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Help with T7RNAP purification


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#1 PaddyS

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Posted 16 October 2009 - 04:16 AM

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

#2 swanny

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Posted 18 October 2009 - 04:24 PM

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

Hi Pat,
What's your source for the enzyme?
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#3 PaddyS

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Posted 20 October 2009 - 02:04 AM

Hi Swanny,

Thanks for your reply.
The source of my protein is from BL21 transformed with the commercial vector pAR1219. This is expressed by myself in LB, induced with 1mM IPTG for 5 hours.

Thanks

Pat

#4 swanny

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Posted 20 October 2009 - 02:56 PM

This may be a bit of a long shot, but the components are all straightforward enough.

1. Treat BL21 (DE3) cells with EDTA. This will lead to the production of T7 RNA polymerase from the DE3 lysogen.
2. Lyse these cells and use them to make up the E coli component of the cell free system.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.




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