Hi all, I have recently tried to transform a plasmid encoding a membrane protein-of-interest to E.coli C41(DE3). The plasmid is kanamycin resistant. It was transformed successfully but when I tried to induce expression using IPTG in small scale experiment, the recombinant protein expression is almost none after staining coomassie staining.
What could be the reasons for this? If the protein is toxic to E.coli, shouldn't the transformed E.coli die in the first place?
Hi there,
Indeed there's really a lot of things that could have gone wrong...maybe answering your specific question first: If your protein of interest is toxic to the bacteria, you start a selection process, i.e. low to no expression is favoured...However, you may find your bacteria to be groing slower than usual
And then here's a selection of other things to consider:
Check your sequence: codon usage, promotor surroundings....
Your protein is a classical transmembrane protein?
Do you need any posttranslational modification?
Is your bacteria strain compatible with your plasmid?
How did you know it was transformed successfully? (Finding clones on plates is not sufficient! Restriction Digests, PCR+sequencing, Northern and/or Southern Blotting to confirm...)
By no means this is a complete list....
Best luck,
Christoph
Membrane proteins are notoriously hard to express. If your 'protein-of-interest' is not an E.coli protein, the host cells may lack the machinery to fold the protein properly, and/or insert it into a membrane. If this is the case it may well be degraded very quickly in the cytoplasm.
As Christopher mentioned, there are a lot of things that may be improved. To your last question: Before you induce by IPTG you expect a very low expression level. Thats why cells may survive. However, you may already selectfor low expression. You may further reduce the level by using E.coli C41(DE3)pLysS or C41(DE3)pLysS.
What vector are you using?
To add to Christopher's comments:
Did you already try different temperatures?
You may include a positive control or/and a construct with a deletion of the "membrane domain".
Hello Zi Hao,
You can continue to optimize conditions for expression of your protein of interest in bacteria, but I ll suggest you to begin to think about expression in mammalian cells too just in case you were not able to express the protein in bacteria. We have literally spent months after months in optimizing expression conditions for expressing some dengue proteins in E. coli and it did not work. From codon optimization to chaperon co-expression, inducer titration to temperatures and trying out different bacterial strains (including the ones mentioned by you and others) nothing worked. We took the same genes, cloned them downstream the IgG kappa signal sequence and expressed under the control of CMV promoter in 293T cells and we got our proteins in the first attempt.
It may well be that your protein is expressed, but inserted in the wrong orientation and protruded outside of the E, coli cell. Have you checked the growth medium of expression for the possible presence of cleaved expressed membrane protein of your interest? Alternatively, you may consider tagging the membrane targeted end of your protein to any of E. coli membrane protein leader sequences, for example OmpA or PelB. Make sure there is a protease cleavable recognition site in between to enable freeing your protein of interest later.
Hi Zi,
Membrane proteins are notoriously difficult to express and occasionally it is necessary to use alternative expression systems (yeast, insect, mammalian, cell-free). Before thinking about this, though, it is worth making absolutely sure that expression does not work in E.coli.
First thing to query is the plasmid. Have you had it sequenced, and is it ok? No deletions or base substitutions? No problems with the reading frame?
Were your transformed colonies fresh? If your colonies had been prepared a few days before you started growing cultures then it is possible that the plasmid got ejected or something. Try freshly transformed cells.
Are you sure that your protein was not expressed? Membrane proteins, especially eukaryotic membrane proteins, often don't "do well" in E.coli. Due to the differences in membrane composition and post-translational modifications eukaryotic membrane proteins will often form inclusion bodies. If this happens then your protein can probably be identified in the insoluble "pellet" fraction of your cell lysate. The protein may also be toxic to your host - if you notice problems with the growth of your bacteria then it may be worth considering ways to reduce toxicity and see if this has a difference.
Membrane proteins also may be expressed at a relatively low level in comparison to cytosolic proteins. As this was only a small-scale culture that you prepared then perhaps Coomassie staining was not sensitive enough for your needs. Check by Western if you have an antibody, or use silver staining.
I hope that helps!
Good luck,
James
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