Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Borrelli G, Trono D (2015) Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int J Mol Sci 16(9):20774–20840

    Article  CAS  Google Scholar 

  • Calderone RA, Fonzi WA (2001) Virulence factors of Candida albicans. Trends Microbiol 9(7):327–335

    Article  CAS  Google Scholar 

  • Chin V, Lee T, Rusliza B, Chong P (2016) Dissecting Candida albicans infection from the perspective of C. albicans virulence and omics approaches on host–pathogen interaction: a Review. Int J Mol Sci 17 (10):1643

  • Dadar M, Tiwari R, Karthik K, Chakraborty S, Shahali Y, Dhama K (2018) Candida albicans-Biology, molecular characterization, pathogenicity, and advances in diagnosis and control–An update. Microbial pathogenesis

  • de Groot NS, Ventura S (2006) Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 580(27):6471–6476. https://doi.org/10.1016/j.febslet.2006.10.071

    Article  CAS  PubMed  Google Scholar 

  • Dharmatti R, Miyatake H, Zhang C, Ren X, Yumoto A, Kiga D, Yamamura M, Ito Y (2018) Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ). Protein Expres Purif 149:17–22

    Article  CAS  Google Scholar 

  • Fekete-Forgács K, Gyüre L, Lenkey B (2000) Changes of virulence factors accompanying the phenomenon of induced fluconazole resistance in Candida albicans. Mycoses 43(7–8):273–279

    Article  Google Scholar 

  • Ghannoum MA (2000) Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbiol Rev 13(1):122–143

    Article  CAS  Google Scholar 

  • Gunsalus KT, Tornberg-Belanger SN, Matthan NR, Lichtenstein AH, Kumamoto CA (2016) Manipulation of host diet to reduce gastrointestinal colonization by the opportunistic pathogen Candida albicans. mSphere 1 (1):e00020–00015

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557–580

    Article  CAS  Google Scholar 

  • Hao Y, Huang X, Mei X, Li R, Zhai Z, Yin S, Huang Y, Luo Y (2008) Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli. Protein Expres Purif 60(2):221–224

    Article  CAS  Google Scholar 

  • Hoover CI, Jantapour MJ, Newport G, Agabian N, Fisher SJ (1998) Cloning and regulated expression of the Candida albicans phospholipase B (PLB1) gene. FEMS Microbiol Lett 167(2):163–169

    Article  CAS  Google Scholar 

  • Huth JR, Weijun F, Ruddon RW (1994) Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin. Biotechnol Bioeng 44(1):66–72

    Article  CAS  Google Scholar 

  • Janson J-C (2012) Protein purification: principles, high resolution methods, and applications, vol 151. John Wiley & Sons

  • Kim J, Sudbery P (2011) Candida albicans, a major human fungal pathogen. J Microbiol 49(2):171

    Article  Google Scholar 

  • Leidich SD, Ibrahim AS, Fu Y, Koul A, Jessup C, Vitullo J, Fonzi W, Mirbod F, Nakashima S, Nozawa Y (1998) Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans. J Biol Chem 273(40):26078–26086

    Article  CAS  Google Scholar 

  • Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60(3):512–538

    Article  CAS  Google Scholar 

  • Mirbod F, Banno Y, Ghannoum MA, Ibrahim AS, Nakashima S, Kitajima Y, Cole GT, Nozawa Y (1995) Purification and characterization of lysophospholipase-transacylase (h-LPTA) from a highly virulent strain of Candida albicans. BBA-Lipids Lipid Metabolism 1257(2):181–188. https://doi.org/10.1016/0005-2760(95)00072-k

    Article  PubMed  Google Scholar 

  • Mukherjee PK, Seshan K, Leidich S, Chandra J, Cole GT, Ghannoum MA (2001) Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo. Microbiology 147(9):2585–2597

    Article  CAS  Google Scholar 

  • Park J, Eom GT, Young OhJ, Hyun Park J, Kim SC, Kwang Song J, Hoon Ahn J (2020) High-level production of bacteriotoxic phospholipase A1 in bacterial host pseudomonas fluorescens via ABC transporter-mediated secretion and inducible expression. Microorganisms 8(2):239

    Article  CAS  Google Scholar 

  • Pierce CG, Lopez-Ribot JL (2013) Candidiasis drug discovery and development: new approaches targeting virulence for discovering and identifying new drugs. Expert Opin Drug Dis 8(9):1117–1126

    Article  CAS  Google Scholar 

  • Price MF, Wilkinson ID, Gentry LO (1982) Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia: J Med Veterinary Mycol 20 (1):7–14

  • Songer JG (1997) Bacterial phospholipases and their role in virulence. Trends Microbiol 5(4):156–161

    Article  CAS  Google Scholar 

  • Tian L, Kong WF, Pan QH, Zhan JC, Wen PF, Chen JY, Wan SB, Huang WD (2006) Expression of the chalcone synthase gene from grape and preparation of an anti-CHS antibody. Protein Expres Purif 50(2):223–228

    Article  CAS  Google Scholar 

  • Wang H, Dai J, Li B, Fan K, Peng L, Zhang D, Cao Z, Qian W, Wang H, Zhao J (2008) Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A. Protein Expres Purif 58(1):140–147

    Article  CAS  Google Scholar 

  • Williams DW, Jordan R, Wei X-Q, Alves CT, Wise MP, Wilson MJ, Lewis M (2013) Interactions of Candida albicans with host epithelial surfaces. J Oral Microbiol 5 (22434.10):3402

  • Yang H, Roberts MF (2002) Cloning, overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D. Protein Sci 11(12):2958–2968

    Article  CAS  Google Scholar 

  • Ying S, Chunyang L (2012) Correlation between phospholipase of Candida albicans and resistance to fluconazole. Mycoses 55(1):50–55

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the Sanya Medical and Health Technology Innovation Project (2017YW15). We thank Professor Yanling Hao (China Agricultural University) for helpful comments and revisions.

Author information

Author notes

  1. *Zigang Zhao, Yaguang Zhou and, Rui Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology, Hainan Hospital of PLA General Hospital, Sanya, Hainan, China

    Zigang Zhao & Yaguang Zhou

  2. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China

    Zhengyuan Zhai

  3. Department of Dermatology, The First Medical Center PLA General Hospital, Beijing, China

    Rui Wang & Fang Xie

Authors
  1. Zigang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yaguang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fang Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhengyuan Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization was contributed by ZGZ, YGZ, and ZYZ; Methodology was contributed by RW and FX; Formal analysis and investigation were contributed by ZGZ, YGZ, and RW; Writing—original draft preparation, was contributed by ZGZ and ZYZ; Writing—review and editing, was contributed by all authors.

Corresponding author

Correspondence to Zigang Zhao.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Ethics approval and consent to participate

Not applicable.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, Z., Zhou, Y., Wang, R. et al. Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli. 3 Biotech 10, 538 (2020). https://doi.org/10.1007/s13205-020-02515-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-020-02515-8

Keywords

Search

Navigation