Purification of Arabidopsis LFR Recombinant Protein in Engineering Bacteria and Preparation of Its Antibody / 生物化学与生物物理进展

ABSTRACT

Genome of Arabidopsis has a kind of genes encoding proteins with ARM repeat domains and some of these proteins are known to play important roles in plant development and responses to hormone. An Arabidopsis mutant lfr with a distinct phenotype was got in leaf and flower development. The gene is predicted to encode a protein with ARM repeat domains. In order to study its function and molecular mechanism, recombination expression plasmid pGEX-2TGST∶LFR was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The expression products were detected by 12% SDS-PAGE. The GST∶LFR fusion protein was existed in soluble form with a relative molecular mass 77 ku, which is fit with the molecular mass supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 ku, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.