Functional expression and purification of Anabaena PCC 7120 XisA protein

• Heterologous expression and purification of Anabaena PCC 7120 XisA protein.
• Confirmation of XisA protein through MALDI-TOF.
• Development of in vivo PCR based method for monitoring recombinase activity of XisA protein.
• Prediction of secondary and 3D structure of XisA protein.

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.