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
