WO1996017942A1 - Production de peptides au moyen de proteines de fusion de recombinaison - Google Patents

Production de peptides au moyen de proteines de fusion de recombinaison Download PDF

Info

Publication number
WO1996017942A1
WO1996017942A1 PCT/US1995/015800 US9515800W WO9617942A1 WO 1996017942 A1 WO1996017942 A1 WO 1996017942A1 US 9515800 W US9515800 W US 9515800W WO 9617942 A1 WO9617942 A1 WO 9617942A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
protein construct
seq
solution
peptide
Prior art date
Application number
PCT/US1995/015800
Other languages
English (en)
Other versions
WO1996017942A9 (fr
Inventor
Bruce E. Partridge
Jay S. Stout
Dennis B. Henriksen
Shane D. Manning
Rebecca S. De La Motte
Barton Holmquist
Fred W. Wagner
Original Assignee
Bionebraska, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bionebraska, Inc. filed Critical Bionebraska, Inc.
Priority to EP95942995A priority Critical patent/EP0796335A1/fr
Priority to AU44158/96A priority patent/AU700605B2/en
Priority to JP8517724A priority patent/JPH10510155A/ja
Publication of WO1996017942A1 publication Critical patent/WO1996017942A1/fr
Publication of WO1996017942A9 publication Critical patent/WO1996017942A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • any foreign protein in any microbial host is theoretically possible, the stability of the protein produced often limits such practice and results in a low yield.
  • small foreign proteins and oligopeptides are not easily overproduced in most cellular hosts.
  • Expression of a small peptide in a host cell raises the possibility that the host will assimilate the polypeptide.
  • small peptides have been expressed as fusion proteins with a second larger peptide, such as a peptide marker (e.g., beta- galactosidase or chloramphenicol acetyl transferase) . While the resulting fusion protein may not be readily assimilated, isolation and purification of the desired protein is often not very efficient or effective.
  • a peptide marker e.g., beta- galactosidase or chloramphenicol acetyl transferase
  • the present invention provides a method for the production of a recombinant peptide which employs a fusion protein construct including a carbonic anhydrase and a variable fused polypeptide.
  • the carbonic anhydrase is a mammalian carbonic anhydrase (mCA) .
  • the carbonic anhydrase and the variable fused polypeptide may be linked together by a cleavage site.
  • the cleavage site is an amino acid or sequence of amino acids which results in the protein construct being selectively cleaved on treatment by a cleavage agent.
  • the cleavage agent may be a chemical reagent, e.g.
  • the cleavage agent may be also be an endopeptidase which cleaves the construct at a specific point in relation to a particular amino acid or sequence of amino acids ("an enzymatic cleavage site") .
  • One embodiment of the invention is directed to a method of producing a peptide which includes precipitating the fusion protein construct.
  • the precipitated construct may be resolubilized.
  • the fragment containing the carbonic anhydrase (“carbonic anhydrase fragment”) may be removed by chromatography, filtration or, preferably, by precipitation.
  • a second embodiment provides a method of producing a peptide which includes (i) cleaving the fusion protein construct to produce a soluble carbonic anhydrase fragment and a soluble variable fused polypeptide fragment, and (ii) precipitating the carbonic anhydrase fragment .
  • the carbonic anhydrase fragment includes the carbonic anhydrase and may also include other amino acids residues, e.g., from an interconnecting peptide or a chemical moiety created by the cleavage reaction.
  • the variable fused polypeptide fragment includes at least one copy of a target peptide and may also include additional amino acid residues such as an N-terminal or C-terminal tail sequence or an intraconnecting peptide. Where the variable fused polypeptide fragment includes more than one copy of a target peptide, the target peptides are typically linked by an intraconnecting peptide which includes a cleavage site.
  • a third embodiment of the invention provides a method of producing a peptide which includes expressing the fusion protein construct as part of an inclusion body in a host cell, e.g., an E. coli host cell, and isolating the fusion protein construct.
  • the fusion protein construct typically includes at least 2 copies of a target peptide.
  • the fusion protein construct includes target peptide corresponding to a peptide selected from the group consisting of GRF(l-44) (SEQ ID NO:20), GLPl(7-34) (SEQ ID NO:21) and PTH(l-34) (SEQ ID NO:22)
  • GRF(l-44) SEQ ID NO:20
  • GLPl(7-34) SEQ ID NO:21
  • PTH(l-34) SEQ ID NO:22
  • GRF(1-41) SEQ ID NO:23
  • GLP1 (7-34) SEQ ID NO:21
  • PTH(l-34) SEQ ID NO:22
  • GRF(1-41) SEQ ID NO:23
  • GLP1 (7-34) SEQ ID NO:21
  • PTH(l-34) SEQ ID NO:22
  • the sequences for amino acids 1-44 of growth hormone releasing factor (GRF(l-44) (SEQ ID NO:20)) and amino acids 1-36 of Glucagon-like Peptide 1 (GLPKl-36) SEQ ID NO:24)
  • Another embodiment of the invention provides a method of producing a peptide which includes cleaving the fusion protein construct to produce a carbonic anhydrase fragment and a variable fused polypeptide fragment.
  • the carbonic anhydrase fragment and the variable fused polypeptide fragment are precipitated.
  • the precipitated fragments may be extacted with with a solvent which includes an organic component to recover the variable fused polypeptide fragment.
  • the invention also provides an inclusion body containing the fusion protein construct expressed in a host cell, e.g., an E. coli host cell.
  • the fusion protein construct includes a carbonic anhydrase and a target peptide.
  • the target peptide preferably includes at least one copy of an amino acid sequence corresponding to a peptide selected from the group consisting of GRF(1-41) (SEQ ID NO:23), GLPK7-34) (SEQ ID N0:21) and PTH(l-34) (SEQ ID NO:22) .
  • the invention also provides a fusion protein construct which includes the carbonic anhydrase and a target peptide.
  • the target peptide includes an amino acid sequence corresponding to a peptide selected from the group consisting of GRF(1-41) (SEQ ID NO:23) , GLPK7-34) (SEQ ID NO:21) and PTH(l-34) (SEQ ID NO:22) .
  • FIG. 1 depicts the various formulas for variable fused polypeptides formed of multiple units of target peptides.
  • the expression of a foreign protein by interaction of recombinant vectors with the biosynthetic machinery of host cells or host organisms is a well-known technique for biochemical protein synthesis.
  • the present invention utilizes a novel modification of this expression technique in combination with a carbonic anhydrase-based fusion protein construct to establish a new, low cost, highly efficient method for the large scale biological synthesis of peptides.
  • the method makes it possible to design processes for the isolation and/or purification of a recombinant peptide based solely on chemical methods, i.e. without requiring affinity chromatography or other chromatographic steps.
  • the fusion protein construct also permits the use of a ligand immobilized affinity separation technique if desired. The method eliminates expensive machinery and reagents, long synthetic times, low reaction efficiency and produces fewer faulty copies and a higher yield of the product peptide, than the solid phase peptide synthesis.
  • the method of the invention incorporates a combination of cellular factors and biologic compositions that enable ready expression of foreign proteins by biological systems. Because the expressed fusion protein construct incorporates a binding protein, carbonic anhydrase, subversion of the particular identity of the target peptide by the expression mechanism of the cell or organism is not permitted. Variation in expression efficiency is minimized. The preferred inductive expression mechanism allows production of peptide constructs that might otherwise be toxic to host cells.
  • the method of the invention also incorporates factors that contribute substantially to the efficiency, capacity and yield of the purification technique.
  • the solubility properties of the carbonic anhydrase and fusion construct including the carbonic anhydrase facilitate a clean, complete separation of the variable fused polypeptide from other constituents.
  • the relatively low molecular weight of the carbonic anhydrase permits a high capacity and efficiency per unit weight of the fusion construct. Because the method does not require any special reagents or reactions, the production of undesirable side products is avoided.
  • the size of the fusion protein construct will vary depending on the nature and number of copies of the target peptide.
  • the fusion protein construct is large enough to avoid degradation by the host cell (e.g., at least about 60 to 80 amino acid residues) and not so large that it can not be effectively expressed by the host cell.
