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).


[img] PDF - Version of Record
Restricted to System admin

Download (1549Kb) | Request a copy


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
Digital Object Identifier (DOI): doi:10.1016/j.pep.2010.01.019
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
Accepted Date and Publication Date:
July 2010In press
Date Deposited: 25 May 2010 10:34
Last Modified: 31 Mar 2016 13:25

Actions (login required)

View Item View Item

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics