soluble protein expression in E. coli

Q. Shang qshang at u.washington.edu
Wed Jan 31 00:52:43 EST 1996



On 18 Jan 1996, D. Hodges wrote:

> Saverio Brogna (Genetics) (sb at mole.bio.cam.ac.uk) wrote:
> : Dear someone,
> : I have expressed e protein in E. coli with differents systems (His-tag, 
> : Glutation-transferase fusion and wild-type) but I can not get it soluble.
> : DO YOU KNOW HOW TO EXPRESS PROTEINS IN A WAY THAT SOME IS SOLUBLE?
> 
> : Thanks
> : -- 
> : Saverio Brogna
> : University of Cambridge, Departement of Genetics
> : Downing Street, Cambridge, CB2 3EH, England (UK)
> : tel +44-223-333970, fax +44-223-333992
> 
> You might like to try one of the systems that direct expression to the 
> periplasm. I have used pASK-60 from Biometra which worked OK. Then there's
> pET12a which should do the same.

I used pET20b which is supposed to direct protein to the periplasm. 
However my proteins still ended up in the inclusion body.  Apparently the 
signal sequence is not sufficient to lead recombinant proteins to the 
periplasm.
--

I came accross a paper (Blackwell and Horgan,FEBS 1991,vol 295,p10-12)) in 
which they expressed a recombinant protein in E.Coli in the presence of 
sorbitol and glycyl betain.  They reported a 427 fold improvement of 
soluble, active protein yield.  In their system LB gives better yield of 
soluble protein than M9(?)salt media and so does lower temperature.

Tanya Shang



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