대장균에서 생산된 항 에이파토시스 재조합 30K 단백질의 정제

Abstract

It has been shown that silkworm hemolymph (SH) inhibits apoptosis in various systems such as insect, mammalian, and human cell systems. And 30K protein was a major apoptosis-inhibiting component in SH. The 30K cDNA was cloned into a pET22b(+) expression vector containing His6-tag and pGEX4T-1 expression vector containing GST-tag. 30K fused GST protein is not efficient in purification process for inactive GST function. On the other hand, the denatured 30K-His6 was purified (92% purity) by single-step immobilized metal ion affinity chromatography (IMAC) and was refolded by dilution with refolding buffer. In purification yield, this method is more efficient than on-column refolding method. The purified 30K protein inhibited the insect cell (Sf9) and mammalian cell (HeLa) apoptosis effectively by addition to the culture medium. For the analysis of apoptosis, cells were stained with Hoechst 33342 and observed with fluorescence microscope. Flow cytometric analysis was carried out for the quantitative analysis of apoptosis.