I am working in Pichia pastoris. I have reached till the expression.
I am using pPIC9 plasmid vector for my protein expression.
For expression, the clones are selected based on the high resistant to G418 and as per the literature high resistant means high copy number. I have gone upto 5mg/ml G418. And we have performed plate assay also to check for its activity , it shows positive.
Then i have taken those clones which are positive in assay for expression
i have checked the expression in YPD, BMMY media , now trying with YNB media . and also at different pH 5 and 6.
There is no expression, confirmed with western blot too.
i have even tried by adding protease inhibitor ..its not working
parameters for expression used are
incubated at 30 degree at 250 rpm.
so what you suggest me to do next to solve my problem?
Please do reply for my question
i will be thanfull to you if you can solve my problem.