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WO1996032487A1 - 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 - Google Patents

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 Download PDF

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Publication number
WO1996032487A1
WO1996032487A1 PCT/NL1996/000160 NL9600160W WO9632487A1 WO 1996032487 A1 WO1996032487 A1 WO 1996032487A1 NL 9600160 W NL9600160 W NL 9600160W WO 9632487 A1 WO9632487 A1 WO 9632487A1
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Prior art keywords
sequence
nucleic acid
protein
expression
lactic acid
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PCT/NL1996/000160
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English (en)
Inventor
Robert Jan Leer
Pieter Hendrik Pouwels
Wilhelmus Johannes Boersma
Original Assignee
Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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Priority to AU51639/96A priority Critical patent/AU5163996A/en
Publication of WO1996032487A1 publication Critical patent/WO1996032487A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a method to construct multi-purpose plasmid vectors that can be used for the introduction, stable maintenance and efficient expression and secretion of foreign genes in a variety of lactic acid strains such as Lactobaci llus strains and Bifidobacteria , as well as to expression vectors to be used in this method.
  • the invention also comprises a method of production of proteins and polypeptides heterologous to lactic acid bacteria at increased levels as well as some novel products.
  • Structural instability of plasmid vectors with strong cloned promoters has been reported by different groups [e.g. Gibson et al, Gene 53. 275" 281 1987] and has also been observed for the promoter of the Lactobac i llus casei Idh gene by the present inventors. This type of instability is explained by assuming that transcription starting at the promoter interferes with efficient replication of the plasmid vector. The instability can, in general, be overcome by insertion of a transcription terminator sequence between the promoter and the origin of replication.
  • European Patent Application EP-B-0.449-770 describes a shuttle vector directed at secretion of homologous and heterologous polypeptide or protein from Lactococcus or Bacillus microorganisms. Secretion is deemed necessary due to inherent problems of internal storage of heterologous expression products requiring refolding of the expression product. None is stated or suggested with regard to instability of a lactic acid bacterial expression product or nucleic acid sequence in a working organism such as E. coli. None is stated or suggested with regard to expression problems of heterologous genes in Lactobacilli or Bifidobacteria. None is stated or suggested regarding amount of expression product produced by a lactic acid bacterium host cell.
  • the problem addressd in the cited patent application differs from the problem addressed in the instant case.
  • Vectors described in the abovementioned European Patent Application do not fall within the category claimed in the instant case.
  • heterologous polypeptides are produced in Lactococcus by means of a T7 or T7- ⁇ ke RNA polymerase gene placed under the control of an inducible promoter effective in the Lactococcal host and a promoter specific for said polymerase upstream of a coding sequence for the desired polypeptide, whereby the promoter directs transcription of said coding sequence selectively as a result of expression of said polymerase.
  • the T7 RNA polymerase system is an E coli system that also works in Lactococcus.
  • the cited patent application does not address the issue of incompatibility of lactic acid bacterial expression products or nucleic acid sequences in a working organism such as E. coli. More specifically a promoter operable in E coli, the T7 promoter is described linked to the signal peptide and protein translation initiation signal of the lactococcal protease gene, whereby the presence of the protease sequence is to ensure secretion of the expression product.
  • the subject invention however is not directed at expression vectors that are only useful in Lactococcus or E coli. Neither is the subject invention directed at improved expression through use of a specific strong promoter.
  • the cited patent application provides a good summary of the state of the art regarding lactic acid bacterial expression of heterologous proteins or polypeptides and clearly illustrates that the problem of low expression using lactic acid bacteria as expression host cells has existed for a long time and that the state of the art has not taught or suggested a solution along the lines indicated by the subject invention which offers a general solution to the problem.
  • Use of the T7 promoter as described in the cited state of the art is not comprised within the subject invention.
  • ⁇ -galactosidase was in fact reduced ten- to twenty-fold (final level 0.1-0.2 ⁇ of total soluble protein) when ⁇ - galactosidase was fused at its N-terminal end with a twenty to thirty amino acid antigenie determinant from the Foot and Mouse Disease Virus capsid protein VP1 or the Rotavirus capsid protein VP7.
  • the cited patent application does not address the issue of incompatibility of lactic acid bacterial expression products or a nucleic acid sequence in a working organism such as E. coli.
  • This cited patent application clearly illustrates that the problem of low expression using lactic acid bacteria as expression host cells has existed for a long time and that the state of the art has not taught or suggested a solution along the lines indicated by the subject invention which offers a general solution to the problem.
  • Use of the SP regulatory elements as described in the cited state of the art is not comprised within the subject invention.
  • WO 92/04451 and US 5,242,821 a number of specific Lactococcus promoters and promoter/secretion promoting signals are disclosed as being useful in production of heterologous and homologous proteins and peptides in E. coli and especially in Gram positive bacteria.
  • the promoter sequences are those found in plasmids pKTH1789, pKTHl ⁇ l ⁇ , pKTHl8l7, pKTHl820, pKTHl821 and pKTHl ⁇ 74.
  • the promoter and secretion promoting signals are those found in plasmids pKTH1797, pKTH1798, pKTH1799, pKTHl ⁇ Ol, pKTHl805 and pKTHl8 ⁇ 6, pKTHl ⁇ 07 and pKTHl809.
  • the promoter/signal promoting sequence is described as being "generally 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".
  • the regulatory elements of these plasmids can according to this document be recombined to produce hybrid expression units which can function together to allow enhanced heterologous expression in E.
  • the description further illustrates how promoters can be picked up by a probe vector lacking a promoter prior to a cat gene and how secretion signal sequences can be picked up using a vector comprising the marker gene ⁇ -lactamase. No indication is given of other heterologous or homologous proteins or polypeptides to be expressed. No comparison of degree of expression with existing systems is given. No illustration with other sequences than Lactococcus sequences is given. No indication is given for different degrees of expression from the various promoters.
  • the invention provides a method for randomly screening for regulatory sequences of Lactococcus. Once such sequences have been ascertained and isolated they can be introduced and applied in other vector systems.
  • No expression vector comprising a lactic acid bacterium promoter followed by at least the first five codons of a lactic acid bacterium gene followed by a further gene product under regulation of the same promoter is disclosed explicitly as such for improving expression of the further gene in lactic acid bacteria in the instant cited application.
