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.
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.