High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit.
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.
Gomez
Rodrigo L
RL
Facultad de Ciencias Bioquímicas y Farmacéuticas, Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Galles
Celina
C
Spampinato
Claudia P
CP
D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D051718:MutS Homolog 2 Protein / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C108984:MSH2 protein, Arabidopsis; D051718:MutS Homolog 2 Protein