i have change an amino acid codon asparagine to aspartic acid in my recombinant protein plasmid then transformed in bl21(de3) and used it for expression of my new mutant protein.I expressed it using 0.6 mMIPTG. WHEN I run in SDS PAGE ,its band was not that clear compared to the wild type recombinant protein. Is it because of the mutation I have done?
Hi,
What do you mean by 'the band was not that clear'? Was the band less focused or had lower intensity? Even a point mutation can affect the expression level of a protein so if you do expression and purification in the same way as the wild type you might see less protein on the SDS-PAGE.
Dear Pushpa:
What do you mean by "band was not clear"? Is it not well defined? Can you see it? If you don't see it is because the concentration protein is lower than the one you expected (due to lower expression or just because you injected lower protein concentration on the SDS-PAGE).
If the protein expression system works fine, a single change of an asparagine to aspartic acid should give you good expression a nice bands on SDS-PAGE.
Good luck,
Rosa María
To give you a combined answer of both those previously, normally you would expect the mutation of asparagine to aspartic acid to have minimal affect on your protein folding. However, I have had similar experiences in the past when substituting an amino acid for a glycine or an alanine, this rendered my protein expression at zero! I could not explain why it would not express, I attempted changing the conditions etc but never had any success. Sometimes these mutations may form essential structures within the core of the protein that severely disrupt the protein folding, The other option is that the destabilised protein is susceptible to degradation!
One thing I would advise is re-sequencing the clone to ensure you have the correct gene sequence, and check the whole ORF while you are at it, there may be another mutation you are not seeing. Otherwise if you have some protein expression then that is great, just work with it from what you have - larger cultures, longer incubation times etc!
Good luck!
Its possible that your new mutant has more propensity to form inclusion bodies - an aggregate of insoluble protein. This is the result of miss-folding or (according to some theories) a deliberate attempt by the host to deal with a protein that is causing it significant metabolic burden.
Inclusion body formation is a relatively common problem with E.coli especially at high IPTG concentrations (0.6mM is high). Peptides in inclusion bodies my be the incorrect length and my not denature completely in SDS - hence they may result in a smear as opposed to a band. Have you tried running soluble vs. insoluble protein fractions on SDS PAGE? This is the definitive way to determine if there is inclusion body formation. There are kits available to prepare these fractions.
If inclusion body formation is to blame, codon optimisation, lower IPTG levels and lower feed rates (in a fermentation environment) may help.
I have sequence my clone and I get good alignment with my wild type. At the begining i got the induction at 0.6mM both for mutant and wild type.Now I m not getting anything.I m attaching the 1st gel which was induced both for mutant and wild type. In the below pic, lane 3 -uninduce, Lane 4,5 and 8 - mutant , and Lane 6,7- wild type. here the band is bigger in wild type than the mutant
When you say you are not getting anything, do you mean that you are not getting any activity? I would definitely look for inclusion bodies using a method like this...
http://camelot.bioc.cam.ac.uk/~marko/methods/ib.pdf
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