US20080287304A1 - Mixture of at Least Two Fusion Proteins as Well as Their Production and Use - Google Patents

Mixture of at Least Two Fusion Proteins as Well as Their Production and Use Download PDF

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US20080287304A1
US20080287304A1 US10/537,588 US53758803A US2008287304A1 US 20080287304 A1 US20080287304 A1 US 20080287304A1 US 53758803 A US53758803 A US 53758803A US 2008287304 A1 US2008287304 A1 US 2008287304A1
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protein
fusion
binding
fusion protein
proteins
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Matthias Paschke
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SEIL PROTEINS GmbH
Navigo Proteins GmbH
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Universitatsklinikum Charite Berlin
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention concerns a protein mixture comprising at least a first fusion protein comprising a protein or protein fragment, and an interaction domain and a protein translocation sequence, which effects that the fusion protein upon expression in a bacterium is translocated through the cytoplasmic membrane in an essentially unfolded state and at least a second fusion protein comprising a protein or protein fragment, and an interaction domain and a protein translocation sequence which effects that the fusion protein is translocated through the cytoplasmic membrane upon expression in a bacterium in an essentially folded state, wherein the interaction domain of the first protein can bind to those of the second protein.
  • Phage display technology is currently used in many areas of biotechnology for identifying proteins with desired properties and enzymatic activities (Forrer, P. et al. (1999) Current Opinion in Struct. Biol. 9:514-520 and Gao, C. et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99:12612-12616).
  • the technology is used to improve, for example, binding properties, the encymatic properties and/or the thermodynamic stability of proteins already known or isolated by phage display technology (Forrer, P. et al. (1999) supra).
  • the basis for the phage display technology lies in the observation that certain so called non-lytic bacteriophage merely infect bacteria and that the phage particles are not released by lysis of the bacterium but rather that the individual parts of the bacteriophage are transported through the cytoplasma into the periplasma and eventually to the bacterial cell surface where the complete phage is assembled which eventually disengages from the bacterial cell.
  • the fusion of the protein of interest with a phage coat protein thus leads to the export of this protein from the bacterial cytoplasma and the presentation on the surface of the bacterium.
  • Phage coat proteins suitable for presentation are for example pIII, pVI, pVII, pVIII and pIX derived from M13 phagemid (Gao, C. et al. (2002) supra).
  • the N-terminus of the phage coat protein is oriented towards the outside and, consequently, the fused protein has to be arranged N-terminally of the phage coat protein in order for it to be presented on the surface.
  • This does not represent a problem, if single already known proteins are fused with one of the indicated phage coat proteins since the START and STOP codons of these proteins are known. It, however, leads to problems if a so called phage library has to be created wherein the phage coat proteins are fused with a cDNA library.
  • the coding nucleic acids comprised in the cDNA library usually comprise translational STOP codons at the 3′-end since the cDNAs resulting from poly(A + ) selection of the mRNA and from subsequent oligo-(dT)-priming always comprise translational STOP codons.
  • a STOP codon will always be located between the cDNA and the phage coat protein upon fusion of an oligo-(dT)-primed cDNA 5′ of the phage coat protein which in turn will inhibit expression of a fusion protein consisting of the cDNA encoded protein and the phage coat protein.
  • a fusion protein was expressed directed by a LacZ promoter which consisted of cJun and a C-terminus and of a phage coat protein (pIII) and, a second fusion protein which consisted of cFos at its N-terminus and of a cDNA library at its C-terminus, wherein also this protein was driven by a second LacZ promoter.
  • a LacZ promoter which consisted of cJun and a C-terminus and of a phage coat protein (pIII)
  • a second fusion protein which consisted of cFos at its N-terminus and of a cDNA library at its C-terminus, wherein also this protein was driven by a second LacZ promoter.
  • Stop-transfer sequences cause through the localized accumulation of positively charged amino acids in the protein sequence that the respective protein becomes stuck in the membrane upon translocation by the Sec transport pathway. Proteins which due to their rapid and/or stable folding cannot be bound in its unfolded form by proteins of the Sec transport pathway, in particular by SecB, are not transported through the Sec translocase complex and remain in the cytoplasma (Yamana et al. (1988) J. Bio. Chem. 263:19690-19696 and Berks, B. C. (1996) Mol. Microbiol. 22:393-404 and Bergs, B. C. et al. (2000) Mol. Microbiol. 35:260-274).
  • Proteins dependent on reducing conditions or which depend for their function on cytoplasmic co-factors like, for example, FeS centres or molybdopterin can also not reach the periplasma via the Sec transport pathway in functional form. Accordingly, many polypeptides due to the lack of compatibility with the Sec transport pathway cannot be presented in a functionally folded state by phage display and subsequently be selected. The translocation of fusion proteins through the Sec transport pathway into the periplasma, thus, represents a significant disadvantage of the phage display techniques known in the prior art.
  • One object of the present invention is, thus, to overcome the limitation of the phage display technology of the prior art and to allow the production of fusion proteins which do not yield functional fusion proteins when produced by the prior art methods.
  • the present invention in one aspect provides a protein mixture comprising: a) at least a first fusion protein comprising: i) a protein or protein fragment, ii) an interaction domain and iii) a protein translocation sequence which effects that the fusion protein upon expression in a bacterium is translocated through the cytoplasmic membrane in an essentially unfolded state and b) at least a second fusion protein comprising i) a protein or protein fragment, ii) an interaction domain and iii) a protein translocation sequence which effects that the fusion protein upon expression in a bacterium is translocated through the cytoplasmic membrane in an essentially folded state, wherein the interaction domain of the first fusion protein can bind to those of the second fusion protein.
  • the protein or protein fragment of the first fusion protein comprises preferably proteins, which are translocated through the cytoplasmic membrane of the bacterium, preferably a Gram negative bacterium in an unfolded state and which accordingly do not require the reducing cytoplasmic environment and/or cytoplasmic co-factors for correct folding and which can also attain an essentially correct folding in periplasma.
  • proteins comprise but are not limited to the immune globulin heavy chains, immune globulin light chains, fragments of these chains, so called “single-chain-antibody” (Bird, R. E. (1988) Science 242:423-6), diabodies (Holliger, P. (1993) Proc. Natl. Acad. Sci. U.S.A.
  • receptors preferably extracellular domains of receptors like, for example, EGFR, PDGFR or VEGFR or receptor ligands like, for example, EGF, PDGF, or VEGF, integrines, preferably their extracellular domains, intimines and their domains, like for example EaeA, carbohydrate binding proteins and domains thereof like, for example, MBP and CBD, album binding proteins and domains or protein A and its domains.
  • the protein or protein fragment of the second fusion protein can be any protein or protein fragment preferred are, however, protein fragments which attain their folding and/or their function only if they are folded in the cytoplasma of a bacterium and which are thus translocated through the cytoplasmic membrane into the periplasma in an essentially folded state.
  • proteins are autofluorescent proteins like, for example, GFP or variants thereof with altered absorption maxima, enzymes like, for example, ⁇ -lactamase, cofactor dependent proteins like, for example, TMAO reductase and horseradish peroxidase, proteins which are encoded by a cDNA derived from a cDNA library or synthetic proteins.
  • the protein or protein fragment of the first fusion protein and the protein translocation sequence is a phage coat protein or a periplasmatic marker enzyme, like PhoA, an intimin, a protein of the outer bacterial membrane or a periplasmatic receptor protein, in particular a carbohydrate binding protein.
  • Preferred phage coat proteins which can be comprised in a protein mixture of the present invention are selected from M13 phagemid coat proteins pIII, pVI, pVII, pVIII and pIX. Out of these phage coat proteins only pIII and pVIII are provided with a known Sec dependent protein translocation sequence while the protein translocation sequences comprised in the remaining phage coat proteins have not been identified as of yet. Since these phage coat proteins are transported into the periplasma of the bacteria in an essentially unfolded state such proteins are considered as proteins which consist of a protein or protein fragment and a protein translocation sequence within the meaning of the invention without identification of the protein translocation sequence.
  • interaction domains which are used in the first and the second fusion protein lead to binding of the first fusion protein to the second fusion protein.
  • interaction domains are preferred which result in a relatively stable interaction between the two proteins, wherein a relatively stable interaction is an interaction which remains stable in the oxidative environment of the periplasma, on the bacterial cell surface or also outside the cell upon secretion of the heterodimer or heteromultimer.
  • Suitable interaction domains of the first and second fusion protein which can be comprised in the fusion protein according to the invention are, for example, a leucine zipper domain and a leucine zipper domain as they have been described for the first time in the two oncoproteins cJun and cFos (Landschulz et al.
  • interaction domain also comprises domains which allow the formation of multimers of more than two fusion proteins.
  • the protein translocation sequence of the first fusion protein effects that the fusion protein is translocated upon expression in a bacterium preferably in a Gram negative bacterium through the cytoplasmic membrane into the periplasma in an essentially unfolded state.
  • a protein sequence potentially suitable as protein translocation sequence which leads to the translocation of a protein fused therewith in an essentially unfolded state, is used with a protein comprising a GFP-myc-TAG.
  • the GFP protein is formed in the cytoplasma of the bacterium which can be detected via the cytoplasmic fluorescence. In this case it does not reach the surface or the media and, thus, the myc-TAG can neither be detected in the medium nor on the surface with an anti-myc-antibody, like for example the monoclonal antibody 9E10.
  • GFP-myc-TAG fusion protein can be detected through an anti-myc-antibody in the medium and/or on the surface of the bacterium.
  • no fluorescence should be detectable in the periplasma since upon translocation of the GFP into periplasma in an essentially unfolded state the protein will not be folded correctly (so called “Sec-incompatibility”).
  • the protein translocation sequences which are preferably used in the first fusion protein are those which are recognized in the Sec dependent transport pathway (Danese, P. N.
  • the second fusion protein comprises a protein translocation sequence which effects that the fusion protein is translocated through the cytoplasmic membrane upon expression in a bacterium, preferably in a Gram negative bacterium in an essentially folded state.
  • a protein translocation sequence with this property is present, if a protein, for example, GFP which can only attain its functional confirmation in the cytoplasma of a bacterium, is transport into the periplasma without a loss of auto fluorescence. This property of the protein translocation sequence of the invention can be assessed with the experiment described above with respect to the first protein translocation sequence.
  • Tat twin-argenine translocation
  • the Tat transport pathway known in the art allows the transport of proteins already folded in the cytoplasma into the periplasma and, thus, the transport of proteins into the periplasma which are incompatible with the Sec transport pathway. Similar to the transport through the Sec transport pathway also the Tat transport is mediated by a specific group of leader sequences (DeLisa, M. P. et al. (2002) J. Biol. Chem. 277:29825-29831).
  • the second fusion protein comprises in a preferred embodiment of the present invention a signal sequence which is recognized by the Tat dependent transport pathway or by a thylakoid- ⁇ -ph dependent transport pathway and which, thus, leads to translocation of the fusion protein in an essentially folded state.
  • a consensus motive of a protein translocation sequence recognized by the Tat dependent transport pathway is described in DeLisa, M. P. et al. ((2002) supra). The sequence is: S/T/RRXFLK.
  • At least a first and at least a second fusion protein are covalently or non-covalently bound to each other.
  • cystein residues or homologes thereof are covalently or non-covalently bound to each other.
  • Covalent bond can, for example, also be effected by the incorporation of amino acids with photoactivatable groups in both fusion proteins and subsequent UV-exposure of the proteins which are initially only bond to each other non-covalently.
  • a further aspect of the present invention is a nucleic acid mixture which encodes a protein mixture of the present invention.
  • a coding nucleic acid within the meaning of the present invention is a nucleic acid sequence which encodes a polypeptide of the invention or a precursor thereof.
  • the nucleic acid mixture is DNA or RNA, preferably a DNA, wherein the DNA can be single stranded or double stranded.
  • the nucleic acid respectively encoding the first or the second fusion protein furthermore comprises promoters which allow the expression of the respective fusion proteins in the host cell. Suitable promoters for the expression in, for example, E.
  • nucleic acids which constitute the respective nucleic acid mixture, are origins of replication (Ori), selective marker genes which, for example, mediate ampicilin or chloramphenicole resistance.
  • origins of replication or selective marker genes which, for example, mediate ampicilin or chloramphenicole resistance.
  • nucleic acids can comprise those elements, which are usually employed in bacterial expression vectors. Someone of skill in the art is aware of a number of such elements as well as vectors like for example pGEM or pUC.
  • the two nucleic acids coding for the first and the second fusion protein are covalently linked to each other, preferably via phosphor diester bond.
  • the nucleic acid molecules which code for the first and the second fusion protein and which comprise suitable regulatory elements are comprised on one plasmid, thus, allowing that the protein mixtures according to the invention can be prepared, for example, in a bacterium already by transfection of only one plasmid and by infection with only one phage, respectively, if the nucleic acid is comprised in a phage.
  • both fusion proteins are expressed under the control of only one promoter as bicistronic cassette.
  • a further aspect of the present invention is a vector comprising a protein mixture of the invention and/or comprising a nucleic acid mixture of the invention.
  • a vector within the meaning of the invention is a protein-nucleic acid mixture, which is capable to introduce the protein mixtures and/or nucleic acid mixtures comprised therein into a cell.
  • the fusion proteins encoded by the nucleic acid mixtures are expressed in the cells and that de novo synthesized fusion proteins can be recovered from the cells and can be presented on the cell surface, respectively.
  • Suitable vectors are, for example non-lytic phages, like M13 phage, fd phage, F1 phage and lytic phage, like ⁇ phage.
  • a further aspect of the further invention is a cell comprising a protein mixture of the invention, a nucleic acid mixture of the invention and/or a vector of the invention.
  • Cells of the invention can be prokaryotic or eukaryotic cells.
  • the cells of the invention are prokaryotic cells, in particular bacteria and more preferably E. coli (TG1, XL-1, JM83, BL21) or B. subtilis.
  • a further aspect of the present invention is a library comprising at least two protein mixtures of the present invention, at least two vectors of the present invention and/or at least two cells of the present invention, wherein the proteins or protein fragments of the respective first or the respective second fusion protein are different from each other.
  • a library can either comprise specifically selected different known proteins or protein fragments or the interaction domain and the protein translocation sequence on the first or the second, preferably the second fusion protein can be fused with a cDNA library, wherein the expression of these nucleic acids leads to a number of different first or second fusion proteins which respectively comprise different proteins or protein fragments.
  • the cDNA part is expressed at the C-terminus of the fusion protein to thereby circumvent the previously described problem with N-terminal fusion of a cDNA.
  • the library comprises a large number of cells of the present invention when each cell produces a different protein mixture, preferably presents it on its surface.
  • the protein or protein fragment and interaction domain of the first protein is a phage co-protein
  • the library of the present invention allows the presentation of a large number of proteins or protein fragments, which are comprised in the second fusion protein.
  • the presentation is, thus, not limited as are the phage display libraries known in the prior art to proteins or protein fragments which fold into their functional form in the periplasma of the cell but also comprises proteins which can attain the functional folding in the cytoplasma.
  • the protein mixtures according to the invention which can form heterodimers or multimeres, wherein the components of the heterodimers or multimeres attain their three dimensional structure in at least two different cellular compartments can now be used in a number of methods comprising among others phage display.
  • a further aspect of the present invention is, thus, a method for identifying substances which can bind to a protein mixture, a vector of the present invention or to a cell of the present invention comprising the step:
  • This method primarily serves the purpose of identifying a substance or substances, which can bind to an already known protein target, for example, to identify an inhibitor, an activator, competitor or modulator of the known protein target.
  • the potentially binding substances the binding of which to a protein mixture of the invention, a vector of the invention and/or the cell of the invention should be measured can be any chemical substance or substance mixture.
  • it can be substances from a peptide library, substances from a combinatorial chemical library, cell extracts, in particular plant cell extracts and proteins or protein fragments.
  • Contacting of the potentially binding substance(s) with a protein mixture, vector or cell of the invention is understood to mean any possibility of interaction between the two components wherein both components can be independently of each other in liquid phase, for example, in solution or in suspension, or can be attached to a solid phase, for example, to an essentially planar surface or can be in the form of particles, pearls or the like.
  • there is a plurality of different potentially binding substances immobilized on a solid surface and is contacted with the protein mixture of the invention, a vector of the invention or cells of the invention and subsequently binding of the substances of the invention to the various positions at which the respective different potentially binding substances are immobilized is measured.
  • Measuring of binding of the protein mixtures, the vectors or the cells of the present invention to potentially binding substances can be carried out by measuring a marker connected to the protein mixture of the invention, the vector of the invention or the cell of the invention wherein suitable markers are known to the person skilled in the art and comprise, for example, fluorescence or radioactive markers.
  • suitable markers are known to the person skilled in the art and comprise, for example, fluorescence or radioactive markers.
  • the protein mixture, the vector, or the cell comprises in addition to the second fusion protein beside the protein or protein fragment the interaction of which with the potentially binding substance is to be investigated, an autofluorescent protein like, for example, GFP or variants thereof.
  • Measuring the binding of the substance can also be detected via the change of electrochemical, in particular redox properties of, for example, the immobilized potentially binding substances after contacting.
  • Suitable methods comprise, for example, potentiometric methods. Further methods for detecting the binding of two molecules or molecular mixtures are known to someone of skill in the art and can all equally be employed for measuring the binding of the potentially binding substance to the protein mixture of the invention, the vector of invention or the cells of the invention.
  • the binding substance can be selected on the basis of, for example, the strength of the bond and can then be used directly, for example, for the inhibition of the known protein target. It is, however, also possible to modify the binding substance by methods known in the art which also comprise methods of combinatorial chemistry. For example, by adding halogen side groups, preferably F or Cl, by adding lower alkyl groups like methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl or tert-butyl groups or by adding amino, nitro, hydroxyl, amido, or carboxylic acid groups.
  • the thus differently modified binding substances can then again be tested for the binding in the method of the invention and can be optimized with respect to the desired binding specificity and the effect caused thereby (for example, activation, inhibition or modulation of the respective activity).
  • a further aspect of the present invention is a method of identifying proteins or protein fragments, which bind to a test substance comprising the steps:
  • protein or protein fragments are selected which can bind to a given test substance.
