Buoyancy activated cell sorting and scalable protein purification using engineered balloon bubbles

Abstract

In one aspect, the present disclosure describes methods useful for purifying a target protein from a sample mixture. The methods disclosed herein can also be used to purify or sort cells from a sample mixture.

Description

[0001] Priority Claim

[0002] This application claims the priority benefit of U.S. Provisional Application Serial No. 63 / 417,928, filed on October 20, 2022, the entire content of which is hereby incorporated by reference.

[0003] Sequence Listing Incorporation

[0004] This application contains a Sequence Listing XML that has been submitted electronically and is hereby incorporated by reference in its entirety. The XML Sequence Listing was created on October 20, 2023, named RICEP0114WO.xml, and is 6,269 bytes in size. Background Art 1. Technical Field

[0005] The present disclosure relates to the fields of biology, biochemistry, molecular biology, biomanufacturing, biotechnology, and medicine. In particular, new methods related to the purification or isolation of enzymes, proteins, and cells are disclosed.

[0006] 2. Related Art

[0007] Cell-based therapies, such as autologous transplantation and engineered T cell therapies (e.g., CAR-T therapy), are a rapidly expanding class of therapies (Bashor et al., 2022). The key to the success of these therapies is the ability to consistently and effectively sort and modify specific cell populations ((Lipsitz et al., 2016). Therefore, effective and economical cell sorting techniques are crucial. Magnetic-activated cell sorting (MACS) is a widely used technique that utilizes magnetic beads modified with antibodies to target cell antigens (Miltenyi et al., 1990; Wang et al., 2022; Wang et al., 2023; Frenea-Robin & Marchalot, 2022). In the presence of a magnetic field, cells that bind to these beads are separated. Although its advantages include high cell recovery and purity, there are concerns about the cost of the magnetic bead reagents and potential downstream cellular effects from the magnetic labeling (Frenea-Robin & Marchalot, 2022).

[0008] Fluorescence-activated cell sorting (FACS) is another method that sorts cells based on the fluorescence intensity and light scattering properties of fluorescence marker-labeled cells through a flow cytometry system (Hu et al., 2016). However, due to the droplet generation rate and scalability, FACS sometimes faces challenges in terms of throughput (Hu et al., 2016). Buoyancy-activated cell sorting (BACS) uses synthetic microbubble-based reagents, demonstrating the potential of buoyancy-based cell sorting processes (Liou et al., 2015). A common challenge for these technologies is the significant cost associated with the production of recombinant proteins, especially antibodies for surface antigen-specific sorting (Wei et al., 2017). The purification and modification costs of recombinant protein reagents play a key role in increasing the total manufacturing cost (Palani et al., 2023).

[0009] Protein purification is a fundamental step in many industrial sectors, such as enzyme catalysis for commodity chemicals (Roe, 2001; Scopes, 1993; Deutscher, 1990), biopharmaceuticals (Rathore et al., 2018; Jozala et al., 2016; Dos Santos et al., 2017; Hanke & Ottens, 2014), agriculture and food industry (Liu et al., 2020; Peydayesh & Mezzenga, 2021), and bioremediation (Viswanath et al., 2014; Cheung & Gu, 2007; Chakroun et al., 2010), with a combined global market size exceeding one billion US dollars. Current in-laboratory and industrial methods include a variety of techniques, such as chromatography, electrophoresis, precipitation, and solubilization (Roe, 2001; Deutscher, 1990; Janson, 2012; Linhult et al., 2005; Flickinger, 2013; Harrison, 1993; Franzreb et al., 2007). Chromatography-based methods are the most commonly used techniques; however, these methods can be time-consuming, require specialized equipment, and often result in low yields (Ghosh, 2022; Gomari et al., 2020). At the same time, methods relying on phase separation, such as precipitation (Labrou, 2014; Burgess, 2009) and oil droplets (Leptihn et al., 2013; Yang & Sagis, 2021), are relatively simple and can be used for large-scale and consistent purification. However, they typically result in protein loss and low purity. Therefore, there is a need to develop new technologies that can combine the scalability of phase separation methods and the selectivity of chromatography.

[0010] The current gold standard in peptide purification at the laboratory scale is affinity chromatography, such as using Ni-NTA columns to purify polyhistidine-tagged proteins of interest (POIs). This technique typically processes samples with volumes less than 100 milliliters. At the industrial scale, where samples of several tens of liters may need to be purified, chromatography becomes costly. For all these reasons, methods capable of purifying protein targets on a large scale are needed.

[0011] Ex vivo cell manufacturing is a critical step and represents a large market in cell-based therapies. Existing cell manufacturing workflows include multiple positive cell selection steps, which create labor-intensive bottlenecks. Current manufacturing methods employ magnetic activated cell sorting (MACS), which involves attaching antibody-labeled target cells to magnetic beads and retaining them in a magnetic column. Due to the magnetic column and required setup, these methods effectively only allow separation in the x-y geometry. Thus, methods that allow for large-scale cell sorting, such as by expanding the x-y geometry of the cell sorting vessel, would represent an improvement over the prior art. Accordingly, there remains a need for cell sorting methods that are more suitable for industrial-scale processes and production. This invention was partially funded by the Robert A. Welch Foundation under Welch Grant No. C-2069-20210327. SUMMARY OF THE INVENTION

[0012] As provided herein, the present disclosure relates to methods of using genetically engineered gas vesicles to purify proteins or using formulated nano- and microstructures containing gas vesicles to separate cells. In some embodiments, the gas vesicles disclosed herein are shown to be capable of selectively binding specific markers, peptides, or proteins of target cells or proteins. In some embodiments, the gas vesicles contain pre-translationally modified proteins, which provide the advantage of reducing processing steps or simplifying the separation of target cells or proteins.

[0013] The use of labeled GVs disclosed herein provides methods for capturing proteins, such as for density-based bioprocesses or for functional modification for targeted applications, with improved control or improved predictability. In some embodiments, the present disclosure provides methods for separating or purifying target proteins that are improved over methods known in the art (such as nickel affinity chromatography). In some embodiments, the present disclosure provides methods for separating and purifying target proteins that have increased yields compared to methods known in the art (such as nickel affinity chromatography). In some embodiments, the present disclosure provides methods for separating and purifying target proteins that have increased purity compared to methods known in the art (such as nickel affinity chromatography). In some embodiments, the present disclosure provides methods for facilitating scalable separation or purification of target proteins or cells for industrial applications. In some embodiments, the methods disclosed herein involve prolonging the flotation of gas vesicle compositions for an extended period of time under normal gravity or over long distances, thereby reducing the need for specialized equipment that may be required in similar methods known in the art.

[0014] The present invention utilizes the buoyancy of the air vesicles used in the methods disclosed herein to modulate the total density of a target protein or target cell once the target protein or target cell binds to the air vesicles, thereby facilitating improved or simplified separation by centrifugation or other density-based separation or purification methods. The air vesicles of the present disclosure, which comprise a protein shell, a hydrophobic interior, and a hydrophilic exterior, have a favorable combination of high physical stability and the ability to repel water (Pfeifer, 2012; Walsby, 1994).

[0015] In some embodiments, the method can be used for cell sorting, such as T cell sorting. In some embodiments, the method includes using density-based fractionation to sort target cells or proteins. The method can involve a variety of air vesicle labeling schemes to bind to target proteins or target cells. In some embodiments, the method utilizes the SpyTag / SpyCatcher protein ligation system using air vesicles comprising SpyTag. In other embodiments, streptavidin-GV binds to biotinylated targets (such as biotinylated antibodies). The method can involve modular functionalization of air vesicles with SpyCatcher fusion proteins. In some embodiments, the method facilitates T cell isolation. In some embodiments, the methods disclosed herein provide a more streamlined and cost-effective method than methods known in the art for performing cell sorting.

[0016] Details regarding these and additional aspects are provided below and in the following sections.

[0017] In some embodiments, the present disclosure provides a method for purifying a target protein from a sample, comprising:

[0018] (A) obtaining a collection of labeled air vesicle compositions, wherein the air vesicles comprise one or more proteins on the surface of the air vesicles, and the one or more proteins have been modified with one or more affinity tags to form the labeled air vesicle compositions; and

[0019] (B) contacting the collection of labeled air vesicle compositions with a sample comprising the target protein for a first period of time; wherein the first period of time is sufficient for the labeled air vesicle compositions and the target protein to bind to form a captured target protein-air vesicle composition; and

[0020] (C) allowing the components of the sample comprising the captured target protein-air vesicle composition to equilibrate for a second period of time; and

[0021] (D) separating the captured target protein-air vesicle composition from the sample mixture.

[0022] In some embodiments, the captured target protein-biovesicle composition forms a floating layer in the sample mixture. In some embodiments, the floating layer forms during a first time period or a second time period.

[0023] In some embodiments, the method includes a target protein on the surface of a cell type in the sample mixture. In some embodiments, the cell is a lymphocyte, such as a T cell. In some embodiments, the cell is derived from a patient.

[0024] In some embodiments, the target protein is present on a single cell type in the sample mixture. In other embodiments, the target protein is present on two or more cell types in the sample mixture.

[0025] In some embodiments, the present disclosure provides a method, wherein the collection of labeled biovesicle compositions comprises two different sets of labeled biovesicle compositions; wherein

[0026] (i) The first set of labeled biovesicle compositions comprises a first affinity tag;

[0027] (ii) The second set of labeled biovesicle compositions comprises a second affinity tag; and

[0028] wherein the first affinity tag and the second affinity tag are complementary.

[0029] In some embodiments, the labeled biovesicle composition further comprises a cell-specific marker. In other embodiments, the cell-specific marker is linked to an affinity tag that is the same as the first affinity tag of the first set of labeled biovesicle compositions.

[0030] In some embodiments, contacting the labeled biovesicle composition with the sample mixture is carried out in two steps, wherein

[0031] (iii) The first step comprises contacting the first set of labeled biovesicle compositions with the sample mixture; and

[0032] (iv) The second step comprises contacting the second set of labeled biovesicle compositions with the sample mixture.

[0033] In some embodiments, the second set of labeled air vesicle compositions is present in excess relative to the first set of labeled air vesicle compositions. In some embodiments, at least one of the affinity tags of the method is a peptide or a protein. In some embodiments, at least one of the affinity tags is a peptide. In some embodiments, at least one of the affinity tags is a protein. In other embodiments, at least one of the affinity tags is an antibody, such as an antibody against a target protein. In other embodiments, the target protein is expressed on the cell surface. In some embodiments, the antibody is an anti-CD3 antibody. In other embodiments, the antibody is an anti-CD28 antibody. In some embodiments, at least one of the affinity tags is streptavidin. In some embodiments, at least one of the affinity tags is biotin. In some embodiments, at least one of the affinity tags is a SnoopTag. In some embodiments, at least one of the affinity tags is a SnoopCatcher. In some embodiments, at least one of the affinity tags is a SpyTag.

[0034] In some embodiments of the method, the labeled air vesicle composition and the target protein bind to form a covalent bond. In other embodiments, the labeled air vesicle composition and the target protein bind to form a non-covalent bond.

[0035] In some embodiments of the methods disclosed herein, the first time period is from about 15 minutes to about 5 hours, such as about 1 hour. In some embodiments of the method, the second time period is from about 15 minutes to about 5 hours, such as about 1 hour or about 2 hours. In some embodiments, the second time period is about 5 hours.

[0036] In some embodiments, the method does not include centrifugation. In other embodiments, the method includes centrifugation. According to some embodiments of the method, separating the captured target protein-air vesicle composition further includes centrifugation. In other embodiments, the centrifugation is conventional centrifugation. In other embodiments, the centrifugation is density gradient centrifugation. In some embodiments, the centrifugation is at 400×g. In some embodiments of the present disclosure, separating the captured target protein-air vesicle composition further includes elutriation.

[0037] In some embodiments, the method further includes separating the target protein from the captured target protein-air vesicle composition after separation from the sample mixture. In some other embodiments, the separation is carried out by cleavage with a protease (such as TEV protease). In some other embodiments, the separation is carried out by intein self-splicing. In some further embodiments, the intein is the ΔI-CM mini-intein. In some embodiments, the intein self-splices upon a change in pH. In some other embodiments, the intein self-splices when the pH changes from about pH 4 to about pH 7. In some other embodiments, the intein self-splices upon a change in temperature.

[0038] In some embodiments, the method further includes detecting the presence of the target protein. In some other embodiments, the detection is detecting the presence of cells.

[0039] In some embodiments, the volume of the sample containing the target protein is about 10 L or greater. In some other embodiments, the volume of the sample containing the target protein is about 100 L or greater. In some other embodiments, the volume of the sample containing the target protein is about 1000 L or greater. In some further embodiments, the volume of the sample containing the target protein is about 10,000 L.

[0040] In one embodiment, a method for purifying proteins using affinity tags on buoyant nanostructures for the large-scale production of biomolecules and enzymes, such as those for commodity, biofuel, or material manufacturing, is disclosed. In another embodiment, a method for formulating buoyant nanostructures and microstructures with antibodies suitable for surface binding or endocytosis by cells for drug applications, such as those for in vitro cell therapy manufacturing, is disclosed.

[0041] It is contemplated that any method or composition described herein can be implemented relative to any other method or composition described herein. For example, a compound synthesized by one method can be used to prepare a final compound according to a different method.

[0042] When used in the claims and / or the specification in conjunction with the term “comprising,” the use of a noun without a quantitative modifier can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “more than one.” The word “about” means plus or minus 5% of the specified number.

[0043] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and the specific examples, while indicating some specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments given herein.

[0045] Figure 1 Shows the genetic modification of an affinity tag onto the surface of gas vesicles.

[0046] Figure 2 Shows the density change of cells expressing single and double target proteins of interest (POI) based on the degree of attachment of gas vesicles for buoyancy-assisted cell sorting.

[0047] Figure 3 Shows the capture and purification of the protein of interest (POI) based on affinity tag interaction and buoyancy separation of the composition.

[0048] Figures 4A to 4H Shows the GV modification strategy and GV characterization. (Figure 4A) shows the wild-type (WT) GV structure of the GvpC annealing pattern. (Figure 4B) NHS-streptavidin chemical modification of GV. (Figures 4C, 4D) Replacement of GvpC on WT GV with modified and recombinantly expressed GvpC fused to SpyTag. (Figure 4E) Covalent modification of GvpC-SpyTag with mWasabi-SpyCatcher fusion protein for surface modification of GV. (Figure 4F) Covalent modification of GvpC-SpyTag with scFv-SpyCatcher fusion protein. (Figure 4G) SDS-Page observation of the ligation between SpyTag and SpyCatcher for GV samples replaced with a series of GvpC-SpyTag replacements (BP - buoyancy purification, mWasa - mWasabi, SpyC - SpyCatcher, SpyT - SpyTag). (Figure 4H) Relative fluorescence intensity of GV modified with decreasing amounts of GvpC-SpyTag after ligation with mWasabi-SpyCatcher, normalized to the sample OD.

[0049] Figures 5A to 5D Provides evidence of the density change of RAW 264.7 macrophages after incubation with GV. (Figure 5A) shows when incubated with PBS (left) or GV at OD 500= Photograph of cell samples incubated together at 5 (right) bottom. (Figure 5B) Percentage of cells recovered in the top fraction across the density series when treated with PBS (grey bars) or GV (blue bars). (Figure 5C) Photograph of the column device for density-medium fractionation of top and bottom cells. Yellow arrows indicate the cell layer captured above the density medium. Visualization of the size of the bottom fraction cell pellet (bottom). (Figure 5D) Photograph of GV-treated cell samples and their separation between the bottom and top fractions.

[0050] Figures 6A to 6G It shows that anti-CD3 scFv-modified SpyTag GV enriches CD4+ and CD8+ cells in the buoyant fraction. (Figure 6A) SpyTag GV is ligated to the anti-CD3 scFv-SpyCatcher fusion protein. (Figure 6B) Schematic diagram of the decrease in cell density corresponding to the GV binding level controlled by CD3 surface antigen recognition. (Figure 6C) Density column insertion system for separating buoyant (upper fraction) and pellet (lower fraction) cells after centrifugation. (Figure 6D) Photograph of different layers observed in the density column after treatment. (Figure 6E) Percentage of T cells in untreated peripheral blood monocular cells (PBMC) or in the upper fraction separated with 1.06 g / mL density medium after incubation with PBS or CD3-GV. (Figure 6F) FSC / SSC profile shows a significant increase in SSC of the GV-treated upper fraction, especially in the FSC cluster containing lymphocytes. (Figure 6G) Distribution of CD4+ or CD8+ T cells in the upper and lower fractions of PBMC with or without GV treatment shows enrichment of T cells.

[0051] Figures 7A to 7B It shows the reference anti-CD3 scFv sequence verified by cell-binding studies. (Figure 7A) Flow cytometry data comparing the staining distribution of the anti-CD3 scFv-mWasabi fusion (top) with a commercially available fluorophore-modified anti-CD3 antibody (OKT3 clone). The digital inlay reports the % of cell staining above the negative staining control. (Figure 7B) Concentration titration of anti-CD3 svFv-mWasabi for staining cells.

[0052] Figures 8A to 8CShows the change in cell density across the PBS-Ficoll-Paque density gradient. (Figure 8A) Percentage of cells depleted in the bottom pellet cell fraction with and without GV treatment. (Figure 8B) CD14+ monocyte loading in the upper and lower fractions of cell samples separated using 1.04 and 1.06 g / mL density media. The numerical inlays represent the percentage of CD14+ cells in the fraction. (Figure 8C) CD14+ monocyte loading in the CD3-GV treated cell fraction.

[0053] Figures 9A to 9B Shows the verification of streptavidin GV modification. (Figure 9A) SDS-Page of unmodified GV (Ana GV), GV after Lightning-Link chemical modification with streptavidin, and GV after two rounds of buoyancy purification (BP) shows the linkage of streptavidin to GV. (Figure 9B) TEM imaging of the clustering and de-clustering states of biotinylated GV and streptavidin-modified GV.

[0054] Figures 10A to 10E Provides evidence that streptavidin-GV can be used with off-the-shelf biotinylated antibodies according to this method. (Figure 10A) Schematic of a two-part cell labeling kit of biotinylated antibody and streptavidin-modified GV. (Figure 10B) Labeling protocol for cells bound with a primary antibody and a second GV. (Figure 10C) FSC / SSC analysis of untreated GV (w / o GV) or FSC / SSC analysis of cells treated with a biotinylated anti-CD3 commercial antibody followed by streptavidin-GV. The inset shows the CD14+ monocyte loading with high GV binding events. (Figure 10D) CD14+ monocyte loading in the upper and lower fractions of PBMCs separated using 1.04 and 1.06 g / mL density media after treatment with PBS. (Figure 10E) CD14+ monocyte loading of PBMCs separated after treatment with antibody + GV.

