Separation of two proteins with similar properties

Marit Valeur Ramstad Marit.V.Ramstad at chem.sintef.no
Mon Dec 16 09:28:06 EST 1996


I am working with an enzyme produced by an recombinant E.coli. This 
enzyme have been purified by ion exchange and hydrofobe chromatography. 
The purified sample has some minor contaminants,but the trouble is that 
it seems like the enzyme is presents in two forms.Most proberly because 
of a minor degradation during the purifications steps.

The teoretical molecular weight of the enzyme is 103.000 and the 
teoretical isoelectric point is 3.9.

SDS-page of the purified sample shows two main proteins, one with 
molecular weight nearly 95.000 and the other nearly 105.000. On a native 
page theese two proteins is separated with a distance about 1 mm.

It is important for me two show if one or both of theese two proteins 
have enzymatical activity.To do this I have to separate the proteins in 
native forms.I have tried with gelfiltration, but the separation was not 
good enough. It is a problem that the proteins most proberly have the 
same properties.

If You have any suggestion on how I can seperate the two proteins and 
solve my problem, I would be very grateful.

Please respond by mail.


Yours sincerely,

Marit Valeur Ramstad




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