  • those are proteins or protein fragments of the second fusion proteins, since this is correctly folded with a higher probability as compared to the proteins or protein fragments of the first fusion proteins which are only correctly folded, if the respective proteins can also attain their native conformation in the oxidative environment of the periplasm.
  • a test substance within the meaning of the present invention can be any chemical substance or a mixture thereof.
  • it is a protein or protein fragment, in particular a receptor or receptor ligand, a transcription factor, an ion channel, a molecule of the signal transduction cascade, a structure or storage protein, a toxin, a light receptor protein and pigment protein.
  • Measuring of the respective binding of the various protein mixtures, vectors and/or cells of the library to the test substance can be carried out as described above via marker dependent or marker independent assay methods.
  • the method of the present invention comprises the further steps: Selecting at least one protein mix, one vector or one cell based on the measured binding and producing a second library wherein the library is produced by modification of the protein or protein fragment, which is comprised in the selected protein mix, in the selected vector or in the selected cell.
  • the selection process of protein mixtures, vector or cells from the library is preferably carried out on the basis of the strength of the bond wherein protein mixtures, vectors or cells are preferred which show the strongest binding to the respective test substance.
  • modification can be generated which respectively lead to minor changes in the amino acid sequence and thus to a multitude of derivates which show a slightly different three dimensional structure in comparison to the starting protein and protein fragment, respectively.
  • modifications can be obtained using methods known in the art like, for example, by random mutagenesis or by targeted substitution of single nucleic acid codons of the nucleic acid coding for the protein or protein fragment. It is thereby preferred that substitutions are so called “conservative” substitutions.
  • a conservative substitution is present if, for example, a nucleic acid codon coding for a basic amino acid is replaced by another nucleic acid codon coding for a basic amino acid, a nucleic acid codon coding for another acidic amino acid is replaced by a nucleic acid codon, coding for a acidic amino acid and a nucleic acid codon coding for a polar amino acid is replaced by another nucleic acid codon coding for a polar amino acid, respectively.
  • the second library newly generated on the basis of the selected protein mixtures, vectors or cells can now again be contacted in a further step with the test substance whereupon in a further step the respective binding of the test substance to the modified protein mixtures, vectors or cells of the second library is measured.
  • the proteins or protein fragments which have been identified or additionally have been optimized with respect to a particular property by above method can now be used as an active agent in a medicament, if they have been, for example, optimized for activation or repression of a particular cellular signal pathway.
  • the methods of the present invention comprise in a preferred embodiment the further step that the selected binding substance or the protein or protein fragment or a variant thereof comprised in the selected protein mix, in the selected vector or in the selected cell is admixed with a pharmaceutical acceptable carrier and/or auxiliary substance.
  • a “variant” of the protein or protein fragment comprises modifications of the N- or C-terminal or modification of amino acid side chains which, for example, increase the stability, solubility or biocompatibility of the proteins or protein fragments.
  • fusion proteins of the proteins or protein fragments identified according to the invention which can comprise as a further component autofluorescent markers like, for example, GFP or cytostatic drugs like, for example, cholera toxin.
  • Pharmaceutically acceptable carriers and/or auxiliary substances comprises substances which stabilize the binding substance and the protein of protein fragments, respectively, or variants thereof, which increase the pharmaceutical tolerance or which are required by the respective form or application like for example tablet, band aid or infusion solution as, for example, preservative, buffer, salt or protease inhibitors.
  • a further aspect of the present invention is a kit for producing a mixture of nucleic acids according to claim 10 comprising:
  • This kit allows the insertion of a chosen nucleic acid sequence 5′ or 3′ of the nucleic acid which codes for the interaction domain and the protein translocation sequence with the result that the resulting nucleic acid codes for a fusion protein which comprises at its C-terminus and/or at its N-terminus a protein or protein fragment encoded by the respectively introduced nucleic acid sequence.
  • the introduced DNA is a cDNA library, wherein this is particularly preferred if it and has been introduced into the nucleic acid by using the 3′-restriction site.
  • the kit comprises the leucine zipper of the cFos protein and in a further preferred embodiment the Tat dependent protein translocation sequence TorA.
  • the kit according to the present invention further comprises at least a second nucleic acid comprising at least one restriction site 5′ and/or 3′ of a nucleic acid coding for a second fusion protein comprising:
  • nucleic acid allows insertion 5′ or 3′ of the nucleic acid encoding for the interaction domain and the protein translocation sequence so that in result the resulting nucleic acid codes for a fusion protein which comprises at is N- or C-terminus a protein or protein fragment coded for by the inserted nucleic acid.
  • nucleic acids coding for a phage coat protein can be inserted into a nucleic acid wherein those are preferably inserted at the 3′ restriction site.
  • nucleic acids coding for phage coat proteins are introduced into the second nucleic acid that the resulting fusion protein upon strong expression of, for example, the gIIIp-fusion protein lead to high toxicity in E. coli cells. For this reason an amber codon is inserted in classical phase display systems 5′ of the gIII-protein. In suppressor strains (e.g. CL-1 Blue) the expression of the gIIIp-fusion protein is thereby reduced by 90%.
  • the amber codon (which is read in non-suppressor strains as STOP codon) enables the easy soluble expression of the protein which was previously fused with a phage protein and presented on the phage by introducing the phagimid into a non suppressor strain (e.g. BL21) and expressing it therein.
  • the first and/or the second nucleic acid comprises in a preferred embodiment either 5′ or 3′ an amber codon.
  • the amber codon is positioned in the first nucleic acid 5′ and in the second nucleic acid 3′.
  • the interactive domain of the second fusion protein is a leucin zipper domain of the cJun protein.
  • the nucleic acid comprises a nucleic acid which codes for a Sec-dependent protein translocation sequence in particular the PelB leader peptide.
  • a further aspect of the present invention is the use of a cell for the production of a protein mix according to the invention as well as the use of a protein mix according to the invention, a vector according to the invention or a cell according to the invention for the preparation of a library according to the invention.
  • a preferred area of using the protein mixes of the invention, the phages of the invention, the cells of the invention in particular the libraries of the invention comprising the above referenced mixtures of proteins, phages and cells as well as of using the kits of the present invention is the presentation of proteins on filamentous phages.
  • a particular focus thereby is on proteins which due to the incompatibility with the Sec transport pathway cannot be presented using the classical phage display technology.
  • this presentation and selection of cDNA expression libraries and the presentation and selection of DNA libraries for directed evolution of proteins also called “protein engineering” are particularly preferred areas of application.
  • a further preferred use is the production of protein conjugates.
  • the use is particularly preferred when the protein or protein fragment of the first fusion protein and the protein or protein fragment of the second fusion protein respectively have different requirements for the cellular environment required for correct folding.
  • the present invention allows the direct fusion of antibodies with marker proteins which would not be correctly folded upon production in bacteria and transport through the Sec-dependent transport pathway and which could, therefore, not be used in standard procedures as marker proteins for marking antibodies.
  • Marker protein antibody fusions the functional expression of which is only enabled by the present invention comprise, for example, fusions of autofluorescent proteins like GFP and immune globulin heavy chains, immune globulin light chains or “single chain antibodies”.
  • FIG. 1 Consensus sequence of Tat-dependent, Sec-dependent, SRP-dependent or YidY-dependent signal sequences wherein X is a random amino acid and # is a hydrophobic amino acid.
  • FIG. 2 Tat-dependent TorA-signal peptide, wherein X is a random amino acid and # is a hydrophobic amino acid.
  • FIG. 3 Underlying principle of the TLF-system, wherein CT represents the pIII domain, pelB the Sec signal sequence, TSS the Tat signal sequence and POI the presented protein.
  • FIG. 4 Restriction map of the plasmid pCD4/GFP24 the nucleic acid sequence of which is depicted in the appendix as SEQ ID NO: 1.
  • FIG. 5 Restriction map of the plasmid pCA1/GFP24 the nucleic acid sequence of which is depicted in SEQ ID NO: 2.
  • FIG. 6 Restriction map of the plasmid pCN1/GFP24 the nucleic acid of which is depicted in SEQ ID NO: 3.
  • FIG. 7 Competitive phage ELISA wherein white bars represent the results with GFP24 presenting phages. GFP24 phages were made with the help of XL-1 blue cells carrying the pCD4/GFP24 plasmid. Grey bars represent the results which were obtained with ⁇ -lactamase carrying phages. The ⁇ -lactamase presenting phages were made in XL-1 blue cells which carried the plasmid pCD4/BLA.
  • FIG. 8 Enzymatic assay of the presentation of ⁇ -lactamase on bacteriophages wherein white circles represent the results with GFP24 carrying phages.
  • the GFP24 phages were made with the help of XL-1 blue cells which carry the pCD4/GFP24 plasmid.
  • Black squares represent the results which were obtained with phages carrying the ⁇ -lactamase.
  • ⁇ -lactamase presenting phages were produced with XL-1 blue cells carrying the pCD4/BLA plasmid. The absorption at 486 nm in relation to the time is shown.
  • pCD4/GFP24 is a cystein display phagimid which is based on the pGP-vector (Paschke M., et al.: (2001) Biotechniques 30: 720-725).
  • pCAI/GFP24 is a cystein display phagimid which is based on pGF-F100. It can be used for the tet o-p controlled expression of proteins as fusion of cFos leucin zipper.
  • the translocation of the cFos-fusion protein into the periplasmatic space is mediated by the TorA leader peptide sequence (Tat-dependent translocation pathway).
  • the tet o-p controlled transcript comprises a second cistron which expresses the c-jun::G3Ps fusion protein.
  • the cJun::G3Ps is directed into the periplasmatic space through the Sec-dependent translocation pathway.
  • Covalent complexes between the cFos-fusion protein and the cJun::G3PS fusion protein are formed in the periplasmatic space due to the dimerization of cJun and cFos and subsequent formation of cystein bonds between the proteins.
  • the phagimid comprises a GFP24 cassette flanked by SfiI restriction sites at positions 148 and 910 and is positioned between the TorA leader peptide and cFos. This cassette has to be replaced by a protein to be presented.
  • pCAI/GFP24 is a cystein display phagimid derived from pCD4, which is based on a pGP vector.
  • pDC4/GFP24 is a cystein display phagimid that is based on the pGP vector (Paschke M., et al.: (2001) Biotechniques 30: 720-725). It can be used for the tet o-p controlled expression of proteins as fusion with the cFos leucin zipper. The translocation of the cFos fusion protein to the periplasmatic space is mediated by the TorA leader peptide (Tat transport pathway).
  • the tet o-p controlled transcript comprises a second cistron, which expresses the cjun::G3Pss fusion protein (G3Pss comprises amino acids 252 to 406 of the mature gIII proteins of the fd phage).
  • G3Pss comprises amino acids 252 to 406 of the mature gIII proteins of the fd phage.
  • the c-jun::G3Pss is directed towards the periplasmatic space through a Sec-dependent transport pathway (pelB leader peptide).
  • Covalent complexes of cFos fusion protein and c-jun::G3Pss are formed due to the dimerization between cJun and cFos in the periplasmatic space and the subsequent formation of cystein bonds between the proteins (Crameri, R. and Suter M. (1993), supra).
  • the phage display of the proteins, which are fused with cFos can be achieved by so-called helper phage rescue.
  • the phagimid pCD4-GFP24 converts chloramphenicol resistance.
  • the resistance gene (CAT) and the tet-repressor (TetR) are under the control of ⁇ -lactamase promoter as a bicistronic cassette.
  • the transcript is terminated in a ⁇ -phage terminator.
  • the Tat-TetR cassette is in a reversed position towards the cFos and the cJun fusion cassette.
  • a GFP24 cassette flanked at the positions 148 and 910 by SfiI restrictions sites is positioned between the TorA leader peptide and cFos. This cassette is replaced by the protein to be presented.
  • pCD4/Bla is a cystein display phagimid derived from pDC4/GFP24 wherein through restriction digest with SfiI the GFP24 fragment has been replaced by the sequence of the mature TEM1 ⁇ -lactamase.
  • the inserted lactamase cloning cassette with 5′ and 3′ terminal SfiI restrictions sites is depicted in SEQ ID NO:4.
  • the infected culture was cultivated for 30 minutes at 37° C. and subsequently mixed with kanamizin at a final concentration of 60 ⁇ g/ml.
  • the culture was cultivated at 25° C. for 10 minutes.
  • the cells were harvested by centrifugation (4000 ⁇ g, 4° C., 5 minutes) and subsequently resuspended in T2Y selection medium comprising 60 ⁇ g/ml kanamizin and 0.5 ⁇ g/ml tetracycline.
  • the culture was cultivated for 5 hours at 25° C. Subsequently, phage preparation from the cell culture supernatant followed as follows: 40-50 ml cells and cell debris each were separated by centrifugation from phage comprising cell culture supernatant (4° C., 10,000 rpm in an A8-24 rotor for 15 minutes). The supernatant was filtered through a 0.45 ⁇ m filter and was mixed with 1 ⁇ 4 volume PEG-NaCl solution (20% w/v PEG 8000, 50% w/v NaCl) and incubated on ice over night or at least for 5 hours. The mixture was then centrifuged at 4° C. for 15 minutes with 15,000 rpm in an A8.24 rotor.
  • the pellet was resuspended in 2.5 ml ice cold PBS and distributed into 2 ml plastic tubes. Then the supernatant was mixed with 1 ⁇ 4 volumes PEG-NaCl solution and incubated on ice for at least one further hour. Then the supernatant was centrifuged at 4° C. and 14,000 rpm for 15 minutes. The phage pellet was dissolved in 0.5-1 ml PBS. If necessary the phage solution was filtered through a 0.45 ⁇ m filter and then stored at 4° C. For long term storage the phage solution was mixed with 1 volume glycerine and stored at ⁇ 70° C.
