The Evolution of Azole Resistance in <i>Candida albicans</i> Sterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions

Abstract

Recombinant Candida albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in Escherichia coli and purified by Ni 2+ -nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC 50 ) using CYP51 reconstitution assays.