No activity of expressed protein obtained from pET32a cloned gene - (Jan/01/2013 )
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
This is very difficult to predict ahead of time. People often do both N and C terminal fusions in the hope that one or the other will produce active product. Other issues may arise, however. Are you certain the protein has folded correctly? Is your assay working? Do you have a positive control for the assay?
Why do you have TRX tag if you are purifying with His Tag? Is it to cleave the His-Tag or what? Are you using ELISA for hte activity assay?
The protein I am working with goes into insoluble fraction on expression. I need protein in soluble form for its activity assay. The Trx-tag is generally use to get the protein into soluble fraction. As Thioredoxin (Trx) itself is highly soluble protein, it brings any protein in-frame to it in soluble fraction. Later, that tag can be cut from the target protein using enterokinase. thus Trx is not a tag for purification of protein but in enhances the solubility of it. For purification I use His-tag.
I work with the pET32a vector for all of my expression constructs. I think it is essential that you remove the Trx tag. I believe it is 109AA! A his tag is 6. With the Trx tag you are working with such a different protein I would never predict that it would function like it should in an assay. The beauty of the pET32a vector is you can cleave that tag off.
Thank you for this help and suggestions.
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.
You can consider to use FPLC systems like AKTA chromatography to purify your recombinant protein after your enterokinase treatment. Use IEX columns.