High throughput screening of ligands across membrane proteins

Abstract

The present invention relates to high throughput screening of at least one ligand of at least one transmembrane protein (TP) of interest embedded in at least one lipid bilayer derived from endogenous microsomes of a plant endoplasmic reticulum (ER), the high throughput screening comprises the steps of: providing at least one endogenous microsome comprising at least one TP embedded in a lipid bilayer, or at least one endogenous microsome fragment comprising at least one TP embedded in a lipid bilayer; providing at least one analyte of interest; contacting the at least one TP with the at least one analyte; and detecting an interaction between the at least one analyte and the at least one TP. The screening is suitable for high multiple levels of TPs and analytes, as well as for rapid and repeatable identification of ligands that are potential candidate drugs. In addition, the invention also provides all basic products, consumables and kits for high-throughput screening.

Description

Technical Field

[0001] The present invention relates to high throughput screening (HTS) of at least one ligand of at least one transmembrane protein (TP) of interest, the transmembrane protein (TP) being embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), the high throughput screening comprising the following steps: providing at least one endogenous microsome comprising at least one TP embedded in the lipid bilayer, or at least one endogenous microsomal fragment comprising at least one TP embedded in the lipid bilayer; providing at least one analyte of interest; contacting at least one TP with at least one analyte; and detecting the interaction between the at least one analyte and the at least one TP. The screening is suitable for high multiplex levels of TPs and analytes, and for rapid and reproducible identification of ligands as potential drug candidates. In addition, the present invention also provides all basic products, consumables and kits for high throughput screening. Therefore, the present invention relates to the technical field of biochemical and biotechnological drug candidate screening for TPs associated with signal transduction that causes or participates in diseases. Background Art

[0002] Currently, as a proof of concept, a cell-free (CF) system based on lysate from tobacco (Nicotiana tabacum) BY-2 cells (containing a Endogenous microsomes of 100 μg / mL were used for TP production. pALiCE02 vector (LenioBio GmbH) was used in Three well-characterized TPs were expressed in the microsomes: hEGF, PVR, TIGIT and ACE2. In addition, TPs with a C-terminal tag were used for proof of concept of HTS according to the present invention and microsomes were captured. Once the TPs were captured, interaction (binding) assays of the TPs and their respective ligands were performed. The CF system was proven to be successful for the production of type I transmembrane proteins PVR, TIGIT, and ACE2. The expression of these proteins facilitated the design of assays to study protein orientation and structure. In addition, the correct folding of PVR and ACE2 and the binding assay confirmed that ACE2 can bind to RBD. The TP expression in the microsomal capture assay demonstrated the utility of the HTS of the present invention for a high-throughput membrane protein drug target screening platform.

[0003] Drug discovery is an arduous, expensive and time-consuming process. The average time to develop a new drug is 10-15 years, and it can cost up to 800 million euros. The SARS-CoV-2 pandemic has shown that this process needs to be accelerated in order to quickly respond to the threat of new pandemics in the coming years. Target-based drug discovery is the most common strategy for new drug development. Traditionally, the most followed paradigm is one drug against one target, but the need for platforms that allow screening of multiple drugs against one target has become essential. Ligand screening for drug discovery is essential for the development of new drugs. Currently, there are two main approaches to drug discovery, target-based screening or phenotypic screening. Phenotypic screening is based on trying to identify molecules that can change the phenotype of a cell to the desired outcome, while target-based screening is based on using recombinant technologies to find targets that play a role in the disease. Since the 1990s, high-throughput screening (HTS) is a focused target screening that is increasingly being used by most pharmaceutical companies to discover new drugs. For example, HTS is frequently used to study potential drugs against neurodegenerative diseases. HTS is based on the screening of multiple molecules / compounds or "hits" against a known target. The increasing use of HTS has replaced the commonly used slow-paced "trial and error" techniques to establish faster methods. However, the throughput of prior art screening methods is limited, indicating that there is a need to develop next-generation drug screening platforms with higher throughput to increase the speed of drug discovery. The main feature of HTS is that the target for drug discovery is known and the testing of multiple molecules acting on the target can be analyzed as potential drugs. The drug target can be a small molecule or any kind of protein that plays a role in a specific disease process and can be addressed by a drug to treat the disease. Therefore, the pharmaceutical industry relies on the selective binding of the "hits" to the target or specific interactions that lead to target modulation.

[0004] However, there are several limitations of HTS that prevent it from realizing its full potential, especially HTS for transmembrane protein (TP) drug candidates, which constitute the majority of drug targets but are underrepresented compared to cytosolic proteins. Currently, more than 60% of existing drug targets are transmembrane proteins (TPs). TPs are involved in important physiological mechanisms that ensure the structural and functional integrity of cells. TPs are key components for many processes such as cell recognition, immune response, signal transduction, and molecular trafficking. The use of TPs constitutes a problem for drug development because expression, post-translational modifications (PTMs), protein folding, and stabilization during HTS are difficult.

[0005] TPs account for 30% of the total proteins encoded by humans, but they are currently not well characterized. TPs are difficult to produce in living eukaryotic cells. One of the main problems in research is that overexpression of TPs has a strong deleterious effect on eukaryotic cell health, thereby hindering the production of correctly folded and physiologically functional TPs. This limitation in protein production is responsible for the delay in the functional and structural characterization of TPs. In addition, prokaryotic cells are not suitable for the production of eukaryotic TPs, and the expression of these proteins leads to the formation of protein aggregates, which need to be dissolved after expression (Sachse et al., 2013). However, there are other protein expression platforms that can produce a variety of types of proteins in a simple way (Khambhati et al., 2019) without relying on the structural integrity of the cell. These platforms are based on cell-free protein synthesis (CFPS).

[0006] Cell-free protein synthesis (CPSF) systems are based on crude lysates that contain the machinery necessary for translation, protein folding, and energy metabolism (Buntru et al., 2014). CFPS is a promising solution for protein production that allows for shorter process times and less proteolysis. A major advantage of CFPS is the ability to express cytotoxic proteins (Khambhati et al., 2019), which has made a significant contribution to the synthesis of difficult-to-express proteins, especially TPs. There are many variations of CFPS, consisting of recombinant transcription-translation machinery or derived from prokaryotic or eukaryotic cell lysates. The most commonly used lysates are Escherichia coli extract (ECE), rabbit reticulocyte lysate (RRL), Chinese hamster ovary cell (CHO) lysate, wheat germ (WGE) lysate, and insect cell extract (ICE). However, the main CFPS platforms currently in use are not entirely ideal, with low overall protein production yields, slow production times, unavailable or inappropriate protein modifications, and not feasible or not feasible enough for the biosynthesis of TPs. To address some of these issues, Buntru et al. (2014) developed a cell-free system using tobacco BY-2 (bright yellow-2) cells that showed higher protein yields in a shorter time. However, there is currently no HTS for accessible and functional TP drug candidates.

[0007] The complexity and barrier provided by the cell membrane raise many difficulties, such as difficulty in standardizing experiments, incompatibility issues and variability. In order to overcome the challenges of TP biosynthesis and TP supply, the prior art uses living cells or artificial components, such as a combination of synthetic lipids and prokaryotic systems (e.g., Escherichia coli extract (ECE)) to obtain correctly folded, modified and stable TPs. In doing so, the above-mentioned disadvantages cannot be overcome, and the biosynthesis is more complicated, more time-consuming and more expensive, and the yield of the desired TP is lower.

[0008] To address these problems, an object of the present invention is to provide a eukaryotic system, preferably a plant cell-free protein synthesis (CFPS) system, as a key tool suitable for working without living cells and without artificial components for the synthesis of correctly folded, modified and stable TPs. The supply of TPs embedded in endogenous microsomal lipid bilayers can provide the HTS platform of the present invention for ligands that are candidate drugs for TPs. Summary of the invention

[0009] Therefore, one object of the present invention is to provide a method for synthesizing TPs embedded in lipid bilayers derived from endogenous microsomes, and to provide TPs of interest embedded in the lipid bilayers for screening of ligands, particularly ligands as candidate drugs. Another object is to provide stable and correctly folded TPs of interest, which are embedded in lipid bilayers derived from endogenous microsomes. In addition, an object of the present invention is to provide endogenous microsomes or fragments thereof comprising at least one TP embedded in the lipid bilayer. Another object of the present invention is to provide TPs embedded in the lipid bilayers, which are captured on the surface for subsequent screening and detection of interactive but still mobile TPs, particularly TPs that are mobile in the medium of the desired detection method, such as in a liquid phase, a gel phase or a gas phase. Alternatively, another object is to provide captured and fixed TPs on a device, such as on a microtiter plate, a microchip, a microarray, a microfluidic device or other consumables for detection purposes. Therefore, another object is to provide suitable consumables for the desired detection method, which comprises the TPs of interest embedded in the lipid bilayer and captured on the consumables. By providing a high yield of TPs embedded in lipid bilayers, it is intended to provide a flexible and adaptable kit for the desired screening design. Another object of the present invention is to provide an optimized and standardized system for high throughput screening (HTS) of ligands of TPs embedded in lipid bilayers, preferably an optimized and standardized system for drug screening. The object of the present invention is to enable, improve, standardize and accelerate HTS of candidate drugs for TPs that are associated with a disease or are associated with the treatment of a disease (e.g., antibodies, chimeric antigen receptors, etc.). To this end, it is an object of the present invention to provide an HTS platform that is easily adaptable to any TP of interest.

[0010] Another object is to provide such a method, wherein the mild conditions in the cell lysate preparation process provide a complete subcellular membrane structure (microsome) derived from ER and / or Golgi apparatus. These membrane vesicles are the key prerequisites for producing functional TPs with their subsequent PTMs. Therefore, another object of the present invention is to provide such a method, wherein TPs are expressed, folded, modified and stabilized in microsomes. PTMs strongly affect the physicochemical properties of nascent polypeptide chains, thereby affecting protein folding, stability and activity. An object of the present invention is to provide an isolated endogenous membrane structure carrying an interested TP.

[0011] The cell-free system of the present invention overcomes the above-mentioned shortcomings of the prior art, and provides rapid acquisition of the TP of interest, low reaction volume and short reaction time, thereby realizing high-throughput TP expression strategy and high-throughput screening using TP. The method of the present invention provides correct folding for full-length TP, and combines co-translational modification and post-translational modification (PTM). Another advantage of the present invention is that there is no need to add artificial membrane structure or exogenous microsomes from other species. The advantage of the present invention is that it provides homologous CFPS for TP. The method, kit and assay according to the present invention are suitable for expression and testing of higher libraries, multiple designs, and allow complex research and high-throughput experiments to run quickly enough, more quickly and at a lower cost. The details of the present invention are explained as follows.

[0012] A first aspect of the present invention is a high throughput screening (HTS for short) of at least one ligand of at least one transmembrane protein (TP) of interest, said transmembrane protein (TP) being embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), in particular, from the ER and the Golgi apparatus, said high throughput screening comprising the following steps:

[0013] - providing at least one endogenous microsome (as defined herein) comprising at least one transmembrane protein (TP) embedded in a lipid bilayer, or at least one endogenous microsomal fragment comprising at least one transmembrane protein (TP) embedded in a lipid bilayer,

[0014] - providing at least one analyte of interest (as defined herein),

[0015] - contacting the at least one TP with at least one analyte, and

[0016] - detecting an interaction between said at least one analyte and said at least one TP.

[0017] Preferably, in an embodiment of the high throughput screening according to the invention, a step of capturing (as defined herein) at least one TP embedded in the lipid bilayer of a microsome or microsome fragment to a biologically inactive surface is performed. The biologically inactive surface is any surface defined in the context of capture.

[0018] The advantage of endogenous microsomes (as defined herein) deriving from plant endoplasmic reticulum (ER) (especially from ER and Golgi apparatus) is that it satisfies all necessary requirements for the correct folding of TP, because such microsomes provide natural environment and complete translocon mechanism for the correct embedding and folding of TP.Most preferably, endogenous microsomes according to the present invention derive from the tobacco BY-2 cell of cultivation.Use HTS of the present invention, desired activity determination and combination determination can be carried out, to identify the active part of regulation and control TP or the binding partner of identification TP.

[0019] In one embodiment, the HTS of the present invention further comprises a step of treating (disrupting or fragmenting) said at least one microsome, in one embodiment free microsome, to obtain microsomal fragments, in one embodiment free microsomal fragments, wherein said at least one TP remains embedded in said lipid bilayer originating from said endogenous microsome. "Free microsomes" within the meaning of the present invention are endogenous microsomes that contain at least one TP embedded in the lipid bilayer of the microsome and are not trapped by the TP to a biologically inactive surface.

[0020] According to the present invention, "treating" microsomes comprising at least one TP embedded in a lipid bilayer means subjecting the entire and intact microsome comprising at least one TP, one or more different TPs, or one or more TP variants of the same TP of interest to chemical and / or physical forces to break down the lipid bilayer. Chemical and / or physical forces are applied that are strong enough to disrupt (destroy) the lipid bilayer, but as small as possible so as not to affect the conformation and activity of the at least one TP. Chemical forces include treatment with detergents. Detergents include maltosides, thiomaltosides, polyoxyethylenes, polyethylene glycols, glucosides, glucosamidopropyls, thiopyranoglucosides, pyranoglucosides, glucamides, methylglucamides, hydroxyethyl sulfoxides, detergents from the maltose-NG family (e.g., lauryl maltose neopentyl glycol (LMNG), amine oxides, n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltopyranoside (DM), n-dodecyl-α-D-maltopyranoside (α-DDM), octyl-β-glucoside (OG), and lauryldimethylamine-N-oxide (LDAO)), CYMAL, detergents GDN-101, OGNG, LDAO, UDAO, and APO109, Tween 20, and Fos-choline detergents (e.g., fos-choline U10-11, cyclofos-6, 12, 13, 14, 15, and 16). Physical forces include shear forces (via shear force generating devices, different mixers), ultra-wave, sonication, ultrasonic. Any treatment method can be used in combination. Thus, the treatment is a chemical treatment (e.g. detergent) and / or a physical or mechanical treatment (e.g. shear force, ultrasonic), wherein the force applied to the microsomes is sufficient to destroy the microsomes to a certain extent, but does not damage the TP or release the TP from the lipid bilayer.

[0021] If necessary, excipients may be used to stabilize the microsomal fragments according to the present invention. Stabilizers or stabilizing excipients are molecules suitable for stabilizing microsomes or microsomal fragments, the microsomes or microsomal fragments comprising at least one TP embedded in the lipid bilayer of the microsomes or microsomal fragments. The stabilizer ensures that the at least one TP remains folded and embedded in the lipid bilayer, and the binding site or epitope of interest remains accessible to the analyte. Stabilizers within the meaning of the present invention include lipid bilayer and / or protein stabilizers, which are well known to those skilled in the art (Sachse et al., 2014). Such excipients include so-called nanodiscs, nanoparticles, scaffold proteins or any combination thereof. Therefore, in another embodiment of the present invention, the HTS may include a step of adding at least one stabilizing excipient (preferably a lipid bilayer stabilizing excipient) to the microsomal fragments, preferably after a treatment step (the treatment step may be before or after capture). The "treatment" described herein does not weaken or destroy the captured state of at least one TP. However, if the strength of the selected capture strategy is not sufficient to withstand the available or preferred treatment methods, it may be necessary to treat before capture.

