I have a problem with expression of a mutant protein in E.coli (BL 21 pLysS strain). Recently I could expressed the native and two other mutants from this protein in the same host by pRSET A. Only one amino acid change in each mutant. I don't know why the last mutant (changing aromatic residue to Alanine) is not expressed like the other ones? I could trace the activity of the expressed protein mutant in E. coli, but protein band is not clear as the other ones in SDS-PAGE. it has correct size before transformation to pLysS.
First of all when you say it is not expressed well do you mean in the soluable form. Have you checked to see if the varient has formed inclusion bodies?. Next have you looked at the codon usage table. Alanine has 4 possible codons, if you use a codon that is not normally used in ecoli there may not be suffiecient tRNA. I would also try and drop the culuture temp after induction. Not much else I can say as without knowing the proteins function it is difficult to advice.
i used GGC in reverse primer and GCC in forward primer and i also tried low temp induction and and also checked whole cell expression but their is no difference between E.coli cell and my transformed cell.
It's not unreasonable that this would happen. Some mutations destabilize proteins. Others don't. One can't really give a general answer as to "why". For some genes that are important in disease, professors' life times have gone into figuring out the mechanistic details (like CFTR, BRCA,...). If you want to find out more, look up how those have been studied... If your gene is important to a grant funding agency, you can make an entire career out of it :)
Hello John Schloendorn
but one amino acid replacement is give so much of decreased or can say no expression.
what is the reason behind this please tell me..
I am assuming you have checked the plasmid and that the protein is in frame and intact, ie the plasmid has been sequenced. Next question is, are there breakdown products. If so try a pLysE strain, also try other Ecoli strains.
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