaac actagtattc tgaacttgcg ttacgagagt 2700 aaccatctga ttgatctgag ccgttacgca tctaaaatca acatcggctc gaaggtgaac 2760 ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact tagaatcgtc aaagattgaa 2820 gttatcttaa aaaatgcgat tgtatataat tcaatgtacg aaaatttctc tacgagcttt 2880 tggattcgta ttccgaaata tttcaacagt atctctttaa acaacgagta tactatcatc 2940 aattgtatgg agaataacag cgggtggaaa gtgagcctta actatggtga aatcatctgg 3000 actctgcagg acactcaaga aattaaacaa cgcgtggtgt ttaaatactc acagatgatt 3060 aacatctcgg attatattaa tcgctggatt tttgtgacaa ttactaacaa ccggctgaac 3120 aacagcaaaa tttacattaa cggtcgcctg atcgatcaga aaccaatcag taatctcggt 3180 aacattcacg catcgaataa tatcatgttc aaactggatg gttgtcgcga cacgcaccgt 3240 tacatttgga tcaaatactt caatttattc gacaaagaac tcaacgaaaa ggagattaag 3300 gatctttatg acaatcagtc taattcgggt attctgaaag acttttgggg tgattacctt 3360 cagtacgata aaccgtatta tatgttaaac ttatatgatc cgaataaata cgttgacgtc 3420 aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc cgcgtggcag cgtgatgacc 3480 actaacattt acttaaactc ctccctctat cgcggtacta aatttattat caagaaatat 3540 gcctctggca acaaggacaa tatcgtacgc aataacgatc gcgtctacat taacgtggtg 3600 gtgaagaata aagaatatcg tctggcgacc aatgctagtc aggcgggcgt ggagaaaatt 3660 ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc aggtggttgt gatgaaaagt 3720 aaaaatgacc aagggatcac caataaatgc aaaatgaatc tgcaagataa caacggcaac 3780 gacattggtt ttatcggctt ccaccaattc aataatatcg cgaaactggt ggcctcaaat 3840 tggtacaacc gtcagattga gcgcagctcc cgcactttag gctgtagctg ggagttcatt 3900 ccggtagatg acggttgggg agaacgccca ttgaaagtcg acaagcttgc ggccgcactc 3960 gagcaccacc accaccacca ctga 3984 <210> 88
<211> 1327 <212> PRT <213> Artificial Sequence <220> <223> BoNT/A-TEV <400 88
Met Pro Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15
Val Asp lie Ala Tyr lie Lys lie Pro Asn Ala Gly Gin Met Gin Pro 20 25 30
Val Lys Ala Phe Lys lie His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160
Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190
Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380
Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
Phe Asn Gly Gin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Gly Gly Gly Gly Glu Asn 435 440 445
Leu Tyr Phe Gin Gly Gly Gly Gly Gly Asp Lys Gly Tyr Asn Lys Ala 450 455 460
Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 465 470 475 480
Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile 485 490 495
Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 500 505 510
Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 515 520 525
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin Leu Glu 530 535 540
Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 545 550 555 560
Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu His 565 570 575
Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 580 585 590
Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 595 600 605
Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gin 610 615 620
Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp 625 630 635 640
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu 645 650 655
Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 660 665 670
Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 675 680 685
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val 690 695 700
Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 705 710 715 720
Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val 725 730 735
Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 740 745 750
Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr Asn Gin 755 760 765
Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu 770 775 780
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 785 790 795 800
Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile 805 810 815
Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 820 825 830
Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gin 835 840 845
Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 850 855 860
Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg Leu Leu Ser Thr 865 870 875 880
Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu 885 890 895
Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys 900 905 910
Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gin 915 920 925
Ile Gin Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys 930 935 940
Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe 945 950 955 960
Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu 965 970 975
Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser 980 985 990
Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gin Asp Thr Gin Glu Ile 995 1000 1005
Lys Gin Arg Val Val Phe Lys Tyr Ser Gin Met Ile Asn Ile Ser Asp 1010 1015 1020
Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn 1025 1030 1035 1040
Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gin Lys Pro Ile 1045 1050 1055
Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu 1060 1065 1070
Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn 1075 1080 1085
Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp 1090 1095 1100
Asn Gin Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu 1105 1110 1115 1120
Gin Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys 1125 1130 1135
Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys 1140 1145 1150
Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser 1155 1160 1165
Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn 1170 1175 1180
Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val 1185 1190 1195 1200
Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala Gly 1205 1210 1215
Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu 1220 1225 1230
Ser Gin Val Val Val Met Lys Ser Lys Asn Asp Gin Gly Ile Thr Asn 1235 1240 1245
Lys Cys Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp Ile Gly Phe 1250 1255 1260
Ile Gly Phe His Gin Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn 1265 1270 1275 1280
Trp Tyr Asn Arg Gin Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser 1285 1290 1295
Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu Lys 1300 1305 1310
Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His 1315 1320 1325 <210> 89 <211> 1044
<212> DNA <213> Artificial Sequence <220> <223> Intervening sequence containing transcriptional and translational sites. <400> 89 aagcttgtgg cctcaaattg gtacaaccgt cagattgagc gcagctcccg cactttaggc 60 tgtagctggg agttcattcc ggtagatgac ggttggggag aacgcccatt gcaccatcat 120 caccatcact gagcggccgc ataatgctta agtcgaacag attgatatgt agctataagt 180 aatcgtattg tacacggccg cataatcgaa attaatacga ctcactatag gggaattgtg 240 agcggataac aattccccat cttagtatat tagttaagta taagaaggag atataccatg 300 ggcgaatctc tgttcaaggg tccgcgtgat tataacccga tatcttcttc tatttgtcat 360 ctgactaacg aaagcgacgg ccacacgact tctctgtacg gtatcggttt cggtccgttc 420 atcattacca acaagcatct gttccgccgt aacaacggta ccctgctggt tcaatctctg 480 cacggcgtct tcaaggtaaa agacaccact acgctgcagc agcacctggt cgacggccgt 540 gacatgatca tcatccgcat gccgaaagat tttccgccgt tcccgcaaaa actgaagttt 600 cgtgaaccgc aacgcgaaga acgtatttgc ctggttacca ccaactttca gaccaaaagc 660 atgtcttcta tggtttccga tacctcttgc accttcccaa gcggtgacgg tattttctgg 720 aaacattgga ttcagaccaa agatggtcag tgcggctctc cgctggtgtc tacgcgtgac 780 ggtttcatcg ttggtatcca ttctgcttct aacttcacta acactaacaa ctactttact 840 tccgttccga aaaacttcat ggagctgctg actaaccaag aggcccagca gtgggtgtcc 900 ggttggcgcc tgaacgcaga ttctgtactg tggggtggtc ataaggtatt catgaacaaa 960 ccggaggagc cgttccagcc ggtcaaagag gcgacccagc tgatgaacga actggtttac 1020 tctcagtaag agctctgtct cgag 1044
<210> 90 <211 >4851 <212> DNA <213> Artificial Sequence <220> <223> BoNT/A-TEV and TEV protease variant 4 open reading frames with the intervening transcription and translation elements. <400 90 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag caaaaccaaa 1320 tccttgggcg gtggtggcga aaacctgtac ttccagggcg gtggcggtgg tgataagggc 1380 tataacaagg ccttcaatga tttatgcatc aaggtgaaca actgggactt gtttttctct 1440 ccatctgaag ataattttac taacgacttg aacaaaggag aggaaattac ttccgatacc 1500 aacatcgaag cagcggaaga gaatattagt ctagatctta ttcaacaata ttacctgacc 1560 tttaattttg ataacgagcc tgagaacatt tccattgaga atctcagctc tgacatcatc 1620 ggccagctgg aactgatgcc gaatatcgaa cgctttccta atggaaagaa atatgaattg 1680 gacaaataca ccatgttcca ctatctccgc gcgcaggagt ttgagcacgg caagtctcgt 1740 attgctctga ccaattcggt aaacgaagcc cttttaaatc cttcgcgtgt gtacaccttt 1800 ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg aagcggcgat gtttttggga 1860 tgggtggaac aactggtata tgactttacg gatgaaactt ctgaagtctc gaccaccgac 1920 aaaattgccg atattaccat tatcattccc tatattggcc ctgcactgaa cattggtaac 1980 atgctgtata aagatgattt tgtgggcgcc ctgatctttt caggcgctgt tatcctgctg 2040 gaatttatcc cggaaatcgc cattccagta ctcggtacct ttgcgctggt gtcctatatc 2100 gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc tcagtaaacg taacgaaaaa 2160 tgggatgagg tgtataagta tattgttacc aactggctcg ctaaagtaaa cacccagatt 2220 gacctgattc gcaagaagat gaaagaagcg ctggaaaacc aagcagaagc gaccaaagct 2280 attatcaact atcaatataa ccagtacaca gaggaagaaa agaataacat caacttcaac 2340 atcgacgact tatcttcaaa gctgaatgaa tctattaaca aagcgatgat taatattaac 2400 aagttcttga accaatgtag tgtcagctat ctgatgaact cgatgatccc ttacggtgtg 2460 aaacgtctgg aagacttcga tgcaagcctt aaagatgccc ttctgaagta tatttacgat 2520 aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag ataaagtcaa caacacgctg 2580 agtacagaca tcccttttca gctgtctaaa tatgtggaca atcagcgcct gctgtccacg 2640 tttacggaat acatcaaaaa catcatcaac actagtattc tgaacttgcg ttacgagagt 2700 aaccatctga ttgatctgag ccgttacgca tctaaaatca acatcggatc caaggtgaac 2760 ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact tagaatcgtc aaagattgaa 2820 gttatcttaa aaaatgcgat tgtatataat tcaatgtacg aaaatttctc tacgagcttt 2880 tggattcgta ttccgaaata tttcaacagt atctctttaa acaacgagta tactatcatc 2940 aattgtatgg agaataacag cgggtggaaa gtgagcctta actatggtga aatcatctgg 3000 actctgcagg acactcaaga aattaaacaa cgcgtggtgt ttaaatactc acagatgatt 3060 aacatctcgg attatattaa tcgctggatt tttgtgacaa ttactaacaa ccggctgaac 3120 aacagcaaaa tttacattaa cggtcgcctg atcgatcaga aaccaatcag taatctcggt 3180 aacattcacg catcgaataa tatcatgttc aaactggatg gttgtcgcga cacgcaccgt 3240 tacatttgga tcaaatactt caatttattc gacaaagaac tcaacgaaaa ggagattaag 3300 gatctttatg acaatcagtc taattcgggt attctgaaag acttttgggg tgattacctt 3360 cagtacgata aaccgtatta tatgttaaac ttatatgatc cgaataaata cgttgacgtc 3420 aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc cgcgtggcag cgtgatgacc 3480 actaacattt acttaaactc ctccctctat cgcggtacta aatttattat caagaaatat 3540 gcctctggca acaaggacaa tatcgtacgc aataacgatc gcgtctacat taacgtggtg 3600 gtgaagaata aagaatatcg tctggcgacc aatgctagtc aggcgggcgt ggagaaaatt 3660 ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc aggtggttgt gatgaaaagt 3720 aaaaatgacc aagggatcac caataaatgc aaaatgaatc tgcaagataa caacggcaac 3780 gacattggtt ttatcggctt ccaccaattc aataatatcg cgaagcttgt ggcctcaaat 3840 tggtacaacc gtcagattga gcgcagctcc cgcactttag gctgtagctg ggagttcatt 3900 ccggtagatg acggttgggg agaacgccca ttgcaccatc atcaccatca ctgagcggcc 3960 gcataatgct taagtcgaac agattgatat gtagctataa gtaatcgtat tgtacacggc 4020 cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc 4080 atcttagtat attagttaag tataagaagg agatatacca tgggcgaatc tctgttcaag 4140 ggtccgcgtg attataaccc gatatcttct tctatttgtc atctgactaa cgaaagcgac 4200 ggccacacga cttctctgta cggtatcggt ttcggtccgt tcatcattac caacaagcat 4260 ctgttccgcc gtaacaacgg taccctgctg gttcaatctc tgcacggcgt cttcaaggta 4320 aaagacacca ctacgctgca gcagcacctg gtcgacggcc gtgacatgat catcatccgc 4380 atgccgaaag attttccgcc gttcccgcaa aaactgaagt ttcgtgaacc gcaacgcgaa 4440 gaacgtattt gcctggttac caccaacttt cagaccaaaa gcatgtcttc tatggtttcc 4500 gatacctctt gcaccttccc aagcggtgac ggtattttct ggaaacattg gattcagacc 4560 aaagatggtc agtgcggctc tccgctggtg tctacgcgtg acggtttcat cgttggtatc 4620 cattctgctt ctaacttcac taacactaac aactacttta cttccgttcc gaaaaacttc 4680 atggagctgc tgactaacca agaggcccag cagtgggtgt ccggttggcg cctgaacgca 4740 gattctgtac tgtggggtgg tcataaggta ttcatgaaca aaccggagga gccgttccag 4800 ccggtcaaag aggcgaccca gctgatgaac gaactggttt actctcagta a 4851 <210> 91 <211> 732
<212> DNA <213> Artificial Sequence <220> <223> TEV variant 7 <400> 91 atgggcgaat ctctgttcaa gggtccgcgt gattataacc cgatatcttc ttctatttgt 60 catctgacta acgaaagcga cggccacacg acttctctgt acggtatcgg tttcggtccg 120 ttcatcatta ccaacaagca tctgttccgc cgtaacaacg gtaccctgct ggttcaatct 180 ctgcacggcg tcttcaaggt aaaagacacc actacgctgc agcagcacct ggtcgacggc 240 cgtgacatga teatcatccg catgccgaaa gattttccgc cgttcccgca aaaactgaag 300 tttcgtgaac cgcaacgcga agaaegtatt tgcctggtta ccaccaactt tcagaccaaa 360 ageatgtett ctatggtttc cgatacctct tgcaccttcc caagcggtga cggtattttc 420 tggaaacatt ggatccagac caaagatggt cagtgcggct ctccgctggt gtctacgcgt 480 gacggtttca tcgttggtat ccattctgct tetaaettea ctaacactaa caactacttt 540 acttccgttc cgaaaaactt catggagctg ctgactaacc aagaggccca gcagtgggtg 600 tccggttggc gcctgaacgc agattctgta ctgtggggtg gtcataaggt attcatgaac 660 aaaeeggagg agccgttcca geeggtcaaa gaggcgaccc agctgatgaa cgaactggtt 720 tactctcagt aa 732
<210> 92 <211> 415 <212> DNA <213> Artificial Sequence <220> <223> Intervening sequence containing transcriptional and translational sites and T7 termination site. <400> 92 aagcttgtgg cctcaaattg gtacaaccgt cagattgagc gcagctcccg cactttaggc 60 tgtagctggg agttcattcc ggtagatgac ggttggggag aacgcccatt gcaccatcat 120 caccatcact gagcggccgc ataatgetta agtcgaacag attgatatgt agetataagt 180 aattgtatga ctgaacctag gctgctgcca ccgctgagca ataactagca taaccccttg 240 gggcctctaa acgggtcttg aggggttttt tgctgatcgt atactctcag gcatctatga 300 gttgtacacg tccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga 360 taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac catgg 415
<210> 93 <211> 4965 <212> DNA <213> Artificial Sequence <220> <223> BoNT/A-TEV and TEV protease variant 4 open reading frames with the intervening transcription and translation elements and termination site. <400> 93 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag caaaaccaaa 1320 tccttgggcg gtggtggcga aaacctgtac ttccagggcg gtggcggtgg tgataagggc 1380 tataacaagg ccttcaatga tttatgcatc aaggtgaaca actgggactt gtttttctct 1440 ccatctgaag ataattttac taacgacttg aacaaaggag aggaaattac ttccgatacc 1500 aacatcgaag cagcggaaga gaatattagt ctagatctta ttcaacaata ttacctgacc 1560 tttaattttg ataacgagcc tgagaacatt tccattgaga atctcagctc tgacatcatc 1620 ggccagctgg aactgatgcc gaatatcgaa cgctttccta atggaaagaa atatgaattg 1680 gacaaataca ccatgttcca ctatctccgc gcgcaggagt ttgagcacgg caagtctcgt 1740 attgctctga ccaattcggt aaacgaagcc cttttaaatc cttcgcgtgt gtacaccttt 1800 ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg aagcggcgat gtttttggga 1860 tgggtggaac aactggtata tgactttacg gatgaaactt ctgaagtctc gaccaccgac 1920 aaaattgccg atattaccat tatcattccc tatattggcc ctgcactgaa cattggtaac 1980 atgctgtata aagatgattt tgtgggcgcc ctgatctttt caggcgctgt tatcctgctg 2040 gaatttatcc cggaaatcgc cattccagta ctcggtacct ttgcgctggt gtcctatatc 2100 gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc tcagtaaacg taacgaaaaa 2160 tgggatgagg tgtataagta tattgttacc aactggctcg ctaaagtaaa cacccagatt 2220 gacctgattc gcaagaagat gaaagaagcg ctggaaaacc aagcagaagc gaccaaagct 2280 attatcaact atcaatataa ccagtacaca gaggaagaaa agaataacat caacttcaac 2340 atcgacgact tatcttcaaa gctgaatgaa tctattaaca aagcgatgat taatattaac 2400 aagttcttga accaatgtag tgtcagctat ctgatgaact cgatgatccc ttacggtgtg 2460 aaacgtctgg aagacttcga tgcaagcctt aaagatgccc ttctgaagta tatttacgat 2520 aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag ataaagtcaa caacacgctg 2580 agtacagaca tcccttttca gctgtctaaa tatgtggaca atcagcgcct gctgtccacg 2640 tttacggaat acatcaaaaa catcatcaac actagtattc tgaacttgcg ttacgagagt 2700 aaccatctga ttgatctgag ccgttacgca tctaaaatca acatcggatc caaggtgaac 2760 ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact tagaatcgtc aaagattgaa 2820 gttatcttaa aaaatgcgat tgtatataat tcaatgtacg aaaatttctc tacgagcttt 2880 tggattcgta ttccgaaata tttcaacagt atctctttaa acaacgagta tactatcatc 2940 aattgtatgg agaataacag cgggtggaaa gtgagcctta actatggtga aatcatctgg 3000 actctgcagg acactcaaga aattaaacaa cgcgtggtgt ttaaatactc acagatgatt 3060 aacatctcgg attatattaa tcgctggatt tttgtgacaa ttactaacaa ccggctgaac 3120 aacagcaaaa tttacattaa cggtcgcctg atcgatcaga aaccaatcag taatctcggt 3180 aacattcacg catcgaataa tatcatgttc aaactggatg gttgtcgcga cacgcaccgt 3240 tacatttgga tcaaatactt caatttattc gacaaagaac tcaacgaaaa ggagattaag 3300 gatctttatg acaatcagtc taattcgggt attctgaaag acttttgggg tgattacctt 3360 cagtacgata aaccgtatta tatgttaaac ttatatgatc cgaataaata cgttgacgtc 3420 aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc cgcgtggcag cgtgatgacc 3480 actaacattt acttaaactc ctccctctat cgcggtacta aatttattat caagaaatat 3540 gcctctggca acaaggacaa tatcgtacgc aataacgatc gcgtctacat taacgtggtg 3600 gtgaagaata aagaatatcg tctggcgacc aatgctagtc aggcgggcgt ggagaaaatt 3660 ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc aggtggttgt gatgaaaagt 3720 aaaaatgacc aagggatcac caataaatgc aaaatgaatc tgcaagataa caacggcaac 3780 gacattggtt ttatcggctt ccaccaattc aataatatcg cgaagcttgt ggcctcaaat 3840 tggtacaacc gtcagattga gcgcagctcc cgcactttag gctgtagctg ggagttcatt 3900 ccggtagatg acggttgggg agaacgccca ttgcaccatc atcaccatca ctgagcggcc 3960 gcataatgct taagtcgaac agattgatat gtagctataa gtaattgtat gactgaacct 4020 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 4080 tgaggggttt tttgctgatc gtatactctc aggcatctat gagttgtaca cgtccgcata 4140 atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccatctta 4200 gtatattagt taagtataag aaggagatat accatgggcg aatctctgtt caagggtccg 4260 cgtgattata acccgatatc ttcttctatt tgtcatctga ctaacgaaag cgacggccac 4320 acgacttctc tgtacggtat cggtttcggt ccgttcatca ttaccaacaa gcatctgttc 4380 cgccgtaaca acggtaccct gctggttcaa tctctgcacg gcgtcttcaa ggtaaaagac 4440 accactacgc tgcagcagca cctggtcgac ggccgtgaca tgatcatcat ccgcatgccg 4500 aaagattttc cgccgttccc gcaaaaactg aagtttcgtg aaccgcaacg cgaagaacgt 4560 atttgcctgg ttaccaccaa ctttcagacc aaaagcatgt cttctatggt ttccgatacc 4620 tcttgcacct tcccaagcgg tgacggtatt ttctggaaac attggattca gaccaaagat 4680 ggtcagtgcg gctctccgct ggtgtctacg cgtgacggtt tcatcgttgg tatccattct 4740 gcttctaact tcactaacac taacaactac tttacttccg ttccgaaaaa cttcatggag 4800 ctgctgacta accaagaggc ccagcagtgg gtgtccggtt ggcgcctgaa cgcagattct 4860 gtactgtggg gtggtcataa ggtattcatg aacaaaccgg aggagccgtt ccagccggtc 4920 aaagaggcga cccagctgat gaacgaactg gtttactctc agtaa 4965 <210> 94 <211> 2697
<212> DNA <213> Artificial Sequence <220>
<223> Open reading frame for NociLHN/A-TEV <400 94 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag caaagaaaac 1320 ctgtacttcc agttcggtgg ttttaccggc gctcgtaaat ctgcacgtaa acgcaagaat 1380 caggctctgg ctggtggcgg tggctctggt ggtggcggta gcggcggtgg cggttctgcg 1440 ctcaatgatt tatgcatcaa ggtgaacaac tgggacttgt ttttctctcc atctgaagat 1500 aattttacta acgacttgaa caaaggagag gaaattactt ccgataccaa catcgaagca 1560 gcggaagaga atattagtct agatcttatt caacaatatt acctgacctt taattttgat 1620 aacgagcctg agaacatttc cattgagaat ctcagctctg acatcatcgg ccagctggaa 1680 ctgatgccga atatcgaacg ctttcctaat ggaaagaaat atgaattgga caaatacacc 1740 atgttccact atctccgcgc gcaggagttt gagcacggca agtctcgtat tgctctgacc 1800 aattcggtaa acgaagccct tttaaatcct tcgcgtgtgt acaccttttt ctcaagcgat 1860 tatgttaaaa aagtgaacaa ggcgaccgaa gcggcgatgt ttttgggatg ggtggaacaa 1920 ctggtatatg actttacgga tgaaacttct gaagtctcga ccaccgacaa aattgccgat 1980 attaccatta tcattcccta tattggccct gcactgaaca ttggtaacat gctgtataaa 2040 gatgattttg tgggcgccct gatcttttca ggcgctgtta tcctgctgga atttatcccg 2100 gaaatcgcca ttccagtact cggtaccttt gcgctggtgt cctatatcgc aaacaaagtt 2160 ttgactgtcc agacgatcga caacgcgctc agtaaacgta acgaaaaatg ggatgaggtg 2220 tataagtata ttgttaccaa ctggctcgct aaagtaaaca cccagattga cctgattcgc 2280 aagaagatga aagaagcgct ggaaaaccaa gcagaagcga ccaaagctat tatcaactat 2340 caatataacc agtacacaga ggaagaaaag aataacatca acttcaacat cgacgactta 2400 tcttcaaagc tgaatgaatc tattaacaaa gcgatgatta atattaacaa gttcttgaac 2460 caatgtagtg tcagctatct gatgaactcg atgatccctt acggtgtgaa acgtctggaa 2520 gacttcgatg caagccttaa agatgccctt ctgaagtata tttacgataa tcgcggaact 2580 cttattggcc aagtggatcg cttaaaagat aaagtcaaca acacgctgag tacagacatc 2640 ccttttcagc tgtctaaata tgtggacaat cagcgccacc atcaccatca ccactaa 2697
<210> 95 <211> 898 <212> PRT <213> Artificial Sequence <220> <223> NociLHN/A-TEV <400> 95
Met Pro Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15
Val Asp lie Ala Tyr lie Lys lie Pro Asn Ala Gly Gin Met Gin Pro 20 25 30
Val Lys Ala Phe Lys lie His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160
Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190
Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380
Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
Phe Asn Gly Gin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Glu Asn Leu Tyr Phe Gin Phe Gly Gly Phe 435 440 445
Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg Lys Asn Gin Ala Leu Ala 450 455 460
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 465 470 475 480
Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 485 490 495
Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile 500 505 510
Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 515 520 525
Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 530 535 540
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin Leu Glu 545 550 555 560
Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 565 570 575
Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu His 580 585 590
Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 595 600 605
Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 610 615 620
Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gin 625 630 635 640
Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp 645 650 655
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu 660 665 670
Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 675 680 685
Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 690 695 700
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val 705 710 715 720
Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 725 730 735
Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val 740 745 750
Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 755 760 765
Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr Asn Gin 770 775 780
Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu 785 790 795 800
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 805 810 815
Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile 820 825 830
Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 835 840 845
Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gin 850 855 860
Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 865 870 875 880
Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg His His His His 885 890 895
His His
<210> 96 <211> 2709 <212> DNA <213> Artificial Sequence <220> <223> Open reading frame for DynLHN/A-TEV <400> 96 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctataa gctcctgtgt gtccgtggta ttatcaccag caaagaaaac 1320 ctgtacttcc agtatggcgg tttcctgcgt cgcattcgtc ctaagcttaa atgggataac 1380 caggctcttg ctggtggtgg tggctctggt ggtggcggta gcggcggtgg tggttctgca 1440 ctcaatgatt tatgtatcaa ggtgaacaac tgggacttgt ttttctctcc atctgaagat 1500 aattttacta acgacttgaa caaaggagag gaaattactt ccgataccaa catcgaagca 1560 gcggaagaga atattagtct agatcttatt caacaatatt acctgacctt taattttgat 1620 aacgagcctg agaacatttc cattgagaat ctcagctctg acatcatcgg ccagctggaa 1680 ctgatgccga atatcgaacg ctttcctaat ggaaagaaat atgaattgga caaatacacc 1740 atgttccact atctccgcgc gcaggagttt gagcacggca agtctcgtat tgctctgacc 1800 aattcggtaa acgaagccct tttaaatcct tcgcgtgtgt acaccttttt ctcaagcgat 1860 tatgttaaaa aagtgaacaa ggcgaccgaa gcggcgatgt ttttgggatg ggtggaacaa 1920 ctggtatatg actttacgga tgaaacttct gaagtctcga ccaccgacaa aattgccgat 1980 attaccatta tcattcccta tattggccct gcactgaaca ttggtaacat gctgtataaa 2040 gatgattttg tgggcgccct gatcttttca ggcgctgtta tcctgctgga atttatcccg 2100 gaaatcgcca ttccagtact cggtaccttt gcgctggtgt cctatatcgc aaacaaagtt 2160 ttgactgtcc agacgatcga caacgcgctc agtaaacgta acgaaaaatg ggatgaggtg 2220 tataagtata ttgttaccaa ctggctcgct aaagtaaaca cccagattga cctgattcgc 2280 aagaagatga aagaagcgct ggaaaaccaa gcagaagcga ccaaagctat tatcaactat 2340 caatataacc agtacacaga ggaagaaaag aataacatca acttcaacat cgacgactta 2400 tcttcaaagc tgaatgaatc tattaacaaa gcgatgatta atattaacaa gttcttgaac 2460 caatgtagtg tcagctatct gatgaactcg atgatccctt acggtgtgaa acgtctggaa 2520 gacttcgatg caagccttaa agatgccctt ctgaagtata tttacgataa tcgcggaact 2580 cttattggcc aagtggatcg cttaaaagat aaagtcaaca acacgctgag tacagacatc 2640 ccttttcagc tgtctaaata tgtggacaat cagcgcctgc tgtccacgca ccatcaccat 2700 caccactaa 2709 <210> 97 <211> 902
<212> PRT <213> Artificial Sequence <220>
<223> DynLHN/A-TEV <400> 97
Met Pro Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15
Val Asp lie Ala Tyr lie Lys lie Pro Asn Ala Gly Gin Met Gin Pro 20 25 30
Val Lys Ala Phe Lys lie His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125
Val Ile Asp Thr Asn Cys Ile Asn Val lie Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160
Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190
Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380
Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
Phe Asn Gly Gin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Glu Asn Leu Tyr Phe Gin Tyr Gly Gly Phe 435 440 445
Leu Arg Arg Ile Arg Pro Lys Leu Lys Trp Asp Asn Gin Ala Leu Ala 450 455 460
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 465 470 475 480
Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 485 490 495
Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile 500 505 510
Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 515 520 525
Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 530 535 540
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin Leu Glu 545 550 555 560
Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 565 570 575
Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu His 580 585 590
Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 595 600 605
Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 610 615 620
Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gin 625 630 635 640
Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp 645 650 655
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu 660 665 670
Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 675 680 685
Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 690 695 700
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val 705 710 715 720
Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 725 730 735
Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val 740 745 750
Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 755 760 765
Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr Asn Gin 770 775 780
Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu 785 790 795 800
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 805 810 815
Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile 820 825 830
Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 835 840 845
Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gin 850 855 860
Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 865 870 875 880
Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg Leu Leu Ser Thr 885 890 895
His His His His His His 900
<210> 98 <211> 320 <212> DNA <213> Artificial Sequence <220> <223> DNA fragment encoding a di-chain loop region comprising an integrated TEV protease cleavage site-Galanin binding domain <400> 98 gaattctaca agctgctgtg cgtggacggc atcattacct ccaaaactaa atctgaaaac 60 ctgtacttcc agggctggac tttgaactct gctggttatc tcctgggccc acatgcggtt 120 gctcttgctg gtggcggtgg ctctggcggt ggcggtagcg gcggtggcgg ttctgcacta 180 gtgcttcagt gtatcaaggt taacaactgg gatttattct tcagcccgag tgaagacaac 240 ttcaccaacg acctgaacaa aggtgaagaa atcacctcag atactaacat cgaagcagcc 300 gaagaaaaca tcagtctaga 320
<210> 99 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Di-chain loop region comprising an integrated TEV protease cleavage site-Galanin binding domain <400> 99
Glu Phe Tyr Lys Leu Leu Cys Val Asp Gly Ile Ile Thr Ser Lys Thr 15 10 15
Lys Ser Glu Asn Leu Tyr Phe Gin Gly Trp Thr Leu Asn Ser Ala Gly 20 25 30
Tyr Leu Leu Gly Pro His Ala Val Ala Leu Ala Gly Gly Gly Gly Ser 35 40 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu Gin Cys 50 55 60
Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn 65 70 75 80
Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn 85 90 95
Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 100 105
<210> 100 <211> 2703 <212> DNA <213> Artificial Sequence <220> <223> Open reading frame for GalLHN/A-TEV <400> 100 atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc 60 tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac 120 aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac 180 ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc 240 gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg 300 acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt 360 agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct 420 gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt 480 atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat 540 ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc 600 gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc 660 ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac 720 cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt 780 gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac 840 gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg 900 aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa 960 tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa 1020 ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc 1080 ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg 1140 aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac 1200 tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc 1260 ggcttgtttg aattctacaa gctgctgtgc gtggacggca tcattacctc caaaactaaa 1320 tctgaaaacc tgtacttcca gggctggact ttgaactctg ctggttatct cctgggccca 1380 catgcggttg ctcttgctgg tggcggtggc tctggcggtg gcggtagcgg cggtggcggt 1440 tctgcactag tgcttcagtg tatcaaggtt aacaactggg atttáttett cagcccgagt 1500 gaagacaact tcaccaacga cctgaacaaa ggtgaagaaa tcacctcaga tactaacatc 1560 gaagcagccg aagaaaacat cagtctagat cttattcaac aatattacct gacctttaat 1620 tttgataacg agcctgagaa catttccatt gagaatetea gctctgacat catcggccag 1680 ctggaactga tgeegaatat egaaegettt cctaatggaa agaaatatga attggacaaa 1740 tacaccatgt tccactatct ccgcgcgcag gagtttgagc acggcaagtc tegtattget 1800 ctgaccaatt cggtaaacga agccctttta aatccttcgc gtgtgtacac ctttttctca 1860 agegattatg ttaaaaaagt gaacaaggcg accgaagcgg cgatgttttt gggatgggtg 1920 gaacaactgg tatatgaett taeggatgaa aettetgaag tctcgaccac cgacaaaatt 1980 geegatatta ccattatcat tccctatatt ggccctgcac tgaacattgg taacatgctg 2040 tataaagatg attttgtggg cgccctgatc ttttcaggcg ctgttatcct gctggaattt 2100 atcccggaaa tcgccattcc agtactcggt acctttgcgc tggtgtccta tatcgcaaac 2160 aaagttttga ctgtccagac gatcgacaac gcgctcagta aaegtaaega aaaatgggat 2220 gaggtgtata agtatattgt taccaactgg ctcgctaaag taaacaccca gattgacctg 2280 attcgcaaga agatgaaaga agcgctggaa aaccaagcag aagcgaccaa agetattate 2340 aactatcaat ataaccagta cacagaggaa gaaaagaata acatcaactt caacatcgac 2400 gaettatett caaagctgaa tgaatetatt aacaaagcga tgattaatat taacaagttc 2460 ttgaaccaat gtagtgtcag ctatctgatg aactcgatga tcccttacgg tgtgaaacgt 2520 ctggaagact tcgatgcaag ccttaaagat gcccttctga agtatattta egataatege 2580 ggaactctta ttggccaagt ggategetta aaagataaag tcaacaacac gctgagtaca 2640 gacatccctt ttcagctgtc taaatatgtg gacaatcagc gccaccatca ccatcaccac 2700 taa 2703 <210> 101 <211> 900
<212> PRT <213> Artificial Sequence <220>
<223> GalLHN/A-TEV <400> 101
Met Pro Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15
Val Asp lie Ala Tyr lie Lys lie Pro Asn Ala Gly Gin Met Gin Pro 20 25 30
Val Lys Ala Phe Lys lie His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160
Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190
Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270
Phe Ile Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350
Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380
Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
Phe Asn Gly Gin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Glu Asn Leu Tyr Phe Gin Gly 435 440 445
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val Ala 450 455 460
Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 465 470 475 480
Ser Ala Leu Val Leu Gin Cys Ile Lys Val Asn Asn Trp Asp Leu Phe 485 490 495
Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu 500 505 510
Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser 515 520 525
Leu Asp Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu 530 535 540
Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin 545 550 555 560
Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr 565 570 575
Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe 580 585 590
Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala 595 600 605
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val 610 615 620
Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val 625 630 635 640
Glu Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr 645 650 655
Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro 660 665 670
Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala 675 680 685
Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile 690 695 700
Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn 705 710 715 720
Lys Val Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn 725 730 735
Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala 740 745 750
Lys Val Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala 755 760 765
Leu Glu Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr 770 775 780
Asn Gin Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp 785 790 795 800
Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn 805 810 815
Ile Asn Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser 820 825 830
Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu 835 840 845
Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile 850 855 860
Gly Gin Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr 865 870 875 880
Asp Ile Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg His His 885 890 895
His His His His 900
<210> 102 <211> 314 <212> DNA <213> Artificial Sequence <220> <223> DNA fragment encoding a di-chain loop region comprising an integrated TEV protease cleavage site-Noci-ceptin binding domain <400> 102 gaattctata agctcctgtg tgtccgcggt attatcaeca gcaaagaaaa cctgtacttc 60 cagttcggtg gttttaccgg egetegtaaa tctgcacgta aacgcaagaa teaggetetg 120 gctggtggcg gtggctctgg tggtggcggt agcggcggtg gcggttctgc gctcaatgat 180 ttatgeatea aggtgaacaa ctgggacttg tttttctctc catctgaaga taattttact 240 aacgacttga acaaaggaga ggaaattact tccgatacca acatcgaagc ageggaagag 300 aatattagtc taga 314 <210> 103 <211> 104
<212> PRT <213> Artificial Sequence <220> <223> Di-chain loop region comprising an integrated TEV protease cleavage site-Nociceptin binding domain <400> 103
Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Thr Ser Lys Glu 15 10 15
Asn Leu Tyr Phe Gin Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala 20 25 30
Arg Lys Arg Lys Asn Gin Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly 35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asn Asp Leu Cys lie Lys 50 55 60
Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr 65 70 75 80
Asn Asp Leu Asn Lys Gly Glu Glu lie Thr Ser Asp Thr Asn lie Glu 85 90 95
Ala Ala Glu Glu Asn Ile Ser Leu 100
<210> 104 <211> 314 <212> DNA <213> Artificial Sequence <220> <223> DNA fragment encoding a di-chain loop region comprising an integrated TEV protease cleavage site-Dynor-phin binding domain <400 104 gaattctata agctcctgtg tgtccgtggt attatcacca gcaaagaaaa cctgtacttc 60 cagtatggcg gtttcctgcg tcgcattcgt cctaagctta aatgggataa ccaggctctt 120 gctggtggtg gtggctctgg tggtggcggt agcggcggtg gtggttctgc actcaatgat 180 ttatgtatca aggtgaacaa ctgggacttg tttttctctc catctgaaga taattttact 240 aacgacttga acaaaggaga ggaaattact tccgatacca acatcgaagc agcggaagag 300 aatattagtc taga 314
<210 105 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Di-chain loop region comprising an integrated TEV protease cleavage site-Dynorphin binding domain <400> 105
Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Thr Ser Lys Glu 15 10 15
Asn Leu Tyr Phe Gin Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys 20 25 30
Leu Lys Trp Asp Asn Gin Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly 35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asn Asp Leu Cys Ile Lys 50 55 60
Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr 65 70 75 80
Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu 85 90 95
Ala Ala Glu Glu Asn Ile Ser Leu 100
<210> 106 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> Open reading frame encoding TEV protease variant 7 <400> 106 ccatggatgg gtggcgaatc tctgttcaag ggtccgcgtg attataaccc gatatcttct 60 actatttgtc atctgactaa cgaaagcgac ggccacacga cttctctgta cggtatcggt 120 ttcggtccgt tcatcattac caacaagcat ctgttccgcc gtaacaacgg taccctgctg 180 gttcaatctc tgcacggcgt cttcaaggta aaagacacca ctacgctgca gcagcacctg 240 gtcgacggcc gtgacatgat catcatccgc atgccgaaag attttccgcc gttcccgcaa 300 aaactgaagt ttcgtgaacc gcaacgcgaa gaacgtattt gcctggttac caccaacttt 360 cagaccaaaa gcatgtcttc tatggtttcc gatacctctt gcaccttccc aagcggtgac 420 ggtattttct ggaaacattg gatccagacc aaagatggtc agtgcggctc tccgctggtg 480 tctacgcgtg acggtttcat cgttggtatc cattctgctt ctaacttcac taacactaac 540 aactacttta cttccgttcc gaaaaacttc atggagctgc tgactaacca agaggcccag 600 cagtgggtgt ccggttggcg cctgaacgca gattctgtac tgtggggtgg tcataaggta 660 ttcatgaaca aaccggagga gccgttccag ccggtcaaag aggcgaccca gctgatgaac 720 gaactggttt actctcagta atgaaagctt 750
<210> 107 <211> 3791 <212> DNA <213> Artificial Sequence <220> <223> ORF encoding p10-TEV variant 7 and polH-DynLHn/A-TEV <400> 107 agatcttatg cggccgcact cgagtcatta gtggtgatgg tgatggtggg ttgacagaag 60 tctctgatta tcaacatact tcgacagttg gaacgggatg tctgttgaca gagtgttgtt 120 taccttatct ttcagacggt caacttggcc aatgagcgtt cccctgttat cgtagatgta 180 cttaagaagg gcgtctttca gcgaggcgtc gaagtcctcc agtctcttta caccatatgg 240 gatcattgag ttcatcaagt atgatacact gcattggttg aggaacttgt taatgtttat 300 cattgccttg tttatgctct cgttgagttt actagagagg tcgtcaatgt tgaagttgat 360 gttgttcttt tcctcctcgg tgtactggtt gtactgatag ttaatgatgg cctttgtcgc 420 ttcagcctgg ttctccagcg cctccttcat ctttttcctg atgaggtcga tttgggtgtt 480 gaccttagcg agccagttag tcacgatgta tttgtacact tcatcccact tctcgtttct 540 ttttgacaga gcattatcga ttgtttggac agtgaggacc ttgttagcaa tgtagctgac 600 caaagcgaag gtaccaagaa caggaatagc gatctctggg atgaactcca acaaaatcac 660 tgctcccgag aaaatcaacg caccgacgaa gtcgtccttg tacagcatat tgccgatgtt 720 aagagcaggt ccaatgtagg gtatgatgat agtgatgtct gcgatcttgt ccgtagtcga 780 aacttcacta gtctcgtcgg tgaaatcgta aaccaactgt tcaacccaac ccagaaacat 840 cgctgcttcg gttgccttat tcaccttctt aacgtaatcc gaactgaaga aggtgtagac 900 acgagaagga ttgagaagag cctcgttgac cgagttagtg agggcgattc tactttttcc 960 gtgctcaaac tcttgagctc tgaggtagtg gaacatcgtg tatttgtcca attcgtactt 1020 cttgccgtta gggaatctct cgatattggg catgagttcc agctgtccga tgatgtcgct 1080 gctcagattt tcgatagaaa tgttttccgg ctcgttatcg aaattgaacg tgagatagta 1140 ctgctgaatc aggtctagac taatattctc ttccgctgct tcgatgttgg tatcggaagt 1200 aatttcctct cctttgttca agtcgttagt aaaattatct tcagatggag agaaaaacaa 1260 gtcccagttg ttcaccttga tacataaatc attgagtgca gaaccaccac cgccgctacc 1320 gccaccacca gagccaccac caccagcaag agcctggtta tcccatttaa gcttaggacg 1380 aatgcgacgc aggaaaccgc catactggaa gtacaggttt tctttgctgg tgataatacc 1440 acggacacac aggagcttat agaattcaaa caggccggtg aaattcttga gctttgtgaa 1500 gttcatgtta ttgatctcgg tattctgacc attgaagtta gccgccaaat tggtgttcct 1560 aaggttaaag ccatcataga tggtgtagtt cacctttggc acgatattga tcttaaacac 1620 agctttgtcg aagttaagat aagtcttgcg gttcaatacc ttgaagaact taacaaagtt 1680 gtcctcggta tagatctctg taagcatttt gtacagcttg tcaaacttga gtttgtccac 1740 ggaaaacttt ccggaggtgt cctcggaaag caagtacttt tccttaaaga cgttcttcat 1800 atactgaagg ctagccgtgg tgccgactat acttttagcc ttattcagcg tactggcaat 1860 atctttgaat ttgttgtagt aatacagtct gaactcattc tcttgcaagg agtcgatgaa 1920 cttagcatcg tgtccaccga aggtacgaag ttcttcgaag gagacttcca gaccggacat 1980 ctcatagtat gcgttggtgt tcaccttgaa aacgcggttt ggattgatgg caattccgta 2040 cagtctatgg cctgcgtgaa tcagctcgtg agccaaggtc accgcgggat ctgtggcgaa 2100 cttgccagcg cccaacaacg gattagtgtc aacctccaat gactcttcga agccgaaagt 2160 gaaatcgggg gaaaacctga tgtattgagt agaaccataa ccgtttctgg tcaggttcag 2220 cacctcatgg ccgaaggact tacattcaaa ctgaatgatg