[0022] The step of treating the at least one microsome may be performed independently (in time and space) of the HTS, it may be a step of the method for the production (biosynthesis) of a TP as described herein according to the invention, or a step of the HTS according to the invention. It may be necessary to produce at least one TP in microsomes, to store the intact microsomes comprising the at least one TP under appropriate conditions, so that the timing and design of the HTS can be determined individually, e.g., to use the HTS of intact microsomes at any time, or to destroy them at any time, so as to provide microsomal fragments immediately before the HTS. In this embodiment, it may even be desirable or necessary to store the microsomes or fragments thereof as defined herein. If necessary, stabilizing excipients may be added.

[0023] In a preferred embodiment of the HTS according to the present invention, the at least one microsome or microsome fragment is derived from the genus Nicotiana of the family Solanaceae, preferably from Nicotiana tabacum, more preferably from BY-2 cell line.

[0024] Nicotiana is the genus of herbaceous plants and shrubs in the Solanaceae. So far, it is known that there are at least 67 species, which are commonly referred to as tobacco plants. Some of them, mainly tobacco, are cultivated to obtain tobacco leaves for the production of tobacco products. Usually, any species that can be cultivated or the species that can be separated from cell lines are all feasible within the meaning of the present invention. Especially, for providing endogenous microsomes according to the present invention, tobacco or its derived cell line is the preferred source of microsomes or microsome fragments, more preferably from the BY-2 cell line of tobacco. In another embodiment of HTS according to the present invention, at least one endogenous microsome or endogenous microsome fragment derived from vegetative endoplasmic reticulum (ER) (especially from ER and Golgi body) is a cell lysate from the Nicotiana plant of the Solanaceae family, preferably from the cell lysate of tobacco, more preferably from the cell lysate of the BY-2 cell line of tobacco.

[0025] The BY-2 cell line and BY-2 cell-free protein synthesis (CFPS) were developed to overcome the shortcomings of most eukaryotic CFPS systems. Compared with wheat germ lysate (WGE) for protein production, BY-2 shows faster lysate production, higher translation activity and higher scale-up potential (Buntru et al., 2014). The results show that functional full-size antibodies can be produced by BY-2 lysate (Buntru et al., (2015)), indicating that BY-2 lysate is a potential expression system for biopharmaceutical production. BY-2 cells contain microsomes derived from the endoplasmic reticulum (ER) and Golgi apparatus of plant cells. By applying appropriate methods, the microsomes can be prepared and isolated for the purposes of the present invention. This is important because one of the challenges when producing TPs is that they need to be integrated into the lipid bilayer so that they can be synthesized in their correctly folded state (Buntru et al., (2015). The present invention transfers the production of TPs into an HTS platform that allows for multiple drug screening against otherwise intractable TP targets. The BY-2-derived HTS, also referred to as BY-2-HTS, is a preferred embodiment of the HTS of the present invention with endogenous microsomes. The endogenous microsomes have the advantage of avoiding complications due to the use of artificial components, artificial membranes or other artificial excipients, and furthermore maintaining the native protein biosynthesis machinery, in particular the native post-translational modification capabilities.

[0026] According to another embodiment of the HTS according to the present invention, preferably BY-2-HTS, at least one TP embedded in the lipid bilayer of the endogenous microsomes and / or the endogenous microsome fragments is captured on a biologically inactive surface, and the endogenous microsomes and / or the endogenous microsome fragments are preferably from tobacco. In another embodiment of the HTS according to the present invention, the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of devices including microtiter plates, microfluidic devices, microarrays, microchips, and / or the biologically inactive surface is any other surface suitable for capturing at least one TP of interest. Any other surface preferably suitable for capture, contact, interaction and detection of the present invention. The proof-of-concept example shows successful capture, interaction and detection according to the present invention, and the results are shown in Figures 10 and 11.

[0027] For all aspects and embodiments of the present invention, a transmembrane protein (TP) is "captured" to a biologically inactive surface such that the TP is controlled during interaction and / or detection of the interaction. The "capture" according to the present invention can be performed by non-specific binding of the at least one TP to the biologically inactive surface, or by capture mediated by at least one tag at the C-terminus and / or N-terminus of the at least one TP. Preferably, the at least one TP is captured to a biologically inactive surface via a C-terminal tag. Suitable tags are well known to the skilled person and include biotin tags, hapten tags, alkaline phosphatase (AP), AU1 epitope, AU5 epitope, bacteriophage T7 epitope (T7 tag), calmodulin binding peptide (CBP), cellulose binding domain (CBP), chitin binding domain (CBD), chloramphenicol acetyltransferase (CAT), choline binding domain (CBD), E2 epitope, FLAG epitope, galactose binding protein (GBP), Glu-Glu (EE tag), glutathione S-transferase (GST), Histidine affinity tag (HAT), HSV epitope, human influenza hemagglutinin (HA), KT3 epitope, LacZ, maltose binding protein (MBP), Myc epitope, PDZ domain, PDZ ligand, polyarginine (Arg tag), polyaspartic acid (Asp tag), polycysteine ​​(Cys tag), polyhistidine (His tag), polyphenylalanine (Phe tag), Profinity eXact, Protein C, S1 tag, S tag, Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep tag, Strep tag II, streptavidin, streptavidin binding peptide (SBP), T7 epitope, tandem affinity purification (TAP), TrpE, Universal and VSV-G. "Captured TP" is always embedded in the lipid bilayer of endogenous microsomes or microsomal fragments, as defined herein. Therefore, "captured TP" is used synonymously for "captured TP embedded in the lipid bilayer of endogenous microsomes or microsomal fragments". "Captured TP" means that the TP embedded in the lipid bilayer is fixed to the at least one biologically inactive surface by the capture step, and the surface carries the TP during the treatment, contact, interaction and / or detection steps of the HTS (preferably BY-2-HTS).

[0028] Therefore, in another embodiment of the HTS of the present invention (preferably BY-2-HTS), the at least one TP comprises at least one tag at the C-terminus and / or N-terminus, preferably the at least one TP is captured on the biologically inactive surface via the C-terminal tag. Any tag is suitable. Preferred are Strep tags, His tags and / or biotin tags ( Figure 1-5 ).

[0029] In one embodiment, at least one microsome comprising at least one TP embedded in a lipid bilayer is first captured, and then the microsome is treated as described herein to obtain microsomal fragments comprising at least one TP embedded in a lipid bilayer, the TP being captured during and after the treatment of the at least one microsome. Alternatively, the at least one microsome is treated in an uncaptured state (preferably a free state) to obtain fragments comprising at least one TP embedded in a lipid bilayer, and then the fragments are captured to the biologically inactive surface by at least one TP. If desired, the microsomal fragments comprising at least one TP are separated from the fragments not containing the TP and / or from the released TP.

[0030] Therefore, in another embodiment, the HTS according to the present invention, preferably BY-2-HTS, further comprises the following steps:

[0031] - capturing said at least one TP embedded in the lipid bilayer of the microsomes onto said at least one biologically inactive surface, and

[0032] - processing the captured at least one microsome to obtain microsomal fragments, wherein the at least one TP remains embedded in the lipid bilayer, or alternatively

[0033] - treating at least one free microsome to obtain free microsomal fragments, wherein the at least one TP remains embedded in the lipid bilayer, and

[0034] - capturing said at least one TP embedded in the lipid bilayer of the microsomal fragments onto said at least one biologically inactive surface.

[0035] Preferably, the treatment is a chemical treatment (e.g., detergent) and / or a physical treatment (e.g., shear force, ultrasound) to an extent sufficient to destroy the microsomes without damaging the TPs in the lipid bilayer. Preferably, the capture is performed using a Strep tag, a His tag, and / or a biotin tag. If desired, the uncaptured microsomes and / or microsome fragments are separated after the capture step. The microsomes and / or microsome fragments that do not contain TPs and / or released TPs are separated from the microsomes and / or microsome fragments that contain at least one TP. The separation can be performed by filtration and / or centrifugation.

[0036] The biologically inactive surface may be the surface of a particle, the surface of a device as defined herein and / or any other surface suitable for capturing the at least one TP of interest. In any case, the "captured TP" is captured in a reaction zone of the above-mentioned surface, wherein the interaction between the at least one TP and the at least one analyte is detected. Therefore, the "biologically inactive surface" according to the present invention refers to any surface that does not dominate or influence the contacting step, the interaction step between the at least one TP and the at least one analyte and / or the detection step of the interaction. The biologically inactive surface may be the surface of a material, in particular a consumable material commonly used for any detection assay, such as a polymer, polystyrene, polypropylene, polycarbonate, alginate / ester, agarose, hydrogel, alginate / ester or agarose hydrogel, polycarboxylate / ester, carboxymethyl dextran hydrogel, glass material, etc. Other materials suitable for biologically inactive surfaces are well known to the skilled person. The biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of devices including microtiter plates, microfluidic devices, microarrays, microchips or any other consumables, and / or the biologically inactive surface is any other surface suitable for capturing and carrying the TP of interest, in particular during the contacting, interaction and / or detection steps. The surface is particularly suitable for capturing and carrying the at least one TP during the HTS according to the present invention, in particular during the processing of the at least one microsome, during the following steps: contacting, interacting and / or detecting the interaction of the at least one TP with the at least one analyte, preferably the binding of the at least one TP to the at least one analyte, without affecting said steps.

[0037] A device within the meaning of the present invention comprises, is coated with or consists of a biologically inactive surface, preferably the device comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 16, at least 16 to 24, at least 24 to 32, at least 32 to 96, at least 96 to 384, at least 384 to 1536 reaction zones (as defined herein) or any other integer between the above ranges. A microtiter plate typically has 6, 12, 24, 48, 96, 384 or 1536 wells, typically arranged in a 2:3 rectangular matrix. The device and particles (as defined herein) are consumables, preferably suitable for use in HTS according to the present invention, and most preferably for the steps of capturing, processing, contacting, interacting and / or detecting the interaction. An example of such a device has been successfully used in proof-of-concept examples, and the results obtained with the device are shown in Figures 10 and 11 and demonstrate that the device (here a microtiter plate) is feasible according to the present invention. In particular, the device can be used for a HTS according to the present invention, preferably a BY-2-HTS, more preferably a multiple BY-2-HTS, and it can be produced by a cell-free production method, in particular, wherein the TP embedded in a lipid bilayer is captured in the device according to the present invention. The same applies to microfluidic devices ( Fig.12 and 16).

[0038] Therefore, another aspect of the present invention is such a device (as defined herein), comprising at least one biologically inactive surface (as defined herein), said surface respectively carrying said at least one captured TP (as defined herein), said TP being embedded in the lipid bilayer of a microsome or a microsome fragment (as defined herein). The device comprises at least two or more reaction zones, said reaction zones respectively comprising biologically inactive surfaces for multiple HTS (preferably multiple BY-2-HTS). In one embodiment, said HTS (preferably BY-2-HTS) comprises the following steps:

[0039] - providing at least one device comprising at least two reaction zones, said reaction zones comprising at least one biologically inactive surface, and

[0040] - capturing (as defined herein) said at least one TP embedded in the lipid bilayer of the microsomes to at least one biologically inactive surface of said device in each reaction zone and, optionally, treating to obtain microsomal fragments as defined herein, or

[0041] - capturing (as defined herein) said at least one TP embedded in the lipid bilayer of the microsomal fragments to at least one biologically inactive surface of said device in said at least one reaction zone.

[0042] - alternatively, providing at least one device comprising at least two reaction zones, said reaction zones comprising at least one biologically inactive surface on which said at least one TP has been captured, wherein said TP is embedded in a lipid bilayer of a microsome or a microsome fragment, and

[0043] - if it is microsomes captured in the device and microsomal fragments are desired for HTS, the processing steps are performed as described herein, or

[0044] - If it is a microsomal fragment captured in said device, the further steps of the HTS of the invention are performed, comprising providing at least one analyte or group of analytes, contacting, interacting and / or detecting.

[0045] If desired, the TP embedded in the lipid bilayer and captured within the device according to the present invention may be covered with a stabilizer during storage prior to performing the HTS.

[0046] According to the present invention, a "reaction zone" is an area where a TP is captured and where the TP comes into contact with the at least one analyte and the interaction with the at least one analyte is tested. The reaction zone comprises a biologically inactive surface defined herein, is coated with a biologically inactive surface defined herein, or is composed of a biologically inactive surface defined herein. The reaction zone is any particle surface of a particle defined herein, or is located on the particle. The reaction zone is a portion of the surface of a device defined herein, a portion of a hole of a microtiter plate, a portion of a channel of an SPR consumable, a portion of any microfluid, a portion of any microfluidic device, or a portion of any other surface of a consumable used in a detection according to the present invention. The device comprises at least two or more such reaction zones, the reaction zone comprising a biologically inactive surface defined herein, is coated with a biologically inactive surface defined herein, or is composed of a biologically inactive surface defined herein.

[0047] In another embodiment of the HTS according to the present invention (preferably BY-2-HTS), the at least one analyte comprises a small molecule, a peptide, a polypeptide, a soluble protein, a membrane protein, a protein complex or any combination thereof.

[0048] An "analyte" within the meaning of the present invention, any aspect and embodiment refers to any molecule that is tested for its interaction with the at least one TP of interest (as further defined herein), preferably for its ability to modulate the activity of the at least one TP (preferably due to signaling or signal transduction as defined herein), and for its binding to a specified domain of the at least one TP and / or a binding domain or epitope on the at least one TP. An analyte may be tested alone or in combination with or dependent on other analytes of the same group (e.g. one, two or more small molecules) or another group (e.g. a small molecule and any protein). Thus, two or more analytes tested in the same reaction zone are considered an "analyte complex" (synonym "analyte combination") and any interaction with the at least one TP of interest is tested in combination. Thus, the screening is suitable for screening at least one "analyte" or "analyte complex" (synonym, analyte combination) for its potential to be a ligand as described herein. Examples of analyte complexes include: a multimeric protein comprising at least two polypeptide chains, at least two analytes combined by covalent or electrostatic forces or hydrogen bonds, and at least one analyte combined with a polymer, nanoparticle, colloid or liposome. The ability of the test analyte to induce signal conduction or signal transduction due to interaction with the at least one TP (preferably the binding of the analyte to the TP). The signal conduction or signal transduction (preferably induced by the binding of the at least one analyte to the at least one TP) can occur through at least one transmembrane domain of the at least one TP (especially the original cytoplasmic domain towards the TP, preferably the TP is captured to a biologically inactive surface in the assay), or on the same side of the interaction of the at least one TP with the at least one analyte. The signal conduction or signal transduction can regulate biological processes, and regulation includes activation, inactivation, inhibition, enhancement, induction or blocking of biological processes. Therefore, the role of the analyte identified as a ligand can be the role of an activator, inhibitor, effector, trigger or regulator of a biological process. Preferably, the biological process is a biological process associated with a disease. "Analyte" within the meaning of the present invention refers to any molecule as described above, which is preferably a natural molecule of microbial, bacterial, fungal, viral, plant, animal and / or mammalian origin. Analytes include small molecules, peptides (e.g., dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, oligopeptides), polypeptides, soluble proteins, membrane proteins and / or protein complexes (of microbial, bacterial, fungal, viral, plant, animal and / or mammalian origin).Analytes within the meaning of the present invention include antibodies, antibody fragments, fc fragments, fab fragments, immunoglobulins (e.g. A, G, D, E, F, M, W, Y), Ig fragments, regulatory proteins (including inhibitors, enhancers, antagonists), membrane proteins, antigens, antigen fragments, chimeric antigen receptors, fragments thereof, receptors, cytokine receptors, and / or analytes are used. According to the present invention, an "analyte panel" comprises two or more analytes or combinations of analytes used in the multiplex HTS, preferably multiplex BY-2-HTS.

[0049] "Small molecules" are defined differently depending on the technical field of application. Currently, small molecules or micromolecules (synonymous) have low molecular weight (≤1000 Daltons) and / or low molecular weight below 900 g / mol. They can modulate biological processes, are of organic origin, and can include secondary metabolites as well as small nucleic acid molecules and small molecule oligonucleotides containing deoxynucleotide monophosphates. Some examples of small molecules include antihistamines, antibiotics, drug candidates, and small molecules from microorganisms and plants, selective serotonin reuptake inhibitors, alicyclic acids, ibuprofen, acetaminophen, morphine, ethanol, psilocybin, L-DOPA, cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, cyclophosphamide, docetaxel, bleomycin, vinblastine, dacarbazine, mustine, vincristine, procarbazine, prednisolone, etoposide, cisplatin, epirubicin, carboplatin, Becitabine, folinic acid, oxaliplatin, gemcitabine, ifosfamide, hydrocodone, metformin, losartan, albuterol, gabapentin, omeprazole, levothyroxine, atorvastatin, brompheniramine, cetirizine, chlorpheniramine, clemastine, diphenhydramine, fexofenadine, loratadine, amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, levofloxacin, citalopram, escitalopram, fluoxetine, paroxetine, sertraline.

[0050] A "ligand" within the meaning of the present invention is any analyte that interacts with at least one TP within the meaning of the present invention, preferably an analyte that binds to said at least one TP. Thus, an "analyte" (or analyte complex) within the meaning of the present invention that is demonstrated to have the ability to induce signal conduction or signal transduction due to the interaction with said at least one TP (preferably the binding of the analyte to said TP) is identified as a ligand. Preferably, due to said interaction (preferably the binding with at least one TP), a desired biological process (preferably a biological process associated with a disease) is regulated. Most preferably, the disease is associated with the tested TP. More preferably, due to said regulation as defined herein, an analyte that is a ligand of the tested TP is considered a candidate drug. The desired ability of the at least one ligand is to be suitable for the prevention or treatment of a disease associated with said at least one TP. The ligand may be an analyte or an analyte complex as defined herein and is demonstrated by the interaction detected according to the present invention.

[0051] Within the meaning of the present invention, an "interaction" between the at least one TP and the at least one analyte (or analyte complex) (particularly after contact) is a molecular interaction that results in a measurable activity or change in activity of the at least one TP induced by the at least one analyte and / or the interaction is a binding between the at least one TP and the at least one analyte. The binding may be associated with a measurable activity, a change in activity or inactivity. Typically, the interaction may result in a modulation (increase, decrease) of the activity, which is detected as a change in activity due to the ability of the analyte to modulate the activity as defined herein. Thus, within the meaning of the present invention, the interaction is demonstrated by detecting activity, such as colorimetric / fluorescence assays, radioactive scintillation proximity assays, atomic force microscopy (AFM), spectrophotometry, mass spectrometry, Raman spectroscopy, membrane potential assays, electrochemical assays, fluorescence resonance energy transfer (FRET) assays, crystallography, cryo-electron microscopy. The binding may be demonstrated by detecting an analyte bound to the TP or by detecting activity after binding. The interaction may be performed and detected in multiple screenings.

[0052] Therefore, another aspect of the HTS of the present invention is a multiplex high throughput screening (multiplex HTS), preferably a multiplex BY-2-HTS, of at least one ligand for at least one TP of interest, providing two or more analytes in two or more reaction zones simultaneously, and / or providing an analyte combination of two or more analytes in two or more reaction zones simultaneously. All definitions apply accordingly to the multiplex design of the HTS of the present invention.

[0053] "Multiplex", "multiplex screening", "multiplex HTS" or "multiplex high throughput screening" (synonyms) means that in the same reaction zone, one or more different TPs are captured to a bio-inactive surface and tested against one analyte or a defined combination of analytes, and the interaction of at least one or more captured TPs with the analyte or combination of analytes is detected. Multiplex also means that in different reaction zones, the same TP is captured to a bio-inactive surface and each TP is tested against two or more different analytes, and the interaction between one captured TP and one analyte or combination of analytes is detected in each reaction zone. Therefore, "multiplex" within the meaning of the present invention means that at least 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 16, at least 16 to 24, at least 24 to 32, at least 32, at least 96 to 384 analytes or more, or combinations of analytes are screened simultaneously for at least one or more TPs of interest. Or any other integer between the above ranges. All other definitions apply accordingly, and any step and combination of steps, including providing microsomes or microsome fragments, providing analytes, processing, capturing, contacting, interacting and / or detecting, and said embodiments of the present invention may be performed in a multiplex HTS, preferably a multiplex BY-2-HTS, as described herein. Multiplex HTS may be performed with any embodiment of a biologically inactive surface, device or consumable as defined herein, respectively.

[0054] The multiplex level is defined or limited by the available or selected detection methods. In particular, it is defined or limited by the available consumables for said detection methods, such as microtiter plates, microfluidic devices, microchips, microarrays or SPR consumables.

[0055] Therefore, an embodiment of multiplex HTS (preferably multiplex BY-2-HTS) comprises the following steps:

[0056] - providing a plurality of endogenous microsomes each comprising at least one transmembrane protein (TP) embedded in a lipid bilayer or a plurality of endogenous microsomal fragments each comprising at least one transmembrane protein (TP) embedded in a lipid bilayer,

[0057] - providing a plurality of analytes or a plurality of combinations of analytes (as defined herein),

[0058] - contacting the plurality of TPs with a plurality of analytes or a plurality of combinations of analytes, and

[0059] - Detecting the interaction of any one analyte and any TP, preferably in a plurality of reaction zones of a device as defined herein, more preferably in a microtiter plate, in a microfluidic device or in an SPR consumable.

[0060] The capture and / or processing steps described with respect to HTS apply accordingly to the multiplex design.

[0061] In another embodiment of the HTS according to the invention (preferably a multiple BY-2-HTS), the interaction between the at least one TP and the at least one analyte (as defined herein), in particular after contact, is detected by immunoassay (preferably enzyme-linked immunosorbent assay (ELISA)), spectrometry (preferably surface plasmon resonance (SPR)), particle size measurement, magnetic measurement and / or optical measurement. These methods are well known to those skilled in the art. In the experiments, the HTS of the invention in combination with ELISA and SPR is demonstrated. These methods are well known to those skilled in the art, and it is clear to those skilled in the art that the analyte or at least one TP or both contain a detection means, or such a means must be added separately to detect any interaction. The detection means include fluorescent molecules, labels, haptens, enzymes or other molecules that allow the detection of interactions. Such means can be encoded by a template encoding the at least one TP, combined with the at least one analyte and / or can be added separately in the detection step. For each of the above detection methods, appropriate detection means are well known, and the examples shown demonstrate feasibility.

[0062] In one embodiment of the invention, the interaction and / or detection of HTS (particularly after contact) is performed with fixed or mobile microsomes or microsome fragments, respectively, each of which contains at least one TP embedded in a lipid bilayer, which TP is captured to a biologically inactive surface. All definitions apply accordingly. However, "captured" is different from "fixed", and a distinction must be made between "captured and mobile" and "captured and fixed". The microsomes or microsome fragments can be captured by the at least one TP, and the microsomes or microsome fragments are mobile or fixed. In contrast to the mobile state, the microsomes or microsome fragments are fixed in the device by the step of capturing the at least one TP on the at least one biologically inactive surface using the biologically inactive surface of the particles defined herein.

[0063] As described herein, TP is very attractive for drug development and constitutes the majority of current drug targets. However, TP is difficult to express poorly characterized proteins. The BY-2 lysate, preferably commercially available System (LenioBio GmbH, e.g. Kits AL0103050, AL0103200, AL0103500) are currently used to show overexpression and production of TPs and to perform HTS according to the present invention. At present, according to the present invention, binding assays for known ligands thereof are tested to show proof of concept for the HTS platform (preferably BY-2-HTS). For this purpose, well-characterized type I transmembrane glycoproteins and their ligands are used. These proteins are angiotensin converting enzyme (ACE2), T cell receptor with Ig and ITIM domains (TIGIT) and its respective ligands, the receptor binding domain (RBD) of the SARS-CoV-1 / 2 spike protein, and the poliovirus receptor (PVR). These TPs are interesting because they have been shown to be "druggable" targets and they are involved in currently very important diseases such as COVID-19 and cancer. Hindering the binding between the relevant TP and its ligand can be effectively used as a possible therapy. The results show that the HTS of the present invention is suitable for studying different "hits" that hinder the binding. Therefore, proof of concept of the HTS platform of the present invention for testing new drugs has been shown.

[0064] Another aspect of the present invention is the cell-free production of at least one transmembrane protein (TP) of interest, embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), in particular from the ER and the Golgi apparatus, preferably based on a plant of the genus Nicotiana of the family Solanaceae, preferably based on the BY-2 cell-free protein synthesis (CFPS) system, said cell-free production comprising the following steps:

[0065] - providing a lysate from a plant comprising at least one endogenous microsome, preferably from a plant of the genus Nicotiana of the family Solanaceae, preferably from a lysate of Nicotiana tabacum, more preferably from a lysate of a BY-2 cell line of Nicotiana tabacum,

[0066] - providing at least one template encoding said at least one transmembrane protein (TP),

[0067] - incubating at least one lysate with at least one template,

[0068] - expressing said at least one transmembrane protein (TP),

[0069] - coupled transcriptional and co-translational translocation of at least one expressed TP into at least one microsome, and

[0070] - obtaining at least one transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome, and

[0071] - Optionally, treating at least one endogenous microsome carrying at least one TP embedded in a lipid bilayer to obtain microsomal fragments comprising at least one TP embedded in a lipid bilayer. If necessary, perform the following steps:

[0072] - Capture (as defined herein) onto a biologically inactive surface either after or before processing.

[0073] The immediately available processing product is: TP embedded in the lipid bilayer of endogenous microsomes or endogenous microsomal fragments, which are free (not trapped) as defined herein; or TP embedded in the lipid bilayer of endogenous microsomes or endogenous microsomal fragments, which are trapped and mobile (e.g. on a particle) or trapped and immobilized (e.g. on a device) as defined herein, respectively.

[0074] In particular, in another embodiment of the cell-free production of at least one transmembrane protein (TP) of interest, the transmembrane protein (TP) is embedded in the lipid bilayer of at least one endogenous microsome according to the present invention, and a step of capturing the at least one TP embedded in the lipid bilayer of the microsome onto the at least one biologically inactive surface is performed. In one embodiment of the method, a capture step is performed and at least one captured microsome is processed to obtain microsomal fragments, wherein the at least one TP remains embedded in the lipid bilayer. In another embodiment of the method, the at least one free microsome is processed to obtain free microsomal fragments, wherein the at least one TP remains embedded in the lipid bilayer, and a step of capturing the at least one TP embedded in the lipid bilayer of the microsomal fragment onto the at least one biologically inactive surface is performed. In particular, for any embodiment of the method, the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of devices including microtiter plates, microfluidic devices, microarrays, microchips, and / or the biologically inactive surface is any other surface suitable for capturing at least one TP of interest.

[0075] Optionally, further steps are necessary and can be combined with the methods described herein, such as separation and / or isolation of the microsomes or microsomal fragments, purification, washing and / or any other steps necessary to provide at least one TP embedded in the lipid bilayer of at least one endogenous microsome or endogenous microsomal fragment. These steps are well known to the skilled person. All embodiments of the capture step (with or without a tag as defined herein), processing, separation and isolation steps described for the HTS are correspondingly applicable to the production method.

[0076] In one embodiment of the cell-free production, the processing step is performed independently in time from the previous step. It may be desirable to produce the TP in microsomes, store intact and free or intact and trapped microsomes (mobile or immobilized), and process the microsomes to obtain microsomal fragments immediately prior to HTS according to the invention, which may be subsequent to the cell-free production of the at least one TP.

[0077] Another embodiment of the cell-free production (CFP) is the production of at least one captured endogenous microsome derived from the plant endoplasmic reticulum (ER), in particular from the ER and the Golgi apparatus, comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of the microsome, the cell-free production comprising the following steps:

[0078] - providing a lysate from a plant comprising at least one endogenous microsome, preferably from a plant of the genus Nicotiana of the family Solanaceae, preferably from a lysate of Nicotiana tabacum, more preferably from a lysate of a BY-2 cell line of Nicotiana tabacum,

[0079] - providing at least one template encoding said at least one transmembrane protein (TP),

[0080] - incubating at least one lysate with at least one template,

[0081] - expressing said at least one transmembrane protein (TP),

[0082] - coupled transcriptional and co-translational translocation of at least one expressed TP into at least one microsome, and

[0083] - obtaining at least one transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome,

[0084] - optionally separating, preferably by centrifugation, the at least one microsome obtained comprising at least one TP of interest as defined above, and

[0085] - capturing (as defined herein) at least one microparticle comprising at least one TP of interest as defined herein, preferably capturing said microparticle onto a biologically inactive surface as defined herein, and

[0086] - obtaining at least one captured microsome comprising at least one TP of interest as defined, preferably captured to a particle or device as defined herein.

[0087] Another embodiment of the cell-free production provides at least one captured endogenous microsomal fragment derived from the plant endoplasmic reticulum (ER), in particular from the ER and the Golgi apparatus, said endogenous microsomal fragment comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of said microsomal fragment, wherein a step of treating the at least one endogenous microsome carrying the at least one TP embedded in the lipid bilayer is further performed to obtain a microsomal fragment comprising the at least one TP embedded in the lipid bilayer.

[0088] All embodiments of the capture (with or without a tag as defined herein), processing or a combination thereof of the HTS described in the first aspect of the present invention are correspondingly applicable to the cell-free production. Therefore, the cell-free production according to the present invention may include capture and / or processing to obtain such particles, devices or other consumables, the particles, devices or other consumables comprising at least one captured endogenous microsome or endogenous microsomal fragment derived from the plant endoplasmic reticulum (ER) (particularly from the ER and the Golgi apparatus), the endogenous microsome or endogenous microsomal fragment comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of the microsome or embedded in the lipid bilayer of the microsomal fragment.

[0089] In another embodiment of cell-free production or cell-free production method, the lysate also comprises plastid, chloroplast, mitochondria or chloroplast and mitochondria. In the further embodiment of cell-free production or cell-free production method, the at least one microsome or microsome fragment derives from the Solanaceae Nicotiana, preferably from tobacco, more preferably from the BY-2 clone. If necessary, the method can include the step of adding a composition comprising bioactive microsome and / or bioactive mitochondria. Preferably, such composition derives from the Solanaceae Nicotiana, preferably from tobacco, more preferably from the BY-2 clone.

[0090] In the coupled transcription and co-translational translocation process, the co-translational translocation of at least one expressed TP into at least one microsome can occur when the expressed TP does not carry a signal peptide, or the co-translational translocation is mediated by at least one appropriate signal peptide. Therefore, in one embodiment of the cell-free production or cell-free production method, the at least one template encodes the at least one TP and a signal peptide, the signal peptide is suitable for mediating the translocation of the TP into the microsome, and is expressed together with the TP from the same template. Other TP translocations do not require signal peptide-mediated translocation. In another embodiment, the template also encodes at least one tag at the N-terminus and / or C-terminus of at least one TP, preferably the template encodes at least one tag at the C-terminus ( Figure 1-5). Preferably, the TP is expressed with a signal peptide and / or at least one tag at the N-terminus and / or C-terminus. In another embodiment of the cell-free production or cell-free production method, the at least one TP is co-translationally translocated with its C-terminus outside the at least one microsome and its N-terminus inside ( Figure 6 ). Preferably, glycosylation, N-glycosylation, other post-translational modifications, lipid modifications and / or folding of the TP occur in the steps of coupled transcription and co-translational translocation and ensure a correctly folded and functional TP. More preferably, the TP is embedded in the lipid bilayer with its transmembrane domain, just as it exists naturally.

[0091] Another aspect of the present invention is an endogenous microsome or at least one endogenous microsome fragment derived from a plant endoplasmic reticulum (ER) (particularly from ER and Golgi apparatus), the endogenous microsome or endogenous microsome fragment comprising at least one transmembrane protein (TP) embedded in a lipid bilayer, preferably obtained by a cell-free production or a cell-free production method according to the present invention. In one embodiment of this aspect, the endogenous microsome or at least one endogenous microsome fragment is free, not captured and not fixed. In another embodiment, the endogenous microsome or at least one endogenous microsome fragment is captured in a particle (as defined herein) but is still mobile and is still suitable for mixing with at least one stabilizer and optionally at least one other excipient to form a composition. In a preferred embodiment, the endogenous microsome or at least one microsome fragment is derived from the genus Nicotiana of the Solanaceae family, preferably from tobacco, more preferably from BY-2 cell line. Another aspect of the present invention is a microsome or microsome fragment as described herein with at least one stabilizer. Therefore, another aspect of the present invention is a composition comprising at least one (free or trapped and mobile) microsome or microsome fragment according to the present invention, at least one stabilizer and optionally at least one further excipient.

[0092] Another aspect of the invention is the use of the microsomes or microsome fragments in the HTS according to the invention or according to any embodiment of the HTS, the microsomes or microsome fragments comprising at least one transmembrane protein (TP) embedded in a lipid bilayer as defined herein. All embodiments of the capture (with or without a tag as defined herein), processing or a combination thereof described in relation to the HTS apply accordingly to the use.

[0093] Another aspect of the invention is a biologically inactive surface comprising at least one captured TP of interest, embedded in the lipid bilayer of at least one endogenous microsome or at least one endogenous microsomal fragment from the plant endoplasmic reticulum (ER), in particular from the ER and the Golgi apparatus, wherein the biologically inactive surface is the surface of a consumable. Preferably, the consumable is a device comprising two or more reaction zones, the reaction zones comprising, consisting of or coated with a biologically active surface as defined herein. Preferably, the device is a microtiter plate, a microchip, a microarray or a microfluidic device, more preferably a microfluidic device (e.g. for SPR) or a microtiter plate (e.g. for ELISA). Another aspect is a consumable as defined herein, preferably a device comprising at least one captured TP of interest, embedded in the lipid bilayer of at least one endogenous microsome or at least one endogenous microsomal fragment from the plant endoplasmic reticulum (ER), in particular from the ER and the Golgi apparatus. The device comprises at least two or more reaction zones, the reaction zones comprising, consisting of or coated with biologically inactive surfaces. It is suitable for use in multiple HTS according to the invention. Preferably, the microsomes or microsome fragments are from plants of the genus Nicotiana of the Solanaceae family, preferably from tobacco, more preferably from lysates of tobacco, most preferably from lysates of the BY-2 cell line of tobacco. Preferably, capture is mediated by a tag as defined herein.

[0094] For all aspects and embodiments of the present invention, "consumables" include, consist of, or are coated with a biologically inactive surface. According to the present invention, such consumables are used in any detection assay. Suitable materials for such consumables include polymers, polystyrene, polypropylene, polycarbonate, alginate, agarose, hydrogels, alginate or agarose hydrogels, polycarboxylates, carboxymethyl dextran hydrogels, glass materials, etc. Other materials suitable for biologically inactive surfaces are well known to those skilled in the art. The definition of the biologically inactive surface applies accordingly. Consumables include particles, microparticles, nanoparticles, magnetic particles, and devices including microtiter plates, microfluidic devices, microarrays, and microchips.

[0095] Preferably, the consumable is for HTS, BY-2-HTS, more preferably multiple BY-2-HTS, or any embodiment thereof according to the present invention. More preferably, the consumable is produced by the production method defined herein. More preferably, the consumable is for use in a contacting process, in a step of processing, interacting and / or detecting an interaction between at least one analyte and at least one TP according to the present invention.

[0096] Another aspect of the present invention is a kit for high throughput screening (HTS) according to the present invention (preferably BY-2-HTS, more preferably multiple BY-2-HTS), wherein the high throughput screening is used for screening at least one ligand of at least one transmembrane protein (TP) of interest, wherein the transmembrane protein (TP) of interest is embedded in the lipid bilayer of at least one endogenous microsome derived from plant endoplasmic reticulum (ER) (particularly from ER and Golgi apparatus), wherein the endogenous microsome is preferably derived from a plant of the genus Nicotiana of the Solanaceae family, preferably from tobacco, preferably from a lysate of tobacco, more preferably from a lysate of tobacco BY-2 cells, the kit comprising:

[0097] - a plant cell lysate comprising endogenous microsomes of endoplasmic reticulum (ER) origin, in particular from the ER and the Golgi apparatus, preferably from a plant of the genus Nicotiana in the family Solanaceae,

[0098] - at least one vector encoding a known TP as a positive control, preferably as a positive control of functional microsomes or microsomal fragments,

[0099] - optionally, at least one carrier as a positive control for the function of the lysate,

[0100] - at least one vector encoding at least one template for at least one transmembrane protein (TP) of interest,

[0101] -optionally, additional excipients, preferably comprising small molecule building blocks, labeled amino acids, cofactors, etc.,

[0102] - optionally, at least one reagent (preferably at least one means) for detecting the interaction between at least one analyte and at least one TP, preferably an antibody for detection,

[0103] - optionally, at least one agent for disrupting at least one microsome (preferably a suitable detergent), and

[0104] - Optionally, at least one analyte or a panel of two or more analytes.

[0105] The kit may comprise additional components, such as a composition comprising biologically active microsomes and / or mitochondria as defined herein. Another additional component may be a consumable for capture as defined herein. Alternatively, the kit comprises a consumable or device according to the invention, which already comprises at least one captured TP. The kit may comprise at least one instruction sheet describing the mixture of components and / or an instruction sheet describing the HTS and / or cell-free production method, with or without capture and / or processing, respectively.

[0106] Preferably, at least one positive control for lysate function is the commercially available pALiCE01-eYFP vector, wherein eYPP represents enhanced yellow fluorescent protein, and at least one positive control for functional microsomes or microsomal fragments is the pALiCE0X-TP vector for TP expression, preferably customized or adapted to the TP of interest. However, any other vector and / or vector and TP combination is suitable, provided that it allows confirmation of the desired function (positive control).

[0107] A preferred embodiment of the kit for HTS (preferably multiplex HTS) further comprises:

[0108] - additional excipients, preferably comprising small molecule building blocks, labeled amino acids, cofactors, etc.,

[0109] - a reagent for detection, preferably an antibody for detection,

[0110] -optionally, an agent for disrupting the microsomes, and

[0111] - Optionally, at least one analyte or a panel of two or more analytes.

[0112] A preferred embodiment of the kit for HTS (preferably multiplex HTS) further comprises:

[0113] - an agent for chemically disrupting the microsomes of the invention, and

[0114] - Optionally, at least one analyte or a panel of two or more analytes.

[0115] For all aspects and embodiments of the present invention, " endogenous microsome " means that it is not artificial, and it is from the cell-free production system of eukaryotic species.Preferably, it comes from the system identical with lysate source and ER, Golgi body, microsome, plastid, chloroplast and / or mitochondrial source.As known in the prior art, " lysate " is the separated biologically active content for protein synthesis." endogenous microsome " comes from the natural system of only a species that derives from the same section.Natural lysate can comprise the component from different species and / or different genera but is derived from the same section.Therefore, the different cell lines of Nicotiana, the different cell lines of tobacco and the different BY-2 cell lines with different genetic modifications can be combined.The microsome mixture that derives from the system that is derived from different cell lines remains the endogenous microsome (mixture) in the meaning of the present invention.The heterozygous lysate that comprises the component from different sections or even different species is excluded, and is not a part of the present invention. A heterogeneous lysate means that eg the ER, Golgi, microsomes, plastids, chloroplasts and / or mitochondrial components are not from the same family, or even from the same species (eg plant, yeast, fungus, prokaryote).

[0116] The microsomes according to the present invention may comprise one or more identical TPs (identical in amino acid sequence and glycosylation) or one or more different variants of the same TP as defined herein. Thus, the microsomes may comprise different variants of the same TP embedded in the lipid bilayer of the same microsome. The variants may have different glycosylation patterns and / or different amino acid sequences. Such microsomes in the embodiments may be used for screening according to the present invention. In another embodiment, the microsomes are treated to obtain microsomal fragments, each of which comprises at least one variant of the same TP embedded in the lipid bilayer. Thus, after treatment, different variants of the TP are separated from each other in different microsomal fragments.

[0117] Eukaryotic CFPS can be divided into two types: those with endogenous microsomal structures and those without endogenous vesicle compartments (Thoring et al., 2019). Currently, the present invention encompasses eukaryotic CFPS containing endogenous microsomal structures for post-translational modification. Therefore, when producing TP, it is preferred to use endogenous microsomes that can achieve correct TP glycosylation.

[0118] For all aspects and embodiments of the invention, a "transmembrane protein" (TP) according to the invention comprises at least one transmembrane domain, at least one cytoplasmic domain and at least one cytoplasmic domain. The TP may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more transmembrane domains. "TP embedded in a lipid bilayer" means that at least one transmembrane domain of the at least one TP is oriented and embedded in the lipid bilayer of the endogenous microsomes or in the lipid bilayer of microsomal fragments derived from endogenous microsomes. TPs within the meaning of the present invention include: type I TPs, type II TPs, type III TPs and type IV TPs with one transmembrane domain, multi-transmembrane TPs, lipid-anchored proteins, GPI-anchored membrane proteins, bitopic TPs, polytopic TPs with α-helical transmembrane domains, multisite TPs with β-folded transmembrane domains, peripheral membrane proteins and monotopic proteins. Preferably, the TP is embedded in a lipid bilayer, as it occurs naturally, and is preferably glycosylated. Examples of TPs are listed below.

[0119]

[0120]

[0121] Some specific examples of potential TPs for HTS according to the present invention include hAQP1 (e.g. Uniprot KB: P29972, 6 TMDs), Mel-hEGFR (e.g. Uniprot KB: P00533, 1 TMD) (at least nine different EGFR mutations have been described), human β-adrenergic receptor, shaker potassium channel, connexin, inositol triphosphate receptor, nicotinic acetylcholine receptor, G protein-coupled receptor GPCR, ion channels, transporters, histidine and tyrosine kinases, cytokine receptors, Toll-like receptors.

[0122] Other specific examples of TP according to the present invention include antigens, preferably antigens associated with malignancies and autoimmune diseases. Preferably, the antigen group includes CD20 as a target for treating B cell malignancies, and CD3, CD22, CD33, CD123 and CD19 as targets for treating B cell malignancies, leukemias, acute myeloid leukemias, chronic lymphocytic leukemias (CLL), acute lymphoblastic leukemias (ALL), chronic myeloid leukemias (CML) and acute myeloid leukemias (AML). In particular, CD20 is a transmembrane protein with four transmembrane domains embedded in the lipid bilayer of the cell membrane. Example 6 shows that as a proof of principle, the expressed CD20 is embedded in the lipid bilayer of endogenous microsomes. The same assay is applicable to any desired antigen for screening ligands.

[0123] Within the meaning of multiplex HTS according to the invention, the contact, interaction (preferably activation and / or binding) between the same TP provided in each of any number of different reaction zones and different analytes is screened. In another embodiment, different variants of the TP of interest are provided in any number of different reaction zones, a corresponding number of analytes are provided, and contact and screening of the interaction of the same or different analytes with different TP variants (per reaction zone) is performed. Variants of the TP comprise any genetic variation, whether or not resulting in a variation in the amino acid sequence, whether or not having an effect on the 3D structure and / or activity, single nucleotide polymorphisms (SNPs), deletions and / or insertions of the TP variants. The above applies to the HTS according to the invention, wherein the at least one TP is captured to a biologically inactive surface, or wherein the at least one TP is not captured to a biologically inactive surface.

[0124] For all aspects and embodiments of the present invention, "mobile microsomes or mobile microsome fragments" comprise at least one TP embedded in the lipid bilayer of the microsomes or the microsome fragments. The mobile microsomes or mobile microsome fragments are captured to the biologically inactive surface of the particles defined herein by the at least one TP. If the at least one biologically inactive surface is located on the particle, the particle carrying the captured TP remains mobile during the processing, contacting, interaction and / or detection steps. However, the TP cannot be free independent of the at least one biologically inactive surface. Therefore, because the biologically inactive surface is mobile, the microsomes or fragments thereof are mobile. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 : Schematic representation of microsomal capture and protein functionality assay using receptor-ligand interactions, as shown for ACE-RBD interaction in Example 1. "Plate" represents a biologically inactive surface according to the present invention.

[0126] Figure 2 : Microsome capture and protein structure determination using antibodies. "Plate" represents a biologically inactive surface according to the present invention.

[0127] Figure 3: a) Schematic diagram of microsome capture and protein functional assay using receptor-ligand interaction, as shown in Example 1 for ACE-RBD interaction. "Plate" represents a biologically inactive surface to which the microsomes have been captured by the expressed TP according to the present invention. b) Functional assay using variants of the receptor. A multiplex design in which expression of variants of a particular TP is performed and different forms of the TP are tested (e.g., in parallel in microtiter plates or other devices or consumables). Thereafter, these versions can be analyzed by high-throughput platforms such as SPR.

[0128] Figure 4 : Schematic representation of protein-protein interactions using different TPs expressed in two separate microsomes (preferably in two separate reaction setups) that are finally brought together (contact step) to study protein-protein interactions, as illustrated by the PVR-TIGIT interaction concept ( FIG. 3 ).

[0129] Figure 5 : Schematic diagram of the HTS assay design according to the present invention, A: for screening of ligands, such as ligands for personalized therapy (different RBD mutants), B: screening for molecules (chemicals, antibodies) that disrupt receptor ligand binding, C: screening for ligands for orphan drugs, D: screening for candidate drugs.

[0130] Figure 6 : A schematic diagram of the directional transformation of TP using an additional transmembrane domain. Ideally, the receptor domain of the TP should be oriented in a manner that is located inside the microsome, and the microsome simulates the oxidative environment of the "extracellular surface" or ER-Golgi apparatus. However, the orientation of the receptor domain can contribute to the research and development of assays in which both receptors and ligands are membrane-bound proteins, and can interact before the microsomes are broken. A possible way to transform this orientation is to use an additional transmembrane domain, so that additional folding can cause the receptor domain of the protein to face the outside of the microsome. The possible disadvantage of this method can be that the misfolding of the external domain is caused by exposure to the reducing cytoplasmic environment of the lysate around the microsome. The stable domain that requires disulfide bonding is exposed to the cytoplasmic environment of the lysate and causes disulfide bond rupture, thereby causing misfolding.

[0131] Figure 7 :exist ACE2, PVR and TIGIT expressed in Western blot analysis of proteins from II. Unpurified proteins and complete lysates expressing the proteins were analyzed directly on SDS-PAGE gels. A. PVR, ACE2 and TIGIT with cSTREP tags at different DNA concentrations (2.5, 5, 7.5 and 10 nM). B. PVR, TIGIT and ACE2 with nSTREP tags at different DNA concentrations (2.5, 5, 7.7 and 10 nM). C. PVR, TIGIT and ACE2 with cSTREP tags and nSTREP tags at previously selected optimal DNA expression concentrations (5 and 7.5 nM). The black triangle heads indicate the optimal concentrations (A and B) and the concentrations selected for subsequent ELISA experiments (C). The red arrows indicate that the expressed proteins may be glycosylated. All proteins on the blot were probed with StrepMAB-classic antibodies coupled to HRP. L: Molecular weight ladder.

[0132] Figure 8 : At 5 and 7.5 nM DNA concentrations Western blot analysis of nHALO-tagged TIGIT and PVR expressed in . PVR_nHALO shows duplicate bands (1 and 2), representing different colonies corresponding to different cloning events. A two-color molecular weight ladder (L) was used to compare protein band sizes. All proteins on the blot were probed with Tag and then with the secondary antibody peroxidase AffiniPure goat anti-mouse IgG.

[0133] Figure 9: Western blot analysis of microsomal lysis assays for PVR, TIGIT and ACE2. A. cSTREP-tagged PVR and ACE2 in different lysis fractions. B. nSTREP-tagged PVR, TIGIT and ACE2 in different lysis fractions. All proteins on the blot were probed with HRP-conjugated StrepMAB-classic antibodies. L: molecular weight ladder; S1: first supernatant; P1: first pellet; S2: second supernatant; P2: second pellet.

[0134] Figure 10: Indirect ELISA for PVR detection using human CD155 / PVR antibody - goat anti-mouse IgG (HRP). A. ELISA graph where the Y-axis represents absorbance and the X-axis represents the serial dilution factor. The more concentrated the sample, the higher the absorbance value displayed, and vice versa. The red curve corresponds to the PVR_cSTREP sample and the yellow line corresponds to the PVR_nSTREP sample. B. Photograph of the ELISA plate where the bright blue color represents the reaction of the detection reagent to the HRP-labeled antibody. The color of the cSTREP sample is more intense compared to the nSTREP.

[0135] Figure 11: Indirect ELISA for ACE2 detection and binding analysis using commercially available RBD-His tag horseradish peroxidase-conjugated antibodies. A. ELISA graph where the Y-axis represents absorbance and the X-axis represents serial dilutions. The absorbance has its highest value when the sample is concentrated and decreases with serial dilutions. ACE2_cSTREP is represented by the red curve and ACE2_nSTREP is represented by the yellow curve. B. Photograph of the ELISA plate after adding the detection reagent, where only the color of ACE2_cSTREP is visible.

[0136] Fig.12 : Comparison of SPR of hEGF produced in BYL or E. coli in Example 2. SDS-PAGE of purified hEGF fractions (lanes ah show loading, washing, elution fractions, etc.) Fig.15 shown.

[0137] FIG. 13 : Summary of LC-ESI-MS / MS analysis of N-glycan structures of RBD and Adalimumab in Example 4. A: RBD (SIVRFPNITNLCPFGE) has the following composition: 63.7% oligomannose, 22.16% heterozygous (Man9+Hex, MM, MMF, MMX, MMXF, GnMX, GnMXF, GnGnF, GnGnXF) and 14.86% unoccupied, B: RBD (VFNATRFASVYAWNRK) has the following composition: 65.99% oligomannose, 32.56% heterozygous (Man9+Hex, MM, MMF, MMXF, GnMF, GnMX, GnMXF, GnGnX, GnGnXF) and 1.43% unoccupied, and C: Humira has the following composition: 69.72% oligomannose, 1.81% heterozygous and 28.47% unoccupied.

[0138] Fig.14 : Binding assay of SARS-CoV-2 antigen of Example 5.

[0139] Fig.15: SDS-PAGE of hEGF purified fractions is shown (lanes ah show loading, washing, elution fractions, etc.). The hEGF gene with an N-terminal Strep-II tag was cloned into pALiCE01 and pALiCE02 to evaluate cytoplasmic and microsomal expression. Microsomal hEGF expression was observed, but cytoplasmic expression was not observed. One-step streptavidin affinity purification is sufficient to extract highly pure soluble hEGF. Lane A: Humira CHO TNFα, 4.53E+05ka(1 / Ms), 8.37E-05kd(1 / s)1.85E-10KD(M); Lane B: Humira CF DAK TNFα, 4.07E+05ka(1 / Ms), 5.01E-05kd(1 / s), 1.23E-10KD(M); Lane C: Humira CF DB3 TNFα, 4.78E+05ka(1 / Ms), 5.83E-05kd(1 / s), 1.22E-10KD(M); Lane D: Humira CF CHJ TNFα, 4.00E+05ka(1 / Ms), 6.24E-05kd(1 / s), 1.56E-10KD(M); Lane E: Humira CHO CD64, 9.59E+05ka(1 / Ms), 1.52E-04kd(1 / s), 1.58E-10KD(M); Lane F: Humira DAK CD64, 7.87E+05ka(1 / Ms), 2.72E-04kd(1 / s), 3.46E-10KD(M); Lane G: Humira DB3 CD64, 6.86E+05ka(1 / Ms), 2.52E-04kd(1 / s), 3.67E-10KD(M); Lane H: Humira CHJ CD64, 7.58E+05ka(1 / Ms), 2.63E-04kd(1 / s), 3.46E-10KD(M)

[0140] Figure 16: Adalimumab SPR. Immobilized antibody molecules were probed with recombinant TNFα and CD64, showing ligand and receptor binding of Example 3, respectively. A: Humira CHO TNFα, B: Humira CF DAK TNFα, C: Humira CF DB3 TNFα, D: Humira CF CHJ TNFα, E: Humira CHO CD64, F: Humira DAK CD64, G: Humira DB3 CD64, H: Humira CHJ CD64

[0141] Fig.17: Depicts the design of plasmid pIB405 used in Example 6, which was used to express CD20 from BY-2 lysate. Plasmid pIB0405 encodes full-length CD20 (297 amino acids; 33.1 kDA), a C-terminal StrepII tag and a melittin signal peptide (MSP) for translocation to microsomes during expression, with other common sequences encoding T7 promoter, AmpR promoter, AmpR and Ori present.

[0142] Fig.18 : At 5, 7.5, 10, 15 and 20 nM DNA concentrations Western blot analysis of StrepII-tagged CD20 expressed in . Expression of CD20 tagged at the C-terminus (plB0405) as used in Example 6 was seen at all DNA concentrations. Double bands were seen, which is sometimes typical of membrane proteins after electrophoresis. The proteins were not purified, and complete lysates expressing the proteins were separated directly on SDS-PAGE gels before blot transfer. All proteins on the blot were probed with StrepMAB-Classic antibody conjugated to HRP. M: Molecular weight marker ladder.

[0143] Fig.19 : Absorbance measured at 620 nm by indirect ELISA (Example 6) is described as [ng / mL] as a function of the binding potency to CD20 expressed from BY-2 lysate at different plasmid concentrations. The control "no lysate" shows no Identify, confirm Specific recognition and binding to the antigen CD20. In the presence of expressed CD20, the absorbance increases as the concentration of the antibody increases. Example

[0144] Example 1

[0145] 1. Materials and Methods

[0146] 1.1 In silico cloning and plasmid design

[0147] First use The human DNA templates for ACE2, PVR, and TIGIT proteins from Gateway cloning were in silico cloned into the vector pALiCE02 using snapgene software (from Insightful Science; available at www.snapgene.com). An in vitro cloning strategy was then determined. II and GST tags were fused to the DNA template at the C-terminus or N-terminus for protein targeting testing purposes. The melittin signal peptide sequence (MSP) was fused at the N-terminus for targeting to microsomes (Stech et al., 2014). All clones are shown in Table 1.

[0148] The template according to the present invention is encoded on a vector, preferably on a pALiCE vector. pALiCE01 and pALiCE02 comprise a basic design (pALiCE) comprising a T7 promoter followed by a tobacco mosaic virus (TMV) 5'ω leader sequence, a gene of interest (GOI, in the present invention encoding a gene of interest to a TP), followed by a TMV 3'UTR. The pALiCE02 vector also comprises an insect melittin signal peptide before the GOI initiation, which allows microsomal targeting and translocation of the TP. In both plasmids, the GOI box consists of an N-terminal Strep-II tag, a subsequent factor Xa cleavage site and eYFP. The design of the vector allows for conventional enzyme-based subcloning of genes into the NcoI and KpnI sites on both sides of the GOI box.

[0149] Table 1: In silico designed clones with respective tags.

[0150]

[0151] The glycosylation sites of the TP were studied using the online N-glycosylation prediction tool NetNGlyc-1.0 (Gupta and Brunak, 2002), as shown in the following schematic diagram:

[0152]

[0153] For proof of concept, PVR, TIGIT, and ACE2 constructs were used with their respective tags and signal peptide sequences (MSPs) and displayed glycosylation sites. PVR consists of 417 amino acids with 8 N-glycan sites: 105, 120, 188, 237, 278, 307, 313, and 405. TIGIT consists of 244 amino acids with 3 N-glycosylation sites: 53, 90, and 236. ACE2 consists of 805 amino acids with 7 N-glycan sites: 53, 90, 103, 322, 432, 546, and 690.

[0154] 1.2 Template amplification, cloning, plasmid extraction and sequencing

[0155] Computer primers were designed as shown below to amplify the gene of interest by PCR.

[0156]

[0157]

[0158]

[0159] Seq-ID No. 1 and 2: universal primers; Seq ID-No. 3 to 33: computer-designed primers.

[0160] The TP gene of interest was amplified using the designed primers and PrimeSTAR GXL DNA polymerase ((1.25 U / μl)*2, TaKaRa Bio) as shown in Tables 2, 3, and 4.

[0161] Table 2: PCR templates and corresponding designed primers for PCR amplification.

[0162]

[0163] Table 3: PCR tube mix for amplification of desired DNA template and vector.

[0164]

[0165] Table 4: PCR settings for amplification of inserts of interest and vectors

[0166]

[0167] Agarose gel (1% (w / v)) containing ethidium bromide (0.5 μg / ml) was run at 150 V for 15 minutes to check the size of PCR amplicons. Bands were visualized using GelDocXR+ (Bio-Rad). The marker used was 1KbPlus DNA ladder (New England Biolabs). After PCR, Gibson use 2xHiFi DNA Assembly Master Mix (NewEngland Biolabs) Gibson Reaction mixture (insert fragment: 2 μl, vector: 1 μl, MiliQ water: 2 μl, 2xHiFi DNA assembly master mix: 5 μl, total volume 10 μl) was incubated in a PCR instrument at 50°C for 30 minutes.

[0168] Plasmid is transformed into competent DH5α Escherichia coli cells.Cells were incubated on ice for 10 minutes, then heat-shocked at 42°C for 30 seconds.Cells were cooled on ice for 1 minute, then in SOC medium (2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4 and 20mM glucose) at 37°C, shaken and incubated for 40 minutes.Eppendorf tubes were centrifuged at 3000rpm for 1 minute in a table centrifuge.Discard supernatant, and precipitation is used for later plating on agar plates containing 100 μl / ml penicillin (Amp).Flat plates were incubated overnight at 37°C.

[0169] Colony PCR was then performed using the specific primers shown in Table 5 and DreamTaq Green premix (2x) (Thermo Fisher Scientific TM ) for amplification. Five colonies were selected from each plate, picked with a toothpick and introduced into five different PCR tubes containing a premix (the contents of a PCR tube for colony PCR, a total volume of 10 μl, consisting of: DreamTaqGreen premix (2x): 5 μl, MiliQ water: 4 μl, forward primer: 0.5 μl and reverse primer: 0.5 μl). The applied PCR settings are summarized in Table 6. As previously described, the PCR amplicon size was confirmed using agarose gel (1% (w / v)) and ethidium bromide (0.5 μg / ml).

[0170] Table 5: Primers used for colony PCR, depending on the fusion tag of each clone.

[0171]

[0172] Table 6: PCR settings for colony PCR. Elongation time 1 min / kb. ACE2 is 3 kb long, PVR and TIGIT are between 1.5-2 kb in length.

[0173]

[0174] For sequencing, follow the manufacturer's instructions. Plasmid purification NoLid (MachereyNagel). Purified plasmids were prepared and sent for sequencing according to the sequencing provider's instructions (Eurofins Genomics). Once the sequence was confirmed, the manufacturer's instructions were followed. Xtra plasmid purification (MachereyNagel) to prepare for future use in The plasmid expressed in .

[0175] 1.3 Protein expression and western blot analysis

[0176] Protein expression:

[0177] Using cell-free systems Protein expression was performed. 96-well cell culture microplate (Greiner Bio-One TM ) were loaded with 50 μl of lysate per well, to which a defined volume corresponding to different DNA concentrations was added. The DNA concentrations used were 2.5 nM, 5 nM, 7.5 nM and 10 nM. Water was loaded in the gaps of the wells to prevent evaporation and maintain humidity. The plate was incubated in an ISF1-X Climo-shaker (Kuhner Shaker) at 25°C for about 20 hours, with continuous stirring at 500 rpm and a humidity of 80%.

[0178] Semi-dry Western Blot:

[0179] Lysate samples were prepared using 4x Laemmli sample buffer (Bio-Rad) supplemented with 10% (v / v) β-mercaptoethanol and incubated at 98°C for 2 minutes. Proteins were separated by electrophoresis using SDS-PAGE gels (MiniProtean TGX Stain-Free 4-20%, Bio-Rad) at 200 V in running buffer (250 mM Tris base, glycine 192 mM, 1% SDS (w / v), pH 8.8) for 30 minutes. Mini Protean was used according to the manufacturer's instructions. The gel was transferred to a nitrocellulose membrane using a Turbo transfer system (Bio-Rad). After transfer, the membrane was blocked with a blocking buffer (5% milk (w / v)-50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween-20 (TBST)) and incubated with shaking for 30 minutes.

[0180] The membrane was then incubated with the corresponding primary antibodies for 1 h at room temperature with shaking: StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences), diluted 1:15,000 in blocking buffer; GST-tagged monoclonal antibody HRP (MA4-004, Thermo Fisher Scientific TM ), diluted 1:2000 in blocking buffer; or Tag (G921A, Promega), diluted 1:10,000 in blocking buffer. After incubation, 3 consecutive 5-min washes were performed with TBST while shaking. The secondary antibody used was only against the N-terminus Peroxidase AffiniPure goat anti-mouse IgG (115-035-003, Jackson ImmunoReseach) was diluted 1:10,000 in blocking buffer and incubated for 1 hour at room temperature with shaking. After incubation, washing with TBST was exactly the same as the previous washing step. Immunodetection was performed by chemiluminescence reaction using HRP-conjugated antibodies. 1:1 SuperSignal TM West Atto Ultra High Sensitivity Substrate (Thermo Fisher Scientific TM ) for HRP antibody detection. TM ) to complete protein band visualization. Use PageRulerTM pre-stained protein molecular weight gradient 10-250kDa as N-terminal and C-terminal II sample markers (ThermoFisherScientific TM ), Precision Plus Protein Dual Color Standard 10-250 kDa (Bio-Rad) was used for the N-terminus and C-terminally GST-tagged samples.

[0181] 1.4 Processing of microsomes to obtain microsomal fragments.

[0182] 20 μl of the incubated lysate was pipetted into a 1.5 ml Eppendorf tube and centrifuged at 16,000 g for 15 minutes. The supernatant or S1 was collected in different test tubes, while the precipitate or P1 (containing microsomes) was resuspended in 1% dodecacyl-b-maltoside (DDM) diluted with PBS (137 mM NaCl, 2.7 mM KCl, Na2HPO4, 1.8 mM KH2PO4) and incubated on ice for 1 hour. P1 was centrifuged at 16,000 g for 15 minutes. Two fractions were collected from the centrifugation: supernatant or S2 (containing dissolved microsomal proteins) and precipitate or P2 (containing microsomal residues). P2 was thoroughly resuspended in PBS. Once all products (S1, P1, S2, P2) were ready, protein samples for western blotting were prepared as described above. A schematic overview of the experimental process is shown below:

[0183]

[0184] 1.5 Capture and protein binding assay of microsomal captures.

[0185] Lysates expressing ACE2 and PVR were used for indirect ELISA. The experimental setup was as follows Figures 1 to 4 shown.

[0186] Indirect ELISA

[0187] Microsome capture:

[0188] The lysate samples were centrifuged at 16,000 g for 15 minutes to concentrate the microsomes, and the pellet was resuspended in PBS. The concentrated microsomal samples were added directly to the Samples were prepared in triplicate in each well of an XT-coated microplate (1-4101-001, Iba). A series of unknown dilutions were performed starting from the concentrated sample (1) until 0 was reached (starting from the concentrated sample 1, 8 dilution steps were performed 1:100: 0.5->0.25->0.125->0.0625->0.03125->0.015625->0.0078125->stop). The plate was incubated at room temperature for 1 hour.

[0189] Protein Binding:

[0190] A PBS solution containing 1% DDM was added to each well and the plate was incubated at room temperature for 15 minutes. The plate was then washed 3 times with PBS-0.05% Tween (v / v) (PBST). 1x blocking buffer (ab126587, Abcam) was added to block the wells to prevent nonspecific binding and incubated at room temperature for 1 hour. A washing step similar to the previous washing step was performed.

[0191] For PVR microsome capture detection, human CD155 / PVR antibody (MAB25301, Bio-Techne) diluted 1:5000 in blocking buffer was added. For PVR protein binding to TIGIT, recombinant human TIGIT Fc chimera (7898-TG, Bio-Techne) was added at 2.5 μg / ml in blocking buffer. For ACE2 microsome capture and protein binding analysis, commercially available RBD SARS-CoV spike protein / RBD protein (RBD with His tag) (40150-V08B2, SinoBiological) and purified RBD SARS-CoV-2 were diluted to a concentration of 2.5 μg / ml in blocking buffer. The plates were incubated at room temperature for 1 hour, followed by a washing step.

[0192] For protein detection, detection antibodies (HRP labeled) bound to proteins were added. For structural antibody detection of PVR, peroxidase AffiniPure goat anti-mouse IgG (115-035-003, Jackson ImmunoResearch) diluted 1:10000 in blocking buffer was added. For PVR-TIGIT binding detection, goat anti-human IgG F (ab') 2: HRP (0500-0099) diluted 1:10000 in blocking buffer was added. For commercially available RBD / purified RBD binding detection of ACE2, His tag horseradish peroxidase-conjugated antibody (MAB050H, Bio-Techne) diluted 1:4000 in blocking buffer and StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences) diluted 1:15,000 in blocking buffer were added. The plate was incubated at room temperature for 1 hour and then washed.

[0193] For protein visualization, the detection reagent TMB ELISA substrate (high sensitivity) (Abcam) was added to each well and incubated for 15 min at 37°C in the dark. Once the wells were developed, the ELISA was performed using a TECAN Infinite M1000 Pro machine and a Tecan i-Control

[0194] 2.0 software was used to measure the absorbance (650 nm) of some colors with the settings shown in Table 8. The absorbance values ​​were analyzed using Excel to construct an absorbance / dilution graph.

[0195] Table 8: Lecture settings for Tecan i-Control 2.0 software.

[0196]

[0197]

[0198] 2. Results

[0199] 2.1. Membrane proteins Successfully expressed.

[0200] To express the TP of interest, in vitro cloning was performed in the pALiCE02 vector as shown in Table 9. The resulting plasmids were used in Proteins were expressed at different plasmid concentrations in and the expression was examined by Western blot analysis.

[0201] Table 9: Clones with inserts successfully cloned in vector pALiCE02.

[0202]

[0203] Proteins with a C-terminal Strep tag showed bands for PVR_cSTREP and ACE2_cSTREP, however, no band was visible for TIGIT_cSTREP ( Figure 7 A). PVR_cSTREP has two visible bands, one of the expected size (49kDa) and another band running higher, which may be a sign of glycosylation. PVR_cSTREP shows bands for all DNA concentrations except 2.5nM. In addition, the highest concentration shows lower band intensity, which may indicate that the system reaches saturation when the DNA concentration is above 7.5nM. ACE2_cSTREP has a visible band of the expected size (96kDa) for all DNA concentrations. At all ACE2_cSTREP concentrations, a higher band corresponding to the glycosylated protein is visible. For TIGIT_cSTREP, there is no visible band. Therefore, the presence of the band on the blot indicates protein expression. A non-specific band of 120kDa was observed in each sample.

[0204] For proteins with N-terminal Strep tags, bands corresponding to their expected sizes were visible for PVR_nSTREP (49 kDa), TIGIT_nSTREP (30 kDa), and ACE2_nSTREP (96 kDa) ( Figure 7 B), which means that the protein was successfully expressed. The PVR_nSTREP band was visible at all concentrations, but with low intensity. In addition, PVR_nSTREP did not show any signs of glycosylation, and only one band was visible on the blot. ACE2_nSTREP showed bands for all concentrations except 2.5nM. The ACE2_nSTREP band ran higher than expected, and two bands were identified at DNA concentrations of 5, 7.5, and 10nM, which may be due to glycosylation. Finally, TIGIT_nSTREP showed a band for all concentrations except 2.5nM, which ran higher than expected, which is a possible sign of glycosylation.

[0205] Blotting experiments were performed using the two optimal DNA concentrations previously analyzed in cSTREP and nSTREP sample blots ( Figure 7C) to test the repeatability of the experiment. Differences in the band intensity of the C-terminal and N-terminal fusions of each construct were observed. PVR_cSTREP showed stronger band intensity and two bands, which may be the result of glycosylation, while PVR_nSTREP had lower intensity and only one band was present. TIGIT_cSTREP did not show any bands, while TIGIT_nSTREP showed very strong bands that were higher than expected, which may be due to glycosylation. Finally, the ACE2_cSTREP band intensity was lower than ACE2_nSTREP, which showed strong and higher than expected bands. The optimal concentration of 5nM was selected from this blot for protein solubilization and ELISA experiments.

[0206] Western blotting was also used to examine the N-termini of PVR and TIGIT. Protein expression. Bands of the expected sizes of PVR (81 kDa) and TIGIT (62 kDa) can be seen on the blot ( Figure 8 ). However, it was seen that these proteins were degraded at a higher level. For PVR, two different clones were used for expression analysis on blots, both showing similar results. Protein expression analysis showed that PVR_c / nSTREP, TIGIT_nSTREP, ACE2_c / nSTREP, PVR_nHALO and TIGIT_nHALO were The results showed that the construct with GST tag and TIGIT_cSTREP were not expressed. II protein constructs have the best protein expression. Therefore, II constructs were used for the following experiments.

[0207] 2.2DDM cannot dissociate MP from microsomal membranes

[0208] To find out whether the microsomal membranes can be disrupted and whether the TP can be solubilized using a mild detergent (1% DDM), a microsomal solubilization assay was performed as described above. Expression of the lysate and analysis of the centrifuged fractions after DDM addition was done by Western blotting. The DNA concentration used was determined by previous Western blot analysis ( Figure 7 C).

[0209] C-terminal Microsomal lysis assays of IIPVR and ACE2 proteins were performed by Western blot analysis ( Fig. 9A). All fractions showed bands of the expected size, but with varying intensities between fractions. As expected, higher band intensities were visible for the P1 and P2 fractions, corresponding to microsomal membrane remnants. However, for the PVR_cSTREP sample, these proteins could also be detected with higher intensity in the S1 fraction. For S1PVR_cSTREP, only one band was visible, meaning that even if protein was present, it corresponded to unglycosylated proteins. The S2 fraction showed faint bands, but the intensity was not sufficient to assume solubility of these proteins.

[0210] In another blot, the N-terminus was analyzed. II PVR, TIGIT and ACE2 ( Fig. 9B ). For all samples, bands of the expected size were only visible on the P1 and P2 fractions. These fractions correspond to microsomal membrane remnants. ACE2_nSTREP showed a low intensity band of S2 that was not strong enough to assume the solubility of the protein.

[0211] When 1% DDM was used ( Figure 8 ), TP is not dissolved and remains in the P1 and P2 fractions where microsomal membrane remnants are expected. Mild detergents like DDM may not be able to dissociate the membrane protein from the microsomal membrane and membrane-bound MP still ends up in the pellet fraction. Stronger detergents may be needed to dissolve TP.

[0212] 2.3 For HTS, correctly folded microsomal PVR can be captured.

[0213] Indirect ELISA experiments were performed to capture microsomes expressing PVR and find out the orientation of the protein. In addition, the protein structure and binding to its ligand (TIGIT) were studied as described above. PVR capture was performed using the C-terminal II was completed. Structural antibodies and commercially available TIGIT were later used to examine protein structure and binding activity.

[0214] By measuring the absorbance, a graph of absorbance and the corresponding dilution factor was constructed ( Fig. 10A ). The more concentrated the sample, the higher the absorbance value. Therefore, the more concentrated the lysate, the more microsomes expressing the protein are captured in the plate, and subsequently more protein can bind to the structural antibody and appear blue. PVR_cSTREP has a higher absorbance value than PVR_nSTREP. By adding a detection reagent to the wells, the sample can be visualized ( Fig. 10B). Color is visible for both samples, however, the color of the PVR_cSTREP sample is stronger than the PVR_nSTREP sample. Therefore, the C-terminally tagged protein is captured to the plate at a higher concentration than the N-terminally tagged protein. For PVR_cSTREP, the second well is colored differently compared to the first and third wells. This may be a result of multichannel pipette error. Protein binding was further tested using the commercially available TIGIT, but the samples showed no color. PVR and TIGIT are not binding.

[0215] Indirect ELISA assays showed that PVR-expressing microsomes were captured in a ready-to-use In addition, the orientation of the protein was consistent with prediction, with the C-terminus located outside the microsome, as samples with the cSTREP-tagged protein showed more staining than samples with the nSTREP-tagged protein ( Fig. 10B ) and higher absorbance values ​​( Fig. 10A ). PVR was correctly folded and displayed color when bound to its structural antibody, but it failed to bind to commercially available TIGIT. Due to the lack of a standard curve, the experiment could not be statistically analyzed, but a qualitative analysis was performed. Future experiments are needed to collect more data and draw a standard curve to quantify the results. In addition, the binding between PVR and TIGIT should be analyzed.

[0216] 2.4 Correctly folded microsomal ACE2 can be captured for HTS and effectively binds to RBD.

[0217] Indirect ELISA experiments were used to capture microsomes expressing ACE2 and study the binding of the protein to its ligand (RBD). II captures ACE2 protein. Commercially available RBD (with His tag) and purified RBD and corresponding detection antibodies were used to determine the structure and binding efficiency of the protein.

[0218] By measuring the absorbance of the sample, a graph is constructed showing the absorbance of the sample and the dilution factor ( Fig.11A ). For ACE_cSTREP, the more concentrated the sample, the higher the absorbance measurement, thus more protein was captured. ACE_nSTREP shows very low measurements, close to 0, for all dilutions. The absorbance values ​​for ACE2_cSTREP are higher than for ACE2_nSTREP. The C-terminally tagged protein was captured on the plate at a higher concentration than the N-terminally tagged samples. Visualize the ELISA plate by adding detection reagent to the sample ( Fig. 11B). A brighter coloration can be clearly observed for ACE_nSTREP, whereas there is little / no blue for ACE_nSTREP. This demonstrates that microsomes expressing the protein with a C-terminal tag are captured. Furthermore, the coloration is a sign of effective binding between ACE2 and RBD.

[0219] When the experimental studies were performed, the purified RBD showed no coloration. The purified RBD was produced in a laboratory and the continuous thawing of the samples may have resulted in inactive protein and thus was the cause of the failure.

[0220] Ready-to-use The indirect ELISA experiment of the plate demonstrated that ACE2 was synthesized as expected, with its C-terminus outside the microsome, allowing capture of the microsome (Figure 11). The ACE2 protein was oriented with its C-terminus outside the microsome and its N-terminus inside. In addition, the addition of a commercially available His-tagged RBD followed by detection with an anti-His antibody revealed that ACE2 was correctly folded and effectively bound to its ligand. Due to the lack of a standard curve, the experiment could not be statistically analyzed. All results provided correspond to the qualitative data analysis obtained from the experiment.

[0221] 3. Discussion

[0222] The results of the proof-of-concept experiments showed that selected TPs such as PVR, TIGIT, and ACE2 play a key role in the CF system. Successful expression is possible in ELISA plates, which are derived from BY-2. II and ACE2 in microsomes. Utilizing TP for microsomal capture is a novel experimental design that allows the study of these TPs and further develops the basis for a high-throughput drug target screening platform. ACE2 was expressed, correctly folded, and showed efficient binding to its ligand, the RBD of SARS-CoV-1.

[0223] The results show that in order to It is feasible to express the TP of interest in the pALiCE02 vector by cloning the gene of interest into the pALiCE02 vector. Four different templates were designed for each TP, two of which were fused to a C-terminal tag and two to an N-terminal tag. Tags are often used to improve protein production or to confer new properties for characterizing the target protein. Currently, tags are also used to capture the microsomes and study the orientation of the protein. II constructs showed good expression. Although GST is frequently used for protein expression, affinity and solubility assays, and is the most common fusion tag in pull-down assays, no expression of any GST-tagged constructs was shown. One hypothesis that could explain the failure to express GST-tagged proteins may be related to the properties of the GST protein. GST tends to dimerize, which can cause aggregation of certain target proteins (especially oligomeric proteins). Therefore, GST is a poor choice for labeling oligomeric proteins. PVR, TIGIT and ACE2 are oligomeric proteins, and when fused to GST, they form large complexes or aggregates, which hinder protein expression.

[0224] Proof-of-concept experiments demonstrate that the present invention overcomes the greatest challenge in producing TPs that integrate into lipid bilayers and are synthesized in a correctly folded structure. In, which is the preferred BY-2 lysate according to the present invention, endogenous microsomes are derived from the ER and the Golgi apparatus. Currently, in order to target the TP to the microsomes, the melittin signal peptide (MSP) is fused to each construct. MSP is able to target the TP to the microsomes (Stech et al., 2014). Currently, TP is effectively targeted to the microsomes and embedded in the microsomal membrane, integrating the TP into the lipid bilayer. However, such an MSP is not mandatory, it depends on whether the TP of interest requires MSP to capture. In addition, ELISA experiments have shown that, consistent with this hypothesis, TP is synthesized with its C-terminus on the outside of the microsomal membrane and its N-terminus on the inside of the microsome. This allows the development of new technologies that can capture microsomes using C-terminally fused tags, and the development of binding and competition assays.

[0225] In particular, for therapeutic proteins, which can be identified as ligands in this article and thus as drug candidates, post-translational modifications (PTMs) such as glycosylation play a crucial role in protein-protein interactions. Achieving glycosylation that can produce functional proteins is another challenge in protein production. Glycosylation of glycoproteins PVR, TIGIT and ACE2 was tested. This is a CFPS system derived from BY-2 tobacco cells, so the PTMs may be different from the native state of TP. Based on the expected differences in protein size, Expression analysis in showed signs of possible glycosylation. In the case of both c / nSTREP constructs of ACE2, TIGIT_cSTREP and PVR_cSTREP constructs. In addition, ELISA studies demonstrated that the proteins were correctly folded, indicating that the correct PTMs occurred, as they could bind to their ligands (ACE2-RBD) or structural antibodies (PVR-anti-CD155). The presence of glycans was tested by treating the protein of interest with PNGase F, which showed the presence of N-glycans. Western blot analysis ( Figure 7 to 1 1) Confirmation of possible PTMs, such as glycosylation, on the expressed protein. Therefore, the present invention can produce efficient, correctly folded proteins without any additional steps (additional steps are necessary in prokaryotic systems (ECE)) to achieve glycosylation of TP.

[0226] Drug discovery and biochemical and structural studies of TPs require the expression of large amounts of protein. To date, in vivo systems have been the preferred system for protein production. Chinese hamster ovary (CHO) cells are the most popular and standardized eukaryotic cell line for protein production (Thoring et al., 2016). However, CHO cells have several limitations with regard to TP production. TPs are difficult proteins to express in living cells, and the production of large amounts of these proteins can have deleterious effects on cell growth and ultimately lead to cell death. Many different approaches to overcome these limitations have been tested on in vivo systems, which ultimately make them more time-consuming and expensive. Now, it has been shown for the first time that BY-2 lysate (commercially available as ) are efficient and can be an alternative to in vivo systems to express the TP of interest without encountering the limitations of in vivo systems. Proven to be a rapid TP production tool where the plasmid of interest and lysate are the only requirements for the experimental setup.

[0227] Treatment of microsomes with mild detergents demonstrated that membrane proteins did not dissociate from the lipid bilayer of the microsomes. Therefore, solubilization of TP is not possible and stronger detergents should be used. Currently, the most commonly used TP solubilization technique involves the use of detergents (Salipro Biotech AB, 2018), however, there are many disadvantages associated with them. For example, the use of detergents is associated with protein instability and poor suitability for structural and biophysical studies (Salipro Biotech AB, 2019). Another technology that has been used for more than 15 years is nanodiscs. Nanodiscs provide a lipid environment but do not bind tightly to transmembrane proteins, which limits their application. Known as New techniques allow reconstitution of purified TPs into a lipid environment and have recently been used for protein purification. is a good system for producing TP, but purification of TP cannot be achieved using mild detergents. It has been demonstrated that TP is stably embedded in the lipid bilayer of the microsomes according to the invention. Using the production system of the invention, preferably as an expression system for TP and combined it with A combination of technologies can be the future of TP characterization.

[0228] Until recently, CFPS systems have not been part of the drug discovery pipeline due to the many problems associated with this technology. However, this will change with the present invention, which provides an optimized and standardized system for high throughput screening (HTS) of ligands of TPs embedded in lipid bilayers. The current results demonstrate the potential of the HTS of the present invention as a TP-based drug target platform. According to the present invention, the expression, microsomal capture and interaction (binding) assays of TPs allow the establishment of a drug discovery platform based on Principle of the HTS drug screening platform of TP in. Competition assays using promising compounds such as monoclonal antibody (mAb) CR3022 (Tian et al., 2020) and chemicals such as corilagin (Yang et al., 2021), which effectively hinder the binding between ACE2-RBD, will be the next step in the development of HTS. In addition, lithyronine (Zhou et al., 2020), which has been shown to hinder the binding between TIGIT-PVR, can be used as a control for PVR-TIGIT assays and help find new potential compounds. CFPS system The combination of TP production in CF systems and the development of binding and competition assays will become the basis for HTS platforms. These platforms can transform the pharmaceutical industry and accelerate the entire drug discovery process. It is a simple and fast system that can replace the existing technology systems for TP production.

[0229] Example 2

[0230] Functional expression of complex peptide human epidermal growth factor (hEGF)

[0231] Human epidermal growth factor (hEGF) has been extensively studied for its potential ability to promote rapid healing of severe injuries such as cuts, burns, and diabetic ulcers. Despite its potential clinical value, hEGF is limited to the treatment of chronic diabetic ulcers due to its high production cost and poor stability. Mature hEGF, a complex peptide of 53 amino acids with three intramolecular disulfide bonds, was chosen as another model protein with one transmembrane domain to challenge the BY-2 system as an expression host for bioactive hEGF.

[0232] To examine the interaction (currently binding) of hEGF, surface plasmon resonance (SPR) was used as spectroscopic method.

[0233] hEGF gene with N-terminal Strep-II tag was cloned into pALiCE01 and pALiCE02 to evaluate cytosolic and microsomal expression ( Fig.15 hEGF expression was observed in microsomes, but not in the cytoplasm, indicating that targeting microsomes is hEGF production is required. A single-step streptavidin affinity purification is sufficient to extract highly pure soluble hEGF ( Fig.15 Binding of the purified protein to the homologous epidermal growth factor receptor (EGFR) was used as a surrogate measure of biological activity and analyzed by SPR ( Fig.12 ). hEGF expressed in BYL showed comparable or improved binding kinetics compared to a commercially available E. coli expressed hEGF reference product, indicating correct folding and disulfide bond formation. Prokaryotic CFPS systems require extensive engineering to achieve disulfide bond formation, which can be circumvented by using BYL microsomes.

[0234] Example 3

[0235] Folding, disulfide bonding, and activity of complex multi-subunit proteins targeted to microsomes

[0236] We have demonstrated that BYL can produce functional eYFP and GOx at various scales, and that functional proteins such as multi-transmembrane GPCRs and small soluble peptides are also possible. Protein function is intrinsically linked to correct folding, but further molecular characterization is needed to demonstrate that microsomes from this eukaryotic CFPS system can produce recombinant proteins of the same quality as cells with batch-to-batch consistency.

[0237] Therefore, a different set of model proteins were selected to further investigate the folding and post-translational modification capabilities, namely: the GOx enzyme, which is a homodimer composed of 80 kDa monomers covalently linked by disulfide bonds and each with eight N-glycosylation sites; the receptor binding domain (RBD) of the spike protein from the SARS-CoV-2 virus, which is a 52 kDa monomer with four intramolecular disulfide bonds and two N-glycosylation sites; and the therapeutic monoclonal antibody adalimumab ( ), which is a complex heterotetramer of 150 kDa, composed of two heavy chains and two light chains, containing a total of two N-glycosylation sites, 12 intramolecular and 4 intermolecular disulfide bonds.

[0238] Adalimumab heavy and light chain genes were cloned directly into the pALiCE02 vector for targeting microsomes, while RBD and GOx genes were similarly prepared using an additional N-terminal Strep-II tag. Triplicate, independent batches of BYL were used to express proteins in 5 mL reactions, and the resulting microsomes were processed by dodecyl maltoside detergent treatment for protein release and subsequent protein A or streptavidin affinity purification. For adalimumab, a 1:1 ratio of heavy and light chain pALiCE02 plasmids was sufficient to produce the expected 150 kDa ratio of full-length IgG molecules. Multiple heavy and light chain assemblies were also visible in non-reducing SDS-PAGE gels, but analytical size exclusion chromatography resolved a single major peak for all three batches. The functional binding activity of these samples to the TNFα ligand and Fc-γ receptor CD64 was evaluated by SPR and compared with commercially available CHO cell-expressed adalimumab reference products (BYL-mAb and CHO-mAb, respectively; Fig.15 ).

[0239] Assay setup:

[0240] CM5 S series chips (Biacore / Cytiva) were functionalized with recombinant protein A (P7837 / lot SLBT1697, Sigma) using an EDC / NHS coupling kit (Biacore / Cytiva BR-1000-50).

[0241] Conditions: temperature 25°C, flow rate 30 μl / min, buffer: HBS-EP, pH 7.4.

[0242] Reagents:

[0243] huCD64 / human FcRI, (R&D Systems 1257FC / lot MOM2119041, 50 kD, reference IgG Adalimumab (Humira) (ABIN5668145), human TNFa, Lenio derived Humira (Alice), 3 lots.

[0244] Dynamics run:

[0245] Serial dilutions of ligands (CD64 and TNFa) starting at 30 nM CD64 and 60 nM TNFa.

[0246] Capture antibody at around 170 RU.

[0247] The association rate constant is 180s and the dissociation rate constant is 420s.

[0248] Two buffer blanks were distributed in each series.

[0249] result:

[0250] For TNFα binding, BYL-mAb showed slightly stronger affinity than CHO-mAb, with an average KD(M) value of 1.34×10 -10 , compared to 1.85×10 -10 In contrast, BYL-mAb showed weaker binding to CD64 receptor (3.53×10 -10 Compared with 1.58×10 -10 ). Considering that the mechanism of action of adalimumab is neutralization of free TNFα, this enhanced ligand binding and weaker receptor binding may combine to improve therapeutic efficacy when this mAb is produced in BYL compared to mammalian cell systems. The data are summarized as follows, Fig.15 As shown:

[0251] picture Determination ka(1 / Ms) kd(1 / s) KD(M) A Humira CHO TNFα 4.53E+05 8.37E-05 1.85E-10 B Humira CF DAK TNFα 4.07E+05 5.01E-05 1.23E-10 C Humira CF DB3 TNFα 4.78E+05 5.83E-05 1.22E-10 D Humira CF CHJ TNFα 4.00E+05 6.24E-05 1.56E-10 E Humira CHO CD64 9.59E+05 1.52E-04 1.58E-10 F Humira DAK CD64 7.87E+05 2.72E-04 3.46E-10 G Humira DB3 CD64 6.86E+05 2.52E-04 3.67E-10 H Humira CHJ CD64 7.58E+05 2.63E-04 3.46E-10

[0252]

[0253]

[0254] Example 4

[0255] exist N-linked glycosylation of glycoproteins expressed in

[0256] Previous reports on glycoproteins produced in eukaryotic CFPS have demonstrated the presence of N-glycosylation using simple SDS-PAGE mobility shifts following PNGase F-mediated deglycosylation. However, detailed analysis of the composition of these N-glycans has not been provided due to the unavailability of sample quantities required for mass spectrometry characterization. The successful scale-up of the BYL system provided sufficient sample for this more in-depth examination, and thus we analyzed the N-glycan structures of three GOx, RBD, and adalimumab formulations that were also used for disulfide bond characterization.

[0257] PNGase F cleavage of N-glycans was followed by liquid chromatography electrospray ionization tandem mass spectrometry identification and structural assignment (LC-ESI-MS / MS; Figure 13). This study revealed high N-glycosylation occupancy and reproducibility between batches, regardless of the protein model. The N-glycan profiles were largely consistent across all N-glycosylation sites and showed a dominant Man8 species. Similar to recombinant proteins produced across Nicotiana species, plant-specific glycosylation signatures of α1,3-fucosylation and β1,2-xylosylation were also observed in some N-glycosylation sites, particularly in the RBD protein. One glycosylation site of the GOx protein was not detected and therefore not characterized. This site is located in a weakly charged peptide of approximately 50 amino acids, which presumably hinders detection within the mass range of the MS instrument.

