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pichia expression - (Jul/23/2008 )

hi all
i am a student working on yeast expression as a part of my research. i am trying to express a 30Kda protein in pichia pastoris. i ve used pPICZ alpha A vector. i ve got successfully few clones in GS115host. i checked those clones for integration by performing PCR with AOX primers. i ve got the desired bands still i am unable to get expression after inducing my clones. what may be the reason? can anybody help me?
have a good day


[quote name='vinita' date='Jul 23 2008, 06:00 AM' post='144863']

Some questions:

Hello vinita, Did you sequencing your construction to be sure the does not have changes in the ORF?

Did you check if the protein is inside the cell instead the supernatant???

How Did you check for the protein presence?


ya i did check my insert by DNA sequencing. it is in correct frame and orientation. i checked both supernatant and lysate by SDS-PAGE analysis.