  • a vector being any one of plasmids pKTH1797. pKTH1798, pKTH1799. pKTHl ⁇ Ol, pKTHl805, pKTHl806, pKTHl807, pKTHl809 and pKTHl889 does comprise a promoter and a signal sequence from lactococcus regulating the ⁇ -lactamase gene.
  • the signal sequence comprises the first codons of the relevant lactococcal gene from which it is derived for secretion purposes of ⁇ -lactamase.
  • Such 7 vectors are however not used for increased expression of heterologous genes in lactic acid bacteria but as probe vectors.
  • the intermediate lactococcal signal sequence could in fact be responsible for increased expression of a heterologous gene linked thereto is also not disclosed. Neither is any indication given of the nature of the intermediate sequence other than that it being a lactococcal secretion signal as described above.
  • the objective of the present invention is to overcome the above mentioned difficulties and to provide per se and in combination i) a method for the construction of plasmid expression and expression- secretion vectors in a working organism other than the lactic acid bacterium to be transformed, for example in the working organism Escherichia coli , ii) a series of expression and expression-secretion vectors for transforming lactic acid bacteria like Lactobaci llus and Bifidobacteria, said vectors being characterized by at least a translation initiation region allowing efficient expression of heterologous nucleic acid encoding foreign proteins or polypeptides, and iii) a series of vectors that efficiently express, secrete and display antigens from pathogens such as influenza virus or rotavirus at the bacterial surface of their host, said vectors preferably being food grade.
  • heterologous proteins or polypeptides in lactic acid bacteria strains.
  • expression of heterologous proteins or polypeptides in Lactobacilli and Bifidobacteria and most preferably in Lactobacilli is an objective of the subject invention.
  • the products thus obtainable are also considered to fall within the scope of the invention.
  • the present invention provides a method to construct by recombination DNA technology a series of expression vectors and expression-secretion vectors in a working organism other than the lactic acid bacterium to be transformed, in particular a non Lactobaci llus working microorganism such as Escherichia coli that can be used to genetically modify a bacterium, in particular a lactic acid bacterium so as to efficiently produce foreign proteins or polypeptides.
  • Especially interesting microorganisms for expression are Lactobacilli and Bifidobacteria.
  • Especially interesting is the production of immunogenic proteins and peptides from pathogenic organisms and proteins e.g. mammalian proteins that can be used for the induction of immunological tolerance.
  • the level of expression of a heterologous protein or polypeptide in a lactic acid bacterium whilst the corresponding nucleic acid is under control of a promoter operative in the lactic acid bacterium, can be considerably increased by insertion of a nucleotide sequence coding for the N-terminal sequence of a gene homologous to a lactic acid bacterium gene, immediately downstream of the translation start codon.
  • the intermediate lactic acid bacterium sequence need only be 5 codons long but can be anywhere in length between 5 _ 15 codons, even 5 ⁇ 34 codons.
  • the fragment can even comprise up to the 5' half of the gene. It will be preferred in general to keep the fragment as short as possible.
  • the protein or polypeptide can be homologous or heterologous to the lactic acid bacterium in which it is to be expressed, with a preference for heterologous.
  • the ' non translated region located between the intermediate sequence and the promoter is preferably homologous to lactic acid bacterium, in particular preferably homologous to the host cell that is to comprise the expression vector for expression of the protein or polypeptide that is to be expressed jointly with the 5' intermediate lactic acid bacterium sequence. It is efficient for the 5' non translated and 5' translated lactic acid bacterium sequences to be derived from the same organism and indeed preferred for them to be derived from tha same gene.
  • promoter regulating the expression of the intermediate sequence and the protein and polypeptide to be expressed in the vector according to the invention.
  • promoter, 5' non translated region and 5' terminal translated fragment of at least codons are all derived from the same lactic acid bacterium and preferably the same gene. The latter saves the person skilled in the art finding the operable sizes and distances of the relevant components for good expression. Naturally however the sizes and distances between the various components can be varied in an attempt to optimise expression. The basic order of the components is however not subject to alteration.
  • the present invention more specifically provides: i) A series of plasmid vectors with which foreign proteins, in particular immunogenic proteins from pathogenic organisms or mammalian proteins that can induce immunological tolerance, can be efficiently expressed in Lactobaci llus . These foreign proteins can optionally be accumulated or excreted and/or secreted either into the culture fluid or subsequently anchored to the membrane and displayed at the surface of bacteria.
  • the present invention is characterized by a novel method to construct the vectors in a non lactic acid bacterium for example in a working organism like Escherichia coli prior to their introduction in a lactic acid bacterium like Lactobaci llus.
  • Application of the invention thus in particular circumvents the structural instability of Lactobaci llus vector material in Escherichia coli caused by cloning of strong Lactobaci llus promoters and/or by cloning and expression in Escherichia coli of Lactobac i llus genes.
  • the construction route in a non lactic acid bacterium for example in a working organism like Escherichia coli involves the insertion of a nucleotide sequence containing a transcription terminator between the promoter controlling the expression of the heterologous nucleic acid and the transcription terminator for the heterologous gene located downstream from the promoter.
  • the newly introduced terminator serves to prevent transcription in Escherichia coli of open reading frames (ORF) that are to be inserted downstream from the promoter sequence so as to prevent their expression in Escherichia coli .
  • ORF open reading frames
  • the newly introduced terminator sequence will be located such that no product controlled by the promoter is produced in the working organism. This can be achieved by locating the newly introduced terminator prior to the first codon of the heterologous expression product.
  • the newly introduced terminator sequence is preferably a strong terminator sequence.
  • the newly introduced terminator sequence must be recognised as such by the working organism in which the vector is to be constructed or manipulated prior to transformation of a lactic acid bacterium.
  • a preferred embodiment comprises a newly introduced terminator sequence that is recognised by the working organism, a non lactic acid bacterium but is not recognised by the lactic acid bacterium in which expression of the heterologous protein or polypeptide is desired.
  • a newly introduced terminator sequence is employed that can also be recognised by a lactic acid bacterium in which the heterologous protein or polypeptide is to be expressed it is necessary for the transcription block to be lifted.
  • the newly introduced terminator sequence is flanked at both sides such that it is excisable from the vector in a manner known per se to a person skilled in the art. This can be the case for example when it is flanked by a nucleic acid sequence encoding one or more very infrequently occurring restriction enzyme sites (e.g. NotI ) which do not occur elsewhere in the plasmid, allowing easy removal of the newly introduced terminator sequence from the vector prior to its introduction into the lactic acid bacterium for example a Lactobaci llus .
