Functional expression and purification of Anabaena PCC 7120 XisA protein

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.

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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

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