EP0550428A1 - Vecteurs sondes de promoteurs pouvant se repliquer en e.coli, b.subtilis, lactococci et lactobacillus, ainsi que leurs utilisations - Google Patents

Vecteurs sondes de promoteurs pouvant se repliquer en e.coli, b.subtilis, lactococci et lactobacillus, ainsi que leurs utilisations

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Publication number
EP0550428A1
EP0550428A1 EP90913051A EP90913051A EP0550428A1 EP 0550428 A1 EP0550428 A1 EP 0550428A1 EP 90913051 A EP90913051 A EP 90913051A EP 90913051 A EP90913051 A EP 90913051A EP 0550428 A1 EP0550428 A1 EP 0550428A1
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European Patent Office
Prior art keywords
plasmid
promoter
plasmids
host cell
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP90913051A
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German (de)
English (en)
Inventor
Ilkka Palva
Mervi Sibakov
Teija Koivula
Atte Von Wright
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Genesit Oy
Valio Finnish Cooperative Dairies Association
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Genesit Oy
Valio Finnish Cooperative Dairies Association
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Application filed by Genesit Oy, Valio Finnish Cooperative Dairies Association filed Critical Genesit Oy
Publication of EP0550428A1 publication Critical patent/EP0550428A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N15/74—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)
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • the present invention relates to the field of molecular biology, and, more particularly, to the fields of recombinant genetics and genetic engineering.
  • the invention further relates to DNA sequences, derived from Lactococcus lactis. which are useful as promoters and promoter/secretion promoting signals for heterologous or homologous expression in bacteria.
  • the invention relates to vectors, such as plasmids, comprising the sequences of the present invention, and to host cells transformed with such vectors.
  • Yet additional aspects of the present invention are related to methods for producing desired heterologous or homologous peptides or proteins employing the sequences, vectors, or transformed hosts of the invention. By means of the invention, greatly improved heterologous and homologous expression and secretion may be achieved.
  • the lactic acid bacteria are of great commercial importance for, among other things, their ability to carry out fermentation, a process in which organic compounds ser e as both electron donors and electron acceptors. Lactic fermentation reduces pyruvate to lactate in a single step reaction catalyzed by NAD-1 inked lactic dehydrogenase, without gas formation, and is the first stage in cheese manufacture. Thus, lactic fermentations are responsible for souring, or acidification, of milk and certain other foods, which allows for anaerobic preservation. Further, these processes are involved in the formation of interesting and desirable food and beverage flavors.
  • the lactic acid bacteria are thus of significant commercial importance. To-date, however, much of the work involving recombinant genetics has been carried out in other bacteria, such as E. coli. One result of this is that the genetics of the lactic acid bacteria are relatively less well understood or characterized. Inasmuch as there is a great deal of practical knowledge relating to the cultivation of lactic acid bacteria for commercial purposes, a continuing need exists for the application of recombinant genetic techni- ques to the understanding of these bacteria. de Vos, Neth. Milk Dairy J. 40:141-154 (1986), and FEMS M crobiol. Rev.
  • European patent application publication number 0 157 441 discloses certain shuttle vectors capable of expression in EL. subtilis. E. coli and Streptococcus lactis, containing the rep!icon from the large Clal fragment of the S. cremoris Wg2 plasmid p VOl. It is stated by the applicants that these vectors can give improved or new properties to lactic acid bacteria transformed therewith. Examples of the use of this system include the expression of genes for a protease and a chymosin precursor in S. lactis. A number of other reports have appeared relating to the characterization of S. cremoris Wg2 protease activity. For example, Kok et al . , Applied Environmental Microbiol.
  • This system was used to express a fusion gene containing the eukaryotic hen egg white lysozyme (HEL) coding sequence in L. lactis.
  • HEL hen egg white lysozyme
  • S. cremoris SK11 contains a non-bitter cell wall-associated proteinase, of which the complete gene has been cloned and sequenced. It is stated that a DNA fragment containing this gene and another proteinase gene was cloned into a lactic streptococcal cloning vector (pNZ521) and expressed.
  • D127 (1988) discloses that recently established host-vector systems have been used to study the organization and expression of plasmid located genes in mesophilic lactic streptococci S. lactis and S. cremoris. It is stated that most attention has been focussed on homologous genes important for use of these strains in industrial fermentation, and on heterologous genes which could be used to construct strains having novel properties. It also is stated that homologous genes encoding lactose and casein degradation events have been analyzed, as well as regulatory control of copy number of S. lactis plasmid pSH71, and that topogenic sequences which direct cellular location of expressed proteins have been identified.
  • the present inventors have discovered, isolated, cloned and sequenced novel promoters and promoter/secretion promoting signals from Lactococcus lactis subsp. lactis, which are useful in the production of heterologous and homologous proteins and peptides in E. coli and, especially, in Gram-positive bacteria.
  • one embodiment of the present invention provides for a promoter probe-vector able to replicate in E. coli. B. subtilis, Lactococci and Lactobacillus, selected from the group consisting of the plasmids pKTH1734 and pKTH1736, the said plasmids constructed as shown in Figure 5, or a functional derivative thereof.
  • promoter probe-vector further comprising multiple cloning sites having nucleotide sequences as shown in Figure 1, or a functional derivative thereof.
  • a promoter probe-vector able to replicate in E. coli, B. subtilis, Lactococci and Lactobacillus. comprising the plasmid pKTH1750, or a functional derivative thereof.
  • E. coli, B. subtilis, Lactococci and Lactobacillus hosts transformed with any of these promoter probe-vectors comprise an additional embodiment of the invention.
  • the present inventors were able to clone and sequence previously unknown and undescribed L. lactis subsp. lactis promoter and promoter/secretion signal promoting nucleotide sequences.
  • the present invention provides for a substantially pure nucleotide sequence as shown in Figures 9, 10, 11, 12, 13, 14, 15, 16, 17 or 19, or a functional or chemical derivative thereof. These sequences may be beneficially incorporated into plasmids, by means of which it has been possible to achieve enhanced heterologous protein expression in E. coli and, especially, in Gra - positive bacteria. Plasmids comprising these nucleotide sequences thus form another embodiment of the present invention.
  • sequences and plasmids of the present invention are those which include L. lactis subsp. lactis-derived promoter sequences, exemplified by the sequences found in plasmids pKTH1789, pKTH1816, pKTH1817, pKTH1820, pKTH1821 and pKTH1874.
  • Other sequences and plasmids of the invention include both the promoter and the secretion promoting signals, and are exemplified by the sequences found in plasmids PKTH1797, pKTH1798, pKTH1799, pKTH1801, pKTH1805, pKTH1806, pKTH1807 and pKTH1809.
  • plasmids and their respective nucleotide sequences form additional embodiments of the present invention.
  • an important teaching of the present invention is the discovery by the present inventors that the regulatory elements of those sequences and plasmids may be recombined to produce hybrid expression units which can function together to allow enhanced heterologous expression in E. coli and, especially, in Gram-positive bacteria.
  • a hybrid expression unit composed of a promoter sequence, exemplified by the any of the sequences found in plasmids pKTH1789, pKTH1816, pKTH1817, pKTH1820, pKTH1821 and pKTH1874, together with a secretion promoting signal derived from sequences and plasmids of the invention including both the promoter and the secretion promoting signals, such as are exemplified by the sequences found in plasmids pKTH1797, pKTH1798, pKTH1799, pKTH1801, pKTH1805, pKTH1806, pKTH1807 and pKTH1809.
  • a hybrid expression unit wherein the promoter sequence is derived from the plasmid pKTH1817, and wherein the secretion signal sequence is derived from the plasmid pKTH1807.
  • the present invention is directed to E. coli and, especially, to Gram-positive host cells transformed with any of the sequences or plasmids of the invention.
  • the plasmids may additionally comprise a nucleotide sequence encoding one or more homologous or heterologous proteins or peptides which it is desired to express primarily in a Gram- positive host.
  • Host cells according to the invention are selected from the group consisting of E. coli and the Gram- positive B. subtilis, Lactococci and Lactobacillus hosts.
  • An additional embodiment of the present invention provides for a method of heterologous or homologous protein or peptide expression, comprising transforming E. coli or a Gram- positive host cell with a plasmid according to the invention (which plasmid also comprises the nucleotide sequence encoding the desired protein or peptide); culturing the transformed host cell in a suitable medium under conditions allowing expression of said protein or peptide, and recovering the expressed protein or peptide from said host cell or said medium.
  • Figure 1 Oligonucleotides used in cloning multiple cloning sites (MCS) in the vector pKTH1736.
  • Figure 2 The size of in vitro synthesized J-lactamase precursors. Lane 1, .-lactamase control; lane 2, pKTH1797; lane 3, pKTH1798; lane 4, pKTH1799; lane 5, pKTH1801; lane 6, M r standard. See text for technical details.
  • Figure 3 RNAs of L. lactis subsp. lactis promoter constructions (panel A) and promoter signal sequence constructions (panel B) obtained by Northern hybridization.
  • Panel A mRNAs were isolated from promoter constructions pKTH1816 (1), pKTH1817 (2), pKTH1820 (3), and pKTH1821 (4) and probed with labeled pPL603. To visualize the bands, X-ray film was exposed 1 h.
