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WO1994000581A1 - Systeme d'expression de lactobacillus utilisant des sequences geniques de proteine capsidique - Google Patents

Systeme d'expression de lactobacillus utilisant des sequences geniques de proteine capsidique Download PDF

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Publication number
WO1994000581A1
WO1994000581A1 PCT/FI1993/000273 FI9300273W WO9400581A1 WO 1994000581 A1 WO1994000581 A1 WO 1994000581A1 FI 9300273 W FI9300273 W FI 9300273W WO 9400581 A1 WO9400581 A1 WO 9400581A1
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ala
thr
leu
sequence
ser
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PCT/FI1993/000273
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Airi M. Palva
Ilkka A. Palva
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Viagen Oy
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Priority to AU43297/93A priority Critical patent/AU4329793A/en
Publication of WO1994000581A1 publication Critical patent/WO1994000581A1/fr

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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Definitions

  • the invention is in the area of molecular biology of the Lactobacilli. Specifically, the invention is directed to Lactobacilli surface coat protein (SP) expression and secretion units and their use for the expression of recombinant genes in Gram-positive bacteria, and especially in Lactobacillus.
  • SP Lactobacilli surface coat protein
  • Lactic acid bacteria including genera Lactobacillus, Pediococcus, Leuconostoc and Lactococcus have a central role in food processing and are of substantial economic importance. Of the above genera, Lactobacillus is the most widely applied.
  • the food products where fermentation by lactic acid bacteria is used include cheese varieties, yogurt, kefir, acidophilus milk, dry sausages, fermented vegetables and soured bread.
  • the main role of lactic acid bacteria in food fermentation is the preservation of the food by the production of lactic acid and other metabolites, and the production of a desired effect on the flavor and texture of the product by these bacteria (Trends in Food Biotechnology: 121-127 (1989), N. Hen and L. Kong, eds.; FEMS Microbiol. Rev. 87:3-14 (1990)).
  • lactic acid bacteria Due to the central role of lactic acid bacteria in industrial processes, a large number of potential applications for these bacteria have been proposed.
  • Use of lactic acid bacteria has been proposed to provide a host with improved resistance to phage, to stabilize the process activity, to enhance flavor production, produce antimicrobials and exocellular polysaccharide, to accelerate cheese ripening and to control bitterness in the final food product.
  • targets for Lactobacillus engineering are either improvements of existing industrial process characteristics and food products, or applications where Lactobacilli are used as new production hosts for food and feed industry related products, especially for those requiring GRAS (Generally Recognized As Safe)-status host systems. Additional applications will no doubt emerge as soon as the molecular characterization of Lactobacilli advances.
  • Figure 1(A-D) depicts the cloning strategy and the primers used for PCR-amplification of the L. brevis DSM20556 SP gene.
  • Figure 1A PCR-Fragments produced from DSM20556 DNA using oligonucleotides shown below in D.
  • Figure IB The physical map of the S-layer region with relevant restriction enzyme sites. The open arrow heads refer to the end points of the DNA-sequence shown in Figure 2B.
  • Figure 1C Cloning strategy of the 5'- and 3'- regions of the SP gene.
  • Figure 1D Oligonucleotides used for synthesis of PCR fragments 1 to 6 [SEQ ID Nos. 1-12].
  • Figure 2(A-B) is the nucleotide sequence the L. brevis SP gene and predicted amino acid sequence.
  • Figure 2A Upstream region of the SP gene from nucleotide position -320 to -1 [SEQ ID No. 13].
  • Figure 2B DNA sequence from the nucleotide + 1 [SEQ ID No. 14] and predicted amino acid sequence [SEQ ID No. 15].
  • the predicted -10 and -35 regions of the promoters PI and P2 are underlined and the 5'- ends of the transcripts found by primer extension (see Fig. 3(A-B)) are marked with arrow heads.
  • the cleavage site of the signal peptide and the mature protein is between amino acids 30 and 31 ( ⁇ ).
  • the N-terminal amino acid sequences of the intact S- layer protein and its tryptic peptides are underlined and numbered from (1) to (5) (see Table 1).
  • the deduced transcription-terminator is shown with arrows.
  • RBS refers to the predicted ribosome binding site.
  • Figure 3(A-B) is an analysis of SP mRNA.
  • Figure 3A Northern blot analysis of transcripts.
  • Total L. brevis GRL1 RNA denatured with glyoxal and DMSO was run in a 0.8% agarose gel using 10 mM phosphate buffer, pH 6.5, followed by blotting to ZETAPROBE ® membrane and hybridization.
  • the [ ⁇ - 32 P] dCTP labelled PCR1 fragment was used as a probe.
  • the filter was washed in 0.5xSSC, 0.1 % SDS at 50°C.
  • RNA molecular weight markers (Bethesda Research Inc.) were used to determine transcript size.
  • Figure 3B Analysis of mRNA-transcripts after the 5 '-end mapping experiment in a 6% sequencing gel. Approximately 5 ⁇ g of total RNA of L. brevis was hybridized to 200 pmol of a 19-mer primer (5'-CTTAGCCATATGAGCCTTA-3', [SEQ ID No. 16] see Fig. 2, position 507-489). After ethanol precipitation, the primer extension of the hybrids was performed in the presence of [ ⁇ - 32 P] dCTP, nonlabelled nucleotides, AMV reverse transcriptase, actinomycin C1 and RNASIN. For determination of the lengths of the extended products, sequencing reactions of PCR2-fragment (see Fig. 1(A)) were performed with the same primer. Summary of the Invention
  • Lactobacillus coat protein is apparently synthesized under a wide variety of culture conditions that support host cell replication, indicating that the surface coat protein promoter must be essentially constitutive and thus devoid of common control repression systems (e.g. catabolite repression, response to nitrogen- or phosphate-limitation or aeration).
  • control repression systems e.g. catabolite repression, response to nitrogen- or phosphate-limitation or aeration.
  • the present invention provides a method to isolate SP, such method providing SP essentially free of natural contaminants and therefore of the requisite degree of purity needed to sequence and clone SP genetic sequences from Lactobacillus sources.
  • cloned constructs that provide recombinant Lactobacillus SP genetic sequences and their regulatory elements have been identified.
  • Lactobacillus SP may be isolated from frozen or fresh Lactobacilli bacteria. As lactobacilli appear to express SP under all growth conditions, any culture method that supports growth or viability may be used. Because the coat protein is so abundant, it may be isolated by electrophoretic methods, utilizing gel electrophoresis as the medium in which to fractionate the proteins.
  • Lactobacillus brevis strain DSM 20556 German Collection of Microorganisms, Braunschweig, FRG
  • MRS-medium Difco
  • the bacteria are then collected by centrifugation at, for example, 10,000 g for 5 min at room temperature, washed once with a neutral, mild buffer such as 50 mM Tris-C1, pH 7.5, and recentrifuged.
  • the cell pellet is suspended in a small volume of the wash buffer and then directly dissolved by addition of SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, ph.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • cells from a 1 ml culture may be suspended in 200 ⁇ l of Laemmli sample buffer and this amount applied to a preparative SDS-PAGE gel (approximately 1 ⁇ l/200 ⁇ l gives a clear visible band in Coomassie Blue staining).
  • Protein staining of the gel will reveal a single major band of about 45-50 kDaltons (kd). Specifically, SP of Lactobacillus brevis reveals a major band at about 46 kd.
  • Coat protein may be visualized on a preparative gel by treating the gel with 1 M KCl rather than Coomassie Blue, so as to allow for subsequent excision of the protein band from the preparative gel for sequence determinations, etc. Protein is eluted from the cut out pieces of such preparative gels by mixing the gel pieces with a buffered strong denaturant in the presence of a chelator.
  • a buffer providing 6 M guanidine hydrochloride, 0.5 M Tris-HCl, 2 mM EDTA, pH 7.5 and mixing for 10 hours, in an end-over-end mixture will suffice to elute the protein from the gel pieces.
  • SP purified by direct excision from the gel in the above manner, or in a manner wherein equivalents of the above sequence of steps are utilized, is purified to an extent capable of being sequenced by techniques known in the art or may be used to raise antibodies.
  • the protein eluted from the gel is dialyzed against a low salt, mildly basic buffer such as 10 mM Tris-HCl, pH 8.5, and freeze dried to concentrate the protein.
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of a hapten which can be recognized and bound by an antibody.
  • An antigen may have one or more than one epitope.
  • An "antigen” is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • the specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al. , J. Nucl. Med. 24:316-325 (1983)).
  • the antibodies of the present invention are prepared by any of a variety of methods.
  • purified Lactobacillus SP, or an antigenic fragment thereof is administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding SP.
  • Cells expressing Lactobacillus SP, or an antigenic fragment thereof, or, a mixture of proteins containing Lactobacillus SP or antigenic fragments thereof can also be administered to an animal in order to induce the production of sera containing polyclonal antibodies, some of which will be capable of binding SP.
  • SP antibody may be purified from the other polyclonal antibodies in the preparation by standard protein purification techniques and especially by affinity chromatography with purified coat or fragments thereof.
  • SP or a fragment of SP may be chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.
  • Monoclonal antibodies can be prepared using hybridoma technology known in the art (Kohler et al., Nature 256:495 (1975); Kohler et al. , Eur. J. Immunol. 6:511 (1976); Kohler et al. , Eur. J. Immunol. 6:292 (1976); Hammerling et al. , in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y. , pp. 563-681 (1981)).
  • such procedures involve immunizing an animal with SP.
  • the splenocytes of such animals are extracted and fused with a suitable myeloma cell line.
  • any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
  • SP 2 O myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, J.R., et al. , Gastro-enterology 80:225-232 (1981).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the SP.
  • Antibodies against both highly conserved and poorly conserved regions of Lactobacillus SP are useful for the identification of the isolated protein as being the coat protein by means of physical analyses such as immunogold electromicroscopy imaging, and also for the identification of clones expressing such protein and studies on the control of biosynthesis and catabolism of coat protein in the native Lactobacillus host and in heterologous hosts.
  • the process for genetically engineering Lactobacillus SP genetic sequences is facilitated through the isolation and sequencing of pure Lactobacillus SP and by the cloning of sequences capable of encoding such SP.
  • the term "genetic sequence” is intended to refer to a nucleic acid molecule such as DNA or RNA, preferably DNA. Genetic sequences which encode Lactobacillus SP may be derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • genomic DNA as genomic DNA will provide not only the SP encoding sequences but also the transcriptional and translational regulatory elements thereof as such, for example, the 5' promoter region of the SP gene, the 3' transcriptional termination region, the genetic sequences which provide the 5' non-translated region of the SP mRNA (if any) and/or the genetic sequences which provide the 3' non-translated region (if any).
  • a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then such regulatory signals may be retained and employed for transcriptional and translational regulation in a host even if such host is not a member of the Lactobacilli.