  • the newly introduced terminator sequence may be flanked by direct repeats such that the terminator sequence is excisable through homologous recombination of the host.
  • the cloned ORF with the heterologous protein or polypeptide will be transcribed from the promoter in the lactic acid bacterium.
  • the working organism need not necessarily be a non lactic acid bacterium; this is merely a preferred embodiment due to the inherent problems of working in a lactic acid bacterium as working organism.
  • the nucleic acid sequence encoding a protein or polypeptide i.e. the product to be expressed need not necessarily be heterologous to a lactic acid bacterium.
  • the system according to the invention may also be employed for homologous nucleic acid sequences. Generally speaking however expression of heterologous sequences normally evokes more problems than expression of homologous sequences.
  • the present invention also provides expression and expression-secretion vectors that allow efficient expression of cloned foreign genes. It was found that the level of expression of a heterologous protein or 12 polypeptide like the Escherichia coli enzyme ⁇ -galactosidase in a lactic acid bacterium such as Lactobaci llus, whilst the corresponding nucleic acid is under control of a homologous promoter of the lactic acid bacterium, for example a Lactobacillus promoter, can be considerably increased by insertion of a nucleotide sequence coding for the N-terminal sequence of a gene homologous to a lactic acid bacterium like a Lactobaci llus immediately downstream of the translation start codon.
  • a and T rich nucleic acid sequences at the beginning of a nucleic acid sequence to be expressed in a lactic acid bacterium is conducive to high expression being achieved from such sequences.
  • lactic acid bacteria like Lactobaci l lus have A and T rich sequences in parts of their genes encoding the N terminal part of their proteins this could be the reason why the presence of such a sequence ensures good expression in a lactic acid bacterium. It is possible that a high number of G and C residues in this region would favour the formation of stable RNA duplexes, which may interfere with translation initiation.
  • Preferred fragments located immediately behind the initiation codon and prior to the protein or polypeptide to be expressed have an AT percentage of 60% or more, preferably higher than 62% , also 65/- or more and even 70 or more. Even higher percentages can be used. AT percentages as high as 8 and 90 even up to 95% and as high as 100 are suitable. As discussed above the length of the fragment is not critical, but must be at least codons. It is preferable to use sequences that naturally occur in a lactic acid bacterium, preferably that occur in the lactic acid bacterium host cell in which the protein or polypeptide has to be expressed.
  • the Lactobacillus microorganism has to date been a most difficult organism in which to achieve good stable expression in particular of nucleic acid heterologous to Lactobacillus or to any lactic acid bacterium and as such such an organism is a preferred host cell for expression vectors according to the invention.
  • said vector In order to obtain good expression in a lactic acid bacterium and optionally surface display of a protein or polypeptide, said vector must comprise an expression promoter sequence and a nucleic acid sequence encoding said protein or polypeptide with expression of said nucleic acid sequence being controlled by said promoter sequence, characterised in that said encoding nucleic acid sequence is preceded by a 5' non- translated nucleic acid sequence comprising at least the minimal sequence required for ribosome recognition and RNA stabilisation followed by a translation initiation codon, said translation initiation codon being immediately followed by a fragment of at least 5 codons of the 5' terminal part of the translated nucleic acid sequence of a gene of a lactic acid bacterium or a structural or functional equivalent of said fragment, said fragment also being controlled by said promoter, thus resulting in an expression product which is a fusion polypeptide.
  • some vectors or plasmids have inadvertently been described in the state of the art comprising the elements minimally required to achieve the desired
  • a structural or functional equivalent of said fragment of at least codons is understood to include fragments encoding the same amino acid sequence but using different codons, fragments derived from other organisms than lactic acid bacteria but encoding corresponding parts of functionally related polypeptides, and equivalents of said fragment having at least 60% or even 80% nucleotide homology and preferably containing at least all the A and T residues of the naturally occurring fragment are covered by this definition. Preferablt the native sequences will be applied.
  • the gene from which the 5' terminal part of the translated nucleic acid sequence is derived is preferably the same gene from which the 5' non- translated region located between the promoter which is used to control the expression of the heterologous nucleic acid is derived.
  • the resulting expression product thus becomes a hybrid of homologous and heterologous amino acid sequences, whereby the presence of the N-terminal sequence of the lactic acid bacterium gene in the resulting expression product does not detract from the functionality of the heterologous product that needs to be expressed. It is not necessary for the homologous N-terminal sequence to be very long.
  • a nucleic acid sequence encoding at least amino acids is sufficient to obtain good expression results. The maximum length to be used will depend on the expression host and the nature of the expression product.
  • the maximum length is not really critical.
  • One embodiment can comprise as small a sequence as possible in order not to risk the loss of functionality of the resulting expression product due to tertiary folding like e.g. proteolytic instability sufficiently to disturb the activity of the heterologous protein or polypeptide.
  • a sequence of ten amino acids has been found to form an embodiment that is capable of achieving the objective of the invention quite adequately.
  • N- terminal sequences with lengths over 20 amino acids are acceptable embodiments as well.
  • longer sequences are contemplated embodiments forming e.g. complete genes, without the corresponding gene terminator.
  • polycistronic mRNA encoding at least two proteins can be envisaged.
  • the expression and expression-secretion vectors of the present invention can comprise the entire non-translated 5' region including the Shine- Dalgarno sequence [Shine and Dalgarno, Proc. Natl. Acad. Sci. USA 71. 5463-5467 (1974)], translation initiation codon and the A/T-rich N- terminal sequences preferably of a lactic acid bacterium gene.
  • the selection of the gene from which the N-terminal sequence and the promoter are to be used will be obvious to a person skilled in the art.
  • the criteria that are relevant are the organism in which expression is desired and the degree of expression that is normal for the gene system of which some components are to be used in the vector when the gene system is functioning normally in its natural environment.
  • Lactobac i llus for example a number of proteins are known or are expected to be expressed to a high degree and any of the relevant components from the corresponding gene systems of Lactobaci llus are suitable for use in vectors according to the invention.
  • a large deal of sequencing has already taken place and a person skilled in the art can quite readily ascertain suitable sequences that can be used.
