RECOMBINANT CANDITROPSIN, AN EXTRACELLULAR ASPARTIC PROTEASE FROM YEAST CANDIDA-TROPICALIS - ESCHERICHIA-COLI EXPRESSION, PURIFICATION, ZYMOGEN ACTIVATION, AND ENZYMATIC-PROPERTIES

A cDNA fragment which encodes the zymogen of canditropsin, the extrace llular aspartic protease from the yeast Candida tropicalis (Togni, G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 2 86, 18 1-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichia coli. Recombinant canditropsinogen (Ctg), which was expressed as inclusion bodies in the cytosol of E. c oli, was refolded by dialysis from an 8 m urea solution and purified t o homogeneity using chromatographies on Sephacryl S-300 and on MonoQ c olumns. The purified Ctg was converted into canditropsin by either aci d activation or trypsin conversion. The specificity of the resulting r ecombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxid ized insulin B chain between Ala-Leu and many other minor cleavage sit es. Canditropsin also hydrolyzes keratin and collagen, which are compo nents of connective tissues known to be hydrolyzed by canditropsin dur ing Candida infections. Canditropsin was strongly inhibited by the uni versal aspartic protease inhibitor pepstatin (K(i) = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. C anditropsin is weakly inhibited by leupeptin and antipain, with an app arent K(i) of 1.74 x 10(-4) M and 1.5 x 10(-5) M, respectively.