Formate–nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives

Protein Expression and Purification 71 (2010) 184–189

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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep

Formate–nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives Katherine S.H. Beckham 1, Jane A. Potter, Shiela E. Unkles * School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK

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Article history: Received 17 November 2009 and in revised form 4 December 2009 Available online 16 December 2009 Keywords: Membrane proteins, Formate–nitrite transporter family Overexpression Circular dichroism Quaternary structure TEV protease

a b s t r a c t The formate–nitrite transporter family is composed of integral membrane proteins that possess six to eight a-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to >95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly a-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Overexpression and purification of integral membrane proteins is notoriously difficult but necessary in order to obtain sufficient protein for structural studies. This intractability is illustrated by the fact that, of over 60,000 known protein structures in the Protein Data Bank [1], integral membrane proteins currently account for only 562 structures, of which only 209 belong to unique proteins [2]. The formate–nitrite transporter family (FNT; TC2.A.44)2 is a group of integral membrane proteins that are, on average, between 256 and 285 residues in length and are predicted to have 6–8 transmembrane domains [3,4]. The FNT proteins are found widely in the prokaryotes, the Pfam database [5] family PF01226 including 747 species, primarily of bacteria (720 species) with 27 sequences from archaeal species. In addition, 64 species of eukaryotes, including yeast, fungi, and protists, possess genes encoding FNT proteins but no clear homologues have been observed in the genomes of higher

* Corresponding author. Fax: +44 1334 463366. E-mail address: [email protected] (S.E. Unkles). 1 Present address: Faculty of Biomedical and Life Sciences, Wolfson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. 2 Abbreviations used: FNT, formate–nitrite transporter; AnNitA, FNT protein of Aspergillus nidulans; TpNirC, FNT protein of Thermofilum pendens; LB, Luria-Bertani medium; CD, circular dichroism; IPTG, isopropyl-b-D-thiogalactopyranoside; DDM, dodecyl-b-D-maltoside; OG, n-octyl-b-D-glucoside; TEV protease, tobacco etch virus N1a protease; IMAC, immobilised metal affinity chromatography; LC/MS/MS, liquid chromatography tandem mass spectrometry. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.12.005

plants or metazoans. Although very few of these transporters have been characterised biochemically, FNT proteins are believed to enable the specific transport of formate and/or nitrite. Prokaryotic FNT proteins that have been characterised include FocA and NirC of Escherichia coli that have been implicated in the transport of formate and nitrite, respectively [3,6]. In the eukaryotes, those from Chlamydomonas reinhardtii belonging to the Nar1 gene family are thought to regulate nitrite assimilation dependent on carbon availability [7] while the FNT protein found in Aspergillus nidulans, AnNitA, mediates specific, high-affinity transport of nitrite [8]. To date, a 3-D structure of any member of the FNT family has yet to be determined. Towards this objective, the eukaryotic AnNitA protein has been overexpressed in the yeast Pichia pastoris. In addition, a homologue, identified by genome analysis of the hyperthermophilic prokaryote Thermofilum pendens [9] and designated TpNirC has been overexpressed in E. coli. Interestingly, the latter protein is one of the largest in the FNT family, containing 382 residues. This study presents optimised conditions for expression and purification of a prokaryotic and an eukaryotic representative of this hitherto structurally-unexplored family of proteins as well as preliminary evidence for their quaternary structure. Materials and methods Chemicals and enzymes The inducer isopropyl-b-D-thiogalactopyranoside (IPTG) was from Melford. NuPAGE gels, reagents and stains (His-tag and Coo-

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massie Brilliant Blue) were from Invitrogen. Dodecyl-b-D-maltoside (DDM) was from Anatrace. Protein purification was conducted using BD TALON™ IMAC Resin (BD Biosciences Clontech). SOL grade (according to manufacturer, >97% pure by HPLC) DDM was used for solubilisation and purification until the final stage of elution from the resin and diafiltration, when ANA grade (according to manufacturer, >99% pure by HPLC) was used. All other chemicals used were of analytical grade and obtained from Sigma, Fluka or BDH. Restriction endonucleases and Phusion DNA polymerase were purchased from New England Biolabs and T4 Rapid Ligation kit from Fermentas. Recombinant His-tagged TEV protease is described in Liu and Naismith [10] and references therein. Plasmid constructs and expression hosts The TpNirC coding region was amplified by PCR from genomic DNA of T. pendens (a generous gift from Dr. C. Reich, University of Illinois) and cloned into vector pTTQ18 [11], downstream of a tac promoter (inducible by IPTG) with a C-terminal RGSH6 tag (RGSHHHHHH) fusion to produce construct pTTQ18TpNirC6H. For amplification of plasmid DNA, E. coli strain DH5a [12] was used and for expression, BL21(DE3) [13], C43(DE3) [14] or Rosetta gami™ (Novagen) E. coli strains. For A. nidulans AnNitA protein expression, the vector pPICZa (Invitrogen) was modified to include the TEV protease recognition and cleavage site between an N-terminal six histidine tag and the multiple cloning site. DNA was PCR amplified from vector pEHISTEV [10] to yield a fragment with SfuI at the 50 end and EcoRI at the 30 end encoding the His-tag and TEV cleavage site. The 50 primer also encoded a mutation such that the nucleotide immediately following the ATG start codon was G to conform more closely to the Kozak consensus [15]. The fragment was inserted into SfuI-EcoRI digested pPICZa providing vector pPICNterm into which was cloned the cDNA encoding AnNitA to yield construct pPICN termAnNitA. TEV protease cleavage of the expressed protein resulted in additional amino acid residues GEF before the start methionine. The plasmid construct was linearised with DraI before electroporation into P. pastoris strain GS115/AtNIA2 [16] with selection for growth on 300 or 400 lg/ml phleomycin. Small scale expression trials in E. coli and membrane preparations Small scale expression trials, as described by Ward et al. [17], were carried out to determine the optimal conditions for TpNirC expression. Briefly, the standard procedure was as follows: 5 ml LB, supplemented with 100 lg/ml carbenicillin and 20 mM glycerol, was inoculated with a single clone of pTTQ18TpNirC6H and grown overnight at 37 °C at 250 rpm. Fifty millilitres of the desired medium (with carbenicillin and glycerol) in 250 ml conical flasks was inoculated with 1 ml of overnight culture. After an initial growth period at 37 °C at 250 rpm until an OD680 of 0.4–0.6, IPTG was added and the cells incubated for a further period at 25 or 37 °C. Cells were pelleted by centrifugation at 3200g at 4 °C, the supernatant discarded, and the pellet frozen at 20 °C. Optimisation of the standard procedure was carried out by varying the following parameters: IPTG concentrations (0, 0.1, 0.25, 0.50, 0.75, 1 mM); induction times (3, 4, 5, 24 h). TpNirC expression was trialed in BL21(DE3), C43(DE3) and Rosetta gami™ E. coli strains, which were grown on LB (Invitrogen), 2xTY (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) and M9 supplemented growth media. The level of TpNirC expression was determined by analysing the levels of the target protein present in crude cell membranes prepared using the water lysis method [17]. The protein content of the membrane preparations was estimated using a bicinchoninic acid assay kit (Pierce) and analysed on NuPAGE gels using both

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His-Tag and Coomassie Brilliant Blue staining. Fortuitously, several standard proteins in the BioRad Low Range marker set stain with the His-tag stain allowing unequivocal size comparison between staining methods. Small scale expression trials in P. pastoris and membrane preparations Six transformant colonies were grown in 50 ml YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 28 °C for 24 h shaking at 250 rpm. Cells were harvested by centrifugation for 5 min at 2500g, washed with 50 ml sterile distilled water and resuspended for expression in minimal medium consisting of yeast nitrogen base lacking amino acids and ammonium sulphate (BD Diagnostics) to which was added 10 mM proline as nitrogen source and 0.5% methanol as inducer of the aox promoter. Growth was continued in 500 ml baffled flasks as above for 24 h before harvesting by centrifugation. Cell pellets were stored at 85 °C. From these trials, a clone was chosen for further optimisation, testing nitrogen source (5 mM ammonium tartrate or 10 mM proline) and induction time (0.5% methanol added at the start of induction and every 24 h subsequently). Crude membrane preparations were obtained by resuspending the cell pellet of 3–5 g in cold extraction buffer (20 mM NaPO4, pH 7.5, 200 mM NaCl, 10% glycerol) to which was added 3 g sterile sand, 1 mM benzamidine and 0.1 mM PMSF. Cells were disrupted by vortexing 12 times for 30 s with 30 s on ice between each vortex, debris removed by centrifugation at 2500g for 15 min at 4 °C, the supernatant fraction centrifuged at 25,000g for 45 min at 4 °C and the resulting crude membrane pellet resuspended in 80 ll extraction buffer. Protein was assayed and AnNitA detected as above for TpNirC. Large scale expression in E. coli and membrane preparations The optimal TpNirC expression conditions were determined and cells were cultured on a large scale using 5 L baffled conical flasks containing 2 L of medium. After growth, cells were harvested by centrifugation at 10,000g for 20 min at 4 °C. The wet weight of the cells was calculated, the cells resuspended with 100 ml of 20 mM NaPO4, 0.5 mM EDTA, 10% glycerol, pH 7.5, to give an approximately 20% w/v suspension as recommended for the cell disruptor (see below) and immediately frozen at 85 °C. Membranes were isolated following the method of Ward et al. [17]. Briefly, the cell pellet resuspension was thawed and passed twice through a cell disruptor (Constant Systems) at 25 Kpsi. Cell debris was removed by centrifugation at 10,000g for 30 min at 4 °C. The supernatant was centrifuged at 131,000g for 2 h at 4 °C, and the resulting pellet containing the membrane fractions was resuspended in 7 ml of 25% sucrose, 20 mM NaPO4, 0.5 mM EDTA using a 20 and 10 gauge needle. The inner and outer membrane fractions were separated on a sucrose gradient containing 10 ml layers of 55%, 50%, 45%, 40%, 35% and 30% (w/v) sucrose in 20 mM NaPO4, 0.5 mM EDTA, pH 7.5. The sample was loaded onto the gradient and centrifuged at 100,000g for 16 h at 4 °C. The inner and outer membrane fractions were removed from the gradient using a syringe. To wash the samples, 20 mM NaPO4 pH 7.5 was added to both the inner and outer membrane fractions, which were centrifuged at 131,000g for 2 h at 4 °C. The supernatant was discarded and the pellet resuspended in the same buffer. The samples were centrifuged at 131,000g for 1 h at 4 °C. The pellets were resuspended in 1–2 ml of buffer, snap frozen using liquid nitrogen, and stored at 85 °C. Large scale expression in P. pastoris and membrane preparation A 300 ml overnight culture grown at 28 °C, 250 rpm was used to inoculate 6  5 L baffled conical flasks containing 1 L YPD and

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growth continued for 24 h. Cells were transferred following centrifugation for 5 min at 2500g and washing in distilled water, to 6  5 L baffled flasks containing 1 L minimal medium (described above for small scale expression in P. pastoris) with 10 mM proline as sole nitrogen source and 0.5% methanol. Growth was continued for 24 h and cells harvested by centrifugation. The cell pellet was stored at 85 °C, thawed and resuspended at 0.5 g/ml in the extraction buffer used for small scale trials. Cells were passed through a disruptor (Constant Systems) at 30 Kpsi and debris removed by two sequential centrifugations at 2500g for 15 min at 4 °C. Membranes were pelleted by centrifugation at 45,000g for 45 min at 4 °C, resuspended in extraction buffer and stored frozen at 85 °C. Solubilisation and purification of TpNirC and AnNitA Membranes at a protein concentration of 10 mg/ml were solubilised by incubation on ice for 30 min using a DDM:protein ratio of 2.5:1 in a buffer composed of 20 mM NaPO4, 0.2 M NaCl, 20% glycerol, pH 7.5. Insoluble material was removed by centrifugation at 45,000g for 45 min at 4 °C. Solubilised protein was applied to BD TALON™ IMAC Resin (BD Biosciences Clontech), incubated with gentle shaking at room temperature for 30 min (TpNirC) or on ice for 4 h (AnNitA) and purification carried out according to the manufacturer’s batch/gravity flow protocol; the wash buffer used contained 20 mM NaPO4, 100 mM NaCl, 10% glycerol, 10 mM imidazole, 0.05% DDM, pH 7.5. The elution buffer (20 mM NaPO4, 200 mM imidazole, 0.01% DDM, pH 7.5) was applied to the TALON resin, the eluate collected in 0.5 ml fractions, and the protein content analysed on a NuPAGE gel by Coomassie staining. The protein was concentrated from the column fractions by diafiltration using an Amicon Ultra-4 centrifugal filter device (Millipore) with a 100 kDa molecular weight cut-off, used according to the manufacturer’s instructions, the filter was washed three times with 20 mM NaPO4, 0.01% DDM, pH 7.5. AnNitA was further purified by digestion with 50 lg TEV protease/mg AnNitA for 4 h at room temperature in a buffer consisting of 20 mM Tris–HCl, 300 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 20 mM imidazole, 0.01% DDM, pH 8.0. After digestion, the sample was subjected to a second round of purification using TALON resin as above, but this time the flow through fractions contained the purified AnNitA, the recombinant TEV protease being retained on the resin. The protein in the flow through fractions was concentrated using Amicon Ultra-4 devices and washed as described above. The percentage expression of AnNitA was estimated from Coomassie-stained gels by densitometry using Un-Scan-It software (Silk Scientific). The concentration of purified TpNirC and AnNitA proteins was determined by BCA assay and by absorbance at 280 nm using a NanoDropTM 1000 Spectrophotometer (Thermo Scientific) with similar results for both methods.

and 0.11 mg/mL TpNirC or 0.13 mg/mL AnNitA. Secondary structure estimates were calculated by the program CONTIN/LL [18] hosted by the CDPro server [19] with reference set SMP50, which contains both soluble and membrane proteins. Data points with high tension voltage values over 400 V were excluded from the analyses. Results and discussion Optimisation of TpNirC expression Small scale expression trials using as host BL21(DE3) indicated that 0.1 mM IPTG was sufficient to induce maximum TpNirC expression. Furthermore, optimal expression was achieved when the cells were induced at 37 °C and incubated for 3 h at 37 °C before harvesting. Induction for longer periods at 37 °C or at 25 °C did not increase the final amount of TpNirC as judged by the appearance of the protein in His-Tag stained NuPAGE gels (data not shown); thus, induction with 0.1 mM IPTG at 37 °C for 3 h were the parameters routinely used in further small scale expression trials. Generally, two major bands were observed in His-Tag stained gels (Fig. 1). The smaller of these at 6.5 kDa was present in all samples regardless of strain or growth conditions, with or without induction, and is likely to be a host protein containing histidine residues accessible to the stain. The larger band at around 32 kDa was present only in induced cells and was verified as TpNirC by LC/MS/MS mass spectrometry (data not shown). This protein was also clearly visible following Coomassie staining of the gel (Fig. 1, lane 12) and, as is common for polytopic membrane proteins, most likely due to their high hydrophobicity, the size of around 32 kDa was less than that predicted from the amino acid sequence (42.7 kDa). TpNirC expression was judged visually to be optimal in the C43(DE3) E. coli host strain when grown on 2xTY medium (Fig. 1). C43(DE3) was originally selected for its ability to tolerate high levels of membrane protein expression, avoiding the often toxic effects of such expression and allowing the cells to grow to high density [14]. Recently the molecular basis for this ability was shown to be due primarily to mutations in the lacUV5 promoter mediating reduced T7 RNA polymerase expression leading to attenuation of transcription of genes under the control of promoters such as tac in pTTQ18 and thus probably preventing the saturation of the Sec translocon with associated cytosolic aggregation of membrane proteins [20]. However, typical final A680 values

Size exclusion chromatography Purified AnNitA and molecular weight standards lactate dehydrogenase, bovine serum albumin and cytochrome c were separated on a HiLoad 16/60 Superdex 200 column (GE Healthcare) connected to a Biologic Duoflow chromatography system (BioRad). Separation of proteins was performed in a buffer composed of 50 mM NaPO4, 100 mM NaCl, 0.01% DDM, pH 7.5. The presence of AnNitA protein in different fractions after chromatography was confirmed by analysis on NuPAGE gels as described above. Circular dichroism spectrophotometry Far UV (180–260 nm) CD spectra were measured using a JASCO J-810 spectropolarimeter with a 0.05 cm pathlength quartz cuvette

Fig. 1. Small scale expression trials of TpNirC run on 4–12% NuPAGE gels. Protein was expressed in C43(DE3) (lanes 1–3); Rosetta gami™ (lanes 4–6); and BL21(DE3) (lanes 7–9) E. coli host strains. The cells were cultured in different growth media: LB (lanes 1, 4, and 7); 2xTY medium (lanes 2, 5, and 8); and M9 supplemented medium (lanes 3, 6, and 9). Lanes 1–9 contain protein from cultures induced with 0.1 mM IPTG for 3 h at 37 °C, lane 10 is protein from uninduced C43(DE3) cells grown in 2xTY medium. The molecular size markers (BioRad Low Range) (lane M) are indicated on the left. Lanes M and 1–10 are stained with His-tag stain. Lanes 11 and 12 show the Coomassie blue stained protein from lanes 10 and 2, respectively. Routinely 15 lg protein was loaded in each lane. Arrow indicates position of TpNirC.

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of around 1.0 for BL21(DE3) cultures were not significantly lower at the end of the induction period than those of C43(DE3), both strains showing a reduction of around 40% compared to uninduced cells grown for the same incubation time, indicating that the expression of the protein was no more toxic in BL21(DE3) than C43(DE3). The increased expression in the latter strain may reflect an additional property within C43(DE3) of more efficient membrane protein folding and insertion [14]. Of the three media tested, M9 minimal medium consistently produced low biomass (data not shown) for TpNirC expression (Fig. 1). Improved levels of TpNirC expression were obtained in 2xTY medium. This was not simply due to an increased biomass on the richer 2xTY medium since typical final A680 values of 0.974 and 0.988 were not significantly different between cultures grown in 2xTY and LB media, respectively. The slightly improved levels of expression seen when grown on 2xTY medium rather than LB may be an additional effect of higher nutrients providing less limitation of components such as chaperones necessary for protein overexpression. TpNirC purification Optimised conditions for TpNirC expression in C43(DE3) host cells in 2xTY medium induced by 0.1 mM IPTG at 37 °C for 3 h were used for large scale cultures yielding on average around 28 g cells from a total of 6 L medium. Following preparation of purified inner membranes, the detergent DDM was chosen for solubilisation of the proteins for two reasons. First, preliminary experiments using either Triton X-100 or n-octyl-b-D-glucoside (OG) had resulted in either very poor solubilisation of the protein (Triton X-100) or protein precipitation during purification (OG) (data not shown) and, second, this detergent has been used successfully for solubilisation and purification of many polytopic membrane proteins. Around 40 mg of total protein was obtained in the inner membrane fraction from the large scale cultures. Following solubilisation, purification by immobilised metal affinity chromatography and concentration by diafiltration, a total yield of 7.3 mg purified protein was obtained (Fig. 2). Optimisation and purification of AnNitA Screening initial transformants of P. pastoris for expression of AnNitA resulted in identification of several strains with variable

