Re: [ccp4bb] protein degradation?

- Protein crystallography

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

Previous message:
Subject: Re: tricky molecular replacement
From: "Randy J {- dot -} Read" rjr27 {- at -} CAM {- dot -} AC {- dot -} UK
Date: 2007-11-04

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

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

On Nov 4, 2007, at 14:23, Eric Dollins wrote:

> 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.

indeed thats often the case; there are commercial cells with plasmids
encoding for rare codons, rosetta-2 work really well for us.

A.

> 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

CCP4bb navigation

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

Previous message:
Subject: Re: tricky molecular replacement
From: "Randy J {- dot -} Read" rjr27 {- at -} CAM {- dot -} AC {- dot -} UK
Date: 2007-11-04

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