Preparation of recombinant carbohydrate deficient active analogs of human chorionic gonadotropin from insect cells.
Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCG beta mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCG beta specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence. -Asn x Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 micrograms/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.
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Department of Biological Sciences, State University of New York at Buffalo 14260, USA.
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D000818:Animals; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins