Hi, everyone!
I have a single-pass transmembrane protein (its functional domain is in cell lumen). Now i want to link a fluorescent protein (e.g. EGFP or dsRed) to its C-terminus. The chimeric protein is not recombinantly expressed in E.coli system and could not be purified. However, expression of the original protein is quite normal. So what is the trick? The centered transmembrane helix could influence expression in bacterial system? Or is there any references that reported a successful procedure like this?
Thanks for all the kindly help! :)
Hi Shen,
You write that the chimeric protein wasn't expressed and couldn't be purified, but that the native protein expressed fine. Could you give some more detail with regards to how you checked for expression of the chimeric protein? Immunostain, GFP fluorescence etc?
One option is that the GFP disrupts the proper folding of the luminal domain of your protein, so that you do not express any functional happy protein. It's very unlikely that the TM-helix would influence it, since you write that the native protein is happily expressed. The TMH is then already inserted in the membrane before the GFP is even produced from the ribosome.
@Ronnie Per-Arne Berntsson
Hi Ronnie,
Thank you for your comments. I ve checked the expression by immunoblotting using the native protein's antibody, but there is not any significant difference between the IPTG-induced group and the control group.
Another confusing phenomenon is that the host cells (E.coli BL21 DE3) were grown very slow. i wonder whether the chimeric protein may be toxic, nevertheless, the cells are not died.
Hi Shen,
assuming that your construct is correctly constructed and the protein expresses even a small amount, you may be able to observe colonies that are expressors by putting the plate with transformed bacteria onto a light box emitting at a frequency that stimulates the fluorophore enough to be visible. Certainly if the fluorophore is EGFP this is a viable means of identifying colonies that express the protein (distinguished from those that are antibiotic-resistant but do not express) by viewing the plate on a blue-light box such as that normally used for DNA agarose gels with CybrSafe or similar dyes. If only a small number of colonies have found a way to express the protein, this may be a better approach to quickly find cells that express than trying a few colonies out of thousands on the plate "blind".
Feel free to discuss this here further.
Good luck! Robin
Hi Shen,
Ok, depending on how the antibody binds the added GFP might interfere with that binding. One way to check is a variant of what Robin suggested. My standard way of screening for membrane protein expression is in-gel fluorescence. Run the whole cells on a SDS-PAGE (do not boil samples before SDS-PAGE!) and then check for GFP fluorescence using whatever source you have available. This will let you know if the protein is expressed, and also whether it is correctly folded (GFP both acts as a expression and folding indicator).
Just to make sure I understand you correctly, you have also over-expressed the native protein (without GFP) in E. Coli? And while doing this you do not see the slow down in cell growth after induction?
the blue light box trick is very useful for any bacteria expressing GFP or GFP fusions to identify candidates for "good" colonies, although you do need to consider that if they've selected for growth while expressing they may have selected for mutations to the expression vector (in which case - perform a mini prep and identify what they have done to 'beat' the toxicity, whether any mutation is in a reading frame or a regulatory element).
Best of luck - please report back to discuss further results.
@Ronnie Per-Arne Berntsson
Hi Ronnie,
Thank you again for your reply. I will try the method that you recommended!
Certainly I ve over-expressed the native protein, and all of the things are quite normal. Well, I'm sorry I didn't make it clear, i meant that the E coli cells (transformed with the chimeric protein) grow slow before induction.
Many thanks! :)
It is plausible that there is some leaky expression and that the protein / fusion is mildly toxic, causing the slow growth before addition of the inducing agent. Could you try adding some glucose to the culture to reduce potential basal lac-operon activity before performing the induction? If this increases growth rate prior to IPTG or auto-induction conditions it may confirm that the reading frame combination isn't tolerated well by the cells, and that suppression of expression makes the cells a bit happier beforehand.
I'll second Robin Dickinsons answer, leaky expression could well be the problem (assuming you are using IPTG inducable promotor that is). Feel free to report back here when you have done a few more experiments to verify what's going on
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