Semi-automated high-throughput medium-scale protein expression method

Abstract

This disclosure relates to a higher-throughput, medium-scale, semi-automated protein expression and screening platform that can be used, for example, for drug discovery research and, among other uses, for testing protein expression conditions. The workflows described herein also enable comprehensive expression and purification screening evaluation of difficult or hard-to-express recombinant proteins in a faster and more efficient manner by delivering small but sufficient amounts of high-quality protein in some embodiments.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 63 / 579,668, filed on 30 August 2023, and U.S. Patent Application No. 63 / 645,754, filed on 10 May 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] field This disclosure relates to a higher-throughput, medium-scale, semi-automated protein expression and screening platform that can be used, for example, for drug discovery research and, among other uses, for testing protein expression conditions. The workflows described herein also enable comprehensive expression and purification screening evaluation of difficult or hard-to-express recombinant proteins in a faster and more efficient manner by delivering small but sufficient amounts of high-quality protein in some embodiments. [Background technology]

[0003] background Recombinant protein production is a crucial component of both basic and drug discovery research. As therapeutic targets become more complex, researchers are constantly discovering innovative expression and purification techniques to improve the production of hard-to-express proteins and difficult multi-protein complexes in a more rapid and cost-effective manner. While high-throughput expression analysis on a small scale is rapid and efficient for triaging most types of protein targets, it often provides insufficient protein yield and information for hard-to-express targets, requiring costly large-scale production for additional characterization, which poses a significant obstacle to drug discovery research. Researchers often need to produce tens or even hundreds of protein variants to tackle such complex drug targets, which are intensive in terms of both resources and time. This necessitates the development of efficient and effective upstream screening strategies to narrow down the optimal constructs as well as expression and purification conditions to support such targets. Furthermore, the traditional toolset for drug discovery is also expanding beyond the traditional small and large molecule boundaries. Emerging therapeutic modalities such as degradation inducers, macrocycle peptides (MCPs), and disulfide-restricted peptides (DCPs) may be explored in parallel at an early stage to address drug-unavailable targets, which often require milligrams of high-quality protein. One exemplary expression approach is to generate and screen a series of constructs using multiple expression systems to identify the most appropriate constructs and systems for producing sufficient quantities of stable and functionally active proteins for structural and functional studies.Several laboratories are developing automated and bioinformatics tools to enable high-throughput small-scale protein expression analysis platforms, allowing for rapid triage of multiple constructs in parallel across different expression systems (Esposito, Garvey, and Chakiath, 2009; Marsischky and LaBaer, ​​2004; Chambers, Austen, Fulghum, and Kim, 2004; Festa, Steel, Bian, and LaBaer, ​​2013; Gileadi et al., 2008; Hunt, 2005; Kraft et al., 2019). However, small-scale approaches have certain drawbacks. For example, the flexibility and screening options offered by small-scale approaches are limited in dealing with underexpressed proteins and multiprotein complexes, particularly those with unstructured domains, prone to aggregation, and requiring additional co-expression partners to be properly expressed, folded, and functional. [Overview of the project]

[0004] overview Therefore, this application provides a novel method for high-throughput protein expression on a larger scale, which can address the shortcomings associated with previous smaller-scale approaches. For example, the method described herein addresses the limitations of small-scale approaches and provides a faster, more effective, higher-throughput triage solution for screening optimal expression and purification conditions for challenging target proteins. In some embodiments, the method herein enables parallel expression, purification, and characterization of 12 to about 100 samples from cell cultures, for example, to identify optimal constructs and appropriate conditions for underexpressed proteins, evaluate co-expression partners in multiprotein complexes, and enable buffer / additive screening for insoluble and agglutinating proteins, among other applications. In addition to protein screening, the method herein also provides small but sufficient quantities of high-quality proteins for screening for desired downstream applications, such as negative staining, biochemical activity assays, DNA binding assays, affinity pulldown, DNA coding compound library (DEL) screening, and surface plasmon resonance (SPR) screening, among others. The methods described herein can also be broadly applied with minimal protocol modifications to all types of targets and expression systems, including intracellular proteins, secretory proteins, and membrane-bound proteins expressed in Escherichia coli, BEVS, and mammalian systems. The methods may also be used to express full-length proteins, specific domains, mutant proteins, or chimeric proteins, and can be used with various peptide affinity tags or fusion partners such as polyhistidine, FLAG, glutathione-S-transferase (GST), and maltose-binding protein (MBP) (see Kimple, Brill, and Pasker, 2013). Furthermore, the methods described herein can provide an efficient construct triage platform in parallel across multiple expression systems for moderately to highly expressed, well-folded single-subunit proteins. For example, gel analysis can be used to confirm the presence of full-length or truncated proteins, identify their molecular weight, and provide information on sample purity.Additionally, the methods described herein may be used to provide recommendations for the best expression systems, cell lines, and tagging strategies for successfully expressing a particular protein or domain on a large scale.

[0005] Exemplary embodiments of this specification include the following: 1. A method for purifying one or more polypeptides from multiple mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples, (a) Growing multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of cells in a cell culture sample, where it is necessary to separate the expressed polypeptide(s) from the cell debris, such as when the polypeptide is not secreted by the cell, and optionally, parts (a) and (b) for each of multiple cell culture samples are performed in the same container. (c) Centrifugation of the lysed cell culture sample and collection of multiple supernatant samples from the cell culture sample, (d) Clarifying multiple supernatant samples by filtration such as deep filtration, (e) Placing multiple clarified supernatant samples in the wells of a multi-well plate, wherein each supernatant sample has a volume of 2 to 30 mL, and the multiple clarified supernatant samples are placed in the wells. (f) Subjecting multiple supernatant samples in the wells of a multiwell plate to affinity chromatography performed using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a multiwell plate (i.e., the same or different multiwell plate), wherein part (f) or part (e) and (f) are performed in parallel on multiple samples, subjecting multiple supernatant samples to affinity chromatography and placing the multiple supernatant samples. Methods that include... 2. Part (f) of the method, or part (e) and (f) The method according to Embodiment 1, wherein the process is automated. 3. The method according to Embodiment 1, wherein parts (d) to (f), parts (e) and (f), or all of parts (a) to (f) are carried out in parallel. 4. The method according to any one of Embodiments 1 to 3, wherein multiple clarified supernatant samples are placed in the wells of a multi-well plate in a volume of 2 to 30 mL per well. 5. A method for purifying one or more polypeptides starting from multiple clarified cell culture supernatant samples in a multiwell plate, wherein the multiple clarified cell culture supernatant samples are obtained from the multiple cell culture samples. (a) Growing multiple mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of cells in a cell culture sample, where it is necessary to separate the expressed polypeptide(s) from the cell debris, such as when the polypeptide is not secreted by the cell, and optionally, parts (a) and (b) for each of multiple cell culture samples are performed in the same container. (c) Centrifugation of the lysed cell culture sample and collection of multiple supernatant samples from the cell culture sample, (d) Clarifying multiple supernatant samples by filtration such as deep filtration, (e) Placing multiple clarified supernatant samples in the wells of a multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL per well. It is obtained by a process that includes, A method comprising subjecting a supernatant sample obtained from processes (f)(a) to (e) to affinity chromatography performed using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a multiwell plate, wherein the method is performed in parallel for multiple clarified supernatant samples, and optionally the method is automated. 6. A method for affinity purification of polypeptides from multiple cell culture supernatant samples expressing one or more polypeptides to be purified, The method involves obtaining a multi-well plate containing multiple clarified cell culture supernatant samples in wells at a volume of 2-30 mL per well, subjecting the multiple clarified cell culture supernatant samples to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a second multi-well plate, wherein the method is performed in parallel for multiple supernatant samples, and optionally the method is automated. A method comprising multiple cell culture supernatant samples obtained from mammalian, insect, and / or bacterial cell culture samples expressing one or more polypeptides, wherein the cell culture supernatants are grown in 20-500 mL increments and subjected to one or more of the following: lysis, centrifugation, and clarification, wherein cell growth and lysis are optionally performed in the same container. 7. The method is (g) Performing size exclusion chromatography (SEC) on the eluate from affinity chromatography, (h) Fractionating polypeptides from SEC into wells of a multiwell plate The method according to any one of embodiments 1 to 6, further comprising, optionally, one or both of parts (g) and (h) being automated. 8. The method according to Embodiment 7, wherein (g) and (h) are automated. 9. The method according to any one of Embodiments 1 to 8, wherein a clarified supernatant sample corresponding to a single cell culture sample is placed in two or more wells of a multiwell plate. 10. The method according to any one of Embodiments 1 to 9, wherein clarified supernatant samples from different cell culture samples are placed in different wells of a multiwell plate. The method according to any one of Embodiments 1 to 10, wherein 8 to 96 clarified supernatant samples, such as 11.8 to 48 samples, 8 to 24 samples, 12 to 48 samples, or 12 to 24 samples, are processed in parallel. 12. The method according to any one of Embodiments 1 to 11, wherein the cells are lysed by adding glass beads while shaking, and / or the cells are not lysed by ultrasonication. 13. The method according to any one of Embodiments 1 to 12, wherein the plurality of cell culture samples are grown at a scale of 30 to 250 mL, 50 to 250 mL, 30 to 200 mL, 50 to 200 mL, or 100 to 200 mL. 14. The method according to any one of Embodiments 1 to 13, wherein the pipette tip comprising the affinity matrix has a volume of 0.5 to 2 mL, such as 1 to 2 mL, or 0.5 to 1.5 mL, or 0.5 mL, or 1 mL, or 1.5 mL, or 2 mL. 15. The method according to any one of Embodiments 1 to 14, wherein the affinity matrix in the pipette tip has a bed volume of 30 to 100 µL, such as 30 to 70 µL, or 40 to 50 µL, or 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, or 100 µL. 16. The method according to any one of Embodiments 1 to 15, wherein the polypeptide is tagged with a polyhistidine, FLAG, streptavidin, glutathione-S-transferase (GST) or maltose-binding protein (MBP) tag, and the affinity matrix recognizes the tag. 17. The method according to any one of Embodiments 7 to 16, wherein the method comprises steps (g) and (h), and SEC chromatography is performed on an SEC matrix comprising particles having a pore size of 140 to 500 angstroms, and / or a particle size of 3 to 5 microns, and / or a molecular weight range of 5 to 700 kDa. 18. The method according to any one of Embodiments 1 to 17, further comprising performing structural or functional analysis on the purified polypeptide, such as cryo-electron microscopy, mass spectrometry, protein-protein interaction assays such as surface plasmon resonance, or homogeneous time-resolved fluorescence assays. 19. The method according to any one of Embodiments 1 to 18, wherein the one or more polypeptides comprise a recombinant protein complex. 20. The method according to any one of embodiments 1 to 19, wherein the one or more polypeptides do not comprise an antibody or an antibody subunit. 21. The method according to any one of embodiments 1 to 20, wherein the cell culture sample is an insect cell culture sample, such as an Sf9 or T.ni cell culture sample. 22. The method according to any one of embodiments 1 to 21, wherein the cell culture sample is a mammalian cell culture sample, such as a HEK293 or CHO cell sample. 23. A system for carrying out the method according to any one of embodiments 1 to 22, wherein the system comprises means for performing automated affinity chromatography in parallel on a plurality of cell culture samples having a volume of 2 to 30 mL, the samples are located in wells of a multi-well plate (e.g., an 8 to 96-well plate), the chromatography is performed using an affinity matrix in a pipette tip, and the system further comprises means for placing an eluate from chromatography into wells of a multi-well plate (such as a second multi-well plate). 24. A kit comprising reagents for carrying out the method according to any one of claims 1 to 22, wherein the kit optionally comprises one or more of: reagents for performing affinity chromatography in parallel on a plurality of 2 to 30 mL samples using an affinity matrix in a pipette tip (e.g., a tip comprising an affinity matrix, and / or one or more buffers such as an equilibration buffer, a wash buffer and an elution buffer), reagents for performing SEC chromatography on a sample (e.g., an SEC column, and / or one or more buffers such as an equilibration buffer, a wash buffer and an elution buffer), a positive control sample, a negative control sample, one or more multi-well plates for holding samples, and instructions for use.

[0006] Further objectives and benefits are partially described in the following description and can be partially understood from the description or acquired through practice. These objectives and benefits are realized and achieved by the elements and combinations specifically indicated in the attached claims. It should be understood that the above general description and the following detailed description are illustrative and descriptive only and do not limit the scope of the claims.

[0007] The accompanying drawings, incorporated into and constituting part of this specification, illustrate specific embodiments and, together with the description, help to further illustrate the specific principles described herein. [Brief explanation of the drawing]

[0008] This U.S. provisional application includes at least one drawing drawn in color. If a non-provisional application or PCT application claiming priority to this U.S. provisional application and incorporating the contents of this provisional application is to be published in the future, a copy of this provisional patent application, including the color drawing, will be provided by the Patent Office upon request and payment of the necessary fees.

[0009] [Figure 1] Diagram of the medium-scale expression and purification process. A schematic workflow outlining the steps involved in the medium-scale expression and purification of recombinant proteins: Insect cell cultures are grown in 500 mL conical tubes, cells are lysed using glass beads, clarified by deep filtration, affinity purification is performed in a 24-well plate using IMCS tips, and the proteins are further purified and characterized by size exclusion chromatography and SDS-PAGE analysis. The purified proteins from this workflow are used for several downstream applications, including negative staining and biochemical assays.

[0010] [Figure 2] A diagram illustrating the visual interpretation of flow cytometry results. Examples of successful and unsuccessful infections based on GP64 positivity and viable cell populations.

[0011] [Figure 3] Diagram of lysate layout for deep filtration. Cell lysates are clarified using a centrifugation and deep filtration process. An Orochem 24-well depth filter is placed on a 24-well deep-well plate, and the lysates are transferred to the filter plate. The filter-collection plate assembly is centrifugated at 980g for 5 minutes. Created with BioRender.com

[0012] [Figure 4] Diagram of lysate rearrangement. After deep filtration, the lysate is transferred to four new 24-well deep-well plates as shown above. Created with BioRender.com

[0013] [Figure 5] Diagram illustrating the deck layout. This diagram shows a Hamilton Star deck layout for medium-scale protein purification using IMCS tips. The deck holds a trough for removing storage buffer from IMCS tips, four racks of 1 mL Hamilton filter tips for transferring buffer to plates, one rack of 1 mL IMCS tips, four troughs for purification buffer, two blotting stations, ten 24-well plates and three 96-well plates used in this method. This purification method has the capacity to process up to 24 unique samples of up to 24 mL volume using up to 96 1 mL IMCS tips. The 24-well sample plates allow for the simultaneous use of up to four 1 mL IMCS tips per well, thereby increasing the amount of resin used to purify each sample. Three washing stations with three associated waste plates are incorporated to provide clean washing reagents for each washing cycle before elution. Blotting stations are performed before the washing and elution steps to remove residual droplets on the IMCS tips.

[0014] [Figure 6]Diagram of the elution plate layout. After affinity purification, the eluates are pooled together from four adjacent wells into a single well before further characterization. Created with BioRender.com

[0015] [Figure 7] A diagram describing the user interface. This custom user interface enables an efficient and accurate protein purification process. Module selection for each step within the "Purification" box allows for run restoration and maximum per-run user customization as needed. The "Sample" selection allows scientists to vary the number of sample binding plates per run. The "Reagent" selection allows for the elimination of reagent additions, which assists in buffer optimization and screening during development runs. The "Exceptions" section allows scientists to perform additional steps before the purification run, including the removal of excess storage buffer from the Integrated Microchromatography System (IMCS) tip into a water-filled trough. This helps improve liquid handling so that carryover storage buffer does not negatively impact the mixing cycle during the protein purification process.

[0016] [Figure 8] Schematic diagram of the flow path of the Thermo Fisher Vanquish® Duo HPLC system. This is a stacked system with a dual gradient pump, dual split autosampler, column compartment, and left and right variable wavelength detectors (VWD). The fraction collectors allow for fraction recovery from plates up to 4 × 96 wells. Two separate fraction collectors are used for the left and right systems. Created at BioRender.com

[0017] [Figure 9]Diagram of Vanquish Chromeleon™ 7 settings. This screen shows the UV left module on the Vanq left system. The lamps are turned on with the toggle switches below the UV and Vis lamps. Select the wavelength as 280 nm for the SEC purification run. Select a data acquisition rate of 10.0 Hz and a response time of 0.5 seconds as the default settings for all purification runs.

[0018] [Figure 10] Diagram of Vanquish Chromeleon® method settings. The Chromeleon® console screen shows how to create a "Sequence" in the "Data" tab for a sample to be run on the Vanquish left arm. For each sample, the position in the autosampler, the volume of sample to be injected into the column, and the instrument method are selected in the sequence. The instrument method is created and the parameters for each module are selected. If the same column is used, this method can be used for all purification runs.

[0019] [Figure 11] Figure showing medium-scale purification data for multiprotein complexes. Chromatogram data from SDS-PAGE gel analysis and size exclusion chromatography of multiprotein complexes purified from insect cells. Protein complexes were purified from Sf9 cells using the affinity purification protocol described for FLAG-tagged proteins. The eluate was concentrated and further washed with SEC at a wavelength of 280 nm. Fractions corresponding to peaks were further analyzed on the gel. A representative chromatogram and analyzed fraction are shown for one of the samples loaded in lane 6 of the first gel.

[0020] [Figure 12]This figure shows the size exclusion chromatography (SEC) chromatogram analysis of an 8-protein multiprotein complex purified from a conventional batch-mode large-scale purification. The column used was Superdex® 200 increase 10 / 300, and the buffer used was 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT. The proteins were purified and concentrated from a 3L culture of Sf9 cells using Affinity M2 anti-FLAG resin and ion exchange, and loaded into SEC for the final purification step. [Modes for carrying out the invention]

[0021] Detailed description of a specific embodiment 1.Definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have meanings that are generally understood by those skilled in the art.

[0022] In this application, the use of “or” means “and / or” unless otherwise specified. In the context of multiple dependent claims, the use of “or” refers only alternatively to multiple preceding independent or dependent claims retroactively. In this application, the articles “a” or “the” preceding an item generally mean that there is “one or more” such items unless the context indicates that there can be only one such item. Also, terms such as “element” or “component” include both elements and components containing one unit and elements and components containing multiple subunits, unless otherwise specified.

[0023] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integers within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0024] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges include the numerical values ​​that define that range. The headings provided herein are not limitations on the various aspects of this disclosure that can be had by referring to the entire specification. Accordingly, the terms defined below are further defined by referring to the entire specification.

[0025] When used in accordance with this disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0026] "Affinity chromatography" refers to a separation method based on specific interactions between molecules on an affinity column and specific polypeptides to be purified, such as the binding of polypeptides to ligands, or the binding of His tags or other peptide tags to metal ions or specific antibodies placed in the chromatography matrix.

[0027] Size exclusion chromatography (SEC) is a chromatography method that separates molecules based on their size.

[0028] The term “matrix” is used herein to refer to chromatographic materials such as affinity chromatography materials or size exclusion chromatography (SEC) materials. In some embodiments, the matrix may include beads or particles containing materials to which polypeptides can be selectively bound, such as those containing chelate ligands bound to nickel. In some embodiments, the matrix of affinity chromatography materials may be arranged in a column through which the material to be purified flows. In other cases, the matrix of affinity chromatography materials may be arranged in a spin column, or on a plate, chip, or other device. In some cases, the matrix may include particles such as beads or magnetic particles that can be separated from the solution, for example, by the introduction of a magnet.

[0029] As used herein, "eluate" refers to material eluted from a chromatographic matrix or column by the application of an elution buffer.

[0030] The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may include natural and / or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. These terms also include polymers of amino acids that have modifications such as glycosylation or sialylation, or that form complexes with other molecules.

[0031] The terms “isolated” or “purified” polypeptide or protein mean polypeptides that have been at least partially isolated from one or more contaminants. In some embodiments, polypeptides are purified to a purity of 80%, 90%, 95%, or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating protein and antibody purity, see, for example, Flatman et al., J.Chromatogr. B848:79-87 (2007).

[0032] As used herein, “automated” or “automatically controlled” processes are processes that can be performed by a computer-controlled system with appropriate software, for example, as opposed to systems that require active manual intervention during or between at least one step, such as moving an analyte-containing sample from one part of the system to another. In some embodiments, the process is automated by software that controls the movement or position of one or more pumps, valves, and / or tees during the course of the process, the movement or position, in turn, controls the flow of buffers and eluents through the system.

[0033] The term “sample” as used herein refers to a certain amount, volume, or portion of a protein purification product or intermediate, such as a portion of a cell culture or a portion of a cell lysate or the supernatant from a cell lysate. The amount, volume, or portion may be up to 100% of the product or intermediate. Thus, a cell culture “sample” refers to a certain amount, volume, or portion of a cell culture produced in or used in a polypeptide purification method, such as those described herein. In some cases, each cell culture sample is grown in a single container such that, in some embodiments, multiple such samples are served in parallel in the methods herein for the purification of polypeptides expressed by cells.

[0034] For example, "multiwell plates" or similar terms, such as 24-well plates or 96-well plates, refer to plates that contain several individual wells from which liquids can be dispensed.

[0035] For example, as used herein for containing affinity matrices, “pipette tip” generally refers to a tube that can be used to dispense liquids. In some cases, the top of the pipette tip can be attached to a pipetting device (e.g., a multipipette) for use in dispensing liquids into and out of the tip.

[0036] The term "plural" refers to two or more. In some embodiments, plural may also include three or more, such as 4 to 96, or a range within those numbers.

[0037] As used herein, a method step performed "in parallel" on multiple samples means that the method step is performed on multiple samples simultaneously. In some cases, the method can be performed on several samples simultaneously, for example, using automated equipment. In other cases, automated equipment is not required. For example, one means of processing samples in parallel includes using a liquid dispensing or recovery device that dispenses or recovers liquid from multiple samples at once, or using a single device such as a multipipette that can be operated manually or automatically as needed.

[0038] 2. Method This disclosure relates, for example, to methods and systems for purifying polypeptides. In some embodiments, for example, the method begins with the growth of several suitable cell culture samples on a “medium scale,” such as 20–500 mL. In some embodiments, the cell culture samples are grown on a scale of, for example, 30–250 mL, 50–250 mL, 30–200 mL, 50–200 mL, or 100–200 mL. The methods herein are adapted for insect cell cultures, mammalian cell cultures, and bacterial cell cultures. The type of cell culture may be selected, for example, based on the polypeptide intended for purification. Thus, in some embodiments, the methods herein include growing several mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples, or alternatively, purifying cell lysate samples obtained from several mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples. Thus, in some embodiments, the method includes growing insect cell culture samples to express polypeptides, or starting from cell lysates obtained from such samples. In some embodiments, the method includes growing a mammalian cell culture sample to express a polypeptide, or starting from a cell lysate obtained from such a sample. In some embodiments, the method includes growing a bacterial cell culture sample to express a polypeptide, or starting from a cell lysate obtained from such a sample. When the same type of cell culture sample is used (for example, multiple cell culture samples are all from insect cell cultures), different cell culture samples may also be grown in parallel for higher efficiency, depending on the conditions, in some embodiments. When different cell culture conditions are compared, such as mammalian cell cultures versus insect cell cultures, the cell culture samples may be grown for different times and under different conditions due to the different needs of each host cell type. In some embodiments, the cells are insect cells such as Sf9 or T.ni cells. In some embodiments, the cells are mammalian cells such as HEK293 or CHO cells.

[0039] The methods described herein are useful for purifying polypeptides from multiple medium-sized cell culture volume samples. For example, multiple different cell cultures, each containing host cells for polypeptide expression, can be grown, and then each cell culture can be purified almost in parallel for medium-sized polypeptide purification of multiple samples. In this way, for example, the methods described herein may be useful for comparing different cell culture conditions for the production of the same polypeptide. They may also be useful in the parallel rapid medium-scale production of multiple different polypeptides, such as polypeptide variants with different mutations, and as a result, all different polypeptides can be tested and compared in downstream assays. Unlike small-scale protein production methods, which may involve cell cultures of about 1-5 mL, medium-sized cell cultures like those described herein may better reflect the conditions of the final, larger cell cultures of 1 liter or more that may be used in later commercial production. Furthermore, the methods described herein enable the production of high-purity proteins on a medium scale and, as described above, allow for the parallel testing of many different cell culture conditions to aid in determining the best conditions for larger-scale production. Furthermore, in some embodiments, the medium-scale production methods described herein enable the production of, for example, 24 to 48 different protein purifies per week, and enable high-throughput analysis of multiple protein samples.

[0040] The methods described herein also allow for the growth of cell culture samples on a medium scale as described above, and enable both growth and lysis in the same container, such as a flask. In some embodiments, cells are grown and lysed in the same container, such as a flask. In other embodiments, cells are grown in one container but lysed in a different container. In some embodiments, cells are lysed using glass beads and shaking. In some such cases, cells are lysed by adding glass beads to the container in which the cells were grown, and thus the cells are grown and lysed in the same container. In some embodiments, cells are not lysed by sonication. For example, glass beads may be added to the cell culture medium at the end of the growth phase, and the container may be agitated in a shaking incubator or similar device to allow for cell lysis. Exemplary protocols for cell growth and lysis are provided in the examples described herein.

[0041] Once cells are lysed, the cell lysate (e.g., lysate) can be centrifuged in either the same container used for proliferation and lysis, or a different container, to separate cell debris from the supernatant containing one or more expressed polypeptides. Thus, multiple medium-sized proliferation, lysis, and centrifugation cell culture samples yield multiple supernatant samples after centrifugation. In some embodiments, centrifugation may be performed in parallel for multiple samples, for example, by placing them in the centrifuge in the same run or in subsequent runs performed on the same day or at approximately the same time. In some embodiments, these samples may be clarified by deep filtration or surface filtration, etc., to further remove contaminants from the expressed polypeptides. In some embodiments, clarification is by deep filtration. This then yields multiple clarified supernatant samples that can be placed in the wells of a multiwell plate and further purified, for example, by chromatography. In some cases, clarification may be performed in parallel for multiple samples. The following examples provide exemplary protocols for performing clarification, for example, by deep filtration. For example, filters may be placed on individual tubes or on the wells of a multiwell plate, or the supernatant collected after centrifugation of cell lysates may be transferred onto the filter, resulting in it flowing through the filter and being collected in the appropriate tube or well below the filter. In this way, multiple samples can be clarified by filtration and placed in tubes or wells of a multiwell plate. In some cases, especially in the case of larger cell cultures, the volume of the supernatant is large enough for one cell culture sample to be divided among several wells of a multiwell plate or among several tubes. In some cases, the supernatant from a single cell culture may be divided among several tubes or wells of the plate, but the supernatants from different cell culture samples are not combined in the same well or tube. Instead, they are processed in separate tubes or wells, for example, in parallel, so that the conditions can be directly compared.

[0042] The clarified supernatant can be recovered from the clarification process, for example, after flowing through a depth filter or other filter into the wells of a multiwell plate or into a separate tube. In this way, the cell culture, lysis, centrifugation, and clarification processes result in multiple clarified supernatant samples that are placed in a set of wells or tubes of a multiwell plate for further processing. In some embodiments, the volume of clarified supernatant placed in each well or tube is 2–30 mL per well or tube. In some cases, the volume is 2–10 mL, 4–10 mL, 4–8 mL, 10–30 mL, 10–20 mL, 5–15 mL, 15–30 mL, or 20–30 mL. In some cases, the clarified supernatant samples are already in the multiwell plate.

[0043] In some cases, cell lysate samples or clarified supernatant samples are then subjected to affinity chromatography to purify one or more polypeptides intended to be expressed by the cells for purification. In some cases, the polypeptides are tagged with appropriate tags that are recognized by affinity reagents, such as antibodies or haptens, located within the affinity matrix used for chromatography. Non-limiting examples of such tags include polyhistidine, FLAG, streptavidin, glutathione-S-transferase (GST), or maltose-binding protein (MBP) tags. An affinity matrix that specifically recognizes the selected tag may be selected. In some cases, if the cells express two or more proteins, one or more proteins may be designed to express the tag so that the affinity matrix holds the tagged protein or a protein complex containing the tagged protein. In some embodiments, the affinity chromatography matrix resides within the pipette tip, simplifying the process of adding and removing liquid from the matrix. As a result, the affinity matrix can be used in conjunction with a multipipette instrument and optionally automated. For example, if the sample is placed in a multiwell plate or tube arranged in a suitable pattern, the liquid in the wells or tube can be drawn up using a multipipette and placed into a pipette tip containing the affinity matrix. In some embodiments, the affinity matrix in the pipette tip can then be washed to remove unbound proteins. In other embodiments, the washing step is not performed. The bound proteins on the matrix can then be eluted. The eluate can optionally be placed into a new set of multiwell plates or tubes, or optionally into the original set of multiwell plates or tubes, for analysis or subsequent processing.In some cases, the pipette tip containing the affinity matrix has a volume of 0.5–2 mL, such as 1–2 mL, 0.5–1.5 mL, 0.5 mL, 1 mL, 1.5 mL, or 2 mL. In some cases, the affinity matrix in the pipette tip has a bed volume of 30–100 μL, such as 30–70 μL, 40–50 μL, or 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, or 100 μL. In some cases, the affinity chromatography process is automated, and as a result, the liquid is added to and removed from the appropriate wells, pipette tips, and tubes in an automated manner using appropriate equipment and software to direct the multipipette or similar device accordingly, if necessary. In other cases, this process is performed manually, but nevertheless, it is performed in parallel with the sample using a multipipette or the like.

[0044] In some cases, depending on the protein being purified, further purification steps may be performed after affinity chromatography as needed. For example, in some cases, size exclusion chromatography (SEC) may be performed after affinity chromatography. In some cases, SEC chromatography is performed on an SEC matrix containing particles having a pore size of 140–500 angstroms and / or a particle size of 3–5 microns and / or a molecular weight range of 5–700 kDa. Proteins may then be obtained from SEC fractions corresponding to appropriately sized polypeptides or polypeptide complexes, which may be monitored, for example, at a wavelength of 280 nanometers. Depending on the available equipment, SEC may be performed sequentially on affinity chromatography eluates from each cell culture sample, or in other cases, in parallel, and the associated liquid partitioning and fraction recovery may be automated.

[0045] Therefore, the methods for purifying one or more polypeptides described herein may begin with the growth of a cell culture sample to express the polypeptide to be purified, or with subsequent steps such as affinity chromatography of multiple cell lysate samples after centrifugation and clarification. An exemplary method described herein is a method for purifying one or more polypeptides from multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples, (a) Growing multiple mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of cells in a cell culture sample, wherein optionally, parts (a) and (b) for each of multiple cell culture samples are performed in the same container, (c) Centrifugation of the lysed cell culture sample and collection of multiple supernatant samples from the cell culture sample, (d) Clarifying multiple supernatant samples by filtration such as deep filtration, (e) Placing multiple clarified supernatant samples in the wells of a multi-well plate, wherein each supernatant sample has a volume of 2 to 30 mL, and the multiple clarified supernatant samples are placed in the wells. (f) Subjecting multiple supernatant samples in the wells of a multiwell plate to affinity chromatography performed using an affinity matrix in a pipette tip, and arranging the eluate from the chromatography in the wells of the multiwell plate, wherein part (f) or part (e) and (f) are performed in parallel on multiple samples, subjecting multiple supernatant samples to affinity chromatography and arranging the multiple supernatant samples. The method includes the following: In some cases, part (f), or parts (e) and (f), or parts (d) to (f) are automated. In some cases, only part (f), or parts (e) and (f), or parts (d) to (f) are performed in parallel (i.e., for multiple samples simultaneously). In some cases, all parts of the method are performed in parallel. In some cases, the multiwell plate may be replaced with an array of suitable tubes for holding liquid samples.

[0046] An additional example is a method for purifying one or more polypeptides by affinity chromatography, starting from multiple clarified cell lysate supernatant samples in a multiwell plate, wherein the multiple clarified cell lysate supernatant samples are derived from multiple cell culture samples. (a) Growing multiple mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of cells in a cell culture sample, wherein optionally, parts (a) and (b) for each of multiple cell culture samples are performed in the same container, (c) Centrifugation of the lysed cell culture sample and collection of multiple supernatant samples from the cell culture sample, (d) Clarifying multiple supernatant samples by deep filtration, (e) Placing multiple clarified supernatant samples in the wells of a multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL per well. It is obtained by a process that includes, The method is (f) The method includes subjecting the supernatant sample obtained from the processes of (a) to (e) to affinity chromatography performed using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a multiwell plate, wherein the method is performed in parallel for multiple clarified supernatant samples, and optionally the method is automated. For example, the method may start with a sample that has been prepared in advance after (a) to (e) above and is ready for affinity chromatography. In some cases, part (f), or parts (e) and (f), or parts (d) to (f) are automated. In some cases, only part (f), or parts (e) and (f), or parts (d) to (f) are performed in parallel (i.e., for multiple samples simultaneously). In some cases, all parts of the method are performed in parallel. In some cases, the multiwell plate may be replaced with an array of suitable tubes for holding liquid samples.

[0047] Further exemplary methods herein include a method for affinity purification of polypeptides from multiple cell culture supernatant samples expressing one or more polypeptides to be purified, comprising: obtaining a multiwell plate containing multiple clarified cell culture supernatant samples in wells at a volume of 2–30 mL per well; subjecting the multiple clarified cell culture supernatant samples to affinity chromatography performed using an affinity matrix in a pipette tip; and placing the eluate from the chromatography into wells of a second multiwell plate, wherein the method is performed in parallel for the multiple supernatant samples, and optionally the method is automated. In some embodiments, the multiple cell culture supernatant samples were obtained from mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples expressing one or more polypeptides, grown on a scale of 20–500 mL. In some embodiments, the cell culture samples were subjected to one or more of lysis, centrifugation, and clarification before use in the method, and optionally, cell growth and lysis (if lysis is performed) were carried out in the same container. For example, in some cases, the clarified cell culture supernatant sample contains polypeptides secreted by the cells, in which case it may not be necessary to lyse the cells. Instead, after sufficient proliferation, the cell culture can be simply centrifuged and / or clarified before affinity chromatography.

[0048] In any of these examples, this method may be used as desired. (g) Performing size exclusion chromatography (SEC) on the eluate from affinity chromatography, (h) Fractionating polypeptides from SEC into wells of a multiwell plate It may also include the following. In some options, parts (g) and (h) are automated.

[0049] In any of these methods, a new multiwell plate may be used at each step. In other cases, the sample may be taken from the multiwell plate, processed, and returned to the same plate. In any of these methods, in some embodiments, clarified supernatant samples corresponding to a single cell culture sample are placed in two or more wells of the multiwell plate. In some cases, clarified supernatant samples from different cell culture samples are placed in different wells of the multiwell plate. In some cases, 8 to 96 clarified supernatant samples, such as 8 to 48, 8 to 24, 12 to 48, or 12 to 24, are processed in parallel. For example, the number of samples that can be processed in parallel during affinity chromatography may depend on the number of wells in the fitted multiwell plate and, correspondingly, the number of pipettes in the corresponding multipipette for transferring the liquid. In some cases, a 24-well plate is selected so that up to 24 samples, i.e., 2 to 24, 8 to 24, 2 to 12, or 12 to 24, can be processed in parallel. In other cases, 48 ​​or 96-well plates may be used. Alternatively, an array of tubes may be used instead of plates.

[0050] In some of the above methods, cells are lysed by adding glass beads while shaking, and / or cells are not lysed by sonication. In some cases, multiple cell culture samples are grown on a scale of, for example, 30–250 mL, 50–250 mL, 30–200 mL, 50–200 mL, or 100–200 mL. In some cases, the pipette tip containing the affinity matrix has a volume of 0.5–2 mL, such as 1–2 mL, or 0.5–1.5 mL, or 0.5 mL, or 1 mL, or 1.5 mL, or 2 mL. In some cases, the affinity matrix in the pipette tip has a bed volume of 30–100 μL, such as 30–70 μL, or 40–50 μL, or 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, or 100 μL. In some cases, polypeptides are tagged with polyhistidine, FLAG, streptavidin, glutathione-S-transferase (GST), or maltose-binding protein (MBP) tags, and the affinity matrix recognizes the tags. In some cases, the method comprises parts (g) and (h), in which SEC chromatography is performed on an SEC matrix containing particles having a pore size of 140–500 angstroms and / or a particle size of 3–5 microns and / or a molecular weight range of 5–700 kDa.

[0051] In some cases, the methods described herein further include performing structural or functional analysis on the purified polypeptide, such as cryo-electron microscopy, mass spectrometry, protein-protein interaction assays such as surface plasmon resonance, or homogeneous time-resolved fluorescence assays.

[0052] In some cases, polypeptides expressed from cell culture samples include recombinant protein complexes. In some cases, polypeptides do not contain antibodies or antibody subunits. In some cases, in the methods herein, the cell culture sample is an insect cell culture sample, such as an Sf9 or T.ni cell culture sample. In some cases, the cell culture sample is a mammalian cell culture sample, such as HEK293 or CHO cells, among other examples. In some cases, a mixture of different cell culture types may be used to compare, for example, insect cell versus mammalian cell protein production. In other cases, the method may be used to compare, for example, the same host cell type grown under different media or conditions. In other cases, the same cell type with optionally the same growth and culture conditions is used, but the polypeptide is varied, for example, to compare various polypeptide variants with each other.

[0053] 3. Systems and Kits This disclosure also relates to systems capable of carrying out the methods described herein, including those described in the sections above, and kits comprising reagents or components useful for carrying out the methods herein. In some embodiments, the system may include a device capable of automating affinity chromatography in a pipette tip. In some embodiments, the system may also include a device capable of transferring multiple cell lysate samples between arrays of wells in a tube or multiwell plate, for example, after centrifugation and clarification, and optionally further capable of performing affinity chromatography on multiple samples in wells of a tube or plate by operation of, for example, a multipipette or similar components used to transfer liquids in and out of wells of a tube or multiwell plate.

[0054] The disclosure herein also includes a kit containing reagents for performing the method herein, namely, a pipette tip containing an affinity matrix, buffers for performing affinity chromatography (including wash buffer, equilibration buffer and / or elution buffer), and a control sample. The kit may also include a suitable plate or tube for holding the sample. The kit may further include instructions for use. [Examples]

[0055] Example 1: Parallel protein expression of 24 samples derived from insect cell cultures Insect cells are widely used as heterologous expression systems for producing high levels of eukaryotic recombinant proteins with simple post-translational modifications such as phosphorylation and glycosylation (Shi and Jarvis, 2007) (Jarvis, 2009). Insect cells possess cellular mechanisms that fold these proteins and target them to correct localization, providing significant amounts of soluble, non-aggregated proteins. This system has proven particularly useful for expressing any class of macromolecular assemblies, including histone methyltransferases, DNA repair enzymes, kinetochores, ubiquitin ligases, and viral capsid complexes (Abdulrahman et al., 2015; Osz-Papai et al., 2015).

[0056] This example focuses on insect cell expression and describes a medium-scale protocol used for successful expression and purification screening of intracellular multiprotein complexes. While protein co-expression using plasmids encoding a single open reading frame (ORF) is widely used for baculovirus-mediated insect cell expression, the use of multi-ORF or polycistronic plasmid vectors offers significant advantages for reconstituting protein complexes with two or more proteins, as this approach uses less virus during co-expression and also ensures uniform expression in insect cells (Snead, Wall, Ambrose, Esposito, and Drew, 2022). Overexpression of multiple proteins from a single plasmid vector can be achieved by driving transcription of each ORF using individual promoters, or by using a polycistronic construct in which a single mRNA transcript encodes multiple proteins. This example also describes the design and implementation of a two-step sequential purification process including affinity capture and size exclusion chromatography (SEC) (Figure 1).

[0057] This process is designed to be an end-to-end solution from culture to protein purification. It enables parallel expression and semi-automated purification of 24 intracellular proteins or complexes from insect cells on a 200 mL scale using affinity purification with a Hamilton STAR® liquid handler, followed by SEC on a Thermo Fisher Vanquish® Duo system. Affinity chromatography using INtip® technology with Integrated Micro Chromatography Systems (IMCS) was performed in a high-throughput manner (Kates et al., 2023) (Kates, Tomashek, Miles, and Lee, 2020). The protocol described in this example utilizes a dispersed micro-extraction INtip® platform that leverages turbulent mixing of resin within a pipette tip to increase the interaction time between resin and sample. This is combined with an automated liquid processing system (Hamilton® Microlab STAR® workstation) to enable parallel rapid purification of recombinant proteins in a consistently high-throughput manner.

[0058] Next, samples were sequentially injected, and SEC was performed on a Thermo Fisher Vanquish® Duo system connected to an autosampler that enabled two simultaneous purifications using a Horizon Dual pump, a separate injection loop, and a separate buffer line with parallel columns and detectors. This method allowed for the purification of high-quality protein complexes, which were further evaluated for downstream applications and assays to identify purified and optimized solutions for large-scale production and purification. More specific protocols related to specific steps within the platform are provided below.

[0059] Basic Protocol 1 describes a method used to generate P1 baculovirus in Sf9 cells using simultaneous transfection of the linearized baculovirus bacmid BestBac® with a target gene subcloned into a baculovirus delivery vector such as pAcGP67. This protocol also describes a method for amplifying baculovirus to generate P2 and P3 stocks in Sf9 cells.

[0060] Basic Protocol 2 describes the infection of Sf9 and T.ni cells on a 200 mL scale with P3 virus to generate biomass expressing the target protein. This also discusses a co-expression approach using simultaneous infection of cells with multiple viruses.

[0061] Basic Protocol 3 describes the preparation steps for cell lysis and INtip® affinity chromatography purification performed on a Hamilton STAR® workstation.

[0062] Basic Protocol 4 describes the SEC method developed for the Thermo Fisher Vanquish™ system to provide analytical and preparative purification on a medium scale.

[0063] Supplemental Protocol 1 describes the glycoprotein 64 (GP64) staining assay used to assess the quality of baculoviruses.

[0064] Auxiliary Protocol 2 describes an automated method and process for medium-scale INtip® affinity purification performed on a Hamilton STAR® workstation.

[0065] Auxiliary Protocol 3 describes a detailed method built using Chromeleon™ software to support the SEC components of a medium-sized platform.

[0066] Basic Protocol 1 Baculovirus production by homologous recombination This protocol assumes that the initial step of cloning the target gene(s) has been achieved. The DNA encoding the target gene was cloned into a modified version of the commercially available baculovirus transfer vector pAcGP67 (BD Biosciences, Brøndby, Denmark), adapted for high-throughput cloning. The recombinant gene was expressed as a fusion protein when cloned to one of the available restriction enzyme sites under the control of a potent baculovirus polyhedrin promoter. The expression cassette was flanked by segments of the baculovirus genome to facilitate transfer to linearized baculovirus DNA via homologous recombination in insect cells (Murphy and Piwnica-Worms, 2001). The GP67 secretion signal sequence in the pAcGP67 vector was removed for intracellular protein cloning and retained for secretion protein cloning. The same expression vector backbone was also used to construct polycistronic or multi-ORF constructs.

[0067] Strategies used to construct constructs containing two or more genes, such as the use of internal ribosome entry sites (IRESs) and self-cleaving 2A peptides, are widely used approaches for constructing polycistronic constructs because both strategies simultaneously express two or more distinct proteins from the same mRNA under the control of a single promoter. Multi-ORF constructs can also be rapidly generated from the pAcGP67 vector by subcloning the target gene using individual promoters, so that the mRNA transcript of each target gene can be generated independently for efficient overexpression of the protein complex. If higher-order protein complexes need to be achieved, the number of promoter systems requiring modification of the pAcGP67 vector can be expanded. Variable multiple gene expression stoichiometry can be achieved by using promoters with different transcription intensities. In insect cells, early or late promoters can be used to control the expression of different genes in the infection cycle. Multiple gene co-expression is context-specific, and different promoters can result in variable multiple gene expression stoichiometry.

[0068] In this example, baculoviruses were generated in insect cells by homologous recombination (Kitts, Ayres, and Possee, 1990; Kitts and Possee, 1993). The protocol described herein provides a step of co-transfection of linearized bacmid with the expression transposition vector pAcGP67 encoding the target gene(s). Co-transfection results in the generation of recombinant baculoviruses, which are then used for recombinant protein expression with higher titers (1 × 10⁻¹⁶). 8 ~1 × 10 9 The samples were amplified to pfu / mL. This protocol is optimized for high-throughput workflows and can handle the generation of up to 96 recombinant baculovirus samples at once.

[0069] material The materials for Section 1 are listed below. Recombinant transfer vector pAcGP67 or similar that expresses the target gene(s) at a concentration of 40 ng / μL (see Background Information / Basic Protocol 1). An empty transfer vector with a 40 ng / μL concentration, with a skeleton matching the vector used for virus generation, will be used as a control. BestBac (trademark) 2.0 linearized bacmid - stock concentration 0.1 mg / mL (Expression Systems #91-002) TransIT (trademark) Insect Transfection Reagent (Mirus #MIR6100) In the logarithmic growth phase, 2 × 10⁶ cells in ESF921 medium (Expression Systems #94-001F) 6 ~7×10 6 Cells derived from the Sf9 cell line, which has a cell density of cells / mL and a viability of over 95%. ESF921 insect cell medium (Expression Systems #96-001-01) heated to 27°C Thermally deactivated FBS (Thermo Fisher #16140-071) 96-well deep-well plate, sterile (Axygen #p-2mL-sq-cs) 96-well round-bottom deep-well plate, sterile (Thompson #93113-S) 24-well Axygen deep-well round-bottom plate, sterile 15mL conical tube AeraSeal(TM)(Millipore Sigma #A9224-50EA) AlumaSeal(TM)(T790080-5) 96-well Duetz sandwich cover with 0.8mm holes (Kuhner Shaker Inc. #104118) Sterile pipette tips (p20, p200, p1000) Matrix tube 1.4 mL (Catalog number 3712-11) U-bottom assay plate (Falcon #353910) Zymo Clean N'Concentrate Kit (Zymo #D4033) Sterilized reagent reservoir 70% EtOH for food disinfection 150mL sterile media bottle

[0070] Materials for Section 2: Selected pipette, preferably 8-channel (p20, p200, p1000) Orbital shaker, set to amplitude 3mm, 1000rpm, 27℃. Tabletop centrifuge (for centrifugating assay plates and DNA plates) Sterile serum pipette Laminar flow hood (Labconco Purifier BSC Class II or equivalent)

[0071] Protocol processes with process annotations: P1 fabrication: Master mixes #1 and #2 were prepared in sterile 15 mL conical tubes within a sterile laminar flow hood. The amount of master mix produced depends on the number of recombinant migration vector DNA constructs to be expressed. At least 10% extra must be prepared to ensure sufficient volume for each sample. Empty vector pAcGP67 was used as a positive control along with BestBac® linearized bacmid DNA. Untransfected cells were used as a negative control. TIFF2026529993000001.tif31170

[0072] 2.5 μL of migration vector DNA was added to the corresponding wells of a 96-well deep-well Axygen plate at a concentration of 40 ng / μl, totaling 100 ng. For larger DNA inserts (>5 kb), doubling the amount of DNA (200 ng) is recommended.

[0073] The DNA plate was centrifuged at 1000g for 1 minute to ensure no air was trapped in the liquid. 25 μL of Master Mix 1 was added to each well and mixed by gently pipetting three times. 25 μL of Master Mix 2 was added to each well and mixed by gently pipetting three times. The plate was sealed with an aluminum seal and incubated at room temperature for 20 minutes to allow the complex to form. The plate was kept in a sterile laminar flow hood during incubation.

[0074] Dissolve the Sf9 cell suspension in ESF921 medium in a 1.0 × 10⁶ solution. 6 The cell suspension was prepared at a density of cells / mL. The volume depended on the number of samples, with 550 μL used per sample. 550 μL of cell suspension was added per well to a total volume of 602.5 μL per well. The plates were sealed with AeraSeal® and covered with Duetz® sandwich covers. The plates were shaken on a 3 mm diameter orbital shaker at 1000 rpm at 27°C for 6 days. Cloth seals such as AeraSeal® allow for maximum culture aeration. Avoid porous plastic seals as they have been shown to limit oxygen intake by the hands. Duetz® sandwich covers reduce evaporation during shaking incubation. Evaporation can negatively affect transfection efficiency. The 6-day incubation period for P1 is the amount of time required for recombinant virus to acquire titer, and shortening this incubation time to less than 5 days is not recommended.

[0075] Preparation of P2 and P3: On day 6, 10 μL of P1 cell suspension was placed in a sterile 96-well Thompson deep-well plate, 1.0 × 10⁶ 6was transferred to 1000 μL of fresh Sf9 cells at cells / mL. 1010 μL is the approximate maximum volume per well possible while maintaining sufficient aeration and avoiding splashing onto the fabric seal cover. The plate was covered with an AeraSeal™ and Duetz™ sandwich cover. The plate was incubated at 1000 rpm for 4 days in a shaker with a 3 mm orbit at 27°C. At the end of the 4-day incubation period, the virus titer is 1×10 8 -1×10 9 pfu / mL should be reached.

[0076] The P1 plate was stored sealed with AlumaSeal at 4°C, so it can be reused for P2 amplification in case of unexpected situations such as contamination or incubator failure. On day 4 of P2 incubation, cells were stained with GP64 antibody to evaluate virus production (see Supplementary Protocol 1). If a flow cytometer is not available, a viral plaque assay can be performed instead of the GP64 assay. The advantage of the GP64 assay is a faster readout within 1 day, compared to 5 to 7 days required to perform a viral plaque assay.

[0077] P2 virus can be stored and used for future generation of P3 (see virus storage).

[0078] P3 virus was generated by transferring 5 μL of P2 cell suspension to 4 mL of fresh Sf9 cells at 1.0×10 6 cells / mL in a sterile 24-well Axygen deep-well plate. The plate was covered with AeraSeal™ and incubated at 300 rpm for 4 days in a 12 mm orbit shaker at 27°C. Generation of P3 virus expands the amount of virus that can be used for medium-scale expression. P3 virus can be stored at 4°C for up to 6 months.

[0079] GP64 Assay (Evaluation of Virus Production Efficiency) Collect 50 μL of cell suspension in a 96-well U-bottom plate, then proceed with GP64 staining and measurement. (See Supplemental Protocol 1)

[0080] Virus storage The plates were sealed with adhesive foil seals inside the hood. A roller was used to ensure a good seal. The plates were rotated at 3,000 × g for 10 minutes to remove cells / debris. The clarified supernatant was transferred to sterile matrix tubes. Thermally inactivated FBS was added to a 10% concentration for storage stabilization (i.e., approximately 111 μL of FBS was added to 1000 μL of clarified supernatant for P2, and approximately 444 μL of FBS was added to 4 mL of clarified supernatant for P3). The plates were stored in the dark at 4°C (baculoviruses can degrade when exposed to light). The viruses remained good for up to 12 months during storage. The viruses can be re-amplified from old stocks before use for expression. Older viruses may have reduced titers, which may affect P3 amplification. (See Table 1)

[0081] Auxiliary Protocol 1 GP64 antibody assay This protocol utilizes fluorescence-activated cell sorting (FACS) to validate high-titer baculovirus stocks. Glycoprotein 64 is a viral protein expressed on the membrane of successfully infected insect cells (Kitts and Green, 1999). This glycoprotein can be tagged with a phycoerythrin-labeled antibody to extrapolate sufficient viral titers (Volkman and Goldsmith, 1988).

[0082] material: The materials are as follows: FACS buffer (see the section on reagents and solutions) 7-AAD Viability Dye (Beckman Coulter #A07704) GP64-PE antibody (Expression Systems #97-201) CytoFLEX® sheath fluid (Beckman Coulter #B51503) or 0.2μm filtration sqH2O (substitute) AlumaSeal(TM)(T790080-5) Tabletop centrifuge (for centrifugating assay plates and DNA plates) A Beckman Coulter CytoFLEX® or similar flow cytometer equipped with a "blue" laser (488 nm). The laser should be usable with phycoerythrin and PC5.5 fluorescent dye samples.

[0083] Protocol process: The protocol used for the GP64 antibody assay is described below.

[0084] Sample preparation: Alicoate 50 μL of sample into a U-bottom 96-well plate. This should be approximately 100,000 cells. The sample volume can be adjusted lower or higher depending on the density of the culture. Cover the plate with a foil seal and rotate at 3000 rpm for 3 minutes. Carefully aspirate the supernatant, taking care not to touch the cell pellet. Dilute PE-anti-GP64 1:125 with FACS Wash (8 μL of PE-anti-GP64 per 1 mL of FACS wash buffer). Resuspend the pellet uniformly in 40 μL of FACS Wash containing PE-anti-GP64. Cover the plate with a foil seal and incubate the sample at 4°C for 20 minutes. At this point, initialize the CytoFLEX so that the system is ready to read the sample after the following steps are completed. For details, refer to the CytoFLEX operating manual. After incubation, add 150 μL of FACS wash buffer to each well. Rotate at 3000 rpm for 3 minutes. Carefully aspirate the supernatant, avoiding contact with the cell pellet. Prepare an appropriate volume of viability / dead cell staining dye mixture. The viability / dead cell staining dye mixture is 40 μL of 7-AAD per 1 mL of FACS wash buffer. Resuspend the pellet in 100 μL of diluted 7-AAD dye. Incubate the plate at room temperature for at least 5 minutes. Proceed with CytoFLEX analysis according to step 7.

[0085] CytoFLEX operation Flow cytometer settings: Set to "high speed" mode flow rate to obtain 10,000 events. Flow rate and endpoint can be adjusted as needed. Set to PE and PC5.5 channels. Ensure the flow cytometer is set to handle U-bottom assay plates.

[0086] Adjust GP64 gating and survival gating as needed to accommodate "GP64 positive / negative" and "live / dead cell" controls (Figure 2). This may drift slightly, so it is important to adjust the gating for positive (empty vector-infected cells) controls and negative (uninfected cells) controls. Allow the dye to enter the damaged cells, bind to the DNA, and stand for approximately 5 minutes until maximum fluorescence is reached. Note: If reading the plate immediately after dye addition, the 7AAD signal may be artificially low in the first few samples.

[0087] Basic Protocol 2 Generation of insect cell biomass expressing target proteins This protocol was used to generate insect cell biomass expressing the target protein. This protocol allows for the generation of 200 mL of Sf9 or T.ni cell paste expressing the target protein using baculovirus generated in Basic Protocol 1.

[0088] Insect cells were infected with baculovirus and incubated for 2-3 days to enable protein expression. The cell pellet was then harvested and frozen before cell lysis and protein extraction. The yield, recoverability, and quality of protein expression may differ between Sf9 and T.ni cells; therefore, it is desirable to test expression in both cell lines. We observed that T.ni cell lines often produce greater amounts of secreted proteins, intracellular proteins, and membrane recombinant proteins per culture volume compared to Sf9. However, Sf9 is often the preferred cell line for intracellular protein expression because a higher percentage of intracellular target protein cleavage is observed in T.ni cells. Infection profiles also differ between these two cell lines and may require separate optimization for optimal protein expression; for example, the initial density at infection, MOI, and incubation length at which optimal protein yield is achieved often differ between the two cell lines. Both cell lines exhibit robust culture characteristics, but the doubling time of T.ni cells in culture is shorter compared to Sf9, which allows for a shorter production timeline and can be an important consideration in resource and reagent usage.

[0089] material: The ingredients included are as follows: Laminar flow hood (Labconco Purifier BSC Class II or equivalent) In the logarithmic growth phase, 2 × 10⁶ cells are growing in ESF921 medium (Expression Systems catalog number 94-001F). 6 ~7×10 6 Sf9 cell line with a cell density of cells / mL and a viability exceeding 95% In the logarithmic growth phase, 2 × 10⁶ cells in ESF921 medium (Expression Systems catalog number 94-002F) 6 ~7×10 6 T.ni cell lines with a cell density of cells / mL and a viability exceeding 95% ESF921 insect cell growth medium (Expression Systems, catalog number 96:001-01) warmed to 27°C Sterile serum pipettes (5 mL, 50 mL), cell counter (Beckman Coulter Vi-CELL XR or BLU or equivalent) 1L Polycarbonate Erlenmeyer Flask with Vent Cap (Corning, Catalog No. 431147) AeraSeal (Millipore Sigma, catalog number A9224-50EA) 500mL cone-bottom polypropylene centrifuge tube (Corning, catalog number 431123) Single-channel pipette (1000 μL) Sterilized 1000μL tip A shaking incubator (Infors or equivalent) with a shaking diameter of 25 mm that maintains 150 rpm and 27°C. Rack for 500mL tubes (Infors, catalog number 66129) Centrifugal separator with oscillating rotor (Beckman or equivalent) Polyetherimide centrifuge tube cushion for 500mL tubes (Corning, catalog number 431124) -80°C freezer (Thermo Fisher or equivalent)

[0090] Protocol process Steps 1-3 of this protocol were performed in a Biosafety cabinet using sterile techniques, as follows:

[0091] Using a 5 mL serum pipette, aspirate 1–2 mL of Sf9 or T.ni cell stock and dispense it into a sample cup in a Vi-CELL counter to measure viable cell density and assess overall culture health by analyzing viability and mean viable cell diameter. Insect cells have a cell density of 7 × 10⁶ cells. 6 While ensuring that the density does not exceed cells / mL, administer 0.7 × 10⁶ every 2-3 days. 6The cultures are maintained in ESF921 medium in Erlenmeyer flasks by periodic division to cell densities of cells / mL or higher. Culture viability must be 95% or higher for Sf9 cells and T.ni cells, respectively, and the diameter of uninfected cells must not exceed 15.5 μm and 19 μm.

[0092] Sf9 or T.ni cells were incubated in ESF921 medium at 27°C for 2 × 10⁶ days. 6 Dilute to cells / mL and dispense 200 mL into each 500 mL tube. To infect Sf9 cells, add 0.5 mL of P3 virus to 200 mL of cells. To infect T.ni cells, add 1 mL of P3 virus to 200 mL of cells. Volume of virus added to insect cell culture (V virus The guide formula for calculating the starting cell density D, expressed in cells / mL units, is culture Culture volume V expressed in milliliters culture MOI (Multiplicity of Infection) expressed in pfu / cell units, and viral titer T calculated in pfu / mL units. virus It depends on [something]. Therefore, it is as follows: V virus =(D culture )(V culture )(MOI) / (T virus ) For newly generated viruses with a high gp64 staining rate, 4 × 10 8 It is permissible to assume a titer of pfu / mL. Furthermore, the inventors experimentally determined that MOIs of 0.5 and 1, respectively, are optimal for most proteins expressed in Sf9 and T.ni cells. Therefore, in the case of Sf9 cells, the following applies: V virus =(2×10 6 cells / mL)(200mL)(0.5pfu / cell) / (4×10 8 pfu / mL = 0.5mL In the case of T.ni cells, the following applies: V virus =(2×10 6 cells / mL)(200mL)(1pfu / cell) / (4×108 pfu / mL = 1mL For the simultaneous expression of proteins from different plasmids, which requires the use of multiple viruses, equal amounts of each P3 virus are added to the cells.

[0093] For Sf9 and T.ni cells, incubate the cultures in tubes at 27°C for 72 hours or 48 hours with shaking at 150 rpm and a shaking diameter of 25 mm. Check cell viability and diameter on day 2 for T.ni and on day 3 for Sf9. If the cell diameter is greater than 17 or 21 μm for Sf9 and T.ni, respectively, and the viability is 50-85%, proceed with harvesting. If the cell diameter is less than 17 or 21 μm for Sf9 and T.ni, respectively, follow the troubleshooting steps outlined in Table 1. If the viability is less than 50%, refer to the troubleshooting steps outlined in Table 1.

[0094] Collect the cells by centrifugation at 2,200g for 10 minutes. Discard the supernatant and freeze the pellet at -80°C. It is recommended to freeze the cell pellet for at least 10 minutes before cell lysis, even if purification is planned for the same day.

[0095] Basic Protocol 3 Medium-scale affinity purification This protocol was used to perform parallel affinity purification of 24 samples containing His-tagged and / or FLAG-tagged proteins using an IMCS chip and a Hamilton STAR liquid handler.

[0096] material The ingredients included are as follows: Equilibration and lysis buffers (see reagent and solution recipes) TCEP-HCl (Pierce, catalog number 20490) Benzonate endonuclease (Sigma, catalog number 101697) Roche Complete (trademark) EDTA protease inhibitor cocktail (Sigma, catalog number 11873580001) Glass beads (Thomas Scientific, catalog number 1177X44 or similar) A 25mm diameter shaking incubator (Infers or equivalent) maintained at 250 rpm and 10°C, using a tray suitable for holding 500 mL tubing. 500mL tube rack (Infors, catalog number 66129) 24-well deep filtration filter plate (Orochem, catalog number OC24DAHL-B) 24-well plate (Axygen, catalog number P-DW-10ML-24-CS) Centrifugal separator with oscillating bucket rotor, such as the Beckman JS-5.3 or equivalent. Microplate carrier for oscillating buckets (Beckman catalog number 368905) Support pads for microplate carriers (Beckman, catalog number 369382) Polyetherimide centrifuge tube cushion for 500mL tubes (Corning, catalog number 431124) Reagent reservoir (Thermo Fisher Scientific catalog number 95128085) Any 1000 μL 12-channel pipette Selected 1000μL tip Selected 1000 μL 8-channel pipette 1000μL IMCS tip filled with 50μL of Ni-NTA resin or 50μL of M2 anti-FLAG resin (IMCS, catalog numbers DP016 or DP017, respectively) 96-well plate (Thermo Scientific, catalog number AB-0932) Elution buffer (see reagent and solution recipes) FLAG peptide (Sigma, catalog number MFCD01863911) Super absorbent pads for heavy-duty blotting systems with dust-free blotting media in omni-trays (V&P Scientific, catalog number VP 540DB) Highly absorbent polypropylene pads cut to fit the OmniTray (V&P Scientific, catalog number VP 540DB1-100) Dust-free blotting media (V&P Scientific, catalog number C VP 540D-100) Hamilton Microlab STAR(TM) Automated dispensing robot with 300mL polypropylene reservoir for buffer solutions (Thermo Fisher Scientific catalog number 12565571) Acid buffer (see reagent and solution recipes) Washing buffer (see reagent and solution recipe) Hamilton CO-RE 1000μL filter tip (Hamilton, catalog number 235940) NanoDrop(TM) Spectrophotometer (ThermoFisher Scientific) NuPAGE® Sample Reducing Agent (10X) (Invitrogen, Catalog Number NP0009) NuPAGE® LDS Sample Buffer (4X) (Invitrogen, Catalog Number NP0007) Thin-walled 200 μL PCR 96-well plate (Axygen or similar) Plate seal (AlumaSeal, catalog number T790080-5 or similar) Use a PCR thermocycle (Fisher Scientific or equivalent) or a 95°C water bath to boil the sample. NuPAGE (trademark) 4-12%, Bis-Tris, 1.0mm, midi-size protein electrophoresis gel (Invitrogen, catalog number WG1402BOX) XCell4 SureLock™ Gel Electrophoresis Tank for MidiCell (Invitrogen, Catalog Number WR0100) NuPAGE (trademark) MES SDS Running Buffer (20X) (Invitrogen, catalog number NP0002) Precision Plus Protein (trademark), Kaleidoscope (trademark), Pre-Stained Protein Standard (Bio-RAD, catalog number 1610375) PowerPac (trademark) (Bio-RAD or equivalent) Gel knife (ThermoFisher, catalog number EI9010 or similar) Tray for washing and staining gels InstantBlue Coomassie Protein Staining Solution (Novus Biologicals, Catalog Number ISB1L) Laboratory rocking platform (VWR or equivalent) Bio-RAD Gel Doc® EZ Imaging System with Image Lab® software or equivalent.

[0097] Protocol process The protocol steps used are as follows:

[0098] Keep the frozen pellet on ice. All cell lysis and rearrangement steps should be performed on ice whenever possible. Leaving cell pellets, lysates, or protein samples at room temperature can accelerate protein degradation.

[0099] Add 24 mL of lysis buffer and approximately 2 mL of glass beads to the frozen pellet in a 500 mL tube and incubate at 10°C with shaking at 250 rpm for 1 hour in an Infors incubator with a shaking diameter of 25 mm. Alternatively, sonication can be used to lyse the cells, but this does not allow for a higher throughput format. The proposed lysis method using beads allows for the incubation and lysis of 24 samples at a time.

[0100] Pre-wet the 24-well Orochem filter with 1 mL of equilibrium buffer by placing the filter plate on top of the 24-well collection plate and centrifugating at 980 g for 2 minutes. Discard the flow-through and return the filter plate to the top of the 24-well plate. The plate assembly can be secured to the side with laboratory tape by centrifugation in step 6. The pre-wet depth filter is essential for efficient collection of the filtrate. Up to four samples can be filtered using one Orochem plate. Alternatively, the lysate can be filtered using conventional PES filters, but these filters tend to clog easily.

[0101] Clarify the lysate by centrifuging at 2,200 g for 10 minutes using a Beckman floor centrifuge. Decant the supernatant into a pre-labeled trough. Discard the pellet.

[0102] Using a 1000 μL 12-channel pipette, transfer the clarified lysates from the trough one at a time to a pre-moistened 24-well filter plate, so that 4 mL of each lysate is transferred to one row (6 wells) of the plate. See the diagram of the lysate layout on the Orochem filter (Figure 3).

[0103] Centrifuge the lysate in the filter plate, which is placed on top of the 24-well collection plate, at 980g for 5 minutes. Discard the filter plate. Lysate filtration helps ensure smooth downstream affinity purification and avoids clogging of the IMCS tip.

[0104] Using a 1000 μL 8-channel pipette, rearrange the clarified and filtered lysate for binding to four new 24-well plates, transferring the lysate at 6 mL / well per column to ensure identical layouts in all four plates. See the diagram of the lysate layout for binding in the 24-well plates (Figure 4).

[0105] Resin-filled IMCS tips are prepared by arranging them on a Hamilton 1 mL tip rack to match the layout of the lysate in a 24-well plate. The amount of resin required for affinity purification can be estimated based on the resin binding capacity and the expected expression yield of the target protein. This protocol allows for the use of 50, 100, 150, or 200 μL of resin per sample. To use 200 μL of resin per purification, four tips are placed in the rack positions corresponding to the wells of that sample in a 24-well plate. This protocol allows for the use of different resins for different samples, enabling the resin to be matched to the purification tags of the target protein. For example, Ni-NTA resin, Sigma M2 anti-FLAG resin, and Streptactin HC resin can be used for polyhistidine-tagged proteins, FLAG-tagged proteins, and Strep-tagged proteins, respectively. The inventors evaluated Phynexus and IMCS tip columns and determined that both perform equally well in medium-scale protein affinity purification. It is important to note that using the Phynexus chip requires a Hamilton STAR configuration that differs from the IMCS chip configuration for proper operation.

[0106] Prepare a 96-well plate containing 300 μL of elution buffer in each well corresponding to the tip layout. The total elution volume for purification performed using four IMCS tips is equal to 1,200 μL.

[0107] Two blotting trays are prepared by assembling the filter paper on top of the pad within the omni-tray.

[0108] Turn on the Hamilton STAR 4°C water cooler to cool the deck that holds the samples during purification.

[0109] The Hamilton STAR® deck is prepared by arranging the chips, buffer, plate, and blotting station as shown in Figure 5.

[0110] material: A water-filled trough for dispensing storage buffer from the IMCS tip. IMCS chip Three racks of CO-RE chips for buffer transfer. Trough filled with acid buffer Trough filled with equilibrium buffer Two troughs filled with washing buffer 96-well plate for acid buffer solutions 96-well plate for equilibrium buffer solution Three 24-well plates for washing buffer. Three empty 24-well plates for dispensing used wash buffer. Two blotting stations (To prepare the blotting stations, dust-free blotting media is placed on a highly absorbent polypropylene pad fitted into an omni-tray.) Four 24-well plates containing filtered lysate (6 mL per well) A 96-well plate containing 300 μL of elution buffer per well.

[0111] Start the IMCS medium-scale protein purification method on Hamilton STAR® (see Supplemental Protocol 2 for method details). After the Hamilton STAR® method is complete, remove all material from the deck. Turn off the water cooler. Note: Daily and weekly maintenance is performed via the "Microlab STAR Maintenance and Validation" application provided by Hamilton Company. In addition, the 96-probe multi-probe head (MPH) and 8-channel pipetting module are wiped with a clean, lint-free, dry cloth dampened with water after each run.

[0112] A separate eluate is pooled for each affinity-purified sample. Refer to the diagram for the eluate pool (Figure 6). A280 measurement is performed using NanoDrop. For the blank, an elution buffer without 3×FLAG peptide is used.

[0113] Prepare a reduced sample for SDS-PAGE analysis by mixing 20 μL of eluate with 3 μL of 10× reducing agent and 7 μL of 4× LDS running buffer in a PCR plate, while maintaining the same sample layout as the pooled elution plate. Heat the sample at 95°C for 5 minutes using a PCR thermocycler. Load 20 μL of sample and 7 μL of pre-stained protein standard onto a NuPAGE gel. Electrophoresis is performed at 180 V for 50 minutes in 1× NuPAGE® MES SDS running buffer. Electrophoresis time can be adjusted based on the expected molecular weight of the protein. For example, proteins smaller than 15 kDa should not be electrophoresed for more than 47 minutes, while very large proteins greater than 200 kDa may require longer electrophoresis times for better resolution.

[0114] Using a gel knife, open the gel cassette and transfer the gel to a tray filled with water. Rinse the gel with water and stain it with InstantBlue Coomassie stain for 1 hour, gently agitating it on a rocking platform. Destain it in water for 2 hours, continuing to agitate it on the rocking platform.

[0115] The gel is scanned and labeled using Bio-RAD Image Lab® software. Scanning is performed using a white tray on the Bio-RAD Gel Doc® EZ imaging system by selecting the default protocol and Coomassie Blue as the application. Image exposure is set to "Faint Bands" or manually set to 0.500 seconds. Image analysis is performed by selecting the "Lane and Bands" tool and the "Analyze Molecular Weight" function in the Image Lab software's analysis toolbox. Bands of interest are annotated using the annotation tool.

[0116] Auxiliary Protocol 2 Automated method for affinity purification on Hamilton STAR® The Hamilton STAR® IMCS medium-scale purification method used in this example involved the following steps:

[0117] Method parameters such as the number of sample binding cycles and elution cycles can be selected on the user interface (Figure 7). Recommended method parameters are listed in Table 2. The acid addition step involves transferring 1 mL of acid buffer from the trough to a 96-well acid plate using a Co-RE tip. Buffer aspiration and dispensing are performed at 250 L / sec and 400 μL / sec, respectively. Note: A 50 μL pre-aspiration of air is performed before each protein purification step. After all mixing cycles have been performed and the final dispensing with a 30-second waiting period is complete, the 50 μL pre-aspiration is used as a blowout on the liquid surface before the final dispensing.

[0118] The equilibration buffer addition step involves transferring 1 mL of equilibration buffer from the trough to a 96-well equilibration plate using a Co-RE tip. Buffer aspiration and dispensing are performed at 250 L / sec and 400 μL / sec, respectively.

[0119] The wash buffer addition step involves transferring 1 mL of wash buffer per channel twice to a 24-well wash plate using a Co-RE tip. This transfer is repeated two more times for two additional wash plates. Buffer aspiration is performed at 250 μL / second. Buffer dispensing is performed at 400 μL / second.

[0120] The storage buffer is released into a water-filled trough using the tip property "MlStarIsCoreHeadSpecialTipPickup".

[0121] The acid washing process involves washing all resin tips with 800 μL of acid buffer by pipetting up and down once at flow rates of 100 μL / sec for aspiration and 30 μL / sec for dispensing, with a 30-second pause after dispensing. Note: The pipetting process using resin-containing IMCS tips is performed at a significantly slower rate than the pipetting process using Co-RE tips. This is done to reduce the pressure rise within the IMCS tip. Varying the aspiration and dispensing rates of the IMCS tip assists in the dispersion and sedimentation of the resin during mixing. Faster aspiration provides more efficient resin dispersion, while slower dispensing rates provide proper resin sedimentation. Furthermore, slower dispensing rates prevent resin from adhering to the tip walls.

[0122] The equilibration process involves washing all resin tips with 800 μL of equilibration buffer by pipetting up and down three times at flow rates of 100 μL / second for aspiration and 30 μL / second for dispensing, including a 2-second pause between each aspiration and dispensing cycle and a 30-second pause after the final dispensing.

[0123] The sample binding step involves loading the protein onto the tip by pipetting 20 times up and down at a flow rate of 100 μL / sec for aspiration and 15 μL / sec for dispensing within each sample plate. This step includes a 2-second pause between each aspiration and dispensing cycle, and a 30-second pause after the final dispensing. Note: Cell lysates are considerably more viscous than the buffers used in this method and require a further reduction in the dispensing rate during the sample binding step.

[0124] The blotting process involves blotting the chips onto absorbent paper after the final bonding process.

[0125] The washing process involves washing all tips with the washing buffer by transferring 300 μL of washing buffer five times from each of the three washing plates, i.e., a total of 15 transfers of 300 μL per tip. A flow rate of 100 μL / second is used for aspiration and a flow rate of 30 μL / second is used for dispensing. This process utilizes a 2-second pause between aspiration and dispensing cycles and a 30-second pause after the final dispensing.

[0126] The elution step involves eluting proteins in the elution buffer by aspirating and dispensing 250 μL twenty times at flow rates of 100 μL / second for aspiration and 15 μL / second for dispensing, with a 2-second pause between each aspiration and dispensing cycle and a 30-second pause after the final dispensing.

[0127] Basic Protocol 4 Size exclusion chromatography (SEC) This protocol describes the second purification step used in SEC, a medium-scale workflow. A Thermo Fisher Vanquish® Duo system (Figure 8) and Phenomenex Yarra® series columns / TSK Super SW columns were used in this protocol. The wavelength used was 280 nm for protein peak analysis.

[0128] material The following materials were used: SEC buffer (see reagent and solution recipe) Millipore Amicon Ultra 0.5 Centrifugal Filter, Ultracel 3kD (Catalog Number UFC500396) Millipore Ultrafree Centrifuge Filter, Dura Free PVDF 0.22μM (Catalog Number UFC306VOO) Thermo Fisher Vanquish Duo HPLC System Phenomenex Yarra series column 3μM SEC-3000, LC column 300×4.6mM (Catalog number 00H-4513-EO) TSKgel SuperSW3000 4.6mm x 30cm (Catalog number 0018675) Waters BEH SEC Protein Standard Mix (Catalog Number 186006518-1) 1x phosphate-buffered saline (PBS) (Invitrogen, 10X, catalog number AM9624) Thermo Scientific WebSeal® well plates with barcodes for Vanquish® UHPLC systems (catalog number 60180-P103B) 96-well microplate, round well, barcoded Thermo Scientific® Plate Seal (Catalog Number 60180-M146) Thermo Scientific (trademark) Abgene 96-well 2.2mL polypropylene deep-well storage plate (catalog number AB0932)

[0129] Protocol steps for size exclusion chromatography using the Thermo Fisher Vanquish Duo system The following protocol steps were used:

[0130] The eluate from affinity purification is concentrated to approximately 130 μL by centrifugation at 13000 g for 20 minutes in a refrigerated centrifuge using a 3 kDa Amicon spin concentrator, followed by filtration using an Amicon 0.2 μm filter tube. Since the Thermo Fisher Vanquish Flex model only offers configurations with 25 μL and 100 μL sample loop sizes, and the maximum loadable sample volume is 100 μL, sample concentration is necessary.

[0131] For higher molecular weight proteins or complexes, a concentrator with a higher molecular weight cutoff can be used. Some proteins tend to precipitate during concentration and cannot be loaded into the SEC. See Troubleshooting Table 1.

[0132] Prepare the BEH molecular weight standard mixture by dissolving it in 0.5 mL of PBS and filtering it through an Amicon 0.2 μm filter. Load the concentrated and filtered sample along with the BEH protein standard mix into a barcoded 96-well ThermoFisher plate, seal the plate, and place it in one of the racks in the autosampler on the Vanquish Duo system (temperature set to 4°C) (Figure 8). The sample can be loaded into any other 96-well microplate as long as it has rounded well bottoms. If using plates from a different source, specify the plate format in the autosampler on the Chromeleon console. Plate sealing is optional but is generally recommended to avoid evaporation of the sample if it is to be held in the autosampler for more than 24 hours.

[0133] System operation and method setup should follow the procedures provided in Auxiliary Protocol 3.

[0134] Equilibrate a Phenomenex Yarra SEC 3000 or TSKgel Super SW3000 column (column volume 5 mL) with SEC running buffer at a flow rate of 0.3 mL / min for 30 minutes. The recommended flow rate for this column is 1 mL / min. However, a flow rate of 0.3 mL / min is recommended to allow for better resolution and avoid pressure buildup. For single-protein purification, the flow rate can be increased to 0.6 mL / min. A TSKgel Super SW3000 column can be used instead of a Phenomenex Yarra SEC 3000 column.

[0135] Create the injection sequence using the appropriate instrument method in the Chromeleon console, as described in Auxiliary Protocol 3. Stop baseline monitoring and start the sequence run. All 24 samples from affinity purification can be loaded into a single plate for SEC purification. It takes approximately 33 minutes for one sample to complete at a flow rate of 0.3 mL / min.

[0136] Fractions are collected using a fraction collector in a Thermo Scientific Abgene® deep-well 96-well plate. The fraction collection mode can be selected as "collection by peak" or "collection by time". The inventors determined that "collection by peak" works better for them, collecting fractions only when peaks are identified (this setting can be adjusted in the method). "Collection by time" collects fractions between peaks, resulting in more than 100 fractions per sample. Alternatively, fraction collection can be turned off if only protein analytical characterization is required. The fraction size can be adjusted by changing the "Tube change duration" parameter in the fraction collector settings. The inventors use a value of 4 seconds for "Tube change duration" to collect fractions of 40-50 μL, as shown in the method script.

[0137] The chromatogram is analyzed to identify peaks of interest and to identify well numbers for fractional analysis by SDS-PAGE. Chromeleon data analysis provides the option to display chromatograms in stacked or overlaid view for comparison of samples. In this invention, it is preferable to analyze the chromatogram using the stacked view option.

[0138] Record the fraction number and dispense 20 μL of each fraction into an Axygen 96-well plate for gel loading. Prepare the reduced sample for SDS-PAGE analysis by mixing 20 μL of sample with 3 μL of 10× reducing agent and 7 μL of 4× LDS running buffer in a PCR plate. First, seal the plate with an aluminum foil seal and boil the sample at 95°C for 10 minutes using a PCR machine. Load 20 μL onto a Novex pre-made gel. Run electrophoresis at 180 V for 50 minutes. The electrophoresis time can be adjusted based on the expected molecular weight of the protein. For example, proteins smaller than 15 kDa should not be run for more than 47 minutes, while very large proteins greater than 200 kDa may require longer electrophoresis times to achieve better resolution.

[0139] Rinse the gel in water, place the gel tray on a rocker, stain with InstantBlue Coomassie stain for 1 hour, then destain in water for 2 hours while agitating on the rocker.

[0140] The gel is scanned and labeled using Bio-RAD Image Lab® software. Scanning is performed using a white tray on the Bio-RAD Gel Doc® EZ imaging system by selecting the default protocol and Coomassie Blue as the application. Image exposure is set to "Weak Stripes" or manually set to 0.500 seconds. Image analysis is performed by selecting the "Lanes and Stripes" tool and the "Analyze Molecular Weight" function in the Image Lab software's analysis toolbox. Bands of interest are annotated using the annotation tool.

[0141] Pool the fractions that contain the desired molecular weight protein of interest.

[0142] Prepare a data report. The protein will be ready for downstream characterization. This purification method yields high-quality purified protein and multiprotein complexes. Yield varies depending on the expression level of the target protein. If the protein is not detected, refer to troubleshooting table T1.

[0143] Auxiliary Protocol 3 Using Chromeleon 7 on Vanquish Duo Open the Chromeleon 7 console on a Windows® computer interfaced with a Vanquish® Duo system.

[0144] If you only need to operate the left system, open the Vanquish Left icon, Vanq_LeftOnly. You can operate both the Vanquish Left and Right systems simultaneously by controlling them from Vanq_Left or Vanq_Right. For simplicity, we will explain only the Vanq_Left system.

[0145] As shown in the screenshot (Figure 9), open the "Equipment" tab in the lower left corner. Turn on the UV lamp and Vis lamp by switching on the toggle switch in the UV left tab. The indicator will change from gray to green in about 15 minutes.

[0146] Open the shutter by selecting the "Open" position from the dropdown options in the UV tab.

[0147] Connect the module and change the column compartment temperature to 10°C in the column compartment tab.

[0148] In the Fraction Collection tab, reset the fraction collector to fraction 1.

[0149] Pump - Set the pump pressure to 350 psi in the left tab. This is calculated by adding the maximum allowable pressure for the column (provided by the manufacturer) and the pressure of the HPLC system. If the pump pressure is higher than the set value, refer to Troubleshooting Table 1. If the pump pressure is not increasing with increasing flow rate, there may be a leak in the system. Refer to Troubleshooting Table 1.

[0150] Adjust the flow rate to 0.3 mL / min using the left tab on the pump. Alternatively, the flow rate can be adjusted to 0.6 mL / min and up to 1 mL / min for Phenomenex Yarra series columns. 0.3 mL / min was chosen for better resolution of protein peaks.

[0151] Load the SEC buffer into the buffer station on the upper stack, insert the left system A_L filter into the buffer, and close the cap. Open the Vanquish left pump valve by turning the knob 360°. This allows purging the system at a high flow rate without increasing the pressure. The default setting for purging is 5 mL / min, with acceleration and deceleration at 0.1 mL / min. These settings can be adjusted in the Options tab. You should hear the purging sound and the rear seal cleaning piston moving.

[0152] After the purge has stopped, close the left pump valve. If the valve is not closed, the motor will not turn on when the system is activated.

[0153] When the motor is turned on via the toggle switch, the system begins operating at the set flow rate.

[0154] Connect the column to the valve and place the column in the column compartment. The column needs to be connected upside down when the flow direction in this system is from bottom to top, i.e., when the pump is at the bottom of the stack and the detector is at the top.

[0155] The baseline is monitored by selecting the 280nm UV wavelength and pump pressure from the dialog box. A280 (absorbance at 280nm) is shown in the fraction acquisition tab, and the pump pressure is shown in the pump left tab. Absorbance at 260nm (for nucleic acid detection) can also be monitored simultaneously, but selecting both wavelengths with this detector will slow down data acquisition.

[0156] Equilibrate a Phenomenex Yarra SEC3000 column or TSK column (column volume 5 mL) with SEC running buffer at a flow rate of 0.3 mL / min for 30 minutes.

[0157] Baseline monitoring is turned off 30 minutes after equilibration.

[0158] Open the "Data" tab in the lower left corner to begin creating a method run. Open "Create New Sequence" from the create icon in the upper left corner (Figure 10).

[0159] Add row entries based on the number of samples.

[0160] For each sample, add the name and location of the sample in the 96-well plate (for example, for the sample in well A12 on the plate in the green position, enter "G:A12").

[0161] Select an instrument method for each sample. If the same column is used and other parameters are not changed, the instrument method can be set up once, as shown below, and can be used for all future purifications.

[0162] Start run and fraction collection.

[0163] After the run is complete, wash the column with buffer and deionized water at a flow rate of 1 mL / min for at least one column volume.

[0164] Remove the column and store it at 4°C. Wash the HPLC line and maintain it by washing with 10% methanol every two months to prevent the growth of contaminants. The rear seal washing line is kept active and operational with 10% methanol and refilled monthly to maintain a long lifespan for the piston and piston seal.

[0165] The reagents and solutions used in the method are as follows: Acid washing buffer (pH 3.0) 100 mL of 1 M glycine-HCl, pH 3.0 (final concentration 100 mM) 30 mL of 5 M NaCl (final concentration 150 mM) 870mL water It must be stored away from corrosive substances and acids. Store at room temperature for up to 12 months. Elution buffer (pH7.5) 50 mL of 1 M Tris pH 7.5 (final concentration 50 mM) 30 mL of 5 M NaCl (final concentration 150 mM) 100 mL of 50% glycerol (final concentration 5%) 125 mL of 2 M imidazole (final concentration 250 mM) 695mL water TCEP (final concentration 1mM) 3×FLAG peptide (final concentration 150 μg / mL) Roche EDTA-free protein inhibitor cocktail (PIC) (1 tablet per 50 mL of final buffer solution) Buffers prepared with Tris, NaCl, glycerol, and imidazole can be stored at 4°C for up to one year. TCEP, 3×FLAG peptide, and PIC must be added fresh before use. Equilibration and dissolution buffers 50 mL of 1 M Tris pH 7.5 (final concentration 50 mM) 30 mL of 5 M NaCl (final concentration 150 mM) 200 mL of 50% glycerol (final concentration 10%) 2 mL of 1 M MgCl2 (final concentration 2 mM) 5 mL of 2 M imidazole (final concentration 10 mM) 713mL water TCEP (final concentration 1mM) Benzonase (0.5 μL per 1 mL of final buffer) Roche EDTA-free protein inhibitor cocktail (PIC) (1 tablet per 50 mL of final buffer solution) Buffers prepared with Tris, NaCl, glycerol, imidazole, and MgCl2 can be stored at 4°C for up to one year. TCEP, benzonase, and PIC must be added fresh before use. SEC buffer 50 mL of 1 M Tris pH 7.5 (final concentration 50 mM) 30 mL of 5 M NaCl (final concentration 150 mM) 920mL water TCEP (final concentration 1mM) Buffers prepared with Tris and NaCl can be stored at 4°C for up to one year. TCEP must be added fresh before use. Washing buffer 50 mL of 1 M Tris pH 7.5 (final concentration 50 mM) 30 mL of 5 M NaCl (final concentration 150 mM) 100 mL of 50% glycerol (final concentration 5%) 10 mL of 2 M imidazole (final concentration 20 mM) 810mL water TCEP (final concentration 1mM) Roche EDTA-free protein inhibitor cocktail (PIC) (1 tablet per 50 mL of final buffer solution) Buffers prepared with Tris, NaCl, glycerol, and imidazole can be stored at 4°C for up to one year. TCEP and PIC must be added fresh before use.

[0166] Multicomponent protein expression Many laboratories are building semi-automated, high-throughput small-scale expression analysis platforms to enable simultaneous analysis of hundreds of constructs with different tags, domains, and multiple cell lines. This allows researchers to efficiently select the best conditions for expressing proteins in good yield in most cases. However, this approach does not work as well when dealing with underexpressed proteins or multi-protein complexes. Therefore, there was a need to build a medium-scale protein expression and characterization platform that enables rapid parallel triage of difficult underexpression and co-expression in a high-throughput manner. We constructed and developed this medium-scale platform based on a small-scale expression analysis platform (Kraft et al., 2019). This is an end-to-end semi-automated workflow from virus generation to protein production, enabling the delivery of 24 different proteins in a single purification run. This includes a two-step purification process including affinity chromatography and size exclusion chromatography. In addition to protein expression and characterization, this workflow has evolved to provide hundreds of micrograms of high-quality purified protein for further downstream applications. The inventors have successfully explored numerous applications using proteins purified from this platform, including negative staining, high-throughput time-resolved fluorescence (HTRF) assays, SPR, affinity pulldown, enzyme assays, and mass spectrometry. Another potential application of this method is screening for optimal buffers or co-expression ratios of different components for reconstituting soluble, stable protein complexes.

[0167] In this example, the inventors focused on the expression and purification of a multiprotein complex having eight component subunits successfully used for negative staining applications. Using this protocol, the results described herein were further tracked for large-scale expression and purification on a 3L scale. Large-scale purification was performed using conventional batch modes of affinity purification with M2 anti-FLAG resin, ion exchange chromatography, and SEC. SEC analysis showed very similar monodisperse peaks for the protein complex, resulting in approximately 0.3 mg of the protein complex per liter of culture (Figure 12). This purified protein was then used to elucidate the high-resolution cryoEM structure of the complex (unpublished). Overall, the end-to-end medium-scale protein screening platform enables parallel expression and purification of 24 samples or conditions, allowing for the delivery of protein in quantities usable for downstream applications.

[0168] Figure 11 shows a successful case study for expressing and purifying multiprotein complexes from insect cells using a medium-scale platform. Protein complexes in insect cells were expressed using co-infection with multiple viruses, each expressing a single protein subunit. For large protein complexes, often containing four or more subunits, co-infection with multiple viruses can lead to increased cellular stress and heterogeneous infection of cells, resulting in incomplete complex formation and / or low protein yield. To reduce the heterogeneity of co-infected viruses, multi-ORF constructs expressing multiple proteins from a single virus can be used to improve the stoichiometry and yield of the complexes. In an attempt to enable the production of an 8-subunit protein complex (proteins A-H), the inventors designed three constructs using the following strategies:

[0169] 1) The largest subunit (protein A, 117.1 kDa) was N-terminus-flag-tagged and expressed on its own vector under a polyhedrin promoter. This was to allow screening of numerous N-terminal truncations as part of a domain walking strategy to increase protein yield.

[0170] 2) Proteins B-E (51.8, 6.1, 34.8, and 47.2 kDa) were generated as untagged proteins, placed on separate vector backbones, and each ORF was placed under the control of a polyhedrin promoter.

[0171] 3) Proteins F~H (39.2, 36.4, 14.5) encoding three different ORFs were placed on a third vector, with each ORF placed under its individual polyhedrin promoter.

[0172] Viruses were prepared from each of the three constructs using the protocols listed above, and all three viruses expressing eight different proteins were co-expressed in a 1:1:1 ratio in Sf9 cells to generate biomass. The biomass was recovered, the cells were lysed, and the proteins were purified using affinity purification and size exclusion chromatography. The first SDS-PAGE gel in Figure 11 shows the results from affinity purification, where all eight protein subunits of the complex were purified using the automated INtip® affinity purification method described above with M2 anti-FLAG resin in an IMCS tip on a Hamilton STAR® workstation. All proteins of the complex were detected at the expected molecular weight, further purified, and analyzed for oligomeric / aggregated state of the complex by size exclusion chromatography using a TSK column. The chromatogram showed a monodisperse peak, and the complex eluted with an elution time of 7.5 minutes corresponding to an elution volume of 2.25 mL. The second gel in Figure 11 shows the analysis of fractions collected from size exclusion chromatography, where all eight proteins of the complex were detected in the analyzed peaks. The total yield of this complex was observed to be approximately 0.43 mg from a 200 mL culture. Peak fractions were collected and analyzed by negative staining electron microscopy. The samples were qualitatively evaluated, and the uniformity of particles in each micrograph was examined. This resulted in a stable and well-behaving protein complex, which helped in identifying the optimal domain boundary of protein A, which appears suitable for structural studies. [Table 1] [Table 2]

[0173] Further comments High-titer virus production Baculovirus production in this protocol relied on successful in vitro recombination of transfer plasmids with linearized baculovirus DNA BestBac® (Expression Systems). The efficiency of homologous recombination should be 90–100%, but this can vary depending on the linearization vector used (Kitts et al., 1990). Many commercially available linearized bacmids exist that utilize in vitro homologous recombination methods (BaculoGold®, BD Biosciences; FlashBAC, Oxford Expression Technologies; BacPAK®, Clontech). However, the final virus stock inevitably contains a mixture of parental and recombinant viruses. The virus should be of high quality and high titer to facilitate expression at medium scale. To avoid the generation of a high proportion of non-recombinant viruses, the virus stock should not be amplified beyond the P3 stage. Although the inventors did not quantify the viral titer, they determined the percentage of cells infected with viral particles using a gp64 assay (Kitts and Green, 1999). Virus stocks with gp64 staining titers below 70% result in unproductive infection and lower yields; therefore, repeated co-transfection is recommended to regenerate the virus.

[0174] insect cell infection Insect cells Sf9 and T.ni should be healthy to undergo productive viral infection and should have diameters of 15.5 and 19 μm, respectively, as measured by Vi-CELL BLU. Culture viability should be 95% or higher for the process to proceed. If cell viability is low or cell diameter is large, fresh cells should be thawed and subculturified for infection. Productive infection of cells is also measured using a Vi-CELL counter. Cells should only be harvested for purification if the cell diameter is greater than 17 or 21 μm for Sf9 and T.ni, respectively, and viability is 50–85%. Otherwise, the infection process is repeated.

[0175] Efficient cell lysis and filtration Cells must be completely lysed using an appropriate lysis method as described above, and the lysate should be filtered to completely remove any residue. There are many physical and chemical methods for lysing cells, including osmotic shock, freeze-thaw, Dounce homogenization, sonication, and lysis with surfactants. Eukaryotic cells have a higher amount of nucleic acids than bacteria, and therefore, nucleases (benzoases) should be included to reduce the viscosity of the cell lysate. The inventors have found that lysis using beads provides complete and efficient lysis of insect cells for higher throughput. Cell residue must be clarified by either high-speed centrifugation or a depth microfiltration system. Orochem's filtration plate is a depth microfiltration system consisting of a filter with an open structure for removing cells and cell residue, and a filter aid material with a tighter pore structure for simultaneously removing colloidal material to deliver a particulate-free feed for downstream processes. If deep filtration is unavailable, the lysate should be clarified before purification using high-speed centrifugation (18,000 g for 30 minutes).

[0176] Purification buffer The buffers for dissolution, washing, and elution in affinity chromatography should be carefully selected according to the isoelectric point and aggregation characteristics of the protein. The most commonly used buffers for purification are the Tris system or the HEPES system. Since higher salt concentrations can interfere with ionic interactions between different components of protein complexes, the salt concentration should be kept between 100 and 150 mM NaCl to purify multiprotein complexes. Since higher salt concentrations result in better purity, the salt concentration can be adjusted to 300 mM to purify single proteins. To purify intracellular proteins, reducing agents such as DTT or TCEP should be added. Since glycerol provides stability to proteins, we include 5-10% glycerol in the buffer.

[0177] Affinity Purification In addition to selecting an appropriate buffer, it is important to select an appropriate resin type for purification and to optimize the automated method for a given resin. Resins can have very different binding capacities. For example, the M2 anti-FLAG and Ni-NTA Superflow® resins used by the inventors have binding capacities of 0.6 mg / mL and 20 mg / mL, respectively, and therefore the number of IMCS chips should be adjusted according to the expected expression level of each sample. Since the binding rate of the target protein is higher or lower depending on the resin, the automated purification method should be adjusted according to the number of sample binding and elution cycles.

[0178] Size exclusion chromatography The inventors tested different chemical columns of different sizes and found that the 5 mL column and the 300 × 4.6 mM TSK column of the Phenomenex Yarra® series performed well for separating purified protein complexes from 200 mL volumes. The columns have a densely packed inert silica-based material with a pore size of 290 Å and a particle size of 3 μm, with a separation range of 5–700 kDa. Another advantage of these columns is that they are compatible with HPLC and UHPLC systems, allow for a fast flow rate of 1 mL / min, and can withstand a pressure of 300 bar.

[0179] Example 2: Medium-scale protein production from mammalian cell cultures Histidine-tagged membrane proteins were co-expressed with one or both of two cytoplasmic protein chaperones, and without co-expression, in several flasks, each with a volume of 30 mL, in EXPi293 cells transformed with an appropriate expression plasmid on day 0, at 37°C. On day 1, the temperature was maintained at 37°C in some flasks and reduced to 30°C in others. On day 4, parallel affinity purification of proteins was performed using a Ni-IMAC nickel column, and the yield of protein and chaperone protein was evaluated for each individual sample, allowing for rapid comparison of different purification conditions (temperature and presence of one or both chaperone proteins).

[0180] Example 3: Medium-scale purification of protein complexes To determine the optimal conditions for protein expression and complex formation, several different medium-scale, parallel protein purifications were performed to purify the E3 ubiquitin ligase protein complex from Sf9 insect cells. Proteins were pulled down in parallel using FLAG tags, followed by size exclusion chromatography and electrophoresis analysis. While the substrate-recognizing protein of the complex did not show expression on its own, co-expression with other members of the complex and chaperone proteins enabled the reconstitution of a stable E3 ligase complex. This example demonstrates that parallel, medium-scale protein purification can help rapidly determine the optimal conditions for purifying protein complexes from cell cultures. References Bahia, D., Cheung, R., Buchs, M., Geisse, S., and Hunt, I. (2005). 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(1993). A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques, 14(5), 810-817. Obtained from https: / / www.ncbi.nlm.nih.gov / pubmed / 8512707 Kost, T.A., Condreay, J.P. and Jarvis, D.L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol, 23(5), 567-575. doi:10.1038 / nbt1095 Kraft, E., Franke, Y., Heeringa, K., Shriver, S., Zilberleyb, I., Kugel, C., ...Bowman, K. (2019). Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins. Methods Mol Biol, 2025, 51-68. doi:10.1007 / 978-1-4939-9624-7_3 Lesley, S.A. (2009). Parallel methods for expression and purification. Methods Enzymol, 463, 767-785. doi:10.1016 / S0076-6879(09)63041-X Marsischky, G. and LaBaer, J. (2004). Many paths to many clones: a comparative look at high-throughput cloning methods. 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Claims

1. A method for purifying one or more polypeptides from multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples, (a) Growing multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of the cells in the cell culture sample, wherein the portions (a) and (b) for each of the multiple cell culture samples are performed in the same container, (c) Centrifuging the dissolved cell culture sample and collecting multiple supernatant samples from the cell culture sample, (d) Clarifying the plurality of supernatant samples by deep filtration, (e) Placing the clarified plurality of supernatant samples in the wells of a multiwell plate, wherein the plurality of clarified supernatant samples have a volume of 2 to 30 mL per well. (f) Subjecting the plurality of supernatant samples in the wells of the multiwell plate to affinity chromatography using an affinity matrix in a pipette tip, and eluting from the chromatography into the wells of the multiwell plate, wherein parts (e) and (f) are performed in parallel for the plurality of samples, subjecting the plurality of supernatant samples to affinity chromatography and arranging them. Methods that include...

2. The above method, part (f), or part (e) and (f) The method according to claim 1, wherein the process is automated.

3. The method according to claim 1, wherein all parts (a) to (f) are performed in parallel.

4. The method according to any one of claims 1 to 3, wherein the plurality of clarified supernatant samples are placed in the wells of the multi-well plate in a volume of 2 to 30 mL per well.

5. A method for purifying one or more polypeptides starting from multiple clarified cell lysate supernatant samples in a multiwell plate, wherein the multiple clarified cell lysate supernatant samples are obtained from multiple cell culture samples, (a) Growing multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples expressing one or more polypeptides to be purified in a volume of 20 to 500 mL, (b) Lysis of the cells in the cell culture sample, wherein the portions (a) and (b) for each of the multiple cell culture samples are performed in the same container, (c) Centrifuging the dissolved cell culture sample and collecting multiple supernatant samples from the cell culture sample, (d) Clarifying the plurality of supernatant samples by deep filtration, (e) Placing the clarified plurality of supernatant samples in the wells of the multiwell plate, wherein the plurality of clarified supernatant samples have a volume of 2 to 30 mL per well. It is obtained by a process that includes, The method comprises subjecting the supernatant sample obtained from the processes of (f)(a) to (e) to affinity chromatography performed using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a multiwell plate, wherein the method is performed in parallel for the plurality of clarified supernatant samples, and optionally the method is automated.

6. A method for affinity purification of polypeptides from multiple cell culture supernatant samples expressing one or more polypeptides to be purified, The method includes obtaining a multi-well plate containing multiple clarified cell culture supernatant samples in wells at a volume of 2 to 30 mL per well, subjecting the multiple clarified cell culture supernatant samples to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluate from the chromatography into the wells of a second multi-well plate, wherein the method is performed in parallel for the multiple supernatant samples, and optionally the method is automated. The method is characterized in that the plurality of cell culture supernatant samples are obtained from mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples expressing one or more polypeptides, which have been grown in a scale of 20 to 500 mL, lysed, centrifuged, and clarified, and the cell growth and lysing are performed in the same container.

7. The method described above is (g) Performing size exclusion chromatography (SEC) on the eluate from the affinity chromatography, (h) Fractionating polypeptides from the SEC into the wells of a multiwell plate It further includes, The method according to any one of claims 1 to 6, wherein optionally, one or both of parts (g) and (h) are automated.

8. The method according to claim 7, wherein parts (g) and (h) are automated.

9. The method according to any one of claims 1 to 8, wherein a clarified supernatant sample corresponding to a single cell culture sample is placed in two or more wells of the multiwell plate.

10. The method according to any one of claims 1 to 9, wherein clarified supernatant samples from different cell culture samples are placed in different wells of the multiwell plate.

11. The method according to any one of claims 1 to 10, wherein 8 to 96 clarified supernatant samples, such as 8 to 48, 8 to 24, 12 to 48, or 12 to 24, are processed in parallel.

12. The method according to any one of claims 1 to 11, wherein the cells are dissolved by adding glass beads while shaking, and / or the cells are not dissolved by sonication.

13. The method according to any one of claims 1 to 12, wherein a plurality of the cell culture samples are grown in a scale of 30 to 250 mL, 50 to 250 mL, 30 to 200 mL, 50 to 200 mL, or 100 to 200 mL.

14. The method according to any one of claims 1 to 13, wherein the pipette tip containing the affinity matrix has a volume of 0.5 to 2 mL, such as 1 to 2 mL, or 0.5 to 1.5 mL, or 0.5 mL, or 1 mL, or 1.5 mL, or 2 mL.

15. The method according to any one of claims 1 to 14, wherein the affinity matrix in the pipette tip has a bed volume of 30 to 100 μL, such as 30 to 70 μL, or 40 to 50 μL, or 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, or 100 μL.

16. The method according to any one of claims 1 to 15, wherein the polypeptide is tagged with a polyhistidine, FLAG, streptavidin, glutathione-S-transferase (GST), or maltose-binding protein (MBP) tag, and the affinity matrix recognizes the tag.

17. The method according to any one of claims 7 to 16, wherein the method comprises parts (g) and (h), and the SEC chromatography is performed on an SEC matrix comprising particles having a pore size of 140 to 500 angstroms and / or a particle size of 3 to 5 microns and / or a molecular weight range of 5 to 700 kDa.

18. The method according to any one of claims 1 to 17, further comprising performing a structural or functional analysis on the purified polypeptide, such as cryo-electron microscopy, mass spectrometry, a protein-protein interaction assay including surface plasmon resonance, or a homogeneous time-resolved fluorescence assay.

19. The method according to any one of claims 1 to 18, wherein the one or more polypeptides comprise a recombinant protein complex.

20. The method according to any one of claims 1 to 19, wherein the one or more polypeptides do not contain an antibody or an antibody subunit.

21. The method according to any one of claims 1 to 20, wherein the cell culture sample is an insect cell culture sample, such as an Sf9 or T. ni cell culture sample.

22. The method according to any one of claims 1 to 21, wherein the cell culture sample is a mammalian cell culture sample.

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