Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies

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.