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 Pichiu pnrt0ri.s 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 "A. 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 Cysto ArgGlu-Pheto 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 the medium detected a major immunoreactive species of M, = 80 K and minor species of M, = 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, = 74 K. N-Glycosidase F treatment converted both the recombinant and plasma CETP to a single species of M, = 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, H., Q. Li, T. Ohnishi, K. W. S. KO, L. B. Agellon, and S. Yokoyama. Expression and secretion of rabbit plasma cholesteryl ester transfer protein by Pichia pastorir. J. Lipid Res. 1996.37: 599-605. Supplementary key words cDNA nucleotide sequence lipid transfer cholesteryl ester CETP in plasma is synthesized in high density lipoprotein (HDL) by 1ecithin:cholesterol acyltransferase (3) and distributed to low density lipoprotein (LDL) and very low density lipoprotein by the action of CETP, mainly in exchange with TG at least in bulk plasma (4,5). This transfer of CE can facilitate the efficient clearance of CE from circulation by receptor-mediated uptake of LDL by the liver. Therefore, CETP may play an important role in the pathway by which peripheral tissue cholesterol is removed by lipoproteins and returned to the liver for catabolism. On the other hand, the action of CETP can also result in lower HDL-cholesterol and higher LDL-cholesterol, a lipoprotein profile considered to be atherogenic and a risk for coronary heart disease (6). Thus, the overall role of CETP in plasma lipoprotein metabolism and development of atherosclerosis is unclear. Rabbit CETP consists of 496 amino acid residues (7) and has a high degree of similarity to the human CETP of 476 amino acid (8). The major difference is the 19-residue insertion near the COOH-terminal in the rabbit protein. Biochemical analysis also show a difference in substrate specificity and distribution in plasma (4, 7, 9). We have previously purified rabbit CETP and shown that human and rabbit CETP have similar specific activities with respect to CE transfer in vitro, although they differ in physicochemical properties, especially in hydrophobicity (10, 11). We also reported the production of novel inhibitory monoclonal antibodies against rabbit CETP that cross-react with human CETP (5). One of the monoclonal antibodies dissociated radioisotopic TG and CE transfer activities of CETP. Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that functions to transfer neutral lipids, such as cholesteryl ester (CE) and triglyceride (TG), among lipoprotein particles (1, 2). The majority of CE Abbreviations: CETP, cholesteryl ester transfer protein; CE, cholesteryl ester; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecylsulfate; DlT, dithiothreitol. 'To whom correspondence should he addressed. Journal of Lipid Research Volume 37, 1996 599 by gest, on July 5, 2011 w w w .j.org D ow nladed fom In order to further dissect the reaction mechanism of CETP, a reliable supply as well as a facile system to introduce specific modifications to the protein is required. To meet these requirements, we have constructed a vector that directs the production of recombinant rabbit CETP in the yeast, Pichia pastoris, which has previously been shown to efficiently express and secrete various recombinant mammalian proteins (12-14). Once the transfected yeast cell line is established, the culture can be grown rapidly, inexpensively, and without the need for a sophisticated culture facility, an advantage compared to the expression systems utilizing mammalian and insect cells ( 1517). MATERIALS AND METHODS Cloning of rabbit CETP cDNA Figure 1 shows the overview of the cloning. Total RNA was extracted from the newborn New Zealand White rabbit liver and poly(A) fraction was separated from total RNA by using oligo(dT) latex (18). Two cDNA fragments (encoding from the N-terminal to amino acid residue 262 of rabbit CETP and from the residue 263 to the C-terminal) were produced by in vitro DNA amplification of cDNA reverse-transcribed from rabbit liver RNA. The region of the rabbit cDNA that encodes the mature polypeptide was obtained by in-frame fusion of the two fragments and cloning in pUC19. The sequence was confirmed by sequencing the cloned amplified products using an Applied Biosystem 373A DNA sequencer. The amplified cDNA sequence differs from the previously published sequence in 8 nucleotides causing six amino acid replacements (Asn + Asp at 110, Asp + Gln at 122, Asn + Lys at 185, Val + Met at 284, Ala + Thr at 434 and Thr + Ala at 435). Interestingly, these replacements are identical to the corresponding amino acid residues in human CETP except for that of the residue 122 (8). The rabbit cDNA was constructed to Nucleotides 1 500 I I contain an EcoRI and Hind111 restriction sites at the 5’ and 3’ ends, respectively. This allows the convenient cloning of the fragment into PHIL-S1 expression plasmid. In addition, the sequence of the 5’ end of the cDNA encoding the mature N-terminal end was designed to allow the in frame fusion with the Pichia pastork acid phosphatase signal sequence encoded by PHIL-Sl. This altered the natural N-terminal residue of the mature CETP from Cys to Arg-Glu-Phe. This modification was necessary to allow the cleavage of the acid phosphatase signal sequence encoded by the PHIL-S1 from the recombinant rabbit CETP. Expression of rabbit CETP in A’chia pastoris Pichia pastoris GSll5 (defective in HlS4), PHIL-Sl expression plasmid and transfection reagents were obtained from Invitrogen (San Diego, CA). Transformation of Pichia pastoris GS115 with the rabbit CETP expression vector was carried out essentially according to the instructions provided by Invitrogen based on the previous publications ( 12-14). A recombination event is designed to occur in vivo between the cassette in PHIL-Sl (includes CETP cDNA and HIS4 between 5’ and 3‘ ends of alcohol oxidase gene (AOXl) regulatory sequences) and those in the genome of Pichia pastoris. The clones with impaired growth in the media containing methanol as the sole carbon and energy source (in which the AOXl structural gene was replaced with the rabbit CETP expression cassette) were selected (18). The modified cells were grown at 30°C in 150 mL of Minimal Glycerol Medium as described by Invitrogen (San Diego, CA). The AOXl promoter is used to drive the expression of the rabbit CETP cDNA by incubating the cells in the medium containing methanol (1%, v/v) at 30°C for 48 h. The conditioned media of the cells was collected for the analysis and partial purification of the recombinant CETP. The cells were washed, resuspended in ice-cold breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM PMSF, 1 mM EDTA, and 5% 100