hi all actually i am facing problem while purifying rec. protein of plant origin.The protein is coming in pellet. i tried to solubilise it, it works but problem of refolding still persist i.e. protein once in open confirmation not refolding itself. so please help me out n suggest an appropriate method to purify this protein.
thanks
Hi,
Are you sure that the problem is due to error in refolding only and there is no other factor involved? Is your protein is an enzyme or some other protein? What is the expression system you are using? What is the nature of your desired protein actually? What extraction protocol your are using? Is it an intracellular or extracellular secretion? And a number of other relevant questions.
All I mean to say is that, a huge no. of factors can cause protein inactivation and one has to find the exact cause of it. So please let us know the relavant information as much as possible. But anyway some general things you can consider are given here.
Most proteins retain activity when handled in neutral aqueous buffers at low temperature (although there are exceptions and these exceptions lend themselves to somewhat different
approaches to purification) This consideration then rules out the use of those techniques
in which the conditions are likely to deviate substantially from the above. With labile
molecules, it is important to plan the purification schedule to contain as few steps as possible
and with minimum requirement for changing buffers , as this willreduce losses of activity.
The second is the presence of specific contaminating compounds that may result in protein degradation or modificationand where the protein of interest is an enzyme the subsequent loss of catalytic activity. Such compounds may be proteinases. Just try to use some protinase inhibitors in extraction methods on routine bases.
Hope this may help.
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