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WO1998003666A1 - Vecteurs de recombinaison - Google Patents

Vecteurs de recombinaison Download PDF

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Publication number
WO1998003666A1
WO1998003666A1 PCT/SE1997/000763 SE9700763W WO9803666A1 WO 1998003666 A1 WO1998003666 A1 WO 1998003666A1 SE 9700763 W SE9700763 W SE 9700763W WO 9803666 A1 WO9803666 A1 WO 9803666A1
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WIPO (PCT)
Prior art keywords
parc
vector
gene
asia
coli
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Application number
PCT/SE1997/000763
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English (en)
Inventor
Umender Sharma
Original Assignee
Astra Aktiebolag
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from SE9602803A external-priority patent/SE9602803D0/xx
Priority claimed from SE9603282A external-priority patent/SE9603282D0/xx
Application filed by Astra Aktiebolag filed Critical Astra Aktiebolag
Priority to JP10506850A priority Critical patent/JP2000514658A/ja
Priority to US08/875,229 priority patent/US6030777A/en
Priority to AU27992/97A priority patent/AU2799297A/en
Priority to EP97922279A priority patent/EP1012315A1/fr
Publication of WO1998003666A1 publication Critical patent/WO1998003666A1/fr

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    • 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
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to recombinant vectors which are useful for direct selection of colonies harboring recombinant plasmids, on basis of the loss of expression of a known gene, the expression of which would normally have resulted in toxicity to the bacterial host.
  • the invention also relates to host cells harboring the said vectors, as well as to methods utilizing the said vectors, for the selection of nucleic acid clones.
  • RNA polymerase In eubacteria, the core RNA polymerase is composed of ⁇ , ⁇ , and ⁇ ' subu its in the ratio 2:1:1.
  • bacteria To direct RNA polymerase to promoters of specific genes to be transcribed, bacteria produce a variety of proteins, known as sigma ( ⁇ ) factors, which interact with RNA polymerase to form an active holoenzyme. The resulting complexes are able to recognize and attach to selected nucleotide sequences in promoters.
  • Antisigma (Asi) proteins i.e. proteins which inhibit the sigma subunit of RNA polymerase, are known in the art.
  • a gene called asiA coding for the 10 kDa antisigma 70 factor of bacteriophage T4 (hereinafter referred to as AsiA), has been identified by Orsini et al. (1993) J. Bacteriol. 175, 85-93.
  • the open reading frame encoded a 90 amino acid protein having the deduced sequence shown as SEQ ID NO: 1.
  • the fls / -encoded protein was overproduced in a phage T7 expression system and partially purified. It showed a strong inhibitory activity towards sigma 0 -directed transcription by RNA polymerase holoenzyme.
  • the nucleotide sequence of gene asiA has been deposited in the GenBank data base under accession no. M99441.
  • proteins regulating the sigma subunit of RNA polymerase are known from other systems such as Salmonella typhimurium (Ohnishi et al. (1992) Mol. Microbiol. 6, 3149-3157) and Bacillus subtilis (Duncan & Losick (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2325-2329; Benson & Haldenwang (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2330-2334).
  • the nucleotide sequences of these antisigma factors do not show any gross similarity with the asiA sequence disclosed by Orsini et al. Therefore, although the different antisigma factors are functionally similar, it is not possible to anticipate that an antisigma factor from E. coh will neutralize a RNA polymerase sigma subunit from another bacterial species.
  • heterologous genes are engineered in such a way that a known marker gene is either interrupted or replaced by the gene. Correct recombinants are selected by screening transformants for the loss of the said marker. This requires screening several hundreds of colonies for the loss of the marker gene. In addition, several of the selected clones generally turn out to be false positives for a variety of reasons.
  • vectors that enable a direct positive selection of correct recombinants.
  • such vectors harbor a gene wherein the encoded product on expression is toxic to the host. This toxicity could be lethal to the host thereby killing the organism or render the host cells to be sick.
  • a heterologous gene interrupts or replaces the toxic gene, the resulting recombinant grows normally in solid media.
