Hi,
I would like to add one thing to the already good list of suggestions.
You can try to express your protein in Lactococcus lactis. We've
earlier had successful expression in lactis where it was none or only
inclusion bodies in E. coli. See for example: Kunji et al. Biochim
Biophys Acta (2003) 97-108, Geertsma and Poolman. Nat Methods (2007)
705-7, Kunji et al. Curr Opin Biotechnol (2005) 546-51.
Cheers,
Ronnie Berntsson
On Jun 29, 2009, at 23:54, Yong, Wei wrote:
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
