Characterization of recombinant heparin cofactor II expressed in insect cells

Protein Expr Purif. 1995 Dec;6(6):806-12. doi: 10.1006/prep.1995.0012.

Abstract

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Chromatography, Affinity
  • Chymotrypsin / antagonists & inhibitors
  • Gene Expression
  • Glycosaminoglycans / pharmacology
  • Glycosylation
  • Heparin Cofactor II / genetics*
  • Heparin Cofactor II / isolation & purification*
  • Heparin Cofactor II / pharmacology
  • Hirudins / pharmacology
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Molecular Weight
  • Nucleopolyhedroviruses / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Spodoptera
  • Thrombin / antagonists & inhibitors

Substances

  • Glycosaminoglycans
  • Hirudins
  • Recombinant Proteins
  • Heparin Cofactor II
  • Chymotrypsin
  • Thrombin