title: Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris.
abstract: Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. CST inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells and to date, no crystal structure of the CST, or any other nucleotide sugar transporter exists. Here we describe the optimised conditions for expression in Pichia pastoris, solubilisation using n-nonyl β-d-maltopyranoside (NM) and single step purification of a functional CST. Importantly we show that despite being able to solubilise and purify the CST using a number of different detergents, only NM was able to maintain CST functionality.
authors: Institute for Glycomics, Griffith University, Gold Coast Campus, QLD 4222, Australia.
authors: Andrea
authors: A
authors: Maggioni
authors: Institute for Glycomics, Griffith University, Gold Coast Campus, QLD 4222, Australia.
authors: Barbara
authors: B
authors: Hadley
authors: Institute for Glycomics, Griffith University, Gold Coast Campus, QLD 4222, Australia.
authors: Mark
authors: M
authors: von Itzstein
authors: Institute for Glycomics, Griffith University, Gold Coast Campus, QLD 4222, Australia. Electronic address: j.tiralongo@griffith.edu.au.
authors: Joe
authors: J
authors: Tiralongo
mesh_terms: D001692:Biological Transport; D015153:Blotting, Western; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D027361:Organic Anion Transporters / Q000096:biosynthesis* / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D003902:Detergents; D027361:Organic Anion Transporters; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C421224:sialic acid transport proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid
keywords: CMP-sialic acid transporter; Detergent solubilisation; Membrane proteins; Nucleotide sugar transporter; Pichia pastoris