Hello! I am in the process of building a protein library using error-prone PCR (epPCR) for directed evolution purposes. However, after assembly of the mutated gene into the pET backbone, I do not see any protein expression. Sequencing does not show any mutation in the promoter and terminator region of the gene. I tried already two different assembly strategies (PCR-based assembly and restriction/ligation) but the result is the same. At the same time, my positive control is highly expressed and activity is nicely observable in the same conditions. The positive control is the unmutated protein whose gene is cloned in the same pET backbone as my library. Do you have any advice on how to fix this issue?
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