With the disclaimer that I have yet to work with a flavoprotein, is there enough FAD around in your culture to allow for proper folding of your protein ? Maybe adding riboflavin to you media, or trying ultimately to refold you protein from inclusion bodies (which will be very pure) in the presence of FAD may work. A quick search in the journal Protein Expression and Purification suggests the latter has been done a few times.
Stephen Weeks 2009/6/29 Yong, Wei <wy...@mcw.edu> > Dear all, > > I know that there are a lot of experts having experience in expressing a > big protein in E.coli or yeast. My project is about a 95kd > covalently-FAD-binding protein (Human protein). I tried very hard but have a > bad luck over the past 1.5 years. I list what I did briefly so far. I am > looking forward to getting suggestions from you. Thanks a ton in advance. > > E.Coli > 1. Express full-length in pET28a (N-6xhis) No > expression > 2. Express full-length in pET21b (no-tag) > Inclusion body > 3. N and C terminus truncated construct > Inclusion body > (tagged and non-tagged) > 4. Co-expression with GroEL/S > Inclusion body > 5. Co-expression with with cofactors > Inclusion body > 6. In pMAL vector > Not sure > > Yeast: (No tag) > 7. In pPICZb vector in Pichia > No expression > 8. In GHB30 vector in Saccharomyces cerevisiae No > expression > > I also tried to use different ways to break cells, use detergent, express > at low temperature (down to 15 degree), modify IPTG concentration and so on. > > I do not know what else I can try. Please give me suggestions. Thank you > very much. > > Best wishes > > Wei Yong >