  • Lactobacillus SP genomic DNA can be extracted and purified from any Lactobacillus cell which naturally expresses SP by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al , eds., Academic Press (1987).
  • Some Lactobacillus species have cell walls that are highly resistant to enzymatic lysis.
  • L. brevis strain DSM 20556 is highly resistant to enzymatic lysis.
  • a novel method has been developed to facilitate membrane lysis. As described below, this method involves the steps of (1) .
  • Na-perchlorate (2.2 ml of 5M Na-perchlorate per 15.75 ml cell volume after the SDS addition) is then added and the cell lysate extracted with chloroform-isoamylalcohol (24: 1). The water phase is removed after centrifugation and nucleic acids are precipitated by bringing the sample to 66% ethanol. Chromosomal DNA is collected around a glass rod and dissolved in an appropriate amount of 10 mM Tris-HCl - 150 mM NaCl, pH 7.5.
  • the chromosomal DNA may be cleaved with restriction enzymes such as HaeIll and fragments of the desired size separated by electrophoresis, such as by agarose gel electrophoresis, using methods known in the art.
  • restriction enzymes such as HaeIll
  • the restriction fragments may be cloned into various commercial vectors, such as, for example, lambda phage vectors, including lambda gt10, according to the manufacturer's instructions, for form a recombinant gene library.
  • mRNA is used as the source of the coat protein encoding sequences
  • such mRNA is preferably enriched in mRNA coding for SP, either naturally, by isolation from cells which are producing large amounts of SP, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
  • cDNA is prepared from the mRNA, and such cDNA may be enzymatically cleaved and ligated into appropriate vectors to form a recombinant cDNA library.
  • a DNA sequence encoding SP, or its gene, or regulatory elements thereof may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., (Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, second edition, 1988) and are well known in the art.
  • Libraries containing sequences coding for SP be screened and a sequence coding for coat protein identified by any means which specifically selects for a sequence coding for coat protein such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of SP, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated SP product produced by the host containing the clone.
  • Oligonucleotide probes specific for SP which can be used to identify clones to this protein can be designed from knowledge of the amino acid sequence of SP.
  • amino acid sequence As used herein, and by convention, when an amino acid sequence is listed horizontally, unless otherwise stated, the amino terminus is intended to be on the left end and the carboxy terminus is intended to be at the right end.
  • the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356- 357).
  • the peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding SP.
  • the probability that a particular oligonucleotide will, in fact, constitute an actual SP encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe, R., et al. , J. Molec. Biol. 183: 1-12 (1985).
  • the suitable oligonucleotide, or set of oligonucleotides, which are capable of encoding at least a fragment of the SP gene (or sequences that are complementary to such coding sequences) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate a cloned SP gene by techniques known in the art.
  • the above-described DNA probe is labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as 32 P, 3 H, 14 C, 35 S, 125 I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • the elucidation of the SP amino acid sequence permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • an oligonucleotide complementary to this theoretical sequence or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides
  • a DNA molecule or set of DNA molecules
  • oligonucleotides were synthesized according to the N-terminal amino acid sequences as shown in Table 1.
  • Table 1 Peptide-sequences of the L. brevis coat protein
  • Deoxyinosine was applied in unknown positions.
  • the oligos were synthesized in such a way that the oligos corresponding to the N-termini of the isolated peptides were in opposite orientation to the oligo corresponding to the N-terminus of the mature SP. These oligos were then used in PCR to generate fragments of the SP gene. The largest of these fragments (1.2 kb) was colinear with the chromosomal coat gene, as shown by Southern blotting, and was used as a probe to screen the gene libraries. The oligos used to generate the 1.2 kb fragment are shown in Figure ID.
  • the SP gene is 1395 bp long and encodes a protein of 49159 daltons. Removal of the deduced signal peptide (30 amino acids) results in a polypeptide of 435 amino acid residues with calculated molecular mass of 45 kDa. This is in excellent agreement with the apparent molecular mass of 46 kDa obtained in SDS-PAGE analysis of SP that is released from the intact cells of Lactobacillus brevis.
  • the full DNA sequence of the SP gene enabled the design of new oligos that result in the synthesis of the complete gene in a single PCR fragment from the Lactobacillus chromosome.
  • the DNA and the deduced protein sequences may be analyzed with standard programs, such as the PC/GENE (Genofit) program.
  • the above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding Lactobacillus SP or fragments of this protein.
  • SP is desirable to express SP.
  • Such expression identifies those clones which express proteins possessing characteristics of SP. Such characteristics may include the ability to specifically bind SP antibody, the ability to elicit the production of antibody which are capable of binding to SP, and the ability to provide a biological activity that is possessed by SP to a cell, among others.
  • L. brevis was particularly chosen to exemplify herein because of its
  • L. buchneri has a SP very similar in size as the L. brevis protein and the L. brevis coat gene hybridizes with the L. buchneri genome.
  • the SP gene may be isolated by combined PCR and reverse PCR as described above. PCR is a preferred method because of the difficulties encountered with the cloning of L. brevis SP using the standard E. coli cloning techniques.
  • the regulatory regions may be characterized by DNA sequence analysis and by determination of the 5' end(s) of the SP gene specific mRNA as exemplified for L. brevis.
  • SP transcriptional and translation or secretory regulatory sequences may be used for the expression of heterologous or homologous recombinant proteins in Lactobacillus.
  • the SP of the invention contains a signal sequence. This is the first instance wherein a sequence of an exported or secreted protein of genus Lactobacillus has been revealed.
  • SP expression and secretion units can be used for expression or secretion of a heterologous protein in members of the genera Lactobacillus. Furthermore, according to the invention, the same expression/secretion units can be applied to drive secretion of a heterologous protein also in members of the genera Lactococci and the genera Bacillus.
  • the secretion unit can consist either of a full SP signal sequence (MQSSLKKSLYLGLAALSFAGVAAVSTTASA, SEQ ID No. 23, bases 1-30 of SEQ ID No.
  • a hybrid signal sequence can be constructed in such a way that the SP signal sequence provides the 5' end of the construct, and it is fused to the 3' end of the signal sequence of the target gene so as to create a hybrid secretion sequence.
  • a signal peptide consists of three domains: a positively charged N-terminal region (N-region), followed by a hydrophobic region (h-region) and a C-terminal region (C-region) which contains the recognition site for the signal peptidase.
  • N-region positively charged N-terminal region
  • h-region hydrophobic region
  • C-region C-terminal region
  • there is a helix breaking residue, Pro or Gly at the end of the h-region for a review see Gierasch, L.M., "Signal Sequences, " Biochemistry 28:923-930 (1989)).
  • hybrid secretion signals may be designed that possess one or more domains of the SP signal sequence and one or more domains of the signal sequence of a different protein.
  • a hybrid signal sequence may contain only the N-region of the SP signal sequence, or only the h-region, or only the C-region; alternatively, a hybrid signal sequence may contain the SP signal sequence's N-region and h-region (the C-region being provided by a different signal sequence), or, the SP signal sequence's N-region and C-region (the h-region being provided by a different signal sequence), or, the h-region and C-region of the SP signal sequence (the N-region being provided by that of a different signal sequence).
  • Each of the hybrid secretory signals of the invention is constructed by recombinant techniques so that the amino acid sequences of the three secretory signal domains, the N-region, the h-region and the C-region, are operably linked to each other, in the order "N-h-C," (the amino terminal end being on the left).
  • the operable linkage is such that the hybrid construct will provide for the secretion of a protein that is operably linked to the carboxy terminus of the C-region domain.
  • such construct provides for both the secretion and cleavage of the hybrid secretion signal, such that the mature protein of interest is present in the culture medium.
  • each of the hybrid secretory sequence's three domains may be heterologous to the other (that is, originate from three different secretory signal sequences), or two of the domains may be derived from the same signal sequence.
  • any combination of the secretion signal domains may be created.
  • a DNA sequence, and especially a DNA sequence encoding the hybrid secretory signal elements of the invention, such as those exemplified below from Bacillus signal sequences, and a Lactobacillus SP signal sequence, or the desired domains thereof, or a specifically desired combination of sequences from each of these signal sequences, may be chemically synthesized using techniques known in the art (for example see Oligonucleotides and Analogues .
  • the promoter and a portion of the signal sequence of a Lactbacillus SP signal sequence is used as the source of the Ndomain and/or h-domain, the promoter without further modification and the signal sequence being altered as described herein as desired to construct the hybrid signal sequence.
  • any promoter capable of functioning in the host cell may be used, and the desired domain sequence of any secretion signal may be used, as long as such sequence provides for the desired secretion function in the host cell.
  • the different junctions between the ⁇ -amylase signal sequence and that of the Lactobacillus signal sequence that may be constructed may be determined in the following manner. As with all other secretion signals, the
  • the SP secretion signal of SEQ ID No. 23 may be broken into an N-region, h-region and C-region, such the the N-region is positively charged, the h-region is hydrophobic and the C-region provides for the recognition site of the signal peptidase.
  • hybrid secretion signal that may be constructed from these domains include sequences containing:
  • junction may affect the protein yield. It is not necessary that the exact native amino acid sequence of a desired secretion element (N-region, h-region, or C-region) be maintained in the hybrid secretion sequence. Changes in the native amino acid sequence of a domain, especially at a junction, such as may be introduced during cloning with restriction enzymes and ligation techniques will not necessarily destroy, or be detrimental to, the functioning of the hybrid secretion sequence.
  • the C-region domain or the helix breaker joint provide a signal peptidase recognition site that is homologous to one expressed in the Lactobacillus host cell. However, this is not necessary if the host signal peptidase will recognize the C-region domain on the hybrid secretion sequence.
  • the helix breaker joint (or C-region) provides a signal peptide recognition site that is homologous to the recombinant protein that is operably linked to the C-region of the hybrid secretory signal sequence, and is recognized by the signal peptidase enzyme in the Lactobacillus host cell (even if heterologous to such host cell).
  • sequence encoding the ribosome-binding site-start-codon- provided by such region originate from an SP gene with a high capacity for efficient translational expression in the desired host cell.
  • Secretion vectors based either on the intact or hybrid signal sequences can be used for production of food or feed industry-related extracellular enzymes, peptides and peptide antimicrobial agents.
  • secretion vector constructs may be designed that retain part of the SP gene encoding the mature SP protein. This would result in a mature protein that is a fusion protein that is secreted. The desired protein could be later released from the carrier SP protein part either by enzymatic or chemical cleavage. As is known in the art, with simple deletions of the SP gene encoding the mature SP it is straightforward to estimate an appropriate number of amino acids to use for the "carrier" part to ensure efficient secretion and/or protection of the required gene product.
  • a third possibility is to create a fusion protein of the complete SP protein joined to the amino acid sequence of a desired protein of interest, so as to produce the desired protein of interest on the cell surface as a part of the surface layer.
  • This product could be, for example, an antigenic epitope and Lactobacillus cells coated with this epitope could then be used as inexpensive animal vaccines or diagnostic tools (e.g., in screening of autoimmune diseases).
  • the product of the cell surface could also be chosen to improve the probiotic value of a Lactobacillus host.
  • the correct insertion position, e.g., for the epitope coding region within the coat gene can be found for example i) by analyzing the coat sequence with an antigenic determinant screening program (e.g.
  • the native SP signal sequence may be used even without a specific proteolytic site within the signal sequence (to allow removal of the signal sequence) if the biological function of the desired protein or fusion protein is not prevented by the presence of the signal sequence.
  • the cloned SP coding sequences, and specifically, the transcriptional and secretory regulatory elements thereof, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to a desired gene coding sequence in an expression vector, and introduced into a Lactobacillus, Lactococcus, or Bacillus host cell to produce recombinant protein under the control of such sequences.
  • the present invention encompasses the expression of a desired protein in Lactobacillus cells, and especially L. brevis.
  • a nucleic acid molecule, such as DNA is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are “operably linked” to the nucleotide sequence which encodes the desired polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • a sequence encoding a desired protein and a coat protein promoter region sequence joined to the 5' end of the sequence encoding the desired protein are said to be operably linked if induction of promoter function results in the transcription of mRNA from the sequences encoding the desired protein and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the coat protein expression and secretion regulatory sequences to direct the expression and secretion of the desired protein, or (3) interfere with the ability of the template encoding the desired protein to be transcribed by the promoter region sequence.
  • a coat protein promoter region would be operably linked to a DNA sequence encoding a desired protein (or antisense RNA) if the coat protein promoter were capable of effecting transcription of that
  • regulatory regions needed for gene expression may vary between species or cell typps, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the -35 and -10 regions known to be necessary for prokaryotic promoters.
  • 5' non-transcribing control sequences will include a region which contains a promoter for transcriptional expression of the operably linked gene.
  • transcriptional control sequences may also include additional activator or repressor sequences as desired.
  • the SP gene has two separate promotes with roughly equal strength under the test conditions.
  • the promoter unit(s) can be used for increasing the efficiency or altering the regulation of metabolic pathways or for intracellular production of enzymes (e.g. , peptidases) or peptides (e.g. milk derived opoidic or taste affecting peptides).
  • enzymes e.g. , peptidases
  • peptides e.g. milk derived opoidic or taste affecting peptides
  • the promoter unit can be used either alone, devoid of the coat gene derived ribosome binding site by using fusions made e.g., at the nucleotide, encoding 5' end of the mRNA, or as a transcription/ translation initiation unit where joints are made directly after the initiation methionine or further down the structural gene yielding fusion proteins.
  • coat protein expression and secretion control sequences signals do not function satisfactorily in a host cell, then sequences functional in the host cell may be substituted as necessary.
  • the vectors of the invention may further comprise other operably linked regulatory elements such as DNA elements which confer antibiotic resistance on a host cell, and which provide for an origin of replication, or for insertion of a desired sequence into the chromosome of a host cell.
  • operably linked regulatory elements such as DNA elements which confer antibiotic resistance on a host cell, and which provide for an origin of replication, or for insertion of a desired sequence into the chromosome of a host cell.
  • RNA non- replicating DNA
  • the expression of the desired protein may occur through the transient expression of the introduced sequence.
  • a non-replicating DNA (or RNA) molecule may be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication.
  • genetically stable transformants may be constructed with vector systems, or transformation systems, whereby DNA encoding the desired protein and the expression and secretion regulatory elements thereof are integrated into the host chromosome.
  • Such integration may occur de novo within the cell or, in a most preferred embodiment be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with transposons or homologous DNA integration which promote integration of DNA sequences in chromosomes.
  • Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly provided on the same vector as that providing the desired DNA gene sequences to be expressed, or such markers may be introduced into the same cell by co-transfection.
  • Factors of importance in selecting a particular plasmid or phage vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species, such as E. coli and Lactobacillus.
  • recipient cells After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein.
  • this expression preferably takes place in a continuous manner in the transformed cells.
  • the expressed protein of interest is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like, according to the biochemical characteristics of the desired protein, for example, by affinity purification with antibodies to a desired protein of interest.
  • conventional conditions such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like, according to the biochemical characteristics of the desired protein, for example, by affinity purification with antibodies to a desired protein of interest.
  • affinity purification with antibodies to a desired protein of interest.
  • the Lactobacillus SP encoding sequences obtained through the methods above, will provide sequences which, by definition, encode the Lactobacillus SP transcriptional, translational, and secretory regulatory elements, and which may then be used to obtain direct the synthesis and secretion of a desired protein in the host cell.
  • Lactobacillus SP encoding sequences will provide sequences which, by definition, encode the Lactobacillus SP transcriptional, translational, and secretory regulatory elements, and which may then be used to obtain direct the synthesis of an antisense RNA (an RNA complementary to protein's mRNA) in the host cell.
  • An antisense RNA sequence will be that sequence found on the opposite, complementary strand of the strand transcribing the protein's mRNA.
  • An expression vector may be constructed which contains a DNA sequence operably linked to a promoter wherein such DNA sequence expresses the SP antisense RNA sequence.
  • Transformation with this vector results in a host capable of expression of a antisense RNA against a specific protein, under the control of the coat protein transcriptional expression signals.
  • antisense RNA interacts with an endogenous DNA or RNA in a manner which inhibits or represses transcription and/or translation of the endogenous protein gene and/or mRNA in a highly specific manner.
