EXPRESSION AND SECRETION OF RABBIT PLASMA CHOLESTERYL ESTER TRANSFER PROTEIN BY PICHIA-PASTORIS

The rabbit cholesteryl ester transfer protein (CETP) was expressed in the methylotrophic yeast Pichia pastoris by introducing the CETP cDNA under the control of the methanol-inducible alcohol oxidase promoter. The cDNA was cloned from in vitro amplified cDNA of rabbit liver mRNA. The nucleotide sequence of the cloned cDNA differed slightly from the previously published sequence that changed the amino acid sequence in six residues. Interestingly, five of these replacements are identical to the corresponding residues in human CETP. In addition, the encoded mature N-terminal sequence was changed from Cys- to Arg-Glu-Phe- to l ink the CETP sequence to the yeast acid phosphatase signal peptide. Th e culture medium of the transformed cells induced with 1% methanol con tained both cholesteryl ester and triglyceride transfer activity compa rable to that of rabbit plasma. Like rabbit plasma, the lipid transfer activity in the medium could be inhibited by monoclonal antibodies th at block CE/TG transfer or TG transfer alone. Immunoblot analysis of t he medium detected a major immunoreactive species of M(r) = 80 K and m inor species of M(r) = 60-100 K. In spite of these differences, the sp ecific transfer activity of the recombinant CETP was indistinguishable from that of rabbit plasma CETP of M(r) = 74 K. N-Glycosidase F treat ment converted both the recombinant and plasma CETP to a single specie s of M(r) = 55 K. Both the plasma and recombinant CETP lost their acti vity after removal of N-linked carbohydrate and sialic acid. A single 55 K component was found in the cell-lysates. The intracellular form o f the recombinant CETP was not modified by N-glycosidase F treatment. In conclusion, the recombinant CETP is synthesized as an inactive poly peptide that is processed and secreted as a functional glycoprotein. I n addition, the N-terminal Cys residue of the plasma CETP is not requi red for its activity.