  • Panel B mRNAs were isolated from promoter signal sequence constructions pKTH1805 (5), pKTH1806 (6), pKTH1807 (7), and pKTH1809 (8), as a probe labeled pKTH78 was used. To visualize the bands, the film was exposed overnight.
  • Figure 4 Construction of vector pKTH1722.
  • Figure 5 Construction of vectors pKTH1734 and pKTH1736.
  • Figure 6 Promoter probe vector pKTH1750.
  • Figure 7 Construction of vectors pKTH1797, pKTH1798, pKTH1799 and pKTH1801 based upon pKTH33, and of vectors pKTH1805, pKTH1806, pKTH1807 and pKTH1809 based upon pVS2.
  • Figure 8 Identification of the 5' end of mRNAs of . lactis subsp. lactis promoter constructions by primer extension. Promoters were from constructions pKTH1817 (panel A, lane 1), pKTH1820 (panel A, lane 2), pKTH1821 (panel B, lane 3), and pKTH1816 (panel B, lane 4). The standard sequence in panel A was from promoter in construction pKTH1817 and in panel B from promoter in construction pKTH1816.
  • Figure 9 Sequence of pKTH1816. The black dot above the sequences indicates the start site of mRNA; if it is in parenthesis it indicates a possible secondary start site (this is true generally for all figures showing plasmid sequences where applicable).
  • FIG. 10 Sequence of pKTH1817.
  • Figure 11 Sequence of pKTH1820.
  • Figure 12 Sequence of pKTH1874.
  • Figure 13 Sequence of pKTH1789.
  • Figure 14 Sequence of pKTH1797.
  • Figure 15 Sequence of pKTH1798.
  • Figure 16 Sequence of pKTH1799.
  • Figure 17 Sequence of pKTH1801.
  • Figure 18 01 igonucleotide primers used in the construction of the hybrid vector of Figure 20.
  • Figure 19 Sequence of pKTH1821.
  • Figure 20 Construction of hybrid vector pKTH1889. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • promoter is meant generally a region on a DNA molecule to which an RNA poly erase binds and initiates transcription.
  • the nucleotide sequence of the promoter determines both the nature of the enzyme that attaches to it and the rate of RNA synthesis.
  • promoter preferably refers to nucleotide sequences derived from L__ lactis subsp. lactis.
  • promoter/signal promoting sequence generally a nucleotide sequence which comprises, in addition to a promoter sequence, a sequence encoding a 16-35 amino acid segment, usually containing hydrophobic amino acids that become embedded in the lipid bilayer membrane, which allows for the secretion of an accompanying protein or peptide sequence from the host cell, and which usually is cleaved from that protein or peptide.
  • promoter/signal promoting sequence preferably refers to nucleotide sequences derived from L. lactis subsp. lactis.
  • hybrid expression unit any combination of the promoter and promoter/signal promoting sequences of the invention to produce a different or distinct sequence which retains expression or expression and secretion functions.
  • the manner and methods of combining the sequences of the invention to produce numerous such hybrid expression units are well known to those of skill, and are described and exemplified herein. Further, those skilled in the art who have fully appreciated the teachings of the present invention will recognize that it will be possible and even desirable to produce such hybrid expression units in order to optimize expression and secretion of given heterologous or homologous proteins or peptides, and that the same will be accomplished using well-known recombinant methods with the exercise of merely routine skill.
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • cDNA is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase) .
  • a "cDNA clone” eans a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism.
  • a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al . , Molecular Cloning: A Laboratory Manual , supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are cell lines of bacteria.
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replica ⁇ tion in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • DNA expression vector is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.
  • DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention, however, are plasmids comprising promoters and promoter-secretion promoting sequences derived from L. lactis.
  • substantially pure any protein of the present invention, or any gene encoding any such protein, which is essentially free of other proteins or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • This term also may be used with reference to the nucleotide sequences encoding the promoters and promoter- secretion promoting sequences of the invention derived from I lactis.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the DNA sequences of the present invention, is meant to refer to any nucleotide subset of the molecule.
  • a “variant” of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be “substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).
  • a “functional derivative" of a gene encoding any of the molecules of the present invention is meant to include “fragments,” “variants,” or “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • a nucleic acid molecule, such as DNA is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, Shine- Dalgarno sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked 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 promoter region sequence to direct the transcription of the heterologous protein gene sequence, or (3) interfere with the ability of the heterologous protein gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the introduced sequence will be incorporated into a plasmid vector capable of autonomous replication in the recipient host.
  • a plasmid vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid 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.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E.
  • coli such as, for example, pBR322, ColEl, pSClOl, pACYC 184, ⁇ VX
  • plasmids are, for example, disclosed by Maniatis, T., et al . (In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli. Academic Press, NY (1982), pp. 307-329).
  • Particularly preferred vectors according to the invention are those which are able to replicate in E. coli, B. subtilis, Lactococci and Lactobacillus.
  • the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate- precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al . , Science 240(4858): 1538 (1988)), etc.
  • biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate- precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran
  • mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al . , Science 240(4858): 1538 (1988)), etc.
  • 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 heterologous or homologous protein, or in the production of a fragment of this protein.
  • the expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyriboc- ytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • the expressed protein will also be secreted from the host cell when any of the promoter/secretion promoting signals of the invention are employed, with the advantage that isolation and purification procedures will be simplified.
  • the expressed heterologous protein or functional derivative thereof may be isolated by the use of antibodies directed against the desired protein or functional derivative. Such antibodies may be obtained by well-known methods.
  • the manner and method of carrying out the present invention may be more fully understood by those of skill by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto.
  • the bacterial strains used are listed in Table 1.
  • pKTH33 A del etion deri vati ve of pHV33 , a chimera between pBR322 and pC194. Described by Palva, Ph.D. thesis, University of Helsinki (1983).
  • Chromosomal DNA from L. lactis subsp. lactis was isolated by the above-described method; only the chromosomal band was collected from CsCl-runs. Further purification of DNA, if needed, was done by CsCl- EtBr density gradient centrifugation, regardless of the source of the DNA preparation.
  • Restriction enzyme digestions were performed according to the manufacturer's recommendations (Boehringer, BRL, Promega) . Selected restriction fragments were obtained by separation of the digested DNA on 0.8% agarose gel electrophoresis (Sharp et al.. Biochemistry 12:3055-3063 (1973)) after which DNA extraction and purification was performed by a phenol-liquid nitrogen freezing method as follows: a slice of agarose containing the desired fragment was transferred to a siliconized Eppendorf tube and mashed with a glass rod. About
  • TE-buffer 250 /il TE-buffer was added together with an equal volume of phenol. After thorough mixing in a Vortex shaker, the tube was immersed in liquid nitrogen until frozen. The phases were separated by centrifugation at 1200 rpm for 15 minutes, after which phenol extraction was repeated and the resulting aqueous layer treated with ether and ethanol-precipitated.
  • the Klenow fragment of DNA polymerase I (Promega) was used.
  • T4 DNA polymerase (Promega) or mung bean nuclease (Promega) also were used.
  • CIP calf intestinal phosphatase
  • T4 polynucleotide kinase (Promega) was used for phosphorylation of the 5'-hydroxyl ends.
  • the ends of the DNA fragments were joined by T4 DNA ligase (Promega). All modifying enzymes were used according to manufacturer's recommendations.
  • Transformation of E. coli cells was accomplished by the method of Hanahan (J. Mol . Biol. 166:557-580 (1983)). ! subtilis cells were transformed by the method of Gryczan et al. (J. Bacteriol. 134:318-329 (1978)). L. lactis protoplast transformation was carried out according to von Wright et al . (Appl. Environm. Microbiol. 50:1100-1102 (1985)). L_ plantarum transformation by electroporation was performed by the method of Aukrust et al . (submitted for publication). The method is described below.
  • Electroporation For electroporation experiments, cells were grown to an optical density of 0.5-1.0 (A5Q0) . chilled on ice, harvested by centrifugation, washed, and resuspended in electroporation buffer (EB) to a cell density of about 10 9 cells/ml. An aliquot of 0.8 ml ice-cold cell suspension was mixed with 0.5-1.0 ⁇ g of plasmid DNA. Cells were kept on ice before and after electroporation in buffer (PEB).
  • Electroporation was performed using a GenePulser " ⁇ apparatus (BioRad Laboratories, Richmond, USA) at a constant capaci ⁇ tance of 25 /iFD, with a field strength between 1250 and 6250 V/cm for whole cells and between 1250 and 5000 V/cm for osmosensitive cells. Electroporation of intact cells was carried out in EB as described in the GenePulser operating instructions (BioRad Laboratories, Richmond, USA). Osmosensitive cells were electroporated in protoplast electroporation buffer (PEB): 0.5 M raffinose, 7 mM sodium phosphate pH 7.4, MgCl 2 up to 50 M.
  • PEB protoplast electroporation buffer
  • ff-lactamase was assayed according to O'Callaghan et al . (Antimicrob. Ag. Chemother. 1:238-288 (1972)). Cell and supernatant fractions were separated by centrifugation after growth in appropriate liquid medium.
  • Chloramphenicol acetyltransferase (CAT) assay Cells were grown to log phase, and 1 ml cultures were collected for enzyme activity analysis. Cells were harvested by centrifugation, washed with 50 mM sodium phosphate buffer pH 7, and suspended in 0.2 ml of the same buffer containing 4 mg/ml lysozyme. Cells were incubated for 30 minutes at 37 ⁇ C, after which they were disrupted by sonication (4 x 15 seconds, using a Bransonic sonicator; after each 15 seconds of sonication, the medium was cooled for 30 seconds in an ice bath). After sonication, the cell debris was pelleted by centrifugation. 50 ⁇ l of the supernatant was used for enzyme assay. CAT-activity was measured according to the method of Shaw, W.V., Meth. Enzvmol . 43:737-755 (1975)).
  • RNA was isolated according to the method of van der Vossen et al . (Appl. Environm. Microbiol. 53:2452-2457 (1987)) . , except that cells were cultured in 10 ml of M17G- medium containing 5 ⁇ g/ml chloramphenicol until Klett 80 was reached, and that the RNA (and DNA) was precipitated with ethanol (the medium was made to 0.5 M with 3 M NaAc, and 3 volumes of ethanol were added). The pellet was dissolved in distilled water.
  • RNAse-free DNAase I Promega
  • 40 mM Tris-HCl pH 7.9
  • the reaction mixture was extracted once with phenol, phenol-chloroform-isoamyl-alcohol (25:24:1, vol/vol) and chloroform-isoamylalcohol (24:1 vol/vol).
  • the RNA was precipitated with ethanol and the pellet was dissolved in 75 ⁇ l of water.
  • RNAs transcribed by the cloned promoter or promoter/signal sequence fragments were run and Northern transfer to nitrocellulose membrane (Schleicher and Schuell) was done according to Williams et al . (in, "Nucleic Acid Hybridization--A Practical Approach,” Hames et al . (eds.), IRL Press, pp. 139-160 (1985)).
  • the nitrocellulose filter was prehybridized in 0.06 M sodium citrate (4 x SSC), 50 mM sodium phosphate buffer (pH 6.5), 5 x Denhardt (Biochem. Biophvs. Res. Com un. .23:641-646 (1966)), 0.2% sodium dodecyl sulphate (SDS), and 200 ⁇ g/ml denatured herring sperm DNA (Sigma). Incubation was done for one to two hours at 65°C. Hybridization was done in the same medium containing nick- translated probe (10 s cpm/ml). After hybridization, the filter was washed (1-2 x) with 0.03 M sodium citrate (2 x SSC), 0.2% SDS, and incubated at 37"C for 30 minutes and for 30 minutes at 55 ⁇ C.
  • the transcriptional start sites were determined by primer extension.
  • 15 ⁇ l RNA (5 to 10 ⁇ g) primer (0.2 pmol of 20 base oligonucleotide) mixture 15 ⁇ l 2 x hybridization buffer (100 mM Tris-HCl, pH 8.3, 2 mM EDTA, 0.8 M NaCl) was added. The mixture was heated to 95"C for 2 minutes and allowed to cool to room temperature over a two-hour period by gradually lowering the thermostat of the water bath.
  • RNA-primer hybrid was precipitated with ethanol, and the pellet was dissolved in 5 ⁇ l of 2 x reaction buffer (100 mM Tris-HCl, pH 8.3 at 42 ⁇ C, 20 mM DTT, 12 mM MgCl 2 , 100 mM KC1, 0.5 M dATP, dTTP and dGTP and 50 ⁇ g/ml actinomycin Cj (Boehringer)).
  • 2 x reaction buffer 100 mM Tris-HCl, pH 8.3 at 42 ⁇ C, 20 mM DTT, 12 mM MgCl 2 , 100 mM KC1, 0.5 M dATP, dTTP and dGTP and 50 ⁇ g/ml actinomycin Cj (Boehringer)
  • RNAsin ⁇ 7 U AMV Reverse transcriptase (Promega) were added, and the total reaction volume was made to 10 ⁇ l with water.
  • the reaction mixture was incubated for 15 minutes at 42 ⁇ C, after which 0.5 ⁇ l of 10 mM dCTP (chase) was added, and incubation was continued at 42 ⁇ C for 1 hour and 45 minutes. Subsequently, the reaction mixture was extracted with phenol and phenol-chloroform-isoamylalcohol (25:24:1), and precipitated with ethanol.
  • the reverse transcriptase reactions were analyzed by electrophoresis on a standard sequencing gel. Sequencing reactions of one of the promoter constructions were used as a size marker and were run in parallel with the reverse transcriptase (RT) reactions.
  • Oligonucleotide synthesis of primers for sequencing and polymerase chain reactions were performed by phosphoramidite chemistry (Beaucage et al . , Tetrahedron Letters£.:1859-1862 (1981)) using Applied Biosystems DNA synthesizer model 381A.
