Toxin induction and protein extraction from Fusarium spp. cultures for proteomic studies

Matias Pasquali, Frédéric Giraud, Jean Paul Lasserre, Sebastien Planchon, Lucien Hoffmann, Torsten Bohn, Jenny Renaut
JoVE. 2010-02-16; (36):
DOI: 10.3791/1690

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1. J Vis Exp. 2010 Feb 16;(36). pii: 1690. doi: 10.3791/1690.

Toxin induction and protein extraction from Fusarium spp. cultures for proteomic
studies.

Pasquali M(1), Giraud F, Lasserre JP, Planchon S, Hoffmann L, Bohn T, Renaut J.

Author information:
(1)Department of Environment and Agro-Biotechnologies, Nutrition and Toxicology
Unit, Centre de Recherche Public-Gabriel Lippmann.

Fusaria are filamentous fungi able to produce different toxins. Fusarium
mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid,
moniliformin, etc… have adverse effects on both human and animal health and
some are considered as pathogenicity factors. Proteomic studies showed to be
effective for deciphering toxin production mechanisms (Taylor et al., 2008) as
well as for identifying potential pathogenic factors (Paper et al., 2007,
Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish
reliable methods for comparing between proteomic studies in order to rely on true
differences found in protein expression among experiments, strains and
laboratories. The procedure that will be described should contribute to an
increased level of standardization of proteomic procedures by two ways. The
filmed protocol is used to increase the level of details that can be described
precisely. Moreover, the availability of standardized procedures to process
biological replicates should guarantee a higher robustness of data, taking into
account also the human factor within the technical reproducibility of the
extraction procedure. The protocol described requires 16 days for its completion:
fourteen days for cultures and two days for protein extraction (figure 1).
Briefly, Fusarium strains are grown on solid media for 4 days; they are then
manually fragmented and transferred into a modified toxin inducing media (Jiao et
al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth
layer. Grinding is performed in a cold chamber. Different operators performed
extraction replicates (n=3) in order to take into account the bias due to
technical variations (figure 2). Extraction was based on a SDS/DTT buffer as
described in Taylor et al. (2008) with slight modifications. Total protein
extraction required a precipitation process of the proteins using Aceton/TCA/DTT
buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally
resolubilized in the protein-labelling buffer and quantified. Results of the
extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to
2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses
on other growing media and other filamentous fungi (Miles et al., 2007).

DOI: 10.3791/1690
PMCID: PMC3152220
PMID: 20160676 [Indexed for MEDLINE]

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