are there any good system to express functional mammalian protein? - I tried using E.coli, but protein is not functional (Aug/30/2006 )
I want get a lot protein, so I can use the protein for SELEX.
I used to systhesis my protein by TNT kits from promega, but the yield is low.
So I tried E.coli. But protein has no activity.
Can somebody recommend me some good system or kits to get a lot functional protein?
thanks very much!
we transfect mammalian cells and extract protein for functional assays. works fine.
is it easy to purify protein? how about the yield? thanks.
we usually have the His-tag in the vector so its easy to purify and yield is quite high.
Expressing eukaryotic proteins in prokaryotes bears some difficulties f.i. lack of processing, chaperons or posttranslational modifications; nevertheless I would try to co-express a chaperone in E. coli, or try a refolding kit such as
a systematic evaluation of various possible conditions to refold and activate your protein even if expressed in E.coli; if you have enough from E. Coli expression there are myriads of eukaryotic - cellular and non-cellular- expression systems
thanks very much!
One of the more successful techniques is baculovirus-mediated transformation of sf9 insect cells.
However, you will need a Class III licence for this work.
yeah, my boss told me that. thanks very much about this infor. I just hope I can activate the proteins I purified from e.coli.
How many plates do you transfect to have good yields fo your protein, What system do you use to purify it?, and what cells you use?
His tag didnt work for me but you made me thing that yields could had been the problem
I am changing to strep tag which suggests transfecting 9x 500 ml flasks to get protein in the range of mg
we use one 10cm plate and transfect 293 cells for expression. We have our constructs in pcDNA with the myc-Histag and purify with the Nickel column. We have been getting like 1-2 mg of protein. Actually one 10cm plate seems to b sufficient for nearly all the experiments including triplicates and controls.
let me know if u need protocol for transfection or purification.