Trivedi Ujwal, Kaushik Shubham, Kunjadia Prashant, Saravanan Matheshwaran, Nagaraja Valakunja, Archana Gattupalli, Nareshkumar Gattupalli
Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, CV Raman Avenue, Bangalore, 560012, India.
Protein Expr Purif. 2016 Feb;118:64-9. doi: 10.1016/j.pep.2015.09.027. Epub 2015 Oct 3.
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 (°)C on LB under static condition.
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