SURF222@kub.nl (Miebet) (09/15/90)
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.BITNETrdonis@crcvms.unl.edu (09/18/90)
-Message-Text-Follows- 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