  • the fusion protein construct will have a molecular weight of up to about 500,000 although larger constructs are also within the scope of the present invention.
  • the size of the fusion protein construct is chosen such that it may be expressed by the host cell so as to avoid introducing errors in the protein sequence. This places practical limitations on the number of copies of the target peptide present in a given construct . The actual number will vary depending on the size and nature of a particular target peptide within the limitations set by the factors discussed above.
  • the carbonic anhydrase may be derived from a variety of sources. Suitable sources include vertebrates and in particular mammalian sources, e.g., a human carbonic anhydrase (hCA) , a rat carbonic anhydrase, a feline carbonic anhydrase, an equine carbonic anhydrase or a bovine carbonic anhydrase.
  • hCA human carbonic anhydrase
  • a rat carbonic anhydrase e.g., a rat carbonic anhydrase
  • feline carbonic anhydrase equine carbonic anhydrase
  • bovine carbonic anhydrase derived from other mammalian species
  • An example of a suitable carbonic anhydrase is human carbonic anhydrase II ("hCAII"; see, Taylor et al., Biochemistry, 9 . , 2638 (1970)) .
  • the carbonic anhydrase may also be a modified functional version of a carbonic anhydrase.
  • the modified functional version will retain a carbonic anhydrase' s capability of being precipitated as a function of solution conditions, e.g. at a particular pH or as a function of metal ion or salt concentration. Modified versions which can be precipitated from a wide variety of solutions by adjusting the pH of the solution to between about 3.2 and about 6.0, more preferably to between about 3.5 and about 5.5, are preferred.
  • the modified functional version typically also retains the ability of a carbonic anhydrase to strongly bind with the inhibitors, benzenesulfonamide, acetazolamide, or derivatives thereof.
  • a detailed discussion of the binding properties of carbonic anhydrases with respect to sulfanilamide and acetazolamide inhibitors is set forth in International Application No. PCT/US91/04511, which is hereby incorporated by reference.
  • suitably modified carbonic anhydrases include functional substitution mutants which (I) do not contain methionine, (II) have all or some glutamates replaced by another negatively charged amino acid, preferably aspartate, (III) have all or some arginines replaced by another positively charged amino acid, preferably lysine, (IV) have all or some asparagines replaced by another amino acid, preferably glutamine, (V) have methionine replaced by another amino acid, preferably alanine, serine, cysteine, threonine or leucine, or (VI) have cysteine replaced by another amino acid, preferably serine, threonine, leucine or alanine.
  • suitably modified versions of carbonic anhydrase also include polypeptides representing a functional fragment of a carbonic anhydrase such as hCAII, such as but not limited to hCAII C-terminated at cysteine 205, asparagine 231, methionine 240, or leucine 250 or N-terminated at proline 21 or glycine 25.
  • the functional fragment retains the ability of a carbonic anhydrase to be precipitated from a wide range of solutions by adjusting the pH.
  • substitution mutants of hCAII and substitution mutants of the functional fragments of hCAII having the following substitutions: i. hCAII with all or some glutamate amino acid residues replaced by another negatively charged amino acid (AA) , preferably an aspartic acid residue, ii. hCAII with all or some arginine AA residues replaced by another positively charged AA, preferably a lysine residue, iii.
  • hCAII or a functional fragment thereof with one or more of the following modifications to the AA positions at [N11X, G12X] (asparagine glycine) , [N62X, G63X] , [N231X, G232X] , M240X (methionine) , or C205X (cysteine) modified as follows (where those AA positions are present in a given fragment) : the asparagine is changed to glutamine or glycine is changed to alanine, methionine is changed to alanine, serine, cysteine, threonine or leucine, and cysteine is changed to serine threonine, leucine or alanine.
  • the carbonic anhydrase and fusion proteins including the carbonic anhydrase preferably are capable of being precipitated from a wide range of solutions by adjusting the pH of the solution to between about 3.2 and about 6.0, preferably between about 3.5 and about 5.5, and more preferably between about 4.0 and about
  • solutions from which the carbonic anhydrase can be precipitated using this method include solutions containing polyethyleneimine, urea (e.g., at least about 2M) or guanidine hydrochloride (e.g., at least about 2M) .
  • the carbonic anhydrase remains soluble in a wide variety of solutions outside of the pH range of about 3.2 to about 6.0.
  • variable fused polypeptide may have several forms (see e.g., Figure 1) .
  • the intraconnecting peptide usually but not necessarily differs in structure and selectivity from the interconnecting peptide (which links the carbonic anhydrase and the variable fused polypeptide) .
  • the intraconnecting peptide When different, the intraconnecting peptide nevertheless has the same general function as the interconnecting peptide so that two different cleavage agents of an enzymatic or chemical nature will separately cleave the variable fused polypeptide from the carbonic anhydrase and cleave the individual target peptides from each other.
  • the third form is a single unit composed of several (i.e., two or more) identical or different target peptides tandemly interlinked together by innerconnecting peptides.
  • the fourth form is composed of repeating multiple tandem units linked together by intraconnecting peptides wherein each unit contains the same series of different individual target peptides joined together by innerconnecting peptides.
  • the fifth form is composed of a series of tandem units linked together by intraconnecting peptides wherein each unit contains several identical or different target peptides joined by innerconnecting peptides and the target peptides do not repeat from unit to unit.
  • the sixth form is composed of identical multiple tandem units wherein each unit contains several identical target peptides joined by innerconnecting peptides.
  • the target peptide may incorporate all or a portion of any natural or synthetic peptide desired as a product, e.g., any desired protein, oligopeptide or small molecular weight peptide.
  • a peptide includes at least two amino acid residues linked by a peptide bond.
  • Suitable embodiments of the target peptide which may appear as single or multiple linked units in the variable fused polypeptide include caltrin, calcitonin, insulin, tissue plas inogen activator, growth hormone, growth factors, growth hormone releasing factors, erythropoietin, interferons, interleukins, oxytocin, vasopressin, ACTH, collagen binding protein, parathyroid hormone, glucagon like peptide, glucagon, proinsulin, tumor necrosis factor, substance P, brain naturetic peptide, individual heavy and light antibody chains, individual antibody chain fragments especially such as the isolated variable regions (VH or VL) as characterized by Lerner, Science, 246. 1275 et . seq. (Dec.
  • polypeptides having physiologic properties such as sweetening peptides, mood altering polypeptides, nerve growth factors, regulatory proteins, functional hormones, enzymes, DNA polymerases, DNA modification enzymes, structural polypeptides, neuropeptides, polypeptides exhibiting effects upon the cardiovascular, respiratory, excretory, lymphatic, immune, blood, reproductive, cell stimulatory and physiologic functional systems, leukemia inhibitor factors, antibiotic and bacteriostatic peptides (such as cecropins, attacins, apidaecins) , insecticidal, herbicidal and fungicidal peptides as well as lysozymes.
  • physiologic properties such as sweetening peptides, mood altering polypeptides, nerve growth factors, regulatory proteins, functional hormones, enzymes, DNA polymerases, DNA modification enzymes, structural polypeptides, neuropeptides, polypeptides exhibiting effects upon the cardiovascular, respiratory, excretory, lymphatic, immune, blood, reproductive
  • variable fused polypeptides are peptides which include an amino acid sequence having the formula:
  • the target peptide may include an amino acid sequence corresponding to GRF(1-41) (SEQ ID NO:23) , GLPK7-34) (SEQ ID NO:21) or PTH(l-34) (SEQ ID NO:22) .
  • the - (CS1) - and -(CS2)- cleavage sites may be a chemical cleavage site or an enzymatic cleavage site.
  • the enzymatic cleavage site may be recognized by an endopeptidase or by an exopeptidase (e.g., where r is 1 and the -(CS2)- is the C-terminal residue of the construct) .
  • the enzymatic cleavage site is recognized by an endopeptidase.
