EXPRESSION AND CHARACTERIZATION OF HUMAN PERLECAN DOMAIN-I AND DOMAIN-II SYNTHESIZED BY BACULOVIRUS-INFECTED INSECT CELLS

We present the in vitro expression and purification of N-terminal frag ments of human perlecan in insect cells. Three tailored fragments of h uman perlecan cDNA were introduced into the polyhedrin locus of baculo virus expression vectors (BEVs) encoding amino acids 1-196 (domain I), 1-404 (domain I+IIa) and 1-506 (domain I+IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restric tion enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [S-35]methionine showed that expression of th e perlecan-(1-404)- and the -(1-506)-peptide was successful, but in th e case of the perlecan-(1-196)-peptide no recombinant protein was prod uced. Immunoblotting showed that both the (1-404)-peptide and (1-506)- peptide are recognized by 95J10, a monoclonal antibody that was previo usly raised against perlecan-(24-404)-peptide expressed in Escherichia coli. Gel permeation and anion-exchange chromatography were applied t o purify the recombinant proteins. Glycosaminoglycans were demonstrate d to be present. Deglycosylation with chondroitinase ABC showed that t he perlecan-(1-404)-peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated f rom the perlecan-(1-404)-peptide were identified as chondroitin sulfat e by agarose gel electrophoresis. Furthermore the perlecan-(1-404)-pep tide showed affinity to immobilized basic fibroblast growth factor. Th e availability of baculovirus-derived recombinant perlecan fragments w ill facilitate domain-specific investigation of the structural and fun ctional properties of perlecan in the future.