EXPRESSION AND PURIFICATION OF RECOMBINANT TICK ANTICOAGULANT PEPTIDE(Y1W D10R) DOUBLE MUTANT SECRETED BY SACCHAROMYCES-CEREVISIAE/

A double mutant of tick anticoagulant peptide (TAP) was cloned as a ch imeric fusion with the yeast alpha-mating factor pre-proleader peptide . Expression in yeast (Saccharomyces cerevisiae) resulted in the secre tion of the TAP mutein into the culture medium. An HPLC-based assay wa s used to screen yeast strains to find those giving highest expression levels, Efficiency of cleavage at the junction of the leader-TAP mute in varied from strain to strain, and a rapid purification method follo wed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme w as developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange c hromatography and reversed-phase HPLC. Scale-up of the process was suc cessful and produced 100 mg of fully functional TAP mutein of >96% hom ogeneity from a 50-L yeast culture. (C) 1998 Academic Press.