Hello, I am trying to express eukaryote protein (36 KDa) in E coli ( BL21 and Rosetta) by using different vectors (pGEX-4T-2 and pET-28a) and tags (GST and His- tag, respectively). However, more than 70% of my protein was cleavaged from the middle! Has somebody faced same issue? Is there any approach to stop cutting my protein? Thanks in advance for any help.
At first it is very interesting, that you have such a problem, which could be a hind for interesting features of your protein.
Maybe it is a good idea to lower the expression temperature. Ofcourse you lower the rate of protein expression, but maybe also the loss due to cleavage. Another way could also be to change the expression system. Why don´t you express a eukaryote protein in a eukaryote. There are a lot of yeast, which could be more suitable for your experiments than E. coli.
Hi!
My suggestion is to verify your cloned gene sequences first through sequencing. Make sure there should be no mutation. Then if everything seems to be fine. Try using glycerol (10%-20%) during inoculation to sonication. Glycerol helps in stabilizing the protein. Hope it works for you. Give your feedback...any kind of help is always welcome.
Good Luck.
Hello!
I have this kind of problems several times; first of all is important to know if the problem is with the protein expresion or with the purification. Making a western-blot of the cells after and before the purification. If the problem is the lysis/purification try less agressive methods( pressure, friction...), adding protease inhibitors... If the protein is breking in the cells, my situation, the final solution was used mammalians cells.
Good luck!!
Thank you very much for all of your helps, and I would like to explain that I have tried different expression conditions (15c-37c and air availability) but did not work that much. I realized that my protein was cleavaged while it is inside of bacterial cells. It was broken before and after purification processes. Also, I prefer to use E coli because I want to produce my protein in large scale and of course in easiest and cheapest way!
Hi Gaurav, First thanks for your suggestions; however, I verified my protein seq. and it was excellent. Could you please explain more what you meant by "Try using glycerol (10%-20%) during inoculation to sonication"?
Hello,
I had similar issues expressing my human membrane protein in e. coli. I have finally overcome the problem using the Single Protein Production (SPP) system. In short, you clone your protein into the pCold vector, that ensures no leakage of expression during the growth phase. At the same time you have co-transformed your cells with MazF, a toxin that cleaves any mRNA that contains the sequence ACA. Of course you should had engineered your DNA clone so that it doesn't contain any ACA. pCold gets activated upon cold shock of your cell cultures at an OD of 0.7, and so the toxin.
We have been using this approach for five different systems, membrane and non membrane proteins, and it always works.
Hi Ahmed Dhamad!!!
Take glycerol in media autoclave that and inoculate with starter culture. Proceed the other protocol as you follows normally (take less concentration of IPTG suppose 0.5 mM). After cell harvesting add 2%-5% glycerol in lysis buffer, wash buffer and elution buffer. You can also add glycerol 5%-10% during the dialysis. Store your protein in 5% glycerol for long term storage at -20 Degree.
Hope it works for you.
Good Luck.
If your protein is being cleaved inside the bacteria, then how many bands of protein are you getting on your PAGE gel ? If after a mass spec, you can identify where your protein is being cleaved inside the bacteria, you could try to identify the cleavage site that is present in your protein and change a residue to its nearest amino acid with similar properties (with a similar functional group like -SH with -OH etc) . Now with this residues change the bacterial cell should no longer be able to cleave your protein and the overall structure of the protein does not get largely affected.
I hope this helps . If the its to be mutated is of importance, I am not very sure what can be done. Have you codon optimised your expressing sequence to bacterial codons ?
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