Kotake H, Li Q, Ohnishi T, Ko K W, Agellon L B, Yokoyama S
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
J Lipid Res. 1996 Mar;37(3):599-605.
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.
通过在甲醇诱导型醇氧化酶启动子的控制下引入胆固醇酯转运蛋白（CETP）cDNA，兔胆固醇酯转运蛋白在甲基营养型酵母毕赤酵母中得以表达。该cDNA从兔肝mRNA的体外扩增cDNA中克隆而来。克隆的cDNA核苷酸序列与先前发表的序列略有不同，这使得六个残基的氨基酸序列发生了改变。有趣的是，其中五个替换与人类CEPT中的相应残基相同。此外，编码的成熟N端序列从Cys-变为Arg-Glu-Phe-，以便将CETP序列与酵母酸性磷酸酶信号肽相连。用1%甲醇诱导的转化细胞培养基中含有与兔血浆相当的胆固醇酯和甘油三酯转运活性。与兔血浆一样，培养基中的脂质转运活性可被阻断CE/TG转运或单独阻断TG转运的单克隆抗体抑制。免疫印迹分析显示分子量为80K的主要条带以及分子量为60 - 100K的次要条带。尽管存在这些差异，但重组CETP的比转运活性与分子量为74K的兔血浆CETP无法区分。N-糖苷酶F处理将重组CETP和血浆CETP都转化为单一的分子量为55K的条带。去除N-连接碳水化合物和唾液酸后，血浆和重组CETP均失去活性。在细胞裂解物中发现了单一的55K成分。重组CETP的细胞内形式未被N-糖苷酶F处理修饰。总之，重组CETP作为无活性多肽合成，经加工后作为功能性糖蛋白分泌。此外，血浆CETP的N端Cys残基对其活性并非必需。
