PMS1 from Arabidopsis thaliana: optimization of protein overexpression in Escherichia coli.
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 pET28a-AtPMS1 plasmid was efficiently expressed in JM109(DE3)-pRIL strain probably due to the genotype features of the cells (endA1, recA1, relA1, Δ(lac-proAB), laqIqZΔM15) 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 His-tagged 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).
Galles
Celina
C
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina.
Gomez
Rodrigo L
RL
Spampinato
Claudia P
CP
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005838:Genotype; D000070956:MutL Proteins; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; D003062:Codon; C496208:PMS1 protein, Arabidopsis; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000070956:MutL Proteins