  • Figure 12 the ribosomal RNA encoding gene rrn, the glyceraldehyde-3-phosphate dehydrogenase encoding gene gapdh are examples of genes that are efficiently expressed in Lactobaci llus.
  • Their promoters and N terminal sequences can quite suitably be employed in vectors according to the invention.
  • the configuration around the translation initiation start site in the expression vectors is chosen so as to warrant translation of foreign proteins in a manner equally efficient to that of the authentic Lactobaci llus.
  • L-lactate dehydrogenase proteinase, ⁇ -amylase, S layer protein, the ribosomal RNA and the glyceraldehyde-3-phosphate dehydrogenase are all proteins that are efficiently translated in Lactobaci llus.
  • L-Lactate dehydrogenase is a key enzyme in the glycolytic pathway of lactobacilli which mediates the conversion of pyruvate to lactate.
  • the proteinase encoded by prtP is an enzyme secreted and anchored to the cell surface through a so-called anchor sequence by a number of Lactococci and Lactobaci lli which enables the bacteria to proteolytically hydrolyse casein and therefore grow on milk.
  • ⁇ - a ylase is an enzyme secreted by Lactobacillus amylovorus that enables the bacterium to grow on starch as sole energy source.
  • nucleic acid sequence encoding a protein or polypeptide i.e. the product to be expressed need not necessarily be heterologous to a lactic acid bacterium.
  • the system according to the invention may also be employed for expression of homologous nucleic acid sequences.
  • the present invention is further in preferred embodiments characterized by the use of fully sequenced, multi-copy plasmid expression and expression-secretion vectors carrying a replicon from the lactic acid bacterium in which the heterologous nucleic acid is to be expressed.
  • Such an organism can be a Lactobaci llus .
  • the vectors preferably have a modular structure facilitating easy manipulation and exchange of cassettes from one vector to another.
  • the cassettes can comprise in addition to the essential components described in the preceding description of embodiments of the invention and those components of neccesity present in a vector in order to be operable a number of additional components.
  • the vector includes in the order 5' to 3' one or more additional components.
  • the additional components follow the constitutive promoter or the strong regulatable promoter, which promoter could e.g. be from a lactic acid bacterium such as Lactobaci llus and/or follow the transcription termination sequence for blocking transcription of the heterologous protein or polypeptide in the working host.
  • the components can be i) a DNA sequence encoding the signal sequence of a secreted protein recognisable by a lactic acid bacterium as such, this could for example be a signal sequence from a lactic acid bacterium, ii) a reporter gene encoding an easily assayable product in the working organism like e.g.
  • said reporter 16 gene can be located either in frame between the initiation codon and the nucleic acid sequence encoding a protein or a polypeptide or in frame after the nucleic acid sequence encoding a protein or a polypeptide under the control of the same promoter as the nucleic acid sequence encoding a protein or polypeptide, iii) a DNA sequence encoding an anchor sequence from a gene of a lactic acid bacterium for example like Lactobaci llus, said DNA sequence being located in frame downstream from the nucleic acid sequence encoding a protein or a polypeptide and iv) a transcription termination sequence of a lactic acid bacterium gene like a Lactobacillus gene located such as to ensure only expression of the desired heterologous protein or polypeptide and v) A DNA sequence encoding a probiotic product.
  • the working host is preferably a non lactic acid bacterium, in particular not a Lactobaci llus and preferably an E coli.
  • Additional embodiments of the invention are further characterized by the presence in the vectors, between the translation initiation region and reporter gene, of a series of restriction enzyme sites (MCS) in three reading frames, allowing in-phase translation of any foreign DNA sequen ⁇ ce, coding for e.g. an antigenic determinant.
  • MCS restriction enzyme sites
  • the advantages of MCS in vectors are well known to a person skilled in the art.
  • the present restriction enzyme sites can be easily manipulated to allow in-phase translation of inserted ORF sequences with the downstream reporter gene; or when the reporter gene is omitted, in-phase fusion with an anchor sequence if immobilisation on the surface of the organism in which the sequence is expressed is required.
  • Such an anchor sequence can be that of prtP of Lactobacillus if Lactobaci llus is to be transformed.
  • phase fusion may also be desirable when the product to be expressed should not contain additional amino acids at the carboxyl end. This can be realised through fusion to the last amino acid codon of the cbh gene.
  • restriction enzyme sites can be easily manipulated so as to allow in-phase translation of the inserted DNA sequence with a downstream reporter gene.
  • a further embodiment of the invention is the presence in the vector of restriction enzyme sites that permit easy deletion of the reporter gene.
  • Another option is the presence of sites such that substitution of the reporter gene by other genes coding for desired proteins, polypeptides or parts thereof can occur.
  • proteins or polypeptide can serve the purpose of an immunogenic carrier, e.g. Escherichia coli ⁇ -galactosidase can be suitably used for this purpose.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram glucuronidase
  • ⁇ -glucuronidase A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention
  • Fusion of an antigenic determinant to a large immunogenic protein e.g ⁇ -glucuroni ⁇ dase
  • the immunogenic protein serves as the carrier.
  • the cassette structure allows deletion of the reporter gene at will, for example for expression of non-fused heterologous proteins.
  • nucleic acid encoding products with health stimulating properties to further enhance the value of a transformed lactic acid bacterium.
  • proteins are immunomodulators, e.g. cytokines, bacteriocines , glutathione-S-transferase, immuno-globulins and levanase.
  • the sequences encoding such proteins are comprised in the state of the art or can be derived without undue burden in a number of cases from clones comprising the relevant sequences.
  • the sequences of such products are hereby enclosed by reference.
  • Embodiments of the present invention can additionally be characterized by the possibility, in the case of the synthesis of proteins that are to be secreted and surface-bound e.g. via the membrane or the S layer protein.
  • the length of the spacer region between the antigenic determinant and the anchor sequence can be varied, depending on the thickness of the peptidoglycan layer and the sensitivity of the fusion protein to proteolytic attack.
  • Embodiments of the present invention can further be characterized by the possibility to replace commonly used antibiotic resistance markers and Escherichia coli DNA sequences of the vectors by a food grade marker e.g. a nutritional selection marker from a food-grade organism, to obtain a food-grade vector comprising elements derived from food-grade organisms only.
  • the food grade marker preferably originates from Lactobaci llus , enabling the construction of vectors to be performed under conditions that are exempt from regulations for recombinant DNA research of the EC.
