[High-level expression of human calmodulin in E. coli and its effects on cell proliferation].
The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template. After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220). The recombinant plasmid was then transformed into E. coli DH5 alpha. 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 SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h 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.
Li
X J
XJ
Center of Medical Laboratory Science of PLA, Nanjing General Hospital of Nanjing Command, Nanjing, 210002.
Wu
J G
JG
Si
J L
JL
Guo
D W
DW
Xu
J P
JP
D000818:Animals; D019046:Bone Marrow Neoplasms / Q000473:pathology; D002147:Calmodulin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004926:Escherichia coli; D006801:Humans; D020014:K562 Cells; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D011994:Recombinant Proteins; D014268:Trifluoperazine