  • Positive selection vectors useful for direct selection of colonies harboring recombinant plasmids, are thus known in the art, e.g. from:
  • Fig. 1 Plasmid map of vector pARC 8173
  • Fig. 2 Plasmid map of vector pARC 8177
  • Fig. 3 Plasmid map of vector pARC 8235
  • Fig. 4 Plasmid map of vector pARC 8233 DISCLOSURE OF THE INVENTION
  • the present invention provides a vector comprising a positive selection cassette, the said positive selection cassette comprising a DNA sequence coding for an antisigma polypeptide, said DNA sequence including a multiple cloning site for cloning of a heterologous gene.
  • the expression of the normally toxic antisigma polypeptide is abolished. Consequently, a positive selection of clones harboring the heterologous gene is achieved.
  • the said vector is a vector wherein the said antisigma polypeptide is an AsiA polypeptide from £. coli bacteriophage T4, having essentially the amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 in the Sequence Listing, or a functionally equivalent variant thereof.
  • the said vector is a vector wherein the said DNA sequence encodes a polypeptide having essentially the amino acid sequence shown as SEQ ID NO: 4 in the Sequence Listing. It will thus be understood that the addition of the multiple cloning site to the gene coding for the AsiA protein will result in the expression of a polypeptide having additional amino acids as compared to the wild-type AsiA protein (cf. Example 6.3 below). It has been shown that such extra amino acids will not abolish the toxicity of the expressed polypeptide (cf. Example 3 below). However, the introduction of a heterologous gene into the cloning site will significantly reduce such toxicity so that a positive selection can be achieved.
  • the vector according to the invention comprises additional features which allows the vector to be used for the expression of a desired protein.
  • Such features can e.g. include - a suitable antibiotic selection marker;
  • a promoter sequence such as a T7 ⁇ 10 promoter sequence, operatively linked to the said DNA sequence coding for an AsiA polypeptide.
  • operatively linked means that the promoter is linked with a structural gene in the proper frame to express the structural gene under control of the promoter.
  • the invention provides a host cell harbouring a vector as described above.
  • the host cell can e.g. be an E. coli cell.
  • the AsiA protein is capable of binding to sigma 70 factors of other bacterial species, such as Salmonella typhimurium.
  • the positive selection vector according to the invention will be useful also in S. typhimurium and other host cells which are suitable for heterologous cloning.
  • a positive selection vector useful in S. typhimurium will be a construct comprising the asiA gene along with a broad host range replicon such as RSF 1010.
  • a further aspect of the invention is a method of positive selection of nucleic acid clones, comprising
  • the said vector is a vector according to the invention as specified above.
  • the said host cell can be e.g. an E. coli cell or a Salmonella typhimurium cell.
  • standard protocols and “standard procedures”, when used in the context of molecular cloning techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the coding sequence for the asiA gene was amplified from the genomic DNA of bacteriophage T4 by PCR using standard methods.
  • the 5'-primer included the sequence of the restriction enzyme Ncol while the 3 '-primer included the sequence for the restriction enzyme BamHl.
  • the amplified fragment was ligated to NcoI-B ⁇ mHI restricted pBR 329 (Covarrubias (1982) Gene 17, 79-89) and transformed into competent E. coli DH5 ⁇ cells. Recombinants were selected at +37°C as chloramphenicol sensitive, ampicillin resistant transformants.
  • One of the transformants having the desired restriction pattern was labelled pARC 8100.
  • the Ncol-Bamlil DNA fragment from pARC 8100 was ligated to pET 8c (Kmr) (Studier et al. (1990) Methods Enzymol. 185, 60-89) and kanamycin resistant transformants with E. coli DH5 ⁇ were selected at +37°C
  • pARC 8115 One of the transformants harboring a plasmid with the expected restriction enzyme profile was labelled pARC 8115.
  • pARC 8115 plasmid DNA was then used to transform the expression host E. coli BL26(DE3) and transformants selected both at +37°C and at +30°C. However, no viable transformants could be obtained at either of the temperatures.
  • the leaky expression from the T7 promoter in pET 8c being much higher than from pET lid (Kmr) (Studier et al., supra) the toxicity of the asiA product could explain the non-transformability.
