Expression and Purification of Recombinant Tick Anticoagulant Peptide (Y1W/D10R) Double Mutant Secreted bySaccharomyces cerevisiae

Abstract A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast α-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.