Expression and purification of the transmembrane domain of fukutin-I for biophysical studies
Marius, P., Wright, J.N., Findlow, Stuart C. and Williamson, P.T.F. (2010) Expression and purification of the transmembrane domain of fukutin-I for biophysical studies. Protein Expression and Purification, 72, (1), 107-112. (doi:10.1016/j.pep.2010.01.019). (In Press).
Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of a-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi.
To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be per- formed. Expressing the N-terminal domain of Fukutin-1 fused to a His6 tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media.
Preliminary biophysical analyses have confirmed the identity of the pep- tide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-struc- tured a-helix as predicted from the sequence.
|Keywords:||expression, purification, transmembrane peptides, solid-state NMR|
|Subjects:||R Medicine > R Medicine (General)|
|Divisions:||University Structure - Pre August 2011 > School of Biological Sciences
|Date Deposited:||25 May 2010 10:34|
|Last Modified:||08 Jun 2012 12:50|
|Contributors:||Marius, P. (Author)
Wright, J.N. (Author)
Findlow, Stuart C. (Author)
Williamson, P.T.F. (Author)
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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