Cook J C, Schultz L D, Huang J, George H A, Herber W K, Ip C, Joyce J G, Mao S S, Markus H Z, Miller W J, Sardana M K, Lehman E D
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania, 19486, USA.
Protein Expr Purif. 1998 Aug;13(3):291-300. doi: 10.1006/prep.1998.0893.
A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.
