Molecular weight of protein is approximate 55 kDa .
I check the different temperature (22, 30, 37) and different time (3, 6, 9... 24).
But I can't see a sharp band on SDS-PAGE.
When the media reached OD=0/72, I divided the media and poured half of it into another flask. Just one of the flask was induced with IPTG (1mM)
both of them incubated at 22 C for 15 hour. After running both sample (IPTG + and IPTG-), I saw a little difference between them based on sharpness of their band. In the other word, the IPTG didn’t affect expression of my protein.
But I saw a so sharp band at the bottom of gel in + IPTG sample. Position of this band is approximately far from my band. I think bacterium put all of their energy to produce this protein. But I don’t know what this band is.
Host bacteria is Bl21
Hi, I would suggest you few things...1) Have you checked whether your sequence has rare codon (codon bias) for E coli?. If it is case, you have probably translation problems due that E coli has not enough tRNAs that recognize those codon (typical problem for heterologous proteins) , so your protein is not fully translated (which could be a explanation for this shorter band that you mentioned, maybe it is your protein but not totally translated) . Try to express your protein on BL21DE3-RIPL (stratagene), which has plasmids expressing those rare tRNAs in Ecoli helping on translation of your protein. 2) If you are sure that your protein is fully expressed, sometimes it is not expressed in high amount, so it is hard to see in whole cell lysate. However, after small scale purification by Niquel column for His-tag you can begin to see your protein more clearly. 3) In case that your protein in quite toxic or unsoluble, try to grow bacteria cultures until OD 0.6 at 37C and after you can add 0.5mM IPTG (for T7 promoter is largely enough) let them grow at 20-22C overnight. 4) If possible at first trial purification, go for denaturing conditions using Urea or GuHCl, those chemicals, specially GuHCl which makes bacteria lysis and solubilization of proteins perfectly (to avoid that your protein go for unsoluble fraction). Good Luck!
hi Jorge
this protein has expressed in Bl21 already and it had good expression ( there aren't rare codon) . just I remove a small domain of this protein.
thank you for good guide
with regards
A simple first question would be have you sequenced your construct to make sure it is definitely in frame?
If it is then another option then is to perform a western, I can't remember the epitope on pET but an antibody is available. This may tell you if there is any degredation which I have seen with some proteins that are susceptible to endogenous proteases.
Did you use any protease inhibitors. If no, there is a possibility that the sharp band at the bottom of the gel maybe protease digested product.
Hi!
I would suggest you to use BLR(DE3) cells which are derived from BL21 cells. But, in this case you will have 6XHIS tagged to your recombinant protein. So, the approx size of your protein in the gel would be 55+25 kDa or almost 80 kDa. I think that should work. Also, look fro codon bias of your sequence in E. coli. I would also suggest to grow the cells in higher temperature than what you are using but for lesser time. Growing for longer time might destroy the antibiotics in the medium and then non-selective bacteria start growing. So, that may be a reason why you are not getting the protein of interest in the solution. While extracting protein for gel loading, use proteinase inhibitor to eliminate any chance of degradation in the soluble phase.
I have exactly the same problem with my gene -I am trying to suppress basal expression with the use of glucose-please read this paper from Bio techniques
(http://www.biotechniques.com/multimedia/archive/00010/00296st03_10412a.pdf)
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