WO1993010247A1

WO1993010247A1 - PHASMID VECTOR IN $i(E.COLI)

Info

Publication number: WO1993010247A1
Application number: PCT/SE1992/000793
Authority: WO
Inventors: Prahalad Dwarakanath; Goutam Das
Original assignee: Ab Astra
Priority date: 1991-11-20
Filing date: 1992-11-19
Publication date: 1993-05-27
Also published as: AU3053692A

Abstract

The construction of a novel phasmid vector which can be used as a general cloning vehicule is described. Upon transformation of E.Coli male specific strains with this novel phasmid containing a recombinant insert, ss phasmid DNA packaged as a viral particle can be produced when the transformants are superinfected with a helper phage. This ss phasmid DNA could also be used as template for ss DNA sequencing and in vitro site directed mutagenesis. At appropriate condition of insertion of the gene and by proper choice of host strain, this novel phasmid has the capability to hyperexpress the inserted gene.

Description

PHASMID VECTOR IN E.COLI

TECHNICAL FIELD

Disclosed is the construction of 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.

BACKGROUND ART

The manufacture of a large number of recombinant DNA molecules have been due to the concurrent development in the construction of vectors for cloning and manipulation of DNA. These vectors are generally characterised by the presence of a genetic determinant, the phenotype of which is altered by insertional inactivation. In addition, 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. For such purposes, 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.

When studying gene structure and function, techniques such as DNA sequencing and site directed in vitro mutagenesis are efficiently carried out on single stranded (ss) DNA templates. In 1981, Dotto et al., (Virology 114, 463-) showed that a plasmid carrying the intergenic region (IG) of filamentous phage f1 could be packaged as ss DNA into a viral particle by a helper phage. On the basis of these observations, a number of cloning vectors have been developed which contain the IG region of a filamentous phage, such as pEMBL (Dente etal., 1983, Nucl.Acid Res. 11, 1645-), Blue scribe (Strategene, San Diego, CA, USA), pGEM (Promega, Madison, Wl, USA), etc.

Utilizing the same logic, a second generation of pUC vectors such as 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-). In a similar fashion, 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. 153, 12-; Peeters, B.P.H., Konings,^' R.N.H. & Schoenmakers, J.G.G. 1987). 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.

The vectors mentioned above are, however, suitable only for general cloning purposes. Phasmid vectors which can be utilized also for expression are very few at present. One of them, pFSE4, employs a λ- phage promotor (P_j_) including the ell ribosome binding site (RBS). Under appropriate conditions, insertion can hypothetical^ be expressed in this vector, although this vector is not considered as a hyperexpression vector (Cloning vectors. A laboratory manual, Pouwels et al. (eds.), 1986). 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).

PURPOSE OF INVENTION

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.

DISCLOSURE OF INVENTION

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.

To achieve the desired vector construction, 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. For the construction of the plasmid according to the invention, pET7 was digested with Pvulϊ and Nru\ and the large fragment was purified. In the next step, 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 PvιA\ and Nm\ digested large fragment of pET7 to generate pARC032 and pARC035 (Figs. 2 and 3).

SPECIAL FEATURES OF THE PHASMID VECTOR

1. pARC032 and pARC035 in E.coli male specific hosts upon superinfection with helper phage R408 (Russel etal., 1986, Gene 45. 333) can package alternative strands of the plasmids as ss DNA into the virus particle. 2. Both the plasmids pARC032 and pARC035 can be used as cloning vectors. 3. 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.

4. The cloned gene can be overexpressed in the presence of a functional T7 RNA polymerase using suitable host systems.

5. Upon introduction of these plasmids into E.coli JM101 , the transformed strains can be superinfected with the helper phage and ss DNA can be isolated for sequencing purposes and/or for other manipulations. 6. Phasmid vector with Kanamycin marker (eg. pARC036) can be used to derive ss plasmid DNA which can be used as a template for in vitro mutagenesis.

EXAMPLE 1 - Construction of pARC036

In order to be able to utilize the ss plasmid DNA, which can be generated from the plasmids according to the invention, 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 HinόlW 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. Upon superinfection with helper phage R408, 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).

EXAMPLE 3 — Hyperexpression of st gene cloned in the novel phasmid vector

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-).

EXAMPLE 4 - ss phasmid DNA as a template for site directed in vitro mutagenesis

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.

THE INVENTION CAN BE SUMMARIZED IN THE FOLLOWING CLAUSES:

1. Vector phasmids pARC032, pARC035 constructed from a starting vector incorporating

(a) in opposite orientation, relative to the T7 0 10 promoter, the replication origin and a morphogenetic signal, or an active mutant thereof, of a filamentous bacteriophage of E.coli bacteria having F pili; and

(b) T7 phage promoter followed by a multiple cloning site.

2. 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. 3. 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. 4. Vector phasmid pARC 032 having the structure shown in Figure 2.

5. Vector phasmid pARC 035 having the structure shown in Figure 3.

6. Vector phasmid pARC032 and pARC035 into which a fragment of double-stranded DNA has been inserted at the multiple cloning site. 7. Vector phasmids pARC032 and pARC035 which can replicate in a bacterial host. 8. 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. 9. 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.

10. 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.

11. 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. 12. Vector phasmids pARC032 and pARC035 wherein the new restriction sites could be introduced into the multiple cloning site by standard method of in vitro site directed mutagenesis. 13. 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. 14. A bacterial cell, transformed by the vector phasmids pARC032 or pARC035, which produces a protein or a peptide encoded by a gene inserted at the multiple cloning site.

