Hi all, I am expressing a bovine protein in E coli dh5a. My native protein expressed very well in bacteria but my double mutant and triple mutant hardly have any expression. only a faint band can be seen on sds page gel after nickel column purification and its hard to concentrate to the desired concentration for crystallization. any suggestions are welcome! thanks in advance!
it's worth checking whether the introduced mutations are introducing stop codons or the protein is becoming more insoluble/any other properties are changed.
Hi there,
If mutations are destabilizing for the protein, it may explain lower expression yield.
Its very common for 'mutant' proteins to not express well. If the mutations compromise folding, then the protein can either be insoluble or be actively degraded by the bacteria. Try expressing in BL21(DE3) cells using a pET-vector. BL21(DE3) cells they have fewer proteases and better overall expression of foreign proteins. The most stringent conditions are expression are at 18 deg C for 24-36 hours with 500 uM IPTG. If it doesn't fold this way, you should re-consider how you are making your mutations.
Hello,
This may be a result of destabilizing mutations, however you should check to see if you may have introduced a rare codon into the plasmid following mutation. You would need to check your cell line to see if the codon you introduced may be the issue.
Hi Mana Mahapatra, thanks for your answer. The DNA sequence is designed and optimized with Geneart of lifetech. I have sequenced it and did not find any problem. I have run the pellet on sds page gel and the protein is still soluble...
Hi Paul Lesbats, the native protein was expressed well in dh5a. I have tried BL21, there was no much difference. Also I have checked the pellet. The protein is still soluble.
Hi Dominique, I think you are right. So any suggestions for designing new double mutated protein? What rules should I follow and what I shouldnot？
Hi Nicole， I think I will redesign the mutated protein. thanks for your answer
Hi Andrew, the new sequence was optimized with Geneart so that should not be the problem.
Cheers
Dear Heng,
As stated already by Dominique, mutation introduced in a protein may destabilize the mutated protein relative to the wild-type one. In the case of a double-mutant, your chance to get an unstable variant is the product (not the sum) of chances with a simple mutation. Quite bad!
What do to do then? For the moment, there is no rules, no laws, almost nothing to help us to decide which position to mutate to not get unstable mutant! So the solution is : try and try again...
There are a few lines of evidence though that indicates that mutations at the surface of the protein could be most of the time less destabilizing than those inside the protein core. And that's all.
There are however another way to get insight into protein structure-function relationships, it is through chimeras. If your protein fits to a system in which two closely related proteins share a similar function. You may, by a variety of techniques, shuffles the sequences of these two parental proteins and thus obtain a collection of chimeric mutants. Depending on the shuffling techniques, from 15 up to 85% of these chimeras will be functional and most (i.e. > 90 %) are stable. Then you do what you have to to link data shifts and this or that sequence shuffling. That way you identify sequence segments which are essential for the function and by placing these identified elements on the 3D structure you can draw interesting conclusions...
Best regards,
Pr Philippe Urban
All things considered, the nature of the mutation is likely the confounding factor. I would recommend a different expression host (instead of DH5a). DH5a is a common cloning strain with many mutations that allow for efficient cloning experimentation. For protein expression, DH5a is less suitable compared to other E. coli hosts. BL21 is common protein expression strain that is lon protease deficient. It might help.
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