I am trying to Induce a construct (1.3kb) from Drosophila in the pGEX-4T1 vector in BL21 cells with IPTG. IPTG induction was done in 3hr grown culture and induce for 3 to 4 hr at 37*C. But there is no induced product visible in SDS-PAGE although empty pGEX-4T1 induced properly. I also check the insert release with RE, which was exactly the same size. Suggest me some way to get rid of the problem. Thank you in advance..
Some items to be checked:
1) Is the construct what it is supposed to be ? RE release of the insert is not proof of this. Did you sequence it throughout to make sure that the reading frame was correct and there was no mutation ?
2) Did you check that the expected product is soluble ? After lysing the cells and centrifuging the lysate, did you resuspend and wash the pellet with a mild detergent (e.g. Triton X-100 1 %) before centrifuging it again and dissolving it in SDS buffer for SDS-PAGE analysis ? By comparing induced and noninduced cells, you should be able to tell whether a significant amout of the recombinant protein is present in the pellet.
3) Looking now at the soluble extract, it may be that the desired protein is produced in too small quantities to be detected as a visible new band against the background of endogenous E. coli proteins. You may try an analytical purification on a small volume of extract by adding a small quantity of glutathion-agarose beads, washing them with buffer and eluting whatever was bound with a small volumeof glutathion-containing buffer, which you can again analyze by SDS-PAGE
4) You mention that the host is BL21 (DE3), I suppose. In the absence of a second plasmid such as pLysS, the strain shows a considerable level of leakage in the absence of inducer. If the recombinant protein has some toxicity, this will lead to counterselection of the hyperproducing phenotype. Many causes possible for this: loss of the plasmid, reduction of its copy number, mutation of the promoter, mutation of the protein etc.
These are just a few basic items. Otherwise, recombinant protein production can be tricky and each protein is different, wth no guarantee of success. For reviews of strategies, see papers by A. de Marco, e.g. PMID: 24176192
Hello, Pierre Béguin
Thank you very much for your suggestions.
1) No, I don't do sequencing for the insert, I just restriction digest the cloned plasmid and get the same (target) size band. based on your suggestion, I will go for sequencing.
2) Basically, to check induction, I collect cells from the induced and uninduced tube and dissolved the pellet in 10ul dH2O, and add DTT and loading dye, then denature at 97*C for 10 min followed by SDS PAGE.
3) Now, I am checking this with a small amount of GST bead, will inform here, whatever the result.
And I will go through the review. Thank you..
Hello, Pierre Béguin
I worked on the problem and noticed that the induced protein remains with the cell debris (cells are lysed properly, observed with proper control). So, what you suggest to isolate the protein from cell debris?
Hello Beppi Sarkar,
What you say shows that your protein forms insoluble aggregates due to incorrect folding of the polypeptide chain, unfortunately a very common problem among recombinant proteins. Unfortunately, there is no recipe guaranteed to work for every protein. There is a vast literature about the expression of "difficult" recombinant proteins. As a first measure, you could try to improve the percentage of correctly folded and suluble protein, for example by inducing the cells at a lower temperature (as low as 14 °C overnight). In some cases, it is possible to dissolve the insoluble aggregates in the presence of a chaotropic agent such as urea or guanidine hydrochloride, followed by renaturation upon removal of the agent by dialysis or dilution. In most cases, this is a tricky procedure that takes a lot of fiddling and optimization of renaturation conditions to avoid reprecipitation of the target protein
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