tcggcagagg gaccgatgat 2280 caccaagttg agttcctctg aacggtagga gccgtcaggt tggatcacgt tgatacagtt 2340 tgtatcgatc actttcagct ctgtatctat ggttgatccg ccccaaaagg ggattccacg 2400 gacgatggaa gtgagcagca tgcgaccgag gtcagtggaa tagatacgct cgaaaagttt 2460 ggtcactccc ttgaggtaat tgtctttctc gttatctgtc gacaagtacg tggagtcata 2520 gtaggacacc ggcacctgct tggcctctgg tggcggattc aaatctcctt cttcggggtt 2580 agtgaaggtg tctctttcgg gaatgaccca tatcttgtta tgaatcttga aggccttaac 2640 aggctgcatt tgaccggcat tcggaatctt gatatacgca atatcgactc cgttgacagg 2700 gtccttatag ttgaattgct tgttgacaaa tcccatggga ttatatttat aggttttttt 2760 attacaaaac tgttacgaaa acagtaaaat acttatttat ttgcgagatg gttatcattt 2820 taattatctc catgatccaa taacctagaa taaaggccga cctttaattc aacccaacac 2880 aatatattat agttaaataa gaattattat caaatcattt gtatattaat taaaatacta 2940 tactgtaaat tacattttat ttacaatcac agatccatat gggcgagtca ttgttcaagg 3000 gaccgagaga ttacaacccc atctcgtcgt caatctgcca cttgacaaac gaatccgacg 3060 gtcacactac ttctctgtac ggtatcggct tcggaccttt catcatcacc aacaagcatt 3120 tgtttaggag aaacaacggt acactccttg tccagtccct gcacggcgta ttcaaagtca 3180 aagataccac gactctgcaa cagcatctgg tcgacggaag ggacatgata atcattcgca 3240 ttcctaaaga cttcccaccc ttccctcaaa agctcaagtt tcgtgagccc cagcgtgagg 3300 agaggatttg tcttgtcacg actaacttcc agaccaaatc tatgtctagc atggtcagcg 3360 atacctcgtg cacttttcca agcggcgatg gaatcttttg gaagcactgg attcagacaa 3420 aggacggcca atgcggttct cctctcgtaa gtacgcgcga cggattcatc gtgggtattc 3480 actccgcttc caacttcacc aacaccaaca actatttcac tagcgtgcca aagaatttca 3540 tggaattgct caccaaccag gaggcccaac aatgggttag tggttggcgt cttaatgcgg 3600 actcagtgct gtggggaggc cataaagttt tcatgaataa gccggaggaa ccttttcaac 3660 ccgtgaagga agcaacacag ctcatgaatg agctggttta ctcacagtga taactcgagc 3720 aatctgatac tagtaataaa agatgtttat tttcattaga tgtgtgtgtt ggttttttgt 3780 ctatagcatg c 3791
Claims 1. An intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of: a) growing a cell comprising a dual expression construct at a first temperature for a certain period of time in order to achieve maximal cell density, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous TEV protease cleavage site; and ii) an open reading frame encoding a TEV protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form. 2. The intracellular method according to Claim 1, wherein the cell is grown in step a) at 37 °C for about 3.5 hours, wherein the dual expression construct comprises: an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising the exogenous TEV protease cleavage site and wherein the cell is grown in step b) at 22 °C for about 16 to about 18 hours, wherein the Clostridial toxin enzymatic domain is a BoNT/A enzymatic domain. 3. The intracellular method according to Claim 1, wherein the cell is grown in step a) at 37 °C for about 3.5 hours, wherein the dual expression construct comprises: an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising the exogenous TEV protease cleavage site and wherein the cell is grown in step b) at 22 °C for about 16 to about 18 hours, wherein the Clostridial toxin translocation domain is a BoNT/A translocation domain. 4. The intracellular method according to Claim 1, wherein the cell is grown in step a) at 37 °C for about 3.5 hours, wherein the dual expression construct comprises: an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising the exogenous TEV protease cleavage site and wherein the cell is grown in step b) at 22 °C for about 16 to about 18 hours, wherein the Clostridial toxin binding domain is a BoNT/A binding domain.
Patentansprüche 1. Intrazelluläres Verfahren des Konvertierens eines einkettigen Proteins in seine zweikettige Form, wobei das Verfahren die Schritte umfasst: a) Wachsenlassen einer Zelle, die ein duales Expressionskonstrukt umfasst, bei einer ersten Temperatur über einen bestimmten Zeitraum, um maximale Zelldichte zu erzielen, wobei das duale Expressionskonstrukt umfasst: i) einen offenen Leserahmen, der ein einkettiges Protein codiert, das eine Zwei-Ketten-Schleifen-Region umfasst, umfassend eine exogene TEV-Protease-Schnittstelle, und ii) einen offenen Leserahmen, der eine TEV-Protease codiert, wobei die Protease die exogene Proteaseschnittstelle spalten kann, die sich in der Zwei-Ketten-Schleife befindet, b) Wachsenlassen der Zelle bei einer zweiten Temperatur über einen bestimmten Zeitraum, um maximale Induktion von Proteinexpression von dem offenen Leserahmen zu erzielen, der das einkettige Protein codiert, wobei das Wachstum in Schritt b) die Expression des einkettigen Proteins und der Protease von dem dualen Expressionskonstrukt induziert, und wobei die hergestellte Protease das einkettige Protein an der exogenen Proteaseschnittstelle spaltet, die sich in der Zwei-Ketten-Schleifen-Region befindet, wodurch das einkettige Protein in seine zweikettige Form konvertiert wird. 2. Intrazelluläres Verfahren gemäß Anspruch 1, wobei die Zelle in Schritt a) etwa 3,5 Stunden lang bei 37°C wachsen gelassen wird, wobei das duale Expressionskonstrukt umfasst: einen offenen Leserahmen, der ein einkettiges Clostridientoxin codiert, wobei das einkettige Clostridientoxin eine enzymatische Domäne, eine Translokationsdomäne, eine Bindungsdomäne und eine Zwei-Ketten-Schlei-fen-Region umfasst, die die exogene TEV-Protease-Schnittstelle umfasst, und wobei die Zelle in Schritt b) etwa 16 bis etwa 18 Stunden lang bei 22°C wachsen gelassen wird, wobei die enzymatische Domäne des Clostridientoxins eine BoNT/A-enzymatische Domäne ist. 3. Intrazelluläres Verfahren gemäß Anspruch 1, wobei die Zelle in Schritt a) etwa 3,5 Stunden lang bei 37°C wachsen gelassen wird, wobei das duale Expressionskonstrukt umfasst: einen offenen Leserahmen, der ein einkettiges Clostridientoxin codiert, wobei das einkettige Clostridientoxin eine enzymatische Domäne, eine Translokationsdomäne, eine Bindungsdomäne und eine Zwei-Ketten-Schlei-fen-Region umfasst, die die exogene TEV-Protease-Schnittstelle umfasst, und wobei die Zelle in Schritt b) etwa 16 bis etwa 18 Stunden lang bei 22°C wachsen gelassen wird, wobei die Translokationsdomäne des Clostridientoxins eine BoNT/A-Translokationsdomäne ist. 4. Intrazelluläres Verfahren gemäß Anspruch 1, wobei die Zelle in Schritt a) etwa 3,5 Stunden lang bei 37°C wachsen gelassen wird, wobei das duale Expressionskonstrukt umfasst: einen offenen Leserahmen, der ein einkettiges Clostridientoxin codiert, wobei das einkettige Clostridientoxin eine enzymatische Domäne, eine Translokationsdomäne, eine Bindungsdomäne und eine Zwei-Ketten-Schlei-fen-Region umfasst, die die exogene TEV-Protease-Schnittstelle umfasst, und wobei die Zelle in Schritt b) etwa 16 bis etwa 18 Stunden lang bei 22°C wachsen gelassen wird, wobei die Bindungsdomäne des Clostridientoxins eine BoNT/A-Bindungsdomäne ist.
