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Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase Frank Jaenecke, Beatrice Friedrich-Epler, Christoph Parthier, and Milton T. Stubbs Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00716 • Publication Date (Web): 03 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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Biochemistry

Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase† Frank Jaenecke,1,2 Beatrice Friedrich-Epler,1 Christoph Parthier1* and Milton T. Stubbs1,2* 1

Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany

2

ZIK HALOmem, Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany

†Dedicated

to the memory of our mentor, colleague and friend Professor Rainer Rudolph, whose work

and vision was the inspiration for these investigations and whose untimely death has been a tragic loss to us and the scientific community. *

address correspondence to: Milton T. Stubbs, Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, Halle/Saale, D-06120, Germany. E-mail: [email protected]; Phone: [+49] (0)345 55 24901; Fax: [+49] (0)345 55 27360 Christoph Parthier, Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, Halle/Saale, D-06120, Germany. E-mail: [email protected]; Phone: [+49] (0)345 55 24898; Fax: [+49] (0)345 55 27360

Funding: This work was supported by the Landesexzellenzinitiative Sachsen-Anhalt “Strukturen und Mechanismen der biologischen Informationsverarbeitung” to M.T.S. Keywords: Membrane protein; folding; renaturation; reconstitution; lipids; anticoagulant List of abbreviations: hVKOR: human vitamin K epoxide reductase; µVKOR: microbial VKOR; IB: inclusion body; POPC: 1-palmitoyl-2-oleoylphosphatidylcholine; POPE: 1palmitoyl-2-oleoylphosphatidylethanolamine; POPS: 1-palmitoyl-2-oleoylphosphatidylserine; DOPC: 1,2-dioleoylphosphatidylcholine; CHAPS: 3-[(3-cholamidopropyl)dimethylammonio] -1-propane-sulphonate;

OG:

n-octyl

β-D-glucopyranoside;

DM:

n-decyl

β-D-

maltopyranoside; DDM: n-dodecyl β-D-maltopyranoside; SDS: sodium dodecyl sulphate Author contributions: B.F.-E. cloned the hVKOR constructs; F.J. expressed, purified, refolded and characterized hVKOR; data were interpreted and discussed by F.J., C.P. and M.T.S, who also wrote the paper.

Jaenecke et al.

In vitro folding of an integral membrane enzyme ACS Paragon Plus Environment

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Abstract Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for haemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in E. coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilised hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding, and paves the way for the provision of pure, active membrane protein for structural studies.

Introduction Vitamin K dependent proteins play a major role in many processes, including blood clotting, bone formation and other regulatory functions.1 The γ-carboxylation of specific glutamate residues within so-called Gla (γ-glutamic acid) domains of these proteins enables them to bind to phospholipid membranes in a Ca2+-dependent manner;2, 3 disorders in γ-carboxylation can result in abrogation of blood clotting or calcification of arteries.4-7 γ-carboxylase activity depends critically on the presence of the reduced cofactor vitamin K, which is converted to vitamin K epoxide during γ-carboxylation (Scheme 1). To replenish the pool of reduced vitamin K, the epoxide is first reduced to the quinone and subsequently to the hydroquinone form of vitamin K. While the conversion of quinone to hydroquinone can probably be carried out by several enzymes,8-11 the complete two-step reduction of vitamin K epoxide to the hydroquinone form via the quinone can only be accomplished by the enzyme vitamin K epoxide reductase (VKOR), making this enzyme crucial to maintenance of the so-called vitamin K cycle.12-14 Scheme 1 Human VKOR (hVKOR) is an 18.2 kDa integral membrane protein of the endoplasmic reticulum containing either three15 or four16,

17

transmembrane helices. A CXXC motif

residing in the final transmembrane segment – found also in the redox centres of thioredoxins and disulphide isomerases – has been shown to be necessary for activity.18, 19 Homologues of Jaenecke et al.


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hVKOR have also been identified in bacteria, plants and archea.20 Although no distinct function has as yet been assigned to microbial homologues (µVKOR), they have been implicated in disulphide bridge formation of secreted proteins, where the redox reaction is thought to be assisted by periplasmatic thioredoxin-like proteins

21, 22

. In several bacteria,

20

µVKOR forms a natural fusion protein with the thioredoxin-like protein, and the structure of one such fused µVKOR homologue from Synechococcus sp. has been described containing a bound ubiquinone molecule in the putative active site.16, 23 It has been proposed that in the course of µVKOR-mediated oxidative disulphide bond formation, the reducing equivalents are transferred to a quinone, which is in turn reduced to the hydroquinone form. Until now, however, neither have quinone epoxides been reported in bacteria, nor is the bacterial µVKOR homologue capable of vitamin K epoxide reduction.16 Interestingly, the hVKOR paralog VKORC1L1 exhibits features similar to µVKOR, with a predominantly antioxidant function and relative insensitivity to warfarin.24, 25 Thus quinone epoxide reduction appears to be a distinct feature of mammalian VKOR.13, 24 hVKOR is inhibited by warfarin and other coumarin-type drugs widely used as anticoagulants in thromboembolic diseases (e.g. myocardial infarct, stroke, thrombosis and pulmonary embolism), and it has been proposed that mutations in the gene VKORC1 (encoding for the protein hVKOR) are responsible for varying degrees of warfarin resistance.26 Due to a narrow therapeutic window between effective dose and onset of life threatening toxic reactions, dosage of the vitamin K antagonists must be carefully tailored to each new patient and closely monitored, making the search for novel inhibitors of hVKOR with enhanced therapeutic benefit an attractive goal. Although some attempts have been made to model the reaction mechanism of hVKOR,27-29 availability of structural information would greatly assist this goal. A major bottleneck to structural studies is the availability of pure and native protein. Although recombinant expression and purification of hVKOR and rat VKOR have been described using insect12,

14

and yeast30-32 cells, structural data for mammalian VKOR remain elusive. The

targeted production of recombinant human proteins in the form of inclusion bodies (IBs) followed by in vitro refolding is now a well-established technique in protein overexpression for structural biology, as the very high IB yields achievable frequently offset downstream losses in achieving the native state.33, 34 Early studies on bacterial porins

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showed that β-

barrel membrane proteins are amenable to refolding from IBs, and that these techniques are also applicable to eukaryotic mitochondrial β-barrel membrane proteins such as VDAC.36 Jaenecke et al.


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Progress on the refolding of α-helical membrane proteins from IBs has been lagging behind however (ref.

37

and references therein); it is thought that their hydrophobic nature renders

them prone to aggregation, hindering the establishment of robust refolding protocols. Promising advances have been made in the production of mammalian GPCRs,38,

39

in

particular using an 'artificial chaperone-based system' that utilises cyclodextrin to facilitate detergent exchange.40 Crucially, determination of the only NMR structure of a GPCR to date (that of CXCR1) was dependent on refolding from prokaryotically expressed inclusion bodies, allowing simple and inexpensive isotopic substitution.39 Here we present the purification, refolding and reconstitution of human VKOR (Figure S1) from a bacterial expression system designed to deliver high yields of biomass. hVKOR has been expressed in E. coli in the form of insoluble IBs as a hexahistidine-tagged ketosteroidisomerase (KSI) fusion protein (Figure 1A). Following solubilisation, chemical cleavage and purification under denaturing conditions, the protein could be refolded using an artificial chaperone system. Refolded hVKOR is enzymatically active when reconstituted into phospholipids, and we show that activity depends critically on the nature of the synthetic lipid environment. Figure 1

Experimental Procedures Construction of vectors and preliminary expression tests An overview of hVKOR expression constructs tested is given in Figure S2. The cDNA of human VKOR (hVKOR) was obtained by gene synthesis (GENEART, Regensburg), codonoptimized for expression in E.coli, (see Figure S3 for sequence) and cloned in the expression vectors pET-11a, pET-15b and pET-19b (Novagen, Madison, WI) using NdeI and BamHI restrictions sites, resulting in constructs for the wild-type protein with or without N-terminal hexa- and decahistidine tags. In addition, fusion constructs of hVKOR containing N-terminal residues from the tryptophan leader peptide (using NdeI and BamHI restriction sites in vector pTCLE),41 fragments of BCL-XL (using AflII and XhoI restrictions sites in vectors pBCL99 and pBCL173)42 and a 125 amino acid ketosteroid isomerase fragment KSI43 (using AlwNI and XhoI restrictions sites in the vector pET-31b(+), Novagen, Madison, WI) were constructed and tested for expression in E. coli BL21(DE3) and E. coli BL21(DE3) Rosetta in LB and TB media at 37°C. Cultures were induced with 1mM IPTG at OD600 ~ 0.6, samples were taken at several time points after induction and analyzed for hVKOR expression using Jaenecke et al.


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SDS-PAGE and Western Blot analysis. With the exception of KSI-hVKOR, none of the tested constructs showed detectable expression, and these were not pursued any further. The N-terminal KSI fusion promotes the expression of target proteins into inclusion bodies.43 To allow separation of hVKOR from the KSI fusion protein in the denatured state, a formic acid cleavage site (-Asp-Pro-) was introduced by site-directed mutagenesis (primers 5’- CAC GCA TGC CAG ATG CTG GAT CCG CTC GAG ATG GGT AGC ACC-3’ and 5’ – GGT GCT ACC CAT CTC GAG CGG ATC CAG CAT CTG GCA TGC GTG -3’) upstream of the XhoI site between the gene sequences for KSI and hVKOR. In later experiments, a secondary formic acid cleavage site in the KSI fragment (-EDPV-) was detected and removed by sitedirected mutagenesis (changed to -EDLV- using primers 5’- GA TGA CGC CAC GGT GGA AGA TCT CGT GGG TTC CGA GCC CAG - 3’ and CTG GGC TCG GAA CCC ACG AGA TCT TCC ACC GTG GCG TCA TC). E.coli BL21(DE3) cells were transformed with the resulting pET-31b-hVKOR plasmid DNA. For transient expression of hVKOR-His6 in HEK293 cells, the coding sequence for a Cterminal His6-tag was inserted into the plasmid pCEP4-VKOR (a generous gift from the group of Prof. Johannes Oldenburg, University Bonn) using the primers 5’-Phos-CAC CAC CAC CAC TGA GCC CTG AAT TCT GCA GAT ATC CAT CAC -3’ (forward) and 5’-Phos-GTG GTG CTC GAG GTG CCT CTT AGC CTT GCC CTG GGG TTC -3' (reverse). Expression of hVKOR fusion protein The KSI-hVKOR fusion protein was expressed in E. coli BL21(DE3) as inclusion bodies using high density fermentation (Figure 1B, C). Cells were cultivated using a 6 L fed-batch process in a complex medium containing 50 g/L yeast extract, 5 g/L glucose, 0.5 g/L NH4Cl, 0.68 g/L MgSO4, and 11 g/L KH2PO4. At an OD600 of 15, cells were fed with yeast extract. Prior to induction, the growth rate was strongly reduced by feed restriction. At an OD600 of 60, the protein expression was induced with 1 mM IPTG and the cells were cultivated for an additional 3 h at a feed-restricted growth rate. Upon induction, the OD600 remained constant, which we attribute to cell growth inhibition following expression of the recombinant membrane protein. Typically, ca. 800 g wet biomass could be harvested from a 6 L culture. Cell disruption and isolation of the inclusion bodies were performed according to established procedures.33,

44

Up to 130 mg of the insoluble 33.3 kDa KSI-VKOR fusion protein were

obtained per litre bacterial culture. The inclusion bodies were solubilized in 50mM Na2HPO4 mM pH 8.0, 15 mM SDS, 1 mM EDTA, and 50 mM DTT for 7 h at room temperature. Purification under denaturing conditions Jaenecke et al.


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The solubilized KSI-hVKOR-His6 fusion protein was dialyzed against 50mM Na2HPO4, 15mM SDS, pH 8 and applied to a 5 ml HisTrap Ni-NTA column (GE Healthcare Life Sciences, Freiburg) at room temperature. Elution was achieved by shifting the pH to 4.5 using the same buffer system. Removal of the N-terminal ketosteroid isomerase fusion partner under denaturing conditions was effected by incubation with 45% (v/v) formic acid for 7 h at 70°C.45 Completeness of cleavage was checked by SDS-PAGE and Western blot analysis (Figure 1D, E). hVKOR was isolated from the KSI fusion partner by a second round of immobilized metal affinity chromatography (IMAC), carried out as before. Mass spectrometry A sample of purified hVKOR was subjected to proteolytic digestion with chymotrypsin and analysed using MALDI-TOF/TOF, resulting in the identification of a C-terminal 21aa fragment containing the C-terminal hexa-histidine tag and two peptides of 16aa and 8aa (Figure S4). In an attempt to improve sequence coverage, chloroform/methanol precipitation of purified hVKOR was conducted followed by purification of the protein dissolved in formic acid using a Sephadex LH-20 column to remove salts and detergents prior to ESI-MS.46 This allowed detection of a fragment of hVKOR comprising the C-terminal 41aa including the active site of the enzyme and the C-terminal hexa-histidine tag. No chemical modifications were detected in the obtained fragments. Refolding Refolding was performed by applying a cyclodextrin-based “artificial chaperone” refolding system

40

in combination with pulsed renaturation at room temperature.47 In brief, protein

refolding was achieved by rapid dilution into a suitable buffer, accompanied by exchange of the strongly denaturing detergent SDS for the milder and non-denaturing detergent n-dodecyl β-D-maltopyranoside (DDM) in combination with CHAPS.[37;42] Pulsed renaturation was used to achieve folding at higher protein concentrations while minimizing the formation of aggregates.47 Purified, solubilized hVKOR (~1mg/ml, determined spectrophotometrically at 280 nm) was diluted into 45 ml of refolding buffer (25mM Na2HPO4, 1M arginine, 20mM methyl-β-cyclodextrin, 0.1% DDM, 0.1% CHAPS, 1mM EDTA) by stepwise addition. A total of 7 pulses were applied at 6 hour intervals. Each pulse resulted in an increase of protein concentration by 3µg/ml. Subsequently, the protein solution was concentrated approximately 7.5-fold using an Amicon filtration unit (10 kDa MWCO, Millipore) and immediately subjected to reconstitution in phospholipids. Reconstitution in phospholipids Jaenecke et al.


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Reconstitution was achieved by detergent-mediated destabilization of prepared liposomes, followed by successive detergent removal in the presence of hVKOR to allow protein incorporation into the lipid bilayer at room temperature.48 Liposomes of various phospholipid composition

(of

1-palmitoyl-2-oleoylphosphatidylcholine

[POPC],

1-palmitoyl-2-

oleoylphosphatidyl-ethanolamine [POPE], 1-palmitoyl-2-oleoylphosphatidylserine [POPS], and / or

1,2-dioleoyl-phosphatidylcholine [DOPC], see Figure S5; note that the phase

transition temperature of POPE/POPC mixed membranes drops significantly in the presence of excess POPC)49 (Avanti Lipids, Alabaster) were prepared in reconstitution buffer (25mM Na2HPO4, 150mM NaCl, pH8.3) by extrusion and destabilized using 22 mM n-octyl-β-Dglucopyranoside (OG).50 1 ml of liposome solution (corresponding to a POPC concentration of 1.3 mM) mixed with 3 ml concentrated refolded hVKOR (ca. 6 µM) was dialyzed extensively against 500 ml of reconstitution buffer for 96 h (3 exchanges of dialysis buffer), reducing the detergent concentration to less than 0.01%. The reconstitution buffer was supplemented in the final two dialysis steps with TCEP (Sigma) or DTT (Roth) to a final concentration of 4 mM. Reconstituted samples were concentrated approximately 4-fold by vacuum evaporation. Enzymatic activity of reconstituted hVKOR Enzymatic activity was assayed based on a previously described protocol

19, 53

employing

several modifications. In brief, 100 µl of VKOR reconstituted in liposomes were diluted with 460 µl of 50 mM Na2HPO4, 150 mM NaCl, 30% glycerol, pH 8.3. 20 µl of 150 mM DTT were added to the sample and pre-incubated for 3 min at room temperature. The reaction was started by addition of 20 µl of 850 µM vitamin K epoxide (dissolved in 98% v/v ethanol) and incubated at 30°C for 1 hour in the dark. The reaction was stopped with 1 ml isopropanol:hexane (3:2 v/v), vitamin K derivatives were extracted and separated by HPLC on a reversed-phase C18 column (Acclaim™ 120 C18, Dionex). Formation of vitamin K quinone/hydroquinone was quantified by peak absorbance at 254 nm. Progress curves of the reaction displayed a linear slope within 60 minutes (data not shown). The enzymatic activity of VKOR reconstituted in POPC liposomes as quantified by peak integration was 0.41x10-3 s-1 and was used as reference (100% relative activity) for all activity measurements. Inhibition of VKOR activity by warfarin was analyzed in the same manner but with addition of 10 µl of 6 mM warfarin (dissolved in DMSO) and pre-incubation on ice for 10 min before initiating the enzymatic reaction with vitamin K epoxide. For determination of IC50 values, data were fitted according to the following equation:54 V(I) = V0/(1+I/IC50 ) Jaenecke et al.


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Experiments for estimation of refolding and reconstitution efficiencies Reconstitution efficiency in proteoliposomes was assessed by flotation assay (Figure S6).51 80 µl of each reconstituted sample were mixed with 50 µl of a 2.5 M sucrose solution and then overlaid with 100 µl 0.75 M sucrose in reconstitution buffer, followed by 50 µl reconstitution buffer. After centrifugation at 250.000 g for 1 hour (TLA-100, BeckmanCoulter), the top 60 µl fraction was carefully aspirated and filled to 80 µl with reconstitution buffer. The amount of reconstituted hVKOR present in 10 µl was estimated using amidoblack staining and compared with 10 µl of the original reconstitution sample prior to flotation.52 Dilution series of lysozyme and purified SDS-solubilised VKOR were used as reference. The enzymatic activity of hVKOR-His6 transiently expressed in HEK293-EBNA cells (Invitrogen, Karlsruhe, Germany) was measured following transfection with the plasmid pCEP4-VKOR-His6 using Lipofectamin® (Invitrogen). Cells were grown and harvested as described previously.55 HEK293 cells (35 µl, corresponding to 500 ng hVKOR-His6) were suspended in 535 µl of assay buffer containing 0.5% CHAPS. 20µl of 150 mM DTT were added and the reaction followed as described for reconstituted hVKOR. Estimation of the amount of hVKOR-His6 present in the assay by Western blot analysis yielded a specific activity towards vitamin K epoxide of 4.9x10-3 s-1. Although this is significantly lower than values recorded previously for hVKOR expressed in insect cells (0.24 s-1 or 0.07 s-1),12, 14 it has recently been demonstrated that vitamin K epoxide reductase activity measurements are strongly dependent on assay conditions, hindering absolute comparisons with published data.56, 57

Results and discussion Various hVKOR constructs were tested for expression (Figure S2) based on a synthetic gene for human VKORC1 optimized for E. coli codon usage (see Figure S3). Of these, only the fusion protein containing the hydrophobic 125 residue N-terminal bacterial ketosteroid isomerase (KSI) sequence and C-terminal hexahistidine tag, encoded on the plasmid pET-31b(+) under the control of the T7-promoter 58 resulted in any significant expression (Figure 1). The KSI-hVKOR fusion protein was found exclusively in the cell lysate pellet fraction in the form of insoluble IBs, which were isolated, washed and solubilized using the strongly denaturing detergent sodium dodecyl sulphate (SDS) before application to an immobilized metal affinity chromatography column in the presence of SDS. Jaenecke et al.


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The eluted protein, possessing an -Asp-Pro- cleavage site between KSI and hVKOR (Figure S3), was subjected to formic acid cleavage under denaturing conditions45 and the reaction products applied to a second round of immobilized metal affinity chromatography to separate the His-tag containing hVKOR from the KSI fragment. According to SDS PAGE analysis, the recombinant hVKOR exhibits more than 90% purity at this stage (Figure 1D, E). Mass spectrometry demonstrated the absence of any chemical modifications for any of the recovered fragments of the protein (Figure S4), demonstrating the integrity of purified hVKOR despite the harsh conditions of the cleavage procedure. Recombinant hVKOR was refolded from the SDS-solubilised state employing an 'artificial chaperone-assisted refolding' protocol.40 Briefly, protein refolding was achieved by rapid dilution into a suitable buffer, accompanied by exchange of the strongly denaturing detergent SDS for the milder and non-denaturing detergent n-dodecyl β-D-maltopyranoside (DDM) in combination (CHAPS).59,

with 60

3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate

Pulsed renaturation was used to achieve folding at higher protein

concentrations while minimizing the formation of aggregates.47 Analysing the enzyme activity using a modified HPLC-based assay,19,

53

the in vitro refolded protein appeared unable to

reduce vitamin K epoxide, showing that hVKOR is inactive in the DDM-containing refolding buffer. Enzymatic activity could only be detected upon reconstitution of refolded hVKOR into phospholipids (Figure 2), delineating this to be a crucial step. SDS-solubilised hVKOR reconstituted directly into POPC phospholipids (i.e. omitting the refolding step) failed to yield any enzymatically active protein. Interestingly, reconstitution of hVKOR refolded in the presence of the detergent combination n-octyl-β-D- glucopyranoside (OG)/CHAPS instead of DDM/CHAPS also failed to result in active enzyme, whereas exclusion of CHAPS from the refolding procedure resulted in significantly reduced enzymatic activity. Thus, refolding and the concomitant detergent exchange from SDS to DDM represent indispensable steps: we have been unable to convert denatured hVKOR into the active form solely by reconstitution into phospholipids. Significantly, vitamin K epoxide reduction was inhibited almost completely by micromolar concentrations of warfarin (IC50 = 2.5 ± 0.4 µM), providing conclusive support for attributing the observed activity to refolded hVKOR. Figure 2 Nevertheless, the measured specific activity of reconstituted hVKOR is ca. one tenth that determined for the enzyme transiently expressed in mammalian cells (0.41x10-3 s-1 vs. Jaenecke et al.


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4.9x10-3 s-1). Determination of the degree of hVKOR incorporation into phospholipids by flotation assay indicates a reconstitution efficiency of around 33% (Figure S6). The refolding yield can therefore be estimated as being between 3% (assuming all correctly folded hVKOR and only a fraction of misfolded hVKOR incorporate into proteoliposomes) and 10% (assuming equal incorporation of both correctly folded and misfolded protein), values that are comparable with those typically obtained for refolding soluble proteins from inclusion bodies. The reduced activity could also be due in part to a statistical distribution of the incorporated enzyme active sites inside and outside the reconstituted proteoliposomes (if transfer of the substrates vitamin K epoxide and DTT across the membrane is slow). In order to test whether such “sidedness” plays a role, the detergent DDM was added to refolded hVKOR reconstituted in POPC phospholipids in order to permeabilise the membrane. Increasing concentrations of DDM led to a monotonic decrease in activity (Figure S7), suggesting that if sidedness plays a part in the observed reduction in activity, it is only minor. In vivo, hVKOR is thought to reside in microsomal membranes,53 which contain phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylserine and cholesterol in an approximate ratio of 10:6:3:1.61 Surprisingly, reconstitution of hVKOR in corresponding synthetic lipid mixtures resulted in reduced enzymatic activities that were significantly lower than that observed for reconstitution in POPC liposomes (Figure 3). Additional experiments in which individual lipid components of the POPC/POPE/POPS/cholesterol mixture were omitted (while keeping a constant ratio of the remaining lipids) revealed increased hVKOR activity upon omission of POPE or cholesterol, although the activity failed to reach the level observed with POPC alone. In contrast, omission of POPS resulted in a similar low activity compared to the full lipid mixture, indicating that POPE and cholesterol are largely responsible for the reduction in activity. No significant difference was observed in the presence of phospholipids comprising one (POPC) (16:0,18:1) or two (DOPC)(18:1,18:1) unsaturated alkyl chains (data not shown). As a single batch of refolded hVKOR was used as starting material for all reconstitution experiments and comparable degrees of protein incorporation were observed (Figure S6), we infer that the different rates of hVKOR activity are due to the constitution of the lipid matrix. Nonetheless, it cannot be absolutely ruled out that alternative lipid compositions might lead to differential incorporation of correctly folded hVKOR into the proteoliposomes or result in different substrate transfer rates across the membrane. Figure 3

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Biochemistry

The procedures presented here demonstrate that hVKOR can be refolded in vitro and is active after reconstitution in a phospholipid bilayer environment. Our results suggest that the enzymatic activity of hVKOR depends on the nature of both the phospholipid head groups and the hydrophobic alkyl chains. This is in accordance with increasing evidence that membrane protein function can be profoundly influenced by the nature of the lipid environment (reviewed recently in refs.62-64). Various modes of action have been discussed in the literature, including general physical properties of the membrane that change with composition (e.g. fluidity or hydrophobic thickness of the lipid bilayer), direct interactions with phospholipids (head groups and/or acyl chains), and membrane curvature. The propensities of lipid mixtures to adopt a wide variety of phase states, as well as to separate into fluid domains, provide a broad continuum for the regulation of membrane protein activity in a biological setting through stabilization or otherwise of defined conformational states. The elucidation of such mechanisms will clearly be a rewarding area of study in the future. The largely synthetic system presented here allows for the production of large quantities of active hVKOR and rational examination of the effects of lipid bilayers of different composition on the embedded membrane enzyme. Moreover, the expression in a prokaryotic system offers the additional advantage of simple and inexpensive isotopic substitution (for NMR experiments) and incorporation of “unnatural” amino acids such as selenomethionine (for MAD phasing in X-ray crystallography) for future structural studies on this eukaryotic αhelical membrane protein.

Acknowledgements We are grateful to Dr. Angelika Schierhorn (Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg) for collection and analysis of the MS data, Prof. Dr. Johannes Oldenburg (Universitätsklinikum Bonn) for initial advice in establishing the hVKOR activity assays and PD Dr. Annette Meister (Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg) for helpful discussions.

Supporting Information Supporting Information is available free of charge online at http://pubs.acs.org: • Figure S1: Schematic overview of hVKOR production procedure • Figure S2: Schematic overview of hVKOR protein constructs used in this work • Figure S3: Nucleotide sequence of the codon-optimized cDNA of hVKOR Jaenecke et al.


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• Figure S4: Mass spectrometric analysis of hVKOR after purification under denaturing conditions • Figure S5: Overview of lipids used in this work • Figure S6: Estimation of reconstitution efficiency by membrane flotation assay • Figure S7: Influence of detergent on the activity of reconstituted hVKOR

References 1.

Danziger,J. Vitamin K-dependent proteins, warfarin, and vascular calcification. Clin J Am. Soc. Nephrol. 3, 1504-1510 (2008).

2.

Soriano-Garcia,M., Padmanabhan,K., de Vos,A.M., & Tulinsky,A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 31, 2554-2566 (1992).

3.

Sperling,R., Furie,B.C., Blumenstein,M., Keyt,B., & Furie,B. Metal binding properties of gamma-carboxyglutamic acid. Implications for the vitamin K-dependent blood coagulation proteins. J. Biol. Chem. 253, 3898-3906 (1978).

4.

Luo,G. et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386, 78-81 (1997).

5.

Oldenburg,J., Marinova,M., Muller-Reible,C., & Watzka,M. The vitamin K cycle. Vitam. Horm. 78, 35-62 (2008).

6.

Price,P.A., Faus,S.A., & Williamson,M.K. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler. Thromb. Vasc. Biol. 18, 1400-1407 (1998).

7.

Tie,J.K. & Stafford,D.W. Structure and function of vitamin K epoxide reductase. Vitam. Horm. 78, 103-130 (2008).

8.

Wallin,R. & Hutson,S.M. Warfarin and the vitamin K-dependent gamma-carboxylation system. Trends in Molecular Medicine 10, 299-302 (2004).

9.

Fasco,M.J. & Principe,L.M. Vitamin-K1 Hydroquinone Formation Catalyzed by DtDiaphorase. Biochemical and Biophysical Research Communications 104, 187-192 (1982).

10.

Wallin,R., Rannels,S.R., & Martin,L.F. Dt-Diaphorase and Vitamin-K-Dependent Carboxylase in Liver and Lung Microsomes and in Macrophages and Type-Ii Epithelial-Cells Isolated from Rat Lung. Chemica Scripta 27A, 193-202 (1987).

11.

Van Horn,W.D. Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductase-like1. Crit Rev Biochem Mol. Biol 48, 357372 (2013).

12.

Chu,P.H., Huang,T.Y., Williams,J., & Stafford,D.W. Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2. Proc. Natl. Acad. Sci. U. S. A 103, 19308-19313 (2006).

13.

Rishavy,M.A., Usubalieva,A., Hallgren,K.W., & Berkner,K.L. Novel Insight into the Mechanism of the Vitamin K Oxidoreductase (VKOR). Electron relay through Cys(43) and Cys(51) reduces VKOR to allow Vitamin K reduction and facilitation of Vitamin

Jaenecke et al.


12

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

K-dependent protein carboxylation. Journal of Biological Chemistry 286, 7267-7278 (2011). 14.

Rishavy,M.A. et al. The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation. J Biol Chem 288, 31556-31566 (2013).

15.

Tie,J.K., Jin,D.Y., & Stafford,D.W. Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms. J Biol Chem 287, 33945-33955 (2012).

16.

Li,W.K. et al. Structure of a bacterial homologue of vitamin K epoxide reductase. Nature 463, 507-512 (2010).

17.

Schulman,S., Wang,B., Li,W.K., & Rapoport,T.A. Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners. Proceedings of the National Academy of Sciences of the United States of America 107, 15027-15032 (2010).

18.

Jin,D.Y., Tie,J.K., & Stafford,D.W. The conversion of vitamin K epoxide to vitamin K quinone and vitamin K quinone to vitamin K hydroquinone uses the same active site cysteines. Biochemistry 46, 7279-7283 (2007).

19.

Rost,S. et al. Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1: evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin. Thromb. Haemost. 94, 780-786 (2005).

20.

Goodstadt,L. & Ponting,C.P. Vitamin K epoxide reductase: homology, active site and catalytic mechanism. Trends Biochem. Sci. 29, 289-292 (2004).

21.

Dutton,R.J. et al. Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin. Proc. Natl. Acad. Sci. U. S. A 107, 297-301 (2010).

22.

Singh,A.K., Bhattacharyya-Pakrasi,M., & Pakrasi,H.B. Identification of an atypical membrane protein involved in the formation of protein disulfide bonds in oxygenic photosynthetic organisms. J. Biol. Chem. 283, 15762-15770 (2008).

23.

Liu,S., Cheng,W., Fowle,G.R., Shen,G., & Li,W. Structures of an intramembrane vitamin K epoxide reductase homolog reveal control mechanisms for electron transfer. Nat Commun 5, 3110 (2014).

24.

Westhofen,P. et al. Human Vitamin K 2,3-Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Mediates Vitamin K-dependent Intracellular Antioxidant Function. Journal of Biological Chemistry 286, 15085-15094 (2011).

25.

Hammed,A. et al. VKORC1L1, an enzyme rescuing the vitamin K 2,3-epoxide reductase activity in some extrahepatic tissues during anticoagulation therapy. J Biol Chem 288, 28733-28742 (2013).

26.

Rost,S. et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 427, 537-541 (2004).

27.

Davis,C.H., Deerfield,D., Wymore,T., Stafford,D.W., & Pedersen,L.G. A quantum chemical study of the mechanism of action of Vitamin K epoxide reductase (VKOR) II. Transition states. J. Mol. Graph. Model. 26, 401-408 (2007).

28.

Deerfield,D., Davis,C.H., Wymore,T., Stafford,D.W., & Pedersen,L.G. Quantum chemical study of the mechanism of action of vitamin K epoxide reductase (VKOR). International Journal of Quantum Chemistry 106, 2944-2952 (2006).

Jaenecke et al.


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

29.

Silverman,R.B. Chemical-Model Studies for the Mechanism of Vitamin-K Epoxide Reductase. Journal of the American Chemical Society 103, 5939-5941 (1981).

30.

Matagrin,B. et al. New insights into the catalytic mechanism of vitamin K epoxide reductase (VKORC1) - The catalytic properties of the major mutations of rVKORC1 explain the biological cost associated to mutations. FEBS Open. Bio 3, 144-150 (2013).

31.

Hodroge,A. et al. VKORC1 mutations detected in patients resistant to vitamin K antagonists are not all associated with a resistant VKOR activity. J Thromb. Haemost. 10, 2535-2543 (2012).

32.

Hodroge,A., Longin-Sauvageon,C., Fourel,I., Benoit,E., & Lattard,V. Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants. Arch. Biochem Biophys 515, 14-20 (2011).

33.

Rudolph,R. & Lilie,H. In vitro folding of inclusion body proteins. FASEB J. 10, 49-56 (1996).

34.

Hwang,P.M., Pan,J.S., & Sykes,B.D. Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli. FEBS Lett 588, 247-252 (2014).

35.

Schmid,B., Kromer,M., & Schulz,G.E. Expression of porin from Rhadopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure. Febs Letters 381, 111-114 (1996).

36.

Hiller,S. et al. Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321, 1206-1210 (2008).

37.

Kiefer,H. In vitro folding of alpha-helical membrane proteins. Biochim. Biophys. Acta 1610, 57-62 (2003).

38.

Baneres,J.L., Popot,J.L., & Mouillac,B. New advances in production and functional folding of G-protein-coupled receptors. Trends in Biotechnology 29, 314-322 (2011).

39.

Park,S.H. et al. Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491, 779-783 (2012).

40.

Rozema,D. & Gellman,S.H. Artificial chaperone-assisted refolding of denaturedreduced lysozyme: modulation of the competition between renaturation and aggregation. Biochemistry 35, 15760-15771 (1996).

41.

Yansura,D.G. Expression as trpE fusion. Methods Enzymol. 185, 161-166 (1990).

42.

Thai,K., Choi,J., Franzin,C.M., & Marassi,F.M. Bcl-XL as a fusion protein for the highlevel expression of membrane-associated proteins. Protein Sci 14, 948-955 (2005).

43.

Baneres,J.L. et al. Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy. J. Biol. Chem. 280, 20253-20260 (2005).

44.

Lilie,H., Schwarz,E., & Rudolph,R. Advances in refolding of proteins produced in E. coli. Curr. Opin. Biotechnol. 9, 497-501 (1998).

45.

Ryan,R.O., Forte,T.M., & Oda,M.N. Optimized bacterial expression of human apolipoprotein A-I. Protein Expr. Purif. 27, 98-103 (2003).

46.

Venter,H., Ashcroft,A.E., Keen,J.N., Henderson,P.J., & Herbert,R.B. Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3). Biochem. J. 363, 243-252 (2002).

Jaenecke et al.


14

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

47.

Winter,J., Lilie,H., & Rudolph,R. Renaturation of human proinsulin--a study on refolding and conversion to insulin. Anal. Biochem. 310, 148-155 (2002).

48.

Karlsson,M. et al. Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS Lett 537, 68-72 (2003).

49.

Cannon,B. et al. Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers. Biophys J 85, 933-942 (2003).

50.

Rigaud,J.L. & Levy,D. Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65-86 (2003).

51.

Wang,C.W., Hamamoto,S., Orci,L., & Schekman,R. Exomer: A coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast. J Cell Biol 174, 973-983 (2006).

52.

Schaffner,W. & Weissmann,C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56, 502-514 (1973).

53.

Wallin,R. & Martin,L.F. Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin. J. Clin. Invest 76, 1879-1884 (1985).

54.

Duggleby,R.G. Determination of inhibition constants, I50 values and the type of inhibition for enzyme-catalyzed reactions. Biochem Med Metab Biol 40, 204-212 (1988).

55.

Rost,S. et al. Novel mutations in the VKORC1 gene of wild rats and mice - a response to 50 years of selection pressure by warfarin? Bmc Genetics 10, (2009).

56.

Bevans,C.G. et al. Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay. Biochim Biophys Acta 1830, 4202-4210 (2013).

57.

Krettler,C., Bevans,C.G., Reinhart,C., Watzka,M., & Oldenburg,J. Tris(3hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity. Anal Biochem 474, 89-94 (2015).

58.

Studier,F.W., Rosenberg,A.H., Dunn,J.J., & Dubendorff,J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89 (1990).

59.

Schmidt,P. et al. Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2. Biotechnol. Prog. 25, 1732-1739 (2009).

60.

Dahmane,T., Damian,M., Mary,S., Popot,J.L., & Baneres,J.L. Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48, 6516-6521 (2009).

61.

Waskell,L., Koblin,D., & Canova-Davis,E. The lipid composition of human liver microsomes. Lipids 17, 317-320 (1982).

62.

Lee,A.G. Biological membranes: the importance of molecular detail. Trends Biochem Sci 36, 493-500 (2011).

63.

Soubias,O. & Gawrisch,K. The role of the lipid matrix for structure and function of the GPCR rhodopsin. Biochim Biophys Acta 1818, 234-240 (2012).

64.

Yeagle,P.L. Non-covalent binding of membrane lipids to membrane proteins. Biochim Biophys Acta 1838, 1548-1559 (2014).

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Figure legends Scheme 1. The Vitamin K cycle. The site-specific carboxylation of glutamate residues in coagulation factor Gla-domains is catalysed by γ-glutamyl carboxylase in a vitamin K hydroquinone dependent manner. VKOR, which reduces the ensuing vitamin K epoxide via the quinone form to replenish the hydroquinone, is the target of the powerful anticoagulant warfarin. R: isoprenoid chain, in animals typically consisting of four isoprene units (Vitamin K2 or menaquinone). Figure 1: Expression, purification and formic acid cleavage of KSI-hVKOR. (A) Schematic representation of the KSI-hVKOR-His6 construct used in this work. Black arrowhead indicates the position of the formic acid chemical cleavage site. (B) A clear band (highlighted with grey arrow at 33 kDa) can be seen in SDS-PAGE for the pET-31b(+) plasmid encoded KSI-hVKOR fusion protein during fermentation of E. coli BL21 (DE3). M: SDS-PAGE protein standard marker. (C) Corresponding Western blot analysis (anti-His6 antibody) demonstrates the identity of the expressed product. M: BenchMark™ His-tagged Protein Standard (Invitrogen). (D) Progress of purification. Lysate: supernatant after inclusion body purification; IB: inclusion body preparation; Ni-NTA: eluate after immobilized metal affinity chromatography (IMAC) of the fusion protein (grey arrow) under denaturing conditions; HCO2H: eluate after IMAC of the formic acid cleaved KSI-hVKOR under denaturing conditions. (E) Anti-His6 Western blot shows that the product (black arrow at ca. 19.5 kDa) corresponds to the His-tagged hVKOR. Figure 2. Influence of the reconstitution procedure upon hVKOR activity towards vitamin K epoxide. Refolding and detergent exchange prior to reconstitution in POPC lipids is required to obtain active hVKOR. The combination of DDM/CHAPS in the refolding step yields higher activity (0.41x10-3 s-1, set to 100%) than refolding in presence of DDM alone. White bars indicate corresponding assays in the presence of 100 µM warfarin. Inset: Dose dependency of hVKOR inhibition by warfarin (IC50 = 2.5 ± 0.4 µM, in agreement with published values).18 Figure 3. Influence of lipid composition upon reconstituted hVKOR activity. Substitution of POPC with a POPC/POPE/POPS/cholesterol lipid mixture corresponding to that found in microsomal membranes (“endosomal mix”) results in significantly lower activity than for POPC alone. By leaving out each phospholipid in turn, the reduced activity can be attributed to the presence of the phosphatidylethanolamine head group and cholesterol. Jaenecke et al.


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Scheme 1. The Vitamin K cycle. The site-specific carboxylation of glutamate residues in coagulation factor Gla-domains is catalysed by γ-glutamyl carboxylase in a vitamin K hydroquinone dependent manner. VKOR, which reduces the ensuing vitamin K epoxide via the quinone form to replenish the hydroquinone, is the target of the powerful anticoagulant warfarin. R: isoprenoid chain, in animals typically consisting of four isoprene units (Vitamin K2 or menaquinone).

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Figure 1: Expression, purification and formic acid cleavage of KSI-hVKOR. (A) Schematic representation of the KSI-hVKOR-His6 construct used in this work. Black arrowhead indicates the position of the formic acid chemical cleavage site. (B) A clear band (highlighted with grey arrow at 33 kDa) can be seen in SDS-PAGE for the pET-31b(+) plasmid encoded KSI-hVKOR fusion protein during fermentation of E. coli BL21 (DE3). M: SDS-PAGE protein standard marker. (C) Corresponding Western blot analysis (anti-His6 antibody) demonstrates the identity of the expressed product. M: BenchMark™ His-tagged Protein Standard (Invitrogen). (D) Progress of purification. Lysate: supernatant after inclusion body purification; IB: inclusion body preparation; Ni-NTA: eluate after immobilized metal affinity chromatography (IMAC) of the fusion protein (grey arrow) under denaturing conditions; HCO2H: eluate after IMAC of the formic acid cleaved KSI-hVKOR under denaturing conditions. (E) Anti-His6 Western blot shows that the product (black arrow at ca. 19.5 kDa) corresponds to the His-tagged hVKOR. Jaenecke et al.


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Figure 2. Influence of the reconstitution procedure upon hVKOR activity towards vitamin K epoxide. Refolding and detergent exchange prior to reconstitution in POPC lipids is required to obtain active hVKOR. The combination of DDM/CHAPS in the refolding step yields higher activity (0.41x10-3 s-1, set to 100%) than refolding in presence of DDM alone. White bars indicate corresponding assays in the presence of 100 µM warfarin. Inset: Dose dependency of hVKOR inhibition by warfarin (IC50 = 2.5 ± 0.4 µM, in agreement with published values).18

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120 100 80 “endosomal mix”

relative VKOR activity (%)

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60 40 20 0

VKOR POPC POPE POPS cholesterol

+ + -

+ + + + +

+ + + +

+ + + +

+ + + + -

+ -

Figure 3. Influence of lipid composition upon reconstituted hVKOR activity. Substitution of POPC with a POPC/POPE/POPS/cholesterol lipid mixture corresponding to that found in microsomal membranes (“endosomal mix”) results in significantly lower activity than for POPC alone. By leaving out each phospholipid in turn, the reduced activity can be attributed to the presence of the phosphatidylethanolamine head group and cholesterol.

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Graphic for the Table of Contents

purification & solubilisation

HCO2H O OH

inclusion bodies

O

O

active enzyme reconstitution

refolding & detergent exchange

Unscrambled enzyme: Human vitamin K epoxide reductase (hVKOR), an integral membrane protein responsible for maintenance of reduced vitamin K pools, has been produced in high yields as an insoluble fusion protein in E. coli. Fusion tag removal by chemical cleavage of detergent solubilized hVKOR, in vitro refolding, detergent exchange and reconstitution in lipids results in active enzyme that is inhibited by the powerful anticoagulant warfarin.

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Figure 1: double column format 214x259mm (300 x 300 DPI)


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Graphic for the Table of Contents Unscrambled enzyme: Human vitamin K epoxide reductase (hVKOR), an integral membrane protein responsible for maintenance of reduced vitamin K pools, has been produced in high yields as an insoluble fusion protein in E. coli. Fusion tag removal by chemical cleavage of detergent solubilized hVKOR, in vitro refolding, detergent exchange and reconstitution in lipids results in active enzyme that is inhibited by the powerful anticoagulant warfarin. 40x19mm (300 x 300 DPI)


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