I would like to share my ideas about expression of extracellular proteins in P. pastoris just to know if you think the same.
As far as I am concerned, it is not so easy that a heterologous protein would be able to be secreted by the secretion machinery of P. pastoris. Even if you use the well-known pre-pro alpha factor of commercial vectors such as pPICZalpha and pPIC9alpha. Sometimes it is necessary to maintain the leader peptide of the heterologous protein downstream the alpha factor, sometimes it is not necessary. And even more difficult, in case your heterologous protein is a pre-pro-protein.
It is very frequent that your protein stays locked in some place in a middle way like ER or Golgi or membrane vesicles. In cases you do not find expression of your heterologous protein in Pichia, I would recommend to perform a western blot to the Pichia pellets and boalá!!, you can find your protein there. Thus, it would not be an expression problem, it would be a proper secretion problem.
Could you share your experience in this regard? thank you very much!!