Revendications 1. Procédé intracellulaire de conversion d’une protéine à chaîne unique en sa forme à deux chaînes, le procédé comprenant les étapes qui consistent à : a) faire croître une cellule comprenant une construction d’expression double à une première température pendant une certaine période de temps afin de parvenir à une densité cellulaire maximale, la construction d’expression double comprenant : i) un cadre de lecture ouvert codant pour une protéine à chaîne unique comprenant une région de boucle à deux chaînes comportant un site de clivage par une protéase TEV exogène ; et ii) un cadre de lecture ouvert codant pour une protéase TEV ; dans lequel la protéase peut cliver le site de clivage par la protéase exogène situé dans la boucle à deux chaînes ; b) faire croître la cellule à une seconde température pendant une certaine période de temps afin de parvenir à une induction maximale de l’expression de la protéine à partir du cadre de lecture ouvert codant pour la protéine à chaîne unique, dans lequel la croissance à l’étape b) induit l’expression de la protéine à chaîne unique et de la protéase à partir de la construction d’expression double ; et dans lequel la protéase produite clive la protéine à une seule chaîne sur le site de clivage par la protéase exogène situé dans la région de boucle à deux chaînes, convertissant ainsi la protéine à chaîne unique en sa forme à deux chaînes. 2. Procédé intracellulaire selon la revendication 1, dans lequel la cellule est soumise à une croissance à l’étape a) à 37 °C pendant environ 3,5 heures, dans lequel la construction d’expression double comprend : un cadre de lecture ouvert codant pour une toxine clostridiale à chaîne unique, la toxine clostridiale à chaîne unique comprenant un domaine enzymatique, un domaine de translocation, un domaine de liaison et une région de boucle à deux chaînes comprenant le site de clivage par la protéase TEV exogène et dans lequel la cellule est soumise à une croissance à l’étape b) à22°C pendant environ 16 à environ 18 heures, dans lequel le domaine enzymatique de la toxine clostridiale est un domaine enzymatique BoNT/A. 3. Procédé intracellulaire selon la revendication 1, dans lequel la cellule est soumise à une croissance à l’étape a) à 37 °C pendant environ 3,5 heures, dans lequel la construction d’expression double comprend : un cadre de lecture ouvert codant pour une toxine clostridiale à chaîne unique, la toxine clostridiale à chaîne unique comprenant un domaine enzymatique, un domaine de translocation, un domaine de liaison et une région de boucle à deux chaînes comprenant le site de clivage par la protéase TEV exogène et dans lequel la cellule est soumise à une croissance à l’étape b) à 22 °C pendant environ 16 à environ 18 heures, dans lequel le domaine de translocation de la toxine clostridiale est un domaine de translocation BoNT/A. 4. Procédé intracellulaire selon la revendication 1, dans lequel la cellule est soumise à une croissance à l’étape a) à 37 °C pendant environ 3,5 heures, dans lequel la construction d’expression double comprend : un cadre de lecture ouvert codant pour une toxine clostridiale à chaîne unique, la toxine clostridiale à chaîne unique comprenant un domaine enzymatique, un domaine de translocation, un domaine de liaison et une région de boucle à deux chaînes comprenant le site de clivage par la protéase TEV exogène et dans lequel la cellule est soumise à une croissance à l’étape b) à22°C pendant environ 16 à environ 18 heures, dans lequel le domaine de liaison de la toxine clostridiale est un domaine de liaison BoNT/A.

Claims (2)

  1. fegyián«« fehérjék IfetMneó tormájóvá történő Igénypontok 1, l|ySáíicú £bÍ^é£tAMááC^fáMpt$''^áklt^^'1ktrácbeőlári$ tnédszefe, amely a 'következő tépéseket fogtatja magában: a) egy kettős expre&amp;sziôs konstrukciót tartalmazó sejt növesztése egy első hőmérsékleten bizonyos ideig a maximális sejtsürüség elérése érdekében, ahol az említett. kettős expresszíés konstrukció a kővetkezőket fogtatja magéban;: fi) égy égyláncn fehérjét kódoló nyitott leolvasási keret, arnely égy kétláncu, egy exögéti f EY proteáx hasítási helyet tartalmazd htnokréglöt foglal magában; és ti) egy Ï1V proteáztkődöíé nyitott leölvasásilkereí, ahot a protéóz képes hasítani a kétláncu hi&amp;kon behüt exogén proteáx hasítási helyet; bj a sejt nő vesztése egy második hőmérsékleten egy bizonyos ideig annak érdedben, hogy a fehérje expresszíójának maximális indukeiőját étpk el az egvláneó fehérjét kódold nyílt leolvasási keretről, ahol a (b) lépésben szereplő növesztés indukálja az egytáncó fehérje és a profeaz expresszióját a kettős espe^feifemirulbióM; és ahol M előállott pöteáz az egyliaei fehérjét a kétiahcd hurokrégiéh belüli exogen prolelz hasítási helyén hasítja,, és :¾ .M h iplypöhf szerinti intraeelhdáris módszer, ahol a sejt növesztésére az ajlépésben 3? *Çsoh komíheln! 3,S érán át kerül sor, ahol a kettos expressziős konstrukció a következőket foglalja magában: egy egyláneu elöstddialis toxint kódoló nyitott, leolvasási keret, ahol az egytáncó ciostrídialis toxin egy élzlmatiküs dömént, egy transzlokáoíés domént, egy .kötő: domént és egykétláneii az exogen TEV proíeáz. hasítási helyét. tanaimaM hurokréglöt foglal magában,, és ahol a sejt növesztése a hjlépésben 22 °C-on körülbelül I Ö~18 örán áf történik, és ahol a clostridiaüs toxin enzirnaukus donién egy BoNT/A enxhftatikiís dómén.
  2. 3. Az L igénypont szerinti i«íraeell|f|a.F|s módszer, ahoi a. sejt a) jéplálpt 37 ®<Aön kötxMbeliil 3,5 érán át feerüksor, és ahol a kettős expressziéi lopstrpkclé a következőket foglalja magában: '^'«g>'tó«Éá'0ctódl^teöMnt kódoló nyitott leolvasási keret, ahol az egyllneé fölostrldtalís Äio egy enzimatikus dootéet, egy transziokáciős domént, egy kötő íióniéní és egy kéttánéís, az exogén TW proteâz ItasMsidiielyét tartalmaié bürekrégiót foglal magában, és ahol a sejt növesztése a b) lépésben 22 !>C-on körülbelül 16-18 6rán ál történik, és ahol a elosíndialis toxin tfanszléfcáéiós; dooiépegy Bpb^MÄszIokäc^'iö^n, 4 A?- i. Igénypont szerinti intracelktíáris módszer, ahol a sejbeóvesztéaére az ai) léplsben 37 °0·οη körülbelül 3,6 órát: át kerül sor, és ahol a kettős expressziös konstrukció a ikövetlíezŐket foglalja magában;;: egy égy láncé el ostAdial ja töálpt kódoló pyttott leolvasási keret, ahol az egyjápeü closindiaiis toxin egy enzirnatlkos döméet, egy ftanszlokáelős domépt, egy kötő döípénfés egy két láncé. az éxogén TfíV proteáz hasítási helyét tartalmazó hürokrégiót foglal magában,, ésiáM a sejt növesztése a b) lépésben 22 “C-on körülbelül 16-18 órán át történik, ahoí a elostridjajis toxlnt köti éopiéü egy BoNT/A kötő dómén,
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