Characterization of Recombinant Heparin Cofactor Ii Expressed in Insect Cells

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 μg/106cells was achieved 2 days postinfection. Approximately 40 μg 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× 10−4M−1min−1) was 2.29 ± 0.36 for rHCII and 3.38 ± 0.34 for pHCII. Chymotrypsin inhibition rates (k2× 10−5M−1min−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× 10−8M−1min−1) for rHCII was 10.4 ± 2.5 at 100 μg/ml heparin and 16.0 ± 4.3 at 1000 μg/ml dermatan sulfate compared to 9.0 ± 0.7 at 200 μg/ml heparin and 18.5 ± 5.3 at 1000 μg/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.