  • the fusion protein construct may include a chemical cleavage site or an enzymatic cleavage site.
  • the cleavage site or sites which may be incorporated into the fusion protein construct will depend upon the identity of the target peptide (s) present.
  • the cleavage site and target peptide are typically selected so that target peptide does not contain an amino acid sequence corresponding to the cleavage site. Secondary considerations will also influence the choice of a particular cleavage site.
  • the cleavage sites may be designed so as to avoid the use of a enzymatic cleavage reaction.
  • a chemical cleavage site such as a site which may by cleaved after treament with an S- cyanylating agent (e.g, 2-nitro-5-thiocyanatobenzoate) or by treatment with an acid having a pK a of no more than about 3.0.
  • an S- cyanylating agent e.g, 2-nitro-5-thiocyanatobenzoate
  • an acid having a pK a of no more than about 3.0 it may be desireable to employ a cleavage site which permits a modification of the target peptide to introduce a specific functional group, e.g., a C-terminal ⁇ -carboxamide group.
  • Enterokinase (Asp) 4 ys GACGACGACGATAAA (SEQ ID NO:2) (SEQ ID NO:l) Factor Xa IleGluGlyArg ATTGAAGGAAGA (SEQ ID NO: ) (SEQ ID NO:3)
  • Host cells transformed with an expression vector carrying a recombinant fusion protein construct gene may be employed to express the fusion protein construct.
  • the host cell may be a prokaryotic or eukaryotic host cell or a cell in a higher organism.
  • the host cell is a microbial host cell such as E. coli .
  • the vector carrying the protein purification construct gene may be prepared by insertion of the DNA segments coding for the fusion protein construct into an appropriate base vector. Methods for expression of single- and multicopy recombinant fusion protein products are disclosed in International Application No. PCT/US91/04511, the disclosure of which is incorporated herein by reference.
  • the expression vector incorporates the recombinant gene and base vector segments such as the appropriate regulatory DNA sequences for transcription, translation, phenotyping, temporal or other control of expression, RNA binding and post-expression manipulation of the expressed product.
  • Structural features such as a promoter, an operator, a regulatory sequence and a transcription termination signal are generally included in the expression vector.
  • the expression vector may be synthesized from any base vector that is compatible with the host cell or higher organism and provides the foregoing features.
  • the regulatory sequences of the expression vector will be specifically compatible or adapted in some fashion to be compatible with prokaryotic or eukaryotic host cells or higher organisms.
  • Post-expression regulatory sequences, which cause secretion of the polypeptide construct can be included in the eukaryotic expression vector.
  • the expression vector exhibit a stimulatory effect upon the host cell or higher organism such that the polypeptide construct is overproduced relative to the usual biosynthetic expression of the host.
  • the recombinant gene is inserted into an appropriate base vector which is used to transform host cells or higher organisms with the resulting recombinant vector.
  • the fusion protein construct may be expressed within the host cell or higher organism as a soluble product or as a product that is insoluble in the cell .
  • the fusion protein construct may be expressed as a secreted product by the host cell or higher organism.
  • the fusion protein construct may be expressed in a host cell such as E. coli as a part of an inclusion body, i.e., an aggregate of insoluble material .
  • Fusion protein constructs which are expressed as part of an inclusion body typically include two or more copies of a target peptide. It has been found that most single copy fusion protein constructs based on a carbonic anhydrase are expressed as a soluble product. For example, single copy fusion protein constructs which include hCAII and a single copy of a target peptide, such as calcitonin, substance P, angiotension, ATPase-IP, £SU ATPase, and asparagine synthetase, are expressed as soluble cell products in E. coli . Surprisingly, it has been discovered that in some instances, however, a single copy carbonic anhydrase- based fusion protein may be expressed as part of an inclusion body.
  • fusion proteins which include hCAII and a single copy of GRF(1-41) (SEQ ID NO:23) , GLPK7-34) (SEQ ID N0:21) or PTH(l-34) (SEQ ID NO:22) are expressed as a part of an inclusion body in an E . coli host cell such as E. coli BL21 F ' ompT r " B m " B (DE3).
  • the present invention provides a method of producing a recombinant peptide which includes precipitating either the fusion protein construct or a fragment of the fusion protein construct including the carbonic anhydrase (collectively a "CA-based protein”) .
  • hCAII is known to precipitate when a solution at about neutral pH of the enzyme in its native form is acidified. It is believed that lowering the pH leads to a zinc cation being stripped out of the native enzyme and substantial conformational changes resulting in its precipitation.
  • CA-based protein typically may be precipitated from a solution simply by adjusting the pH to between about 3.2 and about 6.0, preferably between about 3.5 and about 5.5, and more preferably between about 4.0 and about 5.0.
  • CA-based proteins may also be precipitated from solutions having a high salt concentration, e.g., from solutions which include at least about 5% (w/v) sodium sulfate or at least about 5% (w/v) ammonium sulfate.
  • a recombinant peptide is isolated by a process which includes precipitating the fusion protein construct.
  • the precipitation step may be carried out with a fusion protein construct which has been expressed as a soluble product or with a fusion protein construct which has been expressed as a part of an inclusion body. In the latter instance, the inclusion body is dissolved prior to the precipitation step, e.g., in a solution which includes at least about 0.5M citric acid.
  • the fusion protein construct may also be dissolved in a variety of other solutions, such as a solution containing a chaotropic agent (e.g., guanidine hydrochloride) or a detergent (e.g., SDS) , a solution containing at least about 2 M urea or a solution having a pH of at least about 10.0.
  • a chaotropic agent e.g., guanidine hydrochloride
  • a detergent e.g., SDS
  • the fusion protein construct may be precipitated from the above solutions by adjusting the pH of the solution to between about 3.2 and about 6.0 and preferably to between about 3.5 and about 5.5.
  • the efficiency of the precipitation may be enhanced in some cases by adding a salt to the solution in addition to adjusting the pH.
  • Suitable salts include sodium chloride, potassium chloride, manganese sulfate, sodium sulfate, sodium acetate, and lanthanum chloride.
  • divalent metal ions such as Mg 2* , Ca 2+ , Zn 2 *, Sr 2* or Co 2* together with the pH adjustment may also enhance the efficiency of the precipitation.
  • solubilized fusion protein construct is precipitated from an alkaline solution
  • the pH of the solution is typically adjusted by adding an acid such as acetic acid, formic acid, citric acid, phosphoric acid, and hydrochloric acid.
  • the solubilized fusion protein construct may be also precipitated from solution simply by the addition of a sufficient amount of a salt such as sodium sulfate or ammonium sulfate. In some instances, it may be useful to remove cell debris and nucleic acid material from a crude cell lysate as an initial step in the isolation of a recombinant peptide.
  • PEI polyethyleneimine
  • the precipitated fusion protein may be resolubilized, e.g., by dissolution in a solution having a pH of at least about 10, and preferably at least about 10.5.
  • the precipitated fusion protein may also be redissolved in other types of solutions, such as solutions which include a chaotropic agent, a detergent or at least about 0.5 M citric acid.
  • the fragment containing the carbonic anhydrase (“carbonic anhydrase fragment") may be removed by chromatography, filtration, or preferably, by precipitation.
  • the carbonic anhydrase fragment may be precipitated simply by again adjusting the pH of the solution to between about 3.5 and about 6.0.
  • the carbonic anhydrase fragment may be separated from the cleavage products using a ligand immobilized affinity separation technique.
  • the carbonic anhydrase fragment in native or renatured form, may be removed by contacting a solution of the cleavage products with an inhibitor (e.g., an affinity column) which includes benzenesulfonamide compound or an acetazolamide compound.
  • an inhibitor e.g., an affinity column
  • the benzenesulfonamide compound includes a sulfanilamide compound such as an amino substituted benzenesulfonamide (e.g., 4-amino- benzenesulfonamide) or a derivative thereof.
