Adamu,

Could you clarify what you mean by "no expression"?

How have you checked for your protein of interest, and which fractions did you check in?

You would expect, with these vectors, to at very least see some expression of GST (even with very unfriendly proteins, the cell usually produces some GST that drops off the ribosome before the fusion partner is synthesised). You should also be checking in your whole cell, or cell pellet fractions, in case your protein of interest makes the whole fusion insoluble.

Have you checked to see whether you can get expression of GST alone if you add just pGEX-4T-1 to competent cells, and use the same conditions? It's always best to try a positive control (to check the quality of your reagents) when you are getting null results.

Is it that you see no band appearing compared to non-induced cells? Because expression is often leaky and you might easily miss a protein that is not highly over-expressed. Do you see a separate band for GST appearing? In my experience GST fusions can be unstable and after induction all you get out is GST. In that case you would know induction worked, but you would need to switch the tag.

How big is your protein? Is there a rational for GST? It is a big tag and moreover it tends to dimerize. It could be that it is too costly for the cell to make the fusion or the product is toxic. If you can - try a different tag.

Another option is expression system. I had no luck with BL21 but Rosetta strain did wonders. In my case it appeared to be codon abundance problem.

One more expression protocol you might want to try is autoinduction: Grabski A, Mehler M, Drott D (2005), The Overnight Express Autoinduction System: High-density cell growth and protein expression while you sleep. Nature Methods  2, 233 – 235

In our lab it had worked better for some proteins.

Hello James,

Your system seems fine. As suggested by Nicholas, try to see the expression in whole cell lysate first, by running SDS-PAGE of induced and un-induced E. coli BL21 cell lysates. What is the size of your protein? If it is too big, then during purification it may go to inclusion bodies and you may not get anything by normal purification. if the protein is less than 50 kDa, then this problem should not be there, but check the presence of transmembrane domain and signal peptide, to confirm the localization of your protein. If all these are fine, then check the frame of your clone and also check your primers that you used for cloning. They might have some stop codon before starting the orf of your gene and will lead to ribosomal termination, even if your gene sequence is fine!

Hi, I want to ask the same question. How do you conclude that your protein didn't express. if you just check its expression by SDS-PAGE, some low expression cann't be detected. So I suggest you can prepare 800ml or more E.coli culture, then do routine purification using GST beads. Maybe you will purificate some fusion proteins. you can verify them using MS. If the purification doesn't yield any protein. Then you can draw your conclusion.

in my opinion, there are two major reasons for "no expression" in E.coli, one is codon usage bias which can be solved by choosing different cell types such as Rosetta or performing codon usage optimization. the other is degradation after expression, if this is the case, you can try lower induction temperature and IPTG concentration and handling your protein much more carefully in the process of purification.

Others, you should rule out the possibility your proteins expressed in the form of inclusion body which appeared in precipitate fraction of tatol lysate.

Another option is to use different fusion tag. I think MBP is good choice to promote recombinant protein soluble expression.

Good luck!

Hi James, you said, "By the way my insert is from Mycoplasma and the protein has no mycoplasma stop codon for expression in E. coli." Did you add a stop codon to the mycoplasm gene for expression in E. coli, and did you confirm from the sequencing results that your gene is in-frame with the GST-gene?

Hi James,

is your protein is part of a complex. sometime, proteins need to keep their homo-mer complex in order to stay stable or to avoid fast turn-over in the cells. it might be that your TGS-tag make allosteric interference and break-up the complex. try to fuse the tag to the other side of the protein. I had the same problem with one of the proteins I've studied and I got expression only when the tag was on the N-terminus but not on the C-terminus.

good luck

Thank you all for your contributions. For more information my protein is 1 kb and I used whole cell lysates of the uninduced and induced to run on SDS-PAGE. No expression meant I couldn't detect the desired band when compared between the two. No stop codon was added to the mycoplasma gene.

we recently had the identical problem. zero bands on western blot,(anti GST) but the bacteria grew very well, so must have the vector. My problem was solved by Darrin Kuystermans at the protein core at Sanford-Burnham here in Orlando. They suggested we use 0.05mM IPTG and turn down temp to 15C. Additionally I induced at OD 600 of 1-1.2 rather than the usual 0.6 and had thin but clear bands on western blots as well as correct N-terminal sequencing of the excized band. my protein was apparently being degraded at higher temperatures and level of expression.

You need to have a stop codon at the end of your gene otherwise you will not know what the size of your protein will be and it could have unexpected properties due to the addition of non-native plasmid encoded amino acids to the C-terminus. You can figure out from the plasmid seq. and the frame of your gene where the first in-frame plasmid encoded C-terminal stop codon is. I would figure that out first.