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WO1993010247A1 - VECTEUR PHASMIDIQUE DANS $i(E.COLI) - Google Patents

VECTEUR PHASMIDIQUE DANS $i(E.COLI) Download PDF

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Publication number
WO1993010247A1
WO1993010247A1 PCT/SE1992/000793 SE9200793W WO9310247A1 WO 1993010247 A1 WO1993010247 A1 WO 1993010247A1 SE 9200793 W SE9200793 W SE 9200793W WO 9310247 A1 WO9310247 A1 WO 9310247A1
Authority
WO
WIPO (PCT)
Prior art keywords
vector
parc035
parc032
phasmids
dna
Prior art date
Application number
PCT/SE1992/000793
Other languages
English (en)
Inventor
Prahalad Dwarakanath
Goutam Das
Original Assignee
Ab Astra
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 GB9124556A external-priority patent/GB2250290A/en
Application filed by Ab Astra filed Critical Ab Astra
Publication of WO1993010247A1 publication Critical patent/WO1993010247A1/fr

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Classifications

    • 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
    • C07K14/24—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
    • C07K14/245—Escherichia (G)

Definitions

  • a novel phasmid vector of E.coli which can be used for cloning gene(s) of interest and for hyperexpression of gene products in suitable host bacteria.
  • This vector can, when incorporated into a "male specific" E.coli strain and upon superinfection with helper phage, give rise to single stranded plasmid DNA which can be sequenced directly by dideoxy sequencing methods and which also can be used as a template for site directed in vitro mutagenesis.
  • vectors for cloning and manipulation of DNA are generally characterised by the presence of a genetic determinant, the phenotype of which is altered by insertional inactivation.
  • these vectors contain multiple restriction enzyme cleavage sites allowing the direct cloning of a variety of restriction fragments.
  • the first cloning vector constructed was pBR322 (A survey of Molecular Cloning Vectors and their uses, 1988, Biotechnology Series, Boskin, Butterworth, Rodrigues, M. & Denhardt D. (eds)).
  • This vector could be used only for cloning purposes and not for manipulating the expression of inserted gene.
  • a number of inducible expression systems were developed.
  • An example of such an expression system is the T7 system, developed by Studier & Moffat (1986, J.Mol.Biol. 189, 113-). This system makes use of the high specificity of the T7 RNA polymerase to recognise and transcribe any gene downstream of the T7 0 10 promoter.
  • pUC118 and pUC119 which can give rise to ss plasmid DNA upon superinfection with helper phage have also been developed (Viera & Messing, 1987, Methods Enzymol. 153, 3-).
  • a series of cloning vectors with the property of releasing ss plasmid upon superinfection with helper phage have been developed by Konings et al. (pKUN series) where the specific DNA sequences of the filamentous phage Ff and the like are utilized (Konings et al., 1987, Methods enzymol.
  • phasmid vectors All these plasmid vectors, which have the capability of producing ss plasmid DNA packaged inside a viral coat upon superinfection with another helper phage, are commonly referred to as phasmid vectors.
  • Phasmid vectors which can be utilized also for expression are very few at present.
  • pFSE4 employs a ⁇ - phage promotor (P j _) including the ell ribosome binding site (RBS).
  • P j _ ⁇ - phage promotor
  • RBS ell ribosome binding site
  • insertion can hypothetical ⁇ be expressed in this vector, although this vector is not considered as a hyperexpression vector (Cloning vectors.
  • the other phasmid expression vector belongs to the pSP6 family, where the promoter of a Salmonella phage gene is utilized for the expression of the cloned gene (Cloning vectors. A laboratory manual, Pouwels et al. (eds), 1986).
  • the purpose of the invention is to incorporate the above mentioned individual features of known vectors into a single vector.
  • the invention comprises a phasmid vector which contains the phage f1 origin of replication and which can be used for general cloning, production of single stranded plasmid DNA and for hyperexpression of the cloned gene under appropriate host condition.
  • the phasmid vector according to the invention is a genetic engineering tool, which is used for cloning, sequencing and hyperexpression of a gene.
  • plasmid In order to develop a phasmid vector with the ability to hyperexpress any cloned gene and the ability to produce ss DNA of the said gene, the following features were included in the plasmid: a. Introduction of the origin of replication of the single stranded DNA phage f1 which will enable the ss plasmid DNA to be packaged into the viral particle. b. Introduction of a strong promoter preceding the cloning site(s) for expression of the cloned gene. c. Introduction of an antibiotic resistance marker for proper selection and maintenance of the plasmid in E.coli host. d. Introduction of an E.coli origin of replication for propagation of the plasmid in E.coli.
  • E.coli pET7 plasmid (Fig. 1 ; Studrer and Moffat, 1986, J.Mol.Biol. 189, 113-) was used as a source of the T7 0 10 promoter of Bacteriophage T7 which is specifically recognized by T7 RNA polymerase.
  • pET7 was digested with Pvul ⁇ and Nru ⁇ and the large fragment was purified.
  • the origin of the replication of E.coli l was introduced into this plasmid.
  • the fragment containing the f1 origin of replication was excised out of pGEM7Zf(+) (Promega Corporation Catalogue; Yanish-Perron et al. (1985) Gene 33, 103-109) plasmid by digestion using the restriction enzyme SaU3A ⁇ . Subsequently the restriction fragments were filled-in using the Klenow DNA polymerase. A 788 bp fragment separated on agarose gel contained the f1 origin of replication. This fragment was ligated to the Pv ⁇ A ⁇ and Nm ⁇ digested large fragment of pET7 to generate pARC032 and pARC035 (Figs. 2 and 3).
  • pARC032 and pARC035 in E.coli male specific hosts upon superinfection with helper phage R408 can package alternative strands of the plasmids as ss DNA into the virus particle.
  • Both the plasmids pARC032 and pARC035 can be used as cloning vectors.
  • the gene of interest can be cloned into any of the unique multicloning sites (e.g. SamHI, EcoRV, Hindttl, Cla ⁇ and EcoRI) available following the T7 0 10 promotor element.
  • the cloned gene can be overexpressed in the presence of a functional T7 RNA polymerase using suitable host systems.
  • Phasmid vector with Kanamycin marker eg. pARC036
  • ss plasmid DNA can be used as a template for in vitro mutagenesis.
  • the Ampicillin resistance marker of pARC032 was replaced with the Kanamycin resistance cartridge, since the E.coli CJ236 used in the preparation of the template for in vitro mutagenesis sometimes become spontaneously ampicillin resistant.
  • a plasmid containing the kanamycin resistance gene was digested with Psfl and the 1.3 Kb p fragment containing the Kan gene was purified. The fragment was blunt ended with bacteriophage T4 DNA polymerase.
  • This fragment was ligated to the large fragment of EcoRI and Pst digested pARC032 following T4 DNA polymerase treatment.
  • the ligated product was used to transform E.coli HB101 and kanamycin resistant colonies were selected and the plasmid was characterized.
  • This plasmid pARC036 (Fig. 4) has the T7 0 10 promoter, fl origin of replication, as well as the Kan r marker.
  • the construction of pARC032 and pARC035 is shown in Fig. 5 and that of pARC036 is shown in Fig. 6.
  • EXAMPLE 2 Cloning of E.coli st gene in pARC036
  • the utility of the plasmid pARC036 was checked by inserting an E.coli plasmid gene at the BamHl - Hindttl site of pARC036.
  • the gene cloned was Ecoli st gene as a BamHl - HindlW fragment (Dwarakanath et al., 1989, Gene 81, 219-).
  • the cloning strategy is shown in Fig. 7 and the final construct is shown in Fig. 8.
  • Advantage of this cloning strategy was that if the Hin ⁇ lW sites of the vector pARC036 were used, upon successful cloning in a 3-way ligation process the Kan r property should be restored.
  • the resulting plasmid containing the st gene insert is designated as pARC044 (Fig. 8).
  • the plasmid pARC044 was used to transform E.coli JM101 , BL21 (DE3) and CJ236. The transformed strains maintained the plasmid stably.
  • the JM101 pARC044 produced ss plasmid DNA containing the st gene insert which can be sequenced following the isolation of the ss plasmid DNA according to the known standard procedure (Molecular Cloning, Maniatis etal. (eds) CSH Publ. 1989).
  • E.coli hyperexpression strain BL21 (DE3) which has the T7 RNA polymerase gene cloned under the lac UV5 promoter at the chromosomal locus (Studier and Moffat, 1986, J.Mol.Biol. 189, 113-) was transformed with pARC 044.
  • the transformed clones were characterized and further grown in M9 media (Dwarakanath et al, 1989, Gene 81 , 219-) and were induced to hyperexpress ST as a secreted product according to the procedure previously described by Dwarakanath ef al. (1989, Gene 81 , 219-).
  • the level of secreted ST under induced condition was comparable to the wild-type level as determined by a previously standardized ELISA method (Dwarakanath et al., 1989, Gene 81 , 219-).
  • E.coli CJ236 (dut, ung) was transformed with pARC044 and the transformants were characterized. One of the transformants was grown in liquid medium and was superinfected with helper phage. After overnight incubation at 37°C with vigorous shaking, the culture supernatant containing the phasmid, virions were separated from the bacterial pellet, and the ss plasmid DNA was isolated according to Maniatis. (Molecular Cloning, Maniatis et al., (eds) CSH Publ., 1989). As shown in Fig. 9, this phasmid DNA was used as a template to introduce a mutation in the st gene.
  • Vector phasmids pARC032, pARC035 which contain the replication origin and a morphogenetic signal, or an active mutant thereof, of the filamentous bacteriophage Ff (M13, fd and fl) or a mutant of said bacteriophage.
  • Vector phasmids pARC032 and pARC035 in which the ampicillin resistance phenotype has been replaced by kanamycin resistance phenotype as exemplified by the construction of pARC036 from pARC035.
  • Vector phasmid pARC 032 having the structure shown in Figure 2.
  • Vector phasmid pARC032 and pARC035 into which a fragment of double-stranded DNA has been inserted at the multiple cloning site.
  • Vector phasmids pARC032 and pARC035 which can replicate in a bacterial host.
  • Vector phasmids pARC032 and pARC035 which can produce single stranded DNA of a gene inserted at the multiple cloning site in an appropriate host cell upon superinfection by a helper phage.
  • Vector phasmids pARC032 and pARC035 wherein a synthetic oligonucleotide containing a mismatch flanked by complementary sequences to the gene is inserted at the multiple cloning site.
  • Vector phasmids pARC032 and pARC035 capable of producing ss DNA under standard culture conditions which can be labelled with radioactive elements such as P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA.
  • Vector phasmids pARC032 and pARC035 as in paragraph 1 which upon insertion in a suitable host cell, can be selected by growing the host cell in kanamycin containing medium.
  • Vector phasmids pARC032 and pARC035 which contains a bacteriophage T7 promoter for transcription and which is transcribed in vivo or in vitro by a T7 RNA polymerase.
  • a bacterial cell transformed by the vector phasmids pARC032 or pARC035, which produces a mutagenized protein or peptide encoded by the mutagenized ss DNA.
  • a phasmid capable of producing ss DNA under appropriate culture conditions which can be labelled with radioactive elements such as 32 P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA.
  • a phasmid which upon insertion into a suitable host cell, can be selected by growing the host cell in kanamycin containing medium.
  • a phasmid which contains a bacteriophage T7 promoter for transcription.
  • E.coli HB101 F ⁇ , hsd S20(rB “ ,mB “ ), supE44, recA13, ara14, proA2, rpsL20(Str), xyl-1,mlt-1,);
  • Ecoli JM101 thi, (lac-proAB), [F ⁇ traD36, proAB, laclqZ M15]
  • E.C0II CJ236 dutl, ung1, thi-1, relA1/pCJ105 (cam r F')
  • Ecoli BL21(DE3) hsdS, gal (clts857 indl Sam7 nin ⁇ lacUV5-T7gene1).
  • Plasmids pET7 (a kind gift from Dr. Studier) and pGEM7Zf(+) from Promega.
  • the restriction enzymes EcoRI, BamHl, Hindl ⁇ ,Pst ⁇ , Sau3Al were from Boehringer Mannheim.
  • T4 DNA ligase, T4 DNA polymerase, Klenow fragment were from Boehringer Mannheim.
  • FIGURE 1 E.coli pET7 plasmid
  • FIGURE 3 E.coli pARC035 plasmid
  • FIGURE 8 E.coli pARC044 plasmid

