Trivedi, Ujwal and Kaushik, Shubham and Kunjadia, Prashant and Saravanan, Matheshwaran and Nagaraja, Valakunja and Archana, Gattupalli and Nareshkumar, Gattupalli (2016) Functional expression and purification of Anabaena PCC 7120 XisA protein. In: PROTEIN EXPRESSION AND PURIFICATION, 118 . pp. 64-69.
PDF
Pro_Exp_Pur_118_64_2016.pdf - Published Version Restricted to Registered users only Download (1MB) | Request a copy |
Abstract
Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 degrees C on LB under static condition. (C) 2015 Elsevier Inc. All rights reserved.
Item Type: | Journal Article |
---|---|
Publication: | PROTEIN EXPRESSION AND PURIFICATION |
Publisher: | ACADEMIC PRESS INC ELSEVIER SCIENCE |
Additional Information: | Copy right for this article belongs to the ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA |
Keywords: | nifD element excision; XisA protein; Recombinase activity |
Department/Centre: | Division of Biological Sciences > Microbiology & Cell Biology |
Date Deposited: | 27 Jan 2016 06:18 |
Last Modified: | 27 Jan 2016 06:18 |
URI: | http://eprints.iisc.ac.in/id/eprint/53165 |
Actions (login required)
View Item |