15. A bacterial cell, transformed by the vector phasmids pARC032 or pARC035, which produces a mutagenized protein or peptide encoded by the mutagenized ss DNA.

16. A process for the production of a protein or a peptide, encoded by the gene inserted at the multiple cloning site, by using a bacterial cell transformed by the vector phasmids pARC032 or pARC035. 17. A process for the production of a mutagenized protein or peptide, encoded by the mutagenized ss DNA, by using a bacterial cell transformed by the vector phasmids pARC032 or pARC035.

Further aspects of the invention can be summarized in the following clauses:

1. A phasmid construct containing a DNA phage f1 origin of replication, a T7 phage promoter followed by a multiple cloning site and a kanamycin resistance selection marker. 2. The design of a phasmid as described in Fig. 4 containing features as in paragraph 1 or part thereof.

3. A phasmid to which a gene or a gene segment can be introduced at the multiple cloning site described therein.

4. A phasmid which can replicate in any bacterial host. 5. A phasmid which can produce single stranded DNA of the gene inserted at the multiple cloning site in any host cell upon superinfection by a helper phage. 6. A phasmid wherein a synthetic oligonucleotide containing a mismatch flanked by complementary sequences to the gene inserted at the multiple cloning site, which produces a variant ss

DNA mutagenized at a preselected spot. 7. A phasmid capable of producing ss DNA under appropriate culture conditions which can be labelled with radioactive elements such as ³²P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA. 8. A phasmid which upon insertion into a suitable host cell, can be selected by growing the host cell in kanamycin containing medium.

9. A phasmid which contains a bacteriophage T7 promoter for transcription.

10. Any bacterial cell transformed by the phasmid which produces a protein or a peptide encoded by the gene inserted at the multiple cloning site.

11. Any bacterial cell transformed by the phasmid which produces a mutagenized protein or peptide encoded by the mutagenized ss DNA.

MATERIALS AND METHODS

The bacterial strains used were: 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 ninδ lacUV5-T7gene1).

Helper phage R408.

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.

All DNA manipulations were done by essentially following protocols described in "Molecular cloning - A Laboratory Manual", Maniatis et al. (eds) CSH Publ., 1989.

REFERENCES

1. Rodriguez, R.L., and Denhardt, D.T. (eds) Vectors: A Survey of Molecular Cloning Vectors and their uses 1988, Biotechnology Series, Boston, Butterworth.

2. Studier & Moffat, 1986, J.Mol.Biol. 189, 113.

3. Dotto et al. 1981 , Virology, 114, p. 463.

4. Dotto et al. 1983, Nucl.Acid.Res. 11 , p. 1645.

Peeters, B.P.K., Konings et al., European Patent Application NO.209 204-A1.

6. Viera & Messing, 1987, Methods Enzymol. 153, p.3.

7. Konings et al., 1987, Methods Enzymol. 153, p. 12.

8. Pouwels et al., (eds) Cloning Vectors. A laboratory Manual, 1986.

9. Yanish-Perron et al. 1985, Gene 33, p. 103.

10. Maniatis et al., Molecular Cloning - A laboratory Manual, CSH Publ., 1989.

11. Dwarakanath et al., 1989, Gene 81 , p. 219 BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1 E.coli pET7 plasmid

FIGURE 2

E.coli pARC032 plasmid

FIGURE 3 E.coli pARC035 plasmid

FIGURE 4

E.coli pARC036 plasmid

FIGURE 5

Construction of pARC032 and pARC035

FIGURE 6

Construction of pARC036

FIGURE 7

Cloning of E.coli st gene in pARC036

FIGURE 8 E.coli pARC044 plasmid

FIGURE 9

Strategy for site directed mutagenesis using ss phasmid DNA as a template

Claims

(b) T7 phage promoter followed by a multiple cloning site.

2. 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.

3. 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.

4. Vector phasmid pARC 032 having the structure shown in Figure 2.

5. Vector phasmid pARC 035 having the structure shown in Figure 3.

6. Vector phasmid pARC032 and pARC035 into which a fragment of double-stranded DNA has been inserted at the multiple cloning site.

7. Vector phasmids pARC032 and pARC035 which can replicate in a bacterial host.

8. 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.

9. 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.

10. Vector phasmids pARC032 and pARC035 capable of producing ss DNA under standard culture conditions which can be labelled with p radioactive elements such as P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA.

11. Vector phasmids pARC032 and pARC035 as in Claim 1 which upon insertion in a suitable host cell, can be selected by growing the host cell in kanamycin containing medium.

12. Vector phasmids pARC032 and pARC035 wherein the new restriction sites could be introduced into the multiple cloning site by standard method of in vitro site directed mutagenesis.

13. Vector phasmids pARC032 and pARC035 which contains a bacteriophage 77 promoter for transcription and which is transcribed in vivo or in vitro by a T7 RNA polymerase.

14. A bacterial cell, transformed by the vector phasmids pARC032 or pARC035, which produces a protein or a peptide encoded by a gene inserted at the multiple cloning site.

16. A process for the production of a protein or a peptide, encoded by the gene inserted at the multiple cloning site, by using a bacterial cell transformed by the vector phasmids pARC032 or pARC035.

17. A process for the production of a mutagenized protein or peptide, encoded by the mutagenized ss DNA, by using a bacterial cell transformed by the vector phasmids pARC032 or pARC035.