Re: [ccp4bb] protein degradation?

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: protein degradation?
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2007-11-04

Next message:
Subject: refined vs 'others'
From: Bernhard Rupp bernhardrupp {- at -} SBCGLOBAL {- dot -} NET
Date: 2007-11-04

Subject: Re: protein degradation?
From: Anthony Addlagatta anthony {- at -} UOXRAY {- dot -} UOREGON {- dot -} EDU
Date: 2007-11-04

Hi Vijay,

It is possible that the difference between the two bands is simply a
methionine ! If you are working with a low resolution mass spec, it
may not be very obvious. In most of the newly synthesised proteins
both in prokaryotes and eukaryotes, the initiator methionine is
removed by an enzyme called methionine aminopeptidase (MetAP).
However, next amino acid has to be small and uncharged. Also in large
over expressions, the natural MetAP may not be sufficient to handle
the production rate of the protein of interest.

This problem could easily be ignored because your protein with the N-
terminal His-tag sticks to the metal affinity resin. So you assume
that nothing is wrong with the amino terminus. It is worth looking at
this aspect in addition to several other good suggestions.

Good luck!

Anthony
________________________________
Anthony Addlagatta, PhD
Institute of Molecular Biology
University of Oregon
Eugene, OR-97403
Phone: (541) 346-5867
Fax: (541)346-5870
Web: http://uoregon.edu/~anthony

On Nov 4, 2007, at 4:22 AM, Vijay Kumar wrote:

> Hi,
>
> I have been trying purify a N-ter his-tagged protein over-expressed
> in E.coli. After purification (Ni-NTA or Co-Talon), I find two
> bands in SDS PAGE which are very close each other (top band in the
> right MW and more intense than the lower band). Western blot (for
> his-tag) of the gel gave signal for both the bands. Mass spec
> results confirmed both protein bands are the same. So I think it
> could be C-ter degradation of my protein. Also the 2 bands exist
> after ion-exchange and sizing column.
>
> I use commercially available complete protease inhibitor tablets
> (increasing concentration has no effect) and sonication for lysis.
> I am wondering if people have encountered the same problem and got
> any suggestions?
>
>
> Thanks in advance.
>
> Regards,
>
> Vijay
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: protein degradation?
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2007-11-04

Next message:
Subject: refined vs 'others'
From: Bernhard Rupp bernhardrupp {- at -} SBCGLOBAL {- dot -} NET
Date: 2007-11-04