CEFOBI   05405
CENTRO DE ESTUDIOS FOTOSINTETICOS Y BIOQUIMICOS
Unidad Ejecutora - UE
artículos
Título:
PMS1 from Arabidopsis thaliana: optimization of protein overexpression in Escherichia coli
Autor/es:
GALLES CELINA; GOMEZ RODRIGO; SPAMPINATO CLAUDIA
Revista:
MOLECULAR BIOLOGY REPORTS
Editorial:
SPRINGER
Referencias:
Año: 2011 vol. 38 p. 1063 - 1070
ISSN:
0301-4851
Resumen:
One of the major limitations when attempting to
obtain detailed biochemical, biophysical and immunological
characterization of plant DNA mismatch repair proteins is
their extremely low abundance in vivo under normal growth
conditions. An initial analysis of PMS1 transcript level in
various Arabidopsis thaliana tissues was carried out by
quantitative real-time RT-PCR. For calli, flowers and seedlings,
the corresponding cDNA copies per ng RNA were
66.9, 3.1 and 2.7, respectively. This suggests an important
role of this gene in rapidly dividing tissues. In order to obtain
a high level of PMS1 from Arabidopsis thaliana, the protein
production was successfully optimized in an Escherichia
coli host. The corresponding coding sequence of
PMS1 was inserted into pET28a downstream a hexa-histidyl
leader sequence. The pET28aAtPMS1 plasmid was efficiently
expressed in JM109(DE3)-pRIL strain probably due
to the genotype features of the cells (endA1, recA1, relA1,
D(lac-proAB), laqIqZDM15) and the presence of extra
copies of argU, ileY, and leuW tRNA genes, which encode
the RIL codons. This strategy has allowed us to obtain Histagged
PMS1 at about 7% of the total soluble E. coli cell
protein. The protein was purified by standard Ni? affinity
chromatography procedures and the electrophoretically
homogeneous preparation was used as an antigen for antibody
generation in rabbits. This approach provides effective
tools for a further reconstitution of plant mismatch repair
(MMR) system in vitro and for the analysis of protein
expression and distribution of AtPMS1 in various tissues
after different treatments (e.g. DNA mutagens).