Posted 10 March 2005 - 02:32 AM
Hi to all,
I´ve now read this thread because I also looked for advice to express a protein in Pichia. Thanks to those who told me something new.
Now I have some comments to this theme:
1. Read the literature and be sure to understand what´s written there. The Invitrogen manuals are the first and most important source of information. Very well but expensive is also Methods in Molecular Biology vol. 103 ("Pichia Protocols"), edited by D.R.Higgins and J.M.Clegg. The latter is somewhat of a Pichia Godfather, so look for other papers.
2. It is absolutely mandatory to verify the correct integration of your (linarized) gene into the Pichia genome! When U linearize in the AOX1-promoter site, the gene is recombinated into the Pichia AOX promoter site. Hence there is only one on every allele, maximum will be a "double transformant". Do a PCR with genomic DNA from your transformants, and when you see only a long band (depends on your gene), then you have a good clone: a "homozygous double transformant". This is what you want!
Two bands mean integration just on only one chromosome, and a small band means resistance to zeocin, but no integration. These clones will show little or no expression, friends!
3. I use pPICZB and pPICZalphaB to integrate into the X-33 wildtype strain. I did transformation by the LiCl-PEG-heat shock-method. That gives no maximum efficiency, but one cannot handle more than 20-40 clones at a time, right? The advantage is that you use the competent cells immediately after preparing them. Be also sure to choose the right enzyme to linearize; I´ve used PmeI.
I have not tried other vectors than pPICZ and other integration strategies yet, but multicopy integration (G418 or HIS selection, I´m not sure) is obviously much better.
4. Secretion (by using the pPICZalpha vectors) is favorable because of much easier purification. BUT: the protein can also become oxidized because of contact to air during shaking/fermentation. Any affinity tag is helpful to isolate your protein. If necessary, cleave it with a protease during purification.
5. Fermentation is favorable because of better growth control. In my eyes, the most important parameters are continuous methanol feeding (instead of addition every 12/24 hrs), excessive aeration and better temperature control. This is what my experience shows.
Good luck with expression!