  • DNA fragments was accomplished by GeneAmp DNA Amplification kit as described by Saiki et al . (Science 239:487-491 (1988)) and the DNA Thermal Cycler (both from Perkin Elmer-Cetus) .
  • Taq polymerase was purchased from Perkin Elmer-Cetus. EXAMPLE II
  • a promoter probe-vector able to replicate in E. coli, B. subtilis, Lactococci and Lactobacillus was constructed.
  • the replication origin for the shuttle vector was isolated from the plasmid pSH71.
  • the plasmid pSH71 was digested with restriction enzyme Clal to create two fragments of about 1.7 kb and 0.3 kb, the larger one of which contained the replication origin. The sticky ends were filled in with the Klenow fragment.
  • the mixture was run in an agarose gel to isolate the large DNA fragment, and the DNA was eluted from the gel by electroelution.
  • the gene coding for tetracycline was isolated from the plasmid pBR322, and the gene coding for erythromycin resistance was isolated from the plasmid pVS2.
  • pBR322 was digested with EcoRI and PvuII. The sticky ends created by EcoRI were filled in with the Klenow fragment, the mixture was run in an agarose gel, the tetra ⁇ cycline gene-containing fragment was isolated, and the DNA fragment (about 2 kb) was eluted from the gel by electroelution.
  • the Clal fragment containing the pSH71 replication origin and the DNA fragment containing the tetracycline gene were ligated and transformed into competent E. coli ERF173 cells. Transformants were selected by plating the transformation mixture on Luria-agar p.lates containing 12.5 ⁇ g/ml tetracycline. The structure of the plasmid was verified by restriction enzyme digestions. To this new plasmid, designated pKTH1722 ( Figure 4), the second resistance marker was added. pKTH1722 was linearized by XmnI digestion. The erythromycin gene was isolated from the plasmid pVS2 by Hindlll-Clal digestion, and the sticky ends were filled in with the Klenow fragment.
  • the mixture was run in an agarose gel, the gel fragment containing the erythromycin gene was isolated, and the DNA fragment eluted from the gel by electroelution.
  • the linearized plasmid pKTH1722 and the erythromycin gene-containing DNA fragment were ligated, the ligation mixture was transformed into competent E. coli ERF173 cells, and the mixture was plated on Luria agar plates containing 12.5 ⁇ g/ml tetracycline. Transformants were screened by their ability to grow on Luria-agar plates containing 100 ⁇ g/ml erythromycin. Plasmid isolation was done from erythromycin resistant colonies and the presence of the gene was verified by restriction enzyme digestions.
  • pKTH1734 One correct plasmid construction was named pKTH1734 ( Figure 5).
  • a promoterless gene coding for chloramphenicol acetyltransferase from the plasmid pPL603 was ligated to the plasmid pKTH1734.
  • pKTH1734 was linearized by EcoRI digestion, and the sticky ends were made blunt by the Klenow fragment.
  • the promoterless c t. gene was isolated from the plasmid pPL603 by EcoRI-PvuII digestion, the sticky ends were filled in with Klenow- fragment, and the mixture was run on an agarose gel .
  • the cat gene-containing DNA fragment (about 1.7 kb) was isolated by the phenol-liquid nitrogen freezing method as described above.
  • the linearized plasmid pKTH1734 and the c_at gene- containing DNA-fragment were ligated and transformed to E. coli ERF173 cells.
  • the inserts were screened by isolating plasmids and checking the restriction enzyme recognition patterns by digestions.
  • the plasmid pKTH1736 was obtained ( Figure 5) .
  • MCS multiple cloning sites
  • the plasmid obtained by the above procedure was digested with EcoRI and ligated to itself in a dilute medium and transformed to E. coli ERF173.
  • the promoter probe vector pKTH1750 was obtained from this transformation ( Figure 6).
  • the promoter probe plasmid pKTH1750 can replicate in E. coli, B. subtilis, and L. lactis.
  • the promoters were screened both in B. subtilis and in L. lactis.
  • Lactococcus chromosomal DNA, digested with Sau3A, was ligated with Bglll- digested pKTH1750 in a molar ratio of 2:1 (insert:vector DNA).
  • the mixture was transformed to L. lactis GRS5 cells and plated on M17GS-cm (4 ⁇ g/ml) plates, and also to B. subtilis BRB1 cells and plated on uria-cm (5 ⁇ (spl2hl2vsb6Tg/ml) plates.
  • ⁇ Activity was measured as U/ml of culture medium as described herein.
  • This plasmid was opened with the restriction enzyme Smal for a blunt-end cloning site or with BamHI to generate sticky ends.
