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 alpha-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 His(6) 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 alpha-helix as predicted from the sequence.
Marius
P
P
School of Biological Sciences, University of Southampton, Basset Crescent East, Southampton SO16 7PX, UK.
Wright
J N
JN
Findlow
I S
IS
Williamson
P T F
PT
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014166:Transferases
D011506:Proteins; C491879:Fcmd protein, mouse; D014166:Transferases