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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), pp. 107-112. (doi:10.1016/j.pep.2010.01.019).

Record type: Article

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.

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More information

Accepted/In Press date: July 2010
Published date: July 2010
Keywords: expression, purification, transmembrane peptides, solid-state NMR
Organisations: Molecular and Cellular

Identifiers

Local EPrints ID: 154373
URI: http://eprints.soton.ac.uk/id/eprint/154373
ISSN: 1046-5928
PURE UUID: 0727590d-ab54-475a-afd4-ff2426c95ceb
ORCID for P.T.F. Williamson: ORCID iD orcid.org/0000-0002-0231-8640

Catalogue record

Date deposited: 25 May 2010 10:34
Last modified: 18 Jul 2017 12:47

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Contributors

Author: P. Marius
Author: J. N. Wright
Author: I.S. Findlow

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