I express a recombinant proteins in E. coli (wild type and mutant), the mutant one is much less expressed than the wild type and the culture is slower (time to attain DO 600 of 0.6 before to add IPTG) and I do not have enough quantity. The sequences are ok in both cases. I would like to know ho w to meliorate this protein expression.
Protein expression is an energy dependent process. When expressing exogenous proteins it is an added burden to the host cell and may lead to a decrease in metabolic rate as the new protein requires cellular resources to be produced. Some exogenous proteins are also toxic or inhibitory to certain cell lines, which may also affect the metabolic rate. Your best option for increase protein production would be to grow up larger volumes of culture for extraction, as trying to increase the growth rate my lead to poor quality protein production (inclusions and incorrect folding).
Thanks for answer. My protein, a phusion with GST, shows similar expression in both (wt and mutatnt), but after digestion with Prescission , the expression in mutantt (I tried 3 different clones) is really diminished, It could be also a problem of selective degradation, instability?
It sounds to me like you're not talking about "expression" (which you say is the same as wt), but about "yield" after cleavage. Either it is lost in extraction, or it is not being captured on the column, or it is not being cut. You need to do some troubleshooting to find out where the problem actually occurs.
It seems that either your recombinant protein is toxic to the host cell or host cell is not supporting expression. The post-translational changes may also affect the stability of host cell which lead to the loss of protein.
If you elaborate your question more hope you will get the answer.
Good luck
Generally expression with GST at its N-terminal (is it, right?) give high yelds and it's all fine asl long as your protein is fused with it. When you cleave out the GST, is possible that the mutant protein is not soluble any more. In this case, you would see a pellet if you centrifuge for long enough (say, 10 minutes at max speed of your tabletop centrifuge). If you could tell us also what is your purification strategy could be of help.
Thanks for answer. I send a picture showing the different expression of my prot (Wild type, mutant and control GST) that explains better the trouble.
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