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lost and inactive..protein stuck in pellet:/


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#1 Travica

Travica

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Posted 26 February 2010 - 01:12 AM

Hi!
I've been suffering lately with my protein expression, hope someone can help. <_<

I'm trying to express one of Cytochrome P450 enzymes, membrane protein which has been cloned into pCWori+ vector as truncated, codon optimized and His tagged. I have tried coexpressing it with groEL/groES in JM109 strain, and WB shows very high expression, but the protein stayes 90% in the pellet. Moreover, it is mostly inactive giving spectral peak at 420nm instead of 450nm.
So, I have two problems:
1. big expression, locked up in inclusion bodies probably
2. degraded protein

My protocol includes induction with 1mM IPTG, growing bacteria at 27 degrees, tratment with lyzozyme, protease inhibitors, sonication, and I even tried rescuing protein with 8M urea, but I only got maybe 20% out of the pellet, inactive.. :)

I need the enzyme active and abundant for structural studies..please advize :)
Thank you!!

>>amateur expressionist




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