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).

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Description/Abstract

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.

Item Type: Article
ISSNs: 1046-5928 (print)
Related URLs:
Keywords: expression, purification, transmembrane peptides, solid-state NMR
Subjects: R Medicine > R Medicine (General)
Divisions: University Structure - Pre August 2011 > School of Biological Sciences
ePrint ID: 154373
Date Deposited: 25 May 2010 10:34
Last Modified: 27 Mar 2014 19:12
URI: http://eprints.soton.ac.uk/id/eprint/154373

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