No mRNA or protein made by my mutant gene - (Jul/02/2008 )
Hello,
I am making mutations in a gene that codes for a protein and I found that when I express my gene of interest in E. coli I get no expression as verified by western blot. I also did a northern blot and found that significantly less (almost none) mRNA is made as well. I used all the appropriate controls such as wild type and empty vector. I get robust expression of protein and mRNA in the wild type and none in the empty vector. I sequenced my gene and I found that the sequence is OK. I even tried a mutant E. coli strain that lacked DegP- (periplasmic protease) and I still don't get expression.
Could I be deleting some regulatory element that is mucking up gene expression? It seems unlikely since the mutations are not in or near the promotor.
Any and all suggestions would be helpful. Thanks a lot.
I am making mutations in a gene that codes for a protein and I found that when I express my gene of interest in E. coli I get no expression as verified by western blot. I also did a northern blot and found that significantly less (almost none) mRNA is made as well. I used all the appropriate controls such as wild type and empty vector. I get robust expression of protein and mRNA in the wild type and none in the empty vector. I sequenced my gene and I found that the sequence is OK. I even tried a mutant E. coli strain that lacked DegP- (periplasmic protease) and I still don't get expression.
Could I be deleting some regulatory element that is mucking up gene expression? It seems unlikely since the mutations are not in or near the promotor.
Any and all suggestions would be helpful. Thanks a lot.
What region of the gene are you mutating? Unless you're changing the vector's promoter and regulatory regions, I don't understand the reductions in the mRNA levels. You might have damaged the vector somehow.
Is there a possibility that your protein becomes not soluble ? You may test the non soluble fraction when preparing your extract.
I am making mutations in a gene that codes for a protein and I found that when I express my gene of interest in E. coli I get no expression as verified by western blot. I also did a northern blot and found that significantly less (almost none) mRNA is made as well. I used all the appropriate controls such as wild type and empty vector. I get robust expression of protein and mRNA in the wild type and none in the empty vector. I sequenced my gene and I found that the sequence is OK. I even tried a mutant E. coli strain that lacked DegP- (periplasmic protease) and I still don't get expression.
Could I be deleting some regulatory element that is mucking up gene expression? It seems unlikely since the mutations are not in or near the promotor.
Any and all suggestions would be helpful. Thanks a lot.
What region of the gene are you mutating? Unless you're changing the vector's promoter and regulatory regions, I don't understand the reductions in the mRNA levels. You might have damaged the vector somehow.
Unlikely, because I am using the same vector in my control experiments and the region that contain my mutations is not near the promotor.
My protein is a peripheral membrane protein, but it should show up either way when I am doing a whole cell lysate. One thing I have not tried is putting it under an inducible promotor. It could be that proteases are digesting the protein right when it is made.