Fakhreddine Ababsa
email: fakhreddine.ababsa@ensam.eu

Augmented Reality Application in Manufacturing Industry: Maintenance and Non-destructive Testing (NDT) Use Cases

Keywords: Augmented Reality, Industry 4.0, Manufacturing, Human-machine interaction, Maintenance and quality monitoring

In recent years, a structural transformation of the manufacturing industry has been occurring as a result of the digital revolution. Thus, digital tools are now systematically used throughout the entire value chain, from design to production to marketing, especially virtual and augmented reality. Therefore, the purpose of this paper is to review, through concrete use cases, the progress of these novel technologies and their use in the manufacturing industry.

Introduction

We are in the era of the fourth industrial revolution after that of mechanization, massproduction and automation. Thanks to the digital transformation, factories are more and more connected and appear as part of a global ecosystem that includes the entire production chain. This new paradigm is called Industry 4.0, it aims to maintain and develop a strong and innovative industrial activity. Factories become smarter, more competitive because they are more efficient and safe; their production processes are completely changing. This transformation is mainly based on new technologies such as robotics, virtual and augmented reality, sensor networks, Internet of Things (IoT), data processing, big data, etc. It is a new model that puts the human at the heart of business and organization. Thus, the operator in his factory will accomplish his daily tasks in a different way; he will be equipped with wearable and intelligent devices that allow him to communicate and collaborate with other operators, and to assist him in carrying out his task. At the same time, customer expectations are changing much faster than before. To cope with this, companies need to be able to detect weak signals and feedback this information to anticipate changes in customer behavior. They must also be able to innovate much more quickly, to adapt their offer by adopting an agile organization allowing flexible and therefore completely reconfigurable production modes [START_REF] Kang | Smart manufacturing: Past research, present findings, and future directions[END_REF], able to deliver customized and competitive products. Virtual and Augmented Reality is one of the key components in achieving the goals of the industry 4.0. For example, virtual reality is used during the product creation process to optimize and prototype experiments in order to test new products "in near-use situations". It also allows employees to receive hands on training thanks to virtual environments, which mimic production equipment in a very realistic manner [START_REF] Fischer | Enabling live data controlled manual assembly processes by worker information system and near field localization system[END_REF]. This prevents expensive down time and disruptions in normal operations, and improve the efficiency of skills transfer and increased knowledge retention. On the other hands, augmented reality is a powerful tool that allows operators to better undertake tasks on site, thanks to its ability to visualize virtual objects in the real word. This is the case for tasks such as parts assembly [START_REF] Wang | A comprehensive survey of augmented reality assembly research[END_REF][START_REF] Loch | Comparing video and augmented reality assistance in manual assembly[END_REF], maintenance [START_REF] Paelke | Augmented reality in the smart factory: supporting workers in an industry 4.0. environment[END_REF], quality control or material management [START_REF] Munera | Control kernel in smart factory environments: Smart resources integration[END_REF]. The goal of this paper is to show the practical uses of augmented reality in the manufacturing industry. We will present two innovative solutions to answer the maintenance problem encountered in the manufacturing industry. The paper is organized as follows. Section 2 describes the related projects. Section 3 introduces the Key technologies on augmented reality currently used in the manufacturing industry. Section 4 shows two practical applications of this technology and provides a detailed analysis of its usability in an industrial setting. In Sect. 5, we present the conclusions and future work.

Related Works

The use of Augmented Reality in industry goes back to the 1990s. In 1993 [START_REF] Caudell | Augmented reality: an application of heads-up display technology to manual manufacturing processes[END_REF] Boeing engineers presented one of the first see-through displays integrating a head position estimation approach and a real/virtual registration system, and allowing to increase the operator's vision with digital information relevant to the job he is performing. On the other hand, several companies (EADS, BMW, Airbus, etc.) participated in the ARVIKA project (1999)(2000)(2001)(2002)(2003), which aimed to study the capacity of augmented reality to support work processes in the design, production and service for complex industrial products and plants [START_REF] Wohlgemuth | ARVIKA: Augmented reality for development, production and service[END_REF][START_REF] Friedrich | ARVIKA-augmented reality for development, production and service[END_REF]. The project STARMATE, funded by the EU (2000)(2001)(2002)(2003), provided two main functionalities: User assistance during assembling task of complex mechanical parts and increasing the operator's skills to carry out such processes by passing them training scenarios in augmented reality. The ARTESAS project led by Siemens (2004Siemens ( -2006) ) aimed to develop an augmented reality system for aeronautical and automotive maintenance [START_REF]Advanced Augmented Reality Technologies for Industrial Service Applications) Project[END_REF]. AVILUSplus (2009-2011) is a collaborative project that brought together German basic industries and research institutes to develop innovative systems in the areas of Virtual and Augmented Reality for products life cycle (PLM) services. The main areas are information management, simulation and rendering, tracking, interaction and geometry acquisition [START_REF] Avilusplus | Applied Virtual Technologies Focused Long-range on the Product and Production Equipment Life Cycle[END_REF]. Testia, a subsidiary of Airbus, has developed the SART (Smart Augmented Reality Tool) system for the aerospace industry. A system that allows inspection personnel, as part of quality control, to establish a digital model of the actual part on a tablet or PC, and thus reliably identify non-conforming parts [12]. The European project REPLICATE [START_REF]REPLICATE -cReative-asset harvEsting PipeLine to Inspire Collective AuThoring and Experimentation[END_REF] has improved the human creative process by integrating new user experiences in mixed reality. Thus, experimental solutions such as 3D/4D story-boarding in unconstrained spaces and the expression of new ideas by manipulating objects (assembly/disassembly) in a co-creative workspace, have been developed and tested in an industrial context. In the same way, the European project SPARK (Spatial Augmented Reality as a Key for co-creativity) (2016-2019) aims to realize an intuitive ICT platform based on spatial augmented reality (SAR) in order to show designers and customers new products/concepts in the form of mixed prototypes during brainstorming sessions, with the aim of enriching the design process. It is a collaborative platform that allows users to work freely together, promoting open innovation and creative thinking [START_REF] Spark | Spatial Augmented Reality as a Key for co-creativity[END_REF]. In 2017 Safran Landing Systems launched the Argo (Augmented Reality for Ground Operations) project [START_REF] Argo | Augmented Reality for Ground Operations[END_REF], which aims to deploy augmented reality on an industrial scale at Safran's various MRO sites to help operators with aircraft maintenance tasks and automate the writing of technical operating reports. In this way, Safran hopes to reduce intervention times, which would give a considerable competitive edge as an increase in air traffic goes hand in hand with an increase in MRO activities. In addition, several research works have been conducted over the last years to develop AR systems dedicated to the industry. For example, in [START_REF] Zhu | A context-aware augmented reality assisted maintenance system[END_REF] the authors propose the ACAAR system (Authoring for Context-Aware AR) which allows users to add and organize digital content (text, images, CAD) in 3D space and specify its relationship with the maintenance context. In [START_REF] Palmarini | A systematic review of augmented reality applications in maintenance[END_REF] the authors give a thorough state of the art on the use of AR in industrial maintenance. Future research directions are also proposed, including hardware, monitoring and user interaction. Other studies, such as in [START_REF] Sanna | Using handheld devices to sup port augmented reality-based maintenance and assembly tasks[END_REF], have focused on the use of mobile devices such as smartphones and tablets to perform maintenance and assembly procedures with augmented reality (AR). They evaluated performance by comparing the execution times and errors made when performing a maintenance procedure with an augmented reality tool and paper-based instructions.

Augmented Reality Technologies for Industrial

Applications

Traditionally, augmented reality has been seldom used in the manufacturing industry due to the lack of maturity of the technology, computational and hardware were limited and visualization devices uncomfortable. The advent of tablets and smartphones (Fig. 1) with more capabilities have enabled the development of the first augmented reality applications in industrial environments. However, these devices have not been as successful as expected, as they can divert attention of the operators, who need to have their hands free to perform their tasks. The head-mounted displays (HMDs) developed in recent years overcome these limitations.

The operator equipped with such a device can view spatialized information/instructions in hands-free mode while performing his task. In addition, these helmets over automatic gesture recognition allowing interaction with 3D holograms. Furthermore, the performance of HMDs continues to improve with each release, offering increasingly attractive functionalities for creating immersive and personalized user experiences, which makes it easier to integrate them into industrial processes [START_REF] Bach | The hologram in my hand: how effective is interactive exploration of 3D visualizations in immersive tangible augmented reality?[END_REF]. This explains the new craze for Augmented Reality in the industry in recent years. HMDs are now able to detect information coming from the environment and process/interpret them in real-time. They are more comfortable, less heavy, and easy to use and have a larger field of view. • A diagonal field of view (FoV) of 52°, improving over the 34°field of view of the first generation of HoloLens headset, while maintaining image fidelity at 47 pixels per degree of sight. In practice this, will ensure a better user experience. • An integrated finger and hand movement recognition and tracking system with greater accuracy. This is what makes it possible for users interact directly with 3D content with more natural gestures. • A powerful eye tracking software, allowing users to interact efficiently with the GUI.

• The battery is made of carbon fiber and therefore lighter compared to the HoloLens 1, it is positioned behind the head. It allows 2 to 3 h of active use. The total weight of the helmet is 566 g. • A voice-based interaction thanks to command and control on-device and Cortana natural language with internet connectivity.

Thanks to all these improvements, the Hololens 2 headset is now considered the reference device for the implementation of augmented reality applications in industrial environments.

Programming Library

The development of an industrial application based on augmented reality requires the use of high-performance software capable of tracking 3D objects, reconstructing and understanding the often cluttered and changing work environment. The most frequently used libraries are:

• ArtToolKit: open-source marker-based video tracking library that displays virtual 3D models in the real world. It consists in estimating in real time the pose (position + rotation) of the camera in regard to a planar marker placed into the physical environment. It was originally developed by Hirokazu Kato of Nara Institute of Science and Technology in 1999 The main characteristics of this library are the possibility to customize the targets as long as they remain square in shape, a simplified approach to calibrate the camera, the availability of several platform distributions and to be an open-source system giving access to the whole image processing algorithms. • ArUco Markers: a binary square fiducial markers that can be used for camera pose estimation. ArUco markers are small planar targets containing 2D binary code. Each marker is encoded thanks to the grid which composes it and whose cells are colored in black and white. The ArUco recognition algorithm allows to extract, identify and estimate the pose of the different ArUco markers seen by the camera [START_REF] Garrido-Jurado | Automatic generation and detection of highly reliable fiducial markers under occlusion[END_REF]. • Vuforia SDK: Uses image processing and computer vision approaches to detect and track on the y planar targets and 3D objects. It enables precise alignment of virtual objects to real-world objects. It allows to track several targets simultaneously. It is multi-platform (Windows, Android, IOs) and well suited for mobile devices [START_REF]Vuforia[END_REF]. • ARCore: is the platform developed by Google to provide augmented reality on mobile devices without using markers. The embedded motion tracking technology uses the phone's camera to detect and track the interesting points identified in the surrounding environment. The combination of the movement of these points with data from the phone's inertial sensors allows real-time estimation of the phone's position and orientation as it moves through space. In addition, ARCore can detect at surfaces and estimate the average lighting in its close vicinity [START_REF] Arcore | [END_REF]. • ARKit: allows to build high-detail AR experiences for iPad and iPhone devices. As the device moves, it maps the environment using data coming from on-board sensors (camera, gyroscope accelerometers). The inertial sensor data is fused with the camera information to accurately estimate the location of the device. In addition, ARKit can detect visual characteristics of the environment like planes and tracks motion in conjunction with information from the inertial sensors [START_REF] Arkit | [END_REF] Moreover, compatibility with the Unity 3D game engine is an advantage and should be examined. Indeed, Unity 3D is currently one of the most advanced games on the market and is often used to develop industrial applications using virtual and augmented reality. For example SDKs such as Vuforia, ARCore and ARKit are fully compatible with Unity 3D.

Industrial Augmented Reality Applications

Visualization of NDT Measurements Using Augmented Reality

Non-destructive testing (NDT) is an analytical method used in industry to evaluate the properties of a material, component or system without employing processes that could destroy or affect its properties. NDT is used to detect defects within a component and determine whether the component is damaged or in need of repair. Ultrasonic transducers are often used for NDT of industrial parts in their volume or surface. This consists of emitting acoustic waves and detecting their interaction with defects present in the part. The transmitted waves, like an echo, are then converted, in real time, into a digital image of the defect thus located and characterized. One of the problems of this task concerns the visualization of the measurements in an intuitive and unequivocal way. It is clear that the use of 2D images remains insufficient to illustrate and explain the results of NDT tests. Therefore, more appropriate 3D visualization tools and methods need to be developed in order to improve the understanding and interpretation of NDT data. The objective of this project is to develop an augmented reality interface allowing visualizing directly on the part the measurements resulting from the non-destructive testing. Thus, the operator will be able to visualize the areas requiring further investigation for example. This visualization mode, which is more intuitive than simple numerical data, saves time and significantly reduces the complexity of the inspection task. The part studied in our case is a nacelle of an Airbus A380 (Fig. 3) for which we have a set of data including:

• The nacelle CAD file (Fig. 4-a).

Fig. 4. Example of available data

• Thecoordinatesoftheultrasonictransducersandtheirlocationsonthenacelle(Fig. 4-b).

• The simulation files containing defect coordinates.

Based on this data, our augmented reality application therefore aims to visualize:

• On the real structure, the sensors used for the NDT measurements.

• The structure area with a defect. Sensors whose measurements indicate the presence of a defect will delimit an area. • The precise localization of the defect.

Our Approach. The proposed solution was developed under unity 3D and Vuforia, then deployed and tested on a tablet and a HoloLens 1 headset.

Fig. 5. 3D positioning of transducers

The first difficulty we encountered concerns the import of the nacelle 3D model under unity 3d. Indeed, the provided file is in Catia format which is incompatible with Unity 3D. A step of tessellation and simplification of the model was necessary to avoid overloading the computations of the augmented scene. The second step consists in creating, under 3D unity, the augmented scene. We started by positioning the sensors on the 3D model of the nacelle. It is necessary that this positioning be precise enough to allow the shown result to be coherent with the real measurements. However, the coordinates of these sensors are given in a 2D coordinate system. So, in order to represent them in 3D space, we have implemented a function that projects these positions on the surface of the nacelle whose geometry is known precisely (Fig. 5). The next step is to visualize the defect areas on the 3D model. For this purpose, we have used the NDT measurement results provided by the maintenance department, which indicate the active sensors that have detected a defect. Figure 6 shows an example of this functionality in case four sensors (symbolized here by blue dots) are active. The defect area is displayed as a polygon that passes through all four sensors. In order to reduce the defect search area, we propose to only display the areas generated by at least three and at most five active sensors.

We used Vuforia markers to implement the augmented reality part, a simple and fast solution given the complex geometry of the nacelle (large size and non-textured surface). We used Vuforia's extended tracking which, thanks to the sensors of the mobile device, continues to accurately place the virtual model in the real environment even if Fig. 6. Example of a defect area the marker is lost while moving through the scene. The marker is placed in the center of the horizontal axis of the workpiece, but also on the lower part, which has no sensors. Finally, the interactive interface of the final application allows the user to select one or more sensors in order to view various information such as coordinates or temperature. The user can also choose the defect areas present in the scenario in order to visualize them in augmented reality on the real nacelle. Figure 7 shows an example of a rendering of the latest version of our application.

Fig. 7. View of the final version of our application

Discussion. The first experimental tests have shown that this application works well. Nevertheless, we have noticed that the tracking sometimes becomes unstable, especially when we are very close to the structure or when the marker becomes invisible. To avoid this instability, we have to be at a minimum distance of 2 m from the nacelle, which is not practical for the maintenance technician who has to be as close as possible to the structure. An interesting way to solve this problem is to use markerless tracking based on a simultaneous localization and mapping system (SLAM) to create a geometric reconstruction of the world while estimating the location of the camera [START_REF] Ababsa | Robust Extended Kalman Filtering for camera pose tracking using 2D to 3D lines correspondences[END_REF][START_REF] Maidi | Vision-inertial tracking system for robust fiducials registration in augmented reality[END_REF]. In this case, SDKs such as Google ARCore or ARKit would be more appropriate. In addition, an in-depth study of the usability of the application in real conditions will be carried out. This will enable us to obtain more refined feedback from maintenance operators, particularly on the interface's ergonomics, the affordance of augmented reality interactions, and the contribution of the proposed functionalities to the practical execution of the task. This study would make it possible to improve the application by taking into account the industrial constraints of the project.

Implementation of AR Application in Aeronautical Maintenance

This project is being carried out in collaboration with Safran Landing Systems (SLS) in France. Its objective is to study the feasibility and usability of augmented reality in aeronautical maintenance. Thus, we conducted an experiment on an assembly task to observe the impact of Augmented Reality under industrial conditions. The use case corresponds to the assembly of workpieces or axles, the tightening of individual bolts and the protection of unpainted areas (Fig. 8). The participants in this experiment carried out the task under real working conditions, using two operating modes, the first one using only paper supports according to the classical process, and the second one with an augmented reality support installed on a mobile workstation. The time required to complete the task was registered and the participants' feedback was evaluated by means of questionnaires immediately after the task. We used Diota software [START_REF]Solutions 4.0 for Industry[END_REF] to create our augmented reality solution which was then installed on a Windows 10machine equipped with a high-definition industrial camera. This workstation is placed on a mobile trolley allowing it to be moved as required and easily installed on the assembly line. Thus, the operators involved in the experiment can test the scenario "with augmented reality" in real working conditions without being disturbed. This is important in order not to bias the results of the experiment. Measurement Protocol 9 maintenance operators were enrolled to take part in the experiment. None of them have any prior knowledge of augmented reality technologies. We took into account the level of expertise and knowledge of the participants regarding the task to be performed and aeronautical maintenance in general. We therefore divided them into three groups: Beginner, Confirmed and Expert. The participant begins the experiment using the classical procedure, which consists in searching on the paper support for the relevant information to carry out the assembly of the workpiece. The experiment is repeated with the augmented reality support. For each participant we measure the time needed to understand and achieve the task. We set up two questionnaires that the participants fill in at the end, the NASA-TLX [START_REF] Hart | Development of NASA-TLX (task load index): results of empirical and theoretical research[END_REF] to subjectively evaluate the quality of the experience, notably the mental demand necessary to perform the task with or without augmented reality, and the SUS [START_REF] Brooke | SUS -A Quick and Dirty Usability Scale[END_REF][START_REF] Bangor | Determining what individual SUS scores mean: adding an adjective rating scale[END_REF] to evaluate the degree of facility in using the augmented reality support. The analysis of the quantitative and qualitative results allows, firstly, to measure the impact of augmented reality on the performance of the participants, and secondly, to evaluate the usability of the augmented reality application as well as the cognitive load induced on the participants.

Discussion. The obtained results clearly demonstrate that augmented reality improves performance for all three user profiles. the "with augmented reality" scenario got the best usability score thanks to a better distribution of time between the comprehension phase and the action phase during the assembly task. We have found that augmented reality, because of its ability to facilitate the understanding of the assembly instructions, allows users to concentrate more on the action phase. This is confirmed by the decrease in the cognitive load score of the task and by a better execution time. This supports our hypothesis on the added value of augmented reality in the completion of an industrial process. Furthermore, we have noted that the impact of Augmented Reality is different depending on the level of expertise of the user. Thus, the "Beginner" users benefit the most from augmented reality which considerably reduces their cognitive load, making a strong contribution to their learning. We also noted that these augmented reality tools increase productivity when used by "Confirmed" operators but they have no effect on "Expert" users who have a perfect knowledge of the assembly process. This first experimentation has allowed us to identify and measure the added value of augmented reality on an assembly task. The obtained SUS score shows that users preferred to work with AR rather than with the reference handbook that details the maintenance process. In addition, the execution time for assembly tasks has been reduced by 74% when the AR procedure is carried out, demonstrating that AR has a direct and significant impact on the execution of maintenance tasks. The next step is to extend the experiment to other tasks related to aeronautical maintenance so that the obtained results can be generalized. You can find more details on this study in the paper we presented in 2019 at the EuroVR conference [START_REF] Loizeau | Evaluating added value of augmented reality to assist aeronautical maintenance workers-experimentation on on-field use case[END_REF]. Thus, thanks to this pilot project, Safran plans to deploy Augmented Reality in other MRO departments in Asia and the Americas, and to extend it to other services such as quality control.

Conclusion and Future Directions

The purpose of this study was to present the current state of augmented reality and their practical application in the manufacturing industry. We first outlined a brief history of industrial projects that used augmented reality, we found that such technology is often used in maintenance, training and upstream design in product development. Its use continues to increase, particularly in the context of Industry 4.0, where it represents one of the cornerstones of this paradigm. Then, the paper give the most relevant hardware and software technologies of Augmented Reality actually used in industrial applications. We presented two industrial projects in which we collaborate and whose objectives are the development of augmented reality solutions for aeronautical maintenance, and the study of the usability of such technology in MRO department, taking into account industrial constraints. We have thus implemented a methodology allowing to integrate augmented reality in an industrial task and to measure its added value in a qualitative and quantitative way. Further work will consist on extending these results to evaluate augmented reality on the whole aeronautical maintenance process. Furthermore, in order to address future challenges for the industry 4.0 efforts should focus on improving Augmented Reality HMIs by developing new user-centric approaches that integrate workers into the design loop, and enable on-site and remote collaboration.

Fig. 1 .

 1 Fig. 1. AR mobile device

Fig. 2 .Figure 2

 22 Fig. 2. Microsoft HoloLens 2

Fig. 3 .

 3 Fig. 3. Airbus A380 Nacelle

Fig. 8 .

 8 Fig. 8. The use case equipment


Kenneth Lundstrom
email: lundstrom@flamel.com

Renaud Wagner

Christoph Reinhart

Aline Desmyter

Nadia Cherouati

Thierry Magnin

Gabrielle Zeder Lutz

Melanie Courtot

Cécile Prual

Nicolas André

A1 K Lundstrom

Á G Hassaine

A4 K Lundstrom

A6 R Wagner

Á N Cherouati

Á T Magnin

Á G Zeder-Lutz

Á F Pattus

A10 C Reinhart

Á C Prual

Á N Andre

´á H Michel

Á C Cambillau

A15 K Lundstrom

Gabrielle Zeder-Lutz

Ce ´cile Prual

Nicolas Andre ´gherici Hassaine

Hartmut Michel

Christian Cambillau

Franc Pattus

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems

Keywords: Expression vectors, G protein-coupled receptors, Structural genomics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction G protein-coupled receptors (GPCRs) represent more than 50% of current drug targets, which generate more than $50 billion in annual sales. The functions of GPCRs are many and include various signal transduction pathways but also intercellular mechanisms (Lundstrom and Chiu 2005). GPCRs can be activated through peptides, neurotransmitters, hormones, odors, ions, light, odorants, pheromones, amino acids, amines, nucleotides, nucleosides, prostaglandins and other low weight molecules. Due to the broad spectrum of functions, GPCRs play important roles in various indications such as cardiovascular, metabolic, neurodegenerative, neurological, psychiatric, viral diseases and cancers.

The success in obtaining high resolution structures of membrane proteins has in general been very modest. In comparison to the current number of more than 30,000 entries for soluble proteins in public databases, only 104 unique structures have been solved for membrane proteins (http://blanco.biomol.uci.edu/ Membrane_Proteins_xtal.html). The situation is even worse for GPCRs for which only a single structure has a unique opportunity to evaluate the expression properties of a large number of GPCRs. The selection of GPCRs was preliminary based on ligand availability to be able to monitor the functional activity of receptors expressed in yeast and mammalian cell membranes by radioligand assays and also later on to perform binding assays for purified and solubilized GPCRs as well as refolded receptors from E. coli inclusion bodies. Other criteria were to have representation of different classes and subtypes of GPCRs and to link the targets to human disease. In this context, the majority of GPCRs studied were from class A (95 GPCRs) receptors, which are represented by light (rhodopsin), adrenaline (adrenergic) and olfactory receptors. Only two members of the class B hormone and neuropetide receptors were included in the study. The class C GPCRs with the characteristically large extracellular N-terminal were represented by 4 members. Finally, two yeast receptors were from class D. Of the total number of 104 GPCRs in this study, 82 were of human origin, 9 from rat, 7 from mouse, 2 from bovine, 2 from yeast, 1 from pig and 1 from hamster.

The choice of expression systems was based on previous experience of convenience in handling a large number of clones in parallel, the potential of scale-up and obviously the possibility to obtain yields compatible to structural biology applications. E. coli-based expression was chosen because of the speed, the inexpensive process and the ease of use. The straight forward scale-up to fermentor cultures was also considered as an advantage. E. coli vectors chosen in this study were pET15 vectors with N-and C-terminally engineered 10-histidine tags, respectively. Fiftyfive of the 103 GPCRs were also introduced into 6 Gateway vectors containing various fusion partners. Under the expression conditions used in this study, GPCRs were always produced as insoluble inclusion bodies in the cytoplasm of E. coli. It was decided to use E. coli vectors in this study that uniquely promote expression in bacterial inclusion bodies although relatively high expression have been reported for GPCR expression in bacterial membranes (Tucker andGrisshammer 1996, Weiss andGrisshammer 2002). However, recent improvement in refolding of GPCRs from inclusion bodies [START_REF] Andre | Protein Science[END_REF] was encouraging and the extensive need for construct engineering (mutations, deletions, evaluation of different fusion partners and tags) required for membrane-based expression was not feasible for 100 GPCRs in parallel. For this reason, pET15 vectors with N-and C-terminally engineered 10-histidine tags, respectively, were employed. A large portion of the 103 GPCRs were also introduced into Gateway vectors ) or BamHI-SpeI restriction enzymes when feasible. These fragments were then subcloned into the BamHI-SpeI digested pET15N2, pET15C2, pPIC19KHisFLAGBio, pSFV2genB and pSFV2genC expression vectors (Fig. 1). For Gateway TM (Invitrogen, Carlsbad, CA, USA) cloning PCR fragments were amplified containing the respective attB1 and attB2 recombination sites into Gateway TM destination vectors. Six Gateway TM expression vectors were used (Fig. 1).

U N C O R R E C T E D P R O O F

E. coli based expression in bacterial inclusion bodies

The bacterial expression vectors were transformed into four E. coli strains: BL21(DE3)pLysS and Rosetta(DE3)pLysS from Novagen and C41(DE3) and C43(DE3) from Avidis SA (Saint-Beauzire, France) (Miroux and Walker 1996). Freshly transformed bacteria were cultured in LB medium supplemented with 100 lg/mL ampicillin (34 lg/ml clhloramphenicol for BL21(DE3)pLysS and Rosetta(DE3)pLysS) at 37°C until an OD 600 value of 0.7-0.8 was reached. The expression of recombinant GPCRs was induced by addition of 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) at 37°C for 3 h or at 25°C or 15°C for 16 h.

Cells were harvested at +4°C by centrifugation at 5000 • g for 20 min, lysed in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 0.25 mg/ml lysozyme and 1 mM phenylmethylsulfonyl fluoride and stored at -20°C prior to Western blotting. The optimal growth conditions were applied to 1 L flask and 8 L fermentor cultures. Additionally, a number of GPCRs were analyzed for the localization recombinant receptors in E. coli. Total lysates of bacteria were obtained by lysosome treatment and sonication. Inclusion bodies (pellet) were separated from soluble protein (supernatant) by centrifugation at 10,000 • g for 30 min followed by SDS-PAGE and Western blotting.

Yeast-based expression from Pichia pastoris vectors The yeast expression vector constructs for the 100 GPCRs were linearized with PmeI or SacI and the P. pastoris strain SMD1163 electroporated at 1500 V, 25 lF and 600W using a Gene Pulser I (Bio-Rad, Reinach, Switzerland). Selection of clones was done as previously described (Weiss et al. 1998, Andre ´et al. 2006). In conclusion, recombinant His + clones were first selected on MD agar plates (1.34% yeast nitrogen

U N C O R R E C T E D P R O O F
base without amino acids, 2% dextrose, 0.00004% biotin, 1.5% agar). In a second step the His + clones were cultured on YPD agar containing various concentrations of G418 (1% yeast extract, 2% peptone, 2% dextrose, 2% agar and 0.05-0.5 mg/mL G418) to obtain multicopy transformants. For the production of GPCRs P. pastoris was precultured in BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base without amino acids, 0.00004% biotin, 1% glycerol, 0.1 M phosphate buffer, pH 6) at 30°C and 250 rpm until an OD 600 value of 2-6 was reached. The induction was carried out in BMMY medium (identical to BMGY except 0.5% methanol instead of 1% glycerol) at 30°C from an initial OD 600 value of 1. After 18 h cells were pelleted at 3000 • g for 5 min and subjected to membrane preparation. Cells were washed once with ice-cold lysis buffer (50 mM sodium-phosphate buffer pH7.4, 100 mM NaCl, 5% glycerol, 2 mM EDTA, 1 mM PMSF) and resuspended to 30% wet weight. After 0.5 mm glass beads were added to the cell suspension, yeast cells were broken by vigorous vortexing at 4°C for 10 min. Breaking efficiency was inspected with a light microscope and was usually > 80%. Intact cells and cell debris were separated from the membrane suspension by a low speed centrifugation (3000 • g, 5 min, 4°C). Membranes were then pelleted using an ultracentrifuge (100,000 • g, 45 min, 4°C), resuspended in membrane buffer (50 mM Tris pH8.0, 120 mM NaCl, 20% glycerol, 1 mM PMSF) using a dounce homogenizer. Membrane proteins were quantified applying the BCA method (Pierce, Rockford, IL, USA), using BSA as a

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as previously described (Hovius et al. 1998). Approximately 0.5 g of frozen cell pellets were resuspended in 10 ml of 10 mM HEPES containing 1 mM EDTA (pH 7.4) and homogenized for 10 s with an Ultra-Turrax T25 (IKA, Staufen, Germany) homogenizer. Membranes were pelleted by centrifugation at 27,000 • g for 30 min after a two-fold dilution and resuspended in10 mM HEPES (pH 7.4).

Immunodetection methods

The expression evaluation was visualized by dot blots and/or Western blots. Dot blots. Membranes from P. pastoris cells expressing GPCRs were diluted in TBS buffer containing 0.2% SDS. PVDF membranes (Millipore, 0.45 lm) were washed with methanol and 38 mM Glycine, 10 mM Tris, 20% methanol in a 96 well microfiltration apparatus (Bio-Dot, Bio-Rad, Reinach, Switzerland) and 1 lg of total membrane was added. The PVDF membrane was blocked with 5% low fat powder in TBS for 60 min at room temperature, washed three times for 1 min with TBS and incubated with alkaline phosphatase-conjugated anti-FLAG M2 antibody (Sigma, Buchs, Switzerland).

After three 15 min washes the blots were developed in AP-buffer (100 mM Tris pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) containing 330 lg/mL 5-bromo-4-chloro-indolylphosphate p-toluidinium salt and 165 lg/mL nitroblue tetrazolium chloride. For P. pastoris membranes, a standard curve for the b2 adrenergic receptor corresponding up to 25 pmol receptor per mg protein, was applied as a reference. In this context, signals of a lower intensity than observed for 1 pmol b2 adrenergic receptor were classified as low level (+), intensities corresponding to 1-10 pmol as medium (++) and > 10 pmol as high (+++). However, these extrapolations are only relative as only part of the signals obtained by immunodetection corresponds to functional receptors. Western blots. Samples were subjected to 10% SDS-PAGE and the proteins electrotransferred for 30 min to Hybond ECL nitrocellulose membranes (GE Healthcare, Uppsala, Sweden) and blocked with 5% milk in TBST (TBS with 0.1% Tween 20) at +4°C. The filter was then treated with primary antibodies (anti-FLAG 1:1000 dilution or anti-His 1:1000 dilution) (Sigma, Buchs, Switzerland), washed, and treated with the secondary anti-mouse antibody 1:2000 dilution (Sigma). The anti-His antibody is directly conjugated to peroxidase and requires no secondary antibody use. The visualization of GPCR-specific bands was done with the ECL chemiluminiscence kit from GE Healthcare (Uppsala, Sweden).

376 Radioligand binding assays 377 Membrane fractions were prepared from yeast (Andre ´378 et al. 2006) and mammalian (Hovius et al. 1998) 410 The Pichia pastoris expression vector used in this study 411 has been previously described [START_REF] Andre | Protein Science[END_REF]) and 412 was designed on the basis of previous results obtained 413 for GPCR production (Weiss et al. 1998). To facilitate 414 purification efforts and to improve stability of 415 recombinant GPCRs various tags and fusion partners 416 were also introduced into the expression vectors as 417 illustrated in Fig. 1. A signal sequence from the influ-418 enza HA (hemagglutinin) gene was further introduced 419 into the SFV vector to promote transport and trans-420 location to the plasma membrane as previously dem-421 onstrated for GPCRs expressed in insect cells from 422 baculovirus vectors (Guan et al. 1992). The biotinyla-423 tion domain from Proprionibacterium shermanii

U N C O R R E C T E D P R O O F fractions in E.
coli revealed that the recombinantly expressed GPCRs were uniquely located in inclusion bodies and not inserted into membrane structures, demonstrated by subjecting total bacterial lysates to lysosome treatment and sonication after which inclusion bodies (pellet) were separated from soluble protein (supernatant) by centrifugation and the expressed GPCR verified by SDS-PAGE and Western blotting. Other factors influencing the expression levels were the E. coli strain applied and the growth temperature of bacteria. In this context, the C43 strain was superior to BL1(DE3), Rosetta (DE3) and C41. The initial expression evaluation was carried out at 4 mL scale. Next, 23 well expressed GPCRs were selected for large-scale production in 1 L shake flask cultures. Among the 23 GPCRs 9 were expressed from the pET15N2 vector, 1 from pET12C2 and 15 from Gateway vectors. The yields in flask cultures varied between 2 mg/L and 40 mg/L and the recombinant receptors were only localized in inclusion bodies. Although the yields were relatively high the produced material was not sufficient for extensive purification and refolding exercises. For this reason, 7 of the 23 targets were further subjected to fermentation in 3 or 8 L bioreactors. The yields in the fermentor cultures were generally higher (75-350 mg/L) than in flask cultures and provided now sufficient material for the establishment of purification and refolding conditions.

Only a single vector construct was engineered for P. pastoris (Fig. 1B). Briefly, the vector contained an inducible AOX1 promoter followed by an a-factor signal sequence and FLAG-and deca-His-tags at the N-terminus. The GPCRs were introduced as BamHI-SpeI fragments and flanked by Tev protease cleavage sites. These sites were engineered into the expression vector to allow removal of all tags after purification to avoid any interference of tags during the crystallization procedure. Furthermore, the Bio-tag was engineered at the C-terminal to potentially improve expression and stability of the recombinant GPCRs [START_REF] Reinhart | levels were monitored by meta-434 bolic labeling and saturation binding assays, which 435 indicated similar levels of receptor density for wild 436 type and NK1R-biotin domain constructs[END_REF]. After subcloning of the GPCRs into the P. pastoris vector pPIC9K, a high-throughput screening method based on in situ immunoblotting was established (Magnin et al. manuscript in preparation). This method facilitated significantly the clone selection process of the large number of GPCR constructs studied in parallel. The expression profile in yeast cells applying P. pastoris vectors was quite different from what was obtained in bacteria. The success rate was much higher, partly due to the clone selection process for each individual GPCR, which was established to obtain constructs with optimum number of copies integrated into the yeast genome [START_REF] Andre | Protein Science[END_REF].

U N C O R R E C T E D P R O O F
U N C O R R E C T E D P R O O F
554 and the rat neurokinin-2 receptors. The effect of his-555 tidine addition was favorable for 12 GPCRs and 556 indifferent for 8. To further optimize the expression 557 those parameters with a positive effect were combined, 558 which resulted in doubled production levels for 13 559 GPCRs. The B max values were more than 4 times 560 higher for 5 receptors and the maximum increase of 8.7 561 fold was obtained for the human kappa opioid recep-562 tor. Interestingly, when the expression levels in Dot 563 blots were compared to the B max values, it was evident 564 that optimization did not affect the receptor quantity 565 (Fig. 2). It rather looked like the quality of the re-566 combinant GPCRs was enhanced resulting in larger 567 proportion of receptors with functional binding activ-568 ity. The optimal expression conditions for individual 569 GPCRs have been described in more detail previously 570 [START_REF] Andre | Protein Science[END_REF]). 571 Two expression vectors were designed for SFV-572 based expression (Fig. 1C). In the pSFV2genB vector a 573 Kozak sequence was introduced downstream of the 574 subgenomic SFV 26S promoter to optimize the initia-575 tion of ribosomes (Kozak 1986). A signal sequence 576 from the influenza hemagglutinin (HA) gene was 577 introduced to facilitate the translocation of the GPCRs 578 to the plasma membrane. Furthermore, a FLAG tag 579 was engineered at the N-terminus in front of the full-580 length GPCR flanked by Tev protease cleavage sites. 581 The C-terminal contained a deca-His tag. The second 582 SFV vector, pSFV2genC, was otherwise identical to 583 pSFV2genB except for an additional biotin tag placed 584 downstream of the His-10 tag. One hundred and one 585 GPCRs were evaluated for expression in mammalian 586 cells using SFV-infected host cells. Due to the broad 587 host range of SFV, several mammalian cell lines can be 588 efficiently transduced. However, dealing with such a large number of targets, it was decided to initially limit the expression evaluation to three cell lines, BHK-21 (baby hamster kidney), CHO-K1 (Chinese hamster ovary) and HEK293 (human embryonic kidney) cells, previously shown to express high levels of GPCRs (Lundstrom 2003). A few GPCRs were also expressed in the human T lymphocyte cell line C8166. The success rate for SFV expression was very high with 97 GPCRs showing positive signals and only 4 targets no detectable specific response in Western blots (Table 1).

According to expression evaluation 44 GPCRs were classified as high (+++), 25 as medium (++) and 28 as low (+) expressers. More interestingly, among the GPCRs studied sev-653 eral belonged to the same subfamily of GPCRs. For 654 instance, 4 muscarinic, 4 a-adrenergic, 6 dopamine, 14 655 serotonin, 4 neuropeptide Y, 4 opioid, 5 neurokinin, 4 656 purinoreceptor P2Y and 4 metabotropic glutamate 657 receptors were studied (Table 2). The expressability of 658 the GPCRs was evaluated by immunodetection and 659 grouped as previously into categories of no expression 660 (-), low expression (+), medium expression (++) and 661 high expression (+++). The serotonin receptors, the 662 group with the highest number of subtypes, demon-663 strated no expression for 64.2% of the targets in E. 664 coli, medium expression for 42.9% in P. pastoris and 665 high expression of 57.1% in SFV. Overall, high 666 expression levels were obtained for 31.0% of the 667 serotonin receptors. Another subfamily of receptors 668 that was well expressed for all three expression sys-669 tems, were the neurokinin receptors. Of the 5 neu-670 rokinin receptors tested 60% were expressed at high 671 levels in all three expression systems. None of the 672 neurokinin receptors showed a negative signal. The 8 673 adrenergic receptors showed variable expression lev-674 els. None of them were successfully expressed in E. 675 coli, the expression levels in yeast were either low or 676 medium, whereas in mammalian cells 4 GPCRs 677 showed low, 1 target medium and 3 targets high 678 expression levels. Dopamine receptors were also 679 problematic to express in E. coli as all 6 subtypes were 680 negative. In P. pastoris the majority of dopamine 681 receptors were expressed at medium levels, whereas 682 for SFV 4 of the 6 GPCRs belonged to the group of 683 high expressers.

684 Target size and GC content 685 The size of recombinantly expressed proteins might 686 play an important role in relation to the expression 687 levels obtained. In general, smaller proteins have given 688 better expression yields especially in bacterial systems. 689 In the case of the 100 targets studied here, they rep-690 resented GPCRs ranging in size from a postulated molecular weight of 33.9 kD to 102.3 kD. Comparison of molecular weight to expression levels suggested that size played an important role in bacterial expression. In this context, the largest class A GPCR expressed successfully from E. coli had a postulated molecular weight of 53.6 kD. Similarly, the class B receptor with a size of 54.0 kD gave a positive signal whereas the GPCR of 57.3 kD was negative in Western blots. All 4 class C GPCRs were negative in E. coli. These metabotropic glutamate receptor are large (95-102 kD) and it is therefore not surprising that no expression was observed. Of the two class D yeast STE receptors, only the smaller STE2 (47.8 kD) was expressed, whereas the STE3 (53.7 kD) showed no signal. According to the expression results for the 93 class A GPCRs expressed in E. coli, 54 kD seemed to be the limit where positive signals were detected. In contrast to bacterial expression, when the 100 GPCRs were analyzed for expression in yeast and mammalian cells, no correlation could be observed between size and expressability.

For example, all of the large size class C receptors were expressed in P. pastoris and 3 out 4 of them expressed in SFV. Among factors influencing gene expression in general, the different codon usage in pro-and eukaryotes has been indicated as a cause. This issue has been addressed by developing special bacterial strains for recombinant proteins with poor codon usage for mammalian proteins (Kleber-Janke and Becker 2000). It has also been shown that the frequency of some codons and amino acids correlate with the GC content in the genome (Sueoka 1961). Recently, the trends in codon and amino acid usage in relation to GC composition have been studied (Knight et al. 2001). Here we have investigated whether the GC content has any effect on the expression levels of 103 GPCRs in bacterial, yeast and mammalian cells. The GC content for the 103 GPCRs varied between 36% and 75% for class A receptors. The two class B GPCRs had GC contents of 47% and 63%, respectively. Class C receptors showed a range of GC content from 51% to 61% and the two class D receptors had both a GC content of 38%. In none of the three expression systems was there any correlation between the expression levels and the GC content. Also the GPCRs that showed no signal showed a large variation in GC content. Immunoblotting versus binding activity GPCRs expressed in E. coli inclusion bodies are present in aggregates and require refolding before any functional activity can be restored. In contrast, expression of GPCRs in yeast and mammalian cells

U N C O R R E C T E D P R O O F
762 Table 4. This comparison could obviously only be 763 performed for GPCRs expressed in yeast and mam-764 malian cells as the E. coli expressed GPCRs were lo-765 cated in inclusion bodies and required refolding to 766 regain functional binding activity. In this case, there 767 seemed to be no evident correlation between the 768 intensity of the signals obtained in immunoblots and/or 769 Western blots and the specific binding in agreement 770 with previous observations [START_REF] Andre | Protein Science[END_REF]. More 771 than half (56.3%) of those targets that showed low 772 expression levels by immunodetection also produced 773 specific binding below 1 pmol/mg. 38.9% of the

U N C O R R E C T E D P R O O F
GPCRs demonstrating medium expression levels also showed medium binding activity (1-10 pmol/mg) although almost the same percentage was achieved for binding over 10 pmol/mg (33.3%). Almost half (47.1%) of the GPCRs which showed high expression levels by immunodetection also belonged to the group of targets with high binding activity. The situation for SFV-expressed targets was similar. Among the GPCRs with low expression levels in Western blots also 21.4% showed binding activities below 1 pmol/mg, 41.7% of this group produced specific binding values between 1 pmol/mg and 10 pmol/mg. Those targets that were expressed at medium level generated medium binding activity for 40.0% of the GPCRs. GPCRs with high levels of expression in Western blots, showed an equal distribution (44.0% each) of the specific binding in the groups for medium and high binding activity. However, some GPCRs with only weak signals in Western blots showed high functional binding activity. For example, the rat serotonin 5-HT7 receptor (5H7_RAT) generated no signal by immunodetection, but demonstrated a specific binding of 2.18 pmol/mg. This might be a reflection of the ratio between functional and nonfunctional receptors. A significant effect on the binding level was seen from the expression optimization efforts conducted in P. pastoris. Less efforts have so far been made for optimization of SFV expression. In this context, only limited studies in three cell lines (BHK-21, CHO-K1 and HEK293) were performed in parallel and also the optimal harvest time was established for each GPCR.

