High-Throughput Screening of Ligands for Transmembrane Proteins

Abstract

The present invention relates to a high-throughput screening of at least one ligand 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), comprising the steps of providing at least one endogenous microsome containing at least one lipid bilayer-embedded TP, or at least one endogenous microsome fragment containing at least one lipid bilayer-embedded TP; providing at least one analyte of interest; contacting at least one TP with at least one analyte; and detecting an interaction between at least one analyte and at least one TP. The screening is suitable for the rapid and reproducible identification of high-multiplex grades of TPs and analytes, as well as ligands as potential drug candidates. Further, the present invention provides all essential products, consumables and kits for use in 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 embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), comprising providing at least one endogenous microsome containing at least one lipid bilayer-embedded TP, or at least one endogenous microsome fragment containing at least one lipid bilayer-embedded TP; providing at least one analyte of interest; contacting at least one TP with at least one analyte; and detecting an interaction between at least one analyte and at least one TP. The screening is suitable for the rapid and reproducible identification of high multiplex-grade TPs and analytes, as well as ligands as potential drug candidates. Furthermore, the present invention provides any essential products, consumables and kits for use in high-throughput screening. Accordingly, the present invention relates to the technical field of biochemical and biotechnological drug candidate screening of TPs involved in signal transduction that cause or are involved in diseases.

[0002] Here, as a proof of concept, a cell-free (CF) system based on lysates from Nicotiana tabacum BY-2 cells with endogenous microsomes, called ALiCE®, was used for TP production. The pALiCE02 vector (LenioBio GmbH) was used to express three well-characterized TPs: hEGF, PVR, TIGIT, and ACE2 for use in ALiCE®. Additionally, C-terminally tagged TPs were used for the proof of concept of the HTS according to the present invention, and the microsomes were captured. Once the TPs were captured, interaction (binding) assays of the TPs and their respective ligands were performed. ALiCE® was proven to be a successful CF system for the production of the type I transmembrane proteins PVR, TIGIT, and ACE2. The expression of these proteins contributed to the design of assays to test the orientation and structure of the proteins. Furthermore, correctly folded PVR and ACE2, as well as binding assays, confirmed that ACE2 could bind to the RBD. From the TP expression in ALiCE® and the microsome capture assay, the HTS of the present invention for a high-throughput membrane protein drug target screening platform is proven.

[0003] Drug discovery is a difficult, expensive, and time-consuming process. The average time to develop a new drug is 10 - 15 years and can cost up to 800 million euros. The SARS-CoV-2 pandemic has shown the need to speed up this process for a rapid response to the threat of future pandemics. Target-based drug discovery is the most common strategy for developing new drugs. Conventionally, in the most adhered-to norms, one drug is set for one target, but the need for a platform that enables screening of multiple drugs for one target has become essential. Ligand screening for drug discovery is important for the development of new pharmaceuticals. Currently, there are two main approaches for drug discovery, which are target-based screening or phenotypic screening. Phenotypic screening is based on trying to identify molecules that can change a cell's phenotype towards a desired outcome, whereas target-based screening is based on the use of recombinant techniques to find targets that play some role in a disease. Since the 1990s, high-throughput screening (HTS), which is target-directed screening, has been increasingly used in new drug discovery by most pharmaceutical companies. For example, HTS is frequently used in the research of potential drugs for neurodegenerative diseases. HTS is based on screening multiple molecules / compounds or "hits" against one known target. The increasing use of HTS is replacing the commonly used slow "trial and error" technique to establish a faster method. However, the throughput of the prior art screening process is limited, and there is a need to develop a next-generation drug screening platform with even higher throughput to increase the speed of drug discovery. The main feature of HTS is that the drug discovery target is known and tests using multiple molecules that act on the target can be analyzed as potential drugs. A drug target can be a small molecule or any type of protein that plays some role in a specific disease process and can be addressed by a drug to treat this disease.For this reason, the pharmaceutical industry relies on the selective binding of "hits" to targets or specific interactions that cause the modulation of targets.

[0004] However, high-throughput screening (HTS), especially for membrane proteins (MPs) that make up the majority of drug targets but are under-evaluated compared to cytosolic proteins, exhibits several limitations that prevent it from reaching its full potential. Currently, more than 60% of existing drug targets are membrane proteins (MPs). MPs are involved in essential physiological mechanisms that ensure the structural and functional integrity of cells. MPs are important components for many processes such as cell recognition, immune response, signal transduction, and molecular transport. The use of MPs poses a problem for drug development because expression, post-translational modification (PTM), protein folding, and stabilization during HTS are difficult.

[0005] MPs make up 30% of the total proteins encoded by humans, but they have not been well-characterized. MPs are difficult to produce in living eukaryotic cells. One of the main problems in research is that overexpression of MPs strongly affects the health of eukaryotic cells, thereby preventing the production of correctly folded and physiologically functional MPs. The limitations in the production of this type of protein delay the evaluation of the functional and structural properties of MPs. Furthermore, prokaryotic cells are not suitable for the production of eukaryotic MPs, and the expression of these proteins causes the formation of protein aggregates that need to be solubilized after expression (Sachse et al., 2013). Nevertheless, there are other protein expression platforms that enable the production of many types of proteins in a simple way without relying on the structural integrity of cells (Khambhati et al., 2019). These platforms are based on cell-free protein synthesis (CFPS).

[0006] Cell-free protein synthesis (CFPS) systems are based on crude lysates containing the machinery necessary for translation, protein folding, and energy metabolism (Buntru et al., 2014). CFPS is a promising solution for protein production that enables shortening of the process time and reduction of protein hydrolysis. One of the main advantages of CFPS is the ability to express cytotoxic proteins (Khambhati et al., 2019), which mainly contributes to the synthesis of proteins that are difficult to express, especially TPs. There are many variants of CFPS that are composed of recombinant transcription-translation machinery or are derived from prokaryotic or eukaryotic cell lysates. The most commonly used lysates are Escherichia coli extract (ECE), rabbit reticulocyte lysate (RRL), Chinese hamster ovary (CHO) lysate, wheat germ (WGE) lysate, and insect cell extract (ICE). However, the main CFPS platforms currently in use are not completely ideal, with overall low protein production yields, slow production times, unavailable or inappropriate protein modifications, and being unsuitable or not fully suitable 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, which showed increased protein yields over a shortened period. However, high-throughput screening (HTS) for accessible and functional TP drug candidates is still not available.

[0007] The complexity and barriers posed by the cell membrane cause many difficulties such as experiments that are difficult to standardize, incompatibility problems, and variability. To overcome the challenges in the biosynthesis of TPs and the provision of TPs, in the prior art, synthetic lipids in combination with living cells or artificial components, such as prokaryotic systems (e.g., Escherichia coli extract (ECE)), have been used to achieve correctly folded, modified, and stabilized TPs. Even by doing so, the above-mentioned drawbacks cannot be overcome, and biosynthesis becomes more complex, time-consuming, and costly, reducing the yield of the desired TPs.

[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 an important tool suitable for functioning without using living cells and without using artificial components for synthesizing correctly folded, modified, and stabilized TP. By providing TP embedded within the lipid bilayer of endogenous microsomes, it becomes possible to provide the HTS platform of the present invention for ligands for TP as drug candidates.

[0009] Accordingly, an object of the present invention is to provide a method for synthesizing a TP embedded in a lipid bilayer derived from endogenous microsomes, and in particular, to provide a TP embedded in the lipid bilayer of interest for screening a ligand as a drug candidate. Another object is to provide a stable and correctly folded TP of interest embedded in a lipid bilayer derived from endogenous microsomes. Further, it is an object to provide an endogenous microsome or a fragment thereof containing at least one TP embedded in the lipid bilayer. Another object of the present invention is a TP embedded in the lipid bilayer captured on the surface for subsequent screening and detection methods of interaction, but still a mobile TP, particularly in the medium of a desired detection method, for example, in a liquid phase, a gel phase or a gas phase. Alternatively, another object is to provide a TP captured and immobilized on a device, for example, on a microtiter plate, a microchip, a microarray, a microfluidic device, or other consumables for detection purposes. Accordingly, another object is to provide a suitable consumable for a desired detection method, comprising the TP of interest embedded in the lipid bilayer and captured on the consumable. It is an object to provide a flexible and adaptable kit for a desired screening design by providing a high-yield lipid bilayer-embedded TP. Another object of the present invention is to provide an optimized and standardized system for high-throughput screening (HTS) of ligands for a TP embedded in a lipid bilayer, preferably an optimized and standardized system for drug screening. It is an object to enable, improve, standardize and accelerate HTS of drug candidates for TPs related to diseases or related to the treatment of diseases, for example, by antibodies, chimeric antigen receptors, etc. To that end, it is an object to provide an HTS platform that can be easily adapted to any TP of interest.

[0010] Another object is to provide a method in which gentle conditions during cell lysate preparation provide intact subcellular membrane structures (microsomes) derived from the ER and / or Golgi. These membrane vesicles are an important prerequisite for the production of functionally active TP by subsequent PTM. Thus, another object of the present invention is to provide a method in which TP is expressed, folded, modified, and stabilized within microsomes. PTM strongly affects the physicochemical properties of the nascent polypeptide chain and thus affects protein folding, stability, and activity. The object is to provide an isolated endogenous membrane structure carrying the TP of interest.

[0011] The cell-free system of the present invention overcomes the aforementioned drawbacks of the prior art and provides rapid access to the TP of interest, a low reaction volume, and a short reaction time, thereby enabling high-throughput TP expression strategies and high-throughput screening using TP. The method of the present invention provides full-length TP with correct folding and incorporates co-translational and post-translational modifications (PTM). Another advantage of the present invention is that there is no need to add artificial membrane structures or exogenous microsomes from other species. The advantage of the present invention is to provide homogeneous CFPS for TP. The methods, kits, and assays according to the present invention are suitable for use in the expression and testing of high-level libraries, multiplex designs, and enable complex tests and high-throughput experiments to function fast enough, quickly, and at low cost. The details of the present invention will be described below.

[0012] A first aspect of the present invention is a high-throughput screening (abbreviated as HTS) of at least one ligand 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), particularly the ER and Golgi, · Providing at least one endogenous microsome as defined herein, or at least one fragment of an endogenous microsome, comprising at least one lipid bilayer-embedded transmembrane protein (TP); · Providing at least one analyte of interest as defined herein; · Contacting at least one TP with at least one analyte; · Detecting an interaction between at least one analyte and at least one TP, which is a high-throughput screening.

[0013] Preferably, in one embodiment of the high-throughput screening according to the present invention, a step of capturing at least one TP embedded in the lipid bilayer of a microsome or a microsome fragment on a biologically inert surface (as defined herein) is performed. The biologically inert surface is any surface defined herein with respect to capture.

[0014] The advantage of endogenous microsomes as defined herein, particularly those derived from the plant endoplasmic reticulum (ER), especially ER and Golgi, is that such microsomes provide a natural environment and an intact translocon mechanism for proper embedding and folding of TP, thus meeting all requirements necessary for proper folding of TP. Most preferably, the endogenous microsomes according to the present invention are derived from cultured tobacco-BY-2 cells. In the HTS of the present invention, desired activity assays and binding assays are possible to identify ligands that regulate the activity of TP or to identify binding partners of TP.

[0015] In one embodiment, the HTS of the present invention further includes a step of treating (destroying or crushing) at least one microsome, in one embodiment a free microsome, to obtain microsome fragments, in one embodiment free microsome fragments, wherein at least one TP remains embedded in the lipid bilayer derived from the endogenous microsome. "Free microsome" within the scope of the present invention is an endogenous microsome containing at least one TP embedded in the lipid bilayer of the microsome that is not captured on the biologically inactive surface via the TP.

[0016] "Processing" a microsome comprising at least one TP embedded in a lipid bilayer according to the present invention means that a complete 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 is subjected to chemical and / or physical forces for disrupting the lipid bilayer. Chemical and / or physical forces are applied that are strong enough to rupture (disrupt) the lipid bilayer but are as small as possible so as not to affect the conformation and activity of at least one TP. Chemical forces include treating with detergents. Detergents include maltoside, thiomaltoside, polyoxyethylene, polyoxyethylene glycol, glucoside, glucamidopropyl, thioglucopyranoside, glucopyranoside, glucamide, methylglucamide, hydroxyethylsulfoxide, maltose-NG family of detergents such as lauryl maltose neopentyl glycol (LMNG), amine oxide, n-dodecyl β-D-maltoside (DDM), n-decyl-β-D-maltopyranoside (DM), n-dodecyl-α-D-maltopyranoside (α-DDM), octyl-beta-glucoside (OG), and lauryldimethylamine-N-oxide (LDAO), CYMAL, detergent GDN-101, OGNG, LDAO, UDAO, and APO109, Tween 20, and Fos-choline detergents such as fos-choline U10-11, cyclophos-6, 12, 13, 14, 15 and 16. Physical forces include shear forces (generated by shear force generating devices, different mixers), ultra-wave, sonication, ultrasonic waves. Any treatment methods can be combined. Thus, the treatment is a chemical treatment (e.g., detergent) and / or physical or mechanical treatment (e.g., shear force, ultrasonic wave) in which forces are exerted on the microsome that are sufficient to disrupt the microsome but do not damage the TP or dissolve the TP from the lipid bilayer.

[0017] Optionally, excipients may be used to stabilize the microsomal fragments according to the present invention. The stabilizer or stabilizing excipient is a molecule suitable for stabilizing a microsome or microsomal fragment comprising at least one TP embedded in the lipid bilayer of said microsome or microsomal fragment. The stabilizer ensures that said at least one TP remains folded and embedded within the lipid bilayer and that the binding site or epitope of interest remains accessible to the analyte. Stabilizers within the scope of the present invention include lipid bilayer and / or protein stabilizers 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. Thus, in another embodiment of the present invention, HTS may include the step of adding at least one stabilizing excipient, preferably a lipid bilayer stabilizing excipient, to the microsomal fragment after a processing step which may preferably be before or after capture. "Processing" as described herein does not weaken or disrupt the captured state of at least one TP. However, if the selected capture strategy is not strong enough to withstand the available or preferred processing methods, it may be desirable to perform the processing before capture.

[0018] The step of processing at least one microsome may be performed (temporally and spatially) independently of HTS and may be a step of the method (biosynthesis) for producing said TP described herein according to the present invention, or a step of HTS according to the present invention. It may be desirable to produce at least one TP within the microsome to preserve intact microsomes containing at least one TP under suitable conditions to allow individual decisions regarding the timing and design of HTS. For example, HTS with intact microsomes at any time or with their disruption at any time to provide microsomal fragments immediately prior to HTS. In this embodiment, it may be desirable or even necessary to preserve the microsome or fragment thereof as defined herein. Optionally, a stabilizing excipient may be added.

[0019] In a preferred embodiment of the HTS according to the present invention, at least one microsome or microsome fragment is derived from the genus Nicotiana of the Solanaceae family. Preferably from Nicotiana tabacum, more preferably from the BY-2 cell line.

[0020] Nicotiana is a genus of herbs and shrubs of the Solanaceae family. To date, at least 67 species are known and are generally called tobacco plants. Some of them, mainly N. tabacum, are cultivated for the tobacco leaves for the production of tobacco products. Generally, any species that can be cultured or from which a cell line can be isolated is available within the scope of the present invention. In particular, for the provision of endogenous microsomes according to the present invention. N. tabacum, or a cell line derived therefrom, is a preferred source for microsomes or microsome fragments, more preferably the BY-2 cell line derived from N. tabacum. In another embodiment of the HTS according to the present invention, at least one endogenous microsome or endogenous microsome fragment derived from the plant endoplasmic reticulum (ER), particularly ER and Golgi, is derived from a cell lysate of a plant of the genus Nicotiana of the Solanaceae family, preferably a cell lysate of N. tabacum, more preferably a cell lysate of the BY-2 cell line derived from N. tabacum.

[0021] To overcome the drawbacks presented by most eukaryotic CFPS systems, the BY-2 cell line and BY-2 cell-free protein synthesis (CFPS) were developed. Compared to the lysate of wheat germ (WGE) for protein production, BY-2 shows a faster lysate production with increased translation activity and a higher potential for scale-up (Buntru et al., 2014). It has been shown 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 manufacturing. BY-2 cells contain microsomes derived from the endoplasmic reticulum (ER) and Golgi of plant cells. By applying appropriate methods, the said microsomes can be prepared and isolated for the purposes of the present invention. This is important because one of the challenges in generating TP is the need to incorporate it into the lipid bilayer for synthesis in a correctly folded state (Buntru et al., (2015). The present invention transfers the production of TP to an HTS platform that enables screening of multiple drugs against usually intractable TP targets. The HTS derived from BY-2 is also called BY-2-HTS, which is a preferred embodiment of the HTS of the present invention using endogenous microsomes. The advantage of endogenous microsomes is that the complexity due to the use of artificial components, artificial membranes or other artificial excipients is avoided, and in addition, the natural protein biosynthesis mechanism, especially the natural post-translational modification ability, is maintained.

[0022] Another embodiment of the HTS according to the present invention, preferably BY-2-HTS, is that at least one TP embedded in the lipid bilayer of endogenous microsomes and / or endogenous microsome fragments, preferably derived from N. tabacum, is captured on a biologically inactive surface. 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 a microtiter plate, a microfluidic device, a microarray, a device comprising a microchip, and / or any other surface suitable for capturing at least one TP of interest. Preferably, any other surface suitable for capture, contact, interaction and detection according to the present invention. Successful capture, interaction and detection according to the present invention are shown in proof-of-concept examples, and the results are shown in FIGS. 10 and 11.

[0023] In all aspects and embodiments of the present invention, the transmembrane protein (TP) is "captured" on a biologically inert surface such that the TP is under control during the interaction and / or detection of the interaction. "Capture" according to the present invention may be effected by non-specific binding of at least one TP to a biologically inert surface, or capture is mediated by at least one tag at the C-terminus and / or N-terminus of at least one TP. Preferably, at least one TP is captured on a biologically inert surface via a C-terminal tag. Suitable tags are well known to those skilled in the art and include biotin tag, hapten-tag, 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), HaloTag®, 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), polyaspartate (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. The "captured TP" is always embedded in the lipid bilayer of the endogenous microsomes or microsomal fragments as defined herein.Thus, "captured TP" is used synonymously with "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 immobilized on at least one biologically inactive surface realized by a capture process, and the surface carries the TP during the processes of HTS, preferably BY-2-HTS, treatment, contact, interaction, and / or detection.

[0024] Thus, in another embodiment of the HTS of the present invention, preferably BY-2-HTS, at least one TP contains at least one tag at the C-terminus and / or N-terminus, and preferably, at least one TP is captured on a biologically inactive surface via a C-terminal tag. Any tag is suitable. Strep-tag, His-tag, and / or Biotin-tag are preferred (Figs. 1-5).

[0025] In one embodiment, at least one microsome containing at least one lipid bilayer-embedded TP is first captured, and then the microsome is processed as described herein to obtain a microsomal fragment containing at least one lipid bilayer-embedded TP captured during and after the processing of at least one microsome. Alternatively, at least one microsome is processed in an uncaptured state, preferably in a free state, to realize the said fragment containing at least one lipid bilayer-embedded TP, and then the fragment is captured on the biologically inactive surface via at least one TP. If desired, the microsomal fragment containing at least one TP is separated from the fragment without TP and / or from the dissolved TP.

[0026] Thus, in another embodiment, the HTS according to the present invention, preferably BY-2-HTS, · a step of capturing at least one TP embedded in the lipid bilayer of a microsome on at least one biologically inactive surface, and · processing at least one captured microsome to obtain microsome fragments, wherein at least one TP remains embedded in the lipid bilayer, or alternatively, · processing at least one free microsome to obtain free microsome fragments, wherein at least one TP remains embedded in the lipid bilayer, and · further comprising capturing at least one TP embedded within the lipid bilayer of the microsome fragments on at least one biologically inert surface.

[0027] Preferably, the processing is a chemical treatment (e.g., detergent) and / or physical treatment (e.g., shear force, ultrasound) sufficient to disrupt the microsomes without damaging the TP within the lipid bilayer. Preferably, the capture is performed using a Strep-tag, His-tag, and / or Biotin-tag. If desired, uncaptured microsomes and / or microsome fragments are separated after the capture step. Microsomes and / or microsome fragments without TP and / or dissolved TP are separated from microsomes and / or microsome fragments containing at least one TP. The separation can be performed by filtration and / or centrifugation.

[0028] A biologically-inactive surface can be the surface of a particle, the surface of a device as defined herein, and / or any other surface suitable for capturing at least one TP of interest. In any case, the “captured TP” is captured within the reaction zone of the aforementioned surface where an interaction between at least one TP and at least one analyte is detected. Thus, a “biologically-inactive surface” according to the present invention means any surface that does not affect the steps of contact, interaction, and / or detection of an interaction between at least one TP and at least one analyte. Biologically-inactive surfaces are materials typically used in particular for consumables of any detection assay, for example, polymers, polystyrene, polypropylene, polycarbonate, alginate, agarose, hydrogels, alginate or agarose hydrogels, polycarboxylates, carboxymethyl dextran hydrogels, glass materials, and the like. Other materials suitable for biologically-inactive surfaces are well known to those skilled in the art. Biologically-inactive surfaces are the surfaces of particles including microparticles, nanoparticles, and magnetic particles, the surfaces of devices comprising microtiter plates, microfluidic devices, microarrays, microchips or any other consumables, and / or any other surface particularly suitable for capturing and carrying the TP of interest during the steps of contact, interaction and / or detection in particular. The said surface is particularly suitable for capturing and carrying at least one TP without affecting the said steps during the HTS according to the present invention, in particular during the treatment of at least one microsome, during the steps of contact, interaction and / or interaction, preferably binding detection between at least one TP and at least one analyte.

[0029] Devices within the meaning of the present invention comprise, are coated by, or consist of a biologically-inert 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 aforementioned ranges. Microtiter plates typically have 6, 12, 24, 48, 96, 384 or 1536 wells arranged in a 2:3 rectangular matrix. The devices and particles defined herein are preferably consumables suitable for use in HTS according to the present invention, most preferably suitable for the steps of capture, treatment, contact, interaction, and / or detection of said interaction. An example of such a device has been successfully used in a proof-of-concept example, and the results achieved by said device are shown in FIGS. 10 and 11, demonstrating that said device, here a microtiter plate, is available according to the present invention. In particular, said device is available for use in HTS according to the present invention, preferably BY-2-HTS, more preferably multiplex BY-2-HTS, and can be manufactured by a cell-free production method. In particular, TP embedded in the lipid bilayer is captured by the device according to the present invention. The same applies to microfluidic devices (FIGS. 12 and 16).

[0030] Accordingly, another aspect of the present invention is a device as defined herein comprising at least one biologically-inert surface each carrying at least one defined captured TP embedded in the lipid bilayer of microsomes or microsome fragments as defined herein. The device comprises at least two or more reaction zones each having a biologically-inert surface for use in multiplex HTS, preferably multiplex BY-2-HTS. In one embodiment, the HTS, preferably BY-2-HTS, is · providing at least one device comprising at least two reaction zones each having at least one biologically-inactive surface, and · as described herein, capturing at least one TP embedded within the lipid bilayer of microsomes within each reaction zone on at least one biologically-inactive surface of the device and optionally treating to obtain microsomal fragments as defined herein, or · as described herein, capturing at least one TP embedded within the lipid bilayer of microsomal fragments within at least one reaction zone on at least one biologically-inactive surface of the device.

[0031] · alternatively, providing at least one device comprising at least two reaction zones each having at least one biologically-inactive surface on which at least one TP is already captured, the TP being embedded within the lipid bilayer of microsomes or microsomal fragments, and · treating as described herein, provided that it is a captured microsome within the device and microsomal fragments are desirable for HTS, or · provided that it is a captured microsomal fragment within the device, performing other steps of the HTS of the invention including providing, contacting, interacting and / or detecting at least one analyte or analyte panel.

[0032] Optionally, the TP embedded within the lipid bilayer and captured within the device according to the invention can be covered by a stabilizer during storage prior to performing the HTS.

[0033] The "reaction zone" according to the present invention is a region where TP is captured, contact with at least one analyte occurs, and the interaction with at least one analyte is tested. The reaction zone comprises, is coated by, or consists of a biologically-inert surface as defined herein. The reaction zone is any particle surface of any particle as defined herein, or is located on said particle. The reaction zone is part of the surface of a device, which is the well of a microtiter plate, the channel of an SPR consumable, any microfluidic, any microfluidic device, or any other surface of a consumable used for detection according to the present invention, as defined herein. The device comprises at least two or more of such reaction zones that comprise, are coated by, or consist of a biologically-inert surface as defined herein.

[0034] In another embodiment of 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 of the foregoing.

[0035] "Analyte" within the meaning of the present invention and any aspect and embodiment is a molecule that is tested for its interaction, as defined herein, with at least one target protein (TP) of interest, preferably for binding to a specific domain of at least one TP and / or for binding to a binding domain or epitope on at least one TP, and for its ability to modulate the activity of at least one TP (preferably by signalling or signal transduction as defined herein). An analyte can be tested alone, or in combination with, or dependent on, another analyte of the same group (e.g., one, two or more small molecules), or another analyte of a different group (e.g., a small molecule and any protein). Thus, two or more analytes tested within the same reaction zone are considered an "analyte complex" (synonymous with "analyte combination") and are tested in combination for any interaction with at least one TP of interest. Thus, this screening is suitable for screening at least one "analyte" or "analyte complex" (synonymous with analyte combination) for its potential as a ligand according to the present invention. Examples of analyte complexes include multimeric proteins comprising at least two polypeptide chains, at least two analytes combined by covalent, electrostatic or hydrogen bonds, and at least one analyte combined with a polymer, nanoparticle, colloid or liposome. An analyte is tested for its ability to induce an interaction with at least one TP, preferably signalling or signal transduction resulting from binding of the analyte to the TP. The signalling or signal transduction (preferably induced by binding of at least one analyte to at least one TP) can occur via at least one transmembrane domain of at least one TP (in particular, with respect to the native cytosolic domain of the TP preferably captured on a biologically inactive surface in the assay), or on the same side of the interaction between at least one TP and at least one analyte.The aforementioned signalling or signal transduction may regulate biological processes, and the regulation includes activation, inactivation, inhibition, enhancement, induction or interruption of biological processes. Therefore, the action of an analyte identified as a ligand may be the action of an activator, inhibitor, effector, trigger or regulator of a biological process. Preferably, it is related to a biological process associated with a disease. "Analyte" within the scope of the present invention preferably means any molecule as described above, which is 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 scope 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 an analyte is any protein produced by the use of ALICE®. An "analyte panel" includes two or more analytes or analyte combinations for multiplexed HTS according to the present invention, preferably multiplexed BY-2-HTS.

[0036] "Small molecule" is defined differently depending on the technical field of application. Here, a small molecule or micromolecule (synonyms) has a low molecular weight (≤ 1000 Daltons) and / or a low molecular weight of less than 900 g / mol. They can regulate biological processes, are of organic origin, and can include secondary metabolites, as well as small nucleic acid molecules including deoxynucleotide monophosphates and small molecule oligonucleotides. Some examples of small molecules are antihistamines, antibiotics, drug candidates, as well as small molecules from microorganisms and plants, selective serotonin reuptake inhibitors, alicylic acid, ibuprofen, paracetamol, morphine, ethanol, psilocybin, L-DOPA, cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, cyclophosphamide, docetaxel, bleomycin, vincristine, dacarbazine, mustine, vinblastine, procarbazine, prednisone, etoposide, cisplatin, epirubicin, capecitabine, folic 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.

[0037] A "ligand" within the scope of the present invention is any analyte that interacts with, preferably binds to, at least one TP within the scope of the present invention. Thus, an "analyte" (or analyte complex) within the scope of the present invention that has been demonstrated to have the ability to induce signalling or signal transduction due to interaction with at least one TP, preferably binding of the analyte to the TP, is identified as a ligand. Preferably, due to said interaction, preferably binding to 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 TP being tested. Even more preferably, for said regulation as defined herein, the analyte that is a ligand for the TP being tested is considered a drug candidate. The desired ability of at least one ligand is suitable for the prevention or treatment of a disease associated with at least one TP. A ligand can be one analyte or analyte complex as defined herein and demonstrated by the detected interaction according to the present invention.

[0038] In particular, the "interaction" between at least one TP and at least one analyte (or analyte complex) within the scope of the present invention after contact is a molecular interaction that causes a measurable activity or change in activity of at least one TP induced by at least one analyte, and / or the interaction is a binding between at least one TP and at least one analyte. The binding may or may not be related to a measurable activity, change in activity. Generally, the interaction can cause a modulation (increase, decrease) of activity detected as a change in activity due to the ability of the analyte to modulate the said activity as defined herein. As a result, within the scope of the present invention, the interaction is demonstrated by detection of activity, for example, colorimetric / fluorescent quantification assays, radiometric scintillation proximity assays, atomic force microscopy (AFM), spectrophotometry, mass spectrometry, Raman spectroscopy, membrane potential assays, electrochemical assays, fluorescence resonance energy transfer (FRET) assays, crystal structure analysis, cryo-electron microscopy. The binding can be demonstrated by detection of the analyte bound to the TP or by detection of the activity after binding. The interaction can be carried out and detected in multiplex screening.

[0039] Accordingly, another aspect of the HTS of the present invention is the multiplex high-throughput screening (multiplex HTS), preferably multiplex BY-2-HTS, of at least one ligand of at least one TP of interest, which simultaneously provides two or more analytes to two or more reaction zones and / or simultaneously provides an analyte combination of two or more analytes to two or more reaction zones. Accordingly, all definitions apply to the multiplex design of the HTS of the present invention.

[0040] "Multiplex", "multiplex screening", "multiplex HTS" or "multiplex high-throughput screening" (synonyms) means that within the same reaction zone, one or more different TPs are captured on a biologically inert surface, tested against one analyte or a defined combination of analytes, and the interaction between at least one captured TP and the analyte or combination of analytes is detected. Multiplex also means that within different reaction zones, the same TP is captured on a biologically inert surface, each TP is tested against two or more different analytes, and within each reaction zone, the interaction between one captured TP and one analyte or combination of analytes is detected. Thus, "multiplex" within the scope of the present invention means that for at least one or more TPs of interest, 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 or more analytes, or combinations of analytes, are screened simultaneously. Or any other integer between the aforementioned ranges. Accordingly, any other definition applies, providing microsomes or microsome fragments, providing analytes, any process and combination of processes including processing, capturing, contacting, interacting and / or detecting, and the described embodiments of the present invention can be performed in the multiplex HTS described herein, preferably multiplex BY-2-HTS. Multiplex HTS can be performed by any embodiment of a biologically inert surface, device or consumable as defined herein respectively.

[0041] The multiplex grade is defined or limited by the detection method available or selected. In particular, by consumables available for use in the detection method, such as microtiter plates, microfluidic devices, microchips, microarrays, or consumables for SPR.

[0042] Thus, one embodiment of multiplex HTS, preferably multiplex BY-2-HTS, is · Providing a plurality of endogenous microsomes, each containing at least one lipid bilayer-embedded transmembrane protein (TP), or a plurality of endogenous microsome fragments, each containing at least one lipid bilayer-embedded transmembrane protein (TP); · Providing a plurality of analytes, or a plurality of analyte combinations, as defined herein; · Contacting the plurality of TPs with the plurality of analytes, or the plurality of analyte combinations; · Detecting any one interaction of the analytes and any one interaction of the TPs, preferably within a plurality of reaction zones of a device as defined herein, more preferably within a microtiter plate, a microfluidic device or an SPR consumable.

[0043] Accordingly, for multiplex designs, capture and / or processing steps as described for HTS are applicable.

[0044] In another embodiment of the HTS according to the invention, preferably multiplex BY-2-HTS, in particular the interaction (as defined herein) between at least one TP and at least one analyte after contact is detected by immunoassay, preferably enzyme-linked immunosorbent assay (ELISA), spectroscopic assay, preferably surface plasmon resonance (SPR), particle size measurement, magnetic measurement and / or optical measurement. Such methods are well known to those skilled in the art. Experiments show the HTS of the invention in combination with ELISA and SPR. The methods are well known to those skilled in the art, and it is clear to those skilled in the art that either the analyte or at least one of the TPs, or both, constitute the detection means, or that such means must be added separately to detect any interaction. The detection means includes fluorescent molecules, tags, haptens, enzymes or other molecules that enable the detection of the interaction. Such means may be encoded by a template encoding at least one TP, may be bound to at least one analyte, and / or may be added separately in the detection step. Suitable means for detection are well known for each of the detection methods mentioned, and their feasibility has been demonstrated in the examples presented.

[0045] In one embodiment of the present invention, in particular, the interaction and / or detection after contact with HTS is performed using immobilized or mobile microsomes or microsome fragments, each containing at least one TP embedded within a lipid bilayer, which are captured on a biologically inert surface. Accordingly, all definitions apply. However, "captured" is not the same as "immobilized", and it is necessary to distinguish between "captured and mobile" and "captured and immobilized". The microsomes or microsome fragments can be captured via at least one TP, and the microsomes or microsome fragments are mobile or immobilized. In contrast to the mobile state using the biologically inert surface of the particles defined herein, the microsomes or microsome fragments are immobilized within the device by a process of capturing at least one TP on at least one biologically inert surface.

[0046] As described herein, TPs are of great interest for drug development and constitute most of the current drug targets. Nevertheless, it is difficult to express TPs that have not been well characterized. BY-2 lysates, preferably commercially available ALiCE® systems (LenioBio GmbH, e.g., ALiCE® Kit AL0103050, AL0103200, AL0103500) were used here to show overexpression and production of TPs and to pre-shape the HTS according to the present invention. Here, to demonstrate the proof of concept of the HTS platform according to the present invention, preferably By-2-HTS, a binding assay against its known ligand was tested. For this purpose, well-characterized type I transmembrane glycoproteins and their ligands were used. These proteins are angiotensin-converting enzyme (ACE2), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and their respective ligands, the receptor-binding domain (RBD) of the spike protein of SARS-CoV-1 / 2, and the poliovirus receptor (PVR). These TPs are shown to be targets "leading to the development of new drugs" and are interesting because they are involved in extremely serious current diseases such as COVID-19 and cancer. Interfering with the binding between the said TP and its ligand may effectively act as a possible treatment. The HTS of the present invention was shown to be suitable for testing various "hits" that interfere with the binding. As a result, the proof of concept of the HTS platform of the present invention for testing new drugs is shown.

[0047] Another aspect of the present invention is a 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), particularly ER and Golgi, preferably based on plants of the genus Nicotiana of the Solanaceae family, preferably a cell-free protein synthesis (CFPS) system based on BY-2, ·Providing a plant-derived lysate, preferably a lysate derived from a plant of the genus Nicotiana of the Solanaceae family, more preferably a lysate of the BY-2 cell line derived from N. tabacum, comprising at least one endogenous microsome derived from a plant; ·Providing at least one template encoding at least one transmembrane protein (TP); ·Incubating at least one lysate with at least one template; ·Expressing at least one transmembrane protein (TP); ·Co-transcriptionally and co-translationally translocating 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 carrying at least one TP embedded in the lipid bilayer to obtain a microsome fragment containing at least one TP embedded in the lipid bilayer. If desired, ·As defined herein, the step of capturing on a biologically inert surface is performed after or before the treatment.

[0048] The process products realized immediately are TPs embedded in the lipid bilayer of endogenous microsomes or endogenous microsome fragments that are each free (uncaptured) as defined herein, or endogenous microsomes or TPs embedded in the lipid bilayer of endogenous microsomes that are each captured and are either mobile (e.g., on particles) or captured and immobilized (e.g., on a device) as defined herein.

[0049] In particular, in another embodiment of the cell-free production of at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of at least one endogenous microsome according to the present invention, a step of capturing at least one TP embedded in the lipid bilayer of the microsome on at least one biologically inactive surface is performed. In one embodiment of the method, the capturing step is performed and the at least one captured microsome is processed to obtain microsome fragments, where at least one TP remains embedded in the lipid bilayer. In another embodiment of the method, at least one free microsome is processed to obtain free microsome fragments, where at least one TP remains embedded in the lipid bilayer, and at least one TP embedded in the lipid bilayer of the microsome fragments is captured on at least one biologically inactive surface. In any embodiment of the method, in particular, the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of a microtiter plate, a microfluidic device, a microarray, a device comprising a microchip, and / or any other surface suitable for capturing at least one TP of interest.

[0050] Optionally, further steps are required and may be combined with the methods described herein, for example, the separation and / or isolation, purification, washing of the microsome or the microsome fragments, 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 microsome fragment. Such steps are well known to those skilled in the art. Accordingly, any embodiment of the process of handling the separation and isolation described for HTS (with or without using the tags defined herein) is applicable to the production method.

[0051] In one embodiment of cell-free production, the processing step is carried out in a timely manner independently of the preceding steps. It may be after the cell-free production of at least one TP. In order to obtain the fragment immediately before HTS according to the present invention, it is desirable to produce the TP in microsomes, preserve the intact and free or intact and captured (mobile or immobilized) microsomes, and process them.

[0052] Another embodiment of cell-free production (CFP) is the production of at least one captured endogenous microsome derived from the plant endoplasmic reticulum (ER), particularly ER and Golgi, comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of said microsomes, · providing a plant-derived lysate, preferably a lysate derived from a plant of the genus Nicotiana of the Solanaceae family, more preferably a lysate of the BY-2 cell line derived from N. tabacum, 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 at least one transmembrane protein (TP), · co-transcriptionally and co-translationally translocating 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, · optionally, separating the obtained at least one microsome comprising at least one TP of interest as defined above, preferably by centrifugation, and · capturing at least one microsome comprising at least one TP of interest as defined (as defined herein), preferably capturing said microsome on a biologically inert surface as defined herein, and ·Preferably, it is a production comprising the step of obtaining at least one captured microsome containing at least one TP for a defined purpose, captured by a particle or device defined herein.

[0053] Another embodiment of cell-free production provides at least one captured endogenous microsome fragment derived from a plant endoplasmic reticulum (ER), particularly ER and Golgi, comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of said microsome fragment, wherein further, the step of treating at least one endogenous microsome carrying at least one TP embedded in the lipid bilayer is performed to obtain a microsome fragment containing at least one TP embedded in the lipid bilayer.

[0054] Accordingly, any embodiment of capture (with or without using the tags defined herein), treatment or combinations thereof as described for the HTS of the first aspect of the invention is applicable to cell-free production. Thus, cell-free production according to the invention may include capture and / or treatment to obtain a particle, device or other consumable comprising at least one captured endogenous microsome or endogenous microsome fragment derived from a plant endoplasmic reticulum (ER), particularly ER and Golgi, comprising at least one transmembrane protein (TP) of interest embedded in the lipid bilayer of the microsome or in the lipid bilayer of the microsome fragment.

[0055] In another embodiment of the cell-free production or cell-free production method, the lysate further comprises plastids, chloroplasts, mitochondria, or chloroplasts and mitochondria. In a further embodiment of the cell-free production or cell-free production method, at least one microsome or microsome fragment is derived from a cell line of the genus Nicotiana of the Solanaceae family, preferably N. tabacum, more preferably BY-2. Optionally, the method may include the step of adding a composition comprising biologically active microsomes and / or biologically active mitochondria. Preferably, such a composition is derived from a cell line of the genus Nicotiana of the Solanaceae family, preferably N. tabacum, more preferably BY-2.

[0056] Cotranslational translocation of at least one expressed TP to at least one microsome during cotranscription and cotranslational translocation can occur without the signal peptide of the expressed TP, or cotranslational translocation is mediated by at least one appropriate signal peptide. Thus, in one embodiment of a cell-free production or cell-free production method, at least one template encodes at least one TP and a signal peptide that is expressed together with the TP from the same template and is suitable for mediating translocation of the TP to the microsome. Other TPs are translocated without the need for translocation to be mediated by a signal peptide. In another embodiment, the template further encodes at least one tag at the N-terminus and / or C-terminus of at least one TP, and preferably, the template encodes at least one tag at the C-terminus (Figures 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 a cell-free production or cell-free production method, at least one TP is cotranslationally translocated to the outer C-terminus and inner N-terminus of at least one microsome (Figure 6). Preferably, glycosylation, N-glycosylation of the TP, other post-translational modifications, lipid modifications, and / or folding occur during the processes of cotranscription and cotranslational translocation, ensuring a correctly folded functional TP. More preferably, the TP is embedded by a transmembrane domain within the lipid bilayer as it occurs in nature.

[0057] Another aspect of the invention is a plant endoplasmic reticulum (ER), in particular endogenous microsomes or at least one endogenous microsome fragment derived from ER and Golgi, comprising at least one lipid bilayer-embedded transmembrane protein (TP) obtainable by a cell-free production or a cell-free production method according to the invention. In one embodiment of this aspect, the endogenous microsomes or at least one endogenous microsome fragment are free, not captured and not immobilized. In another embodiment, the endogenous microsomes or at least one endogenous microsome fragment are captured by the particles as defined herein, but are still mobile and are 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 microsomes or at least one microsome fragment are derived from a cell line of the genus Nicotiana of the Solanaceae family, preferably N. tabacum, more preferably BY-2. Another aspect of the invention is a microsome or microsome fragment as described herein, with at least one stabilizer. Thus, another aspect of the invention is a composition comprising at least one (free or captured and mobile) microsome or microsome fragment according to the invention, at least one stabilizer and optionally at least one other excipient.

[0058] Another aspect of the invention is the use of a microsome or microsome fragment comprising at least one lipid bilayer-embedded transmembrane protein (TP) as defined herein in a HTS according to the invention or according to any embodiment of HTS. Accordingly, any embodiment of capture (with or without the tags as defined herein), treatment or combinations thereof as described for HTS applies to this use.

[0059] 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 microsome fragment derived from a plant endoplasmic reticulum (ER), particularly the ER and Golgi, and the biologically-inactive surface is the surface of a consumable. Preferably, the consumable is a device comprising two or more reaction zones that include, consist of, or are coated by 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., in the case of SPR) or a microtiter plate (e.g., in the case of ELISA). Another aspect of the invention 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 microsome fragment derived from a plant endoplasmic reticulum (ER), particularly the ER and Golgi. The device comprises at least two or more reaction zones that include, consist of, or are coated by a biologically-inactive surface. The device is suitable for use in multiplex HTS according to the invention. Preferably, the microsome or microsome fragment is derived from a plant of the genus Nicotiana of the Solanaceae family, preferably N. tabacum, more preferably a lysate derived from N. tabacum, and most preferably a lysate of the BY-2 cell line derived from N. tabacum. Capture is preferably mediated by a tag as defined herein.

[0060] In every aspect and embodiment of the present invention, the "consumable" comprises, consists of, or is coated by a biologically-inert surface. Such consumables are used in any detection assay according to the present invention. Suitable materials for such consumables include polymers, polystyrene, polypropylene, polycarbonate, alginate, agarose, hydrogel, alginate or agarose hydrogel, polycarboxylate, carboxymethyl dextran hydrogel, glass materials, and the like. Other materials suitable for biologically-inert surfaces are well known to those skilled in the art. Accordingly, the definition of a biologically-inert surface applies. Consumables include particles, microparticles, nanoparticles, magnetic particles, microtiter plates, microfluidic devices, devices comprising microarrays and microchips.

[0061] Preferably, the consumable is for use in HTS, BY-2-HTS according to the present invention, more preferably multiplex BY-2-HTS, or any embodiment thereof. More preferably, the consumable is manufactured by the manufacturing method defined herein. More preferably, the consumable is for use during contact in the process of treating and detecting the interaction and / or interaction between at least one analyte and at least one TP according to the present invention.

[0062] Another aspect of the present invention is a kit for use in high-throughput screening (HTS) according to the present invention, preferably BY-2-HTS, more preferably multiplex BY-2-HTS, for at least one ligand 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), particularly from ER and Golgi, preferably from a plant of the genus Nicotiana of the Solanaceae family, preferably N. tabacum, preferably the lysate of N. tabacum, more preferably the lysate of BY-2 cells of N. tabacum, comprising: · A plant cell lysate comprising endogenous microsomes derived from the endoplasmic reticulum (ER), particularly from ER and Golgi, preferably from a plant of the genus Nicotiana of the Solanaceae family; · As a positive control, preferably at least one vector encoding a known TP as a positive control for functional microsomes or microsome fragments, and · Optionally, at least one vector as a positive control for lysate function, and · At least one vector for at least one template encoding at least one TP of interest, and · Optionally, further excipients preferably containing small molecule components, labeled amino acids, cofactors, etc., and · Optionally, at least one agent for detecting the interaction between at least one analyte and at least one TP, preferably at least one means, preferably an antibody for detection, and · Optionally, at least one agent for disrupting at least one microsome, preferably a suitable detergent, and · Optionally, a kit comprising at least one analyte, or an analyte panel of two or more analytes.

[0063] The kit may include additional components such as compositions containing biologically active microsomes and / or mitochondria as defined herein. Another additional component may be a consumable for capture as defined herein. Alternatively, the kit includes a consumable or device according to the invention that already contains at least one captured TP. The kit may include at least one instruction specifying the mixing of components, and / or instructions specifying HTS and / or cell-free production methods, respectively, regardless of the presence or absence of capture and / or processing.

[0064] Preferably, at least one positive control for lysate function is a commercially available pALiCE01-eYFP vector, where eYPP represents enhanced yellow fluorescent protein, and at least one positive control for functional microsomes or microsomal fragments is preferably a pALiCE0X-TP vector for TP expression that is customized or adaptable to the TP of interest. However, any other vector and / or combination of vector and TP is suitable, provided that it allows confirmation of the desired function (positive control).

[0065] A preferred embodiment of a kit for use in HTS, preferably multiplex HTS, is · additional excipients preferably including small molecule components, labeled amino acids, cofactors, etc., and · an agent for detection, preferably an antibody for detection, and · optionally, an agent for disruption of microsomes, and · further optionally includes at least one analyte, or an analyte panel of two or more analytes.

[0066] A preferred embodiment of a kit for use in HTS, preferably multiplex HTS, is · an agent for chemical disruption of the microsomes of the present invention, and · further optionally includes at least one analyte, or an analyte panel of two or more analytes.

[0067] In all aspects and embodiments of the present invention, "endogenous microsomes" means that it is not artificial and is derived from a cell-free production system of a eukaryotic species. Preferably, "endogenous microsomes" are derived from the same system from which the lysate is derived and from which the ER, Golgi, microsomes, plastids, chloroplasts and / or mitochondria are derived. "Lysate" is an isolated biologically active substance for protein synthesis, as known from the prior art. "Endogenous microsomes" are derived from a natural system derived from only one species of the same family. Natural lysates may contain components from different species and / or different genera, but are derived from the same family. Thus, different cell lines of the Nicotiana genus, different cell lines of N. tabacum, and different BY-2 cell lines with different genetic modifications may be combined respectively. A mixture of microsomes derived from systems derived from different cell lines is still an endogenous microsome (mixture) within the meaning of the present invention. Hybrid lysates containing components from different families or even different species are excluded and not part of the present invention. A hybrid lysate means that components such as ER, Golgi, microsomes, plastids, chloroplasts and / or mitochondria are not derived from the same family or even the same species (e.g., plants, yeast, fungi, prokaryotes).

[0068] The microsomes according to the present invention may contain one or more of the same TPs (identical amino acid sequence and glycosylation), or one or more different variants of the same TP as defined herein. Thus, the microsomes may contain 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. In one embodiment, such microsomes can be used for screening according to the present invention. In another embodiment, the microsomes are treated to achieve microsome fragments each containing at least one variant of the same TP embedded in the lipid bilayer. Thus, after treatment, the different variants of the TP are isolated from each other within different microsome fragments.

[0069] Eukaryotic CFPS can be divided into two types: those with an endogenous microsomal structure and those without an endogenous vesicular compartment (Thoring et al., 2019). Here, eukaryotic CFPS with an endogenous microsomal structure for post-translational modification is included in the present invention. Therefore, when producing TP, it is preferable to use endogenous microsomes that enable appropriate TP glycosylation.

[0070] In all aspects and embodiments of the present invention, the "transmembrane protein" (TP) according to the present invention comprises at least one transmembrane domain, at least one cytoplasmic domain, and at least one cytosolic domain. TP can include 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 the lipid bilayer" means that at least one transmembrane domain of at least one TP is oriented and embedded in the lipid bilayer of the endogenous microsome or the lipid bilayer of the microsomal fragment derived from the endogenous microsome. TP within the scope of the present invention includes type I TP, type II TP, type III TP, and type IV TP having one transmembrane domain, multipass TP, lipid-anchored protein, GPI-anchored membrane protein, bitopic TP, polytopic TP having an α-helix transmembrane domain, polytopic TP having a β-sheet transmembrane domain, peripheral membrane protein, and monotopic protein. Preferably, the TP is embedded in the lipid bilayer when occurring naturally and is preferably glycosylated. Examples of TP are listed below.

[0071] [Table 1]

[0072] Some specific examples of potential TPs for HTS according to the present invention include hAQP1 (e.g., Uniprot KB: P29972, 6 TMD), Mel-hEGFR (e.g., Uniprot KB: P00533, 1 TMD), and at least nine different EGFR mutations are described, human beta - adrenergic receptor, shaker potassium channel, connexin, inositol trisphosphate receptor, nicotinic acetylcholine receptor, G - protein - coupled receptor GPCR, ion channel, transporter, histidine kinase and tyrosine kinase, cytokine receptor, Toll - like receptor.

[0073] Other specific examples of TPs according to the present invention include antigens, preferably antigens associated with malignant tumors and autoimmune diseases. Preferably, the group of antigens includes CD20 as a target for the treatment of B - cell malignancies, and CD3, CD22, CD33, CD123, and CD19 as targets for the treatment of B - cell malignancies, leukemia, acute myeloid leukemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). In particular, CD20 is a transmembrane protein having four transmembrane domains embedded in the lipid bilayer of the cell membrane. Example 6 shows as proof - of - principle that CD20 is expressed embedded in the lipid bilayer of endogenous microsomes. The same assay is suitable for any desired antigen for screening ligands.

[0074] Within the scope of meaning of multiplex HTS according to the present invention, the contact, interaction, preferably activation and / or binding between the same TP and different analytes, each provided to any number of different reaction zones, is screened. In another embodiment, different variants of the TP of interest are provided to any number of different reaction zones, a respective number of analytes are provided, contact is made, and the interaction (per reaction zone) between the same or different analytes and different TP variants is screened. Variants of the TP include any genetic variant that causes or does not cause a variant in the amino acid sequence, with or without affecting the 3D structure and / or activity of the TP variant, single nucleotide polymorphism (SNP), deletion and / or insertion. The foregoing is applicable to the HTS of the present invention where at least one TP is captured on a biologically inactive surface or at least one TP is not captured on a biologically inactive surface.

[0075] In any aspect and embodiment of the present invention, a "mobile microsome, or mobile microsome fragment" comprises at least one TP embedded in the lipid bilayer of said microsome or said microsome fragment. The mobile microsome, or mobile microsome fragment, is captured on the biologically inactive surface of the particle as defined herein via at least one TP. Provided that there is at least one biologically inactive surface on the particle, the particle carrying the captured TP remains mobile during the processes of treatment, contact, interaction and / or detection. However, the TP is not free from at least one biologically inactive surface. Thus, the microsome or its fragment is mobile because the biologically inactive surface is mobile. BRIEF DESCRIPTION OF THE DRAWINGS

[0076]

Figure 1

Figure 2

Figure 3a

Figure 3b

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

[0077] Example Example 1 1. Materials and Methods 1.1 In Silico Cloning, Plasmid Design Using SnapGene® software (manufactured by Insightful Science; available at www.snapgene.com), human DNA templates obtained from Gateway cloning of ACE2, PVR, and TIGIT proteins were first cloned in silico into the vector pALiCE02. Then, an in vitro cloning strategy was defined. For the purpose of protein orientation testing, HaloTag®, Strep-Tag® II, and GST tags were fused to the DNA templates at the C-terminus or N-terminus. For microsomal targeting, the melittin signal peptide sequence (MSP) was fused at the N-terminus (Stech et al., 2014). All clones are shown in Table 1.

[0078] The mold according to the present invention is encoded by a vector, preferably a pALiCE vector. pALiCE01 and pALiCE02 include a basic design (pALiCE) that includes a T7 promoter, followed by a tobacco mosaic virus (TMV) 5' omega leader sequence, a gene of interest (GOI that encodes the TP of interest here), and then a TMV 3' UTR. The pALiCE02 vector further includes an insect mellitin signal peptide immediately before the start of the GOI that enables microsomal targeting and translocation of the TP. In both plasmids, the GOI cassette is composed of an N-terminal Strep-II tag followed by a Factor Xa cleavage site and eYFP. This vector design enables conventional enzyme-based subcloning of genes into the NcoI and KpnI sites adjacent to the GOI cassette.

[0079]

Table 2

[0080] The glycosylation sites of TP were tested by using the online N-glycosylation prediction tool NetNGlyc-1.0 (Gupta & Brunak, 2002) and are outlined below:

Number

[0081] For proof of concept, PVR, TIGIT, and ACE2 constructs were used, along with their respective tags and signal peptide sequences (MSP) and the indicated glycosylation sites. PVR is composed of 417 amino acids and has eight N-glycan sites: 105, 120, 188, 237, 278, 307, 313, and 405. TIGIT is composed of 244 amino acids and has three N-glycosylation sites: 53, 90, and 236. ACE2 is composed of 805 amino acids and has seven N-glycan sites: 53, 90, 103, 322, 432, 546, and 690.

[0082] 1.2 Template Amplification, Cloning, Plasmid Extraction and Sequencing In order to amplify the target gene by PCR, in-silico primers were designed as shown below.

Table 3-1

Table 3-2

[0083] As shown in Table 2, Table 3 and Table 4, the target gene of TP was amplified using the designed primers and PrimeSTAR GXL DNA polymerase ((1.25 U / μl) * 2, TaKaRa Bio).

[0084]

Table 4

[0085]

Table 5

[0086]

Table 6

[0087] An agarose gel (1% (w / v)) containing ethidium bromide (0.5 μg / ml) was electrophoresed at 150 V for 15 minutes to confirm the PCR amplicon size. Bands were visualized using GelDocXR+ (Bio-Rad). The marker used was the 1Kb Plus DNA Ladder (New England Biolabs). After PCR, Gibson Assembly® was performed. A Gibson Assembly® reaction mixture (insert: 2 μl, vector: 1 μl, MiliQ water: 2 μl, 2xHiFi DNA Assembly MM: 5 μl, total volume 10 μl) was prepared using NEBuilder® 2xHiFi DNA Assembly Master Mix (New England Biolabs). The mixture was incubated at 50 °C for 30 minutes in a PCR machine.

[0088] The plasmid was transferred into competent DH5α E. coli cells. The cells were incubated on ice for 10 minutes and then subjected to a heat shock at 42 °C for 30 seconds. The cells were cooled on ice for 1 minute and then incubated for 40 minutes in SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) with shaking. The Eppendorf tubes were centrifuged at 3000 rpm for 1 minute in a tabletop centrifuge. The supernatant was discarded and the pellet was used for subsequent plating on an agar plate containing 100 μl / ml ampicillin (Amp). The plate was incubated at 37 °C overnight.

[0089] Following colony PCR, amplification was performed using the specific primers shown in Table 5 below and DreamTaq Green MM (2×) (Thermo Fisher Scientific (trademark)). Five colonies were selected from each plate, swapped, and introduced into five different PCR tubes containing master mix (having a total volume of 10 μl, the content of the PCR tube for colony PCR consisted of DreamTaq Green MM (2×): 5 μl, MiliQ water: 4 μl, primer FOR: 0.5 μl, and primer REV: 0.5 μl) using toothpicks. The applied PCR settings are summarized in Table 6. As described above, the PCR amplicon sizes were confirmed using an agarose gel ((1% (w / v)) containing ethidium bromide (0.5 μg / ml).

[0090]

Table 7

[0091]

Table 8

[0092] For sequencing, NucleoSpin (registered trademark) Plasmid purification NoLid (Macherey Nagel) was performed according to the manufacturer's instructions. The purified plasmid was prepared according to the instructions of the sequencing provider (Eurofins Genomics) and sent for sequencing. After the sequence was confirmed, NucleoBond (registered trademark) Xtra plasmid purification (Macherey Nagel) was performed according to the manufacturer's instructions to prepare the plasmid for subsequent expression in ALiCE (registered trademark).

[0093] 1.3 Protein Expression and Western Blot Analysis Protein Expression: Protein expression was performed using the cell-free system ALiCE®. 96-well cell culture microplates (Greiner Bio-One®) were filled with 50 μl of lysate per well to which a determined volume corresponding to different DNA concentrations was added. The DNA concentrations used were 2.5 nM, 5 nM, 7.5 nM and 10 nM. To prevent evaporation and maintain humidity, the well gaps were filled with water. The plates were incubated at 25 °C for approximately 20 hours with constant agitation at 500 rpm and 80% humidity in an ISF1-X Climo-Shaker (Kuhner Shaker).

[0094] Semi-dry Western Blot: The lysate samples were prepared using 4× 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 an SDS-PAGE gel (MiniProtean TGX Stain-Free 4-20%, Bio-Rad) running in a running buffer (250 mM Tris base, 192 mM glycine, 1% SDS (w / v), pH 8.8) at 200 V for 30 minutes. The gel was transferred to a nitrocellulose membrane using a Mini Trans-Blot® Turbo Transfer System (Bio-Rad) according to the manufacturer's instructions. After transfer, the membrane was blocked using 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 for 30 minutes with shaking.

[0095] Next, while shaking, incubate the membrane with the corresponding primary antibody StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences) diluted 1:15,000 with blocking buffer, GST Tag Monoclonal Antibody HRP (MA4-004, Thermo Fisher Scientific™) diluted 1:2000, or Anti-HALO® Tag (G921A, Promega) diluted 1:10,000 at room temperature for 1 hour. After incubation, wash three times continuously for 5 minutes each while shaking with TBST. Use only the secondary antibody N-terminal HaloTag®, Peroxidase AffiniPure Goat Anti-Mouse IgG (115-035-003, Jackson ImmunoReseach) diluted 1:10,000 with blocking buffer, and incubate at room temperature for 1 hour while shaking. After incubation, wash with TBST in the exact same manner as the previous washing step. Immunodetection was performed by chemiluminescence reaction using HRP-conjugated antibodies. 1:1 SuperSignal™ West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific™) was used for HRP antibody detection. Protein bands were confirmed using ChemStudio PLUS (Analytik Jena™). For N-terminal and C-terminal Strep-Tag® II samples, prestained protein ladder 10 - 250 kDa by PageRuler™ (Thermo Fisher Scientific™) was used as a marker, and for N-terminal HaloTag® samples and C-terminal GST tag samples, Precision Plus Protein Dual Color Standards 10 - 250 kDa (Bio-Rad) was used as a marker.

[0096] 1.4 Microsome Treatment to Achieve Microsome Fragments. 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 into another tube, and the pellet or P1 (containing microsomes) was resuspended in 1% dodecyl-β-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. From this centrifugation, two fractions were recovered, namely, the supernatant or S2 (containing solubilized microsomal proteins), and the pellet or P2 (containing microsomal residue). P2 was completely resuspended in PBS. After the preparation of all the products (S1, P1, S2, P2), the protein samples were prepared for Western blot as described above. A schematic of the experimental procedure is shown below:

Number

[0097] 1.5 Microsome Capture, and Protein Binding Assay Lysates expressing ACE2 and PVR were used for indirect ELISA. The experimental setups are shown in Figures 1 - 4.

[0098] Indirect ELISA Microsome Capture: 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 microsome samples were directly added to each well of a Strep-Tactin® XT coating microplate (1-4101-001, Iba), and the samples were prepared in triplicate. Starting from the concentrated sample (1), a series of unknown dilutions were made until reaching zero (starting from concentrated sample 1 and performing 8 dilution steps of 1:100: 0.5 → 0.25 → 0.125 → 0.0625 → 0.03125 → 0.015625 → 0.0078125 → discard). The plate was incubated at room temperature for 1 hour.

[0099] Protein Binding: 1% DDM in PBS was added to each well, and the plate was incubated at room temperature for 15 minutes. Subsequently, the plate was washed three times with PBS-0.05% Tween (v / v) (PBST). To prevent non-specific binding, 1× blocking buffer (ab126587, Abcam) was added to block the wells, and the plate was incubated at room temperature for 1 hour. The same washing procedure as before was performed.

[0100] For PVR microsome capture detection, Human CD155 / PVR antibody (MAB25301, Bio-Techne) 1:5000 in blocking buffer was added. For the PVR protein that binds to TIGIT, Recombinant Human TIGIT Fc Chimera (7898-TG, Bio-Techne) in blocking buffer at 2.5 μg / ml was added. For ACE2 microsome capture and protein binding analysis, commercially available RBD SARS-CoV Spike / RBD Protein (RBD, 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 plate was incubated at room temperature for 1 hour, followed by the washing procedure.

[0101] For protein detection, a detection antibody (HRP-labeled) that binds to the protein was added. For the detection of the structural antibody of PVR, Peroxidase AffiniPure Goat Anti-Mouse IgG (115-035-003, Jackson ImmunoResearch) 1:10000 in blocking buffer was added. For the detection of PVR-TIGIT binding, Goat anti-Human IgG F(ab’)2:HRP (0500-0099) 1:10000 in blocking buffer was added. For the detection of ACE2-commercial RBD / purified RBD binding, His Tag Horseradish Peroxidase-conjugated antibody (MAB050H, Bio-Techne) 1:4000 and StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences) 1:15,000 in blocking buffer were added. The plates were incubated at room temperature for 1 hour and then washed.

[0102] For protein visualization, Detection regent TMB ELISA substrate (High Sensitivity) (Abcam) was added to each well and incubated at 37 °C for 15 minutes in the dark. After the wells had developed some color, absorbance was measured (650 nm) using a TECAN Infinite M1000 Pro machine and Tecan i-Control 2.0 software with the settings shown in Table 8. The absorbance values were analyzed using Excel to construct an absorbance / dilution graph.

[0103]

Table 9

[0104] 2. Results 2.1 Successful Expression of Membrane Proteins within ALiCE® To express the target TP, in vitro cloning was performed on the pALiCE02 vector as shown in Table 9. Using the generated plasmid, proteins were expressed in ALiCE® at various plasmid concentrations, and the expression was confirmed by Western blot analysis.

[0105]

Table 10

[0106] The C-terminal Strep-tagged proteins showed bands for PVR_cSTREP and ACE2_cSTREP, but no band was visible for TIGIT_cSTREP (Figure 7A). PVR_cSTREP had two visible bands, one of the predicted size (49 kDa) and the other a stronger band that could be a sign of glycosylation. PVR_cSTREP showed bands at all DNA concentrations except 2.5 nM. Furthermore, at the highest concentration, a decreased band intensity was shown, which could indicate saturation of the system at DNA concentrations higher than 7.5 nM. ACE2_cSTREP had a visible band of the predicted size (96 kDa) at all DNA concentrations. A strong band that could correspond to the glycosylated protein was visible at all ACE2_cSTREP concentrations. No band was visible for TIGIT_cSTREP. Thus, the presence of bands on the blot indicates protein expression. A non-specific band of 120 kDa was observed in each sample.

[0107] For the N-terminal Strep-tagged proteins, bands corresponding to their predicted sizes were visible for PVR_nSTREP (49 kDa), TIGIT_nSTREP (30 kDa), and ACE2_nSTREP (96 kDa) (Figure 7B), which indicated successful protein expression. The PVR_nSTREP band was visible at all concentrations, although at low intensity. Furthermore, PVR_nSTREP did not show any signs of glycosylation, and only one band was visible on the blot. ACE2_nSTREP showed bands at all concentrations except 2.5 nM. The ACE2_nSTREP band was stronger than predicted, and two bands were identified at DNA concentrations of 5, 7.5, and 10 nM, which could be due to glycosylation. Finally, TIGIT_nSTREP showed bands stronger than predicted at all concentrations except 2.5 nM, which is a sign of possible glycosylation.

[0108] Blots were performed to test the reproducibility of the experiments using the two best DNA concentrations previously analyzed by blot for the cSTREP and nSTREP samples (Figure 7C). For each construct, a difference in band intensity between the C-terminal and N-terminal fusions was observed. PVR_cSTREP showed stronger band intensity and two bands that could be the result of glycosylation, while PVR_nSTREP was not as strong and had only one band. TIGIT_cSTREP did not show a band, while TIGIT_nSTREP showed a band much stronger than predicted, probably due to glycosylation. Finally, the ACE2_cSTREP band intensity was lower than that of ACE2_nSTREP, where a band stronger than predicted was visible. From this blot, the best concentration (5 nM) was selected and used for protein solubilization and ELISA experiments.

[0109] Western blot was also used to confirm the expression of the N-terminal HaloTag® proteins of PVR and TIGIT. Bands of the predicted sizes of PVR (81 kDa) and TIGIT (62 kDa) were visualized on the blot (Figure 8). However, higher levels of degradation were visible for these proteins. For PVR, two different clones were used for the expression analysis on the blot, and both showed similar results. Protein expression analysis showed that PVR_c / nSTREP, TIGIT_nSTREP, ACE2_c / nSTREP, PVR_nHALO, and TIGIT_nHALO were successfully expressed within ALiCE®. The GST-tagged construct and TIGIT_cSTREP did not express. The results indicated that the Strep-Tag® II protein construct functioned best in protein expression. Therefore, the Strep-Tag® II construct was selected to continue the following experiments.

[0110] 2.2 DDM Cannot Dissociate MP from Microsomal Membranes Microsome solubilization analysis was performed as described above to find out whether the microsomal membranes could be disrupted using mild detergent (1% DDM) and whether TP could be solubilized. Expression of the lysate after addition of DDM and analysis of the centrifuged fractions were performed by Western blot. The DNA concentration used was determined by the previous Western blot analysis (Figure 7C).

[0111] Blot analysis of microsomal solubilization assays of C-terminal Strep-Tag® II PVR and ACE2 proteins was performed (Figure 9A). All fractions showed bands of the predicted sizes, but the intensities differed between fractions. As expected, higher band intensities were seen in fractions P1 and P2 corresponding to microsomal membrane residues. However, proteins could also be detected in fraction S1 with higher intensity for the PVR_cSTREP sample. Only one band was visible for S1 PVR_cSTREP, which, if the protein was present, would have corresponded to the non-glycosylated protein. Fraction S2 showed faint bands but was not strong enough to estimate the solubility of these proteins.

[0112] N-terminal Strep-tag® II PVR, TIGIT, and ACE2 were analyzed on a separate blot (Figure 9B). In all samples, bands of the predicted sizes were only visible in fractions P1 and P2. These fractions corresponded to microsomal membrane residues. ACE2_nSTREP showed low-intensity bands in S2 that were not strong enough to estimate the solubility of the protein.

[0113] TP was not solubilized when 1% DDM was used (Figure 8) and remained in fractions P1 and P2 where microsomal membrane residues were predicted. Mild detergents such as DDM are unable to dissociate membrane proteins from the microsomal membrane, and MPs bound to the membrane may still be in the pellet fraction. More powerful detergents may be required to solubilize TP.

[0114] 2.3 In HTS, Correctly Folded Microsomal PVR Can Be Captured Indirect ELISA experiments were performed to capture microsomes expressing PVR and find the orientation of the protein. Furthermore, the protein structure and its binding to its ligand (TIGIT) were tested as described above. PVR capture was performed using C-terminal Strep-Tag® II. The protein structure and binding activity were confirmed using a structural antibody and commercially available TIGIT later.

[0115] By measuring the absorbance, a graph (Figure 10A) was constructed with the corresponding dilution factors and absorbance. As the sample was concentrated, the absorbance value increased. Thus, the more the lysate was concentrated, the more microsomes expressing the protein were captured on the plate, and subsequently, more protein was able to bind to the structural antibody, resulting in blue coloring. The absorbance value of PVR_cSTREP was higher than that of PVR_nSTREP. The samples were visualized by adding a detection reagent that resulted in blue coloring to the wells (Figure 10B). Color was visible in both samples, but the PVR_cSTREP sample showed a stronger color than the PVR_nSTREP sample. Thus, the C-terminal tagged protein was captured on the plate at a higher concentration than the N-terminal tagged protein. In the case of PVR_cSTREP, the second line of the well showed a difference in coloring compared to the first and third lines. This could have been the result of an error with the multichannel pipette. Further tests regarding protein binding were performed using commercially available TIGIT, but the samples did not show color. PVR and TIGIT did not bind.

[0116] The indirect ELISA experiment showed that microsomes expressing PVR were captured on immediately available Strep-Tactin® ELISA plates (Figure 10). Furthermore, since samples of the cSTREP-tagged protein showed more coloring (Figure 10B) and higher absorbance values (Figure 10A) than samples of the nSTREP-tagged protein, the protein was oriented as predicted at the C-terminus outside the microsomes. PVR was correctly folded and showed color when bound to its structural antibody, but was unable to bind to commercially available TIGIT. The experiment could not be statistically analyzed due to the absence of a standard curve, but a qualitative analysis was performed. Further experiments need to be conducted to compile more data and create a standard curve for quantifying the results. Additionally, the binding between PVR and TIGIT should be analyzed.

[0117] 2.4 In HTS, Correctly Folded Microsomal ACE2 That Effectively Binds to RBD Can Be Captured The indirect ELISA experiment was used to capture microsomes expressing ACE2 and test the binding of the protein to its ligand (RBD). The ACE2 protein was captured using the C-terminal Strep-Tag® II. The structure and binding efficiency of the protein were determined using commercially available RBD (His-tagged) and purified RBD together with the corresponding detection antibody.

[0118] By measuring the absorbance of the samples, a graph representing the absorbance and dilution factor of the samples was constructed (Figure 11A). For ACE_cSTREP, the absorbance measurements increased as the samples were concentrated, indicating that more protein was captured. ACE_nSTREP showed very low values close to 0 for all dilutions. The absorbance values were higher for ACE2_cSTREP than for ACE2_nSTREP. Higher concentrations of the C-terminal tagged protein were captured on the plate than the N-terminal tagged samples. The ELISA plates were visualized by adding the detection reagent to the samples (Figure 11B). A clearer and brighter color could be observed for ACE_nSTREP, while ACE_nSTREP showed little to no blue coloring. This demonstrated that microsomes expressing the C-terminal tagged protein were captured. Furthermore, the coloring was indicative of an effective binding between ACE2 and RBD.

[0119] The purified RBD did not show any coloring when developing the experimental test. The purified RBD was generated in the laboratory and the successive thawing of the samples could result in inactive proteins, which could be the reason for the failure.

[0120] Using an indirect ELISA experiment with a readily available Strep-Tactin® plate, it was demonstrated that ACE2 was synthesized as predicted at the C-terminus outside the microsome, enabling microsome capture (Figure 11). The ACE2 protein was oriented at the C-terminus outside and the N-terminus inside the microsome. Furthermore, after adding a commercially available His-tagged RBD and detecting it with an anti-His antibody, it was revealed that ACE2 was properly folded and effectively bound to its ligand. Since there was no standard curve, the experiment could not undergo statistical analysis. All of the results provided corresponded to the qualitative data analysis obtained from the experiment.

[0121] 3. Discussion The results of the proof-of-concept experiment indicate that in the BY-2-derived CF-based ALiCE®, it is possible to successfully express selected TPs such as PVR, TIGIT, and ACE2. Furthermore, microsomes expressing the C-terminal Strep-tag® II PVR and ACE2 were captured on an ELISA plate. Microsome capture using TPs is a novel experimental design that enabled the testing of these TPs and further development of the basis for a high-throughput drug target screening platform. ACE2 was expressed, properly folded, and showed effective binding to its ligand, the RBD of SARS-CoV-1.

[0122] It has been shown to be available by cloning the gene of interest into the pALiCE02 vector in order to express the target TP within ALiCE (registered trademark). Four different templates were designed for each TP fused with two C-terminal tags and two N-terminal tags. Tags are commonly used to improve protein production or confer new properties for the characterization of target proteins. Here, tags were used to capture microsomes and test the orientation of proteins. The Strep-Tag (registered trademark) II construct showed good expression. Although GST is frequently used in assays of protein expression, affinity and solubility and is the most common fusion tag for pull-down assays, none of the GST-tagged constructs showed expression. One hypothesis that could explain the lack of expression of GST-tagged proteins may be related to the nature of the GST protein. GST has a tendency to dimerize, which can lead to aggregation of certain target proteins, especially oligomeric proteins. Therefore, GST is a poor choice for tagging oligomeric proteins. PVR, TIGIT, and ACE2 are oligomeric proteins, and when fused to GST, they may form large complexes or aggregates that interfere with protein expression.

[0123] In the proof-of-concept experiment, it was shown that the present invention overcomes the greatest challenge of producing a TP that is incorporated into the lipid bilayer and synthesized into a correctly folded structure. In ALiCE (registered trademark), a preferred BY-2 lysate according to the present invention, endogenous microsomes are derived from the ER and Golgi. Here, to target microsomes to TP, a melittin signal peptide (MSP) was fused to each construct. MSP can target microsomes to TP (Stech et al., 2014). Here, microsomes were effectively targeted to TP, TP was embedded in their membranes, and TP was incorporated into the lipid bilayer. However, such an MSP is not essential, and whether an MSP is required for capture depends on the TP in question. Furthermore, in ELISA experiments, it was demonstrated that TP was synthesized at the C-terminus outside the microsomal membrane and at the N-terminus inside the microsomes, in line with the hypothesis. This enabled the development of a novel technique capable of capturing microsomes using a C-terminal fusion tag and developing binding and competition assays.

[0124] In particular, for therapeutic proteins that can be identified as ligands and thus drug candidates in this specification, post-translational modifications (PTMs) such as glycosylation play an important role in protein-protein interactions. Achieving glycosylation that enables functional proteins is another challenge for protein production. Here, the glycosylation of glycoproteins PVR, TIGIT, and ACE2 was tested. Since ALiCE® is a CFPS system derived from BY-2 Nicotiana tabacum cells, PTMs can be different from the native state of TP. In the expression analysis in ALiCE®, signs of possible glycosylation were shown based on the predicted differences in protein size. In the case of ACE2 in the c / nSTREP construct, the TIGIT_cSTREP construct and the PVR_cSTREP construct. Furthermore, the ELISA test demonstrated that the protein was correctly folded, indicating that the correct PTM was performed because the protein could bind to either its ligand (ACE2-RBD) or the structural antibody (PVR-anti-CD155). The presence of glycans was tested by treatment of the protein of interest with PNGase F, which indicates the presence of N-glycans. Western blot analysis (Figs. 7-11) confirmed possible PTMs, such as glycosylation, on the expressed protein. Thus, the present invention produces effectively and properly folded proteins without any additional steps required for glycosylation of TP in the prokaryotic system (ECE).

[0125] Drug development of TP as well as biochemical and structural tests require the expression of large amounts of proteins. In vivo systems have been the preferred systems for protein production so far. Chinese hamster ovary (CHO) cells are the most common and standardized eukaryotic cell line for protein production (Thoring et al., 2016). However, CHO cells have some limitations regarding TP production. TP is a protein that is difficult to express in living cells, and the large-scale production of these proteins has an adverse effect on cell growth and ultimately death. Many different approaches to overcome this limitation have been tested in in vivo systems, but ultimately they are time-consuming and expensive. Here, it was shown for the first time that BY-2 lysate (commercially available as ALiCE®) is effective and can replace the in vivo system for the expression of the target TP without falling into the limitations of the in vivo system. Furthermore, ALiCE® was proven to be a rapid tool for TP production where the target plasmid and the lysate are the only requirements for the experimental setup.

[0126] Treatment of microsomes with mild detergents has proven that membrane proteins do not dissociate from the lipid bilayer of microsomes. Therefore, solubilization of TP is not possible and stronger detergents should be used. Currently, the most used technique for TP solubilization involves the use of detergents (Salipro Biotech AB, 2018), but there are many drawbacks associated with them. For example, the use of detergents is related to protein instability and low applicability in structural and biophysical tests (Salipro Biotech AB, 2019). Another technique that has been used for over 15 years is nanodiscs. Nanodiscs provide a lipid environment but do not bind strongly to transmembrane proteins, thereby limiting their use. A new technique called Salipro® technology enables the reconstitution of purified TP into a lipid environment and has been used in protein purification in recent years. ALiCE® is a good TP production system, but it has not been possible to achieve TP purification using mild detergents. TP has been proven to be stably embedded within the lipid bilayer of microsomes according to the present invention. Using the production system of the present invention, preferably ALiCE®, as an expression system for TP and combining it with Salipro® technology could be a future evaluation of TP characteristics.

[0127] Until recently, due to many problems associated with this technology, CFPS systems were not part of the drug discovery pipeline. However, this will change with the present invention, which provides an optimized and standardized system for high-throughput screening (HTS) of ligands of TP embedded in lipid bilayers. This result demonstrates the potential of the HTS of the present invention as a drug target platform based on TP. The TP expression, microsome capture, and interaction (binding) assays according to the present invention enable setting the principle of the HTS drug screening platform based on TP within ALiCE®. Competitive assays using promising compounds that efficiently blocked the binding between ACE2-RBD, such as the monoclonal antibody (mAb) CR3022 (Tian et al., 2020), and chemicals such as corilagin (Yang et al., 2021), are the next step for developing HTS. Furthermore, lithyronine (Zhou et al., 2020), which was proven to block the binding between TIGIT-PVR, served as a control for the PVR-TIGIT assay and helped to find new potential compounds. Along with the development of binding and competitive assays, the combination of TP production in the CFPS system ALiCE® will form the basis of the HTS platform. These platforms can transform the pharmaceutical industry and accelerate the entire drug discovery process. In conclusion, it is shown here that the use of the CF system ALiCE® is an easy and rapid system that replaces prior art systems for TP production.

[0128] Example 2 Functional Expression of Composite Peptide, Human Epidermal Growth Factor (hEGF) Human epidermal growth factor (hEGF) has been widely studied for its potential ability to promote the rapid healing of severe injuries such as cuts, burns, and diabetic ulcers. Although hEGF has promising potential clinical value, the growth factor is limited in the treatment of chronic diabetic ulcers due to its high manufacturing cost and insufficient stability. Mature hEGF is a 53 - amino - acid complex peptide with three intramolecular disulfide bonds, and mature hEGF was selected as another model protein with a transmembrane domain to induce the ability of the BY - 2 system as an expression host for bioactive hEGF.

[0129] The interaction of hEGF was studied using surface plasmon resonance (SPR) as a spectroscopic assay, where the detection of binding was used.

[0130] The hEGF gene with an N - terminal Strep - II tag was cloned into pALiCE01 and pALiCE02 to evaluate both cytosolic and microsomal expression (Figure 15). Microsomal hEGF expression was observed, but not cytosolic expression, indicating that microsomal targeting is essential for hEGF production from ALiCE®. Single - step streptavidin affinity purification was sufficient to extract soluble hEGF in high purity (Figure 15). The binding of the purified protein to the homologous epidermal growth factor receptor (EGFR) was used as a surrogate measure of bioactivity and analyzed by SPR (Figure 12). hEGF expressed within BYL showed equivalent or improved binding kinetics compared to a commercially available hEGF reference product expressed from Escherichia coli, suggesting correct folding and disulfide - bond formation. The prokaryotic CFPS system requires extensive manipulation to enable disulfide - bond formation, which can be avoided by using BYL microsomes.

[0131] Example 3 Folding, Disulfide Bonding and Activity of Composite Multi-subunit Proteins Targeting Microsomes The inventors have shown that BYL can produce functional eYFP and GOx on various scales, and that diverse functional proteins such as multipass transmembrane GPCRs and small soluble peptides may also exist. Protein function is essentially related to correct folding, but further molecular characterization is needed to demonstrate that the microsomes of this eukaryotic CFPS system can produce recombinant proteins of the same quality as cells and with batch-to-batch consistency.

[0132] Therefore, for further testing of folding and post-translational modification capabilities, a diverse panel of model proteins was selected: the enzyme GOx, a homodimer with eight N-glycosylation sites each, containing 80 kDa monomers covalently linked by disulfide bonds; the receptor-binding domain (RBD) of the spike protein from the SARS-CoV-2 virus, a 52 kDa monomer with four intramolecular disulfide bonds and two N-glycosylation sites; and the therapeutic monoclonal antibody adalimumab (Humira®), a 150 kDa complex heterotetramer containing two heavy chains and two light chains, with two N-glycosylation sites, twelve intramolecular disulfide bonds, and four intermolecular disulfide bonds.

[0133] The genes for the heavy and light chains of adalimumab were directly cloned into the pALiCE02 vector for microsomal targeting, and the genes for RBD and GOx were similarly prepared using an additional N-terminal Strep-II tag. Protein expression was carried out in 5 mL of reaction solution using three independent batches of BYL, and the resulting microsomes were processed by dodecyl maltoside detergent treatment for protein release and subsequent protein A or streptavidin affinity purification. In the case of adalimumab, a 1:1 ratio of heavy and light chain pALiCE02 plasmids was sufficient to produce full-length IgG molecules at the predicted 150 kDa ratio. On non-reducing SDS-PAGE gels, various aggregates of heavy and light chains were also visible, but on analytical size exclusion chromatography, a single major peak was separated for all three batches. In comparison with commercially available adalimumab reference products expressed from CHO cells (BYL-mAb and CHO-mAb, respectively; Figure 15), the functional binding activities of these samples to the TNFα ligand and the Fc-gamma receptor CD64 were evaluated by SPR.

[0134] Assay Setup: CM5 S-Series Chip (Biacore / Cytiva) functionalized with recombinant protein A (P7837 / lot number SLBT1697, Sigma) using an EDC / NHS Coupling Kit (Biacore / Cytiva BR-1000-50). Conditions: Temperature 25°C, flow rate 30 μl / min, buffer: HBS-EP, pH 7.4

[0135] Reagents: huCD64 / human FcRI, (R&D Systems 1257FC / lot number MOM2119041, 50 kD, reference IgG adalimumab (Humira) (ABIN5668145), human TNFα, 3 batches of Humira (Alice) from Lenio.

[0136] Dynamic Experiments: Stepwise dilution of the ligands (CD64 and TNFα), starting with 30 nM of CD64 and 60 nM of TNFα, Capture of the antibody around 170 RU, On-rate 180 s, off-rate 420 s Two buffer blanks distributed across each series.

[0137] Results: For TNFα binding, the BYL-mAb showed a slightly stronger affinity than the CHO-mAb, with an average KD(M) value of 1.34×10-10 compared to 1.85×10-10. Conversely, the BYL-mAb showed a weaker binding to the CD64 receptor than the CHO-mAb (3.53×10-10 compared to 1.58×10-10). Considering that the mechanism of action of adalimumab is the neutralization of free TNFα, this enhanced ligand binding and weak receptor binding may be combined for improved therapeutic effects when this monoclonal antibody is produced in BYL compared to mammalian cell lines. The data are summarized below and shown in Figure 15:

Table 11

Table 12

[0138] Example 4 N-linked Glycosylation of Glycoproteins Expressed within ALiCE® Previous reports of glycoproteins produced by eukaryotic CFPS have demonstrated the presence of N-glycosylation using a simple SDS-PAGE mobility shift after PNGase F-mediated deglycosylation. However, due to the lack of sample amounts required for mass spectrometry characterization, no detailed analysis of the composition of these N-glycans has been provided. The success of the BYL system adjustment has enabled sufficient samples for this more detailed investigation, so the inventors analyzed the N-glycan structures of three GOx, RBD, and adalimumab preparations that were also used for the characterization of disulfide bonds.

[0139] After PNGase F cleavage of the N-glycans, liquid chromatography electrospray ionization tandem mass spectrometry identification and structure assignment were performed (LC-ESI-MS / MS; Figure 13). This test revealed a high degree of N-glycosylation occupancy and reproducibility between batches, regardless of the protein model. The N-glycan profiles were broadly consistent across all N-glycosites, demonstrating the predominant Man8 species. Similar to recombinant proteins produced across Nicotiana species plants, plant-specific glycosylation features such as α1,3-fucosylation and β1,2-xylosylation were also observed at some N-glycosites, particularly within the RBD protein. One glycosylation position of the GOx protein was not detected and was therefore not characterized. This site was located within a weakly charged peptide of approximately 50 amino acids and was presumed to have hindered detection within the mass range of the MS instrument.

[0140] Example 5 Bioactive Expression of Vaccine Subunit Candidate, SARS-CoV-2 Spike Protein Receptor-Binding Domain Here, the inventors showed that the BYL system can produce recombinant SARS-CoV-2 spike protein RBD with suitable disulfide bonds and consistent inter-batch N-glycosylation within 48 hours. This protein is considered an important target for developing SARS subunit vaccines. Indeed, most of the potent neutralizing antibodies against SARS-CoV-2 target the RBD (REF). Reports also suggest that the full S1 subunit of the spike may be a more potent vaccine as it induces higher levels of IgG and IgA antibodies than RBD alone (REF). To challenge this concept, the inventors expressed both the RBD and this S1 subunit, derived from the pALiCE02 vector and having different HaloTag and Strep-II tag conformations.

[0141] Using SPR, initial confirmation of functional RBD binding to the human receptor angiotensin-converting enzyme 2 (hACE2) was obtained for the N-terminal Strep-II-tagged RBD (Figure). From the analysis, it was revealed that the RBD produced within ALiCE binds to the immobilized hACE2 receptor with a higher affinity (Kd = 32 nM) than the RBD proteins produced within mammalian and human cells (Figure). Next, the antigen reactivity against two commercially available antibodies was evaluated and selected based on the recognition of any linear conformational epitope. Similar reactivity of both antibodies against the RBD and S1 recombinantly expressed from both BYL CFPS and HEK293 cells was observed. Importantly, the reactivity against S1 was weaker than that against RBD. Finally, to demonstrate the true biological relevance of the proteins produced within BYL, the inventors found a clear serological reactivity of the antigen samples (Figure 14). A compatible ELISA workflow using SARS-CoV-2 patient serum antibodies showed that the RBD produced within BYL was directly comparable to that of the RBD produced within HEK293 cells. Interestingly, high reactivity was observed in COVID-19 patient samples, but lower reactivity was observed in samples collected from control subjects prior to 2019, and this separation was less important for the S1 antigen. Different C-terminal Strep-II tag and HaloTag configurations did not affect binding, suggesting correct RBD folding independent of fusion protein addition. This experiment suggests that the use of RBD produced from BYL CFPS for therapeutic purposes and the adjusted expression for animal studies are the goals of future research.

[0142] Example 6 1. Expression of CD20 by BY-2 Lysate CD20 is a well-known antigen expressed on B cell lymphocytes throughout their development from the pre-B cell stage to final differentiation into plasma cells. CD20 is a transmembrane protein having four transmembrane domains, two extracellular loops (one of about 44 amino acids and a smaller one of about 7 amino acids), and intracellular N-terminal and C-terminal regions. It exists as a tetramer on the cell surface and contains one disulfide bond at positions 167 - 183, but is thought not to contain a glycosylated side. In the said region, as described by Klein et al. in 2013 and shown in FIG. 1 of Klein et al., various epitopes of CD20 are well-known for antibodies. The core epitope residues (underlined below) within the most relevant epitope region (positions 167 - 183) are shown below:

Table 13

[0143] A detailed description of the above epitopes is found on pages 24 - 29 of Klein et al. and is incorporated herein by reference. The above antibodies are commercially available. Those skilled in the art know further suitable anti-CD20 antibodies.

[0144] 1.1 Plasmid Design The sequence encoding full-length CD20 (297 amino acids; 33.1 kDa) was cloned into the 3175 bp plasmid plB0405 (FIG. 17) and tagged at the C-terminus with StrepII (about 1 kDa) (FIG. 17). For translocation to microsomes during expression, the sequence encoding the melittin signal peptide MSP (about 2.5 kDa) was also cloned into the plasmid. Furthermore, common sequences encoding the T7 promoter, AmpR promoter, AmpR and Ori were incorporated.

[0145] aa sequence of CD20 MTTPRNSVNG TFPAEPMKGP IAMQSGPKPL FRRMSSLVGP TQSFFMRESK TLGAVQIMNG LFHIALGGLL M IPAGIYAPI CVTVWYPLWG GIMYIISGSL LAATEKNSRK CLVKGKMIMN SLSLFAAISG MILSIMDILN IKISHFLKME SLNF IRAHTP YINIYN CEPA NPSEKNSPST QYC YSIQSLF LGILSVMLIF AFFQELVIAG IVENEWKRTC SRPKSNIVLL SAEEKKEQTI EIKEEVVGLT ETSSQPKNEE DIEIIPIQEE EEEETETNFP EPPQDQESSP IENDSSP

[0146] 1.2 Expression by BY-2 Lysate As described above for Example 1, Section 1.3, protein expression and Western blot analysis were performed.

[0147] 1.3 Microsome Capture, and Protein Binding Assay (ELISA) As described above for Example 1, Section 1.5, capture of CD20 embedded in microsomes was performed. Here, CD20 was captured on the surface of the microtiter plate via its C-terminus as described above and shown in Figure 1.

[0148] 1.4 Treatment of Microsomes Containing CD20 After CD20 embedded in microsomes was captured via Strep-Tactin Pate, treatment with DDM was performed as described above for Example 1, Section 1.4 to generate microsome fragments (Figure 1).

[0149] 1.5 Detection of Ligand Binding After capture, treatment and washing, the ligand was added as described above. After incubation and washing (see Example 1, 1.5), an anti-ligand antibody with HRP (see the previous examples above) was added and binding was detected by ELISA as described above.

[0150] 2. Results The expression of CD20 with C-tags is shown in Figure 18. For the ELISA antibody Rituxan®, the epitope of rituximab (see above) was used to detect CD20 in microtiter plates. Various dilutions (Dil) of BY-2 lysates after protein synthesis, 5-fold, 10-fold, 20-fold, 40-fold, and 80-fold dilutions, were used and incubated at various concentrations of Rituxan®. Figure 19 shows that even at an 80-fold dilution, sufficient CD20 embedded in the lipid bilayer of microsomes captured on the surface of microtiter plate wells was detected upon significant binding of Rituxan® to CD20.

[0151] Thus, the above results demonstrate that the assay of the present invention is feasible for complex proteins containing several transmembrane domains such as CD20. While the transmembrane domain is embedded in the lipid bilayer of microsomes by the BY-2 lysate according to the present invention, CD20 can be expressed in its correct orientation and structure. Thereby, the relevant epitope "NPSE" of Rituxan® shown in Figure 1 of Klein et al. is correctly expressed in the correct conformation and is accessible after treatment with DDM for interaction with the antigen-binding domain of Rituxan®. The interaction with Rituxan® and the binding of Rituxan® also demonstrate that the treatment with DDM was strong enough to disrupt the microsomes and gentle enough not to disrupt the transmembrane domain and epitope of CD20.

[0152] Finally, this experiment demonstrates the feasibility of the assay for antigen-antibody screening according to the present invention.

[0153] Example 7 In Example 7, the same methods and materials as described in Example 6 are used. However, the captured microsome fragments are incubated in parallel or simultaneously with a panel of various anti-CD20 antibodies (the "analyte panel" as defined herein). Such a panel includes the antibodies ofatumumab, LT20, 2H7, rituximab, obinutuzumab GA101, B1, and ublituximab listed above. The multiplex assay according to the invention is performed using a panel of various antibodies by means of a microtiter plate, a microfluidic device, or SPR consumables.

[0154] References Buntru, M., Vogel, S., Spiegel, H., & Schillberg, S. (2014). Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC biotechnology, 14(1), 1-11. Buntru, M., Vogel, S., Stoff, K., Spiegel, H., & Schillberg, S. (2015). A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates. Biotechnology and bioengineering, 112(5), 867-878. Gupta, R., & Brunak, S. (2002). Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput. 310 - 22. PMID: 11928486. Henrich, E., Hein, C., Dotsch, V., & Bernhard, F. (2015). Membrane protein production in Escherichia coli cell - free lysates. FEBS letters, 589(15), 1713 - 1722. Khambhati, K., Bhattacharjee, G., Gohil, N., Braddick, D., Kulkarni, V., & Singh, V. (2019). Exploring the potential of cell - free protein synthesis for extending the abilities of biological systems. Frontiers in bioengineering and biotechnology, 7, 248. Klein, C., Lammens, A., Schafer, W., Georges, G., Schwaiger, M., Mossner, E., Hopfner, K - P, Umana, P., and Niederfellner, G. (2013). Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties, mAbs 5:1, 22 - 33; January / February 2013; (C)2013 Landes Bioscience Sachse, R., Wustenhagen, D., Samalikova, M., Gerrits, M., Bier, F. F., & Kubick, S. (2013). Synthesis of membrane proteins in eukaryotic cell-free systems. Engineering in Life Sciences, 13(1), 39 - 48. Stech, M., Hust, M., Schulze, C., Dubel, S., & Kubick, S. (2014). Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments. Engineering in life sciences, 14(4), 387 - 398. Tian, X., Li, C., Huang, A., Xia, S., Lu, S., Shi, Z.,... & Ying, T. (2020). Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerging microbes & infections, 9(1), 382 - 385. 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-ACE2 binding. Phytomedicine, 87, 153591. Zhou, X., Du, J., Wang, H., Chen, C., Jiao, L., Cheng, X.,... & Gao, Y. (2020). Repositioning liothyronine for cancer immunotherapy by blocking the interaction of immune checkpoint TIGIT / PVR. Cell Communication and Signaling, 18(1), 1 - 14

Claims

1. A high-throughput screening of at least one ligand for at least one target transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), - A step of providing at least one endogenous microsome containing at least one lipid bilayer-embedded transmembrane protein (TP), or at least one endogenous microsome fragment containing at least one lipid bilayer-embedded transmembrane protein (TP), - A step of providing at least one analyte, - A step of bringing the at least one TP into contact with the at least one analyte, A high-throughput screening method comprising the step of detecting the interaction between the at least one analyte and the at least one TP.

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

3. The screening according to claim 1 or 2, wherein the step of capturing at least one TP embedded in the lipid bilayer of a microsome or microsomal fragment on a biologically inactive surface is performed.

4. The screening according to claim 1, wherein the at least one TP embedded in the lipid bilayer of the endogenous microsome and / or the endogenous microsomal fragment is captured on a biologically inactive surface.

5. The screening according to claim 4, wherein the biologically inactive surface is the surface of particles including microparticles, nanoparticles and magnetic particles, the surface of a device comprising a microtiter plate, a microfluidic device, a microarray, and a microchip, and / or any other surface suitable for capturing the at least one target TP.

6. The screening according to claim 1, wherein the at least one TP includes at least one tag at its C-terminus and / or N-terminus.

7. The screening according to claim 1, 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 screening according to claim 1, which is a multiple high-throughput screening of at least one ligand of at least one target TP, which simultaneously provides two or more analytes to two or more reaction zones and / or simultaneously provides analyte combinations of two or more analytes to two or more reaction zones.

9. The screening according to claim 1, wherein the interaction between the at least one TP and the at least one analyte is detected by immunoassay, spectroscopic assay, particle size measurement, magnetic measurement and / or optical measurement.

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

11. The screening according to claim 10, wherein the lysate is derived from a BY-2 cell line derived from Nicotiana tabacum.

12. Cell-free production of at least one target transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), - A step of providing a plant-derived lysate containing at least one endogenous microsome. - A step of providing at least one template encoding the at least one transmembrane protein (TP), - A step of incubating the at least one molten material with the at least one mold, - A step of expressing at least one transmembrane protein (TP), - A step of co-transcribed and co-translationally translocating the at least one expressed TP into the at least one microsome, and - A step of obtaining the at least one transmembrane protein (TP) embedded in the lipid bilayer of the at least one endogenous microsome, and Cell-free production, optionally comprising the step of processing the at least one endogenous microsome containing the at least one TP embedded in the lipid bilayer to obtain a microsomal fragment containing the at least one TP embedded in the lipid bilayer.

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

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

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

16. The method according to claim 13, wherein the biologically inactive surface is a surface of particles including microparticles, nanoparticles and magnetic particles, a microtiter plate, a microfluidic device, a microarray, a device comprising a microchip, and / or any other surface suitable for capturing at least one TP of the purpose.

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

18. An endogenous microsome or at least one endogenous microsomal fragment derived from a plant endoplasmic reticulum (ER), comprising at least one lipid bilayer-embedded transmembrane protein (TP) of interest.

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 a plant endoplasmic reticulum (ER), wherein the biologically inactive surface is the surface of a consumable.

20. A kit for high-throughput screening of at least one ligand for at least one target transmembrane protein (TP) embedded in the lipid bilayer of at least one endogenous microsome derived from the plant endoplasmic reticulum (ER), preferably from a plant of the genus Nicotiana in the Solanaceae family, Preferably a plant cell lysate containing endogenous microsomes derived from the endoplasmic reticulum (ER), derived from a plant of the genus Nicotiana in the Solanaceae family, - At least one vector encoding a known TP as a positive control, - At least one vector for at least one template that codes for at least one TP of the above purpose, Optionally, additional excipients, Optionally, at least one active substance for detecting the interaction between the at least one analyte and the at least one TP, Optionally, at least one active agent for disrupting at least one of the microsomes, Optionally, a kit including an analyte panel of at least one analyte or two or more analytes.

21. The kit according to claim 20, further comprising a consumable comprising, consisting of, or coated with a biologically inactive surface, for capturing the at least one TP embedded in the lipid bilayer of a microsome on a biologically inactive surface.

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