Hi,
Do anyone has experience in Fab expression in E coli that ended up with abnormally large pellet (~14g/L, wet weight; and smelly too) which eventually gave low yield (~0.1mg/mL).
My Fabs are encoded on phagemid vector pCES1 and has Amp resistance. They are transformed into BL21(DE3) cells and are grown in 2xYT + 100ug/mL Carbenicillin + 0.1%glucose and induced with 1mM IPTG at 20C for ~16hr.
There was one occasion I got small pellet (est ~4g/L wet wt) and reasonably high yield (>4mg/L). When I try to reproduce it, it failed. Anyone has any clue what might be the reason?
Cheers,
Ted
Hello Ted,
My first question would be the simplest, did you codon optimized for E. coli prior to the expression?
Good questions above. Also do you have ompA or PelB periplasmic leaders on the chains?
@Jennifer - Thanks for your response.
>> did you codon optimized for E. coli prior to the expression?
The answer is no. Indeed, the Fabs are from human, and we should have done this. (Meanwhile, I am trying Rosetta cells this week and waiting to see what happens.)
However, previously the yields were mostly fine (typically ~1mg/L) and I once got 4mg/L. So my guess is that there is something beyond the sequence that affects yield.
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