An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.
The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.
Acharya
A S
AS
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Roy
K B
KB
D001665:Binding Sites; D002455:Cell Division; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D015245:Deoxyribonuclease BamHI