Pichia expression

Francisco P. Chavez Francisco.Alea at cigb.edu.cu
Sat Jun 14 13:11:38 EST 1997


Hi all.
We are trying to express a cellobiose dehydrogenase gene from
Phanerochaeto chrysosporium fungus (codifying for a hemoflavoenzyme
that binds FAD) in  Pichia pastoris.
We have suscesfully cloned the gen into the Invitrogen pPIC3K and
pHILS1 vectors, obtaining some Muts and Mut+ transformants, also we
have got transcripts at the expected size, but we failed to detect
neither enzymatic activity nor polyclonal antibody recognition by
western blotting.
Do you have any experience (positive or negative) on the expression
of proteins bearing a heme domain? Is there any  theoretical
or practical problem with the traduction of such type of gene in
Pichia?

Well any comment or idea woul be very usefull.

Thanks a lot in advance.

Best regards
Francisco Chavez




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