Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris Andrea Maggioni, Barbara Hadley, Mark von Itzstein, Joe Tiralongo ⇑ Institute for Glycomics, Griffith University, Gold Coast Campus, QLD 4222, Australia article info Article history: Received 8 April 2014 and in revised form 10 July 2014 Available online 19 July 2014 Keywords: CMP-sialic acid transporter Pichia pastoris Membrane proteins Detergent solubilisation Nucleotide sugar transporter 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 b-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. Ó 2014 Elsevier Inc. All rights reserved. Introduction Eukaryotic membrane proteins are well established as drug targets of great importance to the pharmaceutical industry [1]. The structural determination of membrane proteins though, espe- cially solute transporters, has been an arduous task, with many groups having limited success ([2,3] and references therein). One of the main bottlenecks in membrane protein structure determination is either poor recombinant protein expression, incomplete solubilisation of expressed protein, and/or low yield of functional protein following purification [4,5]. A range of strat- egies have been tested to try and overcome one or more of these bottlenecks, however no standardised single process or set of experimental conditions have been established that can be applied to all membrane proteins [2,6]. Therefore individual pro- tocols must be established specifically for each membrane protein of interest, including optimisation of expression, detergent solu- bilisation and purification. The cytidine 5 0 -monophosphate (CMP 1 )-sialic acid transporter (CST, SLC35A1), a member of a family of closely related proteins called nucleotide sugar transporters (NSTs), plays a central role in the glyco-protein and -lipid sialylation machinery [7,8]. The CST is a Golgi-resident type III trans-membrane protein with 8–10 pre- dicted trans-membrane domains (TMDs) that is essential for the transport of CMP-sialic acid into the Golgi apparatus of eukaryotic cells [7–9]. The CST is considered to be an attractive target for drug discovery given that its inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells [10]. However, the further development of more potent inhibitors has been hindered by the lack of 2D- or 3D- crystal structure data for any NST including the CST. A significant bottleneck towards achieving this is access to highly pure functional CST. To this end, the CST has been previ- ously expressed in several expression hosts including Saccharomyces cerevisiae [11–13], Escherichia coli [14,15] and Pichia pastoris [16]. However, the expression of the CST in different hosts has never fully addressed the complete precursory conditions required for potential http://dx.doi.org/10.1016/j.pep.2014.07.003 1046-5928/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Tel.: +61 7 5552 7029; fax: +61 7 5552 8098. E-mail address: j.tiralongo@griffith.edu.au (J. Tiralongo). 1 Abbreviations used: CMP, cytidine 5 0 -monophosphate; CST, CMP-sialic acid transporter; NSTs, nucleotide sugar transporters; TMDs, trans-membrane domains; CMC, Critical Micellar Concentration; GeFs, Golgi-enriched fractions; PC, phosphati- dylcholine; IMAC, Immobilised Metal Affinity Chromatography; NM, n-nonyl b-D- maltopyranoside; GeF-UI, Golgi-enriched fractions isolated from methanol-unin- duced; GeF-I, Golgi-enriched fractions isolated from methanol-induced. Protein Expression and Purification 101 (2014) 165–171 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep