I trying to express mammalian membrane protein in ecoli system (with the optimized sequence) with the low copy number plasmid with the malE sequence (Periplasmic region expression).
After expression(1mM IPTG) i am not able to see the any band at the desired place in the gel.
What are all the possible things for this no expression ??? or if there is any low expression how to find that ??????
you can do a western blot of your induced sample by specific antibody (either to tag or protein).
low expression is detected using western blot which is extremely sensitive... maybe try a different plasmid, or IPTG, temperature, cell line (c41, c43, bl21, star, etc). Also change tag from C-ter, N-ter, etc.
A lot of options to check. Even if the expression is low, still you should be able to detect it via western blot, unless the protein size is very small, in that case it can be a bit tricky. If the protein size is very small, let me know, i can give you some suggestions for its detection. In order to increase the expression you can use different concentrations of IPTG and different induction times and temperatures. You may try another type of promoter, maybe arabinose promoters for higher expression. I agree with Toufic, sometime changing the tag position can also help
If the protein is an enzyme, and it has an activity that is not found in the host E. coli, you can test for expression by assaying for activity of the enzyme.
membrane protein is hard to be expressed in E.coli. You could try Rosetta strain (suitable for eukaryotic protein expression) and detect with SDS-PAGE and western-blot. If removal of transmembrane segment will not affect your downstream application. you could try to express the extracellular part.
Try Rosetta 2DE3 strain for expression.
Have u checked the inclusion bodies??
Hey, Many information are missing here. I assume that you are probably a new person in this business. I suggest you to consult GE Healthcare handbook on protein expression. You shall find many tricks there. Any thing can go wrong with yours experiments including the loss of plasmid.
Also, are you trying to check expression after loading the bacterial pellet or what? If you are trying to load bacterial pellet, then make sure that bacteria are properly lysed during the heat treatment. Otherwise, the protein inside the periplasmic space might have stayed with the membranous part, and might not have entered the resolving gel. And if you have centrifuged the sample before loading, then we all know where would be yours protein ;)
Please do provide me the complete information, and i might be able to guess more appropriately.
many reasons... membrane proteins are complicated ,, first check by western the protein
if you have the protein, after you follow all the exprsession protocol go to purification.. because maybe you have your protein in small quantities but with this is enough for your study... if you want an excelent production you have to probe different conditions of expression in order to find the best for your protein.. example you can change temperature ...
Hi Gayathri,
expressing mammalian membrane proteins in E.coli can be an exercise in frustration. If you have the possibility I'd suggest you try some other expression systems in parallel. I'd consider Pichia and Baculovirus to be good systems to look at, mammalian systems might also be ok depending on on the end goal for expression.
-Richard
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