  • the acetazolamide compound is 5-acetamido-l, 3 ,4-thiadiazol- 2-sulfonamide or a derivative thereof.
  • the method of producing a recombinant peptide includes (i) cleaving the fusion protein construct to produce a soluble carbonic anhydrase fragment and a soluble variable fused polypeptide fragment, and (ii) precipitating the carbonic anhydrase fragment.
  • the carbonic anhydrase fragment may include other amino acids residues in addition to the carbonic anhydrase. These other amino acid residues typically are derived from an interconnecting peptide present in the fusion protein construct. Suitable interconnecting peptides for use in the present invention include amino acid sequences containing a chemical or enzymatic cleavage site.
  • the carbonic anhydrase fragment typically includes at least a portion of the interconnecting peptide or a derivative thereof.
  • the derivative generally is produced as a byproduct of the cleavage reaction.
  • a carbonic anhydrase fragment having a C-terminal homoserine residue may be created by cleavage of a fusion protein construct at the C-terminal peptide bond of a methionine residue.
  • the supernatant fraction remaining after the precipitation of the carbonic anhydrase fragment may be isolated by centrifugation.
  • the supernatant fraction contains the variable fused polypeptide fragment and, if desired, the variable fused polypeptide fragment purified further by conventional methods used for the isolation of peptides.
  • the precipitated carbonic anhydrase fragment may be extracted with a solvent which includes an organic solvent.
  • suitable solvents include an organic component such as acetonitrile, propanol, citric acid, polyethyleneglycol, or mixtures thereof.
  • aqueous solutions including an organic solvent e.g., 50% aqueous acetonitrile and 50% aqueous n-propanol, may be used to carry out the extraction.
  • the fusion protein construct Prior to being subjected to the cleavage reaction, the fusion protein construct may be purified by a variety of methods.
  • the fusion protein construct may be purified using a ligand immobilized affinity separation technique or by precipitation. Purification of the fusion protein construct by precipitation is preferred as it allows the recombinant fusion protein construct to be produced by a process which relies solely on wet chemical separation processes such as precipitation, filtration or centrifugation without the need for renaturation or affinity purification.
  • a third embodiment of the invention provides a method of producing a recombinant peptide which includes expressing the fusion protein construct as a part of an inclusion body in an E. coli host cell, and isolating the fusion protein construct.
  • the fusion protein construct typically includes multiple copies of a target peptide.
  • suitable single copy fusion proteins which may be expressed as inclusion bodies in an E.
  • coli host cell are inclusion fusion proteins which include hCAII and a single copy of any one of GRF(1-41) (SEQ ID NO:23) , GRF(l-44) (SEQ ID NO:20) , GLPKl-34) (SEQ ID NO:26) , GLPK7-34) (SEQ ID NO-21) , GLPK7-36) (SEQ ID NO:27) , GLPK7-37) (SEQ ID NO:28) , PTH(l-34) (SEQ ID NO:22) , PTH(l-38) (SEQ ID NO:29) and PTH(l-84) (SEQ ID NO:25) .
  • the inclusion bodies may be isolated from a crude cell lysate by conventional techniques, e.g., by centrifugation.
  • the crude inclusion bodies may be subjected to an initial purification step such as washing the inclusion bodies with a 50mM Tris, ImM EDTA, pH 7.8 solution or a lOOmM EDTA solution to remove any cell debris and/or nucleic acid materials present.
  • the inclusion bodies may be dissolved under a variety of conditions.
  • the inclusion bodies may be dissolved under acidic conditions in a solution having a pH of no more than about 3.2 (e.g., in a solution which includes at least about 500mM citric acid) .
  • the inclusion bodies may be dissolved in a solution which has a pH of at least about 10.
  • the dissolution of the inclusion bodies under basic conditions may be facilitated by the addition of a small amount of a surfactant, such as N-lauroyl sarcosine, to the solution.
  • a surfactant such as N-lauroyl sarcosine
  • the inclusion bodies may also be dissolved in a solution which includes an effective amount of a chaotropic agent such as 6N HCl or urea, or a detergent such as N-lauroyl sarcosine, sodium dodecyl sulfate, sodium octyl sulfate or cetyl trimethyl ammonium bromide (CTAB) .
  • a chaotropic agent such as 6N HCl or urea
  • a detergent such as N-lauroyl sarcosine, sodium dodecyl sulfate, sodium octyl sulfate or cetyl trimethyl ammonium bromide (CTAB) .
  • the solubilized fusion protein construct may be precipitated by adjusting the pH of the solution to between about 3.5 and about 6.0, and preferably to between about 4.0 and about 5.0.
  • the solubilized fusion protein construct may be isolated by a process which includes a ligand immobilized affinity separation technique.
  • Another embodiment of the invention includes cleaving the fusion protein construct to produce a carbonic anhydrase fragment and a variable fused polypeptide fragment and precipitating both the carbonic anhydrase fragment and the variable fused polypeptide fragment.
  • the precipitated fragments may be extacted with a solvent which includes an organic component to recover the variable fused polypeptide fragment.
  • Suitable solvents include an organic component such as acetonitrile, propanol, citric acid, polyethyleneglycol, or mixtures thereof.
  • Example 1 Description of the Expression System Origin, phenotype and genotype of the host cells.
  • E. coli BL21 F “ ompT r " B m ' B was obtained from Novagen, Inc., Madison, WI . These E. coli cells gave high levels of expression of genes cloned into expression vectors containing the bacteriophage T7 promoter.
  • Bacteriophage (DE3) which contains the T7 RNA polymerase gene has been integrated into the chromosomal DNA of the BL21 (DE3) cells. The T7 RNA polymerase gene is controlled by the lacUV5 promoter and the lad gene product.
  • Plasmid pET31FlmhCAII contains the coding region for hCAII (human carbonic anhydrase II) downstream of a bacteriophage T7 promoter in a pUC-derived plasmid backbone.
  • Plasmid pAl was digested with the restriction endonucleases Ssp I and BspE I and the resulting ends were made blunt by treatment with T4 DNA polymerase.
  • the DNA fragment from the pAl digest containing the T7- hCAII-cassette was subcloned into the Sea I restriction site of pBR322 (New England Biolabs) thus conferring tetracycline resistance, but not ampicillin resistance.
  • the resulting plasmid was designated pBNl.
  • the pAl plasmid was opened at the Hind III site and the EcoR V site and the synthetic oligonucleotide, 5' -A GCT GAA TTC AAC GTT CTC GAG GAT -3' (SEQ ID NO: 9) and its complementary sequence 5' -ATC CTC GAG AAC GTT GAA TTC-3' (SEQ ID NO:10) , were cloned into the vector.
  • the insertion of these oligonucleotides provides a T7-hCAII- cassette containing unique EcoR I and Xho I restriction sites at the carboxyl terminal of hCAII.
  • the resulting plasmid was designated pA3.
  • the pBNl vector was digested with EcoR I and the single stranded overhangs were filled in with Polymerase I Large (Klenow) Fragment.
  • the linear plasmid with newly formed blunt ends was religated, thus destroying the EcoR I site.
  • the resulting plasmid was designated pBN3.
  • Plasmid pA3 was digested with the restriction endonucleases, Xba I and BspE I.
  • the DNA fragment from the pA3 digest containing the T7-hCAII-cassette was subcloned into the pBNl vector which had been digested with Xba I and BspE I .
  • the resulting vector was designated plasmid pBN4.
  • Example 2 Inoculum Preparation for hCA-Fusion Constructs L-broth was sterilized in the autoclave at 121°C for 20 minutes on the liquid cycle setting. The glucose and tetracycline stocks were filter sterilized by passage of the solution through a 0.22 ⁇ m filter. Two 250 ml shake flasks were each charged with the following solutions:
  • the media contained the following: 1200.0 g Case amino acids; 300.0 g Yeast extract; 30.0 g NaCl; and 0.10 ml
  • the fermentor was sterilized at 121°C for 25 minutes. The fermentor was cooled to 37°C. Before inoculation, the following solutions were added to the fermentor: glucose (480.0 g in 800.0 ml H 2 0) magnesium (120.0 g MgS0 4 -HO in 250.0 ml H 2 0) phosphates (120.0 g K 2 HP0 4 & 465.0 g KH 2 P04 in
  • the media feed was started.
  • the media feed consisted of 1200.0 g case amino acids and 300.0 g yeast extract dissolved in 5.0 L distilled H 2 0 and sterilized for 20 minutes on liquid cycle .
  • the media feed was added to the fermentor over 1.0-1.5 hours.
  • the fermentation was induced by adding the following solutions to the fermentor: isopropylthiogalactoside (IPTG; 28.8 g in 200 ml distilled H 2 0) ; ZnCl 2 (0.818g in 50 ml distilled H 2 0 with one drop of 6N HCl) .
