And this is what I mailed to him:
Dear Lan,
> Would you please let me know if inclusion body is lethal to cells? I
> found my mutants go to die if I use high concentration induction.
>I think they are not lethal as long as they do not get big enough to
mechanically distort the cell. So some inclusion body formation
would be tolerable, but it is not astonishing that they die when the
IB grow too big. On the other hand, overproducing recombinant
protein is always stress to the cells, so some additional stress
might be harmful.
I must admit that I am not an expert in these cellular things, because
I've studied chemistry and am now in the protein folding field.
> And, whether still have some native comformation protein out of inclusion
> body when inclusion body forms?
Probably there's also native protein in solution. The on-pathway
intermediate is still there, though perhaps in lower concentration,
and is still able to form native protein. But usually under conditions
where inclusion bodies are formed, the amount of soluble protein is
too small to base a purification on that.
I restored the function of one of my
> mutants at 28 C. and also have partial function with low level induction.
> It seems the mutation do not attack the folding important structure.
>
Fine! Now you have to chose which way you try it.
BTW 28°C is not the lowest possible temperature. If you grow the
cells at 37° to an OD of about 1 before induction and then shift to
25 or even 20°, you might get more soluble protein. I observed that
the total amount of protein seemed to be higher at elevated
temperatures, but if your aggregation reaction is temperature-
dependent (which is often the case), you might get more soluble
protein that way
> Since I can not post this, sorry to bother you.
No problem. Okay that I post your and my article?
Bye, Frank
--
Frank Fuerst, Institute of Biochemistry of Potsdam University
Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334; Fax: +49-3371-681339
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