Fig. 2. Coomassie blue stained 4–12% NuPAGE gel showing purification stages of TpNirC. DDM-soluble protein from inner cell membranes (lane 1) was loaded onto an IMAC column, the flow through from this column (lane 2) indicating efficient binding of TpNirC. Following washing, TpNirC was eluted with 0.5 ml fractions (lanes 3–8) and the fractions were concentrated and the protein purified by diafiltration (lane 9). The molecular size markers are indicated on the left.

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expression levels. Following electrophoresis, the AnNitA protein clearly reacted with the His-Tag stain and was subsequently visible also on Coomassie staining of the gel. Examples of protein from high and low expressing strains are shown in Fig. 3, lanes 2 and 3, respectively. As with the TpNirC protein above, the size of the band observed at 31 kDa was less than that predicted from the amino acid sequence (36.9 kDa). The high expression strain (Fig. 3, lane 2) was further tested for the effect of nitrogen source during the induction period and for the length of the induction period. Marginally improved expression was observed when cells were grown with proline (lane 8) as nitrogen source rather than ammonium (lane 7), although continuing the expression period for 48 or 72 h (lanes 9–11) did not result in improvement of the expression level observed after 24 h induction. Thus conditions were chosen such that biomass was obtained during growth at 28 °C in rich YPD medium before switching to minimal medium with proline as nitrogen source for 24 h. From a total of 6 L, 375 g cells were obtained from large scale cultures of which batches of 100 g cells were processed. Fig. 4 shows the AnNitA protein identified by His-Tag stain in crude membranes isolated from the large scale culture and the subsequent the purification steps. Initially present in the crude membrane at around 12% of the total

Fig. 3. Small scale expression trials of AnNitA. Proteins were separated on 4–12% NuPAGE gel, stained with His-Tag stain (lanes1–3) and subsequently with Coomassie blue (lanes 4–11). Lane 1, uninduced cells; lanes 2 and 3, high and low expression, respectively, of AnNitA following induction with methanol for 24 h; lanes 4–6, Coomassie blue stain of lanes 1–3; lanes 7 and 8, induction medium containing 5 mM ammonium tartrate or 10 mM proline as sole nitrogen source, respectively; lanes 9–11, induction period of 24 h, 48 h and 72 h, respectively.

Fig. 4. Stages of purification of AnNitA analysed by electrophoresis on 4–12% NuPAGE gels. Lane 1, His-Tag stained protein from crude membrane preparations; lane 2, Coomassie blue stain of lane 1; lane 3, DDM-soluble protein fraction; lane 4, flow through fraction from first IMAC purification step; lane 5, concentrated protein eluted from first IMAC purification step; lane 6, concentrated protein from the flow through of the second IMAC purification step following TEV protease treatment of the protein in lane 5.

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protein as determined by densitometry of the Coomassie-stained gel (lanes 1 and 2), following solubilisation this percentage of AnNitA increased to around 15% of total protein (lane 3). Some AnNitA (4%) was lost in the flow through fraction from the first IMAC purification (lane 4) and after washing of the resin, elution and concentration, the AnNitA protein comprised 88% of the total protein (lane 5). The yield of protein at this stage was 20 mg. Nevertheless, this level of purity was not regarded as sufficient for crystallisation studies and so a TEV protease digestion step was included. In this second step of purification, AnNitA protein collected in the flow through fraction from the second IMAC purification step after TEV digestion was concentrated to give a final yield of 7.5 mg protein which was >96% pure (lane 6). Although initially more cumbersome in terms of culture conditions, this two-step purification may provide a protein preparation of potentially higher quality for crystallisation studies than that obtained by the onestep method used for TpNirC. One-step purification of membrane proteins expressed in E. coli runs the risk of co-purification of picogram quantities of integral membrane proteins such as the stress induced efflux transporter, AcrB, which can form unwanted, and hence misleading crystals, even at such low concentration [21,22]. A notable feature of the purified protein shown in Fig. 4, lanes 5 and 6 was the presence of a less intensely staining but nevertheless, substantial band of around 60 kDa (lane 5) or 57 kDa following TEV digestion (lane 6). LC/MS/MS mass spectrometry (data not shown) of the 60 kDa band from a NuPAGE gel of purified AnNitA indicated that this band was indeed composed of AnNitA, representing a dimeric form present even under the denaturing conditions of the NuPAGE gel. Quaternary structure of AnNitA Two lines of evidence suggested that the TpNirC and AnNitA proteins may exist as oligomeric structures. First, diafiltration following IMAC purification was carried out using a membrane with a molecular weight cut-off of 100 kDa. If the proteins existed as monomers, taking into account an average micelle size of 64 kDa [23], each protein/micelle would have average sizes of around 107 and 101 kDa, respectively. Therefore, close to the cut-off size, it might have been expected that a significant amount of protein/ micelle would have passed through the membrane into the rejected buffer. However, no protein was detectable by A280 readings in the rejected buffer following centrifugation. Second, electrophoresis of purified AnNitA on NuPAGE gels resulted in two protein bands, one the size expected for the monomeric protein and an-

other band migrating at a size commensurate with a dimeric form (Fig. 4, lanes 5 and 6). Therefore size exclusion chromatography was carried out to investigate the size of purified His-tagged AnNitA relative to known globular proteins (Fig. 5). The AnNitA protein/ micelle complex eluted with a molecular size of around 225 kDa which, following deduction of the average micelle size of 64 kDa [23], yielded an approximation of 161 kDa. This was equivalent in size to 4.36 monomers, given that the predicted size of the His-tagged protein is 36.9 kDa. Only a single protein peak was observed for AnNitA and so the protein was probably present as a tetramer during chromatography. Circular dichroism spectrophotometry The analyses indicated that both proteins were highly helical, giving values of 77% a-helix/1% b-strand for TpNirC and 65% a-helix/4% b-strand for AnNitA. The normalised Root Mean Square Deviation (NRMSD) values were 0.024 and 0.019 for TpNirC and AnNitA, respectively, indicating that the calculated and experimental data were in close agreement. Conclusions This investigation has established methods for the heterologous overexpression of prokaryotic and eukaryotic members of the FNT transporter protein family – the hyperthermophilic, archaeal protein TpNirC and the fungal protein AnNitA – and successful purification of sufficient amounts of these proteins for future structural and biophysical work. In this regard, the inherent thermostability of this archaeal homologue may aid in the crystallisation process. In the course of this work, a commercially available vector for P. pastoris was modified to permit a two step IMAC purification of AnNitA that substantially increased the percentage purity of the final protein yield. CD studies have revealed the a-helical structure of TpNirC and AnNitA, as expected from in silico prediction algorithms. Investigation into the quaternary structure of AnNitA by size exclusion chromatography has indicated the protein is likely to be a tetramer in the native state. Note added to the proof Since submission of this manuscript, two articles describing the structure of a formate transporter, FocA, have been published. These are Wang, et al. [24] and Waight, et al. [25]. Acknowledgments We wish to thank the following for generously providing materials used in this study; Professor P.J. Henderson for vector pTTQ18 and for helpful guidance, Dr. C. Reich for T. pendens genomic DNA and Dr. H. Liu for vector pEHISTEV. References

Fig. 5. Size exclusion chromatography of AnNitA showing relationship between elution volume and molecular size. The position of AnNitA is indicated by the arrow. Molecular size markers used: bovine heart lactate dehydrogenase (140 kDa), bovine serum albumin (66 kDa), equine heart cytochrome C (12.4 kDa).

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