  • the IPTG solution was filter sterilized through 0.22 ⁇ m filter.
  • the ZnCl 2 solution was sterilized for 20 minutes using the liquid cycle in the autoclave.
  • the concentration of IPTG in the fermentor was 2.0 mM and the concentration of ZnCl 2 was 100 ⁇ .
  • a feed of a mixture of amino acids was then started at this point.
  • the amino acid feed consisted of 225.0 g L-serine; 75.0 g L-tyrosine; 74.0 g L-tryptophan; 75.0 g L-phenylalanine; 75.0 g L-proline; and 75.0 g L-histidine; and was dissolved in a mixture of 1.5 L H 2 0 and 500 ml concentrated HCl.
  • the amino acid feed was sterile filtered through a 0.22 ⁇ m filter prior to addition to the fermentation. Induction was allowed to continue for 2.0 hours at which point the fermentation broth was transferred to a harvest tank and chilled to approximately 5-10°C. The fermentation typically yielded between 6.5 to 9.5 kg of wet cell paste (dry cell weight of about 1.0-1.5 kg) .
  • Example 4 Harvest of 60 L Fermentation
  • the cell suspension from the fermentor as desribed above was concentrated over a tangential crossflow membrane to a volume of 10 L.
  • the concentrated cell suspension was diafiltered and washed with 30 L of a cold wash buffer containing 50 mM Tris-S04 pH 7.8, 1.0 mM EDTA, and 0.10 mM phenylmethylsulfonyl fluoride
  • the cell suspension was then concentrated to 8 L. The concentration and washing of the cell suspension typically required 6-10 hours. At this point, the concentrated cell suspension (cell paste) may be bagged and frozen for later processing or transferred to homogenizer holding tanks for cell lysis.
  • the cell paste obtained from the 60 L fermentor was diluted to 32 L in cold wash buffer (see above) and the resulting cell suspension was chilled to 5-10°C.
  • the chilled cell suspension was homogenized at 12,000 psi with a Galin high pressure homogenizer.
  • the homogenized cell paste was passed through a heat exchanger to chill the lysate to 10°C and passed through the homogenizer a second time.
  • Example 6 Preparation of Thrombin Stock Solution
  • the thrombin used in fusion protein cleavage reactions was obtained from Calbiochem in the form of a lyophilized powder. This powder was solubilized prior to use by dissolution in MilliQ water to a concentration of 1 mg/ml as determined by specific activity.
  • the preparation of the DNA segment coding for a single copy PTH(l-34) (SEQ ID NO:22) fusion protein was carried out by preparing an expression vector coding for a PTH-construct which included a DNA segment coding for the hCAII, an interlinking peptide, and PTH(1-34) (SEQ ID NO:22) .
  • the following oligos were obtained from Operon Technologies Inc, 1000 Atlantic Ave. Alameda CA 94501. Oligo 1: 5' CCC AAG CTT CTG TTC GTG GTC CGC GTT CTG
  • Oligo 3 5' TCC AGC TGA TGC ACA ACC TGG GTA AAC ACC
  • Oligo 8 5' CCG GAT ATC TTA GAA GTT GTG AAC GTC CTG
  • CAG (SEQ ID NO:18) The eight synthetic DNA oligos were obtained and the complementary strands phosphorylated and annealed. The four double stranded fragments were used to prepare a DNA fragment coding for the interpeptide linker followed by PTHd-34) (SEQ ID NO:22) . This DNA fragment was digested and inserted into pAl using the restriction sites Hind III and EcoR V creating the vector pAl:PTH(l- 34) .
  • the expression cassette was removed from pAl:PTH(l- 34) by digestion with the restriction endonucleases Xba I and BspE I and inserted into the vector pBNl which had been digested with the same restriction endonucleces. This final vector was desiganted pBNl :PTH (1-34) . E. coli cells transformed with the vector pBNl:PTH(1-34) containing DNA coding for an hCA-PTH fusion protein were prepared, cultured and lysed according to the procedures described above.
  • the hCA- PTH fusion protein included hCAII linked to an amino acid sequence corresponding to PTH(1-34) (SEQ ID NO:22) through a thrombin cleavage site (Gly-Pro-Arg) immediately adjacent the N-terminus of the PTH(1-34) (SEQ ID NO:22) sequence.
  • the hCA-PTH fusion protein was expressed as inclusion bodies in E. coli BL21 F " ompT r B m " B (DE3) cells.
  • the cell lysate from the 60 L fermention run was centrifuged in a continuous flow rotor at 20,000 g at 400 ml/min.
  • the pellet contained 300-500 g of crude inclusion bodies.
  • the crude hCA-PTH inclusion bodies was suspended in 2M citic acid at a concentration of 60 mg solids/ml (4-8 liters) and sonicated at 70% power with a 50% pulse rate.
  • the solubilized inclusion bodies were clarified by centrifugation at 20,000 g.
  • the hCA-PTH fusion protein in the supernatant was precipitated by the addition of 10 N NaOH until the pH reaches 4-5.
  • the precipitated hCA-PTH fusion protein was collected by centrifugation at 20,000 g.
  • the precipitated hCA-PTH fusion protein was then washed with 4 to 8 liters of 5% acetic acid/45% EtOH in Milli-Q water and the precipitated hCA-PTH fusion protein was again collected by centrifugation.
  • the hCA-PTH fusion protein was then washed twice with 4 to 8 liters of 100 mM EDTA and once with 4-8 liters of Milli Q water and the protein collected by centrifugation after each wash.
  • a solution containing 200 ml of 50mM NaOH and 0.25% N-lauroyl sarcosine is added to a bottle containing a pellet of the hCA-PTH fusion protein.
  • the bottles are placed into 37°C water bath to warm pellets (typically for 2-5 minutes) to aid in the solubilization of the pellet.
  • the material is homogenized until all large pieces are disaggregated.
  • the pH is readjusted 11.6 to 11.9 with a solution containing 50mM NaOH and 0.25% N- lauroyl sarcosine.
  • the resulting solution is sonicated until all of the hCA-PTH fusion protein pellet has dissolved.
  • the protein concentration of the reaction solution is determined by absorbance at 280 nm. Ideally the concentration should be 6-7 mg/ml (8-10 liters for a 60 1 fermentation) . If the concentration is greater than 9 mg/ml, the solution is diluted to 6-7 mg/ml with the 50mM NaOH/0.25% N-lauroyl sarcosine solution. The protein solution is stirred vigorously and the pH is adjust to 8-8.2 with 1M Tris-HCl. If solution is hazy, it is filtered through glass fiber filters.
  • the solution is then filtered through 0.45 ⁇ m cellulose acetate membrane and the protein concentration is determined by measuring the absorbance at 280 nm.
  • Solid NaCl is added to a final concentration of 250mM.
  • a thrombin stock solution 1 mg/ml is added to produce a protein to enzyme ratio of 5000:1 w/w.
  • the solution is then stirred in a water bath plate at 37°C.
  • the reaction is monitored by HPLC on a C8 column eluted with a gradient from Buffer A (95% water, 5% acetonitrile (ACN) , 0.1% trifluoroacetic acid (TFA) ) to Buffer B (95% ACN, 5% water, .1% TFA) .
  • Buffer A 95% water, 5% acetonitrile (ACN) , 0.1% trifluoroacetic acid (TFA)
  • Buffer B 95% ACN, 5% water, .1% TFA
  • reaction is complete as determined by the loss of the peak corresponding to the starting material in 46-48 hours.
  • the PMSF is dissolved into 95% EtOH, and added directly to the reaction mixture with vigorous stirring.
  • the thrombin cleavage procedure yields 5.1 g of PTH(1-34) (SEQ ID NO:22) from 60 g of hCA-PTH fusion protein.
  • Both the resulting human carbonic anhydrase (from the cleavage of the hCA-PTH fusion protein) and remaining unhydrolized fusion protein are precipitated from the solution by adding sufficient citric acid to achieve 150mM, leaving the desired peptide (PTH 1-34) (SEQ ID NO:22) in solution.
  • the precipitated material is removed by centrifugation.
  • the supernatant is filtered through 0.45 ⁇ m filter and frozen at -80°C or desalted directly on a C8 column.
  • the yield from the peptide from the step is 85%.
  • the PTH (1-34) may be further purified by chromatography on preparative C8 column using a gradient from 10-70% Buffer B of aqueous acetonitrile/ trifluoroacetic acid (ACN/TFA) buffers, where Buffer A: 0.1% TFA, 95% water, 5% acetonitrile, Buffer B:0.1% TFA, 5% water, 95% acetonitrile.
  • the PTH(l-34) (SEQ ID NO:22) sample may be purified on the preparative C8 column eluting first with a gradient from 10-70% Buffer B; where Buffer A is 5mM HCl, 95% water, 5% acetonitrile and Buffer B.
  • the PTH(l-34) (SEQ ID NO:22) is desalted with a gradient where Buffer A lOmM acetic acid, and Buffer B i- 10 mM acetic acid in 50% aqueous ethanol.
  • Oligopair 1&2 were inserted into pUC19 (commercially available from
  • Oligopair 3&4 was then inserted into this vector between
  • Oligopair 5&6 was ligated into pBluescript IISK+
  • the fragment containing oligopair 5&6 was inserted into pUC19:GLP(7-22) by digesting pUC19 :GLP (7-22) with Nar 1 and EcoR I and digesting PB5 :GLP(21-34)AFA with Cla I and EcoR I and ligating the fragment from PB5:GLP(21- 34)AFA containing oligos 5&6 into the truncated pUC19:GLP(7-22) to yield pUC19 :GLP (7-34)AFA.
  • pUC19:GLP(7-34)AFA was digested with Hind III and the fragment containing the sequence coding for the peptide interlinker followed by GLP1 (7-34) -Ala-Phe-Ala (SEQ ID NO:30) ("GLP1 (7-34)AFA") was transferred to pA4 to yield vector pA4 :GLP (7-34)AFA.
  • the hCAII gene in pA4 had been mutated to change the methionine-240 to a cysteine.
  • the expression cassette was then digested from pA4:GLP(7-
  • the hCA-GLPl-AFA fusion protein included hCAII linked to an amino acid sequence corresponding to GLP1 (7-34) -Ala-Phe-Ala (SEQ ID NO:30) through a methionine residue located immediately adjacent the N-terminus of the GLP1 (7-34) -Ala-Phe-Ala (SEQ ID NO:30) sequence.
  • the hCA-GLPl-AFA fusion protein was expressed as inclusion bodies in E. coli BL21 F " ompT r B m ' B (DE3) cells.
  • the cell lysate was centrifuged in a continuous flow rotor at 20,000 g at 400 ml/min to produce a pellet containing 300-500 g of crude inclusion bodies.
  • the crude hCA-GLPl-AFA inclusion bodies were suspended in lysis buffer at a concentration of 25% solids (1.2-2.0 L) and homogenized in the Galin homogenizer at 13,000 psi .
  • the homogenized inclusion bodies were collected by centrifugation at 20,000 g, resuspended in a solution of 100 mM Na Acetate pH 4.5 and homogenized a second time.
  • the homogenized inclusion bodies were then suspended in distilled water.
  • the resulting suspension is homogenized and the inclusion bodies collected by centrifugation.
  • the purified inclusion bodies are dissolved in 1.5 M citric acid and clarified by centrifugation.
  • the hCA-GLPl-AFA fusion protein was precipitated by the addition of NaOH until the pH is 4-5 and isolated by centrifug
  • the hCA-GLPl-AFA inclusion bodies were dissolved in a solution of 2.0 M Citric Acid at a concentration of about 35-45 mg/ml.
  • the solution was adjusted pH to 1.0 with cone.
  • HCl and 0.2 g cyanogen bromide (CNBr) per g of total protein was added.
  • the protein concentration was determined by HPLC.
  • the protein solution was sparged with pure Argon before addition of the CNBr.
  • the cleavage reaction was allowed to proceed under an Argon atmosphere for 4-5 hours at which time residual CNBr was neutralized by adding 2.2g D,L-methionine per g of CNBr added followed by stirring for 30 minutes.
  • the solution from the CNBr cleavage reaction was diluted 1:1 with a solution of 10% sodium sulfate (w/v) in water to precipitate all protein and peptide from the solution.
  • the sample was centrifuged at 20,000 x g for 15 minutes and the supernatant decanted. Distilled water (200 ml) was added to each centrifuge vial. The resulting mixture was homogenized and centrifuged for 15 min. at 20,000 x g. The supernatant was decanted and a volume of 50% acetonitrile (ACN) was added to the pellet material.
  • ACN 50% acetonitrile
  • the extraction with the 50% acetonitrile solution was repeated two to three times to achieve a 90-95% recovery of GLPl (7-34)AFA (SEQ ID NO:30) in the acetontrile solution.
  • the extraction may also be carried out with 50% n-propanol .
  • the 50% ACN solution is diluted to 12.5% ACN with distilled H 2 0 to facilitate the binding of GLPl (7-34) -AFA (SEQ ID NO:30) to the C8 resin.
  • the peptide solution is loaded onto the C8 resin in an low pressure column and the column is washed with 5 column volumes of 30% (v/v) ACN solution.
  • the GLPl (7-34) -AFA (SEQ ID NO:30) is then eluted with a solution of 50% ACN in distilled H 2 0.
  • the 50% ACN solution is lyophilized to obtain the GLPl (7- 34) -AFA (SEQ ID NO:30) .
  • GLPl (7-34)AFA (SEQ ID NO:30) (1 mmole) is dissolved in a 50/50 (v/v) solution of DMF/H 2 0 which includes 5 mM
  • the GLPl (7-36) -NH 2 may be further purified by chromatography on preparative C8 column using a gradient of aqueous acetonitrile/trifluoroacetic acid (ACN/TFA) buffers.
  • ACN/TFA aqueous acetonitrile/trifluoroacetic acid
  • Example 9 Production of GRF(1-44)NH 7 (SEQ ID NO:20) Eight oligonucleotides containing segments of the linker and the peptide were phosphorylated and complementary oligonucleotide pairs 1&2, 3&4, 5&6, and 7&8 were annealed. Oligonucleotide pairs l ⁇ -2 and 3&4 were simultaneously ligated into the pTZ19R vector (commercially available from Pharmacia Biotech Inc., NJ) between the Hind III and Sal I sites to yield pTZ:GRF(l- 29) .
  • pTZ19R vector commercially available from Pharmacia Biotech Inc., NJ
  • Oligonucleotide pairs 5&6 and 7&8 were simultaneously ligated into a separate pTZ19R vector between the Sal I and EcoR I sites to yield pTZ:GRF(29- 44)A.
  • the gene fragments were cloned adjacent to each other in a single vector by digesting pTZ:GRF(1-29) and pTZ:GRF (29-44)A with the restriction endonucleases Xmn I and Sal I, isolating the 1.9 kb band from the pTZ:GRF(l- 29) vector and the 0.9 kb band of the pTZ :GRF (29-44)A vector and ligating them together to yield the vector pTZ:GRF(l-44)A.
  • the pA2 vector was digested with EcoR V.
  • pTZ:GRF(l-44)A was digested with Dra I and EcoR V.
  • the fragment containing the GRF gene and the linearized pA2 plasmid were ligated together to yield pBN2 :GRF(1-44)A.
  • E. coli cells transformed with vector pBN2:GRF(l- 44)A containing DNA coding for an hCA-GRF fusion protein were prepared, cultured and lysed according to the procedures described above.
  • the hCA-GRF fusion protein included hCAII linked to an amino acid sequence corresponding to GRF (1-44) -Ala (SEQ ID N0:31) through an interconnecting peptide.
  • the interconnecting peptide included a thrombin cleavage site and an enterokinase cleavage site.
  • the enterokinase cleavage site (Asp-Asp- Asp-Asp-Lys) (SEQ ID NO:2) was positioned immediately adjacent the N-terminus of the GRF(l-44) -Ala (SEQ ID NO:31) sequence.
  • the thrombin cleavage site (Gly-Pro- Arg) was located so that after treatment with thrombin, the hCA-GRF fusion protein produced a peptide fragment having an 8 amino acid sequence (Ala-Met-Val-Asp-Asp- Asp-Asp-Lys) (SEQ ID NO:19) connected to the N-terminus of the GRF (1-44) -Ala (SEQ ID N0:31) sequence.
  • the hCA- GRF fusion protein was expressed as inclusion bodies in E. coli BL21 F " ompT r " B m " B (DE3) cells.
  • the cell lysate was centrifuged in a continuous flow rotor at 20,000 g at 400 ml/min to produce a pellet containing 300-500 g of crude inclusion bodies.
  • the crude hCA-GRF-Ala inclusion bodies were suspended in 2M citic acid at a concentration of 60 mg solids/ml (5-9 L) and sonicated at 70% power with a 50% pulse rate.
  • the solubilized inclusion bodies were clarified by centrifugation at 20,000 g.
  • the hCA-GRF-Ala fusion protein in the supernatant was precipitated by the addition of 10 N NaOH until the pH reaches 4-5.
  • the precipitated hCA-GRF-Ala fusion protein was then collected by centrifugation at 20,000 g. After washing with 5% acetic acid/ 45% ethanol in Milli-Q H 2 0, the precipitated hCA-GRF-Ala fusion protein was again collected by centrifugation. The precipitated hCA-GRF- Ala fusion protein was then resuspended twice in 100 mM EDTA and the fusion protein was collected by centrifugation. The washed, precipitated hCA-GRF-Ala fusion protein was suspended in distilled H 2 0. The suspension was homogenized and the hCA-GRF-Ala fusion protein was collected by centrifugation.
  • a 200 ml portion of a solution containing 50mM NaOH and 0.25% N-lauroyl sarcosine was added to a bottle containing a pellet of the hCA-GRF-Ala fusion protein.
  • the bottle was placed into 37°C water bath to warm the pellet.
  • the NaOH solution was swirled around in the bottle to remove all solid material from the bottle sides.
  • the slurry was then poured into a suitable size beaker.
  • the material was homogenized until all large pieces are disaggregated and the pH was monitored. The pH was readjusted to between 11.6 and 11.9 with a solution of 50mM NaOH and 0.25% N-lauroyl sarcosine.
  • the solution was sonicated until all of the inclusion body pellet has dissolved and solution is clear. Sonication times varied according to size of batch. Typically anything over 1 liter was sonicated for 2 minutes, power 10, pulser on, 70% duty cycle (smaller batches required less time) .
  • the protein concentration of the reaction solution was measured. If concentration was greater than 9 mg/ml, the solution was diluted with the 50mM NaOH/0.25% N-lauroyl sarcosine solution to a protein concentration of 6-7 mg/ml. The protein solution was stirred vigorously, and the pH adjusted to 8-8.2 with 1M Tris- HC1. If the solution was slightly hazy at this point, the solution was clarified by filtering through glass fiber filters. The clarified solution was sterile filtered through a 0.45 ⁇ m cellulose acetate membrane. The protein concentration was determined by measuring the absorbance at 280 nm. Sufficient NaCl to produce a 250mM NaCl concentration was added with vigorous stirring.
