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 ca rbohydrate chains have been purified from insect cells by immunoaffini ty chromatography on a monoclonal antibody, B17, column Traces of the hCG beta mutant present, if any, were removed by a second immunoaffini ty 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-glyco sylation. All analogs were overexpressed in High-Five insect cells wit h the expression levels ranging between 1.5 to 15 mu g/ml and were fou nd 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 recep tor assay using rat ovarian membranes. The availability of these analo gs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.