This is not an uncommon problem with frozen glycerol stocks of Pichia. The clone either expresses less than what it did prior to freezing or does not express at all. It has happened to me and I have no scientific reason to explain it.

The only advise I can give is to keep a plate with the clone struck out on it at 4 degrees and restreak about every month. Be sure to take from the heavy growth on the plate and do not pick a single colony when restreaking. I realize this is a pain but it is better than all the time and energy spent making/finding another high expressing clone.

thanks a Lot for your contribution Tom Masi Tom , I've also consulted the troubleshooting service by Invitrogen they told me that one inside the vector after a deep freeze vector can spit off during the growth, remaining resitant to zeocin bt non expressing the protein of interest at all !! Alex

Creating glycerol stocks of Pichia pastoris is a common method for long-term storage of the yeast strain. Here's a general outline of the procedure:

Materials Needed:

Procedure:

Notes:

• When retrieving a glycerol stock for use, it's best to avoid repeated freeze-thaw cycles. Instead, aliquot smaller portions for single-use and discard any unused portion to prevent contamination.
• It's good practice to periodically transfer glycerol stocks to fresh vials to maintain the viability of the strain.
• Ensure proper biosafety practices and compliance with institutional guidelines when handling microbial cultures.

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Thanks Kais to your kind help the problem is to awaken the cell, all the manuals say that the glycerol stocks could be stored for several years, but it's not like they said! For an undetermined scientific reason Pichia cell strain remains resistant to the selective antibiotic, and therefore, but, not capable of producing your recombined protein anymore.

The efficiency of vector integration into the P. pastoris genome might be low, leading to a reduced number of cells expressing the recombinant protein. Perform PCR to confirm the integration of the vector into the P. pastoris genome.

Tha Abigail, but it was all ok before the preparation of the glycerol stocks , the protein was well detected, the same regarding the pcr, amplicons. The worse happened when we decided to awaken the clones , after the 4 years of deep freezing. The clones are correctly selected by zeocin in ypd medium, but the cloned protein wasn’t detect as well as the pcr amplicon.

If the cloned protein wasn't detected, it can be as a results of improper storage conditions affecting vector integrity or protein stability over time.

This was strange becacuse it was all properly detected before the storage zeocine, resistance in the vector detected was present at anytime that’ s why I asked to people…it was so weird.