title: 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 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.
authors: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7035, USA.
authors: A V
authors: AV
authors: Ciaccia
authors: E L
authors: EL
authors: Cunningham
authors: F C
authors: FC
authors: Church
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000494:pharmacology; D006031:Glycosylation; D015844:Heparin Cofactor II / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D006025:Glycosaminoglycans; D006629:Hirudins; D011994:Recombinant Proteins; D015844:Heparin Cofactor II; D002918:Chymotrypsin; D013917:Thrombin