I am working on protein biopharmaceuticals, we have engineered a novel antibodies (4 variants) and take a service from the company to procure our gene of interest in pPIC9k vector. Before confirming the vector we have verified everything regarding our construct and it was all good according to us. After receiving the genes, I proceeded to make its copies and transformed it in to the E. coli DH5a, it was successful as we cross check it with the double digestion and dropout bands were at expected size. Then I further proceeded for P. pastoris transformation. Following steps I have done;

1. Competent cell were prepared with Electroporation Transformation protocol which is given in this article. (Article Transformation

2. Then, I linearized the plasmids with two enzymes as we opted for two restrictions site. ( Variant 1 & 2 have SacI site and Variant 3 & 4 have BglII site)

3. Electroporation was done by using Eporator (Eppendorf Electroporator) at 1700 V. In this we do not have option to set other parameters.

4. Immediately after the electric shock I added 1M sorbitol then incubate it for 2 hours with no shaking then incubate it for recovery.

5. After this I pellet down the cells and thrown the 400 ul sorbitol and again resuspend the cells in remaining media. And then I plated this on MD plate (200 ul).

6. After incubating it at 30 Deg for 4-5 days, we were sometimes able to get colonies and sometimes no colonies were their.

7. The colonies which we get was false positive and were not able to express our desired protein. (Confirmed with SDS & WB)

I have tried multiple protocols (Condensed protocol, used G-Bioscience competent cell preparation kit) and change electroporators also. I also changed my P. Pastoris cells, tried with new cells also, I tried with GS115 cells also, tried with different DTT as well as its concentrations, I tried with different OD from 0.8 to 1.5.

The thing is that from 4 of the variants, I only able to express one variant which was at initial phase only. Also, we have some other molecules (proteins) which we have already expressed it from the same system with the same protocol. Now I am keeping them as my positive control. But now that too have shown the same problem.

Kindly if anyone have any clue regarding this please suggest me. Any small suggestion can make very huge contribution.