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 pET26. Only one amino acid change in each mutant. I don't know why the last mutant (changing N to I) 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. One other surprising thing is that the size of recombinant plasmid containing mutant gene was reduced after extraction from pLysS, but it has correct size before transformation to pLysS.
Im not very sure if I could help, but in my experience, we had the same problem and we expressed in BL21 (DE3) strain just fine with 2 mutants and the other two could not be expressed. All we did was to transform the 2 plasmid to another bacterial strain (such as C43(DE3)) and they expressed fine! But I guess it could be different for your proteins!
Good luck!
Hi, a mutation could also affect the mRNA stability leading to less or no expressed protein.
I would agree with Tuan Hoang, try another strain, but just for scrutinity and based on old self experience, did you sequence the entire sequence that is relevant for the expression. Any cloning can easily introduce by (small) chance some mutations and I have seen point mutations even in the promoter region. N -> I is a single base exchange, so in this case RNA stability may not be a problem.
thanks for your comments, I sent my plasmid for sequencing to ensure about the sequence and also I am trying another host. i hope so it will work.
Once I had another strange experience. A couple of mutants differing in only one amino acid were both produced in high amounts with bacto-tryptone from BD. However, one of them was produced very poorly with bacto-tryptone from another company. The cells were BL21(DE3)CodonPlus.
Try other strains as well like arctic etc...and do play with temperature....
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