Recombinant carbohydrate variant of human choriogonadotropin beta-subunit (hCG beta) descarboxyl terminus (115-145). Expression and characterization of carboxyl-terminal deletion mutant of hCG beta in the baculovirus system.
A recombinant analog of human choriogonadotropin beta-subunit descarboxyl-terminal peptide (115-145 residues, delhCG beta) was obtained by the expression of corresponding beta cDNA in the baculovirus expression system. The efficiency of expression and secretion was high. The recombinant delhCG beta was purified by immunoaffinity using a specific monoclonal antibody against hCG beta and reverse phase high performance liquid chromatography. The hCG beta analog lacked the carboxyl-terminal 31-residue peptide as well as the four O-linked carbohydrates. Also, the N-linked "complex" type carbohydrates in the deletion mutant were modified to the high mannose type. The apparent molecular weights of delhCG beta in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions were found to be 19,000 and 27,500 respectively. delhCG beta on hydrolysis with endo N-acetylglucosaminidase F or H yielded a 17,500 protein band whereas treatment with N-glycanase gave a protein band with a molecular weight of 16,000. The carbohydrate analysis of delhCG beta, calculated on the basis of 4 residues of N-acetylglucosamine, showed 3 or 4 fucose, 0.6 N-acetylgalactosamine, and 11.4 mannose residues, indicating the high mannose type structures of the two N-linked carbohydrate chains. Despite the carbohydrate modification of the N-linked carbohydrates and the carboxyl-terminal deletion, the delhCG beta had about 87% of the immunological activity of the native hCG beta, indicating no significant conformational alteration induced by the mutation. The delhCG beta combined readily with native hCG alpha, and the reconstituted hCG alpha del beta required 0.031 pmol to achieve 50% inhibition of binding of the tracer with rat lutropin/choriogonadotropin receptor compared with 0.039 pmol by native hCG. Like native hCG, hCG alpha del beta also had most comparable ability to stimulate cAMP accumulation and progesterone production in rat Leydig cells. Thus it is clear from the data that the carboxyl-terminal deletion and thereby the deletion of four O-linked carbohydrates had no effect on its in vitro immunological and biological properties.
Chen
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WY
Department of Biological Sciences, State University of New York, Buffalo 14260.
Bahl
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D016367:Baculoviridae / Q000235:genetics; D002241:Carbohydrates / Q000235:genetics; D006063:Chorionic Gonadotropin / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000032:analysis; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011374:Progesterone / Q000032:analysis; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; D013256:Steroids; D011374:Progesterone; D000242:Cyclic AMP