lack of expression of recombinant protein in E.coli host

hi ppl,

i have a problem with expression of my cloned gene in pRSETA vector.i ve tried in different hosts

GJ,BL21,pLysS.- no expression

sequencing results show that gene is in right orientation and reading frame.

i ve also checked for rare codons,well my gene does not have rare codons.

lemme know ur ideas on how to proceed..

thanks

aparnaa

Have you tried growing up the bacteria at a lower temperature?

Also, Merck have some products designed to help get good recombinant protein expression when there is a toxicity issue. They have plasmids that you can grow up at very low copy number (to limit expression during the growth phase) and then boost just before you want to harvest the rec. protein. These products are not marketed through the Merck name but rather through one of the daughter companies.

Hi aparnaa,

By no expression do you mean that you've run a protein gel and see no band of the expected size? One obvious test is to run a construct you know works along with your pRSETA construct to make sure all your conditions (from media to inducible promoter) are working as they should. If you are sure everything is working as it should except getting your protein of interest, does the pRSETA vector along generate any sized protein by its own? If it does you can always use that as a control to see if things are working (you are getting expression of any kind). Finally, do you have any reason to believe that your insert could be highly unstable so you never get enough protein accumulation? 

thanks ivan,
i meant the same,i don c any expression in my gel..my ptn size is 24kda.m able to see host ptn getting induced at the same size, in both induced and uninduced samples of my vector ctrl and clone.
i also did a western to confirm tat the band is not my ptn.it proved to be the same..there was no band at 25kda in western when checked with antiHis.
well is there a probability that an insert could be unstable???

You sound like you are performing this experiment with all the necessary controls. It sounds like you are saying that when you induce the expression of your construct you see the induction of host proteins. This should not be taking place. What kind of inducer are you using? It is possible that your problem is more about how your construct is being induced (i.e. it is not getting induced) and not protein stability. In my experience inducible systems for protein production are very specific and should not show much, if any, host protein induction.

Is it possible that your protein is highly unstable? unfortunately the answer to this is yes. While it may be unlikely, your lack of protein may be due to a very unstable protein.

i ve cloned it in the following site Bamh1 nHind3 in pRSETA.m using BL21 host n inducing with IPTG 1mM.....m sure my construct is fine.wat else could be the possible reason??

i ve cloned it in the following site Bamh1 nHind3 in pRSETA.m using BL21 host n inducing with IPTG 1mM.....m sure my construct is fine.wat else could be the possible reason??

Could the protein be toxic to the bacteria?

Did you also check at the 3' end to make sure that your protein is in frame with a stop codon? If you get run-on translation this could be bad for your protein. 

Also, if your protein precipitates as an inclusion body then you would not see it in your gel. In that case it may be useful to try and re-solubilize it before running a gel. 

I had experience that my protein was toxic to the bacteria, thus the expression level was extremly low.

try western blot to see if there are any expression at all.

also had experience that the protein was in inclusion body. But if you run whole lysate (add SDS sample buffer directly to bactera  pellet) on your SDS gel, it should be good.

hi all

still i ve not expressed my protein.i ve been trying different host and i ve also cloned my gene without the signal sequence(hydrophobic).still no expression.

this protein has a pI 4..is it too acidic?it also has an glutamic acidi domain.

how do i go about..

Here are a couple of things you can try if you haven't already: 1) lower your IPTG concentration (even if you make less protein, that's better than none), 2) grow the cells at 37 deg C until time for induction, then reduce the temperature to 25 deg C or 15 deg C and allow induction to occur and take some time points, say 4 h, 8h, or overnight; spin down a small amount of the induced bacteria (50 ul or so) and resuspend in 100 ul of 1X SDS buffer, and run on an SDS PAGE gel next to uninduced bacteria, 3) clone your protein into a fusion vector, such as one that fuses MBP to the N terminus, or try autoinduction (Protein production by auto-induction in high density shaking cultures, Protein Expression and Purification, 41 (2005), 207-209- in using this protocol, only add the 5052 solution and NPS solutions to your culture at 1X- this procedure does not require IPTG for induction.

The pBad vectors can also be helpful to try out.