| Title: | 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 |
| Author(s): | Guarna MM; Cote HC; Kwan EM; Rintoul GL; Meyhack B; Heim J; MacGillivray RT; Warren RA; Kilburn DG; |
| Address: | "Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada" |
| ISSN/ISBN: | 1046-5928 (Print) 1046-5928 (Linking) |
| Abstract: | "Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity 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 availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) 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 cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro" |
| Keywords: | Amino Acid Sequence Animals Calbindins Carrier Proteins/genetics/metabolism Cell Line Cricetinae Enzyme Activation Factor Xa/genetics/*metabolism Hirudins/genetics/*isolation & purification/*metabolism Humans Maltose-Binding Proteins Mating Factor Oligope; |
| Notes: | "MedlineGuarna, M M Cote, H C Kwan, E M Rintoul, G L Meyhack, B Heim, J MacGillivray, R T Warren, R A Kilburn, D G eng Research Support, Non-U.S. Gov't 2000/10/26 Protein Expr Purif. 2000 Nov; 20(2):133-41. doi: 10.1006/prep.2000.1292" |