  • Phage titre was determined using standard methods employing serial dilutions. The titre usually was in the range of 10 12 and 10 13 cfu/ml.
  • GFP24 is a variant of a green fluorescent protein with a circular permutation which further comprises an epitope of the P24 protein of HIV (Höhne, W. E. et al. (1993) Mol. Immunol. 30:1213-21). GFP24 is bound with high affinity by the anti-P24 antibody CB4-1 (Dr. Scholz, Institute for Biochemistry (Universticiansklinikum Charotti)). Similar to GFP GFP24 cannot be exported through the Sec-transport pathway.
  • a functional GFP24 protein should resulting from expression of the above described pCD4/GFP24 plasmid should, thus, only lead to the presentation of a functional GFP24, if this part of the protein is not transported into the periplasma by a Sec-dependent transport pathway but rather by a Tat-dependent transport pathway.
  • a phage-ELISA was carried out as follows: microtitre plates were coated with 10 ⁇ m/ml anti-P24 antibody C4-1, washed three times with PBS/Tween 0.1% and incubated for 1 to 2 hours per well with Genosys blocking reagent (Sigma-Genosys Ltd., Cambridge, UK) at room temperature under shaking.
  • the microtitre plate was washed three times with PBS/Tween 20 0.1%. Then 50 ⁇ l of GFP24 presenting phage with or without P24 peptide was placed in the well of the micro titre plate and then the presence of phage in the microtitre plate was detected with a horseradish peroxidase coupled anti-phage antibody (Seramun Diagnostica GmbH, Dolgenbrodt, Germany). Signal intensity represented the phage bound to CB4-1.
  • pCD4/GFP24 phage was completely replaced from CB4-1 through the P24 peptide while ⁇ -lactamase presenting phages (pCD4/BLA) which were used as a control did not bind to CB4-1. On top of that no unspecific binding to other antibodies or to the blocking reagents could be detected (see FIG. 7 ).
  • TEM-1- ⁇ -lactamase is a periplasmatic protein which can confer resistance to ampicillin by hydrolysis of the lactam ring of the antibiotic ampicillin.
  • TEM-1- ⁇ -lactamase is usually exported into the periplasma through a Sec-dependent transport pathway.
  • the Sec-signal sequence was removed and replaced by the TorA-sequence.
  • the successful presentation of TEM-1- ⁇ -lactamase was determined with an enzyme assay described in the following, the results of which are depicted in FIG. 8 .
  • nitrocefin stock solution was prepared as follows: 1 mg nitrocefin was dissolved in 100 ⁇ l DMSO. The solution was then mixed with 1.9 ml PBS. The solution was stored at ⁇ 20° C. for a maximum of 2 weeks.

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US20100249377A1 (en) * 2007-12-19 2010-09-30 Bryan Wang ENGINEERED HYBIRD PHAGE VECTORS FOR THE DESIGN AND THE GENERATION OF A HUMAN NON-ANTIBODY PEPTIDE OR PROTEIN PHAGE LIBRARY VIA FUSION TO pIX OF M13 PHAGE
CN111479922A (zh) * 2017-11-03 2020-07-31 内克斯特伊拉股份公司 载体构建体

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WO2008089132A2 (fr) 2007-01-12 2008-07-24 Cornell Research Foundation, Inc. Sélection génétique pour le repliement des protéines et la solubilité dans le périplasme bactérien
DK2193199T3 (da) 2007-08-20 2014-01-13 Nextera As Pvii fag-display
PT2401373E (pt) 2009-02-25 2013-12-16 Nextera As Apresentação em pix de fago independente de sequência sinal
BR112012014194A2 (pt) 2009-12-14 2017-01-10 Scil Proteins Gmbh um método para identificar proteínas ubiquitinas modificadas hetero-multiméricas com capacidade de ligação a ligandos
CA2793424A1 (fr) * 2010-03-18 2011-09-22 Cornell University Preparation d'anticorps correctement plies utilisant la presentation sur la membrane interieure d'intermediaires de translocation twin-arginine
US20240016933A1 (en) * 2020-11-09 2024-01-18 The General Hospital Corporation Methods of Anchoring or Reconstituting Active Molecules on Metabolically Labeled Cells

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US20100249377A1 (en) * 2007-12-19 2010-09-30 Bryan Wang ENGINEERED HYBIRD PHAGE VECTORS FOR THE DESIGN AND THE GENERATION OF A HUMAN NON-ANTIBODY PEPTIDE OR PROTEIN PHAGE LIBRARY VIA FUSION TO pIX OF M13 PHAGE
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