The two SFV vectors used in this study did not show any significant difference in expression levels for the GPCRs tested in parallel (Hassaine et al. 2006). It has also previously been demonstrated that a C-terminally fused Bio-tag had no effect on the expression levels and specific binding of the human neurokinin-1 receptor (Lundstrom et al. 1995). In contrast, some of 812 the GPCRs expressed from the pSFV2genB and 813 pSFV2genC vectors showed significantly lower specific 814 binding activity than previously reported. For instance, 815 the human a2-adrenergic receptor (a 2-AR) showed 816 specific binding of only 0.05 pmol/mg in comparison to 817 previously published values of 176 pmol/mg (Sen et al. 818 2003). Likewise, the human histamine H2 receptor 819 (HH2R) generated 20 pmol/mg receptor compared to 820 another SFV-based study of 100 pmol/mg (Hoffmann 821 et al. 2001). However, in the case of the a 2-AR, an 822 SFV vector with a translation enhancement signal from 823 the SFV capsid vector was used. This has been dem-824 onstrated to increase the expression level by 5-10 fold 825 (Sjo ¨berg et al. 1994). Moreover, in the previously 826 published study on the HH2R, ligand was added to the 827 cell culture medium, which significantly increased the 828 specific binding. Although these differences might 829 explain the discrepancy in the results, also the presence 830 of the influenza HA signal sequence as well as other 831 tags in this study could have an effect. Interestingly, the 832 adenosine A2A receptor generated extreme specific 833 binding values (287 pmol/mg) from the vector with the 834 signal sequence in comparison to 40 pmol/mg from the 835 standard SFV vector (Lundstrom 2000).

836

Conclusions and future prospects 837 In summary, all three systems provided high expression 838 levels of several GPCRs. The bacterial expression 839 allowed production of large quantities of GPCRs albeit 840 in inclusion bodies. This strategy was, however, 841 justified by the positive development of refolding 842 technology on GPCRs and also by the large number of 843 targets evaluated. Expression of GPCRs in bacterial 844 membranes requires substantial engineering of con-845 structs and with the resources available, it was impos-846 sible to perform studies on more than 100 targets in 847 parallel. For this reason, direct comparison between

U N C O R R E C T E D P R O O F
848 the success of GPCR expression in prokaryotes and 849 eukaryotes is inappropriate. Thus, not surprisingly, the 850 yeast-and SFV-based systems generated a much 851 higher number of successfully expressed GPCRs than 852 E. coli. Moreover, the functional activity could be 853 verified by radioligand binding assays at each step of 854 expression and purification. The post-translational 855 modifications and particularly glycosylation was ob-856 served in both yeast and mammalian cells, but the 857 different glycosylation patterns in the two cell types 858 were not studied here. The effect of glycosylation on 859 crystallization will only be discovered later, but from 860 this aspect it might be an advantage to express the non-861 glycosylated GPCRs in E. coli. 862

The current study has provided a massive amount 863 of information on the expression of more than 100 864 GPCRs in three expression systems. Although only 865 50% of the GPCRs were expressed in i, all the 866 recombinant GPCRs could be located in inclusion 867 bodies and for some targets huge amounts, up to 868 350 mg/L, could be produced in fermentor cultures. 869 Plenty of material is therefore available for refolding 870 studies and the development of improved refolding 871 technologies. Although the purpose of this study was 872 not to perform extensive studies on refolding, pre-873 liminary results from trials on several GPCRs suggest 874 that receptor binding activity could be re-established, 875 which will be communicated in future publications 876 in detail. Similarly, several of the GPCRs expressed in 877 P. pastoris were subjected to large-scale production in 878 yeast cell cultures, which generated huge biomasses, 879 providing material for further studies on solubilization, 880 purification and crystallization. Likewise, well ex-881 pressed GPCRs from SFV-infected mammalian sus-882 pension cultures have been subjected to solubilization 883 and purification. Preliminary observations indicated 884 that several GPCRs expressed both from P. pastoris 885 vectors in yeast cells and SFV vectors in mammalian 886 cells can be solubilized and purified to high homoge-887 neity. Detailed results will be presented elsewhere 888 shortly. Selected GPCRs have now been subjected to 889 crystallization screening and optimization. 890 Acknowledgements We are grateful for the technical assistance 891 provided by Juliette Kempf and Tania Steffen (UMR7175, Ill- 892 kirch, France), Gabriele Maul (Max Planck Institute of Biophys- 893 ics, Frankfurt, Germany), Marie-Eve Gravie `re and Ce ´line Huyghe 894 (University of Marseille, France) and Mrs. Nouzha Hassaine 895 (BioXtal, Epalinges, Switzerland). We are also thankful for the 896 help with E. coli scale-up and fermentation from Prof. Rainer 897 Rudolph and Mr. Jan Oschman (Martin-Luther University, Halle, 898 Germany). We would like to thank all those academic and 899 industrial contributors who provided us with cDNAs for several 900 GPCRs used in this study. The financial support received for the 901 MePNet program from the industrial partners is acknowledged.

  Fig. 1 Schematic presentation of expression vectors. a F, yeast a Factor; Bio, biotinylation domain from Proprionibacterium shermanii; FLAG, FLAG-tag; His, 10-histidine tag; GST, Glutathion-S-transferase; K, Kozak consensus sequence; MBP, maltose binding protein; NusA, transcription factor NusA; ss, signal sequence from influenza HA (hemagglutin) gene; Tev (Tobacco Etch Virus), Tev protease cleavage site; Trx, Thioredoxin; white triangle, T7 promoter; grey triangle, yeast AOX1 promoter; black triangle, SFV 26S subgenomic promoter

Fig. 2

 2 Fig. 2 Expression of the human NK2 receptor in P. pastoris: comparison of Western blots to binding activity before and after optimization. Sample 1, 30°C; sample 2, 20°C; sample 3, 20°C + 2.5% DMSO. (A) Western blots using anti-FLAG M2 antibody. Molecular weight marker indicated on the right. (B) Saturation binding using [ 3 H] SR48968

  163 containing various fusion partners. Pichia pastoris 164 vectors were applied for expression in yeast cells as a 165 proven lower eukaryotic system. The P. pastoris system 166 is well establish and fairly rapid to work with and 167 generates large biomasses in fermentor cultures.

	168 Moreover, several GPCRs have been previously suc-
	169 cessfully expressed with this system (reviewed in
	170 Reinhart and Krettler 2006). Finally, the third system
	171 represents expression in mammalian cells. Semliki
	172 Forest virus (SFV) has previously been shown to be
	173 applicable to the expression of a large number of
	174 recombinant proteins, including many GPCRs (Lund-
	175 strom 2003). The expression levels have been very high
	176 with B max values of 150-200 pmol/mg and large-scale
	177 bioreactor production in mammalian suspension cul-
	178 tures has been established for SFV (Blasey et al. 1997).
	179 The inclusion of members of all GPCR families and
	180 several subtypes provided additional interesting infor-
	181 mation in relation to expression patterns. The study of
	182 103 GPCRs also allowed to describe which receptors
	183 were not successfully expressed and in which system.
	193 Materials and methods
	194 Cloning and subcloning of GPCR genes
	195 into expression vectors
	196 Appropriate primers were designed for the 5¢ and 3¢
	197 end coding regions of each GPCR, which were
	198 amplified from plasmid DNA when available or from
	199 commercial polyA + RNA preparations (Clontech,
	200 Palo Alto, CA, USA). In the latter case, RT-PCR
	201 reactions were performed to amplify the full-length
	202 ORFs using the Titan One-Step RT-PCR kit from
	203 Roche (Penzberg, Germany) according to the manu-
	204 facturer's instructions. In both cases, the amplified
	205 fragments were subcloned into the pCR4 Ò Blunt-
	206 TOPO

184 Finally, the expression evaluation study has provided a 185 large number of well expressed targets that currently 186 are subjected to further structural biology exploration. 187 We have previously described the overexpression of 20 188 GPCRs from P. pastoris

[START_REF] Andre | Protein Science[END_REF]

) and 101 189 GPCRs from SFV-infected mammalian cells

(Hassaine 190 et al. 2006

), but this is the first time a comparison of 191 more than 100 GPCRs is presented for three expres-192 sion systems. Ò vector (Invitrogen, Carlsbad, CA, USA) and 207 the accuracy of the sequence verified. In case of dif-208 ferences from the published sequence, the existence of 209 potential polymorphism was investigated. Mutations 210 introduced by the RT-PCR procedure were corrected 211 accordingly. Orientation-defined GPCR fragments with 5¢ end BamHI and 3¢ end SpeI sites were obtained using type IIS restriction endonucleases (Esp31 or BbiI

Table 1

 1 Expression evaluation by immunodetection

		MePNet#	E. coli	P. pastoris	SFV	Size	GC%
	ACM1_HUMAN	23	-	+	+++	51.4
	ACM1_MOUSE	24	-	+	+++	51.3
	ACM2_PIG	26	-	+	+	51.7
	ACM2_HUMAN	25	-	+	+	51.7
	A1AA_HUMAN	104	-	+	+++	51.5
	A1AA_RAT	14	-	+	+++	51.6
	AA1B_HUMAN	15	nd	++	++	56.8
	AA1B_MESAU	105	-	+	+	56.5
	A1AD_RAT	106	-	+	+	59.4
	A2AA_HUMAN	107	-	+	+++	49
	A2AB_HUMAN	16	-	++	+	50
	A2AC_HUMAN	18	-	++	+	49.6
	B3AR_HUMAN	31	-	+	+	43.5
	D2DR_HUMAN	43	-	++	++	50.6
	D2DR_MOUSE	44	-	+++	+++	50.9
	D3DR_HUMAN	45	+	++	+++	44.2
	D4DR_HUMAN	46	+	++	+++	48.4
	DADR_HUMAN	109	-	++	++	49.3
	DBDR_HUMAN	110	-	++	+++	53
	HH2R_HUMAN	61	+	+++	+++	40.1
	HH2_RAT	62	+	nd	+++	40.3
	5H1A_HUMAN	1	-	+++	+++	46.1
	5H1B_HUMAN	2	+	+++	+++	43.6
	5H1D_HUMAN	3	-	+++	+++	41.9
	5H1E_HUMAN	4	-	++	+	41.7
	5H1F_HUMAN	5	-	+++	+++	41.7
	5H2A_HUMAN	6	++	++	+	52.6
	5H2A_MOUSE	7	++	-	+	52.8
	5H2B_HUMAN	8	-	+	+	54.3
	5H2C_HUMAN	9	-	-	+++	51.8
	5H4_HUMAN	144	-	++	+++	43.8
	5H5A_HUMAN	10	-	++	+++	40.3
	5H6_RAT	111	-	++	+++	46.9
	5H7_HUMAN	12	+	+	+++	53.6
	5H7_RAT	117	+++	++	-	49.8
	AG2R_HUMAN	28	++	++	+	41.1
	BRB1_HUMAN	32	++	+	+++	40.4
	BRB2_HUMAN	33	-	+	+++	44.5
	FSHR_HUMAN	131	-	+	+	78.3
	IL8A_HUMAN	63	+	+	++	39.8
	IL8B_HUMAN	136	-	+	+	40.8
	CCR3_HUMAN	39	-	+	+++	40.7
	CCR4_HUMAN	135	nd	nd	+++	39.7
	CKR1_HUMAN	40	+	+++	+++	41.1
	CKR2_HUMAN	41	+	nd	+	41.9
	CKR6_HUMAN	42	+++	+	++	42.5
	CKR7_HUMAN	145	+	+	++	42.9
	CKRB_HUMAN	153	-	+	+++	39.9
	CCKR_HUMAN	38	-	+++	++	47.8
	GASR_HUMAN	57	-	+++	++	48.4
	ET1R_BOVIN	50	+++	+++	-	48.5
	ET1R_HUMAN	51	+++	+	+	48.7
	ACTR_HUMAN	27	+++	++	+	33.9
	ETBR_BOVIN	52	+	++	+	49.4
	MC3R_HUMAN	64	++	+++	+	40
	MC4R_HUMAN	65	+++	+++	+	37
	MC5R_HUMAN	66	++	+++	+	36.6
	NY1R_HUMAN	79	+++	+++	+++	44.4
	NY2R_HUMAN	80	-	+++	++	42.7
	NY4R_HUMAN	81	++	++	++	42.2
	NY5R_HUMAN	82	-	+	nd	52
	OPRD_HUMAN	83	-	+	+	40.4

536 temperature, addition of ligands and additive supple-537 ments to the yeast culture medium strongly influenced 538 the expression levels. In this context, the lowering of 539 the temperature from 30°C to 20°C, addition of specific 540 ligand at concentrations close to 100 times the K d value

Table 3

 3 Immunodetection vs. binding activity of selected GPCRs

					J Struct Funct Genomics
		MePNet#	Pichia IB	Pichia B	SFV ID	SFV BA
	ACM1_HUMAN	23	+	0.23	+++	2.44
	ACM1_MOUSE	24	+	1	+++	2.93
	ACM2_PIG	26	+	1	+	22
	ACM2_HUMAN	25	+	1.5	+	6.1
	A1AA_HUMAN	104	+	0.45	+++	0.25
	A1AA_RAT	14	+	0.25	+++	4.7
	AA1B_HUMAN	15	++	5	++	0
	AA1B_MESAU	105	+	0.14	+	0
	A1AD_RAT	106	+	0.21	+	0
	A2AB_HUMAN	16	++	11	+	0.05
	A2AC_HUMAN	18	++	0.25	+	0.65
	B3AR_HUMAN	31	+	0	+	nd
	D2DR_HUMAN	43	++	39	++	29.2
	D2DR_MOUSE	44	+++	65	+++	21.8
	D3DR_HUMAN	45	++	1.7	+++	1.8
	D4DR_HUMAN	46	++	2	+++	3.47
	DBDR_HUMAN	110	++	0	+++	nd
	HH2R_HUMAN	61	+++	50	+++	20
	5H1A_HUMAN	1	+++	0.9	+++	12.7
	5H1B_HUMAN	2	+++	38	+++	19.2
	5H1D_HUMAN	3	+++	102	+++	11
	5H1E_HUMAN	4	++	0.8	+	0.75
	5H1F_HUMAN	5	+++	0	+++	0.69
	5H2A_HUMAN	6	++	1.8	++	8.5
	5H5A_HUMAN	10	++	24	+++	31
	5H6_RAT	111	++	1	+++	1.4
	5H7_HUMAN	12	+	11.8	+++	2.2
	5H7_RAT	117	++	1.5	-	2.18
	AG2R_HUMAN	28	++	0	+	nd
	BRB1_HUMAN	32	+	0.11	+++	0.19
	BRB2_HUMAN	33	+	0.13	+++	7.9
	ET1R_BOVIN	50	+++	3.5	-	nd
	ET1R_HUMAN	51	+	0.1	+	nd
	ETBR_BOVIN	52	++	0.4	+	nd
	NY1R_HUMAN	79	+++	60	+++	2.3
	NY2R_HUMAN	80	+++	0	+	1.85
	OPRD_HUMAN	83	+	3	+	1.85
	OPRD_MOUSE	84	++	3.7	++	4
	OPRK_HUMAN	85	++	25	++	1.6
	OPRK_MOUSE	86	+++	45	++	1
	SSR1_HUMAN	93	+++	0.1	+++	nd
	SSR3_HUMAN	95	+++	0	+++	nd
	NK1R_HUMAN	72	++	15	+++	56.7
	NK1R_RAT	73	+++	1.9	+	68.9
	NK2R_HUMAN	74	++	39	+++	3.6
	NK2R_RAT	75	+++	162	+++	46.4
	NK3R_HUMAN	76	+++	23	++	26.5
	V1BR_HUMAN	103	+++	0	+++	26.1
	MCR1_HUMAN	60	+++	0.01	nd	nd
	AA1R_HUMAN	19	+	0.8	++	0.4
	AA2A_HUMAN	20	+	180	++	287
	Expression levels as for Table 1
	123

Table 4

 4 Correlation between immunodetection and specific binding

		+	++	+++
	P. pastoris	16	18	17

742 generate receptors located in cell membranes, which 743 can be directly monitored for their functional activity 744 by radioligand binding assays. Selected GPCRs were 745 subjected to binding assays and the correlation 746 between immunodetection and binding activity was 747 evaluated (


Corentin Musa

Antoine Kervoelen

Pierre-Edouard Danjou

Alain Bourmaud
email: alain.bourmaud@univ-ubs.fr

François Delattre

Antoine Kervoëlen

Bio-based unidirectional composite made of flax fibre and isosorbide

Introduction

Since their advent in the thirties and until nowadays, composite materials have experienced a strong development and have con-quered important industrial sectors such as automotive, aerospace, housing, etc. However, current environmental issues are pushing society to make towards to sustainable development. To respond to these changes and replace finite fossil resources, the develop-ment of composites from renewable resources is of particular interest [START_REF] Saba | Recent advances in epoxy resin, natural fiber-reinforced epoxy composites and their applications[END_REF]. Among a large range of reinforcements and due to their availability, environmental assessment and attractive mechanical properties, flax fibres can be considered as a relevant solution to substitute E-glass fibres in Europe [START_REF] Bourmaud | Towards the design of highperformance plant fibre composites[END_REF]. Epoxy resin is the most widely used polymer matrix thermoset resin. Due to its aromatic ring, giving it a high thermal stability, bisphenol A (BPA) is the most commonly used epoxy precursor and is found in 90% of crosslinked epoxy resins. Nevertheless, it is reprotoxic [START_REF] Vandenberg | Human exposure to bisphenol A (BPA)[END_REF] and so requires the development of viable alternatives. Vegeta-ble oils, proteins and polysaccharides have been studied in this way [START_REF] Auvergne | Biobased thermosetting epoxy: Present and future[END_REF]. Isosorbide is considered as a viable candidate to replace BPA in thermoset materials [START_REF] Fenouillot | Polymers from renewable 1,4:3,6-dianhydrohexitols (isosorbide, isomannide and isoidide): A review[END_REF]. The most convenient way to pro-duce diglycidyl ether from an isosorbide precursor is to graft epoxy groups to isosorbide by mixing it with epichlorohydrin in basic medium. This synthesis is well known but produces a significant quantity of oligomers [START_REF] Chrysanthos | Preparation and properties of bio-based epoxy networks derived from isosorbide diglycidyl ether[END_REF]. In a previous study, the synthesis has been improved using high power ultrasound [START_REF] Musa | Ultrasoundassisted heterogeneous synthesis of bio-based oligo-isosorbide glycidyl ethers: towards greener epoxy precursors[END_REF]. This work focuses on the development of new fully bio-based flax-epoxy composites. After a thermal and infrared spectroscopy characterization of the isosorbide-based epoxy resin, its mechanical properties will be investigated thanks to nanoindentation tests. The mechanical properties of unidirectional (UD) composite made of flax fibres, bio-based epoxy precursor cured with a standard diamine will be evaluated and compared to conventional solutions.

Materials and methods

The plant fibres used in this study are scutched textile flax (Aramis variety), cultivated in France in 2015 and obtained from Van Robaeys Frères Company (Killem, France).

The ultrasound-assisted heterogeneous synthesis, chemical and structural characteristics of the bio-based epoxy prepolymers (DGEI) have been previously detailed [START_REF] Musa | Ultrasoundassisted heterogeneous synthesis of bio-based oligo-isosorbide glycidyl ethers: towards greener epoxy precursors[END_REF]. The different resins were obtained by mixing DGEI with isophorone diamine (IPD) hardener in stoichiometric proportions (Table 1) i.e. considering that one active hydrogen of amine function reacts with one epoxy ring. A commercial Epolam 2020 (Axson) was used as a reference.

The thermal properties of epoxy resins were obtained by differ-ential scanning calorimetry (DSC) and were carried out on a TA Instrument DSC Q1000. Samples (5-10 mg) were heated from 100 to 250 °C at 10 °C/min under nitrogen atmosphere. Infrared spectra were recorded on PerkinElmer Spectrum BXII Fourier transform infrared (FTIR) spectrometer over the wavenumber range of 4000-400 cm-1. Micro-UD composites having a 6 2 mm section were manu-factured by hot compression with a targeted fibre volume fraction of 60 ± 2%. The determination of the tensile properties was made in accordance with the NF T 25-501-2 (elementary flax fibre) and ISO-527-5 (UD composites) standards, considering the loading frame compliance. For flax fibres a 10 mm free length was chosen; the frame was clamped on a universal MTStype tensile-testing machine equipped with a 2 N capacity load cell and loaded at a constant crosshead-displacement rate of 1 mm/min up to rupture in a laboratory with a controlled temperature (23 °C) and relative humidity (48%). speed and environmental parameters were used for composite characterisation with using an extensometer to carefully control the displacement. Nanoindentation (NI) measurements were made on epoxy resin after a 1-lm polishing and a three-side pyramid (Berkovich) indenter with a continuous stiffness measurement technique was used. A 3-nm amplitude, 45 Hz oscillations and a 0.05 s-1 loading rate were selected. Measurements were taken at depths to 1500 nm and indentation modulus and hardness were calcu-lated thanks to the Oliver and Pharr method [START_REF] Scheibe | NADP+-malate dehydrogenase in C3-plants: Regulation and role of a lightactivated enzyme[END_REF] by averaging 30 measurements on each sample.

× --

Results and discussion

Thermal and structural characterization of bio-based epoxy resins

The thermal properties of epoxy/amine cured resins are com-pared to literature and standard epoxy resin (Table 1). The decreasing of EEW from 280 to 180 g.eq -1 is associated with the drop of Tg0 and the increasing of the enthalpy from 170 to 332 J.g -1 . Those two elements highlight the increasing of the reactivity between the prepolymers and the hardener with the increasing of the ratio of epoxy ring of prepolymer. The Tgonset and Tgpeak increase in parallel from 46 to 78 °C and 92 to 114 °C, respectively. The increase in epoxy grafting rate produces a higher crosslinking density. Compared with the results obtained by [START_REF] Chrysanthos | Preparation and properties of bio-based epoxy networks derived from isosorbide diglycidyl ether[END_REF], the ultrasonic method allows the Tgpeak to increase from 104 to 114 °C which makes it possible to have thermal properties comparable to the stan-dard epoxy. The overlay of infrared spectra (Fig. 1) shows the characteristic C-O bands of the epoxy groups at 908 and 845 cm-1 and OH group at 3477 cm-1 on DGEI180 spectra. After curing, DGEI180-IPD spectra displays an increase of hydroxyl group (3417 cm-1) a high reduction of epoxy bands and the appear-ance of the characteristic band of primary amine at 1642 cm-1 possibly do to residual hardener. The increasing of OH groups (3017-3383 cm-1) confirms the reaction between the primary amine group with oxirane leading to free hydroxyl groups. The comparison between cured epoxy resins exhibits the increasing of OH due to the increasing of oligomers in prepolymers. The slight increase of epoxy groups (834 cm -1 ) is possibly due to the decrease of the probabilities of meeting between the N-H and oxirane groups, which in agreement with the DSC results.

Nanomechanical characterization of the epoxy resins

Fig. 2 shows average NI modulus and hardness obtained in both commercial Epolam2020 and isosorbid-based epoxy resins. Interestingly, one can notice that DGEI180-IPD exhibits a slightly improved mechanical performance compared to the standard Epo-lam 2020 epoxy resin. These two systems exhibit similar glass transition temperatures, 78 and 80 °C for DGEI180-IPD and Epolam 2020, respectively. NI mechanical properties of home-made resins regularly decrease with the increase of EEW, a drastic drop of NI modulus and hardness ( 94% and 98%, respectively) is noticed for DGEI280-IPD compared to DGEI180-IPD; as expected, a pro-nounced correlation between mechanical performances and glass transition temperature is observed, the NI modulus and hardness being negatively correlated with the EEW values. Indeed, a lower glass transition temperature induces higher polymer chain mobility and a softness behaviour, logically impacting mechanical performance. A more pronounced decrease is noticed for the hardness, compared to the modulus; indeed, hardness calculation is directly linked with the theoretical contact area between the indenter and the material. In case of defects such as voids, porosities within the sample, the hardness can be underestimated inducing low NI values.

Mechanical performances of UD composite

Fig. 3 presents the Young's modulus and tensile strength, according to the fibre volume fraction, of biocomposites performed in this study (in red) and compares them with recent literature data. In view of the thermal and mechanical results presented above, the DGEI180-IPD formulation was selected to develop these composite materials. In addition, the properties of the elementary flax fibres used were determined; with Youn's modulus, stress and elongation at break values of 47.5 ± 11.7 GPa, 965 ± 373 MPa and 2.14 ± 0.73% respectively, they are well correlated to the values measured in the literature [START_REF] Baley | Average tensile properties of French elementary flax fibers[END_REF] on flax fibres with a similar protocol. Interestingly, whether in terms of stiffness or tensile strength, our composites offer performances comparable to literature unidirectional composites made with petroleum-based epoxy resins [START_REF] Bourmaud | Influence of processing temperature on mechanical performance of unidirectional polyamide 11-flax fibre composites[END_REF]. Regarding the stiffness, our values are even among the best com-posites, with values included between 270 and 340 MPa, which shows a good interfacial adhesion between the biobased resin we have developed and the flax fibres. To go further, it would be interesting to investigate the fibre-matrix interfacial shear strength and to sweep a large range of composite fibre volume fractions in a forthcoming work. Nevertheless, the results of this preliminary study validate the use of DGEI180-IPD resin for the development of structural biocomposites.

Conclusion

We have shown that it is possible to synthetize bio-based epoxy prepolymers with high reactivity towards amine hardener. Spectroscopic and calorimetric measurements indicate that their thermal and mechanical properties are at least as good as oil-based epoxy precursors. The introduction of scutched flax fibres in DGEI/amine resins allowed the manufacturing of quasi fully bio-based composites. Mechanical tensile investigations on unidirectional composites have validated their use for structural applications with a Young's modulus and strength at break performance comparable with petroleum-based epoxy/flax fibres materials.

Figure. 1 .

 1 Figure. 1. Overlay of infrared spectra of DGEI180 and epoxy/amine cured resins.

Figure. 2 .

 2 Figure. 2. Comparison of nanoindentation modulus and hardness of epoxy resins cured with different epoxy content and to a standard epoxy Epolam ® .

Figure. 3 .

 3 Figure. 3. Comparison of biobased epoxy-flax composite with literature data (all materials are UD composites made of flax fibres and epoxy matrix) [10].

Table 1

 1 Thermal properties of epoxy resins.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.