  • Lactococcus chromosomal DNA was digested with Sau3A, which yielded fairly large (over 1000 bp) fragments, compatible for ligation with the BamHI-treated vector.
  • the chromosomal DNA was sonicated (Branson Sonifier, Branson Sonic Power Co.) to get 500-600 bp fragments. The extent of sonication was checked by running a small aliquot of treated sample in a 0.8% agarose gel with appropriate controls. The total sonicated DNA was then applied to a 0.8% agarose gel and electrophoresed. Fractions of about 600 bp were extracted and purified by phenol-1 iquid nitrogen treatment.
  • the ends of the DNA fragments were treated with the Klenow fragment as described above.
  • the ligations, in both cases, were performed in a molar ratio of 2:1 (insert:vector DNA) under standard conditions, and the mixtures were transformed into B. subtilis BRBI.
  • DNA was extracted from the positive clones and subjected to plasmid sequencing.
  • Plasmid pKTH33 contains the structural part of TEM- - lactamase gene preceded by an EcoRI linker. Part of the plasmid originates from pBR322, allowing its replication in E. coli. If a sequence bearing an expression/secretion signal is inserted, in frame, with the marker gene ⁇ -lactamase, active enzyme is produced, which renders the transformants resistant to ampicillin. By plating the transformants directly on a picillin plates, a positive selection for signal sequence fragments is obtained.
  • Plasmid pKTH33 was opened with EcoRI, treated with Klenow fragment to obtain blunt-end molecules, and purified by phenol extraction and ethanol precipitation.
  • the ligation mixture was transformed into E. coli ERF173 cells, and plated on Luria-ampicillin (50 ⁇ g/ml) plates.
  • Several transformants were screened for 5-lactamase activity by Nitrocefin assay on icrotiter wells: 200 ⁇ l of Nitrocefin (Glaxo) in 50 mM K-phosphate buffer (pH 7.0) were pipetted into microtiter plate wells. Bacterial colonies were transferred from plates with a toothpick and suspended in Nitrocefin. Positive clones turned red after 1-30 minutes incubation at room temperature, whereas negative clones stayed yellow.
  • the minimal inhibitory concentration (MIC) of ampicillin for the positive clones was determined as described, except that cells were plated on Luria-ap plates containing ampicillin from 50-450 ⁇ g/ml. MIC was the highest concentration still supporting growth.
  • Inserts of pKTH1797, pKTH1798, pKTH1799 and pKTH1801 were sequenced according to the dideoxy method of Sanger, and analyzed for the presence of expression/secretion signals. By matching the three reading frames with the known reading frame of jS-lactamase, the correct reading frame was determined. The length of the precursor proteins was compared with the data obtained from an in vitro transcription-translation assay (Figure 2), in order to confirm the validity of the sequences. ⁇ -lactamase activity of the four constructions was also determined by growing the appropriate strains in liquid medium (Table 6).
  • pKTH33 allows direct selection of the desired fragments, the clones could not, as such, be propagated in Gram-positive bacteria. It was therefore necessary to change the replicon by subcloning the promoter/signal sequence fragments into the plasmid pVS2.
  • the insert plus the entire J-lactamase gene was cleaved off from pKTH1797, pKTH1798, pKTH1799, and pKTHl ⁇ Ol by Clal- PvuII double digestion, and the desired fragments were extracted from a 0.8% agarose gel as previously described and treated with the Klenow fragment to generate blunt ends.
  • the vector pVS2 was opened with Hindlll, and treated with the Klenow fragment as above.
  • Ligation was performed in a molar ratio of 2:1 (insert:plasmid) under standard conditions, and the mixture was transformed into E. coli ERF173 cells and plated on Luria- cm (11 ⁇ g/ml) plates.
  • the production of .-lactamase was checked by the Nitrocefin microtiterwell assay, as described. Rapid isolation of plasmid DNA was done for positive clones, and the size of the insert was verified by restriction enzyme digests.
  • the four secretion vectors were designated pKTH1805, pKTH1806, pKTH1807 and pKTH1809 ( Figure 7).
  • Promoter strength was initially estimated by comparing the promoter's ability to grow on antibiotic plates (cm plates for strains cloned by promoter probe vector; ap plates for strains cloned by promoter/signal sequence vector), its ability to produce high MIC, or its ability to synthesize large amounts of gene product (chloramphenicol acetyl transferase or ⁇ -lactamase). ⁇ 38-
  • the promoter on the expression/secretion plasmid pKTH1807 was replaced by the promoter on the expression plasmid pKHT1817.
  • the promoter was taken from plasmid pKTH1817 by the polymerase chain reaction (PCR) technique, using oligonucleotides A and B as primers ( Figure l ⁇ ).
