Marie-Laure Fogeron

Aurélie Badillo

Vlastimil Jirasko

Jérôme Gouttenoire

David Paul

Loick Lancien

Darius Moradpour

Ralf Bartenschlager

Beat H Meier

François Penin
email: f.penin@ibcp.fr

Anja Böckmann
email: bockmann@ibcp.fr

Roland Montserret

Denis Lacabanne

Jennifer Molle

Célia Boukadida

Sonia Georgeault

Philippe Roingeard

Annette Martin

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins

Keywords: Cell-free protein expression Detergents Hepatitis C virus proteins hepatitis C virus, non-structural protein 2, cell-free protein expression, membrane protein, lipid reconstitution

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cellfree expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Introduction

Cell-free protein expression is an alternative approach to protein production when classical methods using cell-based systems fail (for a recent review see [1,2] and for a recent book [3,4]). As an open system, which allows addition of solubilizing agents, it is highly promising notably for the expression of membrane proteins. Furthermore, the wheat germ system is particularly favorable for the expression of eukaryotic proteins, as the slower ribosomal translation of wheat germ ribosomes better mimics eukaryotic systems than their prokaryotic counterpart. This often enables correct co-translational protein folding [5]. Three expression modes for membrane proteins have been developed in cell-free systems: as precipitate (in the absence of lipid/detergent molecules), solubilized by the presence of detergents, or directly inserted into model membranes [6,7]. While co-translational insertion of the protein into liposomes [8,9] is in principle very attractive, with proteoliposome recovery directly possible by simple centrifugation, this strategy is often difficult to follow because many proteins do not spontaneously insert into the liposome membrane and/or require specific lipid compositions for insertion [8,9]. In addition, this strategy requires high lipid-protein ratios, suffers from poor yields, and presents difficulties for purification. Therefore, protein expression in the presence of detergent [9][10][11][12][13][14] remains the most convenient approach, since proteins may be expressed in their folded, functional, and solubilized form, with high yield, and straightforward downstream purification. Expression in the precipitated form is the least preferred alternative because of often irreversible aggregation, although proteins can sometimes be resolubilized in a folded form using mild detergents [10,15]. When using cell-free expression in the presence of detergent, key parameters to be optimized are the expression yield and, more importantly, the extent of the membrane protein solubilization. While solubilization can be compromised by low detergent concentrations, the expression yields can be dramatically lowered by high detergent concentrations. Both effects depend on the detergent used. Concerning the detergent type, it is well known that charged detergents, such as sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), are efficient for solubilization but generally inhibit protein activity to a large extent. In contrast, non-ionic and some zwitterionic detergents are considered as mild detergents, maintaining native protein structure and activity [16]. Best choices are thus non-ionic or some zwitterionic detergents, and furthermore those which allow complete solubilization before reaching concentrations affecting the translation machinery.

For membrane protein expression using bacterial cell extracts, previous results suggest that mild detergents with a low critical micelle concentration, such as polyoxyethylene-lauryl-ether (Brij) and polyoxyethylene sorbitan monolaurate (TWEEN) derivatives allow successful production and have proven to be optimal with respect to the solubilized protein yield [10,14,15,[17][18][19][20][21][22]. However, their use in wheat germ cell-free expression has shown drawbacks, as evaluated by expression of green fluorescent protein (GFP) as a model to evaluate expression yield [23]. Additionally, for membrane protein expression in the wheat germ system, the available information on the best choice of detergent type and concentration for membrane protein expression is still sparse [9,12,23].

A new class of low CMC detergents are the maltose-neopentyl glycol (MNG) amphiphiles, which have been described recently in the literature [24]. MNG amphiphiles feature a central quaternary carbon derived from neopentyl glycol, two hydrophilic groups derived from maltose, as well as two n-decyl lipophilic tails. They were recently developed for solubilization, stabilization and crystallization of membrane proteins, and have shown promising results in the expression of bacteriorhodopsin in a wheat germ cell-free system [24].

We present here a detergent screen to establish which detergents are compatible with the wheat germ cell-free reaction. Subsequently, we use then the two most promising reagents, C12E8 and MNG-3, for a detailed analysis of the optimal concentration range for protein solubilization. We test this using the hepatitis C virus (HCV) nonstructural proteins 2, 4B and 5A (NS2, NS4B and NS5A), which are membrane proteins notoriously difficult to overexpress due to their toxicity in cell-based systems. The current knowledge about these HCV proteins has been reviewed in [25][START_REF] Moradpour | Replication of hepatitis C virus[END_REF][START_REF] Lohmann | Hepatitis C virus RNA replication[END_REF].

Results

Screening of detergents for compatibility with the cell-free reaction

Detergent screening in the cell-free expression reaction was carried out using the HCV NS2 integral membrane protein fused to a Strep-tag II at the N-terminus, in presence of the following detergents: DPC, DM, DDM, MNG-3, C12E8, LDAO, CHAPS, cholate and OG (listed in Table 1 with the full name). Each detergent was tested at a concentration of 0.05% (w/v) and 0.1% (w/v), i.e. above their CMC (given in Table 1), except for CHAPS, cholate and OG which have a CMC > 0.5% (w/v). For the latter detergents, an additional concentration above their CMC of 1% (w/v) was tested. Three different fractions were analysed by SDS-PAGE and western blotting using monoclonal antibody (mAb) against Streptag II: first, the total reaction mixture resulting from the cell-free reaction (cell-free sample, CFS, see Materials and Methods); second the pellet (P) resulting from centrifugation of this solution at 20,000g for 30 min; and third the supernatant, referred to as SNbeads, as the protein in the supernatant fraction was captured using affinity Strep-Tactin coated magnetic beads.

The results for the HCV NS2 expressed in the presence of the different detergents are shown in Fig. 1 and Table 1. When expressed in the absence of detergent, NS2 is mainly expressed as a precipitate and detected in the pellet. The presence of a small portion of NS2 in the soluble fraction, despite the absence of detergent, could be explained by the residual presence of lipids from the wheat germ extract (WGE) or some low molecular weight, nonnative but soluble, oligomers. Fig. 1 shows that expression yield (as seen in the gels corresponding to the total cell-free sample, CFS) is only maintained at a comparable level to the reaction in the absence of detergent for MNG-3, C12E8, CHAPS, Cholate and OG at 0.1%. At 1%, which was tested for CHAPS, Cholate and OG in order to increase concentration above the CMC, all three fail to support protein expression. As can be seen in Table 1, at 0.05%, MNG-3, C12E8, and DM (which is then below CMC), as well as CHAPS, Cholate and OG (also below CMC) also yield comparable expression. DDM shows reduced expression under all conditions used, and DPC and LDAO fail in all cases.

In order to determine the amount of protein expressed in a soluble state, a comparison between the bands for the pellet (P) and the supernatant (SN-beads) (Fig. 1) has to be made. It can be seen that only MNG-3 and C12E8 allow to obtain the protein in a fully soluble form, without any compromise in yield. CHAPS, Cholate and OG do not allow for better solubility than observed for the reference experiment with no detergent added (column denoted by +). This cannot be improved by adding more detergent, as above the CMC, at 1%, all three detergents inhibit the reaction completely. Table 1 indicates that neither DM nor DDM is efficient in solubilizing the protein at 0.05% or 0.1%. Thus, in order to express NS2 with high yield and in a soluble state, only the two nonionic, low CMC detergents MNG-3 and C12E8 were fully successful.

Expression of NS2 in presence of DDM, MNG-3 or C12E8

In order to determine the minimal amount of detergent required to produce soluble NS2, as well as to assess if higher concentrations affect yield, we tested a concentration range from 0 to 4500/5000 lM for DDM, MNG-3, and C12E8 (Fig. 2 and Table S1).

Although DDM showed a partial inhibitory effect in the preliminary screen, it was tested for comparison, since it is often used as a standard detergent for solubilization and purification of membrane proteins and reconstitution in lipids, and also for its chemical likeness when compared to MNG-3 (Fig. 2D).

As shown in Fig. 2A (CFS, top), with increasing concentrations the presence of DDM progressively alters NS2 expression yields, and fully inhibits it around 3000 lM. Soluble protein is partially observed at all concentrations below 3000 lM, but inhibition of expression occurs before complete solubilization can be reached. In contrast, MNG-3 does not significantly inhibit NS2 expression (Fig. 2B, CFS) and starts to solubilize NS2 around a concentration of 200 lM (SN-beads), and allows to express NS2 in a completely soluble form at a concentration of 650 lM, where all the protein is found in the supernatant fraction (Fig. 2B). The apparent decrease of NS2 present in the supernatant above 3000 lM (Fig. 2B, bottom) is probably due to the fact that MNG-3 starts to inhibit the interaction of the Strep-tag II with the Strep-Tactin coated magnetic beads. Indeed, detergents can affect affinity tag purification, and while we do not observe this for C12E8 even when exceeding the reported limits for this detergent (90 lM, IBA technical info) by a factor of 50, we do observe an effect of MNG-3 when exceeding 2000 lM. Fig. 2C shows that C12E8 has no inhibiting effect on NS2 expression up to a detergent concentration of 4500 lM and allows NS2 expression in a soluble form at 1300 lM, with solubilization starting around 300 lM. The detergent concentration above CMC, as an indicator for the concentration of detergent monomers susceptible to form micelles, is given in Table S1. One can see that soluble protein is obtained around 600 lM detergent in micelle form, while for DDM, the reaction is inhibited before this concentration is reached. Unlike MNG-3, C12E8 does not interfere with affinity capture using Strep-Tactin coated magnetic beads.

Expression of NS2 pro in presence of MNG-3 or C12E8

Like the full-length NS2 protein considered above, its cytosolic protease domain denoted NS2 pro is expressed as a precipitate when protein synthesis occurs without addition of a solubilizing agent (Fig. 3). Indeed, this domain, for which the 3D structure is known [START_REF] Lorenz | Structure of the catalytic domain of the hepatitis C virus NS2-3 protease[END_REF], was recently shown to associate with the membrane surface [START_REF] Lange | Determinants for membrane association of the hepatitis C virus NS2 protease domain[END_REF]. The expression of NS2 pro (N-terminally fused to Strep-tag II) in the presence of MNG-3 or C12E8 is shown in Fig. 3. For each detergent, a concentration range from 0 to 2000 lM was tested. NS2 pro expression is not inhibited by MNG-3 or C12E8 up to the maximum concentration used. MNG-3 allows expression of NS2 pro in a fully soluble form around 2000 lM (Fig. 3A). C12E8 is slightly more efficient, with NS2 pro solubilization being complete at 1300 lM (Fig. 3B).

Expression of NS4B, NS5A and NS5A-DAH in presence of MNG-3

Having identified MNG-3 as an efficient solubilizing agent for the NS2 protein, we further investigated if it allows as well the expression of other HCV proteins in a soluble form. Results for the NS4B and NS5A membrane proteins are shown in Fig. 4, with tested concentration ranges from 0 to 1300 lM (Table S1 and Fig. 4). It can be seen that for NS4B, which is an integral membrane protein (reviewed in [START_REF] Gouttenoire | Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory[END_REF]), solubilization is complete at 650 lM (Fig. 4A), which is similar to the results observed for the full-length NS2 protein. In contrast to NS2 and NS4B, NS5A is a so-called monotopic membrane protein predicted to anchor in-plane to the membrane interface by its N-terminal amphipathic alpha-helix denoted ''AH'' [START_REF] Penin | Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A[END_REF]. As shown in Fig. 4B and C respectively, for both NS5A and NS5A-DAH, solubilization reaches a high level around 300-650 lM, but only shows slightly higher values at the maximum concentration of detergent used. However, the solubilization of NS5A and NS5A-DAH is not complete, even at 1300 lM. The residual insoluble forms of NS5A and NS5A-DAH are likely due to the tendency of this protein to associate in large assemblies [START_REF] Love | Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus[END_REF], which however remain uncharacterized to date.

Analysis of NS2 and NS4B produced and purified in the presence of MNG-3 by exclusion chromatography

Soluble NS2 and NS4B expressed in presence of MNG-3 were purified by affinity chromatography using a Strep-Tactin column (Fig. 5A andB). While the purity level achieved for NS4B is quite high, some contaminants are observed in the NS2 sample. Better purity could be achieved by using a different detergent during purification, e.g., DDM (data not shown). Both proteins were analyzed by size exclusion chromatography (Fig. 5C andD). NS2 was eluted in a single peak at 3.2 ml (Fig. 5B), and no aggregate (which would elute at the void volume at 2.6 ml) was observed. The following three additional peaks come from the reagents present in the Strep-Tactin elution buffer. The elution peak corresponding to NS2 is rather asymmetric, which might be due to the oligomeric nature of NS2 established previously [START_REF] Lorenz | Structure of the catalytic domain of the hepatitis C virus NS2-3 protease[END_REF] and/or interactions with the column chromatographic media. The same observations can be made for NS4B, which is eluted at 3.25 ml, and which is also expected to form oligomers [START_REF] Yu | Palmitoylation and polymerization of hepatitis C virus NS4B protein[END_REF]. For both proteins, superimposition of size exclusion profiles at 280 nm and 220 nm confirm that only the first elution peak corresponds to protein (Fig. S1). These results indicate that for both NS2 and NS4B, expression in MNG-3 yields monodisperse sample.

Discussion

We here tested different detergents for their dual ability to allow efficient expression of membrane proteins in a wheat-germ cell-free reaction and at the same time lead to the production of solubilized proteins. While several detergents tested clearly show an inhibitory effect on the expression, two of them, MNG-3 and C12E8, allowed us to obtain the protein in a soluble, monodispersed form without significant inhibition of the expression yield. With respect to protein yield in cell-free reactions in the presence of detergents, it is unclear mechanistically if it is micelles or detergent monomers that are detrimental for the correct functioning of the cell-free expression. However, we observe that the high detergent monomer concentrations, which occur when detergents with high CMCs are used, cause inhibition of the cell-free reaction. Indeed, for DM and DDM, a relatively low micelle concentration around 2 and 8 lM (0.05% and 0.1%, respectively) already severely inhibits the expression. In contrast, C12E8 concentrations do not inhibit the cell-free reaction up to the highest ones tested (4500 lM, corresponding to a micelle concentration of 37-49 lM). This supports the idea that high monomer concentrations rather than high micelle concentrations inhibit the system. This might explain why low-CMC detergents, which by definition show low concentrations of detergent monomers, are often more successfully used in cell-free expression.

The relevant variables with respect to the detergent concentration are the number of detergent monomers bound to the protein in its fully solubilized state, and the critical micelle concentration. The latter is often well-defined, and should be exceeded in order to yield detergent molecules with an affinity such that they interact with each other (which is the dominating factor [START_REF] Kunji | Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography[END_REF]), and also with the protein [START_REF] Le Maire | Interaction of membrane proteins and lipids with solubilizing detergents[END_REF]. The former parameter is more difficult to assess, as the number of detergent molecules needed to solubilize a protein depends on the hydrophobic sector of the membrane protein to be covered, as well as the cross-sectional area of the detergent and the hydrophobic chain length. Based on calculations, it has been suggested that the protein-detergent complexes might have physicochemical properties quite different from those of detergent micelles. Also, the degree of detergent binding per unit of hydrophobic surface area can vary for different membrane proteins [START_REF] Le Maire | Interaction of membrane proteins and lipids with solubilizing detergents[END_REF]. Empirically, a ratio between 1.0 and 1.4 for the number of detergent molecules in the protein-detergent complex versus the free detergent micelle has been observed for Aac3p solubilized in different detergents [START_REF] Kunji | Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography[END_REF]. This indicates that the increase in the number of detergent molecules in the protein-detergent complex compared to the free detergent micelle might represent only a small factor [START_REF] Kunji | Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography[END_REF], and that the micelle concentration can serve as a good approximation.

The concentration of micelles at which solubilization occurs can be estimated for C12E8 to 10-13 lM (Table S1) for NS2 and NS2 pro , for about 1 lM of protein produced in the reaction mixture. Therefore, under these conditions, about ten detergent micelles per protein molecule produced are available, which is notably comparable to ratios calculated from data revealing the detergent concentration for complete solubilization in the literature. Indeed, three GPCRs [18] as well as an olfactory receptor [START_REF] Kaiser | Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses[END_REF] were successfully solubilized in cell-free reactions mixture using detergent-micelleto-protein ratios between 5 and 8. Thus, in general the ratio seems to be around one order of magnitude, which can be taken into account for future studies to more efficiently screen the range of tested detergent concentrations in cell-free reactions. The observed excess of detergent required for full protein solubilization could be explained by either (i) the detergent interacting as well with other components of the wheat germ extract (the total wheat germ endogenous protein concentration in the reaction is about 2 mg/ml), (ii) the need of more availability of micelles at the exit of the ribosome, or by (iii) the need of more detergent monomers in the mixed protein-detergent micelle compared to the free detergent micelle. Also, one should keep in mind that both detergent CMC and aggregation numbers are dependent on the physico-chemical conditions, and are thus difficult to determine precisely.

For MNG-3, the micelle size is strongly concentration-dependent, and no aggregation number has been determined so far (P. S. Chae, personal communication). The results for NS2 show however that soluble protein is obtained at about the same detergent concentration as for C12E8. MNG-3 has been used in expression of all proteins tested here. It can be seen in Figs. 3A and4 that, even if the order of magnitude remains comparable, not all proteins become soluble at the same concentration threshold. This means that there is probably a dependence on the nature of the protein used, and we describe in the following how the structural features of the different proteins could account for this observation.

NS2 pro is the globular domain of the NS2 protein, which is anchored to the membrane mainly by the N-terminal integral membrane domain [START_REF] Jirasko | Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly[END_REF]. The crystal structure of NS2 pro has been resolved in the presence of detergents and reveals its ability to dimerize [START_REF] Lorenz | Structure of the catalytic domain of the hepatitis C virus NS2-3 protease[END_REF]. It exhibits two helices with a hydrophobic surface that has been shown to interact with membranes [START_REF] Lange | Determinants for membrane association of the hepatitis C virus NS2 protease domain[END_REF]. This rationalizes why NS2 pro is expressed as a precipitate in the cell-free reaction in the absence of detergents. With NS2 pro expressed in somewhat smaller amounts than the full-length protein, proteinspecific factors must be the cause for the higher micelle concentrations required for solubilization. It is possible for example that the absence of a membrane bilayer during the translation of NS2 pro can lower the efficiency of the self dimerization process and prolong the time during which hydrophobic interaction interfaces are exposed which demand higher amount of the detergent to keep the protein in the soluble state. In case of the NS2 full-length protein, the N-terminal domain could contribute to the interaction between monomers and facilitate native protein folding.

The integral membrane protein NS4B (reviewed in [START_REF] Gouttenoire | Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory[END_REF]) expresses very well in the presence of MNG-3, and is solubilized at approximately the same concentration of detergent as NS2. Of note, these two viral proteins both form oligomers and contain several transmembrane passages responsible for their integral membrane character [25][START_REF] Moradpour | Replication of hepatitis C virus[END_REF][START_REF] Lohmann | Hepatitis C virus RNA replication[END_REF].

NS5A is a globular protein displaying an a-helical membrane anchor at its N-terminal end, and a large disordered portion, including the C-terminal D2 and D3 domains (reviewed in [25]), which might also interact with detergent. NS5A is also known to bind to nucleic acids in its hypothesized role of transporting the HCV viral RNA genome to the core protein for encapsidation. Both NS5A and NS5A-DAH are progressively solubilized by the addition of more MNG-3 to the reaction; however, at around 500-650 lM of MNG-3, the pellet fraction seems to remain stable, and no further solubilization is observed. One hypothesis explaining the fact that part of it is invariably found in the pellet, independent of the detergent concentration, might be that the treatment with the endonuclease benzonase is not efficient in accessing the entire amount of nucleic acids present in a particular form of dense NS5A/RNA/DNA assemblies which pellet during centrifugation.

Conclusion

Even if the general use of C12E8 and MNG-3 detergents for a successful expression and solubilization of membrane proteins in the wheat germ cell-free system remains to be established, our results present a further step in devising strategies for the expression of membrane proteins in this cell-free system. Proteins expressed in their detergent-solubilized form can subsequently be used for studies by NMR in solution state and X-ray crystallography, as well as, after reconstitution into membranes, by solidstate NMR and electron microscopy.

Materials & methods

Plasmids

cDNA of NS2 and NS2 pro (HCV strain JFH1; GenBank accession number AB047639), NS4B, NS5A and NS5A-DAH (HCV strain Con1; GenBank accession number AJ238799) were PCR amplified and cloned into pEU-E01-MCS vector (CellFree Sciences, Japan). For full-length NS2 construct, the amino acid (aa) sequence Met-Ala-Ser precedes a Strep-tag II [START_REF] Schmidt | The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins[END_REF] followed by a thrombin cleavage site downstream of a Ser-Gly linker, yielding to the final aa sequence MASWSHPQFEKTGLVPRGSG fused at the N terminus of full-length NS2, For the NS2 pro construct, no thrombine cleavage site has been inserted yielding to the aa sequence MAS-WSHPQFEKSG. For NS4B, NS5A and NS5A-DAH constructs, the Strep-tag II was fused at the C-terminal end of the protein. For NS4B, a thrombin cleavage site followed by a Ser-Ala linker was inserted upstream of the Strep-tag II, yielding to the C-terminal aa sequence LVPRGSAWSHPQFEK. For NS5A and NS5A-DAH, no thrombin cleavage site has been inserted yielding to the aa sequence SAWSHPQFEK. The resulting plasmids were transformed into Escherichia coli TOP10 chemically competent cells (Life Technologies) and DNA was prepared using a NucleoBond Xtra Maxi kit (Macherey-Nagel, France). Plasmids were further purified by a phenol/chloroform extraction according to CellFree Sciences (Yokohama, Japan) recommendations.

Wheat germ cell-free protein expression

Home-made wheat germ extracts were prepared from non-treated durum wheat seeds (Sud Céréales, France) according to Takai et al. [START_REF] Takai | Practical cell-free protein synthesis system using purified wheat embryos[END_REF]. Transcription and translation were carried out separately, as described in [25][START_REF] Moradpour | Replication of hepatitis C virus[END_REF][START_REF] Lohmann | Hepatitis C virus RNA replication[END_REF][START_REF] Takai | Practical cell-free protein synthesis system using purified wheat embryos[END_REF] and in Noirot et al. [START_REF] Noirot | Wheatgerm cell-free production of prion proteins for solid-state NMR structural studies[END_REF]. Transcription was performed in Eppendorf tubes using 100 lg/ml plasmid, 2.5 mM NTP mix (Promega, France), 1 U/ll SP6 RNA Polymerase (CellFree Sciences, Japan) and 1 U/ll RNase Inhibitor (CellFree Sciences, Japan) in transcription buffer (CellFree Sciences, Japan). After incubation for 6 h at 37 °C, mRNA was used directly for translation. Translation was performed in 96-well plates using the so-called bilayer method. The bottom layer, called reaction mix, contains 10 ll mRNA, 10 ll wheat germ extract and 40 lg/ml creatine kinase. The upper layer corresponds to the feeding buffer (200 ll per well) that contains all substrates necessary for protein synthesis: 30 mM Hepes-KOH pH7.6, 100 mM potassium acetate, 2.7 mM magnesium acetate, 16 mM creatine phosphate, 0.4 mM spermidine, 1.2 mM ATP, 0.25 mM GTP, 4 mM DTT and 6 mM amino acids mix. When expression was performed in presence of detergent, the detergent was added in both the reaction mix and the feeding buffer. The reaction sample was incubated at 22 °C for 16 h.

Sample preparation

After protein synthesis, 150 ll of cell free sample (CFS) were incubated with 250 units of benzonase at room temperature (RT) on a rolling wheel for 30 min. CFS was then centrifuged at 20,000g, 4 °C for 30 min. The obtained supernatant was incubated with 5 ll Strep-Tactin magnetic beads (MagStrep ''type2HC'': IBA Lifesciences, Germany) at 4 °C on the wheel for at least 30 min. After three washing steps with 10 volumes of a buffer containing 100 mM Tris-HCl pH8, 150 mM NaCl and 1 mM EDTA, the magnetic beads were directly resuspended in 25 ll 1Â SDS-PAGE loading buffer (62.5 mM Tris-HCl pH6.8, 2% SDS (w/v), 10% glycerol (v/v), 5% b-mercaptoethanol (v/v) and 0.01% bromophenol blue (w/v)). Theses samples were further designated as SN-beads. Note that this step aimed to enrich the sample with the tagged protein and to assess whether the used detergent would interfere with the protein affinity capture. The pellet obtained after centrifugation of the CFS was resuspended in 25 ll 1Â SDS-PAGE loading buffer. In addition, 20 ll CFS were mixed with 5 ll 5X loading buffer for SDS-PAGE analysis.

SDS-PAGE and Western blotting analysis

All expression experiments were assessed using 15% Coomassie blue stained SDS-PAGE gels. Samples are resuspended in a loading buffer containing 62.5 mM Tris-HCl pH6.8, 2% SDS (w/v), 10% glycerol (v/v), 5% b-mercaptoethanol (v/v) and 0.01% bromophenol blue (w/v) and incubated at RT for 15 min before loading. For each sample, 10 ll were loaded on the SDS-PAGE gels. Western-blotting analysis was carried out by protein transfer onto a nitrocellulose membrane using an iBlot Ò gel transfer device (Life Technologies). The nitrocellulose membrane was blocked with 5% non-fat milk powder in PBS-T buffer (12 mM sodium phosphate pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween Ò 20) and, in the case of the pellet and SN-beads samples, incubated with the StrepMAB-Classic, HRP conjugate antibody (IBA Lifesciences, Germany) for 1 h at RT. For a more sensitive detection of the protein of interest in the CFS, the nitrocellulose membrane is incubated with the StrepMAB-Classic primary antibody (IBA Lifesciences, Germany) for 1 h at RT and further incubated with an anti-mouse IgG HRPconjugated secondary antibody (Promega, France) for 1 h at RT. In both cases, epitope-containing bands are visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, France).

Purification of NS2 and NS4B by affinity chromatography

Both NS2 and NS4B were expressed in presence of 1300 lM (0.065%) MNG-3 using the bilayer method as described above. Protein synthesis was upscaled in a 6-well plate (500 ll reaction mix and 5.5 ml feeding buffer per well). After protein synthesis, the cell free sample (CFS) was incubated with benzonase at room temperature (RT) on a rolling wheel for 30 min. MNG-3 concentration was adjusted to 0.1%. CFS was then centrifuged at 20,000g, 4 °C for 30 min. The obtained supernatant was loaded on a Strep-Tactin gravity column (IBA Lifesciences, Germany). Purification was performed as described by the manufacturer, all buffers containing 0.1% MNG-3. Proteins of interest were eluted in 100 mM Tris-HCl pH8, 150 mM NaCl, 1 mM EDTA, 0.1% MNG-3 and 2.5 mM desthiobiotin.

Size exclusion chromatography

For both NS2 and NS4B, 200 ll fractions of protein purified by affinity chromatography were loaded on a KW402.5-4F column (Shodex, USA) using the NGC Chromatography System (Bio-Rad, USA). The column was equilibrated with 50 mM phosphate buffer pH 7.4 containing 0.1% MNG-3 and a flow rate of 150 ll/min was applied. 200 ll affinity elution buffer were also loaded on the column as control.

Introduction

The HCV non-structural protein 2 (NS2) is critical for both HCV polyprotein processing and virus particle formation [1][2][3]. The C-terminal domain functions as a cysteine protease [4] that cleaves the NS2-NS3 junction to liberate fully functional NS3 thus promoting HCV RNA replication . In addition, NS2 is thought to play a central organizing role during HCV assembly, likely involved in a complex network of interactions including E2, p7, NS3, and NS5A [8][9][10][11][12]. NS2 is a 217-amino-acid (aa) integral membrane protein that is bound to intracellular membranes via its hydrophobic N-terminal integral membrane domain (residues 1 to about 100). This domain is believed to comprise 3 transmembrane segments whose atomic structures have been studied separately by NMR (nuclear magnetic resonance) spectroscopy, allowing us to propose a topology model of full-length, membrane-associated NS2 [8,13]. The x-ray crystal structure of the C-terminal protease domain displays a domainswapped homodimer with composite catalytic triads including residues from the two chains [4]. This domain was shown to contribute to membrane association via two α-helices [14].

Although a wealth of in cellulo and functional studies has been carried out for NS2 [8,13,14], its biochemical analysis is hampered by the very low expression level of recombinant full-length NS2 using classical cell-based expression approaches [15]. We here present successful cell-free expression of the protein using wheat germ extracts (WGE) in the presence of detergent . Moreover, we demonstrate that the protein can be obtained in a detergent-solubilized, well-folded, homogenous and functional form, and that it interacts with model membranes.

Results

NS2 can be efficiently produced in an active form using a wheat germ extract (WGE) cell-free expression system

Expression of NS2 with a Strep-tag II (ST) fused at its N-terminus in the WGE cell-free expression system relies on the production of synthetic, in vitro transcribed RNA encoding the protein sequence, which is added to a reaction mixture containing the WGE, together with energy-sources, energy-recycling enzymes and aa needed for protein synthesis [16,21,22].

Using this approach, NS2 from different HCV genotypes comprising isolates Con1 (genotype 1b), H77 (genotype1a), JFH-1 (genotype 2a) and 452 (genotype 3a) was successfully expressed (Figure 1A). In the absence of detergent, the protein is mainly insoluble as indicated by its localization in the pellet fraction. Only a minor portion of NS2 protein was found in the supernatant (denoted "SN-beads" since the tagged protein is concentrated by binding of the NS2 N-terminal Strep-tag II to magnetic Strep-Tactin beads). However, in the presence of 0.1 % lauryl maltose neopentyl glycol (MNG-3) detergent, fully solubilized JFH-1 and Con1 NS2 were obtained (see also reference [16]) while H77 and 452 NS2 partially remained in the pellet. In order to evaluate NS2 functionality in our expression system, we monitored its cysteine-protease activity. To this end we expressed a JFH-1 NS2-NS3pro

Strep-tagged precursor construct (denoted NS2-NS3pro-ST) that include the 213 N-terminal aa of NS3, and detected the cleavage products using antibodies directed either against the NS3pro C-terminal Strep-tag II (denoted NS3pro-ST), or a conformational NS2 antibody.

Note that NS3pro-ST was inactivated (S139A mutation) to avoid unspecific cleavages by this protease. The immunoblot displayed in Figure 1B shows that both cleavage products, being NS3pro-ST and NS2 could be detected in the reaction mixture. Cleavage specificity was confirmed by using the inactive NS2 mutant bearing the C184A mutation in its catalytic site, for which accumulation of uncleaved NS2-NS3pro-ST precursor was observed.

Solubilized NS2 is obtained in a pure and homogenous form

A major challenge in the expression of membrane proteins is to isolate the protein in a pure form, and to avoid formation of aggregates. The N-terminal Strep-tag II allowed us to obtain pure NS2 protein using a single affinity-purification step starting from the supernatant fraction containing the solubilized protein (Figure 2A). It is worth mentioning that the addition of 0.25% DDM before the affinity-purification step not only allowed buffer exchange from MNG-3 to DDM, but resulted in improved NS2 purity and yield. Indeed, the purity of NS2 obtained in the presence of MNG-3 during this step was not satisfactory [16],

as also observed in the presence of lower amounts of DDM (data not shown). With respect to yield, while purity was good in the presence of even higher amounts of DDM (0.5 -1%), the protein yield was substantially reduced under this condition since the high detergent concentration decreases NS2-strep-tag II binding to the strep-Tactin column. The yield of purified NS2 was about 1 mg per mL of WGE. To further characterize purified NS2 protein, we performed size exclusion chromatography. A major peak containing NS2 was observed (Fig. 2B), while the following minor peak is due to reagents present in the Strep-Tactin elution buffer. NS2 protein-detergent complexes eluted with an apparent molecular mass of about 150 kDa (Fig. 2C), indicating the oligomeric nature of recombinant full-length NS2 in detergent micelles.

NS2 is well folded

The far UV circular dichroism (CD) spectrum of full-length NS2 eluted from size exclusion chromatography in 0.1% n-dodecyl β-D-maltoside (DDM) is typical of a well-folded protein (Figure 3). Additionally, the mean molar ellipticity per residue is in the range that can be expected for a protein (few 10,000's). The two minima at 208 and 222 nm, together with a maximum at 192 nm, are typical of α-helical folding. Spectral deconvolution indicated an αhelix content of 52-58 %, while turns represent 12-17%, and β-sheet content is limited to 8-9 %. The latter value is consistent with the content in β-sheet deduced from the crystal structure of the cytosolic domain of NS2 [4]. The limited number of residues folded into α-helices in this crystal structure indicates that the main contribution to helix content in full-length NS2 is due to its membrane domain. This is in agreement with our previous structural analyses of this membrane domain using synthetic peptides, which mostly revealed the presence of α-helical segments [8,13].

NS2 can be reconstituted into model membranes

While expression in cell-free systems in the presence of liposomes might allow for direct membrane insertion of the produced proteins, detergent solubilization and subsequent reinsertion into membranes allows for higher protein yields and purer preparations. Successful

Discussion

NS2 is an important protein in the life cycle of the virus, and is believed to play manifold roles in the progress of virus assembly. Parts of its structure could be studied using different biophysical approaches as x-ray crystallography and solution-state NMR . However, more detailed studies of the protein are missing, as active full-length NS2 has not been available due to the difficulties encountered for overexpression. So far, N-terminally truncated protein has been used to functionally characterize the protease activity, and refolding from inclusion bodies was mandatory [15]. The direct expression in soluble form as presented here circumvents many difficulties linked to purification from inclusion bodies, notably the presence of non-native forms on refolding and protein loss. While many studies have addressed interactions between NS2 with several other viral proteins in cellulo, no invitro assays on these interactions could be carried out in order to gain knowledge on the molecular mechanisms. Indeed, these interactions presumably take place via the hydrophobic membrane-inserted N-terminal region [9,13], and their occurrence might as well rely on the details of the interaction of the protein complex with the membrane. An experimental set-up targeting the identification of these interactions at a molecular level is thus difficult with truncated forms of the protein, and as well might be unsuccessful in detergent-solubilized states. The availability of full-length NS2 in a purified, detergent-solubilized or membranebound forms, in sufficient quantities, opens the perspective for biochemical in vitro structural and functional studies of this enigmatic protein, including structural studies which aim at analyzing the protein in its membrane environment by means of electron microscopy and solid-state NMR. Furthermore, it will allow the probing of interactions with other viral proteins which can also be successfully produced using WGE cell-free expression system [16].

  pEU-E01-StrepII-TCS-NS2fl-Con1 pEU-E01-StrepII-AgeI-TCS-BspEI HCV JFH-1 NS2 94-217 (NS2 pro ) StrepII N-ter AgeI/BamHI pEU-E01-StrepII-TCS-NS2fl-JFH1 pEU-E01-StrepII-AgeI-TCS-BspEI HCV JFH-1 NS2 94-217 (NS2 pro ) StrepII-TCS N-ter BspEI/BamHI pEU-E01-StrepII-TCS-NS2fl-JFH1 pEU-E01-StrepII-AgeI-TCS-

  each) 20 μL CFS + 5 μL Laemmli 5X SDS-PAGE & WB (10 μL each) + benzonase (1 μL / 150 μL CFS) 20,000 g, 4°C CFS membrane protein expressed in the precipitate mode membrane protein expressed in presence of MNG-3 purification on a Strep-Tactin column 0.1% DDM in all buffers 0.1% DDM in all buffers + benzonase (50 μL ≈ 12,500 units / 6 mL CFS) 35 (ratio NS2/A8-35 = 5, w/w) 30 min @ RT, on the wheel overnight @ 4°C without agitation (optional) + Bio-Beads (ratio Bio-Beads/DDM= 20, w/w) 2h @ RT, on the wheel

Fig. 1 .

 1 Fig. 1. Production of full-length NS2 using the wheat germ cell-free expression system in the presence of nine different detergents. Protein samples were analysed by SDS-PAGE followed by Coomassie blue staining (upper panels) and western blotting (lower panels). CFS, cell-free sample; pellet, pellet obtained after centrifugation of CFS; SN-beads, supernatant obtained after centrifugation of CFS and incubated with Strep-Tactin magnetic beads to capture Strep-tag II-tagged NS2 protein; À, negative control (no NS2); +, positive control (NS2 expressed in the absence of detergent). The black arrowheads indicate NS2. CFS -3.6% of total sample (220 ll reaction mix); Pellet and SN beads -27% of the total sample (220 ll reaction mix) loaded on the gel.

Fig. 2 .

 2 Fig. 2. Determination of the optimal DDM, MNG-3 and C12E8 detergent concentration for the expression and solubilization of the NS2 protein. NS2 was expressed in presence of the indicated concentrations of DDM (A), MNG-3 (B) or C12E8 (C). For conditions and labels, see Fig. 1. The black arrow indicates the band corresponding to NS2 protein. Detergent concentrations are given in lM. (D) Chemical structures of DDM, MNG-3 and C12E8.

Fig. 3 .

 3 Fig. 3. Determination of the optimal concentration of MNG-3 and C12E8 detergents for the expression of NS2 pro . For conditions and labels, see Fig. 1. The black arrow indicates the band corresponding to NS2 pro . Detergent concentrations are given in lM.

Fig. 4 .

 4 Fig. 4. Determination of the optimal concentration of MNG-3 for the expression of the NS4B, NS5A and NS5A-DAH proteins. For conditions and labels, see Fig. 1. The black arrow indicates the band corresponding to the protein of interest. Detergent concentrations are given in lM.

Fig. 5 .

 5 Fig. 5. Purification of NS2 and NS4B by affinity chromatography, and analysis by size exclusion chromatography. NS2 (A) and NS4B (B) purified by affinity chromatography were analyzed by SDS-PAGE followed by Coomassie blue staining. CFS, cell-free sample -0.02% of total sample (36 ml reaction mix) loaded on the gel; P, pellet obtained after centrifugation of CFS -0.2% of total sample loaded on the gel; SN, supernatant obtained after centrifugation of CFS -0.02% of total sample loaded on the gel; E1 to E6, elution fractions -1.6% of total fraction loaded on the gel. The black arrow indicates the band corresponding to the protein of interest. Size exclusion chromatography profiles of NS2 (C) and NS4B (D). The profile of the protein sample of interest is drawn in continuous line and the one of the affinity elution buffer in dotted line. Western blotting analyses of the collected fractions are shown above the chromatographic profiles.

  : *:**: .**.**.: . * ::* *** *:*::** :::*.: . **.: *:* * *.:.: **:*.*:*:: :. .**:*:**:*: .** **:***::** ** *:*.***:*******: ******* *:*: *****. **:**

  insertion into membranes is also a good indicator for the structural integrity of the membrane protein. Moreover, homogenous membrane insertion is a prerequisite for further structural studies of the protein by electron microscopy or solid-state NMR. To this end, we analyzed our protein preparations and the possibility to reconstitute NS2 proteoliposomes by electron microscopy. As already indicated by size exclusion chromatography, NS2 solubilized in detergent forms multimers, which can be visualized by electron microscopy using immunolabeling of the N-terminal Strep-tag II fusion protein produced in the presence of detergent (Figure4A). Absence of gold particles on negative control grids lacking NS2 confirmed labeling specificity (data not shown). When detergent-solubilized lipids are added to detergent-solubilized NS2, the protein binds to or integrates into the lipid bilayer upon detergent removal, as shown in Figures4B and 4Cfor L-α-phosphatidylcholine (PC) and a PC/cholesterol mixture (70/30 w/w) respectively. No dispersed labeling of NS2 protein is found, and gold-decorated liposomes are observed, suggesting the tight association/insertion of NS2 into model membranes.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Purification of full-length NS2 (strain JFH-1 of HCV genotype 2a) by affinity chromatography and analysis by size exclusion chromatography. (A) Full-length Nterminally Strep-tag II tagged NS2 was expressed in presence of 0.1% MNG-3, complemented with 0.25% DDM before purification on a Strep-Tactin column in presence of 0.1% DDM. Protein samples were analyzed by SDS-PAGE followed by Coomassie blue staining. CFS, total cell-free sample (8 μL from 108 mL loaded on the gel); P, pellet obtained after centrifugation of CFS (equivalent to 80 μL from 108 mL loaded on the gel); SN, supernatant obtained after centrifugation of CFS and loaded on the affinity column (8 μL from 108 mL loaded on the gel); E1 to E6, affinity elution fractions (8 μL from 2.5 mL loaded on the gel). The black arrowhead indicates the band corresponding to NS2. (B) Size exclusion chromatography of E3 elution fraction using Superdex TM 200 Increase 3.2/100 column. The elution profile of NS2 is drawn in a solid line, and that of the affinity elution buffer in a dotted line. SDS-PAGE and Comassie staining analysis of the collected fractions is shown above the chromatographic profile. Elution volume of NS2 is 1.65 mL. (C) Molecular weight calibration curve of the size exclusion chromatography column established using protein standards (V 0 is 1.25 mL). NS2 Kav is indicated in red on the standard curve.

Figure 3 :

 3 Figure 3: Far UV circular dichroism (CD) spectrum of NS2 after purification by affinity chromatography and size exclusion chromatography. Estimation of the secondary-structure content resulted in an α-helix content of 52-58 %, in 12-17% turns, and in 8-9 % β-sheet.

Figure 4 :

 4 Figure 4: Transmission electron microscopy analysis of full-length NS2 using negative staining and immunogold labeling. (A) NS2 in 0.1% DDM after purification by affinity chromatography and size exclusion chromatography. (B) NS2 reconstituted in egg PC (L-α-phosphatidylcholine). (C) NS2 reconstituted in a mixture of egg PC and cholesterol (70/30, w/w). The scale bar corresponds to 200 nm in all panels.

Table 1

 1 Expression level of full-length NS2 in a cell-free system using wheat germ extract in presence of nine different detergents.

	Detergent Charge	MW	Aggregation CMC		Detergent concentration 0.05%			Detergent concentration 0.1%
			(Da)	Number	lM	%	Det. conc.	Micelle conc.	CFS SN-	Det. conc.	Micelle conc.	CFS SN-
							(lM)	(lM)		Beads	(lM)	(lM)	Beads
	Control								+++ +			+++ +
	DPC	Zwitterionic	351 50-60	900 0.032 1425	9-10	-	-	2849	32-39	-	-
	DM	Nonionic	483 110-140	1800 0.087 1035	-	+++ +	2070	1.9-2.5	+	-
	DDM	Nonionic	511 98	170 0.009	978	8	++	+	1957	18	+	+
	MNG-3	Nonionic	1005 ? ⁄	10 0.001	498	? ⁄	+++ +++	995	? ⁄	+++ +++
	C12E8	Nonionic	539 90-120	90 0.005	928	7-9	+++ +++	1855	15-20	+++ +++
	LDAO	Zwitterionic	229 74	1000 0.023 2183	16	-	-	4367	45	-	-
	CHAPS	Zwitterionic	615 4-14	8000 0.492	813	-	+++ +	1626	-	+++ ++
	cholate	Anionic	431 4	9000 0.388 1160	-	+++ +	2320	-	+++ +
	OG	Nonionic	292 900-1300	12000 0.350 1712	-	+++ +	3425	-	+++ +

DPC, dodecyl-phosphocholine; DM, n-decyl-D-maltoside; DDM, n-dodecyl-D-maltoside; MNG-3, lauryl maltose neopentyl glycol; C12E8, dodecyl octaethylene glycol ether; LDAO, lauryl dimethyl amide oxide; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propansulfonat; cholate, 3, 7, 12-trihydroxy-5-cholan-24-oic acid, monosodium salt; OG, n-octyl-D-glucopyranoside. CFS, cell-free sample; SN-Beads, supernatant obtained after centrifugation of CFS and incubated with Strep-Tactin magnetic beads to capture Strep-tag II tagged proteins. Expression level is indicated as follow: +++, high; ++, medium; +, low; À, no expression. ⁄ No data is available for the aggregation number of MNG-3. Considering protein yields after purification by affinity chromatography of around 0.5 mg NS2 per milliliter of wheat germ extract, +++ represents at least 5 lg of protein in the cell-free sample containing 10 ll of wheat-germ extract.

M.-L. Fogeron et al. / Protein Expression and Purification 105 (2015) 39-46

Acknowledgments

This work was supported in part by a grant from the French ANRS (France Recherche, Nord & Sud, Sida-HIV et Hépatites), an autonomous agency at INSERM, France, and the Swiss National Science Foundation (grant 31003A-138484 to DM). R.B. was supported by the Deutsche Forschungsgemeinschaft (TRR 83, TP13). We acknowledge the platforms Centre Commun de Microanalyse des Protéines and Production et Analyse de Protéines from the UMS 3444 BioSciences Gerland-Lyon Sud. We wish to express our thanks to Marc Le Maire for critical reading of the manuscript and to Pierre Falson for fruitful discussions.

Acknowledgements

This work was supported by a grant from the French ANRS (France Recherche, Nord & Sud, Sida-HIV et Hépatites), an autonomous agency at INSERM, France, and by the ANR (ANR-14-CE09-0024B and ANR-11-BINF-003 Mapping project). R.B. is supported by the Deutsche Forschungsgemeinschaft (TRR 83, TP13). We would like to thank Yaeta Endo for the interesting discussions on protein cell-free expression.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2014.10.003.

Material and Methods

Plasmids cDNA of full-length NS2 from four different HCV strains was PCR amplified and cloned into the pEU-E01-MCS vector (CellFree Sciences, Japan). HCV strains, genotypes (in parenthesis), and their respective GenBank accession numbers are as follows: JFH-1 (2a), AB047639; Con1 (1b), AJ238799; H77 (1a), AF009606; strain 452 (3a), DQ437509. The aa sequence Met-Ala-Ser precedes a Strep-tag II followed by a thrombin cleavage site downstream of a Ser-Gly linker, yielding to the final aa sequence MASWSHPQFEKTGLVPRGSG fused at the N-terminus of the various full-length NS2 proteins [16]. In addition, cDNA of the two following NS2-NS3pro-ST precursors (strain JFH-1) was PCR amplified and cloned into the pEU-E01-MCS vector too. The first construct comprises wild type full-length NS2, and the second the full-length NS2 with a mutation within its catalytic site (C184A mutation, C997A accordingly to HCV polyprotein numbering). Note that, in both constructs, NS3pro serine protease activity was inactived by the S139A mutation (S1169A according to HCV polyprotein numbering) to avoid unspecific cleavages by this protease. The amino acid linker sequence Ser-Gly-Gly precedes a Twin Strep-tag II fused at the C-terminus of NS3pro, yielding to the final aa sequence SGGWSHPQFEK(GGGS) 2 GGSAWSHPQFEK. As detailled in [16], the resulting plasmids were transformed into Escherichia coli TOP10 chemically competent cells (Life Technologies) and DNA was prepared using a NucleoBond Xtra Maxi kit (Macherey-Nagel, France). Plasmids were further purified by a phenol/chloroform extraction according to CellFree Sciences (Yokohama, Japan) recommendations.

Wheat germ cell-free protein expression and sample preparation

Wheat germ cell-free protein expression and sample preparation were performed as described in [16]. In short, home-made wheat germ extracts [22] were prepared according to Takai et al. [START_REF] Takai | Practical cell-free protein synthesis system using purified wheat embryos[END_REF]. Transcription and translation were carried out separately, as described in [25][START_REF] Moradpour | Replication of hepatitis C virus[END_REF][START_REF] Lohmann | Hepatitis C virus RNA replication[END_REF][START_REF] Takai | Practical cell-free protein synthesis system using purified wheat embryos[END_REF] and in Noirot et al. [START_REF] Noirot | Wheatgerm cell-free production of prion proteins for solid-state NMR structural studies[END_REF], and translation was performed using the so-called bilayer method.

When present, MNG-3 detergent was added in both the reaction mix and the feeding buffer.

The reaction mixture was incubated at 22 °C for 16 h. Translation was performed either in 96well plates for small-scale expression test or 6-well plates for larger production.

SDS-PAGE and Western blotting analysis

SDS-PAGE and Western blotting analysis was performed as described in [16]. For activity test using the NS2-NS3pro-ST precursor, the CFS was analyzed by western blotting using antibodies either against the Strep-tag II fused at the C-terminus of NS3pro (StrepMAB-Classic, IBA Lifesciences, Germany) or against the C-terminal domain of NS2.

Purification of full-length NS2 by affinity chromatography

NS2 was expressed in presence of 0.1% MNG-3 using the bilayer method as described previously [16] adapted to wells of 6-well plates (i.e., 0.5 mL of reaction mix containing 0.25 mL mRNA and 40 μg/ml creatine kinase and 0.25 mL of wheat germ extract for the bottom layer, and 5.5 mL of feeding buffer for the upper layer in each well). After protein synthesis, the CFS (total cell-free sample) of 18 wells was pooled (108 mL) and incubated with benzonase at RT on a rolling wheel for 30 min. In addition, DDM was added to CFS at a final concentration of 0.25%, leading to a higher purity level than MNG-3. CFS was then centrifuged at 20.000 g, 4°C for 30 min and the supernatant was loaded on a 5 mL Strep-Tactin high capacity gravity column (IBA Lifesciences, Germany). Purification was performed as specified by the manufacturer, all buffers containing 0.1% DDM. Full-length NS2 was eluted in 100 mM Tris-HCl pH8, 150 mM NaCl, 1 mM EDTA, 0.1% DDM and 2.5 mM desthiobiotin.

Size exclusion chromatography

Size exclusion chromatography of full-length NS2 previously purified by affinity chromatography was performed using a Superdex TM 200 Increase 3.2/100 column (GE Lifesciences). A 50 mM phosphate buffer pH 7.4 containing 0.1% DDM was used as eluent and 150 μg protein were loaded on the column. Calibration curve of the size exclusion chromatography column was established according to le Maire et al. [23] using the following protein standards: cytochrome C, 12.3 kDa ; myoglobin, 17.8 kDa, trypsin inhibitor, 22.1 kDa ; β-lactoglobulin, 35 kDa ; transferrin, 81 kDa ; aldolase, 158 kDa ; ferritin, 440 kDa ; thyroglobulin, 669 kDa. A 50 mM phosphate buffer pH 7.4 containing 0.1% DDM was used as eluent and 100 μg of each protein standard were run separately on the column.

Circular dichroism

Spectra were recorded on a Chirascan spectrometer (Apllied Photophysics) calibrated with 1S-(+)-10-camphorsulfonic acid. Measurements were carried out at 298 K in a 0.1-cm path length quartz cuvette. Spectra were measured in a 180-260 nm wavelength range with an increment of 0.2 nm, bandpass of 0.5 nm and integration time of 1 s. Spectra were processed, baseline corrected, smoothed and converted with the Chirascan software. Spectral units were expressed as the mean molar ellipticity per residue using the protein concentration determined at 280 nm using a NanoDrop spectrometer. Estimation of the secondary structure content was carried out using the CDSSTR, CONTIN and SELCON3 approaches available on the DICHROWEB server (dichroweb.cryst.bbk.ac.uk/).

Lipid reconstitution

Either egg PC (L-α-phosphatidylcholine, 99% pure) or a mixture of egg PC and cholesterol (99% pure, 70/30, w/w) used for lipid reconstitution were purchased from Sigma. The lipids were first solubilized in DDM with a detergent-to-lipid ratio of 10 (mol/mol) [24]. Purified full-length NS2, solubilized in 0.1% DDM, was then mixed with the lipids at a lipid-to-protein ratio of 20 (w/w). Protein-lipid-detergent samples were dialyzed at room temperature (RT) for 6 days against a buffer containing 100 mM Tris-HCl pH8, 150 mM NaCl, 1 mM EDTA, 1 mM DTT and hydrophobic polystyrene beads (Bio-Beads SM-2 adsorbents, Bio-Rad). A dialysis membrane with a molecular weight cut-off of 6-8000 Da (Spectra/Por) was used. A ratio Bio-Beads-to-detergent of 100 (w/w) was applied. Detergent removal by the Bio-Beads leads to the formation of proteoliposomes [25].

Electron microscopy

The material was adsorbed to carbon-and pioloform-coated 300 mesh copper grids (Science Services GmbH) for 10 min at RT, and remaining liquid was drained using a whatman paper.

For immunolabeling, all incubation steps were performed by floating the grids on top of drops. Prior to immunogold labeling, grids were incubated with blocking solution (0.8% bovine serum albumin, 0.1% fish skin gelatin, 50 mM glycine in PBS). Primary mouse anti-Strep-tag II antibody was diluted 1:100 and incubated for 20 min in blocking solution. After five 2-min washes with PBS, grids were incubated with rabbit anti-mouse bridging antibody (Dako Cytomation) diluted 1:150 in blocking solution for 30 min. After five additional wash steps in PBS, bound antibodies were labeled with protein A coupled to 10 nm colloidal gold suspension (Cell Microscopy Center) diluted 1:60 in blocking solution for 30 min. Grids were washed five times with PBS and fixed with 1% glutaraldehyde (Science Services GmbH; Munich, Germany) in PBS for 5 min. Subsequently, grids were washed five times with PBS followed by 5 wash steps in water and negatively stained as follows: grids were washed 3 times for 2 min with water on ice and subsequently, structures were negatively stained using a mixture of 3% aqueous uranylacetate and 2% methyl-cellulose (40:60, v/v) for 8 min on ice.

Grids were looped out and dried for 5 min at RT. Samples were examined by using a Zeiss EM-10 transmission electron microscope (Zeiss, Goettingen, Germany) with a built in Mega View camera (Olympus; Tokyo, Japan).


Bricola Jean-Charles
email: jean-charles.bricola@mines-paristech.fr

Bilodeau Michel

Beucher Serge

A multi-scale and morphological gradient preserving contrast

Keywords: Mathematical morphology, multi-scale gradients, perceptual issues

Introduction

This document outlines an algorithm which extends and enhances the regularised gradient introduced in [START_REF] Rivest | Morphological gradients[END_REF]. The regularised gradient is known to be a thin gradient. It has little noise, is multiscale and has been extensively used, for instance, for the extraction of road markers from particularly image sets [START_REF] Beucher | Road segmentation by watershed algorithms[END_REF]. However, the intensity values taken by the regularised gradient are usually not representative of the perceived contrast across object boundaries. This limitation may prevent hierarchical segmentations based on the waterfalls or synchronous floodings from being meaningful with respect to the actual human perception of saliency. The proposed enhancement of the regularised gradient preserves the thinness and multi-scale properties of the latter whilst taking the actual contrast across different image scales into account.

The Algorithm

The computation of the regularised gradient and the one of its enhanced version is presented in table 1. Common to both variants, the gradient of a grayscale image I may be computed between the minimum and maximum scales λs and λe respectively. Let λ be the size of an isotropic structuring element used within the dilation δ, erosion ε and opening γ operators.

When computing the regularised gradient, the thick gradient in step 1.1 captures the intensity variations of image I at scale λ. The top-hat in step 1.2 eliminates the relief crests having a thickness larger than λ in order to remove the thick contours which have merged due to the presence of thin objects, whilst the erosion in step 1.3 restores the thick residual contours to their original resolution. The following observations, which constitute a starting point for the elaboration of our enhanced regularised gradient, have been made:

• The thick gradient in step 1.1 is by definition highly sensitive to noise. In other words, regions which are supposed to be homogeneous but which are affected by noise will get a significant gradient magnitude. The top-hat in step 1.2 will therefore destroy the dynamic of the crests of interest. To overcome that phenomenon, a strong levelling, also referred to as a sequential levelling alternating dilations and erosions of sizes increasing up to scale λ [START_REF] Meyer | Levelings: theory and practice[END_REF], is applied on the input image (step 2.1 of our algorithm) prior to the computation of the thick gradient at scale λ.

• The white top-hat in step 1.2 is meant to remove the contours of thin objects which, due to the chosen gradient thickness, have merged. These contours reach a thickness of 2λ when scale λ is being considered. When choosing a size of λ, the top-hat tends to remove crests where the curvature is significant as well as junction boundaries, hence the choice of the structuring element of size 2λ-1 in step 2.4.

• In practice, the erosion in step 1.3 destroys the contour dynamics, especially for high scales.

Hence this step has been removed from our algorithm. Instead, we search for the ideal transition points in step 2.2 and superimpose a binary mask on top of the enhanced thick gradient in step 2.5.

function COMPUTEMULTISCALEGRADIENT(I, λs, λe) :

1 : G initialised to the size of I, all pixels set to 0.

2 : for all λ in {λs, λs+1, ..., λe} do: Figure 2 illustrates a smooth change of intensity along a scan-line of image I, of which the actual intensity variation may be captured using a thick gradient of size λ. The transition point corresponds to the intersection of I with the morphological average ½(δλ(I) + ελ(I)) when I is not flat. In practice however, the equality between the input image and its morphological average is rarely verified.

It is necessary to separate the pixels of the input image I into three groups: those above, under or equal to the morphological average. Then, one performs a dilation on I, re-classifies the pixels into the three aforementioned groups and searches for those having a different classification. We consider that I has been "crossed" when a pixel changes from an "under" state to an "above" state. We mark this pixel as a transition point as illustrated in figure 3. Symmetrically, we repeat the same process after performing an erosion on I and take the union of the detections.

Results and Discussion

Figure 1 compares three morphological gradients obtained from a colour image which is subject to noise and blurriness. In order to take account of the colour information, we compute the supremum of the gradients for each considered channel. Instead of using the standard red, green and blue channels, we exploit channels which are representative of the perceived brightness [START_REF] Kalloniatis | Light and dark adaptation. Webvision: The organization of the retina and visual system[END_REF] and the image saturation [START_REF] Demarty | Color segmentation algorithm using an HLS transformation[END_REF] .

Qualitatively, the enhanced regularised gradient clearly highlights the contours of the train, as well as its wheels, its windows and the railway. One can also better distinguish the legs of the character standing in the middle of the scene. The same goes for the radiator and the electrical plug lying in the background. Finally our gradient is the only one among the three which captures well the strong intensity transition between the penguin's leg and the stomach or the foot.

The filtering in step 2.1 clearly contributes to the output of a de-noised gradient for λs>1. It is possible to envisage higher values of λs if one is interested in removing contours which originate from textured objects.

Conclusion

We have proposed a revision of the morphological regularised gradient. In its enhanced version, the regularised gradient reflects the perceived contrast between the objects composing a scene. The core of the multi-scale algorithm is identical for both gradients. The principal changes consist of filtering the input image before computing the thick gradients and replacing the erosion by a detection of brightness transitions for the gradient thinning step. Both the filtering and the binary mask computation are scale-dependant. The results attest to the visual appeal of the enhanced gradient which paves the way for the processing and hierarchical segmentations of difficult images.

▼

  Figure 1. Comparison of different morphological gradients on a colour image

Figure 2 .

 2 Figure 2. Illustrating the relationship between the brightness function and its thick morphological average

  Figure 3. Illustration of the transition point detection

October 2015 Update

October 2015 Update Jean-Charles Bricola, Michel Bilodeau and Serge Beucher

Acknowledgements

This work has been performed in the project PANORAMA, co-funded by grants from Belgium, Italy, France, the Netherlands, and the United Kingdom, and the ENIAC Joint Undertaking.