Cloning, expression and purification of a key A. thaliana DNA mismatch repair protein

GÓMEZ, R.; CLAUDIA PATRICIA SPAMPINATO

Mar del Plata

Congreso; SAIB 2007; 2007

SAIB

The mismatch repair (MMR) system has been highly conserved during evolution and is critical for maintaining the overall integrity of the genetic material, essentially through its post-replicative correction activity. In eukaryotes mismatch recognition is achieved by two different heterodimers which share one subunit, called MSH2. These complexes, MutS-alpha (MSH2-MSH6) and MutS-beta (MSH2-MSH3), are implicated in the first stage of the MMR system and have a special importance in the process. Arabidopsis and other plants present a third heterodimer complex, MutS-gamma, which is formed by an MSH2 subunit and a unique ortholog called MSH7. In this study, we describe the cloning, expression and purification of MSH2 from A. thaliana using E. coli as an expression host. The cDNA fragment of MSH2 was cloned into pET32b, an IPTG-based expression system vector. The protein was expressed as a fusion protein of 120 kDa in E. coli JM109(DE3)RIL strain. Subsequent purification methods had been applied obtaining pure recombinant MSH2. Afterwards, enterokinase treatment generated an intact protein with only one extra amino acid residue at the N-terminal end of the sequence which was used for the induction and subsequent affinity purification of rabbit antibodies against MSH2. The recombinant protein obtained and the antibodies generated will allow further functional and structural studies on plant MMR.A. thaliana using E. coli as an expression host. The cDNA fragment of MSH2 was cloned into pET32b, an IPTG-based expression system vector. The protein was expressed as a fusion protein of 120 kDa in E. coli JM109(DE3)RIL strain. Subsequent purification methods had been applied obtaining pure recombinant MSH2. Afterwards, enterokinase treatment generated an intact protein with only one extra amino acid residue at the N-terminal end of the sequence which was used for the induction and subsequent affinity purification of rabbit antibodies against MSH2. The recombinant protein obtained and the antibodies generated will allow further functional and structural studies on plant MMR.E. coli JM109(DE3)RIL strain. Subsequent purification methods had been applied obtaining pure recombinant MSH2. Afterwards, enterokinase treatment generated an intact protein with only one extra amino acid residue at the N-terminal end of the sequence which was used for the induction and subsequent affinity purification of rabbit antibodies against MSH2. The recombinant protein obtained and the antibodies generated will allow further functional and structural studies on plant MMR.