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Help with T7RNAP purification


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#1 PaddyS

PaddyS

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Posted 16 October 2009 - 04:18 AM

Please note, I have posted this in two discussion groups, so please forgive me for any overlap, I am just hoping desperately for a response :wacko:

Hi everyone,

Could someone please reference or better yet describe any suitable purification strategies of the T7 RNA polymerase, for its eventual use in a cell free system
I would very much like to know what method has been employed for this enzyme which allows for the maintenance of both enzyme activty and achieving high purity.

Your help would be much apprieciated.

I am currently attempting to purify this enzyme according to Schwarz et al. 2007 Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. However during my attempts, I have not been able to achieve an active protein, or at least one that has shown specific activity.

Has anyone figured out a way or any tricks to maintain the activity whilst getting a high quality product?

Thanks

Pat




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