Fusional expression of human cholesteryl ester transfer protein cDNA in recombinant vaccinia virus-infected mammalian cells

Abstract

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 virus-infected 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~ 143B cells. The DNA from the selected virus was ampliHed and expressed in the CV-1 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 GST/CETP was bound on the column. When subjected to a CETP assay.