[0055] Figures 11A to 11C Shows the Ni-NTA purification of POI expressed from Escherichia coli (E. coli). (Figure 11A) Purification of GvpC-SpyTag-His. (Figure 11B) Purification of mWasabi-SpyCatcher-His. (Figure 11C) Purification of anti-CD3 scFv-SpyCatcher-His. WC: Whole cell lysate, S: Supernatant, FT: Flow-through, E: Eluate, W: Wash.

[0056] Figure 12 Provides the assay results for determining the solubility of GvpC. GvpC is depleted from GV after buoyancy purification in the presence of 0 to 6 M urea.

[0057] Figures 13A to 13K Provided evidence of screening via affinity tag insertion, indicating that SnoopTag and Strep-tag II can be introduced into the C-terminus of the shell protein GvpB without disrupting GV assembly. Figure 13A: Structure of the pNL29 operon. The shell protein gene is marked dark blue. Figure 13B: Representative TEM image of pNL29 GVs after disaggregation by 6M urea treatment. Scale bar = 500 nm. c, Cryo-EM structure of the GvpB monomer and the left-handed helical GV shell structure formed by assembled monomers. The schematic shows the helical GV shell lattice with closely packed GvpB monomers, characterized by a lateral spacing of 1.2 nm and a helical pitch of 4.9 nm 1 Figure 13D, Figure 13E: Schematic of the genetic addition of linker and tag to the C-terminus of GvpB. Upon expression and assembly, the GVs are expected to have a surface with an affinity tag. Figure 13F: Table of the major shell proteins with affinity tags and linkers attached to the C-terminal constructs, characterizing their expression and assembly in E. coli. For GV expression and assembly, a single plus (+) indicates that insufficient amounts of intact GVs could hardly be observed by eye after cell lysis, and three pluses (+++) indicate that the number of GVs was sufficient for both TEM and DLS characterization, and a minus (-) indicates that no GVs were detected. Figure 13G, Figure 13H: Representative TEM images of pNL29 GVs with the shell protein GvpB genetically modified with SnoopTag (GvpB::SnoopTag) and StrepTagII (GvpB::StrepTag II). Scale bar = 500 nm. Figure 13I, Figure 13J: Representative DLS measurements of the hydrodynamic diameter and mean hydrodynamic diameter (n = 4 biological replicates) of GV variants. WT: wild-type unmodified pNL29 GV; SnoopTag: pNL29 GV with GvpB genetically modified with SnoopTag (GvpB::SnoopTag); StrepTagII: pNL29 GV with GvpB genetically modified with Strep-tag II (GvpB::Strep-tag II). Error bars represent mean ± standard deviation (STDEV). ** p < 0.01; *** p-value < 0.001. Figure 13K: Schematic of the application of genetically labeled GVs to the purification of target recombinant proteins.

[0058] Figures 14A to 14FIt is shown that 6M urea treatment cannot cause the labeled GVs to de-cluster. Figures 14A and 14B: Representative TEM images show that wild-type pNL29 GVs remain clustered after purification from E. coli (Figure 14A) and transform into a de-clustered state after treatment with 6M urea (Figure 14B). Scale bar = 500 nm. Figures 14C to 14F show representative DLS measurements of the hydrodynamic diameter and average hydrodynamic diameter of GV variants before and after 6M urea treatment (n = 4 biological replicates for WT and Strep-tag II GVs, and n = 5 biological replicates for SnoopTag GVs). Error bars represent mean ± standard deviation (STDEV).

[0059] Figures 15A to 15E Interaction studies of the labeled GVs with their affinity tag binding partners are presented, which show that the surface-expressed tags on the GVs remain functional. Figure 15A: Schematic of an assay using a fluorescently labeled binding partner to confirm the functionality of the affinity tag on the GV surface. 1: Load the labeled GVs into a tube containing a sample of the fluorescently labeled binding partner of the affinity tag. 2: Place the sample on a rotary mixer and incubate at room temperature for 1 hour to allow interaction between the tags on the GVs and the binding partners. Measure the fluorescence intensity of the mixed sample immediately after incubation. 3: Then centrifuge the sample at 400 × g for 10 minutes. 4: After centrifugation, the GVs with the bound fluorescently labeled binding partners float buoyantly to the liquid surface. Use a syringe to remove the liquid and any unbound binding partners located below the GVs. 5: Resuspend the sample in 1× PBS and immediately measure the fluorescence intensity of the resuspended sample. 6: Repeat this process (steps 3 to 5) at least three times, or until the fluorescence intensity of the resuspended sample shows no significant change. Figures 15B to 15E: Percentage of the fluorescence intensity retained on the GVs (n = 3 biological replicates) relative to the initial fluorescence intensity measured immediately after sample incubation, and fluorescence images of the samples after the last round of washing and centrifugation. PBS: Only 1× PBS is present in the tube, without any sample. Error bars represent mean ± standard deviation (STDEV).

[0060] Figures 16A to 16B Calibration between the target protein concentration and the corresponding fluorescence values is shown. The y-axis depicts the fluorescence readings corrected for the blank, while the x-axis shows the protein concentration. The measured values shown include 150 μL of purified His6-tag::SnoopCatcher::sfGFP (Figure 15A) and His6-tag::SnoopCatcher::miniSOG (Figure 15B).

[0061] Figures 17A to 17BFluorescence values of the bottom liquid removed from each round of buoyant separation. The y-axis represents the fluorescence value adjusted for the blank, and the x-axis represents each round of buoyant separation. The presented measurements are from the bottom liquid aspirated with a syringe during the functional testing of the affinity tag assays for purified SnoopTag GV (Figure 17A) and Strep-tag II GV (Figure 17B) (n = 3 biological replicates). Error bars represent mean ± standard deviation (STDEV).

[0062] Figures 18A to 18HProvided is a side-by-side comparison of recombinant protein purification using HisTag / Ni-NTA immobilized metal affinity chromatography (IMAC) and labeled GV-related buoyancy-assisted affinity chromatography (BAAC). Figure 18A: Structure of the His6-tag::SnoopCatcher::miniSOG operon, with a TEV cleavage site (ENLYFQ|S (SEQ ID NO:5)) inserted between miniSOG and SnoopCatcher. Figure 18B: Schematic of how the target protein miniSOG is "eluted" from the surface of labeled GV by cleavage with TEV protease at the recognition site. Figure 18C: Schematic of His-tag / Ni-NTA immobilized metal affinity chromatography (IMAC) and labeled GV buoyancy-assisted affinity chromatography (BAAC). Figures 18D, 18E: Percentage of fluorescence intensity retained on GV relative to the initial fluorescence intensity measured immediately after sample incubation, and fluorescence images of the samples after incubation with TEV protease followed by centrifugation and buoyancy purification to aspirate the supernatant (fluorescent content is boxed). W1 to W4: Percentage of fluorescence intensity retained on GV after the 1st to 4th 1×PBS resuspensions. PE: Percentage of fluorescence intensity retained on GV after incubation with TEV protease followed by buoyancy purification and 1×PBS resuspension (n = 6 biological replicates). Figure 18F: Percentage of miniSOG purified from labeled GV BAAC or His-tag / Ni-NTA IMAC relative to cell lysate. For the labeled GV method, % of purified POI = 100×(fluorescence intensity of miniSOG after removal of TEV protease) / (fluorescence intensity of miniSOG in the cell lysate supernatant). For the Ni-NTA method, % of purified POI = 100×(fluorescence intensity of miniSOG from Ni-NTA eluate) / (fluorescence intensity of miniSOG in the cell lysate supernatant) (for the labeled GV method, n = 6 biological replicates, and for the Ni-NTA method, n = 3 biological replicates). Figure 18G: SDS-PAGE gel of miniSOG purified from clarified E. coli lysate by labeled GV BAAC. Pre: Before induction; Post: After induction; Pellet and Sup.: Pellet and supernatant after cell lysis and centrifugation; W1, W2, W3, and W4: Wash fractions removed by syringe; P TEV : Supernatant of the sample after TEV protease incubation and centrifugation; E: P after removal of TEV protease TEV; The purity of E was determined by densitometry (right), and the gray area under the red line represents the background intensity. Figure 18H: SDS-PAGE gel of miniSOG purified from clarified E. coli lysate by Ni-NTA / His-tag IMAC. Pre: Before induction; Post: After induction; Pellet and Sup.: Pellet and supernatant after cell lysis and centrifugation; Flow-Thru: Flow-through of Ni-NTA chromatography; W1, W2, and W3: Wash fractions of Ni-NTA chromatography; Eluate 1 to 3: Elution fractions of Ni-NTA chromatography; P TEV : After incubation with TEV protease; E: P after removal of TEV protease TEV ; The purity of E was determined by densitometry (right), and the gray area under the red line represents the background intensity. Error bars represent mean ± standard deviation (STDEV).

[0063] Figures 19A to 19GShows "one-step" and "device-free" buoyancy-assisted recombinant protein purification. Figure 19A: Schematic diagram of the "one-step" buoyancy-assisted recombinant protein purification procedure. Escherichia coli cultures containing labeled GV and the target protein are lysed together. Subsequently, centrifugation is performed to separate the target protein bound to the labeled GV (1), the soluble unbound target protein (2), and the inclusion bodies (3). The supernatant containing the soluble unbound target protein and the inclusion bodies is removed by syringe. Then the labeled GV bound to the target protein is resuspended in 1×PBS. This buoyancy purification process is repeated three times, or until the supernatant is clear and contains only the target protein bound to the labeled GV (4). Then the sample is resuspended and incubated with TEV protease at 30 °C for 12 hours. After the final centrifugation, some of the target protein remains on the surface of the labeled GV (5), while some of the target protein is released and becomes soluble in the supernatant (6). Figures 19B and 19C: SDS-PAGE and Western blot results showing the final elution step in the "one-step" buoyancy-assisted recombinant protein purification process. Lane 1 contains the supernatant from the sample after incubation with TEV protease; lane 2 shows the flow-through (eluate 1) after removal of His6-tagged TEV protease by Ni-NTA magnetic beads; lane 3 shows the wash (eluate 2) after rinsing the Ni-NTA magnetic beads; lane 4 is the final eluate from the Ni-NTA magnetic beads. The gray arrow points to His6-tagged TEV protease (in both SDS-PAGE and Western blot) or GvpB with His6-tag::SnoopCatcher (apparently trailing in the Western blot). The green arrow highlights the presence of sfGFP in lanes 2 and 3. d, The amount of the target protein sfGPF produced by two rounds of the "elution" procedure. The amount of sfGFP is calculated from the fluorescence intensity. Figures 19E and 19F: Schematic diagram of a time-lapse photography device for analyzing the buoyant flotation speed of GV: The device captures the initial and final states of well-suspended, labeled GV or GV bound to a fluorescent protein in a 10-cm-long Corning tube. The first photo (left in Figure 19F) shows the GV at the start of the time-lapse sequence, and the last photo (right in Figure 19F) depicts the GV after they have floated to the surface of the liquid, which takes approximately 5.3 hours. Figure 19G: Gray-scale intensity measurements are taken from a selected region of interest on the tube containing GV over a 5-hour period. The resulting data are fit to a single-phase decay non-linear model, as represented by the equation and R 2 (n = 3, biological replicates). Error bars represent mean ± standard deviation (STDEV).

