• perlecan;
  • structure;
  • baculovirus expression;
  • chondroitin sulfate;
  • basic fibroblast-growth factor


  1. Top of page
  2. Abstract
  3. References

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+Iab). 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-(l–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.


Autographa californica nuclear polyhedrosis virus


baculovirus expression vector


basic fibroblast growth factor


glomerular basement membrane


heparin sulfate proteoglycan


Peroxidase (EC


polynucleotide kinase (EC


DNA polymerase (EC


type II site-specific DNase (EC


proteinase K (EC


chondroitin ABC lyase (EC


DNA ligase (EC


  1. Top of page
  2. Abstract
  3. References
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