protein production and purification
malexeye at JAGUAR1.USOUTHAL.EDU
Thu Oct 1 15:40:34 EST 1998
>Chen Ho An <chen at bsm.bioc.ucl.ac.uk> wrote:
>> Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) wrote:
> >system, E.coli, insect cells or eucaryotic system? ( my protein is a
> : > receptor ....)
> :> It depends on your protein. If your protein is a GPCR, forget about
> : >bacteria. If you want to purify it, insect cells are probably easier
> : t>o cultivate in large amounts than higher eukaryotic cells (like 293
> : c>ells).
> >I think this need further clarification. E.coli has been tremondously
> >successful in producing large number of difficult proteins. For example,
> >the TCR (T-cell receptor) has been successfully expressed in E.coli in
> >soluble form. See Mol. Immunol (98) 35(2), 73-81 and J Biol Chem (97)
> >272(51), 32190-7.
>As far as I know, this is only the extracellular domain of the TCR. It
>is true that E. coli can successfully express a lot of soluble proteins
>or protein domains, but has tremendous troubles expressing membrane
>proteins (with a few notable exceptions, for example bacteriorhodopsin
>or lac permease).
>Note that I was specifically talking about G-protein-coupled receptors
>(GPCRs). I know of a single example where a GPCR has been expressed in
>E. coli in inclusion bodies in relatively high yield and then refolded.
>However, it is not clear whether the refolded protein is indeed able
>to productively couple to G-proteins, i.e. it is not clear whether it
>is properly folded at all.
Note that Walker's group described mutants of BL21(DE3), C41 and C43 that
allow for overexpression of previously hard-or-impossible-to-overexpress
proteins in E.coli (the article published in J. Molec. Biol. in 1996, I
beleive. If necessary, I will look up the reference). They provide an
extensive list of membrane proteins that they successfully overexpressed in
these hosts. Of course, this does not guarantee that you will get lucky
with this system, but it is worth a shot...
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