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http://nopr.niscpr.res.in/handle/123456789/15318| Title: | An alternative approach for screening active Bam HI variants: Overexpression in T-7 RNA polymerase based system |
| Authors: | Acharya, Asha S Roy, Kunal B |
| Issue Date: | Oct-2001 |
| Publisher: | NISCAIR-CSIR, India |
| Abstract: | The type II restriction endonuclease. BamHI , has been overexpressed in E. coli by cloning the BamHI 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 BamHI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK 14. Our overexpressing clone, pAABRw in BL21 cell in presence of BamHI methylase in pMAP6 following induction with IPTG yields about 9.2x106 units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH 139 cells even in the absence of T7 polymerase and cognate BamHI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants. |
| Page(s): | 303-308 |
| ISSN: | 0975-0959 (Online); 0301-1208 (Print) |
| Appears in Collections: | IJBB Vol.38(5) [October 2001] |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| IJBB 38(5) 303-308.pdf | 1.31 MB | Adobe PDF | View/Open |
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