Heterologous expression of A. thaliana MutL-alpha using dual-vector strategy

GALLES, C.; CLAUDIA PATRICIA SPAMPINATO

Mar del Plata

Congreso; SAIB 2007; 2007

SAIB

The highly conserved post-replicative mismatch repair (MMR) system is crucial for maintaining genomic stability in all organisms. The main proteins involved in this repair pathway have already been identified, among which are the MutL-type proteins, whose role as molecular switches is critical for the repair reaction to take place. For all plants in general, including, Arabidopsis thaliana,Arabidopsis thaliana, little is known about this process. Our aim is to study the main MutL activity of this organism, MutL-alpha, which consists in a heterodimer of two proteins, PMS1 and MLH1. To the moment, MLH1 and PMS1 DNA sequences have been obtained in our laboratory by reverse transcription followed by PCR via specific primers, using total plant RNA as a template. The cDNA fragments were then cloned into compatible expression vectors and used to transform Escherichia coli JM109(DE3)pRIL strains in order to achieve the recombinant expression of the plant protein. Once the desired expression was observed, we proceeded to purify the recombinant heterodimer and asses its functionality. As an initial step in this direction, purified intact PMS1 protein was used to raise polyclonal rabbit antibodies. The tools generated so far should allow the development of a variety of further studies in Arabidopis,JM109(DE3)pRIL strains in order to achieve the recombinant expression of the plant protein. Once the desired expression was observed, we proceeded to purify the recombinant heterodimer and asses its functionality. As an initial step in this direction, purified intact PMS1 protein was used to raise polyclonal rabbit antibodies. The tools generated so far should allow the development of a variety of further studies in Arabidopis,Arabidopis, consequently shedding some light on how the phenomenon of DNA MMR repair takes place in plants.