title: Functional expression and purification of Anabaena PCC 7120 XisA protein.
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 (°)C on LB under static condition.
authors.affiliation: Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India.
authors.forename: Ujwal
authors.initials: U
authors.lastname: Trivedi
authors.affiliation: Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India.
authors.forename: Shubham
authors.initials: S
authors.lastname: Kaushik
authors.affiliation: Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Kunjadia
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, CV Raman Avenue, Bangalore, 560012, India.
authors.forename: Matheshwaran
authors.initials: M
authors.lastname: Saravanan
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, CV Raman Avenue, Bangalore, 560012, India.
authors.forename: Valakunja
authors.initials: V
authors.lastname: Nagaraja
authors.affiliation: Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India.
authors.forename: Gattupalli
authors.initials: G
authors.lastname: Archana
authors.affiliation: Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, India. Electronic address: gnaresh_k@yahoo.co.in.
authors.forename: Gattupalli
authors.initials: G
authors.lastname: Nareshkumar
mesh_terms: D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016680:Genome, Bacterial; D013058:Mass Spectrometry; D008958:Models, Molecular
chemical_list: D001426:Bacterial Proteins; D004254:DNA Nucleotidyltransferases
keywords: Recombinase activity; XisA protein; nifD element excision