A NOVEL YEAST EXPRESSION SECRETION SYSTEM FOR THE RECOMBINANT PLANT THIOL ENDOPROTEASE PROPAPAIN/

A new high-yield yeast expression/secretion system has been adapted fo r the plant thiol endoprotease papain, The propapain gene, obtained fr om Carica papaya fruit, is expressed in the yeast Saccharomyces cerevi siae. The gene was cloned into a FLAG epitope-tagging expression vecto r downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture su pernatant where it accumulates to similar to 1.7 mg/l. The proregion c ontains three consensus N-linked glycosylation sites, whereas there ar e only two such sites in previously reported cDNA sequences. Removal o f this third N-linked glycosylation site results in a drastic reductio n in the level of protease activity present in the culture supernatant . Two different types of affinity chromatography were used to purify e ither propapain or papain. The propapain precursor is autoproteolytica lly activated to mature papain (M(r) = 24 kDa) using conditions report ed previously. The kinetic parameters obtained agree well with the lit erature values. The yields of active papain are 10-fold higher than th ose previously reported for propapain in other yeast or bacterial expr ession systems. This, together with the ease with which mutant protein s can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.