In addition to other excellent suggestions voiced here (insect cells, refolding, etc.) you can attempt expression in constituitive mode - using a mild always-on promoter. Provided that your protein is not too toxic to the cells this may result in gradual accumulation of folded material rather than sudden accumulation of misfolded junk.
Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Yong, Wei Sent: Monday, June 29, 2009 4:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to express a >95KD FAD protein 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
