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 CEPT. In addition, the encoded mature N-terminal sequence was changed from Cys- to Arg-Glu-Phe- to link the CETP sequence to the yeast acid phosphatase signal peptide. The culture medium of the transformed cells induced with 1% methanol contained both cholesteryl ester and triglyceride transfer activity comparable to that of rabbit plasma. Like rabbit plasma, the lipid transfer activity in the medium could be inhibited by monoclonal antibodies that block CE/TG transfer or TG transfer alone. Immunoblot analysis of M(r) = 80 K and minor species of M(r) = 60-100 K. In spite of these differences, the specific transfer activity of the recombinant CETP was indistinguishable from that of rabbit plasma CETP of M(r) = 74 K. N-Glycosidase F treatment converted both the recombinant and plasma CETP to a single species of M(r) = 55 K. Both the plasma and recombinant CETP lost their activity after removal of N-linked carbohydrate and sialic acid. A single 55 K component was found in the cell-lysates. The intracellular form of the recombinant CETP was not modified by N-glycosidase F treatment. In conclusion, the recombinant CETP is synthesized as an inactive polypeptide that is processed and secreted as a functional glycoprotein. In addition, the N-terminal Cys residue of the plasma CETP is not required for its activity.
Kotake
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Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
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Ohnishi
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Agellon
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Yokoyama
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D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D015870:Gene Expression; D006023:Glycoproteins; D006031:Glycosylation; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014170:Transformation, Genetic; D014280:Triglycerides / Q000378:metabolism
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D011994:Recombinant Proteins; D014280:Triglycerides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase