EXPRESSION OF FUNCTIONAL PAPAIN PRECURSOR IN SACCHAROMYCES-CEREVISIAE - RAPID SCREENING OF MUTANTS

A microbial expression system for the study of the cysteine protease papain has been developed as a more useful alternative to the insect cell/baculovirus expression system we have previously used. A synthetic papain precursor (pro-papain) gene was expressed in the yeast Saccharomyces cerevisiae under the control of the alpha-factor promoter. Efficient expression required fusion of the propapain sequence with the yeast alpha-factor prepro region and a yeast host cell defective in the synthesis of vacuolar proteases. Surprisingly, the glycosylated form of the inactive papain precursor is not secreted, but accumulates within the yeast cell. Complete conversion of the intracellular zymogen into active mature papain could be achieved in vitro. Purified recombinant papain produced by the yeast system has kinetic characteristics similar to those of the natural enzyme. An advantage of the yeast expression system over the baculovirus/insect cell system is that we can perform mutagenesis and screening of papain mutants very efficiently. We have set up a 'one-tube' screening procedure for the simultaneous characterization of numerous mutants of the papain precursor. Yeast cells are grown and lysed in microtiter plate wells and the released papain precursor is then activated to mature papain. This assay allows easy discrimination between proteins with close to wild type properties and proteins that are not functional. We have applied this assay to investigate the spectrum of amino acids which are tolerated at Asn175 of papain using two independently derived libraries of mutants at this position. Many amino acid substitutions at this position are not accepted: only the reintroduction of Asn restored normal function.