I expressed TNFa in E. coli BL21 (DE3) host and got satisfactory expression. The protein is tagless. My question is how do I purify it? Which chromatography techniques can I use and what should be their sequence?
Whatever works..
Most of the tag-less protein purification protocols start with ion exchange and end up with gel filtration.
Hi Shridhar, here are some papers on using recombinant TNFa from E. coli and purification procedures:
http://scholar.google.com/scholar?q=related:TuZgco_8ZcUJ:scholar.google.com/&hl=en&as_sdt=1,21&as_vis=1
Hope this helps!
Whatever works..
Most of the tag-less protein purification protocols start with ion exchange and end up with gel filtration.
Any body have the articles related to anti TNF alpha with the sequence of variable domain??
As Misha wrote, use whatever is suitable and available. If it's DNA binding, you could try some sort of affinity chromatography or IMAC.
Thank you Tomáš and Barbara... :)
@Barbara: are anti-TNF alpha columns commercially available? I did not find any.
First, see the protein characteristics, (% hydrophobic/ hydrophilic AA residues), what is the expression level of your TNF protein?, theromostability.., pH stabillity..solvent stability etc. then since you are working for Gennove Biopharm, hope you have to try for patent non-infringing protocol write?. So check the patents and then design the protocol. Just by posting a question with 2 -3 line and asking for solution will make difficult to answer your query.
@Errana Rajashekhara: Thank you for your suggestions. This protein will be used for internal purposes. I will have to worry about patent infringement of a protocol if and when my company decides to manufacture this protein and commercialize it.Since that is not the plan, I am free to choose any method of purification that is suitable. Since my experiments are at shake flask level, like any other academic lab, magnitude of the purification process is not significant.
By using an antibody column for purification you will select for functional protein. There are many companies that sell TNF alpha antibodies. I suggest you make your own affinity column using something like "amino Link Plus coupling resin"
A Misha said, you should start with ion exchange and then end up at gel filtration. I would like to add one more step in between. If your protein is not destroyed by ammonium sulphate precipitation, you can do precipitation and then redissolve the pellets, load in anion exchanger, elute it with shallow gradient. let see how it looks like. then you can plan further!
if you precipitate protein with AS you cannot use directly ionex because it won't bind. However, it is ideal to use hydrophobic chromatography (octyl sep, phenyl sep etc.) but from my experience one loses lots of activity on those. But that could be just my proteins.
@Birendra and Tomáš: Thanks for your inputs friends... :)
I'm working on this thing and will update the outcomes...
Hello Shridhar,
After precipitation of proteins with ammonium sulphate you have to do dialysis against ion exchange binding buffer.
Most of the tag-less protein purification protocols start with ion exchange. You can isolate by precipitating with ammonium sulphate solution 0.2%. You can demobilize by usingTNFα converting enzyme on a matrix and then extract it from there or use by SUMO-fusion system.You can also isolate by selectin Abs against TNF alpha and can find your proetin from complex dissolution.
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