Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells.
We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus 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, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(1-404)- and the -(1-506)- peptide was successful, but in the case of the perlecan-(1-196)-peptide no recombinant protein was produced. Immunoblotting showed that both the (1-404)-peptide and (1-506)-peptide are recognized by 95J10, a monoclonal antibody that was previously raised against perlecan-(24-404)-peptide expressed in Escherichia coli. Gel permeation and anion-exchange chromatography were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1-404)-peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1-404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan-(1-404)-peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus-derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future.
Groffen
A J
AJ
Department of Pediatrics, University of Nijmegen, The Netherlands.
Buskens
C A
CA
Tryggvason
K
K
Veerkamp
J H
JH
Monnens
L A
LA
van den Heuvel
L P
LP
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000032:analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D017421:Sequence Analysis; D018411:Spodoptera / Q000166:cytology; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D002809:Chondroitin Sulfates; D004262:DNA Restriction Enzymes; D016222:Fibroblast Growth Factor 2; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan