Hey there, currently I am working on threonine synthase enzyme of Leishmania. I have successfully cloned it into E.coli DH5alpha strain. I have also transformed it into E.coli BL21(DE3) strain. I tried induction at 37°C and 25°C with IPTG concentration 0.1mM, 0.5mM and 1mM respectively. At 25°C with IPTG concentration 0.1mM after purification I got very faint band of expected size ~75kDa and it was confirmed by western blot analysis too. But the problem is that yield is very low. How I can maximize the yield? How the recombinant protein expression is increased in E.coli BL21(DE3) strain? Can anyone suggest paper on this ?
Getting no or low expression is very common. If you do a search here on ResearchGate you will likely find hundreds of others asking the same question.
What expression vector you using? Is it appropriate for BL21(DE3)?
Did you sequence verify your clone to be sure it is entirely correct?
Have you retransformed into fresh competent cells and tried testing a few independent transformants? Sometimes you have a transforming that doesn't express well for unclear reasons.
Precise answered by Michael J Benedik.
In my opinion basic step is to check your E.coli efficiency.
Than definitely depends upon your vector type.
Try above Hope it's work.
Hi there,
I would first check if it is a solubility issue (high expression but low solubility). How was it at 37°C? Does IPTG concentration matter or not?
The more obvious things to consider:
lower temperature during induction (we do 15°C overnight in the lab)
coexpression of chaperonins in case solubility is the issue
try auto-inducible medium from Studier
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