Heterologous expression and purification of recombinant cryptococcal CYP51 proteins.

The pCWori⁺:CYP51 constructs were transformed into competent Escherichia coli DH5α cells and expressed as previously described (21). Recombinant CYP51 proteins were isolated according to the method of Arase et al. (22), except that 2% (wt/vol) sodium cholate was used in the sonication buffer and Tween 20 was omitted. The solubilized CYP51 proteins were purified by affinity chromatography using Ni²⁺-nitrilotriacetic acid (NTA) agarose as previously described (21, 23) prior to characterization. Human CYP51 with a deletion of 60 amino acids from the N terminus (Δ60 truncated human CYP51) was expressed and purified as previously described (24) and was shown to be comparable to the full-length human CYP51 in terms of binding to azole antifungal drugs. Protein purities were assessed by SDS-polyacrylamide gel electrophoresis.