No activity of expressed protein obtained from pET32a cloned gene
Posted 01 January 2013 - 03:28 AM
I have a 1.6kb gene cloned in pET 32a vector with N-terminal His-tag. For expression I have transformed the clone in Rosetta (DE3) pLysS cells and induced with 1mM IPTG at 0.6 O.D. Then the culture was transfered to 18 C for 10-12hrs. I have purified the expressed protein using His-tag. When the purified protein was loaded on SDS-PAGE, I could see clear expression at expected size (64KDa protein+19KDa Trx tag= 83KDa). But when I perform activity assay I could not see any product. I would like to know, how much essential is it to remove the Trx tag from the protein to get the activity?
It will be really a great help as I am stuck with this since last 2 months
Posted 01 January 2013 - 09:17 AM
Posted 12 January 2013 - 01:49 PM
Posted 14 January 2013 - 10:59 PM
Posted 16 January 2013 - 11:05 AM
Posted 17 January 2013 - 03:47 AM
Ya, you r right that the Trx tag is ~20KDa size. I have cut the tag using enterokinase, but here the problem is, the tag and the enzyme (enterokinase) remains in the digestion reaction alongwith the protein of interest. Get rid of these is problematic for me as it involves further purification which will result in reduction in the amount of target protein (OR may be activity loss!!!!!). Can you suggest me some protocol which does not include lot of downstream proccessing giving maximum yield of target protein without trx-tag.
Posted 17 January 2013 - 07:24 AM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434