High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit

Abstract

Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His(6)-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals

“…MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSc. Quantitative analysis of transcript levels of representative genes (MSH2 and PMS1) of the pathway indicated that these genes show a higher expression in calli than in seedlings from A. thaliana, thus confirming the essential genome maintenance function performed by MMR system in rapidly dividing tissues [116,117]. Further knowledge of Table 2 iv) Resolution of the D-loop…”

Research on Plants for the Understanding of Diseases of Nuclear and Mitochondrial Origin

“…We successfully overexpressed AtMSH2 and AtPMS1 en Escherichia coli and raised polyclonal-antibodies against these subunits [44, 45]. In addition, in studies to be reported elsewhere, we found that expression of MutL α or MutS γ in Saccharomyces cerevisiae leads to a clear increase in yeast mutator rate, suggesting that the expression of the plant proteins somehow affects yeast MMR mechanism.…”