SBIR-STTR Award

A full-length coding sequence for human protein C will be cloned. This will be done either by obtaining a 5'end clone from a liver CDNA library or by fusing a genomic DNA sequence to CDNA, which lacks an aminoterminal coding sequence. Once this clone is complete it will be introduced into an expression vector designed for high-level expression in mammalian cells. This construction will be transfected into BHK cells and expressed protein C activity measured. Protein C will be assayed by ELISA and for anticoagulant activity.National Heart, Lung, and Blood Institute (NHLBI)National Heart, Lung, and Blood Institute (NHLBI)

Human protein C is a vitamin K-dependent plasma protein involved in the control of blood coagulation and fibrinolysis. An expression system for the production of recombinant human protein C has been established. This system is based on mammalian cells in culture, stably transfected with a DNA construction expressing human protein C. This expression system secretes into the culture media approximately 400 1lg/L of protein C that is gamma carboxylated, completely processed to its two-chain form, and capable of full biological activity.Methodologies, including random integration screening, gene amplification, and dicistronic mRNA constructions will be used in this research to increase the expression level of protein C. Some innovative modifications will be made to the expression construction to produce protein C molecules that will be more efficiently processed, gamma carboxylated, or activated during secretion. The goal is to establish a development-scale expression system to produce human protein C for various therapeutic applications.National Heart, Lung, and Blood Institute (NHLBI)