Related Experiment Video
Updated: Nov 29, 2025
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli
Zigang Zhao1, Yaguang Zhou1, Rui Wang2
1Department of Dermatology, Hainan Hospital of PLA General Hospital, Sanya, Hainan China.
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 15.6 mg from 100 mL of bacterial cell culture, and its concentration was 784 μg/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 68 IU/mg at pH 6.0, 37 °C. Therefore, recombinant PLB1 has potential application in structural analytical studies, or diagnosis of C. albicans infection.
Insights
Researchers successfully produced recombinant Phospholipase B1 (PLB1) from Candida albicans, a key fungal pathogen. This purified PLB1 enzyme shows phospholipase activity and has potential for diagnosing C. albicans infections.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Candida albicans is a significant fungal pathogen causing diverse human infections.
- Phospholipase B1 (PLB1) is implicated in C. albicans pathogenicity and host cell invasion.
- PLB1 is a potential diagnostic marker for C. albicans infections.
Purpose of the Study:
- To express and purify recombinant PLB1 from C. albicans.
- To characterize the enzymatic activity of the recombinant PLB1.
- To assess the potential applications of recombinant PLB1 in diagnostics and structural studies.
Main Methods:
- Expression of PLB1 in an Escherichia coli system.
- Purification using GST-sepharose 4B affinity chromatography, thrombin cleavage, anion-exchange chromatography, and reverse-phase HPLC.
- Assessment of phospholipase activity on egg yolk agar and determination of optimal activity conditions (pH, temperature).
Main Results:
- Recombinant PLB1 was successfully expressed and purified with a yield of approximately 15.6 mg from 100 mL of bacterial culture.
- Purified PLB1 exhibited phospholipase activity, indicated by a precipitation zone on egg yolk agar.
- Optimal activity of PLB1 was observed at pH 6.0 and 37°C, with a specific activity of 68 IU/mg.
Conclusions:
- Recombinant PLB1 was successfully produced and purified.
- The purified enzyme possesses functional phospholipase activity.
- Recombinant PLB1 holds promise for structural analysis and as a diagnostic tool for C. albicans infections.
