Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.

X Lin,J Tang,G Koelsch,M Monod,S Foundling

doi:10.1016/s0021-9258(20)80705-4

Abstract

A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni,G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) 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. coli, was refolded by dialysis from an 8 M urea solution and purified to homogeneity using chromatographies on Sephacryl S-300 and on MonoQ columns. The purified Ctg was converted into canditropsin by either acid activation or trypsin conversion. The specificity of the resulting recombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxidized insulin B chain between Ala-Leu and many other minor cleavage sites. Canditropsin also hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections. Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. Canditropsin is weakly inhibited by leupeptin and antipain, with an apparent Ki of 1.74 x 10(-4)M and 1.5 x 10(-5) M, respectively.

Highlights

A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni, G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichicaoli
Canditropsin hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections
Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 X lo-’ M) and inactivated by two aspartic protease inactivators, DAN

Summary

RESULTS

By proteolytic assay were pooled and further fractionated on an FPLC Expression of RecombinantCanditropsinin E. coli-The anion-exchange monoQ column,(HR5/5) which was equilibrated with 20 mM Tris-HC1, pH 7.0, and eluted by a linear gradient of 1M NaC1. -43/-42 (Fig. 1A).Theexpression vector PET-lla-Ctg, Activation of Canditropsinogen and Purification of the Resulting encodes the putative pro region The resulting canditropsin was further purified with a FPLC MonoQ column that had been equilibrated with 20 mM 1-methylpiperazine, pH 5.0, and eluted with a. By chromatography on a MonoQ column, which produced a single peak at 0.6 M NaCl (results not shown) This material gave rise to a single band in SDS-PAGE (Fig. 2, lane 7). The cDNA and protein sequences of the putative pre-pro-region of canditropsinogen (Ctg) and the relevant structures in the construction of the expression vector. The experimental methods for constructing theexpression vector have been described under “Experimental Procedures.”

CaZtropsinogen Gene

Hg acetate

The assumption that canditropsin belongs to the aspartic

Methods