Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier.
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.
Tiralongo
Joe
J
Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia.
Ashikov
Angel
A
Routier
Françoise
F
Eckhardt
Matthias
M
Bakker
Hans
H
Gerardy-Schahn
Rita
R
von Itzstein
Mark
M
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D051379:Mice; D033704:Nucleotide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D033704:Nucleotide Transport Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; C090989:cytidine 5'-monophospho-(N-acetylneuraminyl-2-8-N-acetylneuraminic acid); D003568:Cytidine Monophosphate