problem with bacterial protein expression

Miebet SURF222 at
Fri Sep 14 18:40:00 EST 1990

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

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