[Yeast] Pichia expression of plant GH43 enzymes

[Faisal A. Chaudhry]

Hello,

  I'm using Pichia pastoris (Invitrogen) to express glycosyl hydrolase enzymes 
from Arabidopsis.  Has anyone expressed something similar?  I'm having 
problems expessing my protein.  I'm using pICZA vector, 25 ml cultures in 
baffled flasks, complex glycerol/methanol media, 4-5 day induction w/ 1% 
methanol.  Various other native Pichia proteins are being expressed, and 
Western blotting (using Sigma H1029 anti poly-His antibody) does not pick up 
any band of appropriate size in pellet or secreted fractions (secreted 
fractions are TCA precipitated), ut works well for a bacterially expressed 
recombinant C-term His tag protein. Control human serum albumin secreted 
beautifully, so I can not say my technique is incorrect.  Does anyone have any 
suggestions.  I'm willing to give more detailed information if requested.  
Thank you!


Faisal Chaudhry