We just want to express one hetero-protein. No matter how we change the parameter of different factors, but it is likely not really work. And we also increased the zeocin concentration for selection the high copy strains, but it does also not work well.
Have you checked the intracellular fraction too? Trafficking through the secretory pathway seems to be the rate-limiting step when expressing secreted proteins in Pichia, not transcription/translation (see e.g. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3369916&tool=pmcentrez&rendertype=abstract). If you find that a significant fraction of the protein remains cell-bound, you might try other alternatives (different signal peptides, eventually ditching the alfa-factor propeptide, co-expression of secretion-enhancing factors and so forth)
By the way, if the target protein is problematic and you are getting it properly folded with all needed PTMs, 5 mg/L is not that bad...
@Alejandro Martin.Thank you very much，Alejandro Martin. It's exceeding helpful for me. And I want to change another vector, do you have any suggestion for me, sir. And my protein is just 10kDa molecular weight and own five disulfide bonds. Meanwhile, just wish you can respond.
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