An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass
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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.
KeywordsFungi Homogenizer Biomass Protein Zirconium beads
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.
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