EP0731837A1 - Bacteries utilisees pour preparer des proteines de fusion stables et procede permettant de les identifier - Google Patents

Bacteries utilisees pour preparer des proteines de fusion stables et procede permettant de les identifier

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Publication number
EP0731837A1
EP0731837A1 EP95905579A EP95905579A EP0731837A1 EP 0731837 A1 EP0731837 A1 EP 0731837A1 EP 95905579 A EP95905579 A EP 95905579A EP 95905579 A EP95905579 A EP 95905579A EP 0731837 A1 EP0731837 A1 EP 0731837A1
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EP
European Patent Office
Prior art keywords
protein
bacteria
proteins
fusion
igass
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EP95905579A
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German (de)
English (en)
Inventor
Thomas Klauser
Joachim Kramer
Thomas F. Meyer
Johannes Pohlner
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Publication of EP0731837A1 publication Critical patent/EP0731837A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to bacteria for the production of stable fusion proteins from a carrier protein and a passenger protein, the bacteria having the genetic marker fpt. Possession of this genetic marker enables the improved production of protein fusions that have a destabilizing effect on bacteria.
  • the present invention relates to bacteria for the production of fusion proteins in which the fusion protein is stably presented on the surface and the bacteria, in addition to the genetic marker fpt, have the markers "ompT and dsbA " .
  • the present invention also relates to methods for identifying bacteria which Present heterologous proteins with an affinity for a binding partner on their surface and methods for constructing vectors encoding these proteins Finally, the present invention also relates to bacteria which stably present at least one fusion protein on their surface and which have the genetic characteristics fpt, ompT and dsbA
  • the method according to the invention allows, for example, the production of protein fusions which are composed of portions of heavy and light antibody domains and the transport protein Igass and their export through the bacterial cell envelope.
  • a fundamental phenomenon of biological systems is specific interactions between receptor molecules and ligands. Even extremely complex processes in highly developed organisms can be reduced to simple molecular interactions and can be carried out on them
  • SPARE BLADE (RULE 26) Level can be analyzed and influenced effectively.
  • the material basis of such a system is formed by both the producer cells and the genetic information, in which the blueprint of a biomolecule is specified.
  • the producer cells must be capable of reproduction and at the same time be able to express heterologous genetic information and to present the corresponding biomolecules on their surface in an accessible manner. Ideally, they are easy to manipulate genetically, physico-chemically and genetically stable and undemanding in terms of their growth conditions.
  • a suitable microrgamsmus that this Escherichia coli is met.
  • Proteins are preferably suitable as biomolecules.
  • the underlying genetic information can range from a few codons to several genes, depending on whether a short peptide, a protein or a receptor consisting of several protein molecules is encoded. With the usual molecular biological methods, a large number of protein variants can be generated at the genetic level and the expression of the heterologous information can be made possible after introduction into the producer cells.
  • an essential prerequisite for the functioning of a cellular selection system is the presentation of relevant protein molecules on the cell surface. Accessibility for potential binding partners must be guaranteed and the binding properties must not be impaired by cellular determinants.
  • Escherichia coli various systems for the presentation of recombinant proteins were processed. The main feature of these systems is the use of protein fusions and the underlying gene fusions.
  • the fusions contain carrier protein portions. The carrier portions enable the export of the protein fusions from the cytoplasm over the two cell membranes of E. coli and their anchoring in the outer membrane.
  • Protein sequences attached to the carboxyl end of such an Lpp-OmpA fusion are accessible after export on the surface of recombinant E. coli cells (Francisco et al., Proc. Natl. Acad. Sci. USA 89 (1992), 2713- 2717).
  • the topological fixation of the amino end of the protein sequences to be presented to the membrane-anchoring OmpA sequence can have a negative effect, especially in cases where a free amino end is necessary for functional reasons.
  • OmpT protease a protein located in the outer membrane. Their activity directed towards the surface of the bacteria leads to the cleavage of Igass fusions and thus to the uncoupling of presented passenger proteins (Klauser et al., EMBO J. 11 (1992), 2327-2335). This activity prevents the physical coupling of binding properties of the passenger proteins with the cell proliferation apparatus. A selection of the producer cells is therefore excluded. This effect can be avoided by using OmpT-negative E. coli host cells.
  • the antibody fragments are in the form of protein fusions with the phage coat proteins g3p or g8p. In both cases it is possible to select from a population of recombinant phages those which have a desired specificity given by the surface-presented antibody fragment (Winter and Milstein, Nature 349 (1991), 293-299).
  • a decisive disadvantage of using phages as carriers of antibody fragments against bacterial cells is that phages do not replicate themselves but require host bacteria that only ensure their multiplication.
  • phages with the desired binding properties therefore requires complex cycles of selection (selection) and infection (phage multiplication). This disadvantage does not appear when whole bacterial cells are used as carriers of recombinant antibody fusions.
  • a method for identifying recombinant antibodies based on the phage Lamba does not allow the selection of specificities but requires complex screening procedures (Lerner et al., Science 252 (1991), 659-667).
  • the Igass domain or C-terminal portions of this domain are predestined as membrane anchors for the presentation of recombinant antibody domains on the surface of Gram-negative bacteria such as E. coli, Salmonella and Neisseries.
  • a bacterial system is characterized by the direct coupling of functional antibodies presented on the surface with the reproductive apparatus of living cells.
  • the antibody fragments can either be obtained from the cytoplasm in the form of inclusion bodies or from Pere periplasma as a soluble protein.
  • the antibody fragments present in the periplasm are correctly folded, therefore biologically active and can be purified directly from fractions of the periplasm.
  • the inclusion bodies present in the cytoplasm consist of denatured antibody fragments which show no biological activity. The latter can be achieved by simply cleaning the antibody fragments by de-and renaturation.
  • the recombinant antibody fragments are able to bind antigen with an affinity that is equivalent to the native antibody molecule.
  • the invention thus relates to a bacterium for producing at least one stable fusion protein from a carrier protein and a passenger protein, the bacterium having the genetic characteristic fpt.
  • the abbreviation fpt denotes a genetically determined property of E. coli which leads to the tolerance of the instability of the bacteria which occasionally occurs during the expression of exported fusion proteins. This marker is located between position 85 and 89 minutes of the gene map of the E. coli genome.
  • the genetic marker fpt can be used to produce bacteria that are tolerant of fusion proteins.
  • the term “carrier protein” denotes amino acid sequences within a fusion protein which do not belong to the passenger protein and which are important for the localization and the stability of the fusion protein in the bacteria.
  • the carrier protein portions include a signal peptide at the amino end of the fusions and the Igass transprotdomain at the carboxyl end of the fusions.
  • the carrier protein portions For fusion proteins that are transported into the periplasm, the carrier protein portions contain a signal peptide at the amino end of the fusions.
  • the term “passenger protein” denotes amino acid sequences within a fusion protein that do not belong to the carrier protein.
  • the passenger protein portions contain those sequences that interact with binding partners.
  • the cause is the formation of intramolecular disulfide bridges in the passenger proteins by the periplasmic disulfide oxidoreductase DsbA. This blockage of the translocation of passenger proteins by the outer membrane is not observed in bacterial cells which carry a mutation in the dsbA gene.
  • the present invention therefore relates to a bacterium with the genetic marker fpt, the fusion protein being stably presented on the surface, the bacterium being gram-negative and also having the genetic markers ompT- and dsbA-.
  • the term “presented on the surface” denotes the localization of a fusion protein or the passenger protein on the side of the outer bacterial membrane facing the medium.
  • Passenger proteins are freely accessible to binding partners in intact gram-negative bacteria.
  • ompT- denotes a genetic modification of E. coli cells that leads to a deficiency in the proteolytic activity of the protease OmpT.
  • dsbA denotes a genetic change in E. coli cells that leads to a deficiency in the activity of the periplasmic oxidoreductase DsbA.
  • the term dsbA is to be understood such that this does not only result in a deficiency in the activity due to a genetic change the oxidoreductase DsbA itself, but also by other factors that indirectly inhibit the activity of this oxidoreductase.
  • E. coli UT5600 has already mutated in one gene (ompT).
  • the transfer of the mutation in the dsbA gene to E. coli UT5600 was carried out by E. coli JCB571 (dsbA :: Kan; zihl2 :: Tnl0) using phage PI transduction. Positive transducers were selected for the cotransduction of a transposon Tn10 insertion near the dsbA gene locus. This also transferred the property determined by adjacent DNA sections that enables an E. coli cell, e.g. VH-Igass to produce stable fusions.
  • the bacterium is used to produce a fusion protein, the carrier protein obtained in the fusion protein containing the Igass protein or a fragment thereof which enables the fusion protein to be secreted.
  • Igass denotes the transport domain located at the carboxyl end of the Neisseria IgA protease precursor protein. This domain mediates the transport at the amino terminal -li ⁇ coupled proteins from the periplasm of gram-negative bacteria through the outer membrane.
  • the passenger protein produced by the bacterium according to the invention is a protein with an affinity for a binding partner, an antibody or an antigen-binding domain of an antibody, an antigen, a protein with enzymatic activity, an inhibitor, a receptor, a ligand or a nucleic acid binding protein.
