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Indian Journal of Microbiology

, Volume 59, Issue 1, pp 100–104 | Cite as

An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass

  • Akhila Krishnaswamy
  • Natasha Barnes
  • Nikita P. Lotlikar
  • Samir R. Damare
Short Communications
  • 78 Downloads

Abstract

Filamentous fungi are ubiquitous eukaryotes having chitin as a major constituent of the cell wall. Chitin is tough to lyse due to which the intracellular fungal proteins are not readily accessible. The problem is further enhanced when the biomass to be analyzed for protein studies is too little due to the extreme experimental parameters under consideration such as increased or lowered pH, temperature, hydrostatic pressure, nutrients, etc. The method described here is capable of obtaining proteins from minuscule quantities of biomass (~5 mg lyophilized biomass). In this study, different lysing conditions and varied composition of extraction buffers were tried to obtain maximum protein of high quality. Lysis with zirconium beads in a combination buffer system (Tris–MgCl2 buffer, urea buffer I and urea buffer II) was best for extracting proteins from the fungal isolates used. The protocol described here provides for a simple and quick method for extraction of high-quality proteins from very less biomass that could be extended to other tough to lyse biological material also.

Keywords

Fungi Homogenizer Biomass Protein Zirconium beads 

Notes

Acknowledgements

Authors thank Director, CSIR-NIO for the infrastructure and other facilities. The first author is thankful to Council of Scientific and Industrial Research (CSIR) for her stipend under the project YSA 1228. The authors are grateful for the research funds received under the CSIR funded project PSC0206. The authors acknowledge the help of Ms. Shruti Shah for testing the robustness of protocol for basidiomycete fungus. The authors thank anonymous reviewers for suggestions to improve the MS substantially. The work is part of the doctoral thesis to be submitted to Goa University at Department of Microbiology. This is CSIR-NIO Contribution Number 6252.

Contribution of authors

The idea was conceived by the corresponding author during the work on deep-sea fungal cultures, where obtaining biomass from fungi growing at elevated hydrostatic pressure is a challenge. The first author was involved in standardization of the method and drafting of the manuscript, and the work is part of her doctoral thesis to be submitted to Goa University. The second and third authors have validated the method by evaluating it for extraction from different fungal cultures. This is an original work by the authors and has not been published elsewhere either completely, in part, or in any other form. The manuscript has not been submitted to another journal. The results described here have been approved for publication by the responsible authority of the institute where the work was carried out, and all the persons entitled to authorship have been named.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12088_2018_752_MOESM1_ESM.doc (582 kb)
Supplementary material 1 (DOC 581 kb)

References

  1. 1.
    Bridge PD, Kokubun T, Simmonds MSJ (2004) Protein extraction from fungi. Methods in molecular biology. In: Cutler (ed) Protein purification protocols, 2nd edn, vol 244. Humana Press Inc., New York, pp 37–46.  https://doi.org/10.1385/1-59259-655-x:37
  2. 2.
    Lakshman DK, Natarajan SS, Lakshman S, Garrett WM, Dhar AK (2008) Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani. Mycologia 100:867–875.  https://doi.org/10.3852/08-065 CrossRefGoogle Scholar
  3. 3.
    Epperson LE, Martin SL (2011) Proteomic strategies to investigate adaptive processes. In: Eckersall PD, Whitfield PD (ed) Methods in animal proteomics. Wiley, Oxford, pp 189–209.  https://doi.org/10.1002/9780470960660.ch8
  4. 4.
    Leite GM, Magan N, Medina Á (2012) Comparison of different bead-beating RNA extraction strategies: an optimized method for filamentous fungi. J Microb Methods 88:413–418.  https://doi.org/10.1016/j.mimet.2012.01.011 CrossRefGoogle Scholar
  5. 5.
    Gibbons LE, Brangs HCG, Burden DW (2014) Bead beating: a primer. Ran Primers 12:1–20Google Scholar
  6. 6.
    Herran NS, Lopez JLC, Perez JAS, Chisti Y (2008) Effects of ultrasound on the culture of Aspergillus terreus. J Chem Technol Biotechnol 83:593–600.  https://doi.org/10.1002/jctb.1821 CrossRefGoogle Scholar
  7. 7.
    Hopkins TR (1991) Physical and chemical cell disruption for the recovery of intracellular proteins. Bioprocess Technol 12:57–83Google Scholar
  8. 8.
    El-Harakany AA, Halim FMA, Barakat AO (1984) Dissociation constants and related thermodynamic quantities of the protonated acid form of tris-(hydroxymethyl)-aminomethane in mixtures of 2-methoxyethanol and water at different temperatures. J Electroanal Chem 162:285–305.  https://doi.org/10.1016/S0022-0728(84)80171-0 CrossRefGoogle Scholar
  9. 9.
    Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307–316.  https://doi.org/10.1002/elps.1150180303 CrossRefGoogle Scholar
  10. 10.
    Berkelman T, Brubacher MG, Chang H (2004) Important factors influencing protein solubility for 2-D electrophoresis. Bioradiations 114:30–32 (BioRad Laboratories Inc) Google Scholar
  11. 11.
    Banasova M, Valachova K, Juranek I, Soltes L (2014) Dithiols as more effective than monothiols in protecting biomacromolecules from free-radical-mediated damage: in vitro oxidative degradation of high-molar-mass hyaluronan. Chem Papers 68:1428–1434.  https://doi.org/10.2478/s11696-014-0591-1 CrossRefGoogle Scholar
  12. 12.
    Gordon A, Barbut S (1992) Effect of chloride salts on protein extraction and interfacial protein film formation in meat batters. J Sci Food Agric 58:227–238.  https://doi.org/10.1002/jsfa.2740580211 CrossRefGoogle Scholar
  13. 13.
    Heukeshovan J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112.  https://doi.org/10.1002/elps.1150060302 CrossRefGoogle Scholar
  14. 14.
    Osherov N, May GS (1998) Optimization of protein extraction from Aspergillus nidulans for gel electrophoresis. Fun Gen Reports 45:38–40.  https://doi.org/10.4148/1941-4765.1262 CrossRefGoogle Scholar
  15. 15.
    Remelli W (2011) Framing the role of rhodanese-like proteins in cell redox balance in two bacterial model systems. Ph.D. Thesis, University of MilanGoogle Scholar
  16. 16.
    Bhadauria V, Peng YL (2010) Optimization of a protein extraction technique for fungal proteomics. Ind J Microbiol 50:127–131.  https://doi.org/10.1007/s12088-010-0072-3 CrossRefGoogle Scholar
  17. 17.
    Bregar O, Mandelc S, Celar F, Javornik B (2012) Proteome analysis of the plant pathogenic fungus Monilinia laxa showing host specificity. Food Technol Biotechnol 50:326–333Google Scholar

Copyright information

© Association of Microbiologists of India 2018

Authors and Affiliations

  1. 1.Biological Oceanography DivisionCSIR-National Institute of OceanographyDona PaulaIndia
  2. 2.Department of BiologyHong Kong Baptist UniversityKowloon TongHong Kong

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