  • SP antisense RNA is expressed, translation and expression of native SP will be reduced or eliminated. This may be desired when it is desired to express a SP fusion protein or other construct on the host surface in place of the native SP protein, or when it is desired to express a coat protein (or other surface protein) that is heterologous to the host.
  • Lactobacillus brevis strain DSM 20556 was used as a source for the isolation of the coat protein.
  • the strain was obtained from the German Collection of Microorganisms (Braunschweig, FRG).
  • MRS-medium Difco
  • the cells were collected by centrifugation at 10,000 g for 5 minutes at room temperature (RT), and washed once with 50 mM Tris-HCl, pH 7.5 buffer.
  • the cell pellet was dissolved in Laemmli sample buffer (Cells from 1 ml culture were suspended in 100 ⁇ l of 50 mM Tris-HCl, pH 7.5 followed by addition of 100 ⁇ l of Laemmli sample buffer), boiled for 5 minutes and analyzed in SDS-PAGE (10%). This resulted in a single major band of M r 46 kd after Coomassie Blue staining.
  • To isolate the protein the same cell sample was applied onto a preparative polyacrylamide gel. After electrophoresis the gel was treated with 1 M KCl on ice to visualize the protein band. The band corresponding to the 46 kd protein was excised from the gel and cut into small pieces.
  • the proteins were eluted from the gel pieces with 6 M guanidine hydrochloride, 0.5 M Tris-HCl, 2 mM EDTA, pH 7.5 by incubating in an end-over mixer at RT for 10 hours.
  • the elute was dialyzed against 10 mM Tris-HCl, pH 8.5 o/n and either freeze dried for protein sequencing or used as such for immunizing rabbits.
  • the resulting antiserum was designated KH1225.
  • the intact cells were analyzed by immunogold electromicroscopy using the antiserum (KH1225) raised against the isolated protein band as follows: the bacterial cells were collected with glass rod from MRS-plates and fixed either with 2.5% glutaraldehyde (Electron Microscopy Sciences, Washington, PA 19034) in 0.1 M phosphate buffer (pH 7.5) or with 3.5 % paraformaldehyde and 0.5 % glutaraldehyde dissolved in 0.1 M phosphate buffer. The fixations were carried out at room temperature for 1 hour. Cells were washed three times with the same buffer.
  • the cells were then dehydrated in a series of ethanol (50, 60, 70 and 90%) washes and treated with 90% ethanol and LR-WHITE (2: 1) for one hour as well as (1: 1) for one hour before the final embedding in LR-WHITE (a hydrophilic acrylic resin of low viscosity; London Resin).
  • LR-WHITE a hydrophilic acrylic resin of low viscosity; London Resin
  • the coat protein was rerun in an inversed gradient SDS-PAGE (Proc. Int. Meeting on Electrophoresis, Ed. Radola, B.J., pp. 293-397 (1989)) with 12% separation gel and a 5 % running gel. From the gel the proteins were transferred electrophoretically onto a polyvinylidene diflouride (PVDF) membrane (In Current Res. in Prot. Chem. Ed., Villafranca, J. Academic Press, 1990)) and degraded in a gas/pulsed liquid sequencer (J. Prot. Chem. 7:242-243 (1988)). The N-terminal sequence of the coat protein is shown in Table 1.
  • PVDF polyvinylidene diflouride
  • peptides derived from the coat protein were sequenced.
  • the freeze dried protein from part 1 was suspended in 300 ⁇ l distilled water and 500 ng of lysylendopeptidase (WAKO, Dallas, TX, USA) was added. The mixture was incubated at 35 °C for 6 hours and the resulting peptides were separated by reverse phase chromatography on a Vydak 218 TPB5 (0.46 ⁇ 15 cm) column connected to a Varian 5000 liquid chromatograph.
  • the peptides were eluted using a linear gradient of acetonitrile (0-60% in 90 min) in 0.15 M trifluoroacetic acid.
  • lysylendopeptidase digested peptides were sequenced as described above after application on polybrene (2 mg) pretreated glass filters.
  • the N-terminal sequences of the isolated peptides are shown in Table 1.
  • the peptide 5 was shown to be identical to the N-terminal peptide of the protein without the N-terminal lysine which was most probably removed by the lysylendopeptidase digestion.
  • L. brevis strain DSM 20556 were highly resistant to enzymatic lysis, the following method was developed to isolate the chromosomal DNA.
  • the cells were grown in MRS-medium (200 ml) at 37 °C to midlogarithmic growth phase (Klett 66 80), collected by centrifugation at 8000 g for 5 minutes at RT and suspended in 3 ml of 3 M guanidine hydrochloride solution. After incubation for 20 minutes at RT, the cells were collected by centrifugation, washed once with 0.15 M NaCl-0.1 M EDTA solution and suspended in 7 ml of the same solution.
  • lysozyme 20 mg of lysozyme, 200 ⁇ l of mutanolysin (15,000 U/ml, Sigma) and 3 ⁇ l of 1 M CaCl 2 were added and the cells were incubated at 55 °C for 2 hours. After addition of 8.75 ml of 20% SDS, followed by incubation of 10 min at 65 °C, 2.2 ml of 5 M Na-perchlorate was added and the cell lysate was extracted with chloroform-isoamylalcohol (24: 1). The water phase was removed after centrifugation and nucleic acid precipitated by bringing the solution to 66% ethanol. The chromosomal DNA was collected around a glass rod.
  • DNA was dissolved in 10 mM Tris-HCl - 150 mM NaCl, pH 7.5.
