Hello, I am currently trying to express the ECD of a transmembrane protein in E.coli. It has multiple disulphide bonds so I co-express it with DsbC and I use a Rosetta-Gami cell line. I was able to get this to work a few times on a small scale, the expression was terrible but western blots confirmed it was there.
I then tried moving to large scale and got complete protein degradation, something I hadn't seen in the small scale experiments. (This was by inducing with 0.4 mM IPTG). I tried autoinduction to make my life easier which I only managed to succeed once in expressing but was generally a failure.
I have made new competent cells from the rosetta-gami and I do a fresh transformation every time before transforming E.coli. The DNA is sequence verified as well. I should also note sometimes the expression fails so completely that not even DsbC is expressed, which usually expresses very highly. Does anyone have any suggestions?
One possibility is that your strain is unstable due to the toxicity of your protein and to leakage of expression in the absence of inducer. Normally, I would recommend to use a strain like BL21AI (arabinose-inducible) that allows a tighter control of expression, but since, to my knowledge, it is not available in the origami/DsbC version, I guess that your best bet would be to grow your preculture in the presence of glucose, leaving out the lactose, then washing the cells to get rid of the glucose and proceeding with induction of the washed cells. This may be preferable to auto-induction, in which lactose is present from the beginning, which may lead to some leakage and segregation of low-producers
hey.. you can troubleshoot as sebastian said. I agree with it and later on you should try slow induction of your protein expression.
good luck
The efficiency of expression at large scale usually decrease. Try expression at low temperatures.
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