Expression and Characterization of Human Perlecan Domains I and II Synthesized by Baculovirus-Infected Insect Cells

Authors


L. P. W. J. van den Heuvel, Dept. of Pediatrics, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands

Abstract

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.

Abbreviations.
AcNPV

Autographa californica nuclear polyhedrosis virus

BEV

baculovirus expression vector

bFGF

basic fibroblast growth factor

GBM

glomerular basement membrane

HSPG

heparin sulfate proteoglycan

Enzymes.
 

Peroxidase (EC 1.11.1.7)

 

polynucleotide kinase (EC 2.7.1.78)

 

DNA polymerase (EC 2.7.7.7)

 

type II site-specific DNase (EC 3.1.21.4)

 

proteinase K (EC 3.4.21.64)

 

chondroitin ABC lyase (EC 4.2.2.4)

 

DNA ligase (EC 6.5.1.1)

Ancillary