The gene (1500 bp) was cloned into plasmid pET28a through NdeI/HindIII sites . a point mutant at 750 bp (G-A) was obained through error-prone PCR and the amino acid changed T-A. Then the plasmid was tranformed into E.coli BL21(De3) for expression of protein induced by IPTG. we found that the protein of mutant expressed much more than the wild type. We used the same plasmid, sites, promoter and induced conditions. The point mutant is the only difference between them. Which factor will affect protein expression ?
Dear Zhou, are you comparing the expression of two genes on the same expression vector, or the mutant on the plasmid with the wt gene on the chromosome? This can be your answer because the plasmid is present in multiple copies. alternatively, if both are on a plasmid, are you using the native promoter or the vector promoter? if the native one, is this protein a regulator of its transcription? This might explain the increase. If the promoter is the one present in the vector, maybe then you are removing a protease site?
Thank you Dr. Matteo Brilli . The two genes were cloned into plasmids pET28a through the same sites, respectively. The protein expression used the vector promoter (T7 promoter) induced by IPTG . We have ruled out that the protease site removment caused the protein expression difference through experimental verification, because we did not find the two proteins were degraded in cells when they were stored at room temperature in 48 h.
All else being equal (including an equal amount of total protein loaded on the gel), the simplest explanation is increase in stability of your mutant protein, which you can confirm by isolating and quantifying mRNA from the two strains (use a housekeeping marker to normalize the signal - same goes fot=r Western blots too). I am assuming that the point mutation has resulted in a change in one amino acid residue.
thank you for a tip Dr. Tausif Alam. Gene expression has been analyzed using real-time quantitative PCR experiments. we found the mRNA was slightly decreased in mutant. Furthermore, when the his-tags were removed from the N terminol of WT protein and MU protein through rebuilding the plasmids, the protein expression of the mutant (MU')is the same with the WT(WT'), and both of the expressing quantity was desreased.
so I speculated that the protein translation may be affected by the secondary structure of mRNA, but how to proof it , we have not found a good method to solve the problem.
Your results point more towards the influence of His-tagging.... to investigate your hypothesis, start with equal amount mRNA for both transcripts and try the cell-free in vitro translation; very direct and straightforward!
Alternatively, you can include SP6 or T3 sequence for transcription and starting with equal amount of template DNA, do a quick combined transcription/translation - depending on the AA composition of your protein, S35-Methionine or a mix of Met+Cys will will you stronger signal. Make sure that mutation does not cause a change in total count of S-AAs. If it does, use 3H-Leu.
Hey Zhou Wei,
based on the information you provided, I think it is not possible to find a simple explanation for the increased expression level of your mutant protein. Is it a soluble protein or membrane protein? Based on the crystal structure or homology model where is your mutation located? In ß-sheet structures or alpha helices? Is your muation in the catalytic center or on the surface of the protein? which kind of activity does your protein possess?
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