a couple of quick things. This sounds like more of a protein-folding
issue. The lack of expression is probably caused by the cells clearing
of aggregated protein. You want to have conditions where the protein can
properly fold.
1) have you considered periplasmic expression by adding a periplasmic
targetting sequence to the N-terminal? It is generally thought, perhaps
anecdotally, that the periplasm is better equipped to handle proteins
likely to misfold (and presumably cause inclusion bodies).
2) Perhaps a heat shock of your cells prior to induction. Incubate the
cells briefly at 42 degrees. This will upregulate the cell's expression
of chaperones and other heat shock proteins which may help avoid the
inclusion body.
3) Are there any cysteines which might be forming non-specific
disulfides? Perhaps this can be resolved by mutagenesis.
4) Last resort. have you tried a snap refold of the inclusion bodies?
Dissolve the pellet in urea. Dialyse it away and see if any amount of
the protein has refolded spontaneously.
good luck
Simon
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
