A bit more information would be helpful. How are you measuring expression (i.e. SDS-PAGE, Western blotting, ELISA...)? Are you only examining samples from culture supernatants, or also trying cellular extracts? If you're looking at culture supernatants, are you concentrating the samples before the assay? Are you doing a time-course experiment or examining only a single time point? What culture medium are you using? How are you inducing the AOX promoter?

In my experience there is no size rule when it comes to heterologous expression Some large proteins behave well no matter what, some small ones are always troublesome. Invitrogen does provide a GS115 derivative expressing B-gal (from E. coli, I think) intracellularly; that's quite a large molecule (116 kDa).

Dear Sir,

Alejandro Martin.

Thanks for editing my question and nice see your valuable suggestion.

We are measuring expression using SDS page only.

We are doing enzyme analysis from culture supernatent without concentrating it and also lysing cells with benazlkonium chloride and lyticase enzyme .

We are running our experimental flasks upto 280 hours to check maximum expression level.

We are using BGY (peptone 2%, glycerol 1%, yeast extract 1%) medium for pichia pastoris growth and replacing medium with (peptone 2%, yeast extract1%) for production of recombinant enzyme.

We are inducing AOX1 promoter by adding 0.5% v/v of 100% methanol twice a day.

Moreover as you said about size rule,

B-gal (3021bp) and Glucose Oxidase (1818bp) both the genes having significant size difference.

And we are not getting expression of both the genes in pichia pastoris.

Now, i will look to check their expression in pichia pastoris GS115 strain.

your early reply will be appreciated.

yours sincerely.

Imtiyaz

SDS-PAGE may not be sensitive enough. You won't see anything in culture supernatants unless expression is good and/or you concentrate your samples, and you won't see a thing in cell extracts in either case. Also, are there any potential N-glycosylation sites in your molecule? Glycosylation makes it harder to spot your protein in SDS-PAGE, as it no longer migrates as a sharp band, so you might want to pre-treat your samples with something like PNGase F or EndoH (both available from NEB).

Enzyme assays are excellent, but will work only if your protein is functional (unlikely if it is trapped along the secretory pathway due to insufficient processing, misfolding, etc). If a suitable antibody were available, I'd try Western blotting some supernatant and cell extract samples to see what is going on.

Did you by any chance optimized the codon usage from the A. niger gene for the codon usage in Pichia pastoris?