  • the chromosomal DNA was partially cleaved with HaeIII and fragments of 4-7 kb size were isolated after agarose gel electrophoresis. These HaeIII-fragments were used to generate a lambda gt10 gene bank using a commercially supplied lambda gt10 system (Promega) according to the manufacturer's instructions.
  • oligonucleotides were synthesized according to the N-terminal amino acid sequences. Deoxyinosine was applied in unknown positions. The oligos were synthesized in such a way that the oligos corresponding to the N-termini of the isolated peptides were in opposite orientation to the oligo corresponding to the N-terminus of the mature coat protein. These oligos were then used in PCR to generate fragments of the coat gene. The largest of these fragments (1.2 kb) was shown to be colinear with the chromosomal coat gene by Southern blotting and was used as a probe to screen the gene libraries. The oligos used to generate the 1.2 kb fragment are shown in Figure ID.
  • RNA was isolated from L. brevis essentially as described by Palva et al. DNA 7:135-142 (1988) except that mutanolysin and lysozyme were used in concentrations of 900 U/ ⁇ l and 20 ⁇ g/ ⁇ l, respectively.
  • RNA gel electrophoresis and Northern blot were as described (DNA 7: 135-142 (1988)) using the 1.2 kb coat gene fragment as a probe.
  • the Northern blot analysis revealed a 1.5 kb mRNA which is in good agreement with the DNA data and indicated that the coat gene is monocistronic.
  • the 5' end(s) of the coat mRNA was determined by primer extension method (Appl. and Environ. Microbiol.
  • integration vectors were constructed for L. brevis, L. Platarum, and L. casei as follows.
  • the vectors were based on the pUC18 plasmid.
  • any other convenient plasmid vector either not able to replicate in Lactobacillus or carrying a replicon that is conditionally non-functional in Lactobacillus (e.g., cold- or thermosensitive), could have been used.
  • a selection marker, functional in Lactobacillus was then added to the pUC18 vector. In this particular case the erythromycin resistance marker (em r ) from S. aureus plasmid pE194 (J.
  • TEM- ⁇ -lactamase was chosen as a model gene to demonstrate the functionality of the coat gene expression and secretion units in various gram-positive hosts, ⁇ -lactamase gene was isolated from pBR322 by PCR using oligos 1 and 2 depicted in Table 3.
  • Positions of primer sequences 3, 4 and 5 for expression and secretion unit in coat sequence are -311 ⁇ -294, 345 ⁇ 326 and 282 ⁇ 260,
  • the PCR fragment contained a DNA sequence encoding the full mature ⁇ -lactamase and four amino acids from the C-terminus of its signal sequence.
  • the fragment was flanked by 5' BamHI and 3' XbaI sites.
  • the expression/secretion unit was isolated by PCR using oligos 3 and 4 (Table 3) and the full length coat gene (see Example 4) as a template.
  • the resulting PCR fragment contained the promoter region starting from nucleotide -311 ( Figure 2A; this fragment also includes a rho independent transcription termination site upstream of the coat promoters to prevent unspecific mRNAs from affecting the system) followed by the ribosome binding site and 30 codons from the signal sequence of the coat gene.
  • the isolated expression/secretion unit was flanked by 5' EcoRI and 3' BamHI sites.
  • the ⁇ -lactamase fragment was ligated to the coat gene expression/secretion unit at the common BamHI site and the ligation mixture was amplified by PCR using oligos 3 and 2 to yield a hybrid ⁇ -lactamase gene under the coat gene expression and secretion signals.
  • This hybrid gene flanked by 5' EcoRI and 3' XbaI sites was then inserted to the Lactobacillus integration vectors pKTH2064 and pKTH2067( ⁇ xample 5) between the respective sites.
  • the ligation mixture was transformed by electroporation in L. plantarum and L. casei hosts.
  • the cells were plated on MRS-em plates (10 ⁇ g/ml) and em-resistant transformant colonies were screened for ⁇ -lactamase activity by using a chromogenic substrate, nitrocefin (Glaxo) that ⁇ -lactamase degrades into a red product (J. Mol. Biol. 126:673-690 (1978)).
  • the transformants were suspended in 250 ⁇ l of nitrocefin solution in microtiter wells and L. plantarum and L.
  • ⁇ -lactamase positive Lactobacillus colonies were grown to mid-logarithmic phase in 1 ml of MRS-em-solution, the cells were removed by centrifugation and suspended in equal volume of 50 mM phosphate buffer, pH 7.0. Equal volumes of the supernatants and cell suspensions were added to the nitrocefin solution. The supernatant samples contained about ten times more ⁇ -lactamase activity than the cell samples (as judged by the change of absorbance during 2 min. incubation), demonstrating that the expression/ secretion signal derived from the coat gene can be used to express and secrete a required protein in Lactobacillus.
  • the hybrid coat/ ⁇ -lactamase gene was also joined as a blunt end fragment to the blunted Clal site of plasmid pVS2 (Appl. Environ. Microbiol 53: 1584-1588 (1987)).
  • the ligation mixture was transformed to B. subtillis Marburg strain IH6064 (Gene 19:82-87 (1982)) by physiological transformation (J. Bacteriol. 134:318-329 (1978)) and to Lactococcus lactis subsp. lactis (J. Bacteriol. 154: 1-9 (1983)) by electroporation (Appl. Environ. Microbiol. 55:3119-3123 (1989)).
  • the selection plates were Luria (Virology 1: 190-206 (1955)) and M 17 glucose (Appl. Environ. Microbiol. 29:807-813 (1975)) supplemented with 5 ⁇ g/ml chloramphenicol for Bacillus and for Lactococcus, respectively.
