I currently plan to use pichia to do heterologous expression of a gene from white rot fungi.
Unfortunatelt, I only have the gDNA sequence of the gene. Can I use it for ligation to pPICZ alpha and more importantly, will it be efficiently spliced once the vector is transformed to pichia?
Anyone with experience of using gDNA to express extracellular protein in pichia system? Or is the use of cDNA mandatory?
Any suggestion or opinion will be welcomed. Thanks! :)
In my experience, if your gDNA does not contain a lot of introns then Pichia can splice out - like in ascomycetes; however in your case it is the basidiomycetes I think Pichia will not be able to process those introns (may be you can check first how many introns your white rot has).
Thx All :) As further info for discussion, I have the predicted cdna sequence of the gene (from genome sequencing result of the fungi). When aligned to the gdna product, it seems like there are 7 intronic sequences all with GT-AG splice site.
My problem is that I cant get the cdna product, only gdna product is available.
Any further suggestions and thoughts are appreciated :)
The laborious way would be to design the intron-splicing primers or overlapping PCR (I am not sure how it is called properly), but you may need to do it one at a time (as I guess some extrons may be quite short as well).
I am not sure about other options, but you can also synthesize the gene - it is not so expensive anymore.
I am not sure about about genetic manipulation of your white rot fungus. Would it be possible to put your gDNA on a multi-copy plasmid or a plasmid which is driven by a strong promotor? If yes, you may overexpress the gDNA including introns in the fungus and let it generate the spliced mRNA in vivo. Using the fungal mRNA you will be able to generate the cDNA in vitro.
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