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Abstract

Un nouveau vecteur phasmidique a été réalisé, et une description est donnée de son utilisation en tant que vecteur général de clonage. Après transformation de souches mâles spécifiques d'E.Coli avec ce nouveau phasmide contenant un segment d'insertion de recombinaison, on peut produire de l'ADN phasmidique à simple brin avec l'encapsidation d'une particule virale, lorsque la cellule transformée est surinfectée avec un phage auxiliaire. Cet ADN phasmidique à simple brin pourrait également être utilisé comme matrice pour le séquençage de l'ADN à simple brin et la mutagénèse in vitro dirigée sur un site. Dans des conditions appropriées d'intersertion du gène et par un choix correct de la souche hôte, ce nouveau phasmide permet une hyperexpression du gène inséré.
PCT/SE1992/000793 1991-11-20 1992-11-19 VECTEUR PHASMIDIQUE DANS $i(E.COLI) WO1993010247A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9124556.3 1991-11-20
GB9124556A GB2250290A (en) 1990-11-21 1991-11-20 Phasmid vectors

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WO1993010247A1 true WO1993010247A1 (fr) 1993-05-27

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026967A3 (fr) * 2000-09-25 2003-01-09 Univ Jefferson Correction de gene cible au moyen d'oligodesoxynucleotides monocatenaires
US6833441B2 (en) 2001-08-01 2004-12-21 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
US7175983B2 (en) 2001-11-02 2007-02-13 Abmaxis, Inc. Adapter-directed display systems

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0354727A2 (fr) * 1988-08-08 1990-02-14 Eli Lilly And Company Vecteurs phasmidiques monocatenaires et procédé de transformation des streptomyces et d'autres actinomycetes
EP0356130A2 (fr) * 1988-08-22 1990-02-28 Genencor International, Inc. Origine de réplication simple brin mobile du phage f1
DE3929822A1 (de) * 1989-09-07 1991-03-14 Max Planck Gesellschaft Vektor und seine verwendung

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0354727A2 (fr) * 1988-08-08 1990-02-14 Eli Lilly And Company Vecteurs phasmidiques monocatenaires et procédé de transformation des streptomyces et d'autres actinomycetes
EP0356130A2 (fr) * 1988-08-22 1990-02-28 Genencor International, Inc. Origine de réplication simple brin mobile du phage f1
DE3929822A1 (de) * 1989-09-07 1991-03-14 Max Planck Gesellschaft Vektor und seine verwendung

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, file 154, Medline, Accession No. 06882389, Medline Accession No. 89184389, MEAD D.A. et al., "Single-Stranded DNA 'Blue' T7 Promoter Plasmids: a Versatile Tandem Promoter System for Cloning and Protein Engineering". *
GENE, Volume 105, 1991, BARRY MARCH FORMAN et al., "pExpress: A Family of Expression Vectors Containing a Single Transcription Unit Active in Prokaryotes, Eukaryotes and In Vitro", page 9 - page 15. *
GENE, Volume 90, 1990, TAKAYASU DATE et al., "Construction of Escherichia Coli Vectors for Expression and Mutagenesis: Synthesis of Human c-Myc Protein that is Initiated at a Non-AUG Codon En EXON 1", page 141 - page 144. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026967A3 (fr) * 2000-09-25 2003-01-09 Univ Jefferson Correction de gene cible au moyen d'oligodesoxynucleotides monocatenaires
US6833441B2 (en) 2001-08-01 2004-12-21 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
US7429652B2 (en) 2001-08-01 2008-09-30 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
US7175983B2 (en) 2001-11-02 2007-02-13 Abmaxis, Inc. Adapter-directed display systems
US7910350B2 (en) 2001-11-02 2011-03-22 Abmaxis, Inc. Adaptor-directed helper systems

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