CEFOBI   05405
CENTRO DE ESTUDIOS FOTOSINTETICOS Y BIOQUIMICOS
Unidad Ejecutora - UE
artículos
Título:
High-Level Production of MSH2 from Arabidopsis thaliana: A DNA Mismatch Repair System Key Subunit
Autor/es:
GOMEZ RODRIGO; GALLES CELINA; SPAMPINATO CLAUDIA
Revista:
MOLECULAR BIOTECHNOLOGY
Editorial:
HUMANA PRESS INC
Referencias:
Año: 2011 vol. 47 p. 120 - 129
ISSN:
1073-6085
Resumen:
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-
His6-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.