[0258] Example 5

[0259] Bioactive expression of the vaccine subunit candidate SARS-CoV-2 spike protein receptor binding domain

[0260] Here we show that the BYL system is able to produce recombinant SARS-CoV-2 spike protein RBD with appropriate disulfide bonds and consistent N-glycosylation from batch to batch within 48 hours. This protein is considered an important target for the development of SARS subunit vaccines. In fact, most of the potent neutralizing antibodies against SARS-CoV-2 target the RBD(REF). Reports also suggest that the intact S1 subunit of the spike may be a more effective vaccine as it induces better levels of IgG and IgA antibodies compared to RBD(REF) alone. To challenge this concept, we expressed the RBD and this S1 subunit in the pALiCE02 vector with different HaloTag and Strep-II tag conformations.

[0261] For the RBD with a Strep-II tag at the N-terminus, SPR was used to preliminarily confirm the functional binding of the RBD to the human receptor angiotensin-converting enzyme 2 (hACE2) (Figure). The analysis showed that the RBD produced in ALiCE bound to the immobilized hACE2 receptor with a higher affinity (Kd=32nM) than the RBD protein produced in mammalian and human cells (Figure). The reactivity of the antigen to two commercially available antibodies was then evaluated, selected based on the recognition of linear or conformational epitopes. We observed that both antibodies had similar reactivity to recombinantly expressed RBD and S1 from BYL CFPS and HEK293 cells. Importantly, the reactivity to S1 was weaker than that to RBD. Finally, to demonstrate the true biological relevance of the proteins produced in BYL, we found that the antigen samples had significant serological reactivity ( Fig.14). An adaptive ELISA workflow utilizing SARS-CoV-2 patient serum antibodies demonstrated that the RBD produced in BYL was directly comparable to that produced in HEK293 cells. Interestingly, higher reactivity was observed for COVID-19 patient samples, but lower reactivity for samples collected from control subjects prior to 2019, and this split was less significant for the S1 antigen. Different C-terminal Strep-II tag and HaloTag configurations did not affect binding, indicating that correct RBD folding is independent of fusion protein addition. This experiment suggests that the use of BYL CFPS-produced RBD for therapeutic purposes and scaled-up expression for animal studies are goals for future studies.

[0262] Example 6

[0263] 1. Expression of CD20 by BY-2 Lysate

[0264] CD20 is a well-known antigen expressed by B-cell lymphocytes throughout their development, from the pre-B cell stage to their final differentiation into plasma cells. CD20 is a transmembrane protein with four transmembrane domains, two extracellular loops (one of approximately 44 amino acids, and a smaller one of approximately 7 amino acids), and intracellular N-terminal and C-terminal regions. It is thought to exist on the cell surface as a tetramer containing a disulfide bond at positions 167-183 but without glycosylated side chains. Within the described region, different epitopes of CD20 are known to be expressed by antibodies, as described by Klein et al. (2013). Figure 1 The core epitope residues (underlined) of the most relevant epitope region (positions 167 to 183) are shown below:

[0265]

[0266] A detailed description of the above epitopes is provided in Klein et al., pages 24-29, and is incorporated herein by reference. The above antibodies are commercially available. Those skilled in the art will be aware of additional suitable anti-CD20 antibodies.

[0267] 1.1 Plasmid design

[0268] The sequence encoding full-length CD20 (297 amino acids; 33.1 kDA) has been cloned into the 3175 bp plasmid pIB0405 ( Fig.17 ) and has a StrepII tag (~1 kDa) at the C-terminus ( Fig.17 ). In order to translocate into microsomes during expression, the sequence encoding the melittin signal peptide MSP (~2.5 kDa) was also cloned into the plasmid. In addition, the universal sequences encoding the T7 promoter, AmpR promoter, AmpR and Ori were also integrated.

[0269] Amino acid sequence of CD20

[0270]

[0271] 1.2 Expression of BY-2 lysate

[0272] Protein expression and Western blot analysis were performed as described in Example 1, Section 1.3 above.

[0273] 1.3 Microsome capture and protein binding assay (ELISA)

[0274] The capture of CD20 embedded in microsomes has been performed as described above in Example 1, Section 1.5. Currently, CD20 has been captured to the surface of a microtiter plate via its C-terminus, as described above, such as Figure 1 shown.

[0275] 1.4 Processing of CD20-containing microsomes

[0276] Treatment with DDM was performed as described above in Example 1, Section 1.4, to obtain microsomal fragments ( Figure 1 ).

[0277] 1.5 Ligand binding assay

[0278] After capture, treatment and washing, ligand was added as described above. After incubation and washing (see Example 1, Section 1.5) anti-ligand antibody containing HRP (see above examples) was added and binding was detected by ELISA as described above.

[0279] 2. Results

[0280] Expression of CD20 with C-tag Fig.18 For ELISA antibodies It targets the epitope of rituximab (see above) and has been used to detect CD20 in microtiter plates. Different dilutions (Dil) of BY-2 lysate were used after protein synthesis: 5x, 10x, 20x, 40x and 80x dilutions and at different concentrations of Incubate under. Fig.19 It was shown that even at 80-fold dilution, While significant binding to CD20 was observed, sufficient CD20 was also detected embedded in the lipid bilayer of microsomes captured to the surface of microtiter plate wells.

[0281] Thus, the results demonstrate that the assay according to the invention is feasible for complex proteins containing multiple transmembrane domains, such as CD20. CD20 can be expressed in its correct orientation and structure by the BY-2 lysate according to the invention, with the transmembrane domains embedded in the lipid bilayer of the microsomes. Thus, The related epitope "NPSE" (such as Klein et al. Figure 1 ) were correctly expressed in the correct conformation and were positive for The interaction of the antigen binding domain of The interaction and binding of CD20 also demonstrated that treatment with DDM was strong enough to disrupt the microsomes, yet mild enough not to disrupt the transmembrane domain and epitopes of CD20.

[0282] Finally, this experiment demonstrated the feasibility of the assay for antigen-antibody screening according to the invention.

[0283] Example 7

[0284] Example 7 uses the same methods and materials as Example 6. However, the captured microsomal fragments are incubated in parallel or simultaneously with different anti-CD20 antibody panels (defined herein as "analyte panels"). Such panels include the above-mentioned antibodies ofatumumab, LT20, 2H7, rituximab, obinutuzumab GA101, B1 and utoximab. Multiplexed assays according to the present invention are performed using a panel of different antibodies using microtiter plates, microfluidic devices or SPR consumables.

[0285] References

[0286] Buntru,M.,Vogel,S.,Spiegel,H.,&Schillberg,S.(2014).Tobacco BY-2cell-free lysate:an alternative and highly-productive plant-based in vitrotranslation system.BMC biotechnology,14(1),1-11.

[0287] Buntru,M.,Vogel,S.,Stoff,K.,Spiegel,H.,&Schillberg,S.(2015).Aversatile coupled cell-free transcription–translation system based on tobaccoBY-2cell lysates.Biotechnology and bioengineering,112(5),867-878.

[0288] Gupta,R.,&Brunak,S,.(2002)Prediction of glycosylation across thehuman proteome and the correlation to protein function.Pac SympBiocomput.310-22.PMID:11928486Henrich,E.,Hein,C., V.,&Bernhard,F.(2015).Membrane protein production in Escherichia coli cell-free lysates.FEBSletters,589(15),1713-1722.

[0289] Khambhati,K.,Bhattacharjee,G.,Gohil,N.,Braddick,D.,Kulkarni,V.,&Singh,V.(2019).Exploring the potential of cell-free protein synthesis forextending the abilities of biological systems.Frontiers in bioengineering andbiotechnology,7,248.

[0290] Klein,C.,Lammens,A., W.,Georges,G.,Schwaiger,M., E.,Hopfner,K-P, P.,and Niederfellner,G.,(2013)Epitope interactions ofmonoclonal antibodies targeting CD20 and their relationship to functionalproperties,mAbs 5:1,22–33;January / February 2013; 2013LandesBioscienceSachse,R.,Wüstenhagen,D., M.,Gerrits,M.,Bier,F.F.,&Kubick,S.(2013).Synthesis of membrane proteins in eukaryotic cell-freesystems.Engineering in Life Sciences,13(1),39-48.

[0291] Stech,M.,Hust,M.,Schulze,C.,Dübel,S.,&Kubick,S.(2014).Cell-freeeukaryotic systems for the production,engineering,and modification of scFvantibody fragments.Engineering in life sciences,14(4),387-398.

[0292] Tian,X.,Li,C.,Huang,A.,Xia,S.,Lu,S.,Shi,Z.,...&Ying,T.(2020).Potentbinding of 2019novel coronavirus spike protein by a SARS coronavirus-specifichuman monoclonal antibody.Emerging microbes&infections,9(1),382-385.

[0293] Yang,L.J.,Chen,R.H.,Hamdoun,S.,Coghi,P.,Ng,J.P.,Zhang,D.W.,...&Wong,V.K.W.(2021).Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2binding.Phytomedicine,87,153591.

[0294] Zhou,X.,Du,J.,Wang,H.,Chen,C.,Jiao,L.,Cheng,X.,...&Gao,Y.(2020).Repositioningliothyronine for cancer immunotherapy by blocking theinteraction of immune checkpointTIGIT / PVR.Cell Communication and Signaling,18(1),1-14。

Claims

1. High throughput screening of at least one ligand of at least one transmembrane protein (TP) of interest, said transmembrane protein (TP) being embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), said high throughput screening comprising the following steps: - providing at least one endogenous microsome comprising at least one transmembrane protein (TP) embedded in a lipid bilayer, or at least one endogenous microsomal fragment comprising at least one transmembrane protein (TP) embedded in a lipid bilayer, - providing at least one analyte of interest, - contacting at least one TP with at least one analyte, and - detecting an interaction between said at least one analyte and said at least one TP.

2. Screening according to claim 1, wherein the at least one microsome or microsome fragment is derived from the genus Nicotiana of the family Solanaceae.

3. Screening according to claim 1 or 2, wherein a step of capturing at least one TP embedded in the lipid bilayer of the microsomes or microsome fragments to a biologically inactive surface is performed.

4. The screen according to any one of claims 1 to 3, wherein at least one TP embedded in the lipid bilayer of the endogenous microsomes and / or the endogenous microsomal fragments is captured onto a biologically inactive surface.

5. The screening according to any one of claims 1 to 4, wherein the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of devices including microtiter plates, microfluidic devices, microarrays, microchips, and / or the biologically inactive surface is any other surface suitable for capturing at least one TP of interest.

6. The screen according to any one of claims 1 to 5, wherein the at least one TP comprises at least one tag at the C-terminus and / or N-terminus.

7. The screen of any one of claims 1 to 6, wherein the at least one analyte comprises a small molecule, a peptide, a polypeptide, a soluble protein, a membrane protein, a protein complex, or any combination thereof.

8. The screen according to any one of claims 1 to 7, wherein the screen is a multiplex high throughput screen of at least one ligand of at least one TP of interest, two or more analytes provided simultaneously in two or more reaction zones, and / or an analyte combination of two or more analytes provided simultaneously in two or more reaction zones.

9. The screen according to any one of claims 1 to 8, wherein the interaction between the at least one TP and the at least one analyte is detected by immunoassay, spectrometry, particle size measurement, magnetic measurement and / or optical measurement.

10. The screening according to any one of claims 1 to 9, wherein at least one endogenous microsome or endogenous microsome fragment derived from plant endoplasmic reticulum is derived from a cell lysate of a plant of the genus Nicotiana of the family Solanaceae.

11. Screening according to any one of claims 1 to 10, wherein the lysate is from the BY-2 cell line of Nicotiana tabacum.

12. Cell-free production of at least one transmembrane protein (TP) of interest, said transmembrane protein (TP) being embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), said cell-free production comprising the following steps: - providing a lysate from a plant comprising at least one endogenous microsome, - providing at least one template encoding at least one transmembrane protein (TP), - incubating at least one lysate with at least one template, - expressing said at least one transmembrane protein (TP), - coupled transcriptional and co-translational translocation of at least one expressed TP into at least one microsome, and - obtaining at least one transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome, and - Optionally, treating at least one endogenous microsome comprising at least one TP embedded in a lipid bilayer to obtain microsomal fragments comprising at least one TP embedded in a lipid bilayer.

13. The method according to claim 12, wherein a step of capturing at least one TP embedded in the lipid bilayer of the microsomes on at least one biologically inactive surface is performed.

14. The method according to claim 13, wherein a capture step is performed and at least one captured microsome is processed to obtain microsomal fragments, wherein the at least one TP remains embedded in the lipid bilayer.

15. The method according to claim 13, wherein at least one free microsome is treated to obtain free microsome fragments, wherein the at least one TP remains embedded in the lipid bilayer, and the at least one TP embedded in the lipid bilayer of the microsome is captured on the at least one biologically inactive surface.

16. The method according to any one of claims 13 to 15, wherein the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of devices including microtiter plates, microfluidic devices, microarrays, microchips, and / or the biologically inactive surface is any other surface suitable for capturing at least one TP of interest.

17. The method according to any one of claims 12 to 16, wherein the at least one microsome or microsome fragment is derived from the genus Nicotiana of the family Solanaceae.

18. Endogenous microsomes or at least one endogenous microsomal fragment derived from the endoplasmic reticulum (ER) of a plant, said endogenous microsomes or endogenous microsomal fragments comprising at least one transmembrane protein (TP) of interest embedded in a lipid bilayer.

19. A biologically inactive surface comprising at least one captured TP of interest embedded in the lipid bilayer of at least one endogenous microsome or at least one endogenous microsomal fragment derived from the plant endoplasmic reticulum (ER), wherein the biologically inactive surface is a surface of a consumable.

20. A kit for high throughput screening of at least one ligand of at least one transmembrane protein (TP) of interest, said transmembrane protein (TP) of interest being embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), said endogenous microsome preferably being derived from a plant of the genus Nicotiana of the family Solanaceae, said kit comprising: - a plant cell lysate comprising endogenous microsomes of endoplasmic reticulum (ER) origin, preferably from a plant of the genus Nicotiana of the family Solanaceae, - at least one vector encoding a known TP as a positive control, - at least one vector encoding at least one template for at least one TP of interest, -optionally, additional excipients, - optionally, at least one reagent for detecting the interaction between said at least one analyte and said at least one TP, - optionally, at least one agent for disrupting at least one microsome, and - Optionally, at least one analyte or a panel of two or more analytes.

21. The kit according to claim 20, wherein the kit further comprises a consumable, the consumable comprising, consisting of or coated with a biologically inactive surface, the biologically inactive surface being used to capture the at least one TP embedded in the lipid bilayer of the microsome to the biologically inactive surface.

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