Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier

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Title Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier
Author Tiralongo, Giuseppe; Ashikov, Angel; Routier, Françoise; Eckhardt, Matthias; Bakker, Hans; Gerardy-Schahn, Rita; von Itzstein, Mark
Journal Name Glycobiology
Year Published 2006
Place of publication United States
Publisher Oxford University Press
Abstract The architectural conservation of nucleotide sugar transport proteins (NSTs) enabled the theoretical prediction of putative NSTs in diverse gene databases. In the human genome, 17 NST sequences have been identified but only six have been unequivocally characterized with respect to their transport specificities. Defining transport characteristics of recombinant NSTs has become a major challenge because true zero background systems are widely absent. Production of recombinant NSTs in heterologous systems has developed multifunctionality for some NSTs leading to a novel level of complexity in the field. Assuming that (1) the specificity of NSTs is determined at the primary sequence level and (2) the proteins are autonomously functional units, final definition of the substrate specificity will depend on the use of isolated transport proteins. Herein, we describe the first report of the functional expression of mouse CMP-sialic acid transporter (CST) in Escherichia coli and thus provide significant progress towards the production of transporter proteins in quantities suitable for functional and structural analyses. Recovery of the active NST from inclusion bodies was achieved after solubilization with 8 M urea and stepwise renaturation. After reconstitution into phospholipid vesicles, the recombinant protein demonstrated specific transport for CMP-N-acetylneuraminic acid (CMP-Neu5Ac) with no transport of UDP-sugars. Kinetic studies carried out with CMP-Neu5Ac and established CMP-Neu5Ac antagonist's evaluated natural conformation of the reconstituted protein and clearly demonstrate that the transporter acts as a simple mobile carrier.
Peer Reviewed Yes
Published Yes
Publisher URI http://glycob.oxfordjournals.org/
Alternative URI http://dx.doi.org/10.1093/glycob/cwj029
Copyright Statement Copyright 2006 authors.This is an open access paper. http://creativecommons.org/licenses/by/3.0/ license that permits unrestricted use, provided that the paper is properly attributed.
Volume 16
Issue Number 1
Page from 73
Page to 81
ISSN 0959-6658
Date Accessioned 2007-02-23
Date Available 2009-05-27T08:31:26Z
Language en_AU
Research Centre Institute for Glycomics
Faculty Institute for Glycomics
Subject PRE2009-Bacteriology; PRE2009-Biochemistry and Cell Biology
URI http://hdl.handle.net/10072/13713
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1

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