Foreign protein expression in E. coli

[rmeck at icbr.ifas.ufl.edu]

In article <9402050924.AA08685 at pclsp2>, vinz at PCLSP2.KUICR.KYOTO-U.AC.JP (Vincenzo Nardi-Dei) writes:
> 
> I have recently cloned an hydrolase of 35,000 M.W. from Pseudomonas
> in to E. coli XL1 Blue, provided from Stratagene.
> The plasmid used is pUC118, the insert is about 1,200 bp
> and the expression is around 10 % of the cell crude extract;
> Unfortunately large amount of the enzyme is found in form
> of inclusion bodies and the cells take 48 hours (!) to reach
> a full growth in LB medium at 37 C and 200 rpm.
> This is not the first time I have
> inclusion bodies and slow growth using XL1 Blue.
> Moreover, a collegue of mine is having the same problem
> with the same strain.
> 
> In this view I would like to change the host: can anyone
> kindly suggest me which strain of E. coli is best fit
> for high expression of this kind of foreign proteins?
> 
> Thank you very much indeed,
> 
> Vincenzo Nardi-Dei
> 
> Laboratory of Microbial Biochemistry
> Institute for Chemicall Research
> Kyoto University
> 611 Japan
> 
Dear Vincenzo
You want to get your hands on a copy of the PET System manual by Novagen.
They'll give some recommendations about avoidence of inclusion bodies
and growth control. E.g. lower temp for growth leads to more protein in 
the soluble fraction, their ref is: Schein and Noteborn 1989 Bio/Technology
7:1141-1148. 
Depending on what you want of this protein you can dissolve the inclusion
bodies with Urea and dialyze this out again, but you might have folding
problems. You can also use and expression vector which exports the protein
into your medium. Or you can make antibodies directly using the inclusion 
bodies.
Keep me posted, as I am embarking on some inclusion body problems too.

Reinhard