  • Primer B for the 3'-end of the promoter fragment was designed so that, at the end of the PCR fragment, a restriction enzyme recognition site for Xbal was created.
  • the signal sequence-?-!actamase (bla) region was taken from plasmid pKTH1807 by PCR, using oligonucleotides C and D as primers.
  • the 5'-end primer (primer C) was designed so that a restriction enzyme recognition site for Xbal was created.
  • Both the promoter fragment and the signal sequence-bla fragment obtained by PCR were digested with Xbal and purified on an agarose gel. They were ligated (as a 1:1 molar concentration ratio of signal sequence-bia to promoter fragment). The ligation of the Xbal site between the promoter and the signal sequence fragments regenerated the authentic 3'- and 5'- sequences at the joint region.
  • the ligation mixture was digested with Bglll and Clal. The digestion mixture was run in an agarose gel, from which the proper fragment—containing the promoter ligated to the signal sequence bl_a--was isolated. This fragment was amplified with PCR and digested with PvuII. It was ligated to a pVS2-vector, which was digested with Hpall and made blunt by the Klenow enzyme.
  • the ligation mixture was transformed into competent E. coli ERF173 cells and plated on Luria-ap (100 ⁇ g/ml) plates. Transformants so obtained were streaked several times on ap plates, in order to get stable cultures.
  • the plasmid was isolated, transformed to L. lactis GRS5 cells, and plated on M17GS-cm (5 ⁇ g/ml) plates. From these transformations, a clone (pKTHl ⁇ 9) was obtained which, as shown in Table ⁇ , produced approximately ten times more 5-lactamase than L. lactis strain pKTHl ⁇ 07, which contained the original promoter/signal sequence combination.

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Abstract

Des séquences d'ADN, dérivées de Latococcus Lactis sous-esp. lactis, sont efficaces comme promoteurs et signaux de promotion de promoteurs/sécrétion pour l'expression hétérologue ou homologue dans des bactéries Gram positif. Dans un autre aspect, l'invention se rapporte à des vecteurs, tels que des plasmides, comportant les séquences de ladite invention, et à des cellules hôtes transformées avec lesdits vecteurs. Cependant, des aspects supplémentaires de ladite invention se rapportent à des procédés de production des peptides ou des protéines hétérologues ou homologues désirés, utilisant les séquences, les vecteurs, ou les hôtes transformés décrits par l'invention. Au moyen de l'invention, on peut réaliser une expression et une sécrétion hétérologues et homologues considérablement améliorées, dans E.coli et des bactéries Gram positif telles que B.subtilis, Lactococci et Lactobacillus.
EP90913051A 1990-08-30 1990-08-30 Vecteurs sondes de promoteurs pouvant se repliquer en e.coli, b.subtilis, lactococci et lactobacillus, ainsi que leurs utilisations Withdrawn EP0550428A1 (fr)

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US5242821A (en) * 1989-07-10 1993-09-07 Valio, Finnish Co-Operative Dairies' Association Lactococcus promoter and signal sequences for expression in bacteria
AU4329793A (en) * 1992-06-30 1994-01-24 Viagen Oy (lactobacillus) expression system using surface protein gene sequences
US5837509A (en) * 1992-12-30 1998-11-17 Bioteknologisk Institut Recombinant lactic acid bacterium containing an inserted promoter and method of constructing same
NZ286635A (en) * 1992-12-30 1997-06-24 Chr Hansen As Recombinant lactic acid bacterium, dna containing a lactic acid promoter
US6143525A (en) * 1994-05-12 2000-11-07 Quest International B.V. Complex inducible promoter system derivable from a phage of a lactic acid bacterium (LAB), and its use in a LAB for production of a desired protein
DK0712935T3 (da) * 1994-11-18 2002-07-15 Stichting Nl I Voor Zuivelonde Fremgangsmåde til styring af genekspressionen i mælkesyrebakterier
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
FR2739629B1 (fr) * 1995-10-06 1997-12-26 Systems Bio Ind Utilisation d'un systeme de secretion sec-dependant pour secreter des proteines normalement secretees par un systeme de secretion sec-independant, bacteries les contenant et leur utilisation
DK0925364T3 (da) * 1996-09-06 2003-02-24 Bioteknologisk Inst Et regulerbart ekspressionssystem til brug i mælkesyrebakterier
EP1206556B1 (fr) 1999-08-06 2007-01-17 Bioneer A/S Peptides signaux de lactococcus lactis
EP1239032A1 (fr) 2001-03-02 2002-09-11 Société des Produits Nestlé S.A. Bactéries lactiques en tant qu'agents pour le traitement et la prévention des allergies

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