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Solubilization and purification of phosphatidylserine synthase from Candida albicans

Systemic infections by Candida spp. are associated with high mortality rates, and this is related, in part, to limitations in current antifungals. In this regard, the phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals. The reasons include: 1) Cho1 is required for oral and systemic Candida infection in the mouse model and is essential for viability in the fungal pathogen Cryptococcus neoformans; 2) this gene and gene product is absent in humans, and 3) it is a highly conserved protein among many human fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Small molecule screening is one way to develop specific inhibitors. This requires protein purification of the drug target. Due to the transmembrane nature of Cho1, therefore, we aim to solubilize and purify the native form of the Cho1 protein. Initially, four nonionic detergents (Digitonin, n‐Dodecyl‐b‐D‐ Maltopyranoside [DDM], n‐Tetradecyl‐b‐D‐Maltopyranoside [TDM], and Triton X‐100) were used to solubilize an HA‐tagged Cho1 protein from the total microsomal fraction. Analysis by Blue Native PAGE (BN‐PAGE) and immunoblotting revealed a single band corresponding to Cho1 protein in these four solubilized fractions. A radiochemical enzymatic assay suggests that the digitonin‐ and DDM‐solubilized fractions have more PS synthase activity than the TDM‐ and Triton X‐100‐ solubilized fractions. Using a pulldown assay for the HA‐tagged Cho1 in the digitonin‐solubilized fraction reveals a single band in BN‐PAGE, and a subsequent 2D gel electrophoresis confirms the purity of this protein following silver staining. The comparative mobility by BN‐PAGE gives an apparent MW of Cho1 of ~178 kDa. Based on the amino acid sequence, this would suggest a hexameric form of the CandidaCho1. This is quite different from the structure of other membrane phospholipid synthases, which all appear to be dimers. Preliminary negative staining and TEM single particle analysis suggests the presence of a hexamer population of Cho1 complex in the pulldown sample. The hexametric Cho1 is predicted to be assembled as a trimer of dimers. As expected, a subsequent 2D particle classification on the TEM images has shown different assemblies of Cho1 dimers, mostly trimers of dimers, strengthening the claim that Cho1 forms native trimers of dimers.