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Protein Expression and Purication 59 (2008) 6978 www.elsevier.com/locate/yprep

Production, characterization and crystallization of the Plasmodium falciparum aquaporin

Kristina Hedfalk a,*, Nina Pettersson b,1, Fredrik Oberg a,2, b a,3 Stefan Hohmann , Euan Gordon
a

Department of Chemistry and Bioscience/Molecular Biotechnology, Chalmers University of Technology, Box 462, S-405 30 Goteborg, Sweden b Department of Cell and Molecular Biology, Goteborg University, Box 462, S-405 30 Goteborg, Sweden Received 25 October 2007, and in revised form 17 December 2007 Available online 26 January 2008

Abstract The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the rst bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to dierent genomic A + T content and dierent codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be conrmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64 mg PfAQP per liter cell culture) PfAQP was puried to homogeneity (18 mg/L) and initial attempts at crystallization of the protein yielded several dierent forms. 2008 Elsevier Inc. All rights reserved.
Keywords: Malaria; Aquaporin; Codon-optimisation; Pichia pastoris; Purication

Malaria is one of the major infectious diseases in the world with 300500 million estimated cases and 12 million fatalities every year [1]. The disease is caused by obligate intracellular parasites of the genus Plasmodium and is transmitted to human by the Anopheles gambiae mosquito. Plasmodium falciparum is the most fatal of the four Plas* Corresponding author. Present address: Department of Chemistry/ Biochemistry, Goteborg University, Box 462, S-405 30 Goteborg, Sweden. Fax: +46 31 786 3910. E-mail address: kristina.hedfalk@chem.gu.se (K. Hedfalk). 1 Present address: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland. 2 Present address: Department of Chemistry/Biochemistry, Goteborg University, Box 462, S-405 30 Goteborg, Sweden. 3 Present address: Structural Chemistry Laboratory, AstraZeneca, Molndal, Sweden.

modia species. Infection occurs when a mosquito bearing the parasite feeds on the hosts blood and in the process releases parasite sporozoite stages into the blood stream. These sporozoites then infect liver cells, where asexual replication occurs to produce large numbers of merozoites, the parasite stage that infects red blood cells. Upon infection of red blood cells this merozoitic parasite rapidly divides, ultimately resulting in cell lysis and production of merozoites ready for a new infectious cycle. It is in this phase that most fatalities occur. P. falciparum has become largely resistant to currently available medicines and hence it is of great importance to search for new ways of combating this disease [2]. Membrane transporters are essential for the parasite for uptake of nutrients, amino acids and biosynthetic precursors; therefore transporters are potential anti-malaria drug

1046-5928/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.01.004

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targets [3,4]. The P. falciparum genome project revealed a high adenosine and thymidine (70.5%) content as well as a low number of genes encoding enzymes and transporters as compared to the genomes of other free living eukaryotes [5]. It is believed that intracellular parasites during evolution have reduced the number of membrane proteins in order to evade the host immune defense. If this assumption is correct, then membrane transporters are likely to be essential for the parasite. Aquaporins are found in all ve kingdoms of life and divided into two subgroups: the orthodox aquaporins, transporting mainly water; and the aquaglyceroporins, which transport various other small, uncharged compounds such as polyols, urea and arsenic [68]. Aquaporins are homotetramers consisting of four monomeric channels. A common membrane domain organization of six alpha helical transmembrane segments, with cytosolic N- and C-termini is also shared by all aquaporins. These six transmembrane helices are connected by ve loops, labeled AE. The loops B and E form helices spanning about half of the membrane bilayer, meeting in the center of the pore. These loops contain the highly conserved NPA motifs which act as lters in the channel. Plasmodium falciparum has one single gene coding for a protein from aquaporin family, PfAQP [9]. Parasites belonging to the phylum Apicomplexa also only contain one aquaporin gene [10], as is the case for Toxoplasma gondii, which contains one bifunctional aquaglyceroporin [11]. The causal agent of sleeping sickness, Trypanosoma brucei, on the other hand, has three aquaporins [12]. It is believed that PfAQP might be of importance for the parasites ability to rapidly proliferate within the erythrocyte. Proliferation is linked to massive production of cell membranes and de novo lipid synthesis requires glycerol to be taken up from the blood serum [9] and the only glycerol uptake mechanism is via PfAQP [10]. PfAQP is bifunctional, transporting glycolytic metabolites, ammonia, polyols of up to ve carbons as well as water [9,13,14]. The PfAQP sequence is 35% identical to that of the well characterized glycerol facilitator (GlpF)4 from Escherichia coli. Quite interestingly, the highly conserved NPA-motifs in loops B and E show variation among eukaryotic microorganisms: the third position in loop B and the second position in loop E often dier, e.g. NPS/ NLA in Fps1 from Saccharomyces cerevisiae [15]. In PfAQP these motifs are NLA/NPS. In both Fps1 and PfAQP, function is retained when the motifs are mutated to NPA, but transport is abolished when NLA/NPS are switched to NPS/NLA in P. falciparum [9,16]. Residue E125, situated in the pore mouth of PfAQP, is crucial for the high water permeability, while pore lining residues aect the permeability of certain polyols [17].

To develop an inhibitor of PfAQP detailed structural and functional data are indispensable and for such studies a high yield of pure protein is required. High-resolution structures are available for aquaporins from bacteria [18,19], mammals [2024], plants [25] and archea [26], but there is yet no aquaporin structure from a eukaryotic microorganism. In this study, PfAQP has been expressed in E. coli and the methylotrophic yeast Pichia pastoris, respectively. Codon optimization of the PfAQP gene was necessary to achieve high-level production. PfAQP produced in P. pastoris was puried, characterized and crystallized and a functional assay was developed in bakers yeast, S. cerevisiae. Materials and methods Plasmids, strains and growth conditions for functional expression The gene for wild type PfAQP (wt-PfAQP, kindly provided by Dr. Eric Beitz, Tubingen, Germany [9]) was codon optimized for E. coli and P. pastoris, respectively, by Geneart (Germany) resulting in the articial genes EOpt-PfAQP and POpt-PfAQP. For cloning in S. cerevisiae, both the wtPfAQP and the POpt-PfAQP (optimized for P. pastoris) genes were amplied by PCR using the primers 50 -CG CCCATGGATGCATATGTTAT-30 (forward) and 50 -CA TCCCGGGCAAATCTACACCATCTTT-30 (reverse) for the wild type gene and 50 -CGCCCATGGATGCACA TGTTGT-30 (forward) and 50 -CTACCCGGGCAAATCA ACACCATCCTT-30 (reverse) for the optimized gene, respectively. Each gene was inserted between the NcoI and XmaI sites of the pYX242 vector (a multi-copy 2 l origin vector, with a constitutive TPI promoter, and a C-terminal HA-tag). The yeast strains used for characterization of wt-PfAQP and POpt-PfAQP were isogenic to W303-1A (MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0) [27]: YSH 642 (gpd1D::TRP1 gpd2D::URA3) [28] and YSH690 (gpd1D::TRP1) [29]. Transformation of plasmids into S. cerevisiae was carried out using the LiCl one-step transformation protocol [30]. Briey, yeast cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose). Growth of transformants was performed in synthetic medium (YNB containing 2% glucose) lacking leucine for selection. For growth assays, cells were pregrown for 2 days on YNB plates, resuspended in YNB to an OD600 = 0.2 and diluted in 10-fold dilution series. Each diluted cell suspension, 5 lL, was spotted onto agar plates supplemented with osmoticum and/or inhibitors as indicated and growth was monitored for 25 days in 30 C. Production of recombinant PfAQP in E. coli and P. pastoris for purication

Abbreviations used: PfAQP, Plasmodium falciparum aquaglyceroporin; wt-PfAQP, wild type PfAQP; IFV, initial fermentation volume; GlpF, glycerol facilitator.

Production of PfAQP in E. coli was carried out using the strain BL21 Star (DE3) (Invitrogen) and autoinduction at

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18 C. Essentially, cloning of the PfAQP genes (wild type [9] and optimized variant (EOpt-PfAQP, Geneart, Germany)) were amplied by PCR using the primer pairs PfnatgwF50 -GGGGACAAGTTTGTACAAAAAAGC AGGCTTCATGCATATGTTATTTTATAAATC-30 and PfnatgwR50 -GGGGACCACTTTGTACAAGAAAGC TGGGTGCAAATCTACACCATCTTTTTC-30 and Pfop tgwF50 -GGGGACAAGTTTGTACAAAAAAGCAGG CTTCCACATGCTGTTCTATAAAAGC-30 and Pfoptg wR50 -GGGGACCACTTTGTACAAGAAAGCTGGG TGCAGATCCACGCCATCTTTTTCG-30 , respectively. The primers contained attB recombination sites and the cloned genes were transferred into pDEST17 vector using the Gateway (Invitrogen) BP recombination reaction. Cloning was conrmed by DNA sequencing before the open reading frame was transferred using the Gateway LR reaction to the pEG1035 vector, a T7 based expression vector which confers a C-terminal 6-Histidine tag (Gordon et al., unpublished data). For production in the methylotrophic yeast P. pastoris, wt-PfAQP [9] and POpt-PfAQP (Geneart, Germany) were cloned in the expression vector pPICZB (Invitrogen). In both cases, PfAQP was produced as fusion with a C-terminal myc- and 6-Histidine tag, respectively. For cloning, the wild type gene was amplied by PCR using forward primer, 50 -TTATTCGAAAAAATGCATATGTTATTTTAT AAATC-30 , and reverse primer, 50 -CCTTCTAGAGCCA AATCTACACCATCTTTTTC-30 introducing a SfuI and an XbaI site (underlined). The POpt-PfAQP gene was cloned directly in pPICZB using EcoRI and XbaI. The resulting plasmids were linearized with PmeI before transformation into the P. pastoris wild type strain X-33 (Invitrogen) using the LiCl method (Invitrogen). To screen for protein production, 10 transformants from each plasmid were selected and analyzed as described previously [31]. One clone of POpt-PfAQP was selected for large-scale growth and purication. For quantitation of the relative production level, three independent cultures were grown at intermediate scale and analyzed as described previously [32]. To optimize growth and production, P. pastoris producing recombinant POpt-PfAQP was grown in a 3 L fermentor (Belach Bioteknik AB). The initial fermentation volume (IFV) of 1.5 L basal salt medium [33] containing 6.53 mL PTM1 trace salts [34] and 4% glycerol was inoculated with 75 mL from an overnight preculture in BMGY (Invitrogen Corporation) resulting in a start OD600 of 0.1. When the initial glycerol feed was consumed after approximately 24 h, the culture was fed with 5% glycerol for 4.5 h (about 50 mL was consumed) to increase biomass. To produce recombinant POpt-PfAQP the culture was fed with 100% methanol for 48 h (340 mL methanol consumed). Water transport assay To test the function of recombinant PfAQP in yeast, water transport in yeast protoplast from S. cerevisiae was

used. To make protoplasts, cells were grown to mid logarithmic phase, harvested, washed once in water and once in 1 M sorbitol, and nally resuspended in SCE buer (1 M sorbitol, 0.1 M sodium citrate, 10 mM EDTA, 0.2 mM b-mercaptoethanol, pH 6.8) containing 2000 U of lyticase (Sigma) per mL of culture and incubated with shaking for 3 h at 30 C. Formation of protoplasts was conrmed microscopically. Protoplasts were harvested, washed twice and nally resuspended in STC buer (1 M sorbitol, 10 mM TrisHCl, pH 7.5, 10 mM CaCl2). For the hypo-osmotic shock, the protoplast solution was diluted to 0.5 M sorbitol and the decrease in the relative OD600 value was monitored at dierent time points up to 120 s and compared to a positive (human AQP1 protoplasts) and a negative control (empty plasmid), respectively. The average of three independent transformants SD was estimated and normalized to the starting optical density of the culture. Immunoblot was used to verify correct localization of recombinant PfAQP produced in S. cerevisiae. Transformed cells were harvested in mid-exponential phase, washed (10 mM TrisHCl pH 7.5, 0.5 M sucrose, 2.5 mM EDTA) and resuspended in homogenization buer (50 mM TrisHCl pH 7.5, 0.3 M sucrose, 5 mM EDTA, 1 mM EGTA, 5 mg/mL BSA, 2 mM DTT, protease inhibitor cocktail (Roche)). Cells were disrupted in a FastPrep (Savant), centrifuged at 10,000g for 10 min and the supernatant was centrifuged further at 100,000g for 90 min to collect the membrane fraction. The membrane pellet was resuspended in buer (10 mM TrisHCl pH 7.0, 1 mM EGTA, 1 mM DTT, 20% (v/v) glycerol, protease inhibitor cocktail), 15 lg total protein was separated by SDSPAGE and transferred to Hybond-ECL membranes (GE Healthcare). Membranes were blocked with PBS5% milk and probed with 1:5000 diluted anti-PfAQP rabbit antibody (kindly provided by E. Beitz) for 1.5 h. The membrane was washed and incubated for 1 h with the secondary antibody (anti-rabbit IgG (Cell Signalling) diluted 1:2500) in PBS5% milk. After a nal wash, the signal was visualized using chemiluminiscence. Purication of POpt-PfAQP For purication, P. pastoris cells containing overproduced POpt-PfAQP were resuspended in buer A (20 mM Tris, pH 8.0, 100 mM NaCl, 500 lM EDTA, 10% glycerol), broken either by an X-Press or a French Press (four passages). The lysate was cleared by centrifugation (15,000g, 45 min). The membrane fraction was isolated from the cleared supernatant by ultracentrifugation in a Ti45 rotor (140,000g, 2 h). The membrane pellet was resuspended in buer B (20 mM Tris, pH 8.0, 100 mM NaCl, 50 lM EDTA, and 10% glycerol) and recentrifuged as before. Finally, the membrane pellet was resuspended in buer B at a concentration of about 200 mg/mL. Before purication, an initial detergent screen was preformed to nd a detergent suitable for solubilization of PfAQP from

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the P. pastoris membranes. In total, eight dierent detergents (5% Cholate, 1.5% DDM, 2% BriJ35, 2% OPOE, 2% b-octylglucoside, 0.5% LDAO, 2% Triton X-100, and 1% Fos-choline-12) resuspended in buer (20 mM Tris, pH 8.0, 100 mM NaCl, 50 mM KCl, 50 lM EDTA, 10% glycerol) were used (1 mL/100 lg membrane). The samples were incubated on ice for 1 h, centrifuged at 100,000g for 30 min and the supernatant applied to 100 lL Ni+-charged NTA resin. After 2 h incubation, the NTA matrix was pelleted at 3000g for 2 min and the supernatant (FT, ow through) was saved for gel analyses. The resin was washed in 1 mL (20 mM Tris, pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol, 0.05% DDM) and the protein eluted in 100 lL wash buer containing 500 mM imidazole. Flow through and elutions, 10 lL of each, was analyzed by SDSPAGE. For large-scale purication and solubilization, about 600 mg of membrane was resuspended in 30 mL of buer B containing 20 mM imidazole, 3% boctylglucoside and mixed with gently agitation for 1 h at 4 C. The solubilized membranes were centrifuged at 15,000g for 20 min, and the supernatant, containing solubilized membranes, transferred to a 50 mL Falcon tube containing 1 mL (packed volume) of Ni+-charged NTA resin. To allow binding to the column, this mixture was incubated at 4 C with gentle agitation for 3 h. The mixture was centrifuged at 3,000g for 2 min to pellet the NTA matrix. The supernatant, i.e. the unbound fraction, was discarded and the pelleted NTA resin resuspended in 50 mL of 20 mM Tris, pH 8.0, 300 mM NaCl, 35 mM imidazole, 10% glycerol, and 0.04% dodecyl-b-D-maltoside (DDM) in order to wash the resin. This wash was repeated twice, and the NTA resin was subsequently taken up in the wash buer and packed into a plastic 2 mL column (Qiagen). The bound protein was eluted by the addition of 6 mL of elution buer (20 mM Tris, pH 8.0, 300 mM NaCl, 750 mM imidazole, 10% glycerol, 0.04% DDM) and fractions of 500 lL were collected. Protein containing fractions were identied by UVvis absorption spectroscopy, pooled and concentrated to 500 lL in a Vivaspin 100 K spin concentrator. The concentrated protein was loaded in to a 10/ 30 HR200 size exclusion column pre-equilibriated with 20 mM Tris, pH 8.0, 50 mM NaCl, 5% glycerol, and 0.04% DDM, eluted with the same buer and fractions of 500 lL were collected. Protein containing fractions were pooled (only the center of the elution peak was selected) and concentrated to about 10 mg/mL in a VivaSpin 100 K concentrator. Crystallization of POpt-PfAQP Before screening for crystallization conditions the concentrated protein solution was centrifuged at 100,000g for 40 min. Crystallization screening was carried out using a combination of sitting drop (Cartesian robot (200 nL drops: 100 nL protein, 100 nL reservoir solution)) and hanging drop methodology with 2 lL nal drop size (1 lL protein, 1 lL reservoir solution). The MemSys and

MemStart kits (Molecular Dynamics) were used as starting points for screening for crystallization conditions, screens were set-up in duplicate at 18 C and 4 C. Results Codon optimized PfAQP is produced in both E. coli and P. pastoris Initially, production of the wild type PfAQP gene was attempted in both E. coli and P. pastoris (Table 1). The level of protein production in both hosts was poor. This is likely due to the GC content, which in P. falciparum is about 29% but about 50% for both E. coli and P. pastoris. Moreover, the Codon Adaptation Index, (a measure of the similarity of the codon usage of a gene to that of the proposed host organism) [35] of wild type PfAQP gene is 0.429 for E. coli and 0.17 for P. pastoris. This indicates that while the PfAQP wild type might be produced in E. coli, highlevel production is unlikely in either host. Hence two articial genes coding for PfAQP (EOpt-PfAQP and POptPfAQP) were designed taking into account the optimal codon usage and GC content for E. coli and P. pastoris, respectively. Gene optimization resulted in changes to 189 (72%) codons for the P. pastoris gene and 147 (58%) for the E. coli gene giving a nal GC content of 50% (E. coli) and 44% (P. pastoris) compared to the wild type gene having 29% GC. Production using the optimized PfAQP genes was tested under dierent expression conditions (Table 1). The production achieved with E. coli was stable and of expected size, but the levels were still low since the protein was divided between the soluble and insoluble fractions (Fig. 1A). However, production of POptPfAQP in P. pastoris was also stable and of expected molecular weight, but the production level was apparently high and the protein was localized to the membrane (Fig. 1B). Correct membrane localization is an indicator of correctly folded and likely active protein. To achieve even higher levels of recombinant protein, the growth was scaled-up in a 3 L fermentor (total volume of 1750 mL, 48 h of induction) to satisfy the high oxygen demand of P. pastoris. Cultivating X-33/POpt-PfAQP under tightly controlled conditions resulted in a substantial increase in the total cell mass of 157 g (wet weight; OD600 = 57). POpt-PfAQP was mainly located in the membrane fraction (data not shown). Notably, growth has a major impact on the production of the recombinant protein in P. pastoris. By cultivating the cells in fermentor (grey bar), the relative production level of POpt-PfAQP in the membrane was increased 26-fold as compared to shake ask cultures (white bar) (Fig. 1C). Codon optimized PfAQP produced in yeast is functionally active To obtain evidence that PfAQP produced in yeast was active we tested its transport capacity in whole cell assays

K. Hedfalk et al. / Protein Expression and Purication 59 (2008) 6978 Table 1 Production of PfAQP in both E. coli and P. pastroris Organism E. coli E. coli E. coli P. pastoris P. pastoris Gene/Construct pWt-PfAQPC-his pWt-PfAQPC-his pRARE pEOpt-PfAQPC-his Wt-PfAQP POpt-PfAQP Protein production /+ /+ + +++

73

Location m/i m/i m/i NA m

Protein production levels insucient for purication is indicated as /+, while +++ indicates high overproduction. Localization is indicated as (i) inclusion body (not in the membrane fraction), or (m), located in the membrane fraction, as assayed by immuno blotting. pRARE is a plasmid that supplies the rare tRNA codons as a supplement to the usual tRNAs present in E. coli as to aid production of eukaryotic proteins in this organism.

Fig. 1. Production of codon optimized PfAQP (Opt-PfAQP) in Escherichia coli and Pichia pastoris. (A) Immunoblot showing that the production of EOpt-PfAQP in E. coli is divided between the soluble and insoluble fractions. (B) Immunoblot showing membrane localized POpt-PfAQP from three dierent P. pastoris shake ask cultures. The reproducible signal indicates a stable production of POpt-PfAPQ in the membrane. (C) Quantitation of the relative production levels of POpt-PfAQP in the P. pastoris membrane when cultured in shake asks (n = 3 SD), white bar, and fermentor, grey bar, respectively. The relative production level of POpt-PfAQP in the membrane is increased 26-fold in fermentor-grown cells.

