I am working on fungal lectins. I am required to purify a cytoplasmic lectin from a opportunistic fungal pathogen species. The yield has been very low in the past few months. I want to know if there is any molecule/treatment that can be given to the fungus to induce the synthesis of the native protein?
Hi Suhas
To obtain large amounts of protein it is usually necessary that the gene be expressed at a high level. Your gene may be expressed constitutively at low or high level or it may be induced under specific conditions. To find out this information you would need to study the gene expression by northern blot or RT-PCR under a range of conditions using a comparitor gene with known expression level (eg beta-tubulin, actin and so forth). If you find that your gene is induced under particular conditions you can grow your fungus under these conditions and should get more protein, assuming there are no translational controls.
The alternative is to clone your lectin gene so that it expressed under the control of a highly expressing promoter. gpdA or glucoamylase promoters have been shown to work in several fungi. Finally you could clone your gene into a bacterial or yeast expression system - there are many of these available.
good luck
You can use Pichia pastoris expression system to produce large amounts of fungal proteins. As Pichia is an yeast the modification will be similar.
Look for some stress condition because this protein produced during pathogenesis.
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