E. coli protein after NiNTA

EK khatipovNO-SPAM at NO-SPAMuchicago.edu
Sat Oct 19 23:01:59 EST 2002


"Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
news:Pine.SGI.4.33.0210191343270.5934524-100000 at mole.bio.cam.ac.uk...
> On Fri, 18 Oct 2002, EK wrote:
>
> >
> > "Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
> > news:Pine.SGI.4.33.0210181257520.6010164-100000 at mole.bio.cam.ac.uk...
> > > Dear Peter,
> > >           I made a beautiful powerpoint slide for my group on just
this
> > > subject.  One of the proteins I did N-terminal sequencing of was:
> > >
> > > P04475.  HSP70.  69kDa.
>
> > And it does not seem to have strings of histidines in it's sequence,
thus
> > the question is why does it co-purify on IMAC that AFAIK requires
> > availability of consecutive his residues?
> > -Emir
>
> . . . proteins do fold up!  For P04475 I could find no corresponding
> structure, but for another Ni-NTA sticky protein P17169...
>
> Glucosamine-6-P Synthase. 67kDa. pI 5.7. 1GDO.
> CGIVGAIAQR DVAEILLEGL RRLEYRGYDS AGLAVVDAEG HMTRLRRLGK VQMLAQAAEE
> HPLHGGTGIA HTRWATHGEP SEVNAHPHVS EHIVVVHNGI IENHEPLREE LKARGYTFVS
> ETDTEVIAHL VNWELKQGGT LREAVLRAIP QLRGAYGTVI MDSRHPDTLL AARSGSPLVI
> GLGMGENFIA SDQLALLPVT RRFIFLEEGD IAEITRRSVN IFDKTGAEVK RQDIESNLQY
> DAGDKGIYRH YMQKEIYEQP NAIKNTLTGR ISHGQVDLSE LGPNADELLS KVEHIQILAC
> GTSYNSGMVS RYWFESLAGI PCDVEIASEF RYRKSAVRRN SLMITLSQSG ETADTLAGLR
> LSKELGYLGS LAICNVPGSS LVRESDLALM TNAGTEIGVA STKAFTTQLT VLLMLVAKLS
> RLKGLDASIE HDIVHGLQAL PSRIEQMLSQ DKRIEALAED FSDKHHALFL GRGDQYPIAL
> EGALKLKEIS YIHAEAYAAG ELKHGPLALI DADMPVIVVA PNNELLEKLK SNIEEVRARG
> GQLYVFADQD AGFVSSDNMH IIEMPHVEEV IAPIFYTVPL QLLAYHVALI KGTDVDQPRN LAKSVTVE
>
> ...where once again the best string is only two histidines, the
> corresponding structure (1GDO) shows quite a few clusters of histidines
> in 3D space.
>
> Regards, Mike.
>

Yes, I also thought about His patches, but I don't remember seeing anywhere
that Ni can bind a discontinuous 6his epitope. Any references for that?
Dima's explanation sounds very plausible, too. As for Glucosamine-6-P
Synthase, it is possible that since it has a his-rich patch, it might stick
to say negative patches or loops on misfolded proteins. BTW, there are a
couple of putative Ni/Fe hydrogenases reported for E.coli that might also be
the source of contamination for IMAC.
Emir





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