  • the cm-resistant transformants were screened by nitrocefin assay as described above and also in the case positive transformants grown to mid-logarithmic phase in L-broth (Bacillus) or M17-glc-broth (Lactococci) secreted over 90% of the ⁇ -lactamase activity to the culture medium.
  • the promoter region of the coat gene was isolated as a PCR fragment starting from nucleotide -311 ( Figure 2A) and containing both promoters, the ribosome binding site and the four first codons from the 5' end of the signal sequence. The fragment was flanked by a 5' EcoRI site and a 3' BamHI site. The relevant oligos 3 (5') and 5 (3') are shown on Table 3.
  • a promoterless cloramphenicol acetyltransferase gene (cat) in a promoter probe vector pKTH1750 (Appl. Environ. Microbiol.
  • GGT GGC AAG TCT GAC ACT GCC TTT GCT GGT GGT ATC AAG TCT GCT GAA 675 Gly Gly Lys Ser Asp Thr Ala Phe Ala Gly Gly Ile Lys Ser Ala Glu

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Abstract

Systèmes très efficaces d'expression/sécrétion destinés à la manipulation par recombinaison de Lactobacillus, Lactococcus et Bacillus. Ces systèmes utilisent des éléments de régulation de l'expression et de la sécrétion de la protéine capsidique de Lactobacillus (SP) dans l'expression des gènes recombinés.
PCT/FI1993/000273 1992-06-30 1993-06-24 Systeme d'expression de lactobacillus utilisant des sequences geniques de proteine capsidique WO1994000581A1 (fr)

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Cited By (9)

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NL9401935A (nl) * 1994-11-18 1996-07-01 Nl Zuivelonderzoek Inst Werkwijze voor het regelen van de gen-expressie in melkzuurbacteriën.
WO1996032486A1 (fr) * 1995-04-11 1996-10-17 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede de construction de vecteurs pour bacteries d'acide lactique comme lactobacillus, qui permet aux bacteries d'exprimer, de secreter et de faire apparaitre des proteines en surface
WO1997006265A3 (fr) * 1995-08-07 1997-05-22 Perkin Elmer Corp Systeme de selection de clone de recombinaison
EP0712935A3 (fr) * 1994-11-18 1997-11-26 Stichting Nederlands Instituut Voor Zuivelonderzoek Méthode de contrÔle de l'expression des gènes des bactéries lactiques
US5843656A (en) * 1995-08-07 1998-12-01 The Perkin-Elmer Corporation Recombinant clone selection system
WO1999051631A1 (fr) * 1998-04-03 1999-10-14 Timo Korhonen Une region proteique responsable de la liaison avec des types de cellules epitheliales et une sequence d'adn codant pour cette region
US6100043A (en) * 1995-08-04 2000-08-08 The Perkin-Elmer Corporation Recombinant clone selection system
JP2013039065A (ja) * 2011-08-12 2013-02-28 Nagasaki Univ 自己炎症疾患又は自己免疫疾患関連遺伝子及びその利用
JP2021521898A (ja) * 2018-05-04 2021-08-30 メディトックス インク． 標的蛋白質をコーディングするポリヌクレオチドを含む組み換え微生物に由来する細胞外小嚢及びその用途

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL9401935A (nl) * 1994-11-18 1996-07-01 Nl Zuivelonderzoek Inst Werkwijze voor het regelen van de gen-expressie in melkzuurbacteriën.
EP0712935A3 (fr) * 1994-11-18 1997-11-26 Stichting Nederlands Instituut Voor Zuivelonderzoek Méthode de contrÔle de l'expression des gènes des bactéries lactiques
US5914248A (en) * 1994-11-18 1999-06-22 Stichting Nederlands Instituut Voor De Zuivelinderzoek Method for controlling the gene expression in lactic acid bacteria
WO1996032486A1 (fr) * 1995-04-11 1996-10-17 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede de construction de vecteurs pour bacteries d'acide lactique comme lactobacillus, qui permet aux bacteries d'exprimer, de secreter et de faire apparaitre des proteines en surface
WO1996032487A1 (fr) * 1995-04-11 1996-10-17 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Nouveau procede de creation de vecteurs pour bacteries d'acide lactique comme lactobacillus, qui permet aux bacteries d'exprimer, de secreter et de faire apparaitre des proteines en surface
US6100043A (en) * 1995-08-04 2000-08-08 The Perkin-Elmer Corporation Recombinant clone selection system
WO1997006265A3 (fr) * 1995-08-07 1997-05-22 Perkin Elmer Corp Systeme de selection de clone de recombinaison
US5843656A (en) * 1995-08-07 1998-12-01 The Perkin-Elmer Corporation Recombinant clone selection system
US6090562A (en) * 1995-08-07 2000-07-18 The Perkin-Elmer Corporation Recombinant clone selection system
WO1999051631A1 (fr) * 1998-04-03 1999-10-14 Timo Korhonen Une region proteique responsable de la liaison avec des types de cellules epitheliales et une sequence d'adn codant pour cette region
JP2013039065A (ja) * 2011-08-12 2013-02-28 Nagasaki Univ 自己炎症疾患又は自己免疫疾患関連遺伝子及びその利用
JP2021521898A (ja) * 2018-05-04 2021-08-30 メディトックス インク． 標的蛋白質をコーディングするポリヌクレオチドを含む組み換え微生物に由来する細胞外小嚢及びその用途

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