  • binding partner denotes a molecule, a chemical compound or a macromolecule. Binding partners can be either freely soluble, bound to a matrix or associated with a biological membrane.
  • the term “antigen-binding domain” denotes at least the portion of an antibody molecule that is sufficient for the specific binding of an antigen.
  • variable antibody domains are exported as a monomeric ("single-chain Fv") scFv-Igass fusion, in which the domains VL and VH are covalently linked by a short peptide (eg a (Gly 4 Ser) 3 linker) or as two separate VL and VH Igass fusions within a bacterial cell. In the latter case, the antibody components of both fusions accumulate on the cell surface to form a functional Fv fragment.
  • a short peptide eg a (Gly 4 Ser) 3 linker
  • the bacterium used to produce the fusion protein is the E. coli JK 321 strain. This strain was obtained on December 22, 1993 from the DSM (German Collection of Microorganisms, 38124 Braunschweig, Ma ⁇ cher or Weg lb, Germany) under the deposit number DSM 8860.
  • Another object of the present invention is to provide the genetic marker fpt for producing a tolerance of bacteria to fusion proteins.
  • strains containing this marker which enables the stable surface exposure of fusion proteins, offers a number of possible uses. For example, it is conceivable to select a particular E. coli clone with certain properties from any immunoglobulin Igass library using any antigen.
  • a further object of the present invention is therefore to provide a method for the identification of bacteria which stably present proteins with an affinity for a binding partner on their surface, the method comprising the following steps:
  • step (c) culturing the bacterium from step (b), so that the bacteria of the culture obtained present the fusion protein or the fusion proteins stably on their surface;
  • the presentation of more than one fusion protein on the cell surface may, for example, be desirable in those cases in which individual chains of multimeric proteins are expressed separately and these then separate assemble the cell surface into a functional complex.
  • a bank of DNA sequences is used to construct the vector in step (a), in which the respective DNA sequences encode variants of the passenger protein or proteins, or variants of the carrier protein or the carrier proteins.
  • the term “variants of passenger proteins” denotes amino acid sequences which differ from passenger proteins and which have modified binding properties with respect to a binding partner.
  • variants of carrier proteins denotes carrier proteins modified in their amino acid sequence, which have changed transport properties with respect to coupled passenger proteins.
  • variants of the passenger protein for example, an antibody fragment with a given specificity could be optimized for a specific application by directed mutagenesis.
  • An evolution-like adaptation to an external factor through undirected mutations is possible through the continuous cultivation of E. coli cultures.
  • enzymatic activities can be presented, selected and modified.
  • the generation of variants of the carrier protein can result in a better adaptation to a given passenger protein or its variants, which can facilitate the secretion of the fusion protein.
  • the bacterium is grown in step (c) under conditions in which the DNA sequence encoding the fusion protein or the fusion proteins is mutated, so that the bacteria of the culture obtained variants of the Present fusion protein or the fusion proteins on their surface.
  • mutating conditions denotes cultivation conditions under which the addition of chemical substances and / or the action of ionizing radiation leads to an increased mutation rate in bacterial cultures.
  • the carrier protein contained in the fusion protein contains the Igass protein or a fragment thereof, which enables the fusion protein to be secreted.
  • the protein with an affinity for a binding partner is an antibody or an antigen-binding domain of an antibody, an antigen, a protein with enzymatic activity, an inhibitor, a receptor, a ligand or a nucleic acid. binding protein.
  • the bacteria which stably present the desired fusion protein on their surface can by interacting with the binding partner of the passenger protein bound to a matrix, by interacting with a fluorescence-labeled binding partner or by interacting with the Magnetic particle-bound binding partner of the passenger protein can be isolated.
  • Another object of the present invention is to provide gram-negative bacteria which stably present at least one fusion protein on their surface and which have the genetic markers fpt, ompT- and dsbA-.
  • a preferred embodiment relates to bacteria in which the carrier protein contained in the fusion protein contains the Igass protein or a fragment thereof which secretes of the fusion protein and / or the passenger protein enables a protein with an affinity for a binding partner, an antibody or an antigen-binding domain of an antibody, an antigen, a protein with enzymatic activity, an inhibitor, a receptor, a ligand or a nucleic acid-binding Is protein.
  • a further object of the present invention is the use of these bacteria for diagnostic purposes, for the production of proteins with an affinity for a binding partner, for the production of detergents, for the development of raw materials, for food processing or for the degradation or enrichment of pollutants.
  • the bacteria presenting specific antibody fragments according to the invention can be used to obtain these antibody fragments and these can then be used in the diagnostic or therapeutic field, if appropriate after purification.
  • Recombinant antibody fragments which, for example, specifically recognize surface structures of tumor cells, can be used in conjunction with a radionuclide or an effective toxin for therapeutic applications.
  • Binding specificities selected with the method according to the invention can be implanted ("humanized") by genetic engineering of the CDR (complementarity determining regions) regions in human immunoglobulin chains and used for the therapy of diseases.
  • Fig. 1 Schematic representation of the strategy for the construction of the VH-igass fusion in plasmid pJK165.
  • the VH gene fragment was fused at the 5 'end with the signal sequence of the ompA gene and at the 3' end with the igass gene fragment.
  • the DNA fragments required for this were linked together in a three-fragment ligation.
  • the non-coding region and the signal sequence of the ompA gene from E. coli DH5a were synthesized using chromosomal DNA as a template and the oligonucleotides JK20 and JK21 (Fig. 8) by PCR.
  • the PCR fragment obtained was inserted directly into the pCRIOOO vector without prior incubation with restriction enzymes (pJK129).
  • the VH gene fragment was also synthesized by PCR, in which case the corresponding cDNA served as a template and the oligonucleotides JK09 and JK17 (Fig. 8) were used.
  • the PCR fragment obtained was then incubated with Klenow poly erase to fill in protruding ends and, after restriction with EcoRI, inserted into the HincII / EcoRI sites of the pEMBL ⁇ vector.
  • the XhoI / EcoRI VH gene fragment was isolated from the plasmid pJK92 obtained and ligated together with the Clal / Xhol ompA gene fragment and an XhoI / EcoRI vector fragment from pTK59.
  • the plasmid pJK165 formed encodes a VH-igass fusion whose "-10 region" of the promoter was reconstituted in accordance with the starting plasmid pTK59.
  • Fig. 2 Schematic representation of the strategy for the construction of the VL-ig- necessarily fusion in the plasmids pJK78 and pJK257.
  • the plasmid pJK78 was created by incorporating a DNA fragment with the VL gene fragment of a monoclonal antibody into the Ndel / EcoRI vector fragment of the plasmid pTK59.
  • the VL gene fragment was generated using the oligonucleotides JK08 and JK14 (Fig. 8) in a polymerase chain reaction and cloned into the pCRIOOO vector (pJK56).
  • the corresponding DNA fragment of the VL gene was processed at the Ndel and EcoRI interfaces introduced by the oligonucleotides at the 5 'and 3' ends and ligated to the Ndel / EcoRI vector fragment of the plasmid pTK59.
  • the resulting plasmid was named pJK78. b.
  • the exchange of the gene of the b-lactamase (bla) and the ColEl replication origin on the plasmid pJK78 took place in two stages.
  • the gene of the chloramphenicol acetyl transferase (cat) of plasmid pACYC184 was amplified by PCR using the oligonucleotides JK32 and JK33 (Fig. 8).
  • the PCR fragment obtained was cloned into pJK78 vector fragment cut with HindIII.
  • the resulting plasmid pJK250 is characterized by the presence of two resistance genes (bla and cat) and the ColEl origin of replication.
  • the vector fragment (bla and ColEl) was deleted from pJK250 by restriction with Clal and Nhel and substituted from the plasmid pACYC184 by a Clal / Nhel question (pl5A origin).
  • the resulting plasmid pJK257 encodes a VL-igass fusion and allows its coexpression with the plasmid pJK165 (VH-iga ß ) within E. coli JK321 cells.
  • VH-Igass construct pJK165 was created by substituting the Clal / EcoRI fragment of pTK59 with the VH gene fragment and a DNA fragment that encodes the first 204 base pairs of the E. coli ompA transcript.
  • the VH-Igass fusion has at its N-terminus the signal peptide of the OmpA precursor protein, (ii)
  • the VL-Igass construct pJK78 was created by inserting the VL gene fragment into the Ndel / EcoRI interfaces of pTK59; the VL-Igass fusion formed in this way consequently carries the CtxB signal peptide.
  • E. coli JK321 Mode of action of a-protein on the surface of antibody-presenting E. coli JK321 by immunofluorescence labeling.
  • the cell suspension was placed in a rotary incubator for a further 30 min and then washed once in PBS.
  • Surface-bound antigen was fixed with paraformaldehyde (1%) / glutardialdehyde (0.5%) and detected with antigen-specific, polyclonal serum (anti-Fp80) and FITC-coupled mouse anti-rabbit IgG serum.
  • Fig. 6 Schematic representation of a method for the procedure of the antibody-presenting E. coli JK321.
  • Fig. 7 Selective enrichment of VL / VH-Igass coexpressing E. coli JK321 by antigen-labeled mitrocellulose. After separation of a-protein (about 10 ng / lane) by SDS-PAGE, the antigen was then blotted onto nitrocellulose. A nitrocellulose strip was developed with anti-Fp80 as an immunoblot in order to make the exact position of the a-protein band visible. To carry out the binding experiment, the E. coli strains UT5600 (pTK59; Rif S ), JK321 (pTK59; Rif R ) and JK321 (pJK165 / pJK257; Rif R ) were grown overnight as liquid cultures.
  • the two recombinant, Rif-resistant JK321 strains were diluted in a ratio of about 1: 2000 against the Rif-sensitive strain UT5600 (pTK59). After incubation of the bacterial suspension in PBS / BSA (1%) for 30 minutes, 1 ml of this was placed in 15 ml of PBS and placed in an empty petri dish together with a strip of nitrocellulose. After incubation with gentle shaking for 30 minutes at RT was washed three times with PBS (20 ml) and then the nitrocellulose was incubated on Rif-containing nutrient plates.
  • (+) and (-) refer to the coding or the complementary
  • VH-Igass fusion protein exerts a cytotoxic effect on the E. coli K12 irt ⁇ cells.
  • JK321 a section of the genome of an E. coli strain (JCB571) (Bardwell, loc. Cit.), Which carries a mutation in an unknown gene, was transferred to another E. coli strain (UT5600) ( Earhart, loc. Cit.).
  • UT5600 Earhart, loc. Cit.
  • Pl-phage transduction is usually used to map genes on the chromosome (Taylor and Trotter, Bacteriol. Reviews 31 (1967), 332- 344).
  • Pl phages multiply in bacterial cells, they pack not only viral DNA, but occasionally a piece of the host cell genome Virus envelope.
  • the length of the packed bacterial DNA is up to 2 minutes of the E. coli genome, which enables the co-transduction of two distant genetic markers. If this host cell DNA gets into another bacterial cell in a subsequent PI infection, this DNA fragment can recombine there into the chromosome.
  • E. coli JCB571 (d ⁇ bA :: Kan, zihl2 :: Tn10) was selected as the donor and E. coli UT5600 (ompT-) as recipient strain.
  • E. coli UT5600 (ompT-) was selected as the donor and E. coli UT5600 (ompT-) as recipient strain.
  • positive transductants which had reduced the mutated unknown gene locus in conjunction with the mutated d ⁇ bA gene and integrated into their chromosome, were grown with growth medium and growth medium on culture medium Tetracycline selected.
  • the strain JK321 After transformation with the plasmids pJK78 (VL-Igass) and pJK165 (VH-Igass), the stable expression of these Igass fusion proteins in E. coli JK321 could be shown (Fig. 4).
  • the strain JK321 also enables simultaneous coexpression of the plasmids pJK78 (VL-Igass) and pJK165 (VH-Igass) within a bacterial cell.
  • oligonucleotide JK21 (Fig. 8) was provided with an Xhol cleavage site, which enabled fusion with the 5 'end of the VH gene fragment.
  • a VH gene fragment was amplified by PCR using the oligonucleotides JK17 and JK09 (Fig.
  • the plasmid formed from this was designated pJK165 (Fig. 1).
  • E. coli JK321 After transformation of E. coli JK321 with plasmid pJK165 a high stability of the recombinant bacteria is observed. This effect does not occur in recombinant E. coli K12 wild-type cells.
  • E. coli K12 wild-type cells which carry the plasmid pJK165 a destabilization of the recombinant bacteria and the formation of mutants which no longer synthesize the VH-Igass fusion protein are observed due to a cytotoxic effect of the VH-Igass fusion protein.
  • the molecular background of these mutations is the breakdown of the plasmid pJK165 into a smaller derivative, from which the VH-igass fusion is no longer expressed.
  • VL-igass fusion was constructed in two stages (plasmids pJK78 and pJK257): in the first step, the VL domain was connected to a signal peptide at the N-terminus and to the Igass domain at the C-terminus (plasmid pJK78). The signal peptide ensures the transport of the fusion protein through the cytoplasmic membrane and the Igass domain subsequently mediates the transport of the VL antibody domain through the outer membrane.
  • the ColE1 replication origin and the gene of the ⁇ -lactamase (bla) in the plasmid pJK78 were replaced by the pA15 replication origin and the gene of the chloramphenicol acetyl transferase (cat) of the cloning vector pACYC184 (Rose, Nucl. Acid Res 16 (1978), 355) in plasmid pJK257.
  • the detailed construction of both plasmids is described in the legends to Fig. 2a / b and Fig. 3.
  • the use of different replication origins and resistance genes in the plasmids pJK165 and pJK257 enables the copropagation of both plasmids within one bacterial cell. '
  • the developed E. coli strain JK321 enables the Igass-mediated exposure of variable antibody domains on the cell surface and the binding of the specific antigen.
  • VH or VL Igass fusion proteins e.g. pJK165 or pJK257
  • the simultaneous production of the VL and VH Igass fusions of two plasmids e.g. pJK165 and pJK257
  • the two fusion proteins are transported separately from one another into the outer membrane, where the individual VL and VH domains of the fusion proteins assemble to form an Fv fragment.
  • the VL and VH Igass fusions are exposed as an Fv-Igass heterodimer on the cell surface of E. coli JK321. Recombinant E.
  • E. coli JK321 which export heterodimeric VH / VL-Igass fusion proteins to the cell surface, bind the specific antigen via their immunoglobulin components (Fig. 5). Furthermore, recombinant E. coli JK321 cells, which coexpress the VH and VL-Igass fusion proteins within a bacterial cell of two plasmids, can be used by using the specific antigen from a dilution against E. coli JK321 cells, the other, non-specific Igass fusion proteins (e.g. export CtxB-Igass, plasmid pTK59), enrich (Fig. 7).
  • Example 5 Development of a method for the detection of the enriched, antibody-presenting E. coli JK321 cells.
  • E. coli JK321 cells have a resistance gene against the antibiotic Rifa picin. This was in E. coli JK321 cells have been transformed into a DNA fragment that bears such a resistance gene. The DNA fragment recombined into the genome of E. coli JK321 and led to the expression of the Rifa picin resistance.
  • a specific genetic marker eg antibiotic resistance
  • E. coli JK321 host cells only allows the selective growth of the recombinant E. coli clones on Rifa picin-containing medium, which simultaneously present VL and VH Igass fusions with a specificity selected before the experiment.
  • the recombinant control cells used eg E. coli UT5600
  • the use of host cells that are resistant to rifampicin (JK321) or sensitive (UT5600) enables the specific, antibody-presenting cells to be clearly identified (Fig. 6).

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  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Peptides Or Proteins (AREA)
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Abstract

L'invention concerne des bactéries utilisées pour la préparation de protéines de fusion stables, à partir d'une protéine de transport et d'une protéine transportée, lesdites bactéries comportant le marqueur génétique fpt. Le fait de comporter ce marqueur génétique permet de parvenir à une meilleure production de fusions de protéines, ce qui a un effet déstabilisant sur les bactéries. Cette invention concerne notamment des bactéries utilisées pour préparer des protéines de fusion, où la protéine de fusion est présentée de manière stable sur la surface et où les bactéries comportent en plus du marqueur fpt, les marqueurs ompT- et dsbA-. L'invention concerne en outre des procédés permettant d'identifier des bactéries qui présentent sur leur surface, des protéines hétérologues ayant une affinité avec des partenaires de liaison, ainsi que des procédés permettant de créer par recombinaison des vecteurs codant ces protéines. Enfin, l'invention concerne des bactéries qui présentent sur leur surface, de manière stable, au moins une protéine de fusion et qui comportent les marqueurs génétiques fpt, ompT- et dsbA-, ainsi que leur utilisation, par exemple, à des fins de diagnostic.
EP95905579A 1993-12-23 1994-12-22 Bacteries utilisees pour preparer des proteines de fusion stables et procede permettant de les identifier Withdrawn EP0731837A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4344350 1993-12-23
DE4344350A DE4344350C2 (de) 1993-12-23 1993-12-23 Bakterien zur Herstellung stabiler Fusionsproteine und Verfahren zu deren Nachweis
PCT/EP1994/004286 WO1995017509A1 (fr) 1993-12-23 1994-12-22 Bacteries utilisees pour preparer des proteines de fusion stables et procede permettant de les identifier

Publications (1)

Publication Number Publication Date
EP0731837A1 true EP0731837A1 (fr) 1996-09-18

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Application Number Title Priority Date Filing Date
EP95905579A Withdrawn EP0731837A1 (fr) 1993-12-23 1994-12-22 Bacteries utilisees pour preparer des proteines de fusion stables et procede permettant de les identifier

Country Status (5)

Country Link
US (1) US6040141A (fr)
EP (1) EP0731837A1 (fr)
JP (1) JPH11514201A (fr)
DE (1) DE4344350C2 (fr)
WO (1) WO1995017509A1 (fr)

Families Citing this family (9)

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Publication number Priority date Publication date Assignee Title
DE4041045A1 (de) * 1990-12-20 1992-07-02 Max Planck Gesellschaft Zweiphasen-systeme fuer die produktion und praesentation von fremdantigen in hybriden lebendimpfstoffen
US6100043A (en) * 1995-08-04 2000-08-08 The Perkin-Elmer Corporation Recombinant clone selection system
US5843656A (en) * 1995-08-07 1998-12-01 The Perkin-Elmer Corporation Recombinant clone selection system
AU6844696A (en) * 1995-08-07 1997-03-05 Perkin-Elmer Corporation, The Recombinant clone selection system
EP0886678B1 (fr) * 1996-03-15 2005-06-29 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Systemes d'exportation pour proteines recombinees
WO2002070645A2 (fr) * 2001-03-02 2002-09-12 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Affichage en surface fonctionnel de polypeptides
DE10314413B4 (de) * 2003-03-28 2015-08-06 ZYRUS Beteiligungsgesellschaft mbH & Co.Patente I KG Verfahren zum Nachweis der Expression rekombinanter Proteine an einer Zelloberfläche und zur Immobilisierung und Aufreinigung von Zellen und Proteinen
KR101387693B1 (ko) * 2008-12-04 2014-04-21 한국생명공학연구원 고분비성 단백질의 스크리닝 및 재조합 단백질의 생산을 위한 융합 파트너로서 그의 용도
CN108359676A (zh) * 2013-03-13 2018-08-03 自动显示生物技术有限公司 功能性蛋白在范围广泛的革兰氏阴性细菌中的改善的表面展示

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Publication number Priority date Publication date Assignee Title
NZ236819A (en) * 1990-02-03 1993-07-27 Max Planck Gesellschaft Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions
DE4122598C1 (fr) * 1991-07-08 1992-07-30 Deutsches Krebsforschungszentrum Stiftung Des Oeffentlichen Rechts, 6900 Heidelberg, De
US5348867A (en) * 1991-11-15 1994-09-20 George Georgiou Expression of proteins on bacterial surface

Non-Patent Citations (1)

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Title
See references of WO9517509A1 *

Also Published As

Publication number Publication date
US6040141A (en) 2000-03-21
DE4344350C2 (de) 1995-09-21
JPH11514201A (ja) 1999-12-07
WO1995017509A1 (fr) 1995-06-29
DE4344350A1 (de) 1995-06-29

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