I'm screening a random mutated protein to improve its expression and solubility level in E. coli.
So my idea of screening is, fusing it up-stream of RFP or GFP CDS, and by detecting the intensity of color of each colony, I could expect the expression and solubility level.
Now, I'm thinking this way can be work in case of expression, (if it expressed efficiently, subsequently the RFP will expressed and the intensity of color will increase) but I'm not sure what will happen if my protein was insoluble, is it will be affected with presence of RFP to be soluble (and give me false positive) or it will lead to insoluble RFP also and the results will be (negative)?
Mohamed: protein overexpression in the bacterial system is both a fundamental and a practical problem for researchers, especially when the protein is of a mammalian origin. In addition, GFP itself is a difficult one for solubility in the E. coli, making it unfit for a routine screening. Prior to mutagenesis or fusion with GFP of your protein, I'd suggest to use different signal peptide(s) for a better folding and hopefully secretion, or at least not deposited in the inclusion body. Good luck.
Thanks Zhao for your answer, what do you think about LacZa instead of GFP :)
LacZ is no different from GFP. You need to produce your protein successfully by itself first, then put a reporter protein like GFP for mutagenesis work.
You may want to try the Intein expression system. We have had very good luck with expression in E.coli. However, you would want to make both an N-and C-terminal construct because, for unknown reasons, one will often express better than the other. Also, on column cleavage of native protein is simple.
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