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

Expression and purification of the transmembrane domain of fukutin-I for biophysical studies
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 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 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 a-helix as predicted from the sequence.
expression, purification, transmembrane peptides, solid-state NMR
1046-5928
107-112
Marius, P.
2022328d-4410-4ac3-902d-5e1c87b3c958
Wright, J. N.
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
Findlow, I.S.
14e4601d-1383-42ef-8872-2cacadce4903
Williamson, P.T.F.
0b7715c6-b60e-4e95-a1b1-6afc8b9f372a
Marius, P.
2022328d-4410-4ac3-902d-5e1c87b3c958
Wright, J. N.
e53ee4b9-10f5-4365-a9f2-5a7b0eacd86d
Findlow, I.S.
14e4601d-1383-42ef-8872-2cacadce4903
Williamson, P.T.F.
0b7715c6-b60e-4e95-a1b1-6afc8b9f372a

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).

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 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 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: 14 Mar 2024 02:52

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Contributors

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

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