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

Abstract

The present disclosure relates to a higher throughput, medium scale, semi-automated protein expression and screening platform useful, for example, in drug discovery studies and in addition for testing protein expression conditions, among other uses. In some embodiments, the workflows described herein also enable a comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a faster and efficient manner by delivering small but sufficient amounts of high quality proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Application No. 63 / 579,668, filed August 30, 2023, and U.S. Application No. 63 / 645,754, filed May 10, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The disclosure relates to a higher-throughput, medium-scale, semi-automated protein expression and screening platform that can be used, for example, in drug discovery research and, in addition, for testing protein expression conditions, among other uses. In some embodiments, the workflow described herein also enables comprehensive expression and purification screening of challenging or difficult-to-express recombinant proteins in a faster and more efficient manner by delivering small but sufficient quantities of high-quality protein. Background Technology

[0004] Recombinant protein production is a crucial component of basic research and drug discovery. As therapeutic targets become increasingly complex, researchers are constantly seeking innovative expression and purification techniques to improve the production of difficult-to-express proteins and challenging multi-protein complexes in a faster and more cost-effective manner. While small-scale high-throughput expression analysis can rapidly and efficiently classify most types of protein targets, it often fails to provide sufficient protein yields and information for challenging targets, requiring expensive large-scale production for further characterization and posing a significant bottleneck to drug discovery research. Researchers typically need to produce dozens to hundreds of protein variants to address such resource-intensive and time-consuming complex drug discovery targets. This necessitates the development of efficient and effective upstream screening strategies to narrow down the scope to optimal constructs and expression and purification conditions to support such targets. Furthermore, the classic toolbox of drug discovery is extending beyond traditional small and large molecules. Emerging therapeutic modalities (such as degradative agents, macrocyclic peptides (MCPs), and disulfide-bound peptides (DCPs)) can be explored in early parallel to address undrugable targets that typically require milligrams of high-quality proteins. One exemplary expression approach involves using multiple expression systems to generate and screen a range of constructs to identify the most suitable constructs and systems for producing sufficient quantities of stable and functionally active proteins for structural and functional studies. Several laboratories have developed automated and bioinformatics tools to enable high-throughput, small-scale protein expression analysis platforms that can rapidly classify multiple constructs in parallel across different expression systems (Esposito, Garvey, and Chakiath, 2009; Marsichky 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 methods have certain drawbacks. For example, it offers limited flexibility and screening options to address low-expressed proteins and multi-protein complexes, particularly those with unstructured domains, prone to aggregation, and requiring additional co-expression partners for proper expression, folding, and function. Summary of the Invention

[0005] Therefore, this application provides a novel method for high-throughput protein expression at a larger scale, addressing the drawbacks associated with previous smaller-scale methods. For example, the method described herein overcomes the limitations of smaller-scale approaches and provides a faster and more efficient higher-throughput classification solution for screening optimal expression and purification conditions for challenging target proteins. In some embodiments, the method described herein allows for the parallel expression, purification, and characterization of, for example, a dozen to one hundred samples from cell cultures, to identify optimal constructs and suitable conditions for poor protein expression, evaluate co-expression couples in multi-protein complexes, and enable buffer / additive screening for insoluble and easily aggregated proteins, among other uses. In addition to protein screening, the method described herein 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 pull-down, DNA-encoded chemical library (DEL) screening, and surface plasmon resonance (SPR) screening, among others. The method described herein can also be widely adopted with minimal protocol modifications for all types of targets and expression systems, including intracellular, secreted, and membrane-bound proteins expressed in *E. coli*, BEVS, and mammalian systems. This method can 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 couplers, such as multihistidine, FLAG, glutathione S-transferase (GST), and maltose-binding protein (MBP), etc. (see Kimple, Brill, and Pasker, 2013). Furthermore, the method described herein can provide an efficient construct classification platform that can be used in parallel across multiple expression systems for the medium to high expression of appropriately folded single-subunit proteins. For example, in some cases, gel analysis can be used to confirm the presence of full-length or truncated proteins, identify molecular weight, and provide information about sample purity. Additionally, the method described herein can be used to recommend optimal expression systems, cell lines, and labeling strategies for the successful large-scale expression of specific proteins or domains.

[0006] Exemplary embodiments of this document include the following:

[0007] 1. A method for purifying one or more peptides from multiple mammalian cell culture samples, insect cell culture samples, and / or bacterial cell culture samples, the method comprising:

[0008] (a) Growing multiple mammalian, insect, or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL;

[0009] (b) If it is necessary to isolate the expressed polypeptide from cell debris, such as lysing the cells of the cell culture sample if the polypeptide is not secreted by the cells, optionally wherein for each of the plurality of cell culture samples, part (a) and (b) are performed in the same container;

[0010] (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample;

[0011] (d) Optionally clarify the plurality of supernatant samples by filtration such as depth filtration;

[0012] (e) Placing multiple clarified supernatant samples into the wells of a multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL / well; and

[0013] (f) subjecting the plurality of supernatant samples in the wells of the multi-well plate to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluent from the chromatography in the wells of the multi-well plate (i.e., the same or different multi-well plates); wherein part of (f) or part of (e) and (f) are performed in parallel on the plurality of samples.

[0014] 2. The method according to Example 1, wherein the following parts of the method are automated:

[0015] Part (f) or part (e) and (f).

[0016] 3. The method according to Example 1, wherein portions (d) to (f), portions (e) and (f), or all of portions (a) to (f) are performed in parallel.

[0017] 4. The method according to any one of Examples 1 to 3, wherein the plurality of clarified supernatant samples are placed in the wells of the multi-well plate at a volume of 2 to 30 mL / well.

[0018] 5. A method for purifying one or more peptides from a plurality of clear cell culture supernatant samples in a multi-well plate, said plurality of clear cell culture supernatant samples being obtained from the plurality of cell culture samples by a process comprising:

[0019] (a) Growing multiple mammalian, insect, and / or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL;

[0020] (b) If it is necessary to isolate the expressed polypeptide from cell debris, such as lysing the cells of the cell culture sample if the polypeptide is not secreted by the cells, optionally wherein for each of the plurality of cell culture samples, part (a) and (b) are performed in the same container;

[0021] (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample;

[0022] (d) Optionally clarify the plurality of supernatant samples by filtration, such as by depth filtration;

[0023] (e) A plurality of clarified supernatant samples are placed in the wells of the multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL / well;

[0024] The method includes: (f) subjecting the supernatant sample obtained from the process of (a) to (e) to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluent from the chromatography into the wells of a multi-well plate; wherein the method is performed in parallel on the plurality of clarified supernatant samples, and optionally wherein the method is automated.

[0025] 6. A method for affinity purification of peptides from multiple cell culture supernatant samples expressing one or more peptides for purification, the method comprising:

[0026] A multi-well plate is obtained, the multi-well plate containing a plurality of clear cell culture supernatant samples with a volume of 2 to 30 mL / well in the wells of the multi-well plate, and the plurality of clear cell culture supernatant samples are subjected to affinity chromatography using an affinity matrix in a pipette tip, and the eluent from the chromatography is placed in the wells of a second multi-well plate; wherein the method is performed in parallel on the plurality of supernatant samples, optionally wherein the method is automated.

[0027] The plurality of cell culture supernatant samples are obtained from mammalian, insect, and / or bacterial cell culture samples expressing one or more polypeptides, the cell culture samples being grown at a scale of 20 to 500 mL and subjected to one or more of lysis, centrifugation, and clarification, optionally wherein cell growth and lysis are performed in the same container.

[0028] 7. The method according to any one of Examples 1 to 6, wherein the method further comprises:

[0029] (g) Size exclusion chromatography (SEC) is performed on the eluent of the affinity chromatography; and

[0030] (h) Separating the polypeptide from the SEC into the wells of a multi-well plate, optionally one or both of (g) and (h) are automated.

[0031] 8. The method according to Example 7, wherein parts (g) and (h) are automated.

[0032] 9. The method according to any one of Examples 1 to 8, wherein a clear supernatant sample corresponding to a single cell culture sample is placed in more than one well of the multi-well plate.

[0033] 10. The method according to any one of Examples 1 to 9, wherein a clear supernatant sample from different cell culture samples is placed in different wells of the multi-well plate.

[0034] 11. The method according to any one of Examples 1 to 10, wherein 8 to 96 clarified supernatant samples are processed in parallel, such as 8 to 48, 8 to 24, 12 to 48, or 12 to 24.

[0035] 12. The method according to any one of Examples 1 to 11, wherein cells are lysed by adding glass beads accompanied by oscillation and / or wherein the cells are lysed without sonication.

[0036] 13. The method according to any one of Examples 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.

[0037] 14. The method according to any one of Examples 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.

[0038] 15. The method according to any one of Examples 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.

[0039] 16. The method according to any one of Examples 1 to 15, wherein the polypeptide is tagged with a multihistidine, FLAG, streptavidin, glutathione S-transferase (GST) or maltose-binding protein (MBP) tag, and wherein the affinity matrix recognizes the tag.

[0040] 17. The method according to any one of Examples 7 to 16, wherein the method comprises portions (g) and (h), and wherein the SEC chromatography is performed on an SEC matrix comprising particles having a pore size between 140 and 500 angstroms and / or a particle size between 3 and 5 micrometers and / or a molecular weight range of 5 to 700 kDa.

[0041] 18. The method according to any one of Examples 1 to 17, wherein the method further comprises 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.

[0042] 19. The method according to any one of Examples 1 to 18, wherein the one or more polypeptides comprise a recombinant protein complex.

[0043] 20. The method according to any one of Examples 1 to 19, wherein the one or more polypeptides do not include antibodies or antibody subunits.

[0044] 21. The method according to any one of Examples 1 to 20, wherein the cell culture sample is an insect cell culture sample, such as an Sf9 or T.ni cell culture sample.

[0045] 22. The method according to any one of Examples 1 to 21, wherein the cell culture sample is a mammalian cell culture sample, such as HEK293 or CHO cells.

[0046] 23. A system for performing the method according to any one of Examples 1 to 22, wherein the system includes a method for performing automated affinity chromatography in parallel on a plurality of cell culture samples of 2 to 30 mL volume, wherein the samples are placed in the wells (e.g., 8 to 96 wells) of a multi-well plate, wherein the chromatography is performed using an affinity matrix in a pipette tip, and the system further includes a method for placing the eluent from the chromatography into the wells of a multi-well plate (such as a second multi-well plate).

[0047] 24. A kit comprising reagents for performing the method according to any one of claims 1 to 22, wherein the kit optionally comprises one or more of the following: reagents for performing affinity chromatography on a plurality of 2 to 30 mL samples in parallel using an affinity matrix in a pipette tip (e.g., a pipette tip containing the affinity matrix, and / or one or more buffers, such as equilibration, washing, and elution buffers); reagents for performing SEC chromatography on the samples (e.g., an SEC column, and / or one or more buffers, such as equilibration, washing, and elution buffers); a positive control sample; a negative control sample; one or more multi-well plates for holding the samples; and instructions for use.

[0048] Further objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. These objects and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and explanatory, and are not intended to limit the claims.

[0049] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments and, together with the specification, serve to further explain certain principles described herein. Attached Figure Description

[0050] This U.S. provisional application includes at least one color drawing. If a non-provisional or PCT application that asserts priority to this U.S. provisional application and is incorporated herein by reference is published in the future, this Office will, upon request and at the necessary cost, provide a copy of this provisional patent application, including the color drawing.

[0051] Figure 1. Medium-scale expression and purification steps. A schematic workflow illustrating the steps involved in the medium-scale expression and purification of the recombinant protein: Insect cell cultures are grown in 500 mL conical tubes; cells are lysed using glass beads and clarified by deep filtration; affinity purification is performed in 24-well plates using IMCS tips; the protein is further purified and characterized by size exclusion chromatography and SDS-PAGE analysis. Several downstream applications were performed using the protein purified from this workflow, including negative staining and biochemical assays.

[0052] Figure 2. Guidelines for visual interpretation of flow cytometry results. Examples of successful and unsuccessful infections based on GP64-positive and viable cell populations.

[0053] Figure 3. Lysate layout for deep filtration. Cell lysates were clarified using a centrifugation and deep filtration process. An Orochem 24-well deep filter was placed on top of a 24-well deep plate, and the lysates were transferred into the filter plate. The filter collection plate assembly was centrifuged at 980 g for 5 minutes. Created using BioRender.com

[0054] Figure 4. Rearrangement of the pyrolytes. After depth filtering, the pyrolytes are transferred to four new 24-well deep-hole plates, as shown above. Created using BioRender.com.

[0055] Figure 5. Workbench Layout Illustration. This figure illustrates the Hamilton Star workbench layout for medium-scale protein purification using IMCS tips. The workbench features a slot for removing storage buffer from the IMCS tips, four holders with 1 mL Hamilton filter tips for transferring buffer to the plate, one holder with 1 mL IMCS tips, four slots for purification buffer, two blot stations, ten 24-well plates, and three 96-well plates for this method. This purification method enables the processing of up to 24 unique samples of up to 24 mL volume using up to ninety-six 1 mL IMCS tips. The 24-well sample plates allow for the simultaneous use of up to four 1 mL IMCS tips per well, increasing the amount of resin available for purifying each sample. During each wash cycle prior to elution, three wash stations are combined with three associated waste plates to provide clean wash reagent. Blot stations are implemented before the wash and elution steps to remove residual droplets from the IMCS tips.

[0056] Figure 6. Elution plate layout. Following affinity purification, the eluent was pooled from four adjacent wells into a single well before further characterization. Created using BioRender.com.

[0057] Figure 7. User Interface Description. This customizable user interface enables efficient and accurate protein purification processes. Modular selections for each step in the "Purification" box allow for run recovery and maximum user-customizable runs (if needed). The "Sample" selection allows scientists to change the number of sample binding plates for consecutive runs. The "Reagent" selection allows exclusion of reagent additions. This aids in buffer optimization and run screening. The "Anomalies" section allows scientists to perform an additional step, which includes pouring excess storage buffer removed from the Integrated Microchromatography System (IMCS) tip into a water-filled tank prior to the purification run. This supports improved liquid handling, ensuring that excess storage buffer does not negatively impact mixing cycles during protein purification.

[0058] Figure 8. Schematic diagram of the flow path of the Thermo Fisher Vanquish™ Duo HPLC system. This is a stacked system with dual gradient pumps, dual-separation autosamplers, column chambers, and left and right variable wavelength detectors (VWD). The fraction collectors allow for fractionation collection of up to 4 x 96-well plates. The left and right systems use two separate fraction collectors. Created using BioRender.com

[0059] Figure 9. Vanquish Chromeleon™ 7 Setup. This screen shows the UV Left module on the Vanq Left system. Turn on the lamp using the toggle switch below the UV and visible light lamps. Select 280 nm wavelength 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.

[0060] Figure 10. Vanquish Chromeleon™ 7 Method Setup. The Chromeleon™ console screen shows the method for creating a "Sequence" for a sample in the "Data" option to run on the Vanquish left arm. For each sample's sequence, select the location in the autosampler, the volume of sample to be injected into the column, and the instrument method. Create the instrument method and select the parameters for each module. If using the same column, this method can be used for all purification runs.

[0061] Figure 11. Medium-scale purification data of the multi-protein complex. SDS-PAGE gel analysis and size exclusion chromatography chromatogram data of the multi-protein complex purified from insect cells. The protein complex was purified from Sf9 cells using an affinity purification protocol described for FLAG-labeled proteins. The eluent was concentrated and further cleaned using SEC at 280 nm. Fractions corresponding to the peaks were further analyzed on the gel. A representative chromatogram and analytical fractions of one of the samples loaded in lane 6 of the first gel are shown.

[0062] Figure 12 shows the size exclusion chromatography (SEC) chromatograms of eight protein-polyprotein complexes purified from conventional batch purification in a large-scale purification process. The column used was a Superdex® 200 increase 10 / 300, and the buffer used was 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT. Proteins were purified from 3 L cultures of Sf9 cells using affinity M2-ant FLAG resin, followed by ion exchange, concentration, and loading onto the SEC for the final purification step. Detailed Implementation

[0063] Detailed description of certain embodiments

[0064] 1. Definition

[0065] Unless otherwise defined, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0066] In this application, unless otherwise stated, “or” is used to mean “and / or”. In the context of multiple dependent claims, the use of “or” is only used alternatively to refer back to more than one preceding independent or dependent claim. In this application, the articles preceding an item, “a / an(a)” or “the”, generally mean “one or more / a combination of such items”, unless the context specifies that only one such item may exist. Furthermore, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components comprising one unit, as well as elements and components comprising more than one subunit.

[0067] As stated herein, unless otherwise noted, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (such as one-tenth and one-hundredth of an integer).

[0068] Units, prefixes, and symbols are represented in their forms accepted by the Système International de Unites (SI). Numerical ranges include the numbers defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the entire specification. Thus, by referring to the entire specification, the terms defined immediately thereafter are defined more fully.

[0069] As used in accordance with this disclosure, unless otherwise specified, the following terms shall be understood to have the following meanings:

[0070] "Affinity chromatography" refers to a separation method based on specific interactions between affinity column molecules and specific peptides to be purified, such as the binding of peptides to ligands, the binding of His tags or other peptide tags to metal ions or specific antibodies placed in the chromatographic matrix.

[0071] Size exclusion chromatography (SEC) is a chromatographic method for separating molecules based on their size.

[0072] 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 comprise beads or particles containing materials in which peptides can selectively bind, such as those containing chelating ligands that bind to nickel. In some embodiments, the matrix of affinity chromatography materials may be placed in a column through which the material to be purified may flow. In other cases, it may be placed in a centrifuge column, or on a plate, chip, or other device. In some cases, the matrix may comprise beads or particles (such as magnetic particles) that can be separated from solution by, for example, the introduction of a magnet.

[0073] As used in this article, “elution buffer” refers to material eluted from a chromatographic matrix or column by applying an elution buffer.

[0074] The terms “peptide” and “protein” are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain natural and / or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. These terms also include amino acid polymers that have been modified (e.g., glycosylated, sialylated, etc.) or are complexed with other molecules.

[0075] The terms “isolated” or “purified” polypeptide or protein refer to a polypeptide that has been at least partially separated from one or more contaminants. In some embodiments, the polypeptide is purified to a purity greater than 80%, 90%, 95%, or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0076] As used herein, an "automated" or "automatically controlled" process is one that can be operated, for example, by a computerized control system with appropriate software (as opposed to a system that requires active, manual intervention during or between at least one step), such as to move a sample containing an analyte from one part of a 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 process, which in turn controls the flow of buffer and eluent through the system.

[0077] The term "sample" as used herein refers to the 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, for example, a supernatant from a cell lysate. The amount, volume, or portion can be up to 100% of the product or intermediate. Thus, a cell culture "sample," for example, refers to the amount, volume, or portion of a cell culture produced or used in a peptide purification method, as described herein. In some cases, each cell culture sample is grown in a single container, such that in some embodiments, multiple such samples are subjected in parallel to the methods described herein to purify peptides expressed by cells.

[0078] For example, "multi-well plate" or similar terms, such as 24-well plate or 96-well plate, refer to a plate that includes multiple individual wells into which liquid can be dispensed.

[0079] As used herein, "pipette tip," for example, for containing an affinity matrix, generally refers to a tube that can be used to dispense liquids. In some cases, the tip of a pipette tip can be attached to a pipette instrument (e.g., a multipipette) for delivering liquids into or out of the tip.

[0080] The term "multiple" in this document means two or more. In some embodiments, "multiple" may also include more than two, such as 4 to 96, or a range within these numbers.

[0081] As used herein, for example, method steps performed "in parallel" on multiple samples means that the method steps are performed on multiple samples simultaneously. For example, in some cases, automated equipment can be used to perform the method on multiple samples at the same time. In other cases, automated equipment is not required. For example, one way to process samples in parallel involves using a liquid dispensing or collecting device that dispenses or collects liquid from multiple samples at once, or using an instrument such as a multipipette that can be operated manually or automatically as needed.

[0082] 2. Methods

[0083] This disclosure relates to methods and systems for, for example, purifying peptides. In some embodiments, the method, for example, begins with growth from multiple suitable cell culture samples at a “medium scale” (such as 20 to 500 mL). In some embodiments, the cell culture samples are grown at, for example, scales of 30 to 250 mL, 50 to 250 mL, 30 to 200 mL, 50 to 200 mL, or 100 to 200 mL. The methods described herein are compatible with insect cell cultures, mammalian cell cultures, and bacterial cell cultures. For example, the type of cell culture may be selected based on the peptide intended for purification. Thus, in some embodiments, the methods described herein include growing multiple mammalian, insect, and / or bacterial cell culture samples, or alternatively purifying cell lysate samples obtained from multiple mammalian, insect, and / or bacterial cell culture samples. Thus, in some embodiments, the method includes growth using insect cell culture samples for expressing peptides, or beginning with cell lysates obtained from such samples. In some embodiments, the method includes growth using mammalian cell culture samples for expressing peptides, or beginning with cell lysates obtained from such samples. In some embodiments, the method includes growth using bacterial cell culture samples for expressing peptides, or starting with cell lysates obtained from such samples. When using the same type of cell culture samples (e.g., multiple cell culture samples all derived from insect cell cultures), in some embodiments, different cell culture samples may also be grown in parallel for greater efficiency, depending on the conditions. For example, when comparing different cell culture conditions (such as mammalian cell cultures versus insect cell cultures), cell culture samples may be grown at 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.

[0084] The methods described herein can be used to purify peptides from multiple medium-sized cell culture volumes of samples. For example, multiple different cell cultures, each containing host cells for peptide expression, can be grown, and each cell culture can then be purified substantially in parallel to achieve medium-scale peptide purification from multiple samples. In this way, for example, the methods described herein can be used to compare, for instance, different cell culture conditions used to produce the same peptide. They can also be used, for instance, to rapidly, at a medium scale, produce multiple different peptides, such as variants of peptides with different mutations, allowing all the different peptides to be tested and compared in downstream assays. Unlike small-scale protein production methods that may involve approximately 1 to 5 mL of cell culture, the medium-scale cell cultures described here can also better reflect the conditions for a final, larger-scale cell culture of 1 liter or more that can be used for later commercial production. Furthermore, the methods described herein allow for the production of highly purified proteins at a medium scale, and, as described above, the testing of many different cell culture conditions in parallel to help determine the optimal conditions for larger-scale production. Moreover, in some embodiments, the medium-scale production methods described herein allow for the production of, for example, 24 to 48 different protein purifications per week, enabling high-throughput analysis of multiple protein samples.

[0085] The methods described herein also allow for the growth of cell culture samples at a medium scale, as described above, and for growth and lysis within the same container, such as a flask. In some embodiments, cells are grown and lysed within 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, glass beads are used and agitation is employed to lyse the cells. In some such cases, cells are grown and lysed within the same container by adding glass beads to the container in which the cells are grown. In some embodiments, sonication is not used to lyse the cells. For example, glass beads may be added to the cell culture solution at the end of the growth phase, and the container may be agitated in a shaking incubator or using similar equipment to allow for cell lysis. Exemplary schemes for cell growth and lysis are provided in the embodiments herein.

[0086] Once the cells are lysed, the cell lysis solution (e.g., lysate) can be centrifuged either in the same container used for growth and lysis or in different containers to separate cell debris from a supernatant containing one or more expressed peptides. Thus, multiple cell culture samples (various to medium scales) that have been grown, lysed, and centrifuged produce multiple supernatant samples after centrifugation. In some embodiments, multiple samples can be centrifuged in parallel, such as by placing them in a centrifuge in the same run or in a subsequent run on the same day or at approximately the same time. In some embodiments, these samples can then be clarified, such as by depth filtration or surface filtration, to further remove contaminants from the expressed peptides. In some embodiments, clarification is performed by depth filtration. This then produces multiple clarified supernatant samples, which can be placed in the wells of a multi-well plate and further purified, for example, by chromatography. In some cases, clarification can be performed in parallel on multiple samples. The following examples provide an exemplary scheme for clarification, for example, by depth filtration. For example, a filter can be placed on a single tube or the well of a multi-well plate, and the supernatant collected after centrifuging cell lysates can be transferred to the filter, allowing it to flow through and be collected in the appropriate tube or well below the filter. In this way, multiple samples can be clarified by filtration and placed in the wells of a tube or multi-well plate. In some cases, particularly for larger-scale cell cultures, the volume of supernatant is large enough that a single cell culture sample will be separated in several wells of a multi-well plate or in several tubes. In some cases, while the supernatant from a single cell culture can be separated in the wells of several tubes or plates, the supernatants from different cell culture samples are not combined into the same well or tube. Instead, they are processed in separate tubes or wells, e.g., in parallel, allowing for direct comparison of conditions.

[0087] The clarified supernatant can be collected from the clarification step, such as after flowing through a depth filter or other filter into the wells of a multi-well plate or into a separate tube. In this way, the cell culture, lysis, centrifugation, and clarification processes produce multiple clarified supernatant samples, which are placed in the wells of a multi-well plate or in a set of tubes for further processing. In some embodiments, the volume of clarified supernatant placed in each well or tube is 2 to 30 mL per well or tube. In some cases, the volume is 2 to 10 mL, 4 to 10 mL, 4 to 8 mL, 10 to 30 mL, 10 to 20 mL, 5 to 15 mL, 15 to 30 mL, or 20 to 30 mL. In some cases, the clarified supernatant sample has already been placed in the multi-well plate.

[0088] In some cases, the cell lysate sample or clarified supernatant sample is then subjected to affinity chromatography to purify one or more peptides that the cells intend to express for purification. In some cases, the peptide is tagged with an appropriate tag that is recognized by an affinity reagent (such as an antibody or hapten) located in the affinity matrix used for chromatography. Non-limiting examples of such tags include multihistidine, FLAG, streptavidin, glutathione S-transferase (GST), or maltose-binding protein (MBP) tags. An affinity matrix that specifically recognizes the selected tag can be selected. In some cases, if the cells express more than one protein, one or more proteins can be designed to express the tag such that the affinity matrix will retain the tagged protein or a protein complex containing the tagged protein. In some embodiments, the affinity chromatography matrix is ​​present in the pipette tip, thereby simplifying the transfer of liquid into and out of the matrix and allowing the affinity matrix to be used with multi-pipette instruments, and optionally, making it automated. For example, if the sample is placed in a multi-well plate or tube arranged in a suitable manner, liquid can be aspirated into the wells or tube using a multipipette and placed into a pipette tip containing an affinity matrix. In some embodiments, the affinity matrix in the pipette tip can then be washed to remove unbound proteins. In other embodiments, no washing step is performed. Proteins bound to the matrix can then be eluted. In some cases, the eluent can be placed in a new multi-well plate or tube set, or in some cases, in the original multi-well plate or tube set, for analysis or subsequent processing. In some cases, 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. In some cases, 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. In some cases, the affinity chromatography step is automated, allowing liquid to be added to and removed from appropriate wells, pipette tips, and tubes in an automated manner, as needed, using appropriate equipment and software, thus guiding a multipipette or similar device accordingly. In other cases, this step is performed manually but still in parallel with the sample, such as when using a multipipette.

[0089] In some cases, further purification steps are performed after affinity chromatography, depending on the protein to be purified. For example, in some cases, size exclusion chromatography (SEC) can be performed after affinity chromatography. In some cases, SEC chromatography is performed on an SEC matrix containing particles having a pore size between 140 and 500 angstroms and / or a particle size of 3 to 5 micrometers and / or a molecular weight range of 5 to 700 kDa. The protein can then be obtained from the SEC fraction corresponding to the appropriate size of the peptide or peptide complex, which can be monitored, for example, at a wavelength of 280 nanometers. Depending on the available equipment, in some cases, affinity chromatography eluents from each cell culture sample can be SECed sequentially, or in others, SEC can be performed in parallel, and in some cases, the associated liquid dispensing and fraction collection can be automated.

[0090] Therefore, methods for purifying one or more peptides described herein can begin with the growth of cell culture samples to express the peptide for purification, or from subsequent steps such as affinity chromatography of multiple cell lysate samples, such as after centrifugation and clarification. Exemplary methods described herein include a method for purifying one or more peptides from multiple mammalian cell culture samples, insect cell culture samples, or bacterial cell culture samples, the method comprising:

[0091] (a) Growing multiple mammalian, insect, and / or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL;

[0092] (b) Lysing the cells of the cell culture sample, optionally wherein for each of a plurality of cell culture samples, part (a) and (b) are performed in the same container;

[0093] (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample;

[0094] (d) Clarify the multiple supernatant samples by filtration such as depth filtration;

[0095] (e) Placing multiple clarified supernatant samples into the wells of a multi-well plate, wherein the supernatant sample has a volume of 2 to 30 mL / well; and

[0096] (f) Multiple supernatant samples in the wells of the multi-well plate are subjected to affinity chromatography using an affinity matrix in a pipette tip, and the eluent from the chromatography is placed in the wells of the multi-well plate; wherein part (f) or parts (e) and (f) are performed in parallel on multiple samples. In some cases, part (f) or parts (e) and (f) are automated, or parts (d) through (f) are automated. In some cases, only part (f) or parts (e) and (f) or parts (d) through (f) are performed in parallel (i.e., simultaneously on multiple samples). In some cases, all parts of the method are performed in parallel. In some cases, the multi-well plate may be replaced by a suitable tube array for holding liquid samples.

[0097] Other examples include a method for purifying one or more peptides by affinity chromatography from multiple clear cell lysate supernatants obtained from multiple cell culture samples via a process including:

[0098] (a) Growing multiple mammalian, insect, and / or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL;

[0099] (b) Lysing the cells of the cell culture sample, optionally wherein for each of a plurality of cell culture samples, part (a) and (b) are performed in the same container;

[0100] (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample;

[0101] (d) Clarify the multiple supernatant samples by deep filtration; and

[0102] (e) Place multiple clarified supernatant samples into the wells of a multi-well plate, wherein the supernatant sample has a volume of 2 to 30 mL / well;

[0103] The method includes:

[0104] (f) The supernatant sample obtained from processes (a) to (e) is subjected to affinity chromatography using an affinity matrix in a pipette tip, and the eluent from the chromatography is placed in the wells of a multi-well plate; wherein the method is performed in parallel on the plurality of clarified supernatant samples, and optionally wherein the method is automated. Thus, for example, the method can begin with a sample prepared for affinity chromatography, which was previously prepared after sections (a) to (e) above. In some cases, section (f) or sections (e) and (f) are automated, or sections (d) to (f) are automated. In some cases, only sections (f) or sections (e) and (f) or sections (d) to (f) are performed in parallel (i.e., simultaneously on multiple samples). In some cases, all sections of the method are performed in parallel. In some cases, the multi-well plate can be replaced by a suitable tube array for holding liquid samples.

[0105] Further exemplary methods of this document include an affinity purification method for a plurality of cell culture supernatant samples expressing one or more peptides for purification, the method comprising: obtaining a multi-well plate containing a plurality of clarified cell culture supernatant samples in wells of the multi-well plate at a volume of 2 to 30 mL / well; subjecting the plurality of clarified cell culture supernatant samples to affinity chromatography using an affinity matrix in a pipette tip; and placing the eluent from the chromatography into wells of a second multi-well plate; wherein the method is performed in parallel on the plurality of supernatant samples, optionally wherein the method is automated. In some embodiments, the plurality of cell culture supernatant samples are obtained from mammalian, insect, and / or bacterial cell culture samples expressing one or more peptides, the cell culture samples being grown at a scale of 20 to 500 mL. In some embodiments, the cell culture samples are subjected to one or more of lysis, centrifugation, and clarification prior to use in this method, optionally wherein cell growth and lysis (if lysis) are performed in the same container. For example, in some cases, the clarified cell culture supernatant sample contains peptides secreted by the cells. In such cases, it may not be necessary to lyse the cells. Instead, the cell culture can be simply centrifuged and / or clarified before affinity chromatography after sufficient growth.

[0106] In any of these examples, the method may further include, if desired:

[0107] (g) Size exclusion chromatography (SEC) was performed on the eluent of affinity chromatography; and

[0108] (h) Separate the peptides from the SEC into the wells of the multi-well plate. In some options, parts (g) and (h) are automated.

[0109] In any of these methods, a new multiwell plate may be used at each step. In other cases, samples may be obtained from, processed, and returned to the same multiwell plate. In some embodiments of any of these methods, a clear supernatant sample corresponding to a single cell culture sample is placed in more than one well of the multiwell plate. In some cases, clear supernatant samples from different cell culture samples are placed in different wells of the multiwell plate. In some cases, 8 to 96 clear supernatant samples are processed in parallel, such as 8 to 48, 8 to 24, 12 to 48, or 12 to 24. For example, the number of samples that can be processed in parallel during affinity chromatography may depend on the number of wells in the compatible multiwell plate and, correspondingly, on the number of pipettes in the respective multipipettes used for transferring the liquid. In some cases, a 24-well plate is chosen so that up to 24 samples can be processed in parallel, i.e., 2 to 24, 8 to 24, 2 to 12, or 12 to 24, etc. In other cases, 48 ​​or 96-well plates may be used. Alternatively, a tube array can be used to replace the board.

[0110] In some of the methods described above, cells are lysed by adding glass beads accompanied by oscillation, and / or the cells are not lysed by sonication. In some cases, multiple cell culture samples are grown at scales of 30 to 250 mL, 50 to 250 mL, 30 to 200 mL, 50 to 200 mL, or 100 to 200 mL. In some cases, pipette tips containing an affinity matrix have volumes 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. In some cases, 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. In some cases, the peptide is tagged with multiple histidines, FLAG, streptavidin, glutathione S-transferase (GST), or maltose-binding protein (MBP), and the affinity matrix recognizes the tag. In some cases, the method includes portions (g) and (h), and SEC chromatography is performed on an SEC matrix containing particles having a pore size between 140 and 500 angstroms and / or a particle size of 3 to 5 micrometers and / or a molecular weight range of 5 to 700 kDa.

[0111] In some cases, the methods described herein further include structural or functional analysis of the purified peptides, such as cryo-electron microscopy, mass spectrometry, protein-protein interaction assays such as surface plasmon resonance, or homogeneous time-resolved fluorescence assays.

[0112] In some cases, the peptides expressed from cell culture samples contain recombinant protein complexes. In some cases, the peptides do not contain antibodies or antibody subunits. In some cases of the methods described herein, the cell culture samples are insect cell culture samples, such as Sf9 or T. ni cell culture samples. In some cases, the cell culture samples are mammalian cell culture samples, such as HEK293 or CHO cells, and other examples. In some cases, mixtures of different cell culture types can be used, for example, to compare protein production in insect cells versus mammalian cells. In other cases, the method can be used to compare, for example, the same host cell type grown in different culture media or conditions. In still other cases, the same cell type optionally having the same growth and culture medium conditions but with variations in the peptides is used, such as to compare various peptide mutants with each other.

[0113] 3. System and reagent kit

[0114] This disclosure also relates to systems capable of performing the methods described herein (including those described in the preceding sections), and kits containing reagents or components that can be used to perform the methods described herein. In some embodiments, the system may include devices capable of automatically performing affinity chromatography in pipette tips. In some embodiments, the system may further include means for transferring and removing multiple cell lysate samples (e.g., after centrifugation and clarification) from tubes of a well array or multiwell plate, and optionally further capable of performing affinity chromatography on multiple samples in the wells of a tube or plate, for example, by operation of a multipipette or similar component for transferring liquid into and from the wells of a tube or multiwell plate.

[0115] This disclosure also covers kits containing reagents for performing the methods described herein, such as reagents for affinity chromatography, i.e., pipette tips containing an affinity matrix, buffers (including washing, equilibration, and / or elution buffers) for affinity chromatography, control samples, etc. The kits may also contain suitable plates or tubes for holding the samples. The kits may further include instructions for use.

[0116] Example

[0117] Example 1: Parallel protein expression from 24 samples from insect cell culture

[0118] Insect cells are widely used as a heterologous expression system to produce 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 for folding and targeting these proteins correctly and provide a large quantity of soluble, non-aggregating proteins. This system has proven particularly useful for expressing any type of macromolecular assembly, including histone methyltransferases, DNA repair enzymes, kinetocytes, ubiquitin ligases, and viral capsid complexes (Abdulrahman et al., 2015; Osz-Papai et al., 2015).

[0119] This example focuses on insect cell expression and describes a medium-scale protocol for the successful expression and purification screening of intracellular multi-protein complexes. While co-expression of proteins using a single open reading frame (ORF)-encoding plasmid is widely used for baculovirus-mediated insect cell expression, the use of multi-ORF or multicistronic plasmid vectors offers significant advantages for recombinant 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 using a single promoter to drive transcription of each ORF or by using multicistronic constructs, where 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).

[0120] This process is designed to be an end-to-end solution from culture to protein purification. It enables the expression of 24 intracellular proteins or complexes from insect cells at a 200 mL scale and affinity purification using a Hamilton STAR™ liquid processor, followed by semi-automated purification in parallel using SEC on a Thermo Fisher Vanquish™ Duo system. Affinity chromatography was performed in a high-throughput manner using INtip™ technology via an integrated microchromatography system (IMCS) (Kates et al., 2023) (Kates, Tomashek, Miles, and Lee, 2020). The protocol described in this example uses the dispersive microextraction INtip™ platform, which utilizes turbulent mixing of resin within the pipette tip to increase the interaction time between the resin and the sample. This is coupled with an automated liquid handling system (Hamilton® Microlab STAR™ workstation) to enable consistent and high-throughput parallel and rapid purification of recombinant proteins.

[0121] SEC is then performed on a Thermo Fisher Vanquish™ Duo system coupled with an autosampler that sequentially injects the protein and allows for simultaneous two purifications using separate buffer lines with Horizon Dual pumps, separate injection loops, and parallel columns and detectors. This method allows for the production of high-quality purified protein complexes, further evaluation of downstream applications and assays to identify purification and optimization solutions for large-scale production and purification. More specific protocols related to specific steps within the platform are provided below.

[0122] Basic Protocol 1 describes a method for generating P1 baculovirus in Sf9 cells using co-transfection of linearized baculovirus baculovirus baculosomes BestBac™ with genes of interest subcloned into baculovirus transfer vectors (such as pAcGP67). This protocol also describes how to amplify the baculovirus to generate P2 and P3 stock solutions in Sf9 cells.

[0123] Basic scheme 2 describes the infection of Sf9 and T.ni cells with P3 virus at a scale of 200 mL for the production of one or more biomass expression proteins of interest. Co-expression methods via co-infection of cells with multiple viruses are also discussed here.

[0124] Basic Protocol 3 describes the preparative steps for cell lysis and INtip™ affinity chromatography purification performed on a Hamilton STAR™ workstation.

[0125] Basic scheme 4 describes the SEC method developed on the Thermo Fisher Vanquish™ system to provide medium-scale analytical and preparative purification.

[0126] Supporting protocol 1 describes a glycoprotein 64 (GP64) staining assay for assessing the quality of baculoviruses.

[0127] Support Scenario 2 describes an automated method and procedure for medium-scale INtip™ affinity purification performed on a Hamilton STAR™ workstation.

[0128] Support Scenario 3 describes a detailed approach to building using Chromeleon™ software to support SEC components for mid-sized platforms.

[0129] Basic Plan 1

[0130] Baculoviruses are generated through homologous recombination

[0131] This protocol assumes that the initial steps of cloning one or more genes of interest have already been completed. DNA encoding the gene of interest is cloned into a modified version of the commercially available baculovirus transfer vector pAcGP67 (BDBiosciences, Brøndby, Denmark), suitable for high-throughput cloning. The recombinant gene is expressed as a fusion protein when cloned into one of the available restriction enzyme sites controlled by a strong baculovirus polyhedrome promoter. A fragment of the baculovirus genome is side-attached to the expression cassette for transfer into linearized baculovirus DNA via homologous recombination in insect cells (Murphy and Piwnica-Worms, 2001). The GP67 secretion signal sequence in the pAcGP67 vector is removed for cloning intracellular proteins, while the secretion signal sequence is retained for cloning secreted proteins. The same expression vector backbone is also used to generate polyci / trans or multi-ORF constructs.

[0132] Strategies for generating constructs containing two or more genes (such as using internal ribosome entry sites (IRES) and self-cleaving 2A peptides) are widely used methods for generating multi-cis-trans constructs because both strategies simultaneously express two or more separate 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 gene of interest with a single promoter, enabling the independent generation of mRNA transcripts for each target gene for efficient overexpression of the protein complex. When higher-order protein complexes are required, the number of promoter systems can be expanded, requiring modification of the pAcGP67 vector. Promoters with varying transcriptional strengths can be used to achieve variable multi-gene expression chemometry. For insect cells, early or late promoters can be used to control the expression of different genes during the infection cycle. Multi-gene co-expression is context-specific, and different promoters can lead to variable multi-gene expression chemometry.

[0133] In this example, baculoviruses were generated in insect cells via homologous recombination (Kitts, Ayres, and Possee, 1990; Kitts and Possee, 1993). The protocol described herein provides steps for co-transfection of linearized baculoviruses with the expression transfer vector pAcGP67 encoding one or more genes of interest. Co-transfection resulted in the production of recombinant baculoviruses, which were then amplified to higher titers (1 x 10⁻⁶). 8 Up to 1 x 10 9 (pfu / mL), making it suitable for recombinant protein expression. This protocol is optimized for high-throughput workflows and can generate up to 96 recombinant baculovirus samples at a time.

[0134] Material

[0135] The materials used in the first part are listed below:

[0136] Recombinant transfer vector pAcGP67 or similar expressing one or more target genes at a concentration of 40 ng / µL (see Background Information / Basic Protocol 1).

[0137] An empty transfer vector with a backbone matching that used for virus generation was used as a control at a concentration of 40 ng / µL.

[0138] BestBac™ 2.0 linearized rod-shaped particles, stock solution concentration 0.1 mg / mL (Expression Systems #91-002)

[0139] TransIT™ Insect Transfection Reagent (Mirus #MIR6100)

[0140] In ESF921 medium (Expression Systems # 94-001F), at 2 x 10 6 With 7 x 10 6 Cells / mL from the Sf9 cell line in the logarithmic growth phase and with >95% viability

[0141] ESF921 insect cell culture medium (Expression Systems #96-001-01) heated to 27°C

[0142] Heat-inactivated FBS (Thermo Fisher #16140-071)

[0143] 96-well deep-well plate, sterile (Axygen #p-2mL-sq-cs)

[0144] 96-well round-bottom deep-well plate, sterile (Thompson # 93113-S)

[0145] 24-well Axygen deep-hole round bottom plate, sterile

[0146] 15 mL conical tube

[0147] AeraSeal™ (Millipore Sigma #A9224-50EA)

[0148] AlumaSeal™ (T790080-5)

[0149] 96-hole Duetz sandwich cap with 0.8 mm holes (Kuhner Shaker Inc. #104118)

[0150] Sterile pipette tips (p20, p200, p1000)

[0151] 1.4 mL matrix tube (catalog number 3712-11)

[0152] U-shaped bottom measuring plate (Falcon # 353910)

[0153] Zymo Clean N' Concentrated Kit (Zymo #D4033)

[0154] sterile reagent reservoir

[0155] 70% EtOH for disinfection of cleanroom hoods

[0156] 150 mL sterile culture flask

[0157] Part Two Materials:

[0158] Choose a pipette, preferably an 8-channel one (p20, p200, p1000).

[0159] Orbital oscillator, 3 mm eccentricity, set to 1000 rpm at 27°C.

[0160] Benchtop centrifuge (for centrifugation sedimentation assay plates and DNA plates)

[0161] sterile serum pipette

[0162] Laminar flow hood (Labconco purifier BSC Class II or equivalent)

[0163] Solution steps with step annotations:

[0164] P1 is generated:

[0165] In a sterile laminar flow hood, prepare premixes #1 and #2 in sterile 15 mL conical tubes. The amount of premix to be produced depends on the number of recombinant transfer vector DNA constructs to be expressed. At least 10% extra should be prepared to ensure sufficient volume for each sample. Use empty vector pAcGP67 along with BestBac™ linearized rod-like DNA as a positive control. Use untransfected cells as a negative control.

[0166]

[0167] Add a total of 100 ng of 2.5 μL of transfer vector DNA at a concentration of 40 ng / µL to the corresponding wells of a 96-well Axygen plate. For larger DNA inserts (>5 kb), it is recommended to double the amount of DNA (200 ng).

[0168] Rotate the DNA plate at 1000 g for 1 minute to ensure no air remains in the liquid. Add 25 μL of premix 1 to each well and gently pipette 3 times to mix. Add 25 μL of premix 2 to each well and gently pipette 3 times to mix. Seal the plate with the aluminum seal and incubate at room temperature for 20 minutes to allow complex formation. Keep the plate in a sterile laminar flow hood during incubation.

[0169] In ESF921 medium at 1.0 x 10 6 Prepare Sf9 cell suspensions at a density of cells / mL. The volume depends on the number of samples, using 550 μL per sample. Add 550 μL of cell suspension to each well, for a total volume of 602.5 μL / well. Seal the plate with an AeraSeal™ and cover it with a Duetz™ sandwich cap. Shake the plate on a 3 mm diameter orbital shaker at 1000 rpm at 27°C for 6 days. Fabric seals, like AeraSeal™, allow for maximum culture aeration. Avoid using porous plastic seals, as they have been shown to limit oxygen uptake in this experiment. The Duetz™ sandwich cap reduces evaporation during shake incubation. Evaporation can negatively impact transfection efficiency. The 6-day incubation period for P1 is the necessary amount of time for the recombinant virus to acquire titers, and it is not recommended to shorten this incubation time by at least 5 days.

[0170] Generate P2 and P3:

[0171] On day 6, 10 µL of P1 cell suspension was added to sterile 96-well Thompson deep-well plates at a concentration of 1.0 x 10⁻⁶. 6 Transfer 1000 µL of fresh Sf9 cells / mL to 1010 µL of the plate. 1010 µL is approximately the maximum possible volume per well to maintain adequate aeration and avoid splashing onto the fabric seal. Cap the plate with an AeraSeal™ and Duetz™ sandwich cap. Incubate the plate at 1000 rpm for 4 days in a 3 mm diameter shaker at 27°C. At the end of the 4-day incubation period, the viral titer should reach 1 x 10⁻⁶ cells / mL. 8 Up to 1 x 10 9 pfu / mL.

[0172] Store P1 plates sealed with AlumaSeal at 4°C for reuse in P2 amplification under unforeseen circumstances (such as contamination, incubator malfunction, etc.). On day 4 of P2 incubation, stain cells with GP64 antibody to assess viral production (see Supporting Protocol 1). If flow cytometry is unavailable, viral plaque assays can be performed instead of GP64 assays. The advantage of GP64 assays is faster readout, within one day, compared to the 5 to 7 days required for viral plaque assays.

[0173] P2 viruses can be stored and used for future P3 generations (see Save Virus).

[0174] 5 µL of P2 cell suspension was added to sterile 24-well Axygen deep-well plates at a concentration of 1.0 x 10⁻⁶. 6 P3 virus was prepared by transferring 1 cell / mL to 4 mL of fresh Sf9 cells. The plate was capped with AeraSeal™ and incubated for 4 days at 27°C on a 12 mm shaker at 300 rpm. The generated P3 virus can be scaled up to a volume suitable for medium-scale expression. P3 virus can be stored at 4°C for up to 6 months.

[0175] GP64 assay (to assess viral production efficiency)

[0176] Collect 50 µL of cell suspension in a 96-well U-shaped plate and perform GP64 staining and measurements. (See Supporting Protocol 1)

[0177] Preserving viruses

[0178] Seal the plate in a purification hood using an adhesive foil seal. Use a roller to ensure a good seal. Rotate the plate at 3,000 xg for 10 minutes to remove cells / debris. Transfer the clear supernatant to a sterile matrix tube. Add heat-inactivated FBS at a concentration of 10% for storage stabilization (i.e., for P2, add approximately 111 µL of FBS to 1000 µL of clear supernatant; for P3, add approximately 444 µL of FBS to 4 mL of clear supernatant). Store the plate in the dark at 4°C (baculoviruses degrade upon light exposure). The virus remains well in storage for up to 12 months. The virus can be re-amplified from older stock solutions before use for expression. Older viruses may have a reduced titer, which may affect P3 amplification. (See Table 1.)

[0179] Support Option 1

[0180] GP64 antibody assay

[0181] This protocol utilizes fluorescence-activated cell sorting (FACS) to validate high-titer baculovirus stock solutions. Glycoprotein 64 is a viral protein expressed on the membrane of successfully infected insect cells (Kitts and Green, 1999). This glycoprotein can be labeled with a phycoerythrin-labeled antibody to infer sufficient viral titers (Volkman and Goldsmith, 1988).

[0182] Material:

[0183] The materials are as follows

[0184] FACS Buffer (Reference Reagents and Solutions Section)

[0185] 7-AAD reactivity dye (Beckman Coulter #A07704)

[0186] GP64-PE antibody (Expression Systems #97-201)

[0187] CytoFLEX™ sheath fluid (Beckman Coulter #B51503) or 0.2 µm filtered sqH20 (alternatives)

[0188] AlumaSeal™ (T790080-5)

[0189] Benchtop centrifuge (for centrifugation sedimentation assay plates and DNA plates)

[0190] A Beckman Coulter CytoFLEX™ or similar flow cytometer equipped with a "blue" laser (488 nm). The laser should be compatible with phycoerythrin and PC5.5 fluorescent dye samples.

[0191] Solution steps:

[0192] The following describes the protocol used for GP64 antibody assay.

[0193] Sample preparation:

[0194] Aliquot 50 µL of sample into 96-well U-bottom plates. This should be approximately 100,000 cells. The sample volume can be adjusted lower or higher depending on the culture density. Cover the plate with a foil seal and centrifuge at 3000 rpm for 3 minutes. Carefully aspirate the supernatant to avoid contact with the cell pellet. Dilute PE-anti-GP64 1:125 in FACS wash buffer (8 µL PE-anti-GP64 / 1 mL FACS wash buffer). Resuspend the pellet evenly in 40 µL of FACS wash buffer containing PE-anti-GP64. Cover the plate with a foil seal and incubate the sample at 4°C for 20 minutes. Initialize CytoFLEX at this time so that the system is ready to read the sample after completing the following steps. Refer to the CytoFLEX manual for details. After incubation, add 150 µL of FACS wash buffer to each well. Centrifuge at 3000 rpm for 3 minutes. Carefully aspirate the supernatant to avoid contact with the cell pellet. Prepare an appropriate volume of viable dye mixture for staining live / dead cells. The viable dye mixture is 40 µL 7-AAD / 1 mL FACS wash buffer. Resuspend the pellet in 100 µL of diluted 7-AAD dye. Incubate at room temperature for at least 5 minutes. Analyze on CytoFLEX according to step 7.

[0195] CytoFLEX Operations

[0196] Flow cytometer settings: Set the flow rate to 10,000 events in "Fast" mode. The flow rate and endpoint can be adjusted as needed. Set the PE and PC5.5 channels. Ensure the flow cytometer is set to process U-bottom assay plates.

[0197] If necessary, adjust the GP64 gating and viability gating to suit the "GP64 positive / negative" and "live / dead cell" controls (Figure 2). This may drift slightly, so adjusting the gating relative to the positive (empty vector-infected cells) and negative (uninfected cells) controls is crucial. Allow approximately 5 minutes for the dye to enter the damaged cells, bind to the DNA, and reach maximum fluorescence. Note: If the plate is read too early after dye addition, the 7AAD signal in the first few samples may be artificially reduced.

[0198] Basic Plan 2

[0199] Production of insect cell biomass expressing one or more target proteins

[0200] This protocol is used to produce insect cell biomass expressing the target protein. It enables the production of Sf9 or T. ni cell paste expressing the target protein on a scale of 200 mL using the baculovirus produced in Basic Protocol 1.

[0201] Insect cells were infected with baculovirus and incubated for 2 to 3 days to allow protein expression. The cell pellet was then harvested and frozen, followed by cell lysis and protein extraction. Protein expression yield, recyclability, and quality may differ in Sf9 and T. ni cells, and therefore, it is recommended to test expression in both cell lines. We observed that the T. ni cell line typically produces higher amounts of secreted, intracellular, and membrane recombinant proteins per culture volume compared to Sf9. However, Sf9 is generally the preferred cell line for intracellular protein expression due to the higher intracellular target protein cleavage rates observed in T. ni cells. The infection characteristics of the two cell lines also differ and may require individual optimization to achieve optimal protein expression; for example, the initial infection density, multiplicity of infection (MOI), and incubation length typically differ between the two cell lines when achieving optimal protein yield. While both cell lines exhibit robust culture characteristics, the shorter doubling time of T. ni cells in culture compared to Sf9 can shorten production timelines and may be an important consideration in terms of resource and reagent usage.

[0202] Material:

[0203] Materials include:

[0204] Laminar flow cleanroom hood (Labconco Purifier BSC II or equivalent)

[0205] Sf9 cell lines in the logarithmic growth phase and >95% viability (in ESF921 medium (Expression Systems catalog number 94-001F) at 2 x 10⁻⁶ ppm) 6 With 7 x 10 6 (between cells / mL)

[0206] T. ni cell lines in the logarithmic growth phase and >95% viability (in ESF921 medium (ExpressionSystem catalog number 94-002F) at 2 x 10⁻⁶ ppm) 6 With 7 x 10 6 (between cells / mL)

[0207] ESF921 insect cell growth medium (Expression Systems, catalog number 96-001-01) warmed to 27°C

[0208] Sterile serum pipettes (5 mL, 50 mL) and cell counters (Beckman Coulter Vi-CELL XR or BLU or equivalent).

[0209] 1 L polycarbonate conical flask with vent cap (Corning, catalog number 431147)

[0210] AeraSeal (Millipore Sigma, catalog number A9224-50EA)

[0211] 500 mL conical bottom polypropylene centrifuge tubes (Corning, catalog number 431123)

[0212] Single-channel pipette (1000 µL)

[0213] Sterile 1000 µL pipette tip

[0214] A shaking incubator with a 25 mm oscillation diameter, maintained at 150 rpm and 27°C (Infors or equivalent).

[0215] 500 mL tube support (Infors, catalog number 66129)

[0216] Centrifuges with oscillating rotors (Beckman or equivalent)

[0217] Polyetherimide centrifuge tube pads for 500 mL tubes (Corning, catalog number 431124)

[0218] -80℃ refrigerator (Thermo Fisher or equivalent)

[0219] Solution Steps

[0220] Steps 1 through 3 of this protocol are performed using aseptic techniques in a biosafety cabinet, as shown below:

[0221] Using a 5 mL serum pipette, aspirate 1 to 2 mL of Sf9 or T. ni cell stock solution and dispense into the sample cup of a Vi-CELL counter to measure viable cell density and assess overall culture health by analyzing viability and mean viable diameter. Maintain insect cells in ESF921 medium in conical flasks, periodically dividing every 2 to 3 days until they reach a size of at least 0.7 x 10⁻⁶.6 A cell density of 10 cells / mL is required, while cells are not allowed to exceed 7 x 10⁻⁶. 6 The density should be cells / mL. For Sf9 and T.ni cells, respectively, the culture viability should be >= 95%, and the diameter of uninfected cells should not exceed 15.5 and 19 µm, respectively.

[0222] Dilute Sf9 or T.ni cells to 2 x 10⁻⁶ in ESF921 medium at 27°C. 6 1 cell / 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. The volume of virus to be added to the insect cell culture (V) is calculated in milliliters. 病毒 The guiding formula depends on the initial cell density D expressed in cells / mL. 培养物 Culture volume V, expressed in milliliters 培养物 MOI (multiple of infection) expressed in pfu / cell and viral titer T calculated in pfu / mL 病毒 .therefore,

[0223] V 病毒 = (D 培养物 (V) 培养物 (MOI) / (T) 病毒 )

[0224] For newly generated viruses with a high percentage of gp64 staining, assuming a titer of 4 x 10⁻⁶... 8 pfu / mL is acceptable. Furthermore, we experimentally determined that MOIs of 0.5 and 1, respectively, were optimal for most proteins expressed in Sf9 and T. ni cells. Therefore, for Sf9 cells,

[0225] V 病毒 = (2 x 10 6 (cells / mL)(200 mL)(0.5 pfu / cell) / (4 x 10) 8 pfu / mL) = 0.5 mL

[0226] For T.ni cells,

[0227] V 病毒 = (2 x 10 6(1 cell / mL)(200 mL)(1 pfu / cell) / (4 x 10) 8 pfu / mL) = 1mL

[0228] For cases where multiple viruses are required to co-express proteins from different plasmids, equal amounts of each P3 virus are added to the cells.

[0229] For Sf9 and T. ni cells, culture in tubes at 27°C with shaking at 150 rpm for 72 or 48 hours with a 25 mm shaking diameter. Check cell viability and diameter of T. ni on day 2 and Sf9 on day 3. For both Sf9 and T. ni, harvest cells if the cell diameter is greater than 17 or 21 µm and the viability is between 50% and 85%. For both Sf9 and T. ni, if the cell diameter is less than 17 or 21 µm, proceed with troubleshooting as described in Table 1. If the viability is less than 50%, refer to troubleshooting as described in Table 1.

[0230] Harvest cells by centrifugation at 2,200 g for 10 minutes. Discard the supernatant and freeze the pellet at -80°C. Even if purification is planned for the same day, it is recommended to freeze the cell pellet for at least 10 minutes before cell lysis.

[0231] Basic Plan 3

[0232] Medium-scale affinity purification

[0233] This protocol was used to perform parallel affinity purification of 24 samples containing His and / or FLAG-tagged proteins using IMCS pipette tips and a Hamilton STAR liquid processor.

[0234] Material

[0235] Materials include:

[0236] Equilibration buffer and lysis buffer (see formulations in Reagents and Solutions).

[0237] TCEP-HCl (Pierce, catalog number 20490)

[0238] Benzoylase endonuclease (Sigma, catalog number 101697)

[0239] Roche Complete TMEDTA-free protease inhibitor mixture (Sigma, catalog number 11873580001)

[0240] Glass beads (Thomas Scientific, catalog number 1177X44 or similar)

[0241] A shaking incubator with a 25 mm oscillation diameter, maintained at 250 rpm and 10°C, with suitable supports to hold 500 mL tubes (Infors or equivalent).

[0242] 500 mL tube holder (Infors, catalog number 66129) 24-well depth filter plate (Orochem, catalog number OC24DAHL-B)

[0243] 24-well plate (Axygen, catalog number P-DW-10ML-24-CS)

[0244] Centrifuges with basket-type rotors, such as the Beckman JS-5.3 or equivalent.

[0245] Microporous plate carrier for suspended baskets (Beckman catalog number 368905)

[0246] Microporous plate carrier support pad (Beckman catalog number 369382)

[0247] Polyetherimide centrifuge tube pads for 500 mL tubes (Corning, catalog number 431124)

[0248] Reagent storage container (Thermo Fisher Scientific catalog number 95128085)

[0249] 1000 µL 12-channel pipettes are available.

[0250] 1000 µL suction tips are available.

[0251] 1000 µL 8-channel pipettes are available.

[0252] 1000 µL IMCS pipette tips (IMCS, catalog numbers DP016 or DP017) filled with 50 µL Ni-NTA resin or 50 µL M2 anti-FLAG resin.

[0253] 96-well plate (Thermo Scientific, catalog number AB-0932)

[0254] Elution buffer (refer to the formulation in the reagents and solutions).

[0255] FLAG peptide (Sigma, catalog number MFCD01863911)

[0256] Heavy-duty imprinting system with lint-free imprinting media in a universal tray - Super Absorbent Pad (V&P Scientific, catalog number VP 540DB)

[0257] Super absorbent polypropylene pad cut to fit universal tray (V&P Scientific, catalog number VP 540DB1-100)

[0258] Lint-free imprinting media (V&P Scientific, catalog number C VP 540D-100)

[0259] Hamilton Microlab STAR™

[0260] Automated 300 mL polypropylene reservoir for buffer solutions (Thermo Fisher Scientific catalog number 12565571)

[0261] Acidic buffer solution (see formulation in Reagents and Solutions)

[0262] Washing buffer (see formulation in Reagents and Solutions)

[0263] Hamilton CO-RE 1000 µL filter tip (Hamilton, catalog number 235940)

[0264] NanoDrop™ Spectrophotometer (ThermoFisher Scientific)

[0265] NuPAGE™ Sample Reducing Agent (10X) (Invitrogen, Catalog No. NP0009)

[0266] NuPAGE™ LDS Sample Buffer (4X) (Invitrogen, Catalog No. NP0007)

[0267] 200 µL thin-walled PCR 96-well plates (Axygen or similar)

[0268] Plate seals (AlumaSeal, catalog number T790080-5 or similar)

[0269] PCR thermal cycler (Fisher Scientific or equivalent) or a 95°C water bath for boiling samples.

[0270] NuPAGE™ 4% to 12%, Bis-Tris, 1.0 mm, medium protein gel (Invitrogen, catalog number WG1402BOX)

[0271] XCell4 SureLock™ Protein Gel Run Kit (Invitrogen, Catalog No. WR0100)

[0272] NuPAGE™ MES SDS Run Buffer (20X) (Invitrogen, Catalog No. NP0002)

[0273] Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards (Bio-RAD, Catalog No. 1610375)

[0274] PowerPac™ (Bio-RAD or equivalent)

[0275] ThermoFisher (catalog number EI9010 or similar)

[0276] Tray for cleaning and staining gel

[0277] InstantBlue Coomassie staining (Novus Biologicals, catalog number ISB1L)

[0278] Laboratory swing platform (VWR or equivalent)

[0279] Bio-RAD Gel Doc™ EZ imaging system or equivalent with Image Lab™ software.

[0280] Solution Steps

[0281] The steps used are as follows:

[0282] Keep frozen precipitates on ice. All cell lysis and rearrangement steps should be performed on ice whenever possible. Leaving cell precipitates, lysates, or protein samples at room temperature may lead to accelerated protein degradation.

[0283] Add 24 mL of lysis buffer and approximately 2 mL of glass beads to the cryoprecipitate in a 500 mL tube and incubate at 10 °C with shaking at 250 rpm for 1 hour in an Inforrs incubator with a 25 mm oscillation diameter. Alternatively, sonication can be used to lyse cells, but this will not allow for higher throughput formats. The proposed bead-based lysis method allows for the lysis of 24 samples in a single incubation.

[0284] Pre-wetting the 24-well Orochem filter with 1 mL of equilibration buffer is done by placing the filter plate on top of the 24-well collection plate and centrifuging at 980 g for 2 min. Discard the flow-through and place the filter plate back on top of the 24-well plate. In step 6, the plate assembly can be secured to one side with laboratory tape by centrifugation. Pre-wetting the depth filter is crucial for efficient filtrate recovery. One Orochem plate can be used to filter up to four samples. Alternatively, conventional PES filters can be used to filter the lysates; however, these filters are prone to clogging.

[0285] Clarify the lysate by centrifuging at 2,200 g for 10 min using a Beckman floor-standing centrifuge. Pour the supernatant into a pre-labeled tank. Discard the precipitate.

[0286] Using a 1000 µL 12-channel pipette, transfer the clarified lysates one by one from the tank to a pre-wetted 24-well filter plate, such that each lysate is transferred to one row (6 wells) of the plate, 4 mL / well. See the illustration of the lysate layout in the Orochem filter (Figure 3).

[0287] Centrifuge the lysate at 980 g for 5 min in a filter plate placed on top of a 24-well collection plate. Discard the filter plate. Lysate filtration ensures smooth downstream affinity purification and helps avoid clogging of the IMCS pipette tip.

[0288] Using a 1000 µL 8-channel pipette, rearrange the clarified and filtered lysate into four new 24-well plates, transferring the lysate column by column, 6 mL / well, ensuring that all four plates have the same layout. See the illustration of the layout of the lysate in the 24-well plate for binding (Figure 4).

[0289] Resin-filled IMCS tips were prepared by arranging resin-filled IMCS tips in a Hamilton 1 mL tip holder to match the lysis product layout in a 24-well plate. The amount of resin required for affinity purification could be estimated based on resin binding capacity and the expected expression yield of the target protein. In this protocol, 50, 100, 150, or 200 µL of resin could be used per sample. For 200 µL of resin per purification, four tips were placed in the holder positions corresponding to the wells of that sample in the 24-well plate. This protocol allows for the use of different resins for different samples to match the resin to the purification tag of the target protein. For example, Ni-NTA resin, Sigma M2 anti-FLAG resin, and Streptactin HC resin could be used for multihistidine-labeled proteins, FLAG-labeled proteins, and Strep-labeled proteins, respectively. We evaluated the Phynexus and IMCS tip columns and determined that both performed equally well in medium-scale protein affinity purification. It is important to note that using the Phynexus tip requires different settings on the Hamilton STAR than the IMCS tip in order to perform the procedure correctly.

[0290] Prepare 96-well plates containing 300 µL of elution buffer in the wells corresponding to the pipette tip layout. The total elution volume for purification using four IMCS tips will be equal to 1,200 µL.

[0291] Prepare two trays for imprinting by assembling filter paper on top of the liner in a universal tray.

[0292] Turn on the 4°C water cooler on the Hamilton STAR to cool the worktable that will hold the sample during purification.

[0293] As shown in Figure 5, the Hamilton STAR™ workbench is prepared by arranging pipette tips, buffer solution, plates, and blot stations.

[0294] Material:

[0295] - A water-filled trough for dispensing storage buffer from IMCS pipette tips.

[0296] -IMCS suction head

[0297] - Three CO-RE pipette tips for buffer transfer

[0298] - A tank filled with acidic buffer solution

[0299] - The tank is filled with equilibration buffer.

[0300] - Two tanks filled with washing buffer

[0301] - 96-well plates for acidic buffer solutions

[0302] - 96-well plate for equilibration buffer

[0303] - Three 24-well plates for washing buffer

[0304] - Three empty 24-well plates for dispensing used wash buffer.

[0305] - Two imprinting stations (To prepare the imprinting stations, place the lint-free imprinting media on top of the super absorbent polypropylene pad mounted in the universal tray)

[0306] - Four 24-well plates containing filtration lysate (6 mL / well)

[0307] - 96-well plates with 300 µL elution buffer per well

[0308] Initiate the IMCS medium-scale protein purification method on the Hamilton STAR™ (see Support Protocol 2 for method details). After the Hamilton STAR™ method is complete, remove all materials from the worktable. Turn off the water cooler. Note: Perform daily and weekly maintenance using the "Microlab STAR Maintenance and Validation" application provided by Hamilton. Additionally, after each run, wipe the 96-probe multi-probe head (MPH) and 8-channel pipetting module with water and a clean, lint-free, dry cloth.

[0309] For each affinity-purified sample, use a separate elution chamber. See the illustration of the elution chambers (Figure 6). Perform A280 measurements using NanoDrop. For the blank, use elution buffer without 3x FLAG peptides.

[0310] Reduced samples for SDS-PAGE analysis were prepared by mixing 20 µL of elution buffer with 3 µL of 10x reducing agent and 7 µL of 4x LDS run buffer in a PCR plate, maintaining the same sample layout as in the mixed elution plate. The samples were heated at 95°C for 5 min using a PCR thermal cycler. 20 µL of sample and 7 µL of pre-stained protein standards were loaded onto a NuPAGE gel. The gel was run at 180V in 1x NuPAGE™ MES SDS run buffer for 50 min. Run times 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 larger than 200 kDa may require longer run times for better separation.

[0311] Using a gel scalpel, open the gel container and transfer the gel to a tray filled with water. Rinse the gel in water and stain in Instant Blue Coomassie stain for 1 hour, gently agitating on a shaking platform. Destain in water for 2 hours, continuing to agitate on a shaking platform.

[0312] The gel was scanned and labeled using Bio-RAD Image Lab™ software. On the Bio-RAD Gel Doc™ EZ imaging system, a white tray was used to perform the scan by selecting the default scheme and Coomassie Blue as the application. The image exposure was set to "Weak Bands" or a manual exposure of 0.500 seconds. Image analysis was performed by selecting the "Lane and Band" tool and the "Analyze Molecular Weight" feature in the Image Lab software's analysis toolbox. The bands of interest were annotated using the annotation tool.

[0313] Support Option 2

[0314] Automated methods for affinity purification on Hamilton STAR™

[0315] The Hamilton STAR™ IMCS medium-scale purification method used in this example employs the following steps:

[0316] Method parameters, such as the number of sample binding cycles and the number of elution cycles, can be selected on the user interface. Figure 7Recommended method parameters are listed in Table 2. For the acid addition step, use a Co-RE pipette tip to transfer 1 mL of acid buffer from the well to a 96-well acid plate. Buffer aspiration and dispensing are performed at 250 and 400 μL / sec, respectively. Note: Perform a 50 µL air pre-aspiration before each protein purification step. After all mixing cycles have been completed and a final dispensing has been completed with a 30-second wait time, use the 50 µL pre-aspiration as a spray above the liquid surface.

[0317] The equilibration addition step involved transferring 1 mL of equilibration buffer from the well to a 96-well plate using a Co-RE pipette tip. Buffer aspiration and dispensing were performed at 250 and 400 μL / sec, respectively.

[0318] For the wash buffer addition procedure, use a Co-RE pipette tip to transfer 1 mL of wash buffer (per channel) twice into a 24-well wash plate. Repeat the transfer twice for two additional wash plates. Aspirate the buffer at 250 μL / sec. Dispense the buffer at 400 μL / sec.

[0319] Use the “MlStarIsCoreHeadSpecialTipPickup” tip feature to dispense the storage buffer into a tank filled with water.

[0320] The acid washing step involves washing all resin tips with 800 µL of acid buffer, followed by one up-and-down pipetting operation at a rate of 100 µL / sec aspirate and 30 µL / sec dispensing, with a 30-second pause after each dispensing. Note: Pipetting with resin-containing IMCS tips should be performed at a significantly slower rate than when using Co-RE tips. This is to reduce pressure buildup on the IMCS tips. Variations in aspiration and dispensing rates of the IMCS tips support resin dispersion and sedimentation during mixing. Faster aspiration provides more efficient resin distribution, while slower dispensing rates provide adequate resin sedimentation. Furthermore, slower dispensing rates prevent resin from adhering to the tip walls.

[0321] The equilibration step uses 800 µL of equilibration buffer to perform three up-and-down pipetting cycles at a flow rate of 100 µL / sec for aspiration and 30 µL / sec for dispensing to wash all resin tips, and includes a two-second pause between each aspiration and dispensing cycle and a 30-second pause after the final dispensing.

[0322] The sample binding step involves pipetting 700 µL into each sample plate 20 times at a flow rate of 100 µL / s for aspiration and 15 µL / s for dispensing, to load the protein onto the pipette tip. 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 significantly more viscous than the buffer used in this method, necessitating a further reduction in the dispensing rate during the sample binding step.

[0323] After the final bonding step, the imprinting step imprints the suction cup onto the absorbent paper.

[0324] The washing step involved washing all pipette tips with wash buffer by transferring 300 µL of wash buffer five times from each of the three wash plates, for a total of 15 transfers of 300 µL / pipettes. The flow rate for aspiration was 100 µL / sec, and the flow rate for dispensing was 30 µL / sec. This step included a 2-second pause between each aspiration and dispensing cycle and a 30-second pause after the final dispensing.

[0325] The elution step elutes proteins in the elution buffer by aspirating and dispensing 250 µL 20 times at a flow rate of 100 µL / s for aspiration and 15 µL / s for dispensing, and includes a 2-second pause between each aspiration and dispensing cycle and a 30-second pause after the final dispensing.

[0326] Basic Plan 4

[0327] Size exclusion chromatography (SEC)

[0328] This protocol describes SEC, the second purification step used in a medium-scale workflow. The protocol utilizes a Thermo Fisher Vanquish™ Duo system (Figure 8) and a Phenomenex Yarra™ series column / TSKSuper SW column. The wavelength used for analyzing protein peaks is 280 nm.

[0329] Material

[0330] Use the following materials:

[0331] SEC buffer (see formulation in Reagents and Solutions)

[0332] Millipore Amicon Ultra 0.5 centrifugal filter, Ultracel 3kD (catalog number UFC500396)

[0333] Millipore Ultrafree centrifugal filter, Dura Free PVDF 0.22 µM (catalog number UFC306VOO)

[0334] Thermo Fisher Vanquish Duo HPLC System

[0335] Phenomenex Yarra Series 3 µM SEC-3000, LC column 300 x 4.6 mm (Catalogue No. 00H-4513-EO)

[0336] TSKgel SuperSW3000 4.6 mm X 30 cm (Catalog No. 0018675)

[0337] Waters BEH SEC Protein Standard Mixture (Catalogue No. 186006518-1)

[0338] 1X Phosphate-Buffered Saline (PBS) (Invitrogen, 10X, Catalog No. AM9624)

[0339] Thermo Scientific™ WebSeal™ plates for barcoding Vanquish™ UHPLC systems (catalog number 60180-P103B)

[0340] 96-well microplate, round holes, barcode-enabled.

[0341] Thermo Scientific™ Plate Seals (Catalogue No. 60180-M146)

[0342] Thermo Scientific™ Abgene 96-well 2.2 mL Polypropylene Deep Well Storage Plate (Catalogue No. AB0932)

[0343] Procedure for size exclusion chromatography using the Thermo Fisher Vanquish Duo system

[0344] Use the following steps:

[0345] The eluent was concentrated from affinity purification to approximately 130 µL by centrifugation at 13000 g for 20 min in a refrigerated centrifuge using a 3 kDa Amicon centrifuge concentrator and filtered using an Amicon 0.2 µm filter tube. Sample concentration was necessary because the Thermo Fisher Vanquish Flex model only offers sample loop sizes of 25 µL and 100 µL, therefore the maximum sample volume that can be loaded is 100 µL.

[0346] For proteins or complexes with higher MW, a higher MW cutoff concentrator can be used. Some proteins tend to precipitate during concentration and cannot be loaded onto the SEC. See Troubleshooting Table 1.

[0347] The BEH molecular weight standard mixture was prepared by dissolving in 0.5 mL PBS and filtering through an Amicon 0.2 µm filter. The concentrated and filtered sample, along with the BEH protein standard mixture, was loaded into barcoded 96-well ThermoFisher plates, sealed, and placed on one rack of the autosampler on the Vanquish Duo system (temperature set to 4 °C) (Figure 8). Samples can be loaded onto any other 96-well microplate as long as it has a circular well bottom. If using plates from other vendors, specify the plate format in the autosampler on the Chromeleon console. Plate sealing is optional, but it is generally recommended to use plate sealing to prevent sample evaporation if the sample will be stored in the autosampler for more than 24 hours.

[0348] Follow the steps provided in Support Scheme 3 to operate the system and set up the methods.

[0349] Equilibrate a Phenomenex Yarra SEC 3000 or TSKgel Super SW3000 column (5 mL column volume) with SEC run 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, for better separation and to avoid pressure buildup, we recommend a flow rate of 0.3 mL / min. For the purification of single proteins, the flow rate can be increased to 0.6 mL / min. The TSKgel Super SW3000 column can be used as an alternative to the Phenomenex Yarra SEC 3000 column.

[0350] As described in Support 3, create a series of injections using the appropriate instrumentation method in the Chromeleon console. Stop monitoring the baseline and begin the sequence run. All 24 samples from affinity purification can be loaded onto a plate for SEC purification. It takes approximately 33 minutes to complete one sample at a flow rate of 0.3 mL / min.

[0351] Fractions were collected using the fraction collector in a Thermo Scientific Abgene™ deep-well 96-well plate. The fraction collection mode can be selected as "Collect by Peak" or "Collect by Time." We have determined that "Collect by Peak" is more effective for us, and fractions are only collected when a peak is identified (this setting can be adjusted in the method settings). "Collect by Time" collects fractions between peaks and results in more than 100 fractions per sample. Alternatively, fraction collection can be turned off when only protein analysis characterization is required. The fraction size can be adjusted by changing the "Tube Change Duration" parameter in the fraction collector settings. We collected 40 to 50 µL fractions using a value of 4 seconds for "Tube Change Duration," as shown in the method script.

[0352] Analyze the chromatograms and identify peaks of interest to determine the number of wells used for fractionation analysis via SDS-PAGE. Chromeleon data analysis offers the option to stack or overlay chromatograms for sample comparison. We prefer to use the stacking option for chromatogram analysis.

[0353] Record the fraction number and aliquot 20 µL for each fraction in the Axygen 96-well plate used for gel loading. Prepare a reduced sample for SDS-PAGE analysis by mixing 20 µL of sample with 3 µL of 10x reducing agent and 7 µL of 4x LDS run buffer in a PCR plate. Seal the plate with aluminum foil and boil the sample at 95°C for 10 min using a PCR machine. Load 20 µL onto a Novex pre-prepared gel. Run at 180V for 50 min. The run 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 larger than 200 kDa may require longer run times for better separation.

[0354] Rinse the gel in water, then place the gel tray on a rocker and stain in InstantBlue Coomassie stain for 1 hour, and then destain in water for 2 hours while rocking on the rocker.

[0355] The gel was scanned and labeled using Bio-RAD Image Lab™ software. On the Bio-RAD Gel Doc™ EZ imaging system, a white tray was used to perform the scan by selecting the default scheme and Coomassie Blue as the application. The image exposure was set to "Weak Bands" or a manual exposure of 0.500 seconds. Image analysis was performed by selecting the "Lane and Band" tool and the "Analyze Molecular Weight" feature in the Image Lab software's analysis toolbox. The bands of interest were annotated using the annotation tool.

[0356] This includes pooling of proteins of the desired molecular weight.

[0357] Create a data report. The protein is ready for downstream characterization. This purification method produces high-quality purified proteins and multi-protein complexes. Yield varies depending on the expression level of the target protein. If no protein is detected, refer to troubleshooting table T1.

[0358] Support Option 3

[0359] Operating Chromeleon 7 on Vanquish Duo

[0360] Open the Chromeleon 7 console on a Windows™ computer that is connected to the Vanquish™ Duo system.

[0361] If you only need to operate the left-side system, simply open the Vanquish Left icon (Vanq_Left). You can operate both the Vanquish Left and Right systems simultaneously via the Vanq_Left or Vanq_Right controls. For simplicity, we will only describe the Vanq_Left system.

[0362] Open the "Instruments" tab in the lower left corner, as shown in the screenshot (Figure 9). Turn on the UV lamp and visible light lamp under the UV left tab by turning on the toggle switch. The switch will change from gray to green in about 15 minutes.

[0363] Open the shutter by selecting the open position from the drop-down options under the UV tab.

[0364] Connect the module and change the column chamber temperature to 10°C under the column chamber tab.

[0365] Under the Class Collection tab, reset the Class Collector to Class 1.

[0366] In the Pump_Left tab, set the pump pressure to 350 psi. This is calculated by adding the maximum allowable pressure of the column (provided by the manufacturer) and the pressure of the HPLC system. If the pump pressure is displayed higher than the set value, refer to Troubleshooting Table 1. If the pump pressure does not increase with the flow rate, there may be a leak in the system. Refer to Troubleshooting Table 1.

[0367] In the Pump Left tab, adjust the flow rate to 0.3 mL / min. Alternatively, for Phenomenex Yarra series columns, the flow rate can be adjusted to 0.6 mL / min, with a maximum of 1 mL / min. We chose 0.3 mL / min for better protein peak separation.

[0368] Load the SEC buffer onto the top stack of buffer stations, insert the left system A_L filter into the buffer, and close the cap. Open the Vanquish left pump valve by rotating the knob 360°. This allows the system to be purged at a high flow rate without pressure buildup. The default purging rate is 5 mL / min, with acceleration and deceleration at 0.1 mL / min. These settings can be adjusted under the tab. You should hear the purging and back-sealing wash piston moving.

[0369] After exhausting, close the left pump valve. If the valve is not closed, the motor will not turn on when you run the system.

[0370] Turn on the motor by switching on the toggle switch, and the system will start running at the set flow rate.

[0371] Connect the column to the valve and place it in the column chamber. The column needs to be connected in reverse because the flow direction in this system is from bottom to top, i.e., the pump is at the bottom and the detector is at the top of the stack.

[0372] To monitor the baseline, select the 280 nm UV wavelength and pump pressure from the dialog box. A280 (absorbance at 280 nm) will be displayed below the fractional collection tab, and the pump pressure will be displayed on the Pump Left tab. Absorbance at 260 nm can also be monitored simultaneously (for nucleic acid detection), but data collection will be slower when selecting both wavelengths using this detector.

[0373] Equilibrate a Phenomenex Yarra SEC3000 column or TSK column (5 mL column volume) with SEC run buffer at a flow rate of 0.3 mL / min for 30 minutes.

[0374] Baseline monitoring will be turned off after 30 minutes of balancing.

[0375] Open the "Data" tab in the lower left corner and begin the creation process. Open "Create New Sequence" from the creation icon in the upper left corner (Figure 10).

[0376] Add row items based on sample quantity.

[0377] Add the sample name and location in the 96-well plate for each sample (e.g., for the sample in well A12 on the plate located in the green position, enter "G:A12").

[0378] Select an instrument method for each sample. The instrument method can be set up once as shown below, and can be used for all future purifications if the same column is used and no other parameters are changed.

[0379] Start running and hierarchical collection.

[0380] 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.

[0381] Disconnect the column and store at 4°C. Wash the HPLC tubing and maintain this process every 2 months with 10% methanol to prevent any contaminant growth. Use 10% methanol to keep the post-sealed wash tubing active and operable, and refill monthly to maintain a long lifespan for the piston and piston seal.

[0382] The reagents and solutions used in this method are as follows:

[0383] Acid washing buffer (pH 3.0)

[0384] 100 mL 1 M glycine-HCl pH 3.0 (final 100 mM)

[0385] 30 mL 5 M NaCl (final 150 mM)

[0386] 870 mL water

[0387] It is corrosive and must be stored with acid.

[0388] Store at room temperature for up to 12 months

[0389] Elution buffer (pH 7.5)

[0390] 50 mL 1 M Tris pH 7.5 (final 50 mM)

[0391] 30 mL 5 M NaCl (final 150 mM)

[0392] 100 mL 50% glycerol (final 5%)

[0393] 125 mL 2 M imidazole (final 250 mM)

[0394] 695 mL water

[0395] TCEP (final 1 mM)

[0396] 3x FLAG peptides (final 150 µg / mL)

[0397] Roche EDTA-Free Protein Inhibitor Mixture (PIC) (Final 1 tablet / 50 mL buffer)

[0398] The buffer solution made from Tris, NaCl, glycerol, and imidazole can be stored at 4°C for up to 1 year. TCEP, 3XFLAG peptide, and PIC must be added fresh before use.

[0399] Equilibration and lysis buffer

[0400] 50 mL 1 M Tris pH 7.5 (final 50 mM)

[0401] 30 mL 5 M NaCl (final 150 mM)

[0402] 200 mL 50% glycerol (final 10%)

[0403] 2 mL of 1M MgCl2 (final 2 mM)

[0404] 5 mL 2 M imidazole (final 10 mM)

[0405] 713 mL water

[0406] TCEP (final 1 mM)

[0407] Benzoic acid enzyme (final 0.5 µL / 1 mL buffer)

[0408] Roche EDTA-Free Protein Inhibitor Mixture (PIC) (Final 1 tablet / 50 mL buffer)

[0409] The buffer solution made from Tris, NaCl, glycerol, imidazole, and MgCl2 can be stored at 4°C for up to 1 year. TCEP, benzoic acid enzyme, and PIC must be added fresh before use.

[0410] SEC buffer

[0411] 50 mL 1 M Tris pH 7.5 (final 50 mM)

[0412] 30 mL 5 M NaCl (final 150 mM)

[0413] 920 mL water

[0414] TCEP (final 1 mM)

[0415] The buffer solution made from Tris and NaCl can be stored at 4°C for up to 1 year. TCEP must be added fresh before use.

[0416] Washing buffer

[0417] 50 mL 1 M Tris pH 7.5 (final 50 mM)

[0418] 30 mL 5 M NaCl (final 150 mM)

[0419] 100 mL 50% glycerol (final 5%)

[0420] 10 mL 2 M imidazole (final 20 mM)

[0421] 810 mL water

[0422] TCEP (final 1 mM)

[0423] Roche EDTA-Free Protein Inhibitor Mixture (PIC) (Final 1 tablet / 50 mL buffer)

[0424] Buffer solutions made from Tris, NaCl, glycerol, and imidazole can be stored at 4°C for up to 1 year. TCEP and PIC must be added fresh before use.

[0425] Multicomponent protein expression

[0426] Many laboratories have established semi-automated, high-throughput, small-scale expression analysis platforms to allow the simultaneous analysis of hundreds of constructs with different tags, domains, and in multiple cell lines. This enables researchers to efficiently select the optimal conditions for expressing their proteins in most cases. However, this approach is less effective for challenging poorly expressed proteins or multi-protein complexes. Therefore, there is a need to establish medium-scale protein expression and characterization platforms that allow for parallel and rapid classification of challenging low-expression and co-expressed proteins in a high-throughput manner. We constructed and developed this medium-scale platform based on the small-scale expression analysis platform (Kraft et al., 2019). It is an end-to-end semi-automated workflow from virus generation to protein production and is capable of delivering 24 different proteins in a single purification run. It involves two-step purification, including affinity chromatography and size exclusion chromatography. In addition to protein expression and characterization, this workflow is continuously being developed to provide hundreds of micrograms of high-quality purified protein for further downstream applications. We have successfully explored numerous applications of proteins purified using this platform, including negative staining, high-throughput time-resolved fluorescence (HTRF) assays, SPR, affinity pull-down, enzymatic assays, and mass spectrometry. Another potential application of this method is screening for optimal buffer or co-expression ratios of different components to reconstruct soluble, stable protein complexes.

[0427] In this embodiment, we focus on the expression and purification of a multi-protein complex with eight component subunits, which has been successfully used in negative staining applications. Further follow-up to the results described herein using this protocol was performed for large-scale expression and purification at a 3 L scale. Large-scale purification was performed using conventional batch mode affinity purification with M2 anti-FLAG resin, ion exchange chromatography, and SEC. SEC analysis showed very similar monodisperse peaks for the protein complexes, yielding approximately 0.3 mg of the protein complex per liter of culture (Figure 12). This purified protein was then used to resolve the high-resolution cryo-electron microscopy structure of the complex (not disclosed). Overall, the end-to-end medium-scale protein screening platform enables parallel expression and purification of 24 samples or conditions and allows for the delivery of usable quantities of protein for downstream applications.

[0428] Figure 11 illustrates a successful case study of expressing and purifying a multi-protein complex from insect cells using a medium-scale platform. The protein complex in insect cells was expressed through co-infection with multiple viruses, each expressing a single protein subunit. Typically, for large protein complexes containing four or more subunits, co-infection with multiple viruses can lead to increased cellular stress and uneven infection, potentially resulting in incomplete complex formation and / or low protein yield. To reduce viral heterogeneity after co-infection, multi-ORF constructs expressing multiple proteins from a single virus can be used to improve complex chemometry and yield. To generate 8-subunit protein complexes (proteins A through H), we designed three constructs using the following strategy:

[0429] 1) The largest subunit (protein A, 117.1 kDa) is FLAG-tagged at the N-terminus and expressed under a polyhedral promoter on its own vector. This is to enable screening for many N-terminal truncated variants as part of a domain expansion strategy to increase protein yield.

[0430] 2) Proteins B through E (51.8, 6.1, 34.8, and 47.2 kDa) were generated as unlabeled proteins and placed on separate vector backbones, with each ORF under the control of a polyhedral promoter.

[0431] 3) Proteins F to H (39.2, 36.4, 14.5) encoding three different ORFs were placed on a third vector, with each ORF under a separate polyhedral promoter.

[0432] Viruses were generated from each of the three constructs using the protocols listed above, and all three viruses expressing eight different proteins were co-expressed in Sf9 cells at a 1:1:1 ratio to produce biomass. Biomass was harvested, cells were lysed, and proteins were purified using affinity purification and size exclusion chromatography. The first SDS-PAGE gel in Figure 11 shows the results of 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 pipette tip on a Hamilton STAR™ workstation. All proteins of the complex were detected at the expected molecular weight, further purified, and the oligomeric / aggregate state of the complex was analyzed on size exclusion chromatography using a TSK column. The chromatogram shows monodisperse peaks, and the complex eluted at an elution time of 7.5 min, corresponding to an elution volume of 2.25 mL. The second gel in Figure 11 shows the analysis of the fractions collected from size exclusion chromatography, and 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 200 mL of culture. Peak fractions were collected and analyzed using negatively stained electron microscopy. Qualitative evaluation of the sample was performed, observing the uniformity of particles in each micrograph. This helped us identify the optimal domain boundaries of protein A, resulting in a stable and well-behaved protein complex that appeared suitable for structural studies.

[0433] Table 1. Troubleshooting Guidelines for Large-Scale Expression and Purification in Insect Cells

[0434]

[0435] Table 2. Parameters for Automated Affinity Purification

[0436]

[0437] *In the final mixing cycle, add a 30-second hold time after the aspiration step.

[0438] Further notes

[0439] High-titer virus production

[0440] In this protocol, baculovirus production depends on successful in vitro recombination of the transfer plasmid with linearized baculovirus DNA from BestBac™ (Expression systems). Homologous recombination efficiency should be between 90% and 100%, but can vary depending on the linearized vector used (Kitts et al., 1990). Many commercially available linearized baculoviruses are available for use with in vitro homologous recombination methods (BaculoGold™, BD Biosciences; FlashBAC, Oxford Expression Technologies; BacPAK™, Clontech). However, the final viral stock inevitably contains a mixture of parental and recombinant viruses. The virus should be of high quality and titer for moderate-scale expression. To avoid generating a high percentage of non-recombinant virus, the viral stock should not be amplified after the P3 phase. Although we did not quantify the viral titer, we used a gp64 assay to determine the percentage of cells infected with the viral particles (Kitts and Green, 1999). Viral stock solutions with titers below 70% gp64 staining will result in nonproductive infection and low yields, and therefore we recommend repeated co-transfection to allow for viral regeneration.

[0441] Insect cell infection

[0442] Insect cells Sf9 and T. ni should be healthy, productively infected with the virus, and their diameters should be 15.5 µm and 19 µm, respectively, as measured by Vi-CELL BLU. Culture viability should be >= 95% to proceed. If cells have low viability or large diameters, fresh cells should be thawed and passaged for infection. Productive infection of cells is also measured using a Vi-CELL counter. Cells are harvested for purification only if the diameters of Sf9 and T. ni cells are greater than 17 or 21 µm, respectively, and their viability is between 50% and 85%, respectively. Otherwise, the infection process is repeated.

[0443] Highly efficient cell lysis and filtration

[0444] As mentioned above, complete cell lysis requires the use of appropriate lysis methods, and the lysate should be filtered to completely remove debris. Numerous physical and chemical methods exist for cell lysis, including osmotic shock, freeze-thaw cycles, Dounce homogenization, sonication, and detergent-based lysis. Eukaryotic cells have a higher amount of nucleic acid compared to bacteria and therefore should contain nucleases (benzoic acidases) to reduce the viscosity of the cell lysate. We have found that bead-based lysis provides complete and efficient lysis of insect cells in a higher flux manner. Cell debris must be clarified by high-speed centrifugation or a deep microfiltration system. Orochem's filter plates are a type of deep microfiltration system consisting of a filter with an open pore structure (removing cells and cell debris) and a filter aid with a denser pore structure (simultaneously removing colloidal substances) to deliver a particle-free feedstock for downstream processes. If deep filtration is unavailable, the lysate should be clarified by high-speed centrifugation (18,000 g, 30 min) prior to purification.

[0445] Buffer for purification

[0446] Based on the isoelectric point and aggregation properties of proteins, the lysis, washing, and elution buffers used for affinity chromatography should be carefully selected. The most commonly used buffers for purification are Tris-based or HEPES-based. Salt concentrations should be maintained between 100 and 150 mM NaCl for purifying multi-protein complexes, as higher salt concentrations may interfere with ionic interactions between different components of the protein complex. Salt concentrations can be adjusted up to 300 mM for purifying single proteins, as higher salt concentrations result in better purity. Reducing agents, such as DTT or TCEP, should be added for purifying intracellular proteins. Our buffers contain 5% to 10% glycerol, as glycerol provides stability to proteins.

[0447] Affinity purification

[0448] In addition to selecting the correct buffer, choosing the right type of resin for purification and optimizing the automated method for a given resin is also important. Resins can have very different binding capacities. For example, the M2 anti-FLAG and Ni-NTA Superflow™ resins we use have binding capacities of 0.6 mg / mL and 20 mg / mL, respectively, and therefore the number of IMCS tips should be adjusted accordingly based on the expected expression level for each sample. Some resins have higher or lower rates of binding to target proteins, and therefore the automated purification method should be adjusted accordingly based on the number of sample binding and elution cycles.

[0449] Size exclusion chromatography

[0450] We tested various chemical columns of different sizes and found that the 5 mL column from the Phenomenex Yarra™ series and the TSK column with dimensions of 300 x 4.6 mm were ideal for separating protein complexes purified on a 200 mL scale. The columns are tightly packed with inert silica-based material with a pore size of 290 Å and a particle size of 3 µm, providing a separation range of 5 to 700 kDa. Another advantage of these columns is their compatibility with HPLC and UHPLC systems, allowing for rapid flow rates of 1 mL / min and withstanding pressures up to 300 bar.

[0451] Example 2: Medium-scale production of proteins from mammalian cell cultures

[0452] In several different flasks, histidine-labeled membrane proteins were expressed with or without one or both of two cytoplasmic protein chaperones, each in a 30 mL volume in EXPi293 cells transfected with an appropriate plasmid, for expression at 37°C on day 0. On day 1, the temperature was maintained at 37°C in some flasks and lowered to 30°C in others. On day 4, parallel affinity purification of the proteins was performed using a Ni-IMAC nickel column, and the yield of protein and chaperone proteins in each individual sample was evaluated, allowing for rapid comparison of different purification conditions (temperature and the presence of one or both chaperone proteins).

[0453] Example 3: Medium-scale purification of protein complexes

[0454] To purify the E3 ubiquitin ligase protein complex from Sf9 insect cells, several different protein purifications were performed in parallel at a medium scale to determine the optimal conditions for protein expression and complex formation. Proteins were pulled down in parallel using FLAG tags, followed by size exclusion chromatography and electrophoresis analysis. The substrate recognition protein of the complex itself did not show expression, but co-expression with other members and chaperone proteins of the complex enabled the reconstructing 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 culture.

[0455] References

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[0472] Kitts, P. A., Ayres, M. D., and Possee, R. D. (1990). Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res, 18(19), 5667-5672. doi:10.1093 / nar / 18.19.5667

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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, the method comprising: (a) Growing multiple mammalian, insect, or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL; (b) Lysing the cells of the cell culture sample, wherein for each of the plurality of cell culture samples, portions (a) and (b) are performed in the same container; (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample; (d) Clarify the plurality of supernatant samples by deep filtration; (e) Place multiple clarified supernatant samples into the wells of a multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL / well; as well as (f) subjecting the plurality of supernatant samples in the wells of the multi-well plate to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluent from the chromatography into the wells of the multi-well plate; wherein portions (e) and (f) are performed in parallel on the plurality of samples.

2. The method of claim 1, wherein the following portion of the method is automated: Part (f) or part (e) and (f).

3. The method of claim 1, wherein all of 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 at a volume of 2 to 30 mL / well.

5. A method for purifying one or more peptides from a plurality of clear cell lysate supernatants in a multi-well plate, said plurality of clear cell lysate supernatants being obtained from a plurality of cell culture samples by a process comprising: (a) Growing multiple mammalian, insect, or bacterial cell culture samples expressing one or more polypeptides for purification at a scale of 20 to 500 mL; (b) Lysing the cells of the cell culture sample, wherein for each of the plurality of cell culture samples, portions (a) and (b) are performed in the same container; (c) Centrifuge the lysed cell culture sample and collect multiple supernatant samples from the cell culture sample; (d) Clarify the plurality of supernatant samples by deep filtration; (e) A plurality of clarified supernatant samples are placed in the wells of the multi-well plate, wherein the supernatant samples have a volume of 2 to 30 mL / well; The method includes: (f) subjecting the supernatant sample obtained from the process of (a) to (e) to affinity chromatography using an affinity matrix in a pipette tip, and placing the eluent from the chromatography into the wells of a multi-well plate; wherein the method is performed in parallel on the plurality of clarified supernatant samples, and optionally wherein the method is automated.

6. A method for affinity purification of peptides from multiple cell culture supernatant samples expressing one or more peptides for purification, the method comprising: A multi-well plate is obtained, the multi-well plate containing multiple clear cell culture supernatant samples with a volume of 2 to 30 mL / well in the wells of the multi-well plate, and the multiple clear cell culture supernatant samples are subjected to affinity chromatography using an affinity matrix in a pipette tip, and the eluent from the chromatography is placed in the wells of a second multi-well plate; wherein the method is performed in parallel on multiple supernatant samples, optionally wherein the method is automated. The plurality of cell culture supernatant samples are obtained from mammalian, insect, or bacterial cell culture samples expressing one or more polypeptides, the cell culture samples being grown at a scale of 20 to 500 mL and subjected to lysis, centrifugation, and clarification, wherein cell growth and lysis are performed in the same container.

7. The method according to any one of claims 1 to 6, wherein the method further comprises: (g) Size exclusion chromatography (SEC) is performed on the eluent of the affinity chromatography; as well as (h) Fractionate the peptides from the SEC into the wells of the multi-well plate. Optionally, one or both of (g) and (h) are automated.

8. The method of claim 7, wherein portions (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 more than one well of the multi-well 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 multi-well plate.

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

12. The method according to any one of claims 1 to 11, wherein the cells are lysed by adding glass beads accompanied by oscillation and / or wherein the cells are lysed without sonication.

13. The method according to any one of claims 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 claims 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 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 multihistidine, FLAG, streptavidin, glutathione S-transferase (GST) or maltose-binding protein (MBP) tag, and wherein the affinity matrix recognizes the tag.

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

18. The method according to any one of claims 1 to 17, wherein the method further comprises 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 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 include antibodies or antibody subunits.

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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