using the yeast S. cerevisiae, in which systems for testing of aquaporins have been developed [8,36]. The correct targeting of POpt-PfAQP protein to the membrane was conrmed by Immunoblot on isolated cell membranes, using a polyclonal antibody directed against the protein (Fig. 2A). When S. cerevisiae cells were exposed to hyperosmotic stress (0.5 M NaCl), transformants containing the optimized PfAQP gene, but not the wild type gene, showed decreased growth, indicating the presence of a functional glycerol channel (Fig. 2B). This experiment was done using a yeast strain lacking GPD1, encoding one of the two Gpd isoenzymes required for biosynthesis of glycerol. This strain is by itself slightly osmosensitive and hence eects are amplied. The observed osmosensitivity was due to osmotic and not salt stress since it was observed also with high concentrations of the osmolytes KCl and sorbitol (Fig. 2C). Addition of CuSO4, an inhibitor of aquaporins, diminished osmosensitivity mediated by expression of rat AQP3, but not that of POpt-PfAQP, pointing to a specic eect (Fig. 2D).

Functionality was also tested in a system, where glycerol uptake is indirectly monitored as a function of the cells ability to grow in presence of very high glycerol levels. This system is based on the use of a yeast strain decient in glycerol production (gpd1Dgpd2D). Cells unable to produce glycerol are highly osmosensitive. However, if hyperosmotic stress is mediated by glycerol, uptake of glycerol via a membrane channel can rescue cells which regain turgor and continue to grow. This was observed for cells transformed with POpt-PfAQP, but not with the wild type gene (Fig. 2E). Analysis of the capacity of PfAQP to transport water when expressed in S. cerevisiae cells was carried out with protoplasts from cells expressing POpt-PfAQP, human AQP1 or an empty plasmid. Protoplasts containing water channels burst faster than control cells upon a hypo-osmotic shock (Fig. 2F for human AQP1). However, POptPfAQP did not mediate water transport in this assay, although such capacity was previously reported in the Xenopus oocyte system [9]. Water transport has previously

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Fig. 2. Biological activity test of recombinant PfAQP in yeast. (A) Membrane localization of POpt-PfAQP shown by Western blot analysis of the total cell membrane fraction from yeast cells expressing POpt-PfAQP using an anti-PfAQP antibody. (B) For growth tests, cells were spotted in 1:10 dilution series on synthetic growth media supplemented with the indicated compounds. At high osmolarity (0.5 M NaCl) yeast cells (gpd1D) expressing POpt-PfAQP show decreased growth as compared to cells transformed with wild type PfAQP or empty vector. (C) The same growth phenotype is observed at high concentrations of KCl and sorbitol, indicating that the observed eect is due to high osmolarity and not sodium toxicity. (D) The hyperosmotic growth defect of POpt-PfAQP is not suppressed by addition of 1 mM CuSO4, which is a known inhibitor of AQP3. (E) Expression of POpt-PfAQP, but not wild type PfAQP or control cells, suppresses the growth defect of a yeast strain decient in glycerol production (gpd1Dgpd2D) at high glycerol levels. (F) Yeast protoplasts expressing an orthodox aquaporin (human AQP1) burst faster than protoplats expressing POpt-PfAQP or an empty plasmid when exposed to hypo-osmotic stress. Protoplast bursting was monitored for 2 min as a decrease in optical density (600 nm). The starting value of each transformant was set to 1.0 and all data were normalized to this value. An average SD of three independent experiments is presented.

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been observed in the yeast system for rat AQP5 and yeast Aqy2 ([36] and Pettersson et al., unpublished results). Highly pure POpt-PfAQP is achieved by a two step purication process A detergent screen was carried out to determine the optimal conditions for solubilization and purication of POpt-PfAQP from the P. pastoris membranes (Fig. 3A). Of the eight detergents tested, both DDM and OG showed a high degree of solubilization, extracting the majority of the protein from the membrane fraction. We chose to use b-octylglucoside based partially on its previous successful use with aquaporins (e.g. GlpF and SoPIP2;1 [25,37]). After solubilization of the membrane fraction with 3% octylglucoside, POpt-PfAQP was selectively puried by the use of the C-terminal His6 tag. The detergent b-octylglucoside was exchanged under the washing steps to DDM as we observed some instability of the protein when stored in b-octylglucoside for prolonged periods. This behavior was not apparent in the presence of the detergent DDM. After elution from the NTA resin and concentration, the protein was further puried by size exclusion chromatography. A typical trace of the elution prole is shown in Fig. 3B. The protein

eluted as two peaks, which have the same appearance when analyzed on SDSPAGE (data not shown), and which probably represent the tetrameric and monomeric forms of the protein. After size exclusion chromatography the protein was essentially pure as seen on SDSPAGE analysis (Fig. 3B). The yield after purication was estimated to 18 mg pure POpt-PfAQP per liter cell culture, which corresponds to 1.9 mg POpt-PfAQP/g crude membranes and 0.14 mg POpt-PfAQP/g wet cells, by protein determination on the pure fraction (Table 2). Ordered crystals are formed from highly pure POpt-PfAQP Gel ltration-puried POpt-PfAQP protein was concentrated in a Vivaspin 100 K cut-o centrifuge concentrator. The large cut-o size was chosen to prevent the inclusion of detergent micelles (DDM micelles are $70 kDa). The protein was concentrated to approximately 15 mg/mL (calculated using the predicted extinction coecient of 34,280 M/L). Crystallization conditions were explored by using the commercial kits MemSys and MemStart with duplicate set-ups at 4 C and at 18 C. Crystals were obtained after 4 days of incubation at room temperature and 7 days at 4 C (Fig. 4).

Fig. 3. Solubility and purication of POpt-PfAQP. (A) Solubility screen for POpt-PfAQP. Total cell membrane was solubilized and puried protein was analyzed by SDSPAGE (FT, ow through, and E, eluted fractions). Detergents used were: (1) Cholate, (2) DDM, (3) BriJ35, (4) OPOE, (5) boctylglucoside, (6) LDAO, (7) Triton X-100, (8) Fos-choline-12. (B) Purication of POpt-PfAQP by size exclusion chromatography. PfAQP puried by anity chromatography was applied to a Sephadex HR200 column, and the column developed by washing with 1.5 column volumes of 0.04% DDM in 20 mM Tris, pH 8.0, 100 mM NaCl, 5% glycerol. POpt-PfAQP was eluted as two peaks, which likely represent the tetrameric and dimeric forms.

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Table 2 Purication of POpt-PfAQP from P. pastoris Purication Step Membrane Washed membrane Ni-NTA Gel Filtration Total protein (mg) 1310 1030a 24a 18a
a

POpt-PfAQP (mg) 64 54b 20b 18

b

Purity 0.048 0.052 0.95 1

Recovery 1 0.85 0.35 0.28

Fold of purication 1 1.1 20 21

Purication steps and estimated yield from 1 L yeast culture. a Total protein estimated with Bio-Rad protein assay using BSA as a standard. b Amount of POpt-PfAQP estimated by immunoblot using puried POpt-PfAQP as a standard.

Fig. 4. Examples of protein crystals of puried PfAQP obtained by screening; conditions were explored by using the commercial kits MemSys and MemStart.

Discussion Here we report the high-level overproduction of PfAQP in the methylotrophic yeast P. pastoris as well as the generation of ordered crystals for structural studies. Since the protein appears to be functional in S. cerevisiae we assume that it is likely that the protein produced in the related species P. pastoris is also functional. To our knowledge this is the rst reported overproduction and crystallization of a putative membrane-localized drug target from P. falciparum. Successful heterologous production of eukaryotic membrane proteins leading to high-resolution structures is far from trivial. To our knowledge it has only been reported seven times [22,25,3842]. To achieve high yields of PfAQP it was necessary to synthesize genes with optimized codon and A + T content since rst expression trials with the wild type gene indicated that production occurred at very low or undetectable levels both in bacteria and in P. pastoris. Both the wild type and optimized constructs shared identical 50 and translation start sequences. Instead of carrying out repeated rounds of site directed mutagenesis we resynthesized the entire coding sequence, optimizing one construct for E. coli and one for P. pastoris. The methods applied to optimize genes by Geneart are described in patent WO2004059556 (Method and device for optimizing a nucleotide sequence for the purpose of expression of a protein). These include the removal of cis-active sequence motifs, substancial regions of homology in the target DNA sequence, inverse repeats etc. The choice of actual codon (where multiple codons are available) is not clear,