[0064] Figures 20A to 20GShows a side-by-side comparison of sfgGFP purification using HisTag / Ni-NTA immobilized metal affinity chromatography (IMAC) and labeled GV-related buoyancy-assisted affinity chromatography (BAAC). Figures 20A, 20B: Percentage of retained fluorescence intensity from sfGFP on GV relative to the initial fluorescence intensity measured immediately after sample incubation (Figure 20A), and fluorescence image of the sample after incubation with TEV protease followed by centrifugation and buoyancy purification to aspirate the supernatant with a syringe (fluorescent content is boxed) (Figure 20B). W1 to W4: Percentage of retained fluorescence intensity on GV after the 1st to 4th 1×PBS resuspension. PE: Percentage of retained fluorescence intensity on GV after incubation with TEV protease, followed by buoyancy purification and 1×PBS resuspension (n = 5 biological replicates). Error bars represent mean ± standard deviation (STDEV). Figures 20D, 20E: Percentage (Figure 20D) and amount (Figure 20E) of sfGFP purified from labeled GV BAAC or His-tag / Ni-NTA IMAC relative to the cell lysate. For the labeled GV method, % of purified POI = 100×(fluorescence intensity of sfGFP after removal of TEV protease) / (fluorescence intensity of sfGFP in the cell lysate supernatant). For the Ni-NTA method, % of purified POI = 100×(fluorescence intensity of sfGFP from the Ni-NTA eluate) / (fluorescence intensity of sfGFP in the cell lysate supernatant) (n = 5 biological replicates). The amount of sfGFP was calculated from the fluorescence intensity. Figure 20F: SDS-PAGE gel of sfGFP purified from clarified E. coli lysate by labeled GV BAAC. Pre: Before induction; Post: After induction; Pellet and Sup.: Pellet and supernatant after cell lysis and centrifugation; W1, W2, W3, and W4: Wash fractions removed by syringe; P TEV : Supernatant of the sample after TEV protease incubation and centrifugation; E: P after removal of TEV protease TEV ; The purity of the eluate was determined by densitometry (right), and the gray area under the red line represents the background intensity. Figure 20G: SDS-PAGE gel of concentrated sfGFP after removal of TEV protease by Ni-NTA / His-tag IMAC or SnoopTag GV BAAC. The purity of each was determined by densitometry (right), and the gray area under the red line represents the background intensity.

[0065] Figure 21Shows the loss of POI in the buffer exchange procedure for His-tag / Ni-NTA purification. After performing the buffer exchange procedure to remove high concentrations of imidazole from the elution step in His-tag / Ni-NTA purification, the percentage of miniSOG and sfGFP loss was calculated as: % POI loss = 100 × (fluorescence intensity of POI before buffer exchange - fluorescence intensity of POI after buffer exchange) / (fluorescence intensity of POI before buffer exchange) (for miniSOG, n = 3 biological replicates, and for sfGFP, n = 5 biological replicates). The total volume of POI was always kept consistent. Error bars represent mean ± standard deviation (STDEV).

[0066] Description of Exemplary Embodiments

[0067] The present disclosure relates to the development of methods for separating cells using buoyant microbubbles. Microbubbles dissipate in solution within a few minutes, require gentle handling protocols, and are limited in the multiplex capabilities of the technology.

[0068] Methods for purifying target cells from a mixed sample have been provided herein. In some embodiments, the target protein is present on the cell surface. Thus, in some embodiments, the methods of the present disclosure have been shown to purify or sort cells from a mixed sample by buoyancy assisted cell sorting (BACS). Compared to the more demanding and time-consuming conditions required by similar techniques known in the art, the methods described herein require low-speed centrifugation, elutriation, or gravity flotation. Additionally, the compositions described in the present disclosure methods exhibit improved stability over those of similar techniques.

[0069] In some embodiments, the present method provides a greater degree of control in target cell or target protein capture compared to known methods. For example, traditional magnetic bead-based cell sorting techniques are binary in terms of how cells are captured - yes or no. A single antibody-modified bead needs to bind to the target cell for its retention. On the other hand, multiple GVs (e.g., thousands) are required to alter the density of a single cell, and this can be affected by: the labeling density on the GV surface, the use of multiple types of labels, altering the GV structure / volume, or using a clustered formulation.

[0070] Methods for sorting a mixed population of mammalian cells during cell manufacturing have been described herein. In an improvement over known cell sorting methods, the methods of the present disclosure do not require an external selection force, such as a magnetic column.

[0071] According to some embodiments of the present disclosure, GVs are purified and labeled to induce protein-protein ligation by way of biotin-streptavidin interaction with an antigen-specific tag or other GVs. In some embodiments, the labeled GVs can be incubated with a heterogeneous cell population and bind to the target subpopulation labeled with an antibody or other antigen-specific biomolecule (such as a nanobody or an aptamer). In some embodiments, the buoyancy acting on the GVs provides an upward lift force to float the GV-cell complex to the surface while non-target cells precipitate. These and more details will be discussed in more detail below.

[0072] The present disclosure also relates to the development of methods for separating proteins and enzymes in the industry for manufacturing goods. Current methods such as large-scale chromatography, protein secretion, non-specific binding of cells to vesicles are either too costly or have limitations.

[0073] Described herein are methods that have been shown to purify target proteins from a mixed sample of cell lysates. These and more details will be discussed in more detail below.

[0074] I. Gas Vesicles and Buoyancy-Assisted Purification

[0075] Gas vesicles (GVs) are protein-shelled nanobubbles that are naturally produced in Anabaena flos-aquae or Haloarchae salinarum, or heterologously produced in Escherichia coli. The GV gene cluster for E. coli expression includes a major shell protein gvpB (the basic construct of GV) and ten other accessory proteins gvpRNFGLSKJTU (SEQ ID NO: 6). The methods of the present disclosure provide for at least one modification of at least one of the shell proteins of GV. In some embodiments, gvpB is modified. In some embodiments, gvpA is modified. In some embodiments, gvpC is modified. Modifications of any of the ten accessory proteins are also contemplated. The methods may also relate to recombinant forms of the shell proteins. For example, the GV shell protein sequences can be rationally modified to introduce additional functionality or to alter GV geometry, rigidity, and clustering state. The recombinant shell proteins can include genetic fusion elements (such as tags) that can be expressed, purified, and interchangeably swapped onto wild-type GVs. Recombinant expression can also be used to assemble GVs by transforming a modified variant of the gvp gene cluster that includes the structural shell proteins (GvpA, GvpC) and the accessory proteins required for GV formation in a non-native host.

[0076] The modification of the shell proteins as described above can include adding at least one affinity tag to the wild-type or recombinant shell proteins, such as at gvpB ( Figure 1) or add an affinity tag to the C-terminus of gvpC (Figure 4). The affinity tags used herein are any moieties that bind tightly to the target with an affinity constant below 13×10 -6 M. In some embodiments, the affinity tag binds to its target by a covalent bond. In other embodiments, the affinity tag binds to its target by a non-covalent bond. In some embodiments, the affinity tag can be a peptide. Some specific examples of such peptide affinity tags known in the art include, but are not limited to, SnoopTag, SnoopTagJr, SnoopCatcher (also known as monomeric streptavidin or mSA), SpyTag, SpyCatcher, DogTag, DogCatcher, or Strep-tacin (also known as Strep-tag II). The GV with the attached affinity tag is referred to herein as a labeled GV. Additional examples and details related to affinity tags are provided in the following sections.

[0077] A variety of labeling strategies can be utilized to modify GV. In one embodiment, amino acid residues on the surface of GV, such as lysine residues, can be modified, for example, by an EDC / NHS reaction, to attach an affinity tag to the surface of GV. For example, GV can be surface-modified to contain biotin or streptavidin. Thus, biotin-GV and streptavidin-GV are each considered embodiments of a labeled GV. According to this method, streptavidin-GV can bind to a wide catalog of commercially available biotinylated targets (such as an antibody that has been biotinylated). In some embodiments, the labeled GV can be generated using commercially available one-step NHS-ester modification reagents (e.g., NHS-fluorophore, NHS-streptavidin). In some embodiments, the methods of the present disclosure provide for the conjugation of one or more antibodies to the surface amino acids of GV by methods known to those skilled in the art. In such embodiments, the one or more antibodies are the affinity tag and the corresponding antigen is the target protein as described herein. In some embodiments, the GV-antibody conjugate is a labeled GV of the present disclosure.

[0078] In some embodiments, the present method provides improved production process efficiency and cost advantages over traditional bead or resin-based systems. A notable feature of GVs is their adaptability to pre-translational modifications. By directly genetically encoding specific ligands or proteins onto the GV capsid protein, modified GVs can be expressed directly in a bacterial system. This results in the co-expression and assembly of functional GVs in a single step, thus simplifying the production process. Such a method eliminates the need for subsequent conjugation steps, which are typically required in bead or resin systems. Traditional methods generally require chemical synthesis and modification of beads, followed by separate expression and purification of the desired recombinant protein. Once expressed and purified, the protein must be chemically conjugated to the beads, adding multiple layers of complexity and cost. In contrast, due to their buoyancy, GVs can be easily separated in the buoyant fraction, simplifying purification and eliminating the need for secondary resins or other downstream processes. This integrated bioprocess workflow not only enhances process efficiency but also provides significant cost savings by reducing the need for multiple raw materials and extensive purification steps.

[0079] In some embodiments, the methods of the present disclosure include adding a plurality of independently selected affinity tags to at least one protein of the GV( Figure 2 ). Accordingly, the present method can be used for multiplex purification of more than one target protein. As a non-limiting example, in some embodiments, the GV is dual-labeled with two different, independently selected affinity tags: one affinity tag for EpCAM present on low-count circulating tumor cells (CTCs), and one affinity tag for T cells, such as a CD3 antibody. Accordingly, the present disclosure provides a method for purifying tumor-reactive T cells according to the methods of the present disclosure.

[0080] In some embodiments, the present method can purify cells containing a certain combination of target proteins. In some embodiments, two or more different collections of independently labeled GVs can be prepared or obtained such that the affinity tag of one collection of labeled GVs binds to at least one of the affinity tags of a different other collection of labeled GVs.

[0081] In some embodiments, the methods of the present disclosure can optionally include expressing or linking to the GV a moiety that is different in purpose from that which binds to peptides, proteins, or cells in the sample mixture. In some embodiments, the moiety can be an antibody, such as CD28. Accordingly, the present disclosure provides a method for, for example, simultaneously purifying and activating T cells. Accordingly, the methods of the present disclosure represent an improvement in the cell manufacturing life cycle relative to methods known in the art, where such activation is typically performed in a post-processing step after initial sorting.

[0082] In some embodiments, according to the present method, GVs serve as bioprocessing reagents for biologics, and cell or gene therapy. For example, an Fc binding protein (FcBP) can be fused to the GV capsid protein for buoyancy-based antibody purification (Choe et al., 2016). Currently, there is significant interest in improving the purification and clearance process for viral vector purification. GVs that recognize the capsid or associated tags can be used for separation by buoyancy. Alternatively, bioactive proteins can be functionalized onto GVs to act as nanoparticle carriers. The advantage of dealing with buoyant particles is that when centrifuged, they rapidly float while all other substances tend to pellet. Soluble impurities can be depleted by cycling the floating layer separation and resuspension in fresh buffer (Lakshmanan et al., 2017). The equipment required for cell separation would be a centrifuge, where the GV reagent serves as a universal "bead" or resin that is suitable for multiple modification modes. Thus, the present method provides a simplified or more economically favorable method for bioprocessing biologics or cell or gene therapy.

[0083] The method of the present disclosure involves incubating labeled GVs with a sample mixture. The sample mixture can comprise bacteria or mammalian cells, a lysed cell solution, proteins or peptides. The sample mixture can comprise a peptide or protein of a target or target protein that contains an affinity tag for the labeled GV. In some embodiments, the sample mixture can comprise a cell population that expresses the target or target protein on the cell surface of the sample mixture. The peptides, proteins, and cells of the sample mixture can naturally express the target or target protein. In other embodiments, a subset or the entirety of the peptides, proteins, and cells of the sample mixture can be modified using the principles and techniques of biochemistry or molecular biology applied by those skilled in the art to have a target or target protein of the affinity tag of the GV. Further modification of the peptides, proteins, or cells of the sample mixture is contemplated, including any target or target protein that any of the foregoing substances have, to improve solubility. A non-limiting example of such a modification is the addition of maltose-binding protein (MBP) to a target peptide added to the peptides, proteins, or cells of the sample mixture by the principles and techniques applied by those skilled in the art to the sample mixture.

[0084] In some embodiments, the population of peptides of a target or target protein comprising an affinity tag of labeled GV may represent the majority of the population of peptides of the sample mixture. In some embodiments, the population of proteins of a target or target protein comprising an affinity tag of labeled GV may represent the majority of the population of proteins of the sample mixture. In some embodiments, the population of cells of a target or target protein comprising an affinity tag of labeled GV may represent the majority of the population of cells of the sample mixture. In some embodiments, the population of peptides of a target or target protein comprising an affinity tag of labeled GV may represent the minority of the population of peptides of the sample mixture. In some embodiments, the population of proteins of a target or target protein comprising an affinity tag of labeled GV may represent the minority of the population of proteins of the sample mixture. In some embodiments, the population of cells of a target or target protein comprising an affinity tag of labeled GV may represent the minority of the population of cells of the sample mixture.

