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Re: [ccp4bb] protein degradation?
 

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CCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007
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Subject: Re: protein degradation?
From: Wataru Kagawa wkagawa {- at -} JOTA {- dot -} GSC {- dot -} RIKEN {- dot -} GO {- dot -} JP
Date: 2007-11-04 		Next message:
Subject: ΄πΈ΄: protein degradation?
From: Jiang Yu jyu {- at -} MAIL {- dot -} USTC {- dot -} EDU {- dot -} CN
Date: 2007-11-04


Subject: Re: protein degradation?
From: Eric Dollins d {- dot -} e {- dot -} dollins {- at -} GMAIL {- dot -} COM
Date: 2007-11-04
Are you expressing a eukaryotic protein? If so, you might want to
check for rare codons. There are a number of websites where you can
put in your coding sequence and check. I recently had this issue and
it turned out to be incomplete/stalled translation rather than
proteolysis as I had several rare codons in succession. You can
circumvent this by adding an antibiotic selectable plasmids encoding
the rare tRNAs.
Good luck
Eric


On 11/4/07, 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
>
>


--
D. Eric Dollins, Ph.D.
C266 LSRC, Research Dr.
Duke University Medical Center
Durham, NC 27710
(919) 681-1668, d.e.dollins@gmail.com
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