Cloning and prokaryotic expression in TaCaM2-3 of wheat and preparation of antiserum

Abstract

Multiple calmodulin (CaM) isoforms exist in plant organisms and vary by their primary structures of 148 amino acids. They have different expression patterns and/or target enzyme activation abilities. To further understand the biological significance of TaCaM isoforms, total RNA was isolated from mature leaves of wheat and then TaCaM2-3 gene was amplified by PCR after reverse transcription. The PCR product was generated into T-easy vector to subsequently sequence. Then the recombinant expression vector (pET28a-TaCaM2-3) was constructed and transformed into E. coli strain BL21 to obtain a high level expression vector of CaM. SDS-PAGE analysis showed that the recombinant E. coli could express an approximate 20 kD protein. A western blotting analysis showed an anti-CaM monoclonal antibody specifically bound to the 20 kD band of expressed product. TaCaM-II was purified by Ni-NTA affinity chromatography from recombinant bacterial lysate. TaCaM-II protein was used to immunize New Zealand white rabbits to produce a polyclonal antiserum. The specificity of the anti-TaCaM-II antiserum was successfully verified by western blotting analysis.