  • thrombin stock solution (1 mg/ml) to produce a protein to enzyme ratio of 5000:1 was added and the resulting mixture stirred in a water bath on top of stir plate at 37°C.
  • the reaction was monitored by HPLC on a C8 column eluted with a gradient of H 2 0/ACN/TFA buffers.
  • the reaction was stopped by the addition of PMSF to a final concentration of 0. ImM when the fusion construct peak was essentially gone (usually 46-48 hours) .
  • the PMSF was dissolved into 95% EtOH and added directly to the reaction mixture with vigorous stirring.
  • Citric Acid Precipitation of the hCA-Fragment The human carbonic anhydrase fragment from the cleavage of the fusion protein and any residual uncut fusion protein were removed from solution by precipitation with 90mM citric acid, leaving the intermediate peptide, Ldr-GRF (1-44 ) -Ala, in solution.
  • the volume of the thrombin cut solution was measured in a graduated cylinder. A stock solution of 1M citric acid was added to final concentration of 90mM citrate. The addition was made slowly while with vigorously stirring the solution. The aggregated material was centrifuged and the supernatant was filtered through a 0.45 ⁇ m filter. The filtered supernatant was either frozen at -80°C or run directly onto a C8 column for desalting.
  • the pellets from the precipitation were washed with a solution of 90 mM citrate pH 3.0-3.1. Between 150-200 ml of citrate solution was added to each pellet of 1-2 grams of hCA. The pellets were homogenized and the supernatant from the wash was filtered through a 0.45 ⁇ m filter and saved. The washed pellets were discarded. The supernatant was either frozen at -80°C or desalted directly on a C8 column.
  • the 90 mM citric acid solution of Ldr-GRF (1-44) -Ala was loaded directly onto a preparative C8 column.
  • the column was eluted with a gradient of aqueous ethanol/acetic acid buffers.
  • the fractions containing the Ldr-GRF (1-44) -Ala were collected (typically 90-95% pure) and the solution concentrated by evaporation to 21-25 mg/ml.
  • the resulting solution was stored at -80°C prior to transpeptidation.
  • the concentration Ldr-GRF (1-44) -Ala peptide was determined by C8 HPLC using 1 mM GRF standard.
  • the peptide solution was diluted to a concentration of 3 mM Ldr-GRF(1-44) -Ala (18.0 mg/ml) with water.
  • EDTA and sodium phosphate were added (to 5 mM; 1.9 mg/ml and 25 mM; 3.6 mg/ml respectively) and the pH was adjusted to 6.0 with 1 M NaOH.
  • 2-Nitrophenylglycinamide [ONPGA] 250 mM; 89.0 m g/ml was added and the pH was again adjusted to 6.0 with 1 M NaOH.
  • Carboxypeptidase-Y (“CPD-Y”; 2 ⁇ l/ml) was the added and stirring was in the dark at 35-40 °C. The extent of reaction to produce Ldr-GRF-ONPGA was monitored by HPLC. After about 1-2 hours the reaction was stopped by the addition of acetonitrile to a final concentration of 15% v/v.
  • the sample from the CPD-Y transpeptidation was loaded onto a C8 HPLC column and rinsed with a 20% ethanol, 10 mM sodium phosphate, pH 6.8 buffer to remove unreacted ONPGA.
  • the column was then eluted with a 50% EtOH, 10 mM Sodium Phosphate, pH 6.8 buffer and the Ldr- GRF-ONPGA peak was collected.
  • the concentration of the Ldr-GRF-ONPGA peptide was determined by C8 HPLC using 1 mM GRF standard and the concentration was diluted to 1 mM Ldr-GRF-ONPGA (6.0 mg/ml) with 50% ethanol.
  • Sodium bisulfite (to 5 mM) and sodium benzoate (to 50 mM ) were added and the solution is adjusted to pH 9.5 with 1 M NaOH.
  • the mixture was irradiated (wavelength >305 nm; 200-210 W medium pressure mercury lamp) while being maintained in a 20-25 °C circulating water bath. During the reaction a constant flow of Argon was maintained and the reaction was constantly stirred. The reaction was monitored by HPLC. After about 1-2 hours reaction was stopped by lowering the pH to 5.5 with acetic acid.
  • Ldr-GRF-NH2 Enterokinase Cleavage Protocol
  • the solution of Ldr-GRF-NH2 from the photolysis reaction was diluted 1:5 with water (1.0 mg/ml peptide) .
  • Triton X-100 was added to a final concentration of 0.1%.
  • Succinic acid and calcium chloride were added to produce concentrations of 50 mM (5.9 mg/ml) and 2 mM (0.3 mg/ml) respectively and the solution pH was adjusted to 5.5 with 10 M NaOH. After the solution was filtered through a 0.45 ⁇ m membrane, 5.0 mg/ml Dowex 1 resin was added.
  • the Dowex 1 resin an anion exchange resin, was added to the reaction to bind the peptide containing the Asp-Asp- Asp-Asp (SEQ ID NO:32) sequence. This was found to increase both the rate of the reaction and the overall yield, as well as reduce the concentration of enterokinase required while improving substrate specificity.
  • a 1:3000 ratio of enterokinase enzyme (1 ul per 10 mg peptide) was added and the reaction was maintained in a 35-40 °C water bath with constant stirring. After 20-24 hours, the cleavage reaction which converts the Ldr-GRF-NH2 into GRF (1-44) -NH2 (SEQ ID NO:20) reached 70-80% completion.
  • DNA coding for a hCA-9AA fusion protein may be prepared and cultured according to the procedures described above.
  • the hCA-9AA fusion protein includes hCAII linked to the amino acid sequence Thr-Asn-Thr-Gly-Ser-Gly-Thr-
  • Pro-Ala (SEQ ID NO:33) through an interconnecting peptide.
  • the interconnecting peptide includes an enterokinase cleavage site positioned immediate adjacent the N-terminus of the 9AA (SEQ ID NO:33) sequence.
  • the hCA-9AA fusion protein may be expressed as a soluble protein in E. coli BL21 F " ompT r ' B m " B (DE3) cells.
  • the cell paste from the fermentor is diluted with cold wash buffer and chilled to 5-10°C.
  • the chilled cell suspension is homogenized at 12,000 psi with Galin high pressure homogenizer.
  • the cell paste is passed through a heat exchanger and chilled to 10°C prior to a second pass through the homogenizer.
  • a PMSF stock solution in 95% ethanol is added to a final concentration of 0.05 mM PMSF.
  • the lysate is cooled to about 8°C, passed through the homogenizer at 12,000 psi and drained into a holding tank. After passing the lysate through the homogenizer at 12,000 psi yet another time, a 5% v/v pH 7.5 solution of polyethyleneimine (PEI) may be added to a total concentration of 0.35% PEI.
  • PEI polyethyleneimine
  • the resulting mixture is stirred for 20 minutes and clarified by Westfalia at 20,000 x g. The clarified solution is filtered through a 0.22 micro ⁇ m Pall filter system.
  • a affinity column including p-aminomethylbenzene- sulfonamide-agarose resin is equilibrated with a 0.1M Tris-S0 4 pH 8.7 solution.
  • the clarified supernatant from the PEI precipitation is loaded onto the column and the column is eluted with a series of Tris-S0 4 buffer solutions.
  • the column fractions containing the fusion protein construct are pooled.
  • the protein construct is precipitated from the pooled fractions by lowering the pH of the solution to 4.0 with glacial acetic acid.
  • the precipitated fusion protein construct may be isolated by centrifugation and lyophilized.
  • the precipitated fusion protein construct is dissolved in a ph 8.0 buffer solution containing 50mM Tris and ImM CaCl 2 .
  • the mixture is sonicated until all the fusion protein construct has been dissolved.
  • 5.0 mg/ml Dowex 1 resin was added.
  • a 1:3000 ratio of enterokinase enzyme (1 ul per 10 mg peptide) was added and the reaction was maintained in a 35-40 °C water bath with constant stirring for 20-24 hours.
  • the reaction mixture was filtered to remove the Dowex 1 and the reaction was stopped by the addition of acetonitrile to a final concentration of 15%.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (i) ORIGINAL SOURCE: (xi)
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention décrit un procédé pour isoler et/ou purifier un peptide de recombinaison à l'aide d'une protéine de fusion de synthèse incluant une anhydrase carbonique et un polypeptide fusionné variable. Ce procédé comporte la précipitation, soit de la protéine de fusion de synthèse, soit d'un fragment de celle-ci incluant l'anhydrase carbonique. L'invention décrit aussi des corps d'inclusion qui incluent la protéine de fision de synthèse et un procédé de production d'un peptide incluant l'expression de la protéine de fusion de synthèse comme faisant partie d'un corps d'inclusion. Les protéines de fusion de synthèse qui incluent une anhydrase carbonique et certains peptides cibles sont également décrits.
PCT/US1995/015800 1994-12-07 1995-12-07 Production de peptides au moyen de proteines de fusion de recombinaison WO1996017942A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP95942995A EP0796335A1 (fr) 1994-12-07 1995-12-07 Production de peptides au moyen de proteines de fusion de recombinaison
AU44158/96A AU700605B2 (en) 1994-12-07 1995-12-07 Production of peptides using recombinant fusion protein constructs
JP8517724A JPH10510155A (ja) 1994-12-07 1995-12-07 組み換え融合蛋白構築物を用いるペプチドの製造方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35053094A 1994-12-07 1994-12-07
US08/350,530 1994-12-07

Publications (2)

Publication Number Publication Date
WO1996017942A1 true WO1996017942A1 (fr) 1996-06-13
WO1996017942A9 WO1996017942A9 (fr) 1996-08-15

Family

ID=23377117

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/015800 WO1996017942A1 (fr) 1994-12-07 1995-12-07 Production de peptides au moyen de proteines de fusion de recombinaison

Country Status (5)

Country Link
EP (1) EP0796335A1 (fr)
JP (1) JPH10510155A (fr)
AU (1) AU700605B2 (fr)
CA (1) CA2206848A1 (fr)
WO (1) WO1996017942A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1016723A1 (fr) * 1998-12-21 2000-07-05 Eli Lilly And Company Fabrication recombinante de beta-Lipotropine et autres peptides
WO2003076456A2 (fr) * 2002-03-12 2003-09-18 Dsm Ip Assets B.V. Procede de recuperation de proteines par precipitation
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US6703484B2 (en) 1997-01-08 2004-03-09 Invitrogen Corporation Methods for production of proteins
EP1539210A2 (fr) * 2002-09-06 2005-06-15 Bayer Pharmaceuticals Corporation Agonistes modifies du recepteur glp-1 et leurs procedes pharmacologiques d'utilisation
WO2008122089A1 (fr) * 2007-04-05 2008-10-16 The University Of Queensland Procédé de purification de peptides recombinés par précipitation sélective
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US7847079B2 (en) 2001-12-21 2010-12-07 Human Genome Sciences, Inc. Albumin fusion proteins
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US9733212B2 (en) 2006-07-21 2017-08-15 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
CN113340691A (zh) * 2021-06-05 2021-09-03 清华大学 一种提取海洋沉积物中不同赋存形态磷的方法
US11261472B2 (en) 2016-12-30 2022-03-01 Universidad De Chile Peptide sequence of a guide protein for the production of a peptide of interest, an expression vector, a host cell, and a method for the production of a peptide of interest

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200643033A (en) * 2005-03-08 2006-12-16 Chugai Pharmaceutical Co Ltd Conjugate of water-soluble modified hyaluronic acid and glp-1 analogue

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007085A1 (fr) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Procede de purification de polypeptides recombinants
WO1988007086A1 (fr) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Sequences d'amorce pour la production de proteines recombinantes
WO1992000993A1 (fr) * 1990-07-10 1992-01-23 California Biotechnology Inc. Isolation et purification de proteine de surfactant pulmonaire
WO1992001707A1 (fr) * 1990-07-16 1992-02-06 Bionebraska, Inc. Clonage de peptides destine a la purification
EP0528686A2 (fr) * 1991-08-19 1993-02-24 Suntory Limited Procédé pour la production de peptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007085A1 (fr) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Procede de purification de polypeptides recombinants
WO1988007086A1 (fr) * 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Sequences d'amorce pour la production de proteines recombinantes
WO1992000993A1 (fr) * 1990-07-10 1992-01-23 California Biotechnology Inc. Isolation et purification de proteine de surfactant pulmonaire
WO1992001707A1 (fr) * 1990-07-16 1992-02-06 Bionebraska, Inc. Clonage de peptides destine a la purification
EP0528686A2 (fr) * 1991-08-19 1993-02-24 Suntory Limited Procédé pour la production de peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VAN HEEKE, GINO ET AL: "Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: Expression of the Escherichia coli F1-ATPase.epsilon. subunit", PROTEIN EXPRESSION PURIF. (1993), 4(4), 265-74 CODEN: PEXPEJ;ISSN: 1046-5928, 1993, XP000568594 *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7223566B2 (en) 1997-01-08 2007-05-29 Invitrogen Corporation Methods for production of proteins
US9409942B2 (en) 1997-01-08 2016-08-09 Life Technologies Corporation Methods for production of proteins
US8519099B2 (en) 1997-01-08 2013-08-27 Life Technologies Corporation Methods for production of proteins
US8389681B2 (en) 1997-01-08 2013-03-05 Life Technologies Corporation Methods for production of proteins
US6703484B2 (en) 1997-01-08 2004-03-09 Invitrogen Corporation Methods for production of proteins
US8012715B2 (en) 1997-01-08 2011-09-06 Life Technologies Corporation Methods for production of proteins
US7265206B2 (en) 1997-01-08 2007-09-04 Invitrogen Corporation Methods of production of proteins
US7259242B2 (en) 1997-01-08 2007-08-21 Invitrogen Corporation Methods for production of proteins
US7189827B2 (en) 1998-10-23 2007-03-13 Amgen Inc. Modified peptides as therapeutic agents
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US7169905B2 (en) 1998-10-23 2007-01-30 Amgen Inc. Modified peptides as therapeutic agents
US7166707B2 (en) 1998-10-23 2007-01-23 Amgen Inc. Modified peptides as therapeutic agents
US9534032B2 (en) 1998-10-23 2017-01-03 Amgen Inc. Thrombopoietic compounds
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
EP1016723A1 (fr) * 1998-12-21 2000-07-05 Eli Lilly And Company Fabrication recombinante de beta-Lipotropine et autres peptides
US7847079B2 (en) 2001-12-21 2010-12-07 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US8252739B2 (en) 2001-12-21 2012-08-28 Human Genome Sciences, Inc. Albumin fusion proteins
US8513189B2 (en) 2001-12-21 2013-08-20 Human Genome Sciences, Inc. Albumin fusion proteins
US8071539B2 (en) 2001-12-21 2011-12-06 Human Genome Sciences, Inc. Albumin fusion proteins
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
WO2003076456A3 (fr) * 2002-03-12 2003-12-24 Dsm Ip Assets Bv Procede de recuperation de proteines par precipitation
WO2003076456A2 (fr) * 2002-03-12 2003-09-18 Dsm Ip Assets B.V. Procede de recuperation de proteines par precipitation
EP1539210A2 (fr) * 2002-09-06 2005-06-15 Bayer Pharmaceuticals Corporation Agonistes modifies du recepteur glp-1 et leurs procedes pharmacologiques d'utilisation
EP1539210A4 (fr) * 2002-09-06 2006-06-07 Bayer Pharmaceuticals Corp Agonistes modifies du recepteur glp-1 et leurs procedes pharmacologiques d'utilisation
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US10188740B2 (en) 2005-08-12 2019-01-29 Amgen Inc. Modified Fc molecules
US11266744B2 (en) 2005-08-12 2022-03-08 Amgen Inc. Modified Fc molecules
US9733212B2 (en) 2006-07-21 2017-08-15 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
US10302591B2 (en) 2006-07-21 2019-05-28 Life Technologies Corporation Sharply resolving labeled protein molecular weight standards
WO2008122089A1 (fr) * 2007-04-05 2008-10-16 The University Of Queensland Procédé de purification de peptides recombinés par précipitation sélective
US11261472B2 (en) 2016-12-30 2022-03-01 Universidad De Chile Peptide sequence of a guide protein for the production of a peptide of interest, an expression vector, a host cell, and a method for the production of a peptide of interest
CN113340691A (zh) * 2021-06-05 2021-09-03 清华大学 一种提取海洋沉积物中不同赋存形态磷的方法
CN113340691B (zh) * 2021-06-05 2023-12-12 清华大学 一种提取海洋沉积物中不同赋存形态磷的方法

Also Published As

Publication number Publication date
CA2206848A1 (fr) 1996-06-13
JPH10510155A (ja) 1998-10-06
AU4415896A (en) 1996-06-26
AU700605B2 (en) 1999-01-07
EP0796335A1 (fr) 1997-09-24

Similar Documents

Publication Publication Date Title
AU605291B2 (en) Process for the purification of recombinant polypeptides
AU710695C (en) Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs
CA2237296C (fr) Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine
US7105638B1 (en) Product and process for the production, isolation, and purification of recombinant polypeptide
AU700605B2 (en) Production of peptides using recombinant fusion protein constructs
WO1996017942A9 (fr) Production de peptides au moyen de proteines de fusion de recombinaison
NZ338004A (en) Process for producing peptide with glucagon like peptide-1 (GLP-1) activity by incorporating a protective and helper component to facilitate purification
JP2005515167A (ja) インスリン分泌性ｇｌｐ−１（７−３６）ポリペプチドおよび／またはｇｌｐ−１類似体を生成する方法
US6051399A (en) Production of C-terminal amidated peptides from recombinant protein constructs
US20160168226A1 (en) Process for production of insulin and insulin analogues
CA2159079C (fr) Methodes et systemes d'expression de l'adn, utilises pour la surexpression de proteines dans des cellules hotes
US5334532A (en) PDGF-B fusion protein, vectors, and host cells for use in production thereof
CA2324513A1 (fr) Proteine chimere contenant une sequence de type chaperon intramoleculaire et son application dans la production d'insuline
US20030144471A1 (en) Method for making acylated polypeptides
CN114380903B (zh) 一种胰岛素或其类似物前体
KR100368073B1 (ko) 융합파트너를 이용한 재조합 단백질의 제조방법
JP4386722B2 (ja) アシル化されたポリペプチドの製造方法
US20090035815A1 (en) Synthetic Gene for Enhanced Expression in E. Coli
KR20040007892A (ko) 재조합 인간 훼리틴 단백질 및 그 제조방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 41-51,DESCRIPTION,REPLACED BY NEW PAGES 41-52;PAGES 52-61,CLAIMS,RENUMBERED AS PAGES 52/1 AND53-61;DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2206848

Country of ref document: CA

Ref country code: CA

Ref document number: 2206848

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1995942995

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1995942995

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1995942995

Country of ref document: EP