[0085] In the methods of the present disclosure, the population of peptides of a target or target protein having an affinity tag of labeled GV can be about 10%. About 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the total population of peptides in the sample mixture, or any range derivable therefrom. In some embodiments, the population of proteins of a target or target protein having an affinity tag of labeled GV can be about 10%. About 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the total population of proteins in the sample mixture, or any range derivable therefrom. In some embodiments, the population of cells of a target or target protein having an affinity tag of labeled GV can be about 10%. About 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the total population of cells in the sample mixture, or any range derivable therefrom.

[0086] In some embodiments, the population of peptides of a target or target protein having an affinity tag of labeled GV may be less than 0.1% to 10% of the total population of peptides in the sample mixture. In some embodiments, the population of proteins of a target or target protein having an affinity tag of labeled GV may be less than 10% of the total population of proteins in the sample mixture. In some embodiments, the population of cells of a target or target protein having an affinity tag of labeled GV may be less than 10% of the total population of cells in the sample mixture.

[0087] According to the method of the present disclosure, the labeled GV is incubated with a sample mixture of a peptide, protein, or cell containing a target or target protein having an affinity tag for the labeled GV for a first period of time such that the peptide, protein, or cell of the sample mixture of the target or target protein having an affinity tag for the labeled GV binds to the labeled GV. Accordingly, the present disclosure provides a method for forming a captured target protein - gas vesicle composition. In some embodiments, the first period of time is less than one hour. In some embodiments, the incubation of the labeled GV with the sample mixture is carried out at about 21 °C or about room temperature. In some embodiments, the above incubation can be carried out at about 4 °C. For some embodiments, the corresponding captured target can be a soluble protein within a cell, which will be released upon cell lysis. For embodiments of the present disclosure where the target or target protein is on the surface of an intact cell, the corresponding captured target protein - gas vesicle composition can equivalently or interchangeably be referred to as a captured target cell - gas vesicle composition. For some embodiments, the method of the present disclosure does not require isolation or purification of the target protein on the cell surface. For some embodiments, the captured target is an intact cell internalized by GV, which can be mediated by binding to surface proteins or by the natural endocytosis process of the cell alone without relying on any binding to proteins.

[0088] In some embodiments, the captured target protein - gas vesicle composition can have a density different from that of the target or target protein of the labeled GV or the affinity tag of the labeled GV. In some embodiments of the present disclosure where the target or target protein is on the cell surface, the density of the captured target protein - gas vesicle composition can have a density different from that of the uncaptured cells. The natural cell density tends to be between 1.06 and 1.07 g / cm (Miltenyi et al., 1990; Wang et al., 2022; Wang et al., 2023; Frenea - Robin et al., 2022; Hu et al., 2016; Liou et al., 2015; Weil et al., 2017; Palani et al., 2023; Zipursky et al., 1976). In some embodiments, the cell density is reduced by contacting (e.g., binding) with the labeled GV. In some embodiments, due to binding to the labeled GV, the cell density is reduced by about 0.005 g / cm 3 、about 0.01 g / cm 3 、about 0.015 g / cm 3 、about 0.02 g / cm 3 、about 0.025 g / cm 3 、about 0.03 g / cm 3 、about 0.035 g / cm 3, about 0.04 g / cm 3 or any range derivable therefrom. In some embodiments, due to binding to labeled GV, the cell density is reduced by about 0.02 g / cm 3 , about 0.025 g / cm 3 , about 0.03 g / cm 3 , about 0.035 g / cm 3 , about 0.04 g / cm 3 or any range derivable therefrom. In some embodiments, due to binding to labeled GV, the cell density is reduced by about 0.04 g / cm 3 . In some embodiments, due to binding to labeled GV, the cell density is reduced by more than 0.04 g / cm 3 . In some embodiments, when using the prepared density medium, the reduction in cell density due to binding to labeled GV according to the present method promotes the formation of buoyant and non-buoyant fractions. Any commercially available density medium or any medium prepared with a specific density according to methods known in the art is contemplated for use in the methods disclosed herein. In some embodiments, the density of the medium is about 1.005 g / mL, about 1.01 g / mL, about 1.02 g / mL, about 1.03 g / mL, about 1.04 g / mL, about 1.05 g / mL, about 1.06 g / mL, about 1.07 g / mL, about 1.08 g / mL or any range derivable therefrom. In some embodiments, the present method involves using a medium with a density of about 1.04 g / mL. In other embodiments, the present method involves using a medium with a density of about 1.06 g / mL.

[0089] The methods described herein provide a second time period during which the captured target protein-buoyant vesicles can be separated in the sample mixture according to density. In some embodiments, the second time period can occur simultaneously with the above-described first time period. In some embodiments, the second time period can overlap with the first time period. In other embodiments, the second time period can occur after the first time period.

[0090] In some embodiments, the sample mixture is left undisturbed during a second time period. In some embodiments, the sample mixture is left undisturbed for at least 15 minutes during the second time period. In some embodiments, the sample mixture can be left undisturbed for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, or any range derivable therefrom during the second time period. In some embodiments, the second time period is from about 4 hours to about 6 hours. In some embodiments, the second time period is about 5 hours. The sample mixture can be left undisturbed overnight during the second time period, or for any amount of time until particle accumulation at the sample mixture-air interface has been observed. In some embodiments, particle accumulation at the sample mixture-air interface is indicated by the formation of a white layer of air bubbles.

[0091] The second time period can include a centrifugation-assisted flotation step ( Figure 3 ). The sample mixture containing the captured target protein-air bubble composition can be centrifuged, for example, at 400×g. The sample mixture containing the captured target cell-air bubble composition can be centrifuged for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or any range derivable therefrom. The second time period can include more than one centrifugation-assisted flotation step. In some embodiments, the methods of the present disclosure include one, two, three, four, or five centrifugations.

[0092] In some embodiments, the methods disclosed herein optionally include density gradient centrifugation or counterflow elutriation of the sample mixture containing the captured target protein-air bubbles. Elutriation is a countercurrent centrifugation device that separates cells based on size and density, balancing gravity and the drag force introduced from fluid flow (Coulais et al., 2012; Stroncek et al., 2014). While it can effectively separate peripheral blood mononuclear cells (PBMCs) from red blood cells, it cannot separate lymphocytes from each other due to their similar size and density. GVs