Nucleic Acids Research, Vol. 18, No. 14 4295
Three component system consisting of host bacteria, phage and plasmid, for efficient gene expression controlled by the SP6 promoter L.A.Zheleznaya, R.S.Savchenko1 and N.I.Matvienko1 Institute of Biophysics and 'Institute of Protein Research, USSR Academy of Sciences, Pushchino, Moscow Region, USSR Submitted May 14, 1990
The system consisting of a plasmid with the T7 promoter and phage X with the T7-RNA polymerase gene is very promising for efficient expression of cloned genes controlling production of toxic proteins for E. coli as expression starts only after infection of the bacteria with the recombinant X phage (1, 2). We have constructed a similar system on the basis of the recombinant phage M13 with the SP6 polymerase gene (Ml3tgl31-SP6-RNApol). The whole SP6 RNA polymerase gene has been cloned in phage M13tgl31 under the control of the lac-promoter. The cloned fragment is deleted of the SP6 promoter downstream of the polymerase gene and of the host RNA polymerase promoters upstream of the gene but the fragment preserves the SD-sequence in from of the gene as well as the T terminator after the gene (3). To check the efficiency of the new system plasmids pSP65 and pGEM-1 were used in which the Cmr gene and the LacZ and Cmr genes, respectively, were inserted downstream of the SP6 promoter. Extensive expression of the cloned genes was observed in the presence of IPTG after infection with recombinant M13 phage of E. coli F+ bacteria harboring these plasmids
(Fig. 1). REFERENCES 1. Studier,F. and Moffatt,B.A. (1986) J. Mol. Biol. 189, 113-130. 2. West,D.K. et al. (1989) J. Biol. Chem. 264, 10343-10346. 3. Savchenko,R.S., Zheleznaya,L.A. and Matvienko,N.I. (1988) Dokl. Akad. Nauk SSSR 300, 735-739.
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6 a b
Figur 1. IPT'G-induction of chloramphenicol acetyltrnsferase and (3-galactosidase after infection of E. ccli XL1I with phage M 13tg 13 1-SP6pol. 1, 2, 3 - E. coli XLI (pSP65-Cmr); 4, 5, 6 - E. ccli (pGEMI-LacZ, Cm'); 2, 3, 5, 6 infection with Ml13tgl13l1-SP6pol; 2, 5 - without IPTG; 3, 6 - with IPTG (40 jig/ml). a - f3-galactosidase, b - chloramphenicol acetyltransferase.
Three component system consisting of host bacteria, phage and plasmid, for efficient gene expression controlled by the SP6 promoter L.A.Zheleznaya, R.S.Savchenko1 and N.I.Matvienko1 Institute of Biophysics and 'Institute of Protein Research, USSR Academy of Sciences, Pushchino, Moscow Region, USSR Submitted May 14, 1990
The system consisting of a plasmid with the T7 promoter and phage X with the T7-RNA polymerase gene is very promising for efficient expression of cloned genes controlling production of toxic proteins for E. coli as expression starts only after infection of the bacteria with the recombinant X phage (1, 2). We have constructed a similar system on the basis of the recombinant phage M13 with the SP6 polymerase gene (Ml3tgl31-SP6-RNApol). The whole SP6 RNA polymerase gene has been cloned in phage M13tgl31 under the control of the lac-promoter. The cloned fragment is deleted of the SP6 promoter downstream of the polymerase gene and of the host RNA polymerase promoters upstream of the gene but the fragment preserves the SD-sequence in from of the gene as well as the T terminator after the gene (3). To check the efficiency of the new system plasmids pSP65 and pGEM-1 were used in which the Cmr gene and the LacZ and Cmr genes, respectively, were inserted downstream of the SP6 promoter. Extensive expression of the cloned genes was observed in the presence of IPTG after infection with recombinant M13 phage of E. coli F+ bacteria harboring these plasmids
(Fig. 1). REFERENCES 1. Studier,F. and Moffatt,B.A. (1986) J. Mol. Biol. 189, 113-130. 2. West,D.K. et al. (1989) J. Biol. Chem. 264, 10343-10346. 3. Savchenko,R.S., Zheleznaya,L.A. and Matvienko,N.I. (1988) Dokl. Akad. Nauk SSSR 300, 735-739.
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5
6 a b
Figur 1. IPT'G-induction of chloramphenicol acetyltrnsferase and (3-galactosidase after infection of E. ccli XL1I with phage M 13tg 13 1-SP6pol. 1, 2, 3 - E. coli XLI (pSP65-Cmr); 4, 5, 6 - E. ccli (pGEMI-LacZ, Cm'); 2, 3, 5, 6 infection with Ml13tgl13l1-SP6pol; 2, 5 - without IPTG; 3, 6 - with IPTG (40 jig/ml). a - f3-galactosidase, b - chloramphenicol acetyltransferase.
