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Re: [ccp4bb] Two off-topic questions
 

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CCP4bb <-- 2008 <-- May 2008 <-- 12 May 2008
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From: Emily Scott eescott {- at -} KU {- dot -} EDU
Date: 2008-05-12 		Next message:
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From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-05-13


Subject: Re: Two off-topic questions
From: Ashley Buckle ashley {- dot -} buckle {- at -} MED {- dot -} MONASH {- dot -} EDU {- dot -} AU
Date: 2008-05-12
Are they Lancashire Pigs?

On 13/05/2008, at 12:12 AM, Yong, Wei wrote:

> Dear all,
>
> I am sorry to bother you with two off-topic questions.
>
> Question one:
> I have solved a pig mitochondrial protein crystal structure whose
> molecular weight is about 95kd. However, I do not have the sequence
> information of this protein. It is weird that I could not blast its
> sequence in pig genome. Because pig sequences of known proteins are
> very similar to their human homologous, I used this protein's human
> homologous sequence to fit into electron density. Currently, I am
> planning to sequence the full length sequence of the protein.
> However, I was not able to successfully extract mRNA from pig liver
> using Qiagen RNeasy kit (I only tried once). Does any body have
> ideas about how to extract mRNA from pig live (or animal tissue in
> general)? Are there any other database I can search for pig protein
> sequence information?
>
> Question two:
> At the same time, I was trying to express the human homologous of
> the protein in E.Coli so that I would be able to do some functional
> studies. I tried pET21b and pET28a vectors. Protein was not
> expressed when pET28a construct was used but expressed in insoluble
> form with pET21b vector construct. I tried different strains, lower
> IPTG concentrations (down to 0.1 mM), lower temperatures (down to 15
> degree), adding cofactor, detergent extraction and so on. I even
> truncated the first 17 residues. I was not able to obtain soluble
> human protein at all. Does any body have experiences working with
> human mitochondrial proteins?
>
> There is another question which might be very naive. I saw people
> publishes important rat protein crystal structures although
> sequences of their human homologous are known. Does it mean that rat
> proteins are more stable than human ones?
>
> Any suggestions would be greatly appreciated.
>
> Thank you for your suggestions in advance!
>
> Best wishes
>
> Wei Yong



Ashley Buckle Ph.D
NHMRC Senior Research Fellow
The Department of Biochemistry and Molecular Biology
School of Biomedical Sciences, Faculty of Medicine &
Victorian Bioinformatics Consortium (VBC)
Monash University, Clayton, Vic 3800
Australia

http://www.med.monash.edu.au/biochem/staff/abuckle.html
iChat/AIM: blindcaptaincat
skype: ashley.buckle
Tel: (613) 9902 0269 (office)
Tel: (613) 9905 1653 (lab)

Fax : (613) 9905 4699




CCP4bb navigation

CCP4bb <-- 2008 <-- May 2008 <-- 12 May 2008
Previous message:
Subject: crystallography job posting
From: Emily Scott eescott {- at -} KU {- dot -} EDU
Date: 2008-05-12 		Next message:
Subject: Beamtime available; Deadline for applications : May 15.
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-05-13


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