TY - JOUR
T1 - Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells
AU - Groffen, Alexander J.A.
AU - Buskens, Carin A.F.
AU - Tryggvason, Karl
AU - Veerkamp, Jacques H.
AU - Monnens, Leo A.H.
AU - Van Den Heuvel, Lambert P.W.J.
PY - 1996
Y1 - 1996
N2 - 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 baculovirusderived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future.
AB - 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 baculovirusderived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future.
KW - Baculovirus expression
KW - Basic fibroblast-growth factor
KW - Chondroitin sulfate
KW - Perlecan
KW - Structure
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U2 - https://doi.org/10.1111/j.1432-1033.1996.00827.x
DO - https://doi.org/10.1111/j.1432-1033.1996.00827.x
M3 - Article
C2 - 8944771
SN - 0014-2956
VL - 241
SP - 827
EP - 834
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 3
ER -