Factor X fusion proteins: Improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing aff inity tags to aid in the downstream processing. After purification, the aff inity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availabili ty from plasma. To develop a recombinant source of FXa, we have expressed t wo novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepro peptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBDCe x) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the c atalytic domain of FX and a polyhistidine tag. Both FX variants were secret ed into the medium, their affinity tags were functional, and following acti vation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBDCex fusion protein was 10-fold higher than that of FX-CBDC ex and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha -factor-hirudin fusion protein secreted by Saccharomyces ce revisiae. After cleavage, the released hirudin demonstrated biological acti vity in a thrombin inhibition assay, suggesting that this method may be app licable to the production of toxic or unstable proteins. The availability o f novel FX derivatives linked to different affinity tags allows the develop ment of a versatile system for processing fusion proteins in vitro. (C) 200 0 Academic Press.