Details

Autor(en) / Beteiligte

• Li, X J
• Wu, J G
• Si, J L
• Guo, D W
• Xu, J P

Titel

High-level expression of human calmodulin in E. coli and its effects on cell proliferation

Ist Teil von

• Shi yan sheng wu xue bao, 2001-06, Vol.34 (2), p.131

Ort / Verlag

China

Erscheinungsjahr

2001

Quelle

MEDLINE

Beschreibungen/Notizen

• 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