Purification of enzymatically active protein from PAGE - (Oct/04/2005 )
I am trying to purify the digested product of His-tagged fusion proteins (pET System – Novagen) produced in E. coli (BL219DE3)) to compare their kinetics of different isoforms of an oxidative decarboxylase (soluble, malic enzyme, E.C. 1.1.1.40). My original idea was to His purified the fusion proteins, digest them, run them again through the affinity column and get rid of the enterokinase.
The problem I first encountered was that not only the full length (100 kD) but also truncated versions, of much lower MW (between 20-50 kD) are produced. I tried to fix this problem using host strains that carry rare codon tRNAs (Rosetta2 – Novagen) but without success. I was thinking that a good way to obtain the digested product and get rid of the rest (i.e., uncut fusion, truncated forms, enterokinase) would be to run an exclusion chromatography after digestion to separate the species by size. This would most likely work for the truncated forms and enterokinase since they are much smaller than the fusions, at a cost of protein loss.
On the other hand, somebody suggested to run a preparative SDS-PAGE and then cut the right “band” and elute the protein. Would it be possible to get good quality protein using this procedure and have “publishable” results? I wonder if this would be an acceptable procedure to obtain and analyze protein kinetics. What protocol for protein elution from SDS-PAGE (or native?) and subsequent refolding would you reccommend?
in one word NO! you will not be able to get any activity back
BUT
you can get G50column it much faster and nothing happened to the activity
First, do you know that the his tag fusion will interfer with your results? If the fused protein works, why bother removing the his tag at all?
I've used his-tagged enzymes (purified with magnetic beads) in in vitro assays (see for example, Science 18 March 2005; 307:1778-1781), and there was no problem...
'Course I've also had enzymatic reactions fail... 
BUT
you can get G50column it much faster and nothing happened to the activity
sorry i brought this thread up again...but im having same problem now as my protein is degraded after purification....i understand from these replies that it is better not to try purify it from some native PAGE with refolding but use G50 column instead.....actually we have G50 here but on the datasheet its written <700 Da....
...but monsa's protein was around 100KDa....and mine is also around 90KDa so my question is: can G50 be used for these large protein purifications? thank you a lot. ...but monsa's protein was around 100KDa....and mine is also around 90KDa so my question is: can G50 be used for these large protein purifications? thank you a lot.you can use g-50 if you don't mind your protein being in the void peak. you will be able to separate your protein from the fractionated proteins as long as you don't overload.
It means that all what is higher than 700 Da won't be separated and you will be able to collect them in the void volume., then the proteins lower than 700 Da will be separated.
the question is : what do you want to separate your protein from?
are the truncated forms lower than 700 Da? If not, they will be also in the void volume and you won"t be able to separate the complete protein from the truncated one.
Same problem if you want to separate your protein from the tagged protein. (but in this case you could pass them on a NTA column, and the digested protein will be in the wash fraction and the non digested in the elution fraction).
If you really want to purify, you should use a column such as Superdex 200 from amersham.
you can have a look here
http://www5.amershambiosciences.com/aptrix...=71501796AF.pdf
the question is : what do you want to separate your protein from?
are the truncated forms lower than 700 Da?
actually, the fractionation range of g-50 is 1500-30000 for globular proteins. so, it could be okay for their purpose (g-75 might be better if they want to use the void to separate theirs from the other proteins) .
but, i agree that superdex 200 would be a better choice, if available.
thank you very much for your replies, actually my protein is around 90KDa and its degraded product is only about 4KDa different so its around 86KDa....we dont have superdex 200 which i want to use so much.....so i guess since my both products are very large i wont be able to seperate them on G-50... 
...i dont want any chromatogram after that i just want to detect the non-degraded one by SDS-PAGE>.....actually all I want is to have the non-degraded protein band on SDS-PAGE without its degradation products...its crucial for my experiment....
please can you also tell me if I can use HPLC with C18 column for this purpose....its available here in the lab...thanx a lot again
you might be able to use c-18 to separate your protein but...
c-8 or c-4 or phenyl are the ligands normally used to separate proteins, c-18 is normally used for small peptides but may be used for some proteins. also, you need to use large pore (300A or larger) or non-porous stationary phase to handle molecules as large as proteins.
good luck
thank you very much for your help! 