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KMID : 0578319950050020107
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Molecules and Cells
1995 Volume.5 No. 2 p.107 ~ p.113
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Fusional Expression of Human cholesteryl Ester Transfer Protein cDNA in Recombinant Vaccinia Virus-infected Mammalian Cells
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Yoon, Woo Hyun
Jang, Moon Kyoo/Bok, Song-Hae/Park, Yong Bok
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Abstract
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Biologically active human plasma cholesteryl ester transfer protein (CETP) was directed to produce as a fusion protein with glutathione-S-transferase (GST) in recombinant vaccinia virusinfected mammalian cells. Complementary DNAs for GST and CETP were ligated side by side, and introduced into vaccinia viral transfer vectors. The viral vectors were transfected into CV-1 cells infected previously with wild-type vaccinia viruses. Recombinant viruses were generated by the homologous recombination and selected with BUdR and X-gal in human TK^(-) l43B cells. The DNA from the selected virus was amplified and expressed in the CV-I cells. The GST/CETP fusion protein (86 kDa) was identified on the SDS-PAGE followed by Western blot analysis using polyclonal antibody against the C-terminal active region of CETP fused with GST. The virus-infected cell lysates were loaded on the glutathione-Sepharose affinity column and the CETP was eluted from the column by cleavage with thrombin while the CST/CETP was bound on the column. The purified CETP showed biological activity when subjected to a CETP assay.
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KEYWORD
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