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From: rdonis@crcvms.unl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: problem with bacterial protein expression
Message-ID: <1990Sep17.234803.18570@hoss.unl.edu>
Date: 17 Sep 90 22:43:31 GMT
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Organization: UNL Computing Resource Center
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In article <9009161555.AA25938@genbank.bio.net>, SURF222@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@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@crcvms.unl.edu
rdonis@unlvax1.bi