  • Transformants could however be obtained using E. coli BL21(DE3)/pLysS (Studier et al., supra) as host, in which the leaky expression is additionally repressed by the T7 lysozyme expressed from pLysS.
  • the NcoI-B ⁇ mHI 366 bp DNA fragment was obtained from pARC 8100 and ligated to the NcoI-B ⁇ mHI sites of pET lid (Kmr) and transformed into E. coli DH5 ⁇ . Transformants were selected for kanamycin resistance. Plasmid preparations from individual transformants were digested with restriction enzymes and the correct transformant that released the 284 bp fragment after NcoI-B ⁇ mHI digest was labelled pARC 8101. The transformants were selected at +37°C and appeared normal. pARC 8101 DNA was then used to transform the expression host E.
  • BL26(DE3)/pLysS colonies were healthy at +37°C and +30°C indicating that tight regulation of expression in the BL26(DE3)LysS strain was the reason for the non- toxicity of the asiA product in this strain.
  • Transformants obtained from E. coli DH5 ⁇ were healthy both at +37°C and +30°C indicating that the gene when transformed into a non-expression host was non- toxic to the host.
  • the asiA gene was excised from pARC 8101 as a Xbal-BamHl DNA fragment to include the sequence for the ribosome binding site and ligated to the Xbal-BamHl cleaved low copy vector pWKS129 (Wang & Kushner (1991) Gene 100, 195) and the ligation mix transformed to E. coli DH5 ⁇ .
  • Recombinant plasmid harboring the asiA gene was identified by restriction profile and labelled pARC 8114. This plasmid DNA when transformed into E. coli BL21(DE3) gave viable transformants both at +37°C and +30°C. This is because the low copy number of the plasmid reduces the level of the AsiA protein expressed through leaky expression.
  • the coding sequence for the asiA gene was excised as an Ncol - B ⁇ mHI fragment from pARC 8100 and ligated to Ncol - Bglll cleaved pARC 0499.
  • the plasmid pARC 0499 (Deposited under the Budapest Treaty with accession no. NCIMB 40664) has a Ncol site in frame with the glutathione-S-transferase encoding sequence, enabling fusion of the N-terminus of asiA sequences.
  • the ligation mix was transformed into E. coli DH5a and transformants selected at +37°C and +30°C. Viable colonies were obtained at both temperatures indicating the N-terminal fusion reduces the toxicity of the protein.
  • the recombinant plasmid obtained with the sequences encoding GST- AsiA was labelled pARC 8105.
  • the construct pARC 8105 when transformed into DH5 ⁇ yielded healthy colonies both at +37°C and at +30°C
  • pARC 8105 was transformed into BL26(DE3) the result was healthy colonies at +30°C, but sick colonies at +37°C. Consequently, it was surprisingly found that the GST fusion construct retained the toxicity of the AsiA product. This also suggests that addition of extra amino acids at the N- terminal of the AsiA protein does not lead to loss of activity. This was an important observation for the construction of a positive selection vector, since it is essential that the addition of nucleotide stretches in the form of a multiple cloning site should not alter the in vivo toxicity properties of the asiA gene product.
  • E. coli RNA polymerase assay was standardized following the protocol of Orsini et al. (J. Bacteriol. 175, 85-93, 1993) using T4 phage DNA as template to quantify sigma 70 dependent transcription.
  • E. coli RNA polymerase core enzyme was purified following the protocol of Burgess and Jendriask (1975) Biochemistry 14, 4634-4638.
  • RNA polymerase could be >80% inhibited by 0.07 ⁇ g of purified AsiA protein.
  • RNA polymerase activity quantified As shown in Table 1, addition of 4 ⁇ g of S. typhimurium sigma 70 could restore the activity of the RNA polymerase.
  • the gene encoding ⁇ subunit of E. coli RNA polymerase was amplified from a standard E. coli strain by PCR, using a 5'-primer which introduced a Ncol site at the 5' -end of the PCR product; and a 3'-primer which introduced a B ⁇ mHI site at the 3'-end of the PCR product.
  • the 1 kb PCR product was cut with Ncol and B ⁇ mHI and cloned into pARC 8101 (an AsiA clone) at the NcoI-B ⁇ mHI site essentially replacing the asiA gene with the new gene and transformed into E. coli BL 26(DE3).
  • the construct was designated pARC 8113.
  • Six healthy colonies out of 100 sick colonies were recombinants carrying the plasmid encoding the gene for the ⁇ subunit of RNA polymerase. Consequently, a vector could be generated where loss of toxicity to the asiA gene indicated insertion of the desired gene into the cloning site.
  • T4 chromosomal DNA was amplified by PCR using a 5'-primer having the sequence shown as SEQ ID NO: 5, which provided Ncol and HmdIII sites at the 5'- end of the product.
  • the 3'-primer, having the sequence shown as SEQ ID NO: 6, provided the loss of the EcoR and introduction of EcoRV site at the 3'-end of the full length asiA gene.
  • the resulting 250 bp fragment was digested with Ncol-EcoRV and ligated to
  • Ncol-EcoRV digested pBR 329 (Covarrubias (1982) Gene 17, 79-89).
  • the construct was transformed into E. coli DH5 ⁇ .
  • the correct recombinant was identified by restriction digestion of plasmid preparations and identified as pARC 8169.
  • a 63 bp fragment which contained restriction sites for multiple restriction enzymes was obtained by digesting pTrc99A with NcoI-H dIII.
  • the vector pTrc99A comprises a 55-bp EcoRI-H dIII polylinker fragment (Amann et al. (1988) Gene 69, 301).
  • the said 63 bp fragment thus provided a multiple cloning site, which can be used for cloning of heterologous proteins, to the construct.
  • the fragment was purified by known methods.
  • the plasmid pARC 8169 was digested with Ncol-Hi nd ⁇ ll and the major fragment was purified and ligated to the 63 bp fragment as obtained from pTRC 99A.
  • the ligation mix was transformed into E. coli DH5 ⁇ .
  • the correct recombinant was selected by restriction digestion of the plasmid made by a miniprep and was identified as pARC 8172.
  • the plasmid pARC 8172 was digested with Ncol-EcoRV and the resulting 295 bp fragment was purified by standard methods and ligated to pET lid (Studier et al., supra) digested with EcoRI, ends filled in by Klenow polymerase and again digested with EcoRI.
  • the ligation mix was transformed into E. coli DH5 ⁇ .
  • the correct recombinant was identified by restriction of plasmid preparation of randomly selected transformants and was named as pARC 8173 (Fig. 1) (Deposited under the Budapest Treaty under accession No. NCIMB 40784).
  • the plasmid pARC 8173 was transformed into E. coli BL26(DE3) and the transformation yielded only sick colonies at +37°C confirming that asiA gene was fully expressed in pARC 8173.
  • Plasmid pET8c (Studier et al., supra) was digested with EcoRI, ends were filled using Klenow Polymerase and again digested with Ncol. This was ligated to the 295 bp Ncol-EcoRV fragment obtained from pARC 8172 (see above) and transformed into E. coli DH5 ⁇ . Correct recombinant was identified by restriction analysis of plasmids obtained by mini prep analysis of several transformants and was identified as pARC 8177 (Fig. 2) (Deposited under the Budapest Treaty under accession No. NCIMB 40785). The plasmid pARC 8177 was transformed into E. coli BL26(DE3) and the transformation yielded no colonies confirming that the asiA gene was fully expressed in pARC 8177. 6.3. Amino acid sequence of the expressed AsiA protein
  • the plasmids pARC 8173 and pARC 8177 are useful as recombinant vectors for cloning of heterologous proteins.
  • the plasmids pARC 8173 and pARC 8177 may be restricted with any of the restriction enzymes defined by the multiple cloning site as shown in Figs. 1 and 2, respectively, and the heterologous gene suitably inserted using standard methods.
  • Preference of any one plasmid over the other would depend on the purpose.
  • either construct can be used.
  • pETlld based pARC 8173 should be used, since the ⁇ lO-Lac promoter is tightly regulated and the expression can be modulated by the addition of IPTG.
  • ColEI based vectors are a series of vectors useful for cloning heterologous genes into E. coli.
  • the 700 bp Bglll-Xmnl fragment from pARC 8173, encompassing the T7 promoter and the asiA gene was cloned into pACYC 184 (Chang, A.C.Y. and Cohen, S.N. (1978) J. Bacteriol. 134, 1141-1156) that has the pl5A origin of replication.
  • the resulting recombinant construct pARC 8235 (Fig. 3) was transformed into BL26(DE3). Transformation gave no colonies at +37°C, indicating the expression of the asiA gene.
  • the construct pARC 8235 was used to test the principle of positive selection by the following procedure: pARC 8235 was digested with B ⁇ mHI-HmdIII and ligated to 1.1 kb B ⁇ mHI-Hindlll fragment from the construct pARC 0356, which encodes the o mraY gene of E. coli, and transformed into E. coli BL26(DE3). Recombinants were selected for chloramphenicol resistance (10 ⁇ g/ml) at +37°C Correct clone was confirmed four out of six healthy colonies by restriction analysis of plasmids.
  • TELEPHONE +46-8 553 260 00
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  • TELEX 19237 astra s
  • TITLE Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • NCIMB National Collections of Industrial and Marine Bacteria Limited

