Optimization of a protein extraction technique for fungal proteomics

Bhadauria, Vijai; You-liang, Peng

doi:10.1007/s12088-010-0072-3

Cited by 7 publications

(11 citation statements)

References 9 publications

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“…[ 17 ]. Here, two types of mechanic cell disruption were compared: beads and grinding in liquid nitrogen, the latter often being used for the extraction of fungal proteins [ 39 – 41 ]. While grinding in liquid nitrogen ensured a better cooling of the sample, cell disruption via beads has a very low personal deviation.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

et al. 2015

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The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

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Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

et al. 2015

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“…Total protein extraction from M. alpina was performed using the Mg/CHAPS protein extraction method [29]. Total fungal protein inhibition was determined by the Coomassie Blue (Bradford) protein assay method [30,31] with bovine serum albumin as a standard.…”

Section: Total Protein Extraction and Inhibition Methodsmentioning

confidence: 99%

Using Mortierella alpina as a Novel Platform for Lipid Synthesis Inhibitor Screening

Azam¹,

Chen³

et al. 2015

ABB

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The screening method for discovering new lipid synthesis inhibitors has not been well developed. Lipid synthesis genes are responsible for the synthesis of fatty acids in normal and tumor cells. Mortierella alpina(M. alpina) fungus has been found to produce large amounts of fatty acids because it possesses a full complement of lipid synthesis genes. We therefore hypothesized that M. alpina could be a good screening tool to find new compounds that inhibit fatty acid synthesis. We developed a M. alpina liquid culture based method to analyze the inhibitory effects of lipid synthesis inhibitors. We applied a color indicator method to monitor the oil production using some well documented lipid inhibitors (C75, Cerulenin) to verify the system, and analyzing pictures using image analysis software. The experimental drug nocodazole inhibited the lipid production in M. alpina almost to the same extent as the control inhibitors. Compared with biomass and protein levels, there was a profound effect on fatty acid level. We report the development of a fast and effective method for screening lipid synthesis inhibitors which can be used against obesity and cancer. This method can further be used to screen additional lipid and fatty acid synthesis inhibitors from natural compound libraries.

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“…For total protein extraction from T. virens mycelia, the methodology described by Bhadauria and Peng (2010) was implemented with modifications. Frozen mycelia (0.5 g) were ground into a fine powder using liquid nitrogen in precooled conditions.…”

Section: Mycelia Protein Extractionmentioning

confidence: 99%

TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes

et al. 2019

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Species of the genus Trichoderma are ubiquitous in the environment and are widely used in agriculture, as biopesticides, and in the industry for the production of plant cell wall-degrading enzymes. Trichoderma represents an important genus of endophytes, and several Trichoderma species have become excellent models for the study of fungal biology and plant-microbe interactions; moreover, are exceptional biotechnological factories for the production of bioactive molecules useful in agriculture and medicine. Next-generation sequencing technology coupled with systematic construction of recombinant DNA molecules provides powerful tools that contribute to the functional analysis of Trichoderma genetics, thus allowing for a better understanding of the underlying factors determining its biology. Here, we present the creation of diverse vectors containing (i) promoter-specific vectors for Trichoderma, (ii) gene deletions (using hygromycin phosphotransferase as selection marker), (iii) protein localization (mCherry and eGFP, which were codon-optimized for Trichoderma), (iv) gene complementation (neomycin phosphotransferase) and (v) overexpression of encoding gene proteins fused to fluorescent markers, by using the Golden Gate cloning technology. Furthermore, we present the design and implementation of a binary vector for Agrobacterium-mediated transformation in Trichoderma to increase the homologous recombination rate and the generation of a novel selection marker based on carboxin resistance.

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Optimization of a protein extraction technique for fungal proteomics

Cited by 7 publications

References 9 publications

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

Using Mortierella alpina as a Novel Platform for Lipid Synthesis Inhibitor Screening

TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes

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