Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies
Marius, P., Wright, J. N., Findlow, I.S. 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). (PMID:20117215).
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 α-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 performed. 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 peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured α-helix as predicted from the sequence.
|Keywords:||expression, purification, transmembrane peptides, solid-state nmr|
|Subjects:||Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
|Divisions:||Faculty of Natural and Environmental Sciences > Biological Sciences > Molecular & Cellular
|Date Deposited:||13 Mar 2012 13:36|
|Last Modified:||13 Mar 2012 13:36|
|Contributors:||Marius, P. (Author)
Wright, J. N. (Author)
Findlow, I.S. (Author)
Williamson, P.T.F. (Author)
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
Actions (login required)