  • pGEM-3 Basic vector construction for a vector suitable for Lactobacillus Firstly as starting material for the construction of the expression and expression-secretion vectors, pGEM-3 was used.
  • the strategy for the construction of the expression vector is as follows. Expression from strong Lactobac i llus promoters like the Lactobaci l lus case i Idh promoter in E. coli frequently leads to structural instability which can be overcome by inserting a terminator directly behind the promoter sequence. Consequently, the transcription terminator (e.g. T s _ h ) was first inserted in pGEM-3 before a Lactobaci llus promoter sequence was introduced.
  • T s _ h the transcription terminator
  • nucleotide sequence of pGEM-3 Promega
  • pGEM-3 Promega
  • pLPE323 Lactobaci llus plasmid vector
  • All other elements used for the construction of the expression vectors such as promoter sequences, terminator sequences etc, is fully known. All elements were provided with flanking nucleotide sequences harbouring restriction enzyme sites which do not occur elsewhere in the plasmid, permitting easy manipulation of the elements.
  • the final set of expression cassettes was equipped with a number of unique restriction enzyme sites permitting further modification of the vectors, if wanted.
  • a 260-bp Hpal-Hindlll fragment containing the last amino acid codon and transcription terminator sequence of the cbh gene of Lactobaci llus plantarum 80 [Christiaens et al , Appl. Environm. Microbiol. 8, 3792-3798 1992] was ligated between the Hindi and Hf dlll sites of pGEM-3. Subsequently, the H ⁇ ndlll site was converted into an E ⁇ gl site using a synthetic linker sequence.
  • the remaining multiple cloning sequence was replaced by a chemically synthesized oligonucleotide (PRESS) containing the following restriction enzyme sites, EcoRI, Bglll , Spel , SphI , B ⁇ mHI, BstEll .
  • the BstEII site is adjacent to the Hindi site used for cloning of the cbh fragment.
  • the resulting vector which was designated pGTC3 ( Figure 1) was used for the insertion of three DNA fragments containing expression and expression- secretion sequences.
  • promoter bearing DNA fragments were inserted into the new PRESS of pGTC3 ( Figure 1).
  • the promoter, translation initiation region and first 8 specific codons of Lactobaci llus casei Idh, which are present on a 513 ⁇ bp S ⁇ 3AI fragment were cloned between the Bglll and B ⁇ mHI sites of pGTC3, leaving the B ⁇ mHI site 3' from the promoter sequence intact.
  • a DNA fragment containing the 5' part of the Lactobaci llus casei Idh gene [Kim et al, Appl. Environm. Microbiol. 57.
  • the first 38 amino acids of the primary translation product (preproprotein) comprise the typical tripartite structure of a Sec-dependent signal sequence.
  • the vector constructed in this way was called pGAM3 ( Figure 2B-b) .
  • the terminator sequence of the Lactobaci llus casei Idh gene was inserted in the vectors.
  • a synthetic oligonucleotide was inserted encompassing the transcription terminator of the Lactobac i llus casei Idh gene flanked by two Notl sites and followed by a nucleotide sequence containing B ⁇ mHI and Ncol sites.
  • E. coli uidA gene coding for ⁇ -glucuronidase was isolated from pN0M2 [Roberts et al, Curr. Genet. 15. 177-180 1989] and cloned into a ⁇ UC19 derivative.
  • the uidA gene was extended with two codons by insertion of a hexanucleotide comprising an Xhol site ( Figure 4).
  • a BstEII site was introduced 6 nucleotides downstream from the translation stop codon. All changes, which were introduced by PCR, were confirmed by sequence analysis.
  • the series of vectors was further extended by insertion of a PCR- generated XhoI-BstEII fragment of the prtP gene from Lactobaci llui. casei ATCC 393 encoding 117 C-terminal amino acids ( Figure 5-c) .
  • This C- terminal sequence is, in analogy with similar sequences from lactococcal proteases, thought to be responsible for anchorage to the bacterial cell surface.
  • the vectors can be further adapted to accomodate differences in thickness of the peptidoglycan layers of different bacterial species by extension of the cell wall spanning domain.
  • the cell wall spanning peptide can be easily extended by replacement of the present XhoI-BstEII fragment by a larger XhoI-BstEII fragment encoding additional aminoacid sequences, or insertion into the Xhol site of a sequence encoding additional amino acids of the cell wall spanning domain.
  • the anchored protein will be closer to or in direct contact with the cell surface.
  • a synthetic oligonucleotide comprising restriction enzyme sites for the following enzymes, C ⁇ l, B ⁇ mHI, Muni , Kpnl , Hindlll , Smal , Bbsl , EcoRI, Sail, Apal , Ncol (MCS) was inserted between the B ⁇ mHI and TVcoI sites of one of the expression cassetttes.
  • the original B ⁇ mHI site was destroyed using this procedure, but a new B ⁇ mHI site was present within the newly inserted synthetic oligonucleotide ( Figure 6-b) . From these prototype cassettes the MCS sequences can easily be transferred to other cassettes as all cassettes contain unique B ⁇ mHI and Ncol sites.
  • any open reading frame can be aligned in-frame with that of the N-terminal sequence of the ami/, Idh or prtP genes.
  • ORF's that are cloned in one of the sites of the MCS or between two sites of the MCS, can be easily aligned with the uidA gene. Alignment of the reading frame of the new ORF's to sequences downstream from the MCS can be achieved by adjusting the sequence in the distal part of this MCS.
  • the Bbsl restriction enzyme recognition sequence was incorporated within the synthetic oligonucleotide (MCS) .
  • the enzyme Bbsl recognizes the non-palindromic sequence GAAGAC and cleaves the DNA strand 2 nucleotides downstream from this sequence. Cleavage in the anti- parallel strand occurs 6 nucleotides downstream from the recognition sequence ( Figure 7) •
  • the resulting sequence shows again a +1 frameshift of the sequences upstream of and including the Bbsl site, relative to the TVcoI site ( Figure 8-c).
  • the Bbsl recognition sequence is not affected by this procedure.
  • the Bbsl recognition sequence in the MCS can also be used to clone EcoRI compatible sequences in the MCS.
  • the nucleotides for the MCS directly following the Bbsl site were chosen to yield compatible termini after Bbsl and EcoRI cutting. The latter enzyme will not be used in practice since it is not unique in the expression vectors.
  • the presence of unique Xhol and BstEII sites in the expression cassettes allows variation at will of the length of the anchor sequence, so as to adjust the peptidoglycan spanning region to the thickness of the peptidoglycan layer of the host bacterium.
  • the nucleotide sequence with the terminator, T ldh was removed from the pLP400-T and pLP500-T series of plasmids by digestion with TVotI and religation.
  • the religated plasmids were used to transform Lactobaci l ⁇ lus, resulting in plasmids pLP4 ⁇ l, pLP402, pLP403, and pLP501, pLP502 and pLP503 ( Figure 9a and 9b).
  • the applicability of the vectors can be further improved by removal of the Escherichia coli DNA sequences and replacement of the erythromycin- resistance gene by a food-grade selection marker, resulting in a food- grade vector.
  • These alterations can be achieved by digestion of plasmids of the pLP 00 and pLP500 series with Bglll and Vhel and insertion of a DNA fragment comprising the xylR, xylA/B genes and a replicon for Lactobaci llus ( Figure 10).
  • Lactobaci llus casei or Lactobaci llus plantarum transformed with vectors harbouring these xyl genes can be selected on plates with xylose as sole energy source.
  • Beta-glucuronidase activity of transformants expressing the uiA gene was determined semi-quantitatively in a plate assay using the chromogenic substrate X-gluc. In such assays colonies of Lactobaci llus transformants that express uidA stain blue, whereas colonies of transfor ⁇ mants that do not express the uidA gene are white. Beta-glucuronidase activity of transformants expressing the uidA gene can be quantitatively and reproducibly assayed enzymatically or immunologically using anti- ⁇ - glucuronidase antibodies.
  • a method for production comprises use of an expression vector and/or host cell comprising such a vector according to the invention in a manner known per se. This involves transformation of a lactic acid bacterium with an expression vector according to the invention comprising the nucleic acid sequence encoding the product of interest. A number of such nucleic acid sequences have been indicated elsewhere in the description. Following introduction of the expression vector in the lactic acid bacterium, the host cell must be cultured in a medium in a manner to be determined in a manner known per se by a person skilled in the art best suited to the kind of host cell, the promoter and/or nucleic acid sequence to be expressed such that expression occurs. Determination of the optimal conditions lies within reach of a person skilled in the art without requiring inventive step or undue experimentation.
  • the method for constructing an expression vector according to the invention also falls within the scope of protection.
  • Such a method comprises introduction of a vector according to the invention for maintenance and manipulation in a working organism other than the strain of lactic acid bacterium in which expression is to occur, manipulating the vector in a manner known per se to obtain desired characteristics in said working organism and finally introducing the vector comprising a nucleic acid sequence encoding a protein or polypeptide in the strain of lactic acid bacterium in which expression is to occur, said sequence preferably being heterologous to the strain of lactic acid bacterium in which expression is to occur.
  • the method may be useful when the working organism is a non lactic acid bacterium.
  • the vector when the vector comprises the transcription terminator sequence located between the promoter and the first codon of the nucleic acid sequence encoding a protein or a polypeptide the vector can be treated in a manner known per se for excising nucleic acid sequences to excise the terminator sequence in order to allow transcription of the nucleic acid sequence encoding a protein or a polypeptide. This can occur e.g. by treatment of the vector with restriction enzyme for cutting the vector at the sites flanking the terminator sequence.
  • vector for maintenance is introduced into the expression host prior to the excision and due to the presence of direct repeats flanking the terminator sequence and treatment rendering homologous recombination possible subsequently the expression can be rendered possible due to excision of the terminator sequence in such a fashion.
  • a vector according to the invention for maintenance and manipulation in a working organism of a nucleic acid sequence encoding a polypeptide or protein said vector comprising an expression promoter sequence and a cloning site for said nucleic acid sequence encoding a polypeptide or protein with the cloning site being located such that said nucleic acid sequence when present in the vector is or will be controlled by said promoter sequence and is or will be present in an open reading frame in phase with a translation initiation region, is characterised in that a terminator sequence recognisable as such by the working organism in which the vector is to be maintained prior to introduction into a lactic acid bacterium is located on the vector such that the complete polypeptide or protein encoded by the nucleic acid sequence cannot be expressed.
  • the working organism is a non lactic acid bacterium.
  • a multiple cloning site will be preferable as cloning site.
  • a vector according to the invention will comprise the nucleic acid sequence encoding the protein or polypeptide at the appropriate cloning site.
  • the terminator sequence is present in one particular embodiment between the promoter sequence and the first codon of the nucleic acid sequence encoding a protein or polypeptide.
  • the terminator sequence is present between the promoter sequence and the translation initiation region preceding the nucleic acid sequence.
  • any of the preceding embodiments may further comprise the transcription terminator sequence such that it is excisable from the vector in a manner known per se to a person skilled in the art e.g when the transcription terminator sequence is flanked by restriction enzyme sites uniquely present in the vector at such flanking position or is flanked by direct repeats e.g. excisable by homologous recombination by the host.
  • a vector may further comprise one or more of the components: i) a replicon for a lactic acid bacterium ii) a DNA sequence encoding the signal sequence of a secreted protein recognisable by a lactic acid bacterium as such this could e.g. be a signal sequence from a lactic acid bacterium, said signal sequence being located in frame between the initiation codon and the nucleic acid sequence encoding a protein or a polypeptide, iii) a reporter gene encoding an easily assayable product, such a reporter gene e.g.
  • a transcription terminator sequence recognisable by a lactic acid bacterium, said sequence being located downstream of the nucleic acid sequence encoding a protein or polypeptide, v) a DNA sequence encoding an anchor sequence from a lactic acid bacterium gene and said DNA sequence being located in frame downstream from the nucleic acid sequence encoding a protein or a polypeptide and vi) a DNA sequence encoding a product with health stimulating properties. vii) solely foodgrade components.
  • Example 1 The expression and expression-secretion vectors pLP4 ⁇ l,2,3 ⁇ T and pLP501,- 2,3-T, that were constructed in Escherichia coli, were digested with TVotl and ligated, in order to obtain vectors in which the T ldh sequence was omitted. Lactobaci llus casei ATCC 393 was transformed with the ligation mixtures using electroporation [Posno et al, Appl. Envionm. Microbiol. 57, 1822-1828 (1991)]. Transformants were selected on selective media.
  • Selective media contained 10 g of proteose pepton, 5 g neat extract, 5 g yeast extract, 1 ml Tween 80, 5 g Na-acetate.3H 2 0, 0.2 g MgS0_,.7H 2 0, 50 mg MnSO / ,.4H 2 0) , 2 g diammoniumcitrate, 17 g agar per 800 ml of deionized water. After autoclaving (20 min at 120°C) 200 ml 0.5 M potassium- phosphate buffer pH 7.1 was added.
  • erythromycin and X-gluc final concentrations 5 and 4 ⁇ g/ml, respectively
  • an energy source glucose or cellobiose; 0.5-1-0%)
  • the media were poured into petri dishes (13 cm diameter; 25-30 ml medium) , after solidification the agar containing plates were dried for 20 min at 60 ⁇ C and used immediately. Lactobaci llus transformants containing vectors from the pLP400 or pLP500 series were streaked onto the surface of the agar plates. Colonies and their phenotypical characteristics (blue or white) could be seen within 24 hrs of incubation at 37 fl C.
  • the white phenotype was correlated with structurally instable plasmids (size smaller than starting material).
  • Escherichia coli transformants with a blue phenotype were suspended in physiological salt solution and streaked onto agar plates with selective medium, most colonies showed a white phenotype, indicating plasmid instability.
  • a DNA fragment encoding the SA epitope (Site A; Wiley et al, Nature 89, 373-378 (1981)) of influenza virus comprising 18 amino acids (VTAACS- HAGKSSFYRNLL) was inserted between the B ⁇ mHI and TVcoI sites of plasmids pLP4 ⁇ l,2,3-T and plasmids pLP501,2,3-T. No structural instability of these vectors was observed in Escherichia coli .
  • Lactobaci l lus casei ATCC 393 was transformed with the resulting plasmids pLP4 ⁇ l-SA, pLP402-SA, pLP403-SA and with pLP501-SA, pLP502-SA and pLP503 ⁇ SA.
  • Transformants were selected on selective media as described in Example 1. When transformants of the pLP400-SA series were plated on indicator plates with X-gluc containing cellobiose as energy source all colonies were light blue, but were white when glucose was used as energy source. When transformants of the pLP500-SA series were plated on indicator plates all colonies were dark blue, irrespective of whether cellobiose or glucose was used as energy source.
  • a DNA fragment encoding the SA epitope of influenza virus comprising 18 amino acids was inserted between the B ⁇ mHI and TVcoI sites of derivatives of plasmids pLP4 ⁇ l and pLP501 that lack the uidA gene, resulting in plasmids pLP4 ⁇ l-SA-l and pLP501-SA-l.
  • the SA encoding sequence is directly fused, in-frame, to the anchor region encoding sequence.
  • Lactobaci llus casei ATCC 393 was transformed with plasmids pLP4 ⁇ l-SA-l and pLP501-SA-l. Transformants were selected on selective media as described in Example 1. Western analysis shows that transformed bacteria synthesize a product of the expected size that specifically reacts with antibodies against the SA epitope.
  • expression-secretion vectors pLP4 ⁇ l-T-SA-3 and pLP501- T-SA-3 with 3 copies of the SA epitope sequence fused, in-frame, with the uidA gene sequence were obtained.
  • Lactobaci llus casei ATCC 393 was transformed with the resulting plasmids pLP4 ⁇ l-SA-l,2,3 and pLP501-SA-l,2,3. Transformants were selected on selective media as described in Example 1. When transformants of the pLP400-SA series were plated on indicator plates with X-gluc containing cellobiose as energy source all colonies were light blue, but were white when glucose was used as energy source. When transformants of the pLP500- SA series were plated on indicator plates all colonies were dark blue, irrespective of whether cellobiose or glucose was used as energy source. Western analysis shows that transformed bacteria synthesize a product of the expected size that specifically reacts with antibodies against the SA epitope and antibodies against ⁇ -glucuronidase.
  • a DNA fragment encoding the hacket epitope of influenza virus comprising
  • Lactobaci llus casei ATCC 393 was trans- formed with the resulting plasmids pLP4 ⁇ l-HA, pLP402-HA and pLP503-HA and Lactobaci llus plantarum strains ATCC 14917, ATCC 8014, NCIB 8826, 80, NCDO 1193 with pLP402-HA and pLP503-HA. Transformants were selected on selective media as described in Example 1. When transformants of the pLP400-HA series were plated on indicator plates with X-gluc containing cellobiose as energy source all colonies were light blue, but were white when glucose was used as energy source.
  • the amount of fusion protein synthesized by pLP503 _ HA was quantitatively determined by measuring the ⁇ -glucuronidase activity and by quantitative E1ISA. The enzymatic assay and the ELISA determination indicate that the fusion protein amounts to 1-3% of total cellular protein.
  • Example 6 A DNA fragment encoding pig rotavirus VP ⁇ polypeptide comprising the first 248 amino acids of the VP4 protein (this part corresponds with the region of the VP4 protein that arises after trypsic cleavage [Lopez et al, 65, 3738-3745 (1991)]) was inserted between the B ⁇ mHI and TVcoI sites of plasmids pLP4 ⁇ l-T, pLP402-T, pLP501-T and pLP502-T, resulting in plasmids pLP401-T-R08, pLP402-T-R08, pLP501-T-R08 and pLP502-T-R0 ⁇ .
  • Lactobac i llus casei ATCC 393 was transformed with plasmids pLPi*01-R08, pLP*02-R08, pLP501-R0 ⁇ , and pLP502-R08. Transformants were selected on selective media as described in Example 1. When transformants of the pLPiJOO-RO ⁇ series are plated on indicator plates with X-gluc containing cellobiose as energy source all colonies are light blue, but are white when glucose is used as energy source. When transformants of the pLP500-R0 ⁇ series are plated on indicator plates all colonies are dark blue, irrespective of whether cellobiose or glucose is used as energy source.
  • a Structure of plasmid pGTC3- T gbh , transcription terminator of Lactobaci llus plantarum cbh gene.
  • b Nucleotide sequence comprising PRESS, poly restriction enzyme site sequence, and part of the sequence containing the cbh terminator.
  • Figure 2 Schematic representation of the construction route of the expression vectors.
  • Figure 3 Sequence of the synthetic oligonucleotide duplex encompassing the transcription terminator of the L-l h gene of Lactobaci llus casei .
  • Inverted repeats capable of forming a stem loop structure are marked by horizontal arrows.
  • the overhanging terminal sequences on the left and right side of the sequences are compatible to the B ⁇ mHI and BstEII sites, respectively, of the promoter-terminator cassette vectors.
  • Figure 4 Nucleotide sequence of the 3'end of the uidA gene before (a) and after (b) mutagenesis by PCR.
  • An Xhol site was introduced immediately upstream of the translation stop codon to allow in-frame fusion of amino acid encoding sequences of the uidA gene with an ORF to be fused with the 3' end of this uidA gene.
  • Figure 5 a Physical map of promoter-double-terminator cassette vector pPTT3.
  • P stands for P ⁇ h , P ldh -ss nrtP , P ⁇ - ⁇ ss, or P ⁇ , T j ⁇ and T ⁇ : transcription terminators of the Lactobac i l lus casei Idh and Lactobac i llus plantarum cbh gene, respectively.
  • b Physical map of promoter-terminator cassette in which an adjusted Escherichia coli uidA gene was cloned.
  • c. As b. The reading frame of the uidA gene is fused to that of the 3' end of the prtP gene coding for the anchor sequence.
  • Nucleotide sequences of the region between the Idh gene transcription terminator sequence and the uidA gene of the expression cassette vectors are provided. a. Nucleotide sequence between the B ⁇ mHI and TVcoI sites in pGTT3; this sequence is part of the synthetic oligonucleotide sequence encompas ⁇ sing the Lactobaci llus casei Idh transcription terminator signal. b. Nucleotide sequence of MCS inserted between B ⁇ mHI and TVcoI sites of pPTUT. The original B ⁇ mHI was destroyed using this strategy but a new B ⁇ mHI site is present in the MCS. Starting from this MCS two new, slightly modified MCS were obtained in which the B ⁇ mHI site and following sites are frame-shifted.
  • c A vector containing the MCS as depicted in b) was digested with CZ ⁇ l and the resulting DNA termini were filled in with dNTP's using T4 DNA polymerase. After ligation the MCS contained a B ⁇ mHI site shifted two nucleotides with respect to G.
  • Nucleotide sequence of a MCS obtained by replacing the Cl ⁇ l- B ⁇ mHI sequence in b) by a Cl ⁇ l-BspEI-B ⁇ mHI sequence. The B ⁇ mHI site is shifted four nucleotides with respect to G.
  • DNA fragments can be cloned in MCS depicted in b, c or d depending on the reading frame needed.
  • restriction enzyme sites just upstream of the TVcoI site can be modified with other restriction enzymes (see elsewhere in the description).
  • Figure ⁇ a) Nucleotide sequence of MCS sequences as depicted in Figure 6-b,c,d. b) Nucleotide sequence of MCS after filling up the Bbsl restriction enzyme generated termini of MCS depicted in a) with dNTP's using T4 polymerase and ligation. c) Nucleotide sequence of MCS after filling up the Bbsl restriction enzyme generated termini of MCS depicted in b) with dNTP's using T4 polymerase and ligation. The line between G-G corresponds with the nucleotide sequences of Figure 6-b,c,d.
  • Figure 9 a Schematic representation of expression vector.
  • b Schematic structure of cassettes present in plasmids of the pLP400 and pLP500 series.
  • P j ⁇ and P amv represent the promoters of the Idh gene of
  • Lactobacillus casei and amy promoter of the ⁇ -amylase gene of Lactobaci l ⁇ lus amylovo ⁇ us respectively.
  • uidA ⁇ -glucuronidase gene of Escherichia coli .
  • T ldh transcription terminator of the Idh gene of Lactobaci llus casei .
  • T cbh transcription terminator of the cbh gene of Lactobac i llus plantarum.
  • Anchor prtP sequence from Lactobacillus casei encoding the membrane anchor sequence and peptidoglycan spanning region.
  • MCS multicloning region
  • the B ⁇ mHI site, and all other sites of the MCS are present in three reading frames, allowing in-frame translation of any DNA sequence fused to the N-terminal sequence of LDH, ⁇ -amylase or PrtP.
  • the presence of a unique cloning sites in the MCS permits easy alignment, if necessary, of the reading frames of the antigenic determinant encoding sequence and of uidA .
  • Nucleotide sequence of the distal part of the MCS containing the TVcoI and Bbsl recognition sites The recognition sequences for B ⁇ mHI, TVcoI and Bbsl are indicated with bold letters.
  • the nucleotide sequence of S layer protein in particular the promoter region of I. acidophi lus slpA .
  • ORGANISM fragment with 3'end of uidA gene
  • ORGANISM Bamhl-Ncol fragment of pPTUT with mcs frameshifted 4 nts
  • Glu Glu Asp Arg lie Asn Ser Gin Val Asp Arg Ala Leu Ala Met 1 10 15
  • ORGANISM mcs nucleotide sequence of seq 6,7 and ⁇ between first two G's
  • Lys Lys Thr Glu Leu lie Asn Ser Gin Val Asp Arg Ala Leu Ala Met 1 10 15
  • MOLECULE TYPE DNA (genomic)
  • Met Lys Lys Asn Leu Arg lie Val Ser Ala Ala Ala Ala Ala Ala Leu Leu 1 5 10 15

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Abstract

La présente invention concerne un mode de création de vecteurs plasmide à usages multiples, pouvant servir à introduire, à maintenir dans des conditions de stabilité et à exprimer et sécréter efficacement des gènes ou des fragments de gènes étrangers dans une variété de souches d'acide lactique telles que les souches de Lactobacillus, et Bifidobacterium. Elle concerne aussi les vecteurs d'expression à utiliser selon ce procédé, ainsi qu'un procédé de production accrue de protéines et de polypeptides, hétérologues et homologues des bactéries d'acide lactique. Elle concerne enfin des bactéries d'acide lactique contenant ces vecteurs d'expression et produisant ces protéines ou ces polypeptides.
PCT/NL1996/000160 1995-04-11 1996-04-11 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 WO1996032487A1 (fr)

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US9452205B2 (en) * 2012-09-24 2016-09-27 Montana State University Recombinant Lactococcus lactis expressing Escherichia coli colonization factor antigen I (CFA/I) fimbriae and their methods of use
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