Hi Wei Yong,
Sounds like a tricky situation.
A couple of things come to mind:
1) Have you tried expression from a synthetic gene? Sometimes the
mRNA is unstable and improving mRNA stability through optimization
(synthetic gene) helps.
2) Are you able to look at either human isoforms or orthologs from
other species?
3) Have you tried expressing with an N-terminal SUMO tag? I have seen
expression with an N-terminal SUMO tag in some cases where expression
was previously undetected.
4) Have you tried cell-free expression?
5) You might also consider insect cell expression or expression from
mammalian cells
6) Do you get enough protein in some cases that you might try to
attempt some refolding from inclusion bodies?
Just a couple of avenues to think along.
Good luck!
Raji
On Jun 29, 2009, at 5:54 PM, 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