however, there is a trade o in any codon optimization between increased GC content (which is in general has a positive eect on mRNA half life) and codon choice. Out of curiousity we made a comparision of the Genart calculated sequence and a home made gene sequence using the JCAT webserver (http://www.jcat.de/Start.jsp) [43]. The JCAT server produced a gene optimized albeit for S. cerevisiae which was 93% identical while the JCAT version of the E. coli gene was 80% identical to the gene optimized by Genart, with the majority of codon choices in the Geneart case belonging to class I (intermediate to highly expressed genes), and the JCAT version class III (catabolic genes). After optimization, production in E. coli was still poor, indicating that the problem was more likely related to folding and membrane insertion, a phenomenon we recently observed also with mammalian AQP6 in bacteria. On the contrary, in P. pastoris production levels were very high and the protein was localized to the membrane fraction. Previous attempts with heterologous production of the antimalarial drug resistance protein Pfcrt [44] in P. pastoris and S. cerevisiae yielded promising results with an optimized coding sequence. Others have also reported increased yields of Plasmodium proteins after codon optimization [45,46]. The bacterium E. coli is the most common host for the heterologous production of soluble proteins, especially for proteins of bacterial origin. There is increasing use of E. coli for the production of bacterial membrane proteins [47]. Despite these advances there is still limited success in the application of bacteria for production of eukaryotic integral membrane proteins, an observation that could

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explain the lack of success producing aquaporin from P. falciparum, a eukaryotic microorganism, in E. coli. In comparison, the yeast P. pastoris has been used previously to produce large amounts of recombinant proteins where yields up to 1 g/L culture have been reported for a soluble protein [48]. In addition, eukaryotic integral membrane proteins, such as the spinach aquaporin SoPIP2;1 and hAQP1 [25,32], have been produced to high levels in this host. The main advantages of P. pastoris are due to it being a eukaryote and that the protein is produced from the powerful and tightly regulated AOX1 promoter. In addition, P. pastoris grown under tightly controlled regimes in fermentors reaches very high cell densities as well as improves the production level of the recombinant protein. The functionality of the overproduced protein is vital for the validation of any future structural data. Owing to a lack of genetic systems for P. pastoris we used S. cerevisiae to test whether the recombinant PfAQP was functional in yeast. The wild type PfAQP gene was not functional when tested in S. cerevisiae, while the optimized gene conferred the ability to take up and release glycerol after dierent osmotic shock regimes, as monitored by growth assays that have previously been correlated with actual glycerol transport data [8] (Fig. 2B and E). We also showed that POpt-PfAQP is not inhibited by CuSO4, which is previously known to inhibit aquaporins [49]. This nding, together with the previous notion that CuSO4 selectively inhibits AQP3 and AQP5 but not AQP9, is promising in the view of nding specic drugs for this family of proteins since CuSO4 does not seem to inhibit POpt-PfAQP. The well-known water channel inhibitor, HgCl2 could not be used in this study as it has proved toxic to yeast cells [36]. Previous studies by Hansen et al., suggest that PfAQP, when expressed in Xenopus oocytes, has water transport activity [9]. In our system, using yeast protoplasts, we could not conrm water transport, which has been demonstrated in this system for AQP1, AQP5 and the yeast aquaporin Aqy2 ([36] and Pettersson et al. unpublished data). We cannot exclude post translational modication of the protein (yielding an altered functional form), but Western blots against the POpt-PfAQP derived from P. pastoris and S. cerevisiae indicate no size dierences and mass spectrometry analysis of the overproduced protein showed no modications (data not shown). Interestingly, Hansen et al. note that PfAQP produced in oocytes does not migrate at the expected MW on SDSPAGE gels [9], a phenomenon we did not observe. When POpt-PfAQP was puried in the absence of protease inhibitors a stable smaller proteolytic fragment of unknown characteristics appeared. The reconstitution of the overproduced channel into liposomes and direct assay of water transport activity will be a major direction of our future work. Finally, we have succeeded in obtaining the rst crystals of PfAQP, which provide a promising lead to the determination of the 3D structure of this putative drug target.

Acknowledgments This work was supported by the European Commission via contracts QLG2-CT-2002-00988 (SPINE) to Lena Gustafsson and Roslyn M. Bill and LSHP-CT2004012189 (MalariaPorin project) to Stefan Hohmann. We also acknowledge the Chalmers Bioscience Initiative and the Wallenberg Foundation for their support of the Membrane Protein Centre, Lundberg Laboratory, Goteborg, Sweden. References
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