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Abstract

Cette invention a trait à des vecteurs de recombinaison utilisés pour sélectionner directement des colonies hébergeant des plasmides hybrides, en se fondant sur l'absence d'expression d'un gène codant pour un polypeptide AsiA issu du bactériophage T4 de Escherichia coli, l'expression de ce gène devant normalement se révéler toxique pour l'hôte bactérien. L'invention porte également sur des cellules hôtes hébergeant lesdits vecteurs ainsi que sur des techniques d'utilisation de ces derniers aux fins de la sélection de clones d'acide nucléique.
PCT/SE1997/000763 1996-05-17 1997-05-07 Vecteurs de recombinaison WO1998003666A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP10506850A JP2000514658A (ja) 1996-07-18 1997-05-07 組換えベクター
US08/875,229 US6030777A (en) 1996-05-17 1997-05-07 Recombinant vectors
AU27992/97A AU2799297A (en) 1996-07-18 1997-05-07 Recombinant vectors
EP97922279A EP1012315A1 (fr) 1996-07-18 1997-05-07 Vecteurs de recombinaison

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE9602803A SE9602803D0 (sv) 1996-07-18 1996-07-18 Recombinant vectors
SE9603282A SE9603282D0 (sv) 1996-09-10 1996-09-10 Recombinant vectors
SE9603282-6 1996-09-10
SE9602803-0 1996-09-10

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WO1998003666A1 true WO1998003666A1 (fr) 1998-01-29

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PCT/SE1997/000763 WO1998003666A1 (fr) 1996-05-17 1997-05-07 Vecteurs de recombinaison

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EP (1) EP1012315A1 (fr)
JP (1) JP2000514658A (fr)
AU (1) AU2799297A (fr)
WO (1) WO1998003666A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014819A1 (fr) * 1991-02-26 1992-09-03 E.I. Du Pont De Nemours And Company Vecteur de selection positive pour un systeme de clonage de p1 bacteriophage
DE4204103A1 (de) * 1992-02-12 1993-08-19 Hoechst Ag Expression cytotoxischer gaba-permeasegene, verfahren zu ihrer isolierung und ihre verwendung

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014819A1 (fr) * 1991-02-26 1992-09-03 E.I. Du Pont De Nemours And Company Vecteur de selection positive pour un systeme de clonage de p1 bacteriophage
DE4204103A1 (de) * 1992-02-12 1993-08-19 Hoechst Ag Expression cytotoxischer gaba-permeasegene, verfahren zu ihrer isolierung und ihre verwendung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BACTERIOLOGY, Volume 175, No. 1, January 1993, G. ORSINI et al., "The asiA Gene Bacteriophage T4 Codes for the Anti-q70 Protein", pages 85-93. *

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AU2799297A (en) 1998-02-10
JP2000514658A (ja) 2000-11-07

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