Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli.

Abstract

Candida albicans is an important fungal pathogen that causes a wide variety of human infections, ranging from mucocutaneous infections to life-threatening systemic infections. Phospholipase B1 (PLB1) has been reported to be directly responsible for C. albicans pathogenicity and is likely to be involved in the early steps of host invasion. Therefore, PLB1 could be a potential marker for diagnosis of C. albicans infection. In this study, PLB1 was expressed using an Escherichia coli expression system. Recombinant PLB1 is found in inclusion bodies and constitutes up to 38.4% of total insoluble protein. After refolding in a GSH/GSSG redox system, GST-tagged PLB1 was purified by GST-sepharose 4B affinity chromatography and then cleaved with thrombin to remove the GST-tag. The recombinant PLB1 was further purified by anion-exchange chromatography and reverse phase HPLC. The final yield of purified PLB1 was approximately 15.6mg from 100mL of bacterial cell culture, and its concentration was 784μg/μL. The recombinant PLB1 could form a white precipitation zone on egg yolk agar plate, suggesting its phospholipase activity. Moreover, the maximum activity of PLB1 was 68IU/mg at pH 6.0, 37°C. Therefore, recombinant PLB1 has potential application in structural analytical studies, or diagnosis of C. albicans infection.