problem with bacterial protein expression

rdonis at crcvms.unl.edu rdonis at crcvms.unl.edu
Mon Sep 17 17:43:31 EST 1990


-Message-Text-Follows-
In article <9009161555.AA25938 at genbank.bio.net>, SURF222 at kub.nl (Miebet) writes...
>Hi out there,
>Anyone present who could help me with a nice little problem?
>I am trying to express a protein fragment in E. coli using Studier's T7
>system. The expression vector has been made, including the correct
>coding region under the control of the T7 promoter. As a host, I use
>BL21(DE3) /pLysS. If I analyse lysed whole bacteria on a SDS-PAGE
>gel, large amounts of protein appear to be synthesized (app. 10-20
>ug/ml). However, if I lyse the bacterial pellet (either by freeze/thaw or
>by adding .1% Triton X-100), only 10% or less of the protein is in the
>supernatant, the rest is pelleted with the bacterial debris. Would anyone
>have a suggestion or useful tip as to how I could solubilize this
>precipitated protein in such a way, that it is in it natural conformation
>(again)? Would anyone know what is the reason the protein is not
>soluble in the first place? Or maybe someone has an idea on how I
>could prevent the protein from precipitating? Any suggestion, idea, hint
>or or other useful information would be most welcome to
> 
>                           Han Schilthuis
>                           Hubrecht Laboratory
>                           Utrecht, the Netherlands
>                           E-mail: SURF222 at HTIKUB5.BITNET

Some have found that growing the bugs at lower temperature (25-30 C) 
will reduce the formation of insoluble aggregates.
Good luck with this common problem to all those doing this kind of thing.
Ruben Donis
Rdonis at crcvms.unl.edu
rdonis at unlvax1.bi



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