An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.

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dc.contributor.authorAcharya, A Sen_US
dc.contributor.authorRoy, K Ben_US
dc.date.accessioned2001-10-12en_US
dc.date.accessioned2009-05-27T09:36:59Z
dc.date.available2001-10-12en_US
dc.date.available2009-05-27T09:36:59Z
dc.date.issued2001-10-12en_US
dc.description.abstractThe 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.en_US
dc.description.affiliationCentre for Biotechnology, Jawaharlal Nehru University, New Delhi, India.en_US
dc.identifier.citationAcharya AS, Roy KB. An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system. Indian Journal of Biochemistry & Biophysics. 2001 Oct; 38(5): 303-8en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/28042
dc.language.isoengen_US
dc.source.urihttps://https://www.niscair.res.in/ScienceCommunication/ResearchJournals/rejour/ijbb/ijbb0.aspen_US
dc.subject.meshBinding Sitesen_US
dc.subject.meshCell Divisionen_US
dc.subject.meshCells, Cultureden_US
dc.subject.meshDNA-Directed RNA Polymerases --metabolismen_US
dc.subject.meshDeoxyribonuclease BamHI --geneticsen_US
dc.subject.meshMutationen_US
dc.subject.meshPlasmidsen_US
dc.subject.meshRecombinant Proteins --isolation & purificationen_US
dc.subject.meshViral Proteinsen_US
dc.titleAn alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.en_US
dc.typeJournal Articleen_US
dc.typeResearch Support, Non-U.S. Gov'ten_US
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