E. coli protein after NiNTA
dk at no.email.thankstospam.net
Fri Oct 18 22:29:31 EST 2002
"EK" <khatipovNO-SPAM at NO-SPAMuchicago.edu> 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. pI 4.6. GKIIGIDLGT TNSCVAIMDG TTPRVLENAE
>> GDRTTPSIIA YTQDGETLVG QPAKRQAVTN PQNTLFAIKR LIGRRFQDEE VQRDVSIMPF
>> KIIAADNGDA WVEVKGQKMA PPQISAEVLK KMKKTAEDYL GEPVTEAVIT VPAYFNDAQR
>> QATKDAGRIA GLEVKRIINE PTAAALAYGL DKGTGNRTIA VYDLGGGTFD ISIIEIDEVD
>> GEKTFEVLAT NGDTHLGGED FDSRLINYLV EEFKKDQGID LRNDPLAMQR LKEAAEKAKI
>> ELSSAQQTDV NLPYITADAT GPKHMNIKVT RAKLESLVED LVNRSIEPLK VALQDAGLSV
>> SDIDDVILVG GQTRMPMVQK KVAEFFGKEP RKDVNPDEAV AIGAAVQGGV LTGDVKDVLL
>> LDVTPLSLGI ETMGGVMTTL IAKNTTIPTK HSQVFSTAED NQSAVTIHVL QGERKRAADN
>> KSLGQFNLDG INPAPRGMPQ IEVTFDIDAD GILHVSAKDK NSGKEQKITI KASSGLNEDE
>> IQKMVRDAEA NAEADRKFEE LVQTRNQGDH LLHSTRKQVE EAGDKLPADD KTAIESALTA
>> LETALKGEDK AAIEAKMQEL AQVSQKLMEI AQQQHAQQQT AGADASANNA KDDDVVDAEF EEVKDKK
>> which is the protein you are thinking of isn't it?
>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?
His binds Ni2+ best but other aromatic residues do well too.
hsp70 is a heat shock protein, a chaperone. It's very function is to stick
to misfolded crap, the kind of stuff that is in abandon when
overexpressing proteins. No wonder then it is frequently found
as contaminant in various tag-based purifications.
>> Regards, Mike.
>> On 17 Oct 2002, Peter Cherepanov wrote:
>> > Hi everybody!!!
>> > does anyone in the world know the identity of the E. coli 70 kDa protein
>> > that co-purifies on Ni columns?
>> > The expression and yields of the protein I work with are not very high,
>> > this band becomes a very significant contamination after first
>> > chromatography.
>> > It stays bound on NiNTA at least upto 40 mM imidazol. It is not a real
>> > problem, since it does not bind to SP sepharose, or Heparin seph, which
>> > use on a later stage, but I still would like to know, and would
>> > any hint. (I recall something about heat shock proteins - can it be the
>> > one???? HSP70????).
>> > thanks in advance,
>> > Peter
>> > ---
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