HIGH - LEVEL EXPRESSION OF HUMAN CALMODULIN IN E. COLI AND ITS EFFECTS ON CELL PROLIFERATION
LI Xiao Jun WU Jian Guo SI Jun Lin GUO Da Wen XU Jian Ping (Center of Medical Laboratory Science of PLA , Nanjing General Hospital of Nanjing Command , Nanjing ,210002)
The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was used as template. After inserting the hCaM Ⅲ cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA - re-combinant expression vector (hCaM3/pBV220 ). The re-combinant plasmid was then transformed into E. coli DH5α. After heat induction, a high level expression of CaM protein was obtained. SDS - PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti - CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl - sepharose CL - 4B affinity chromatography from recombinant bacterial lysate. 3 - 4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2 /O cells were seeded in 24 - well or 96 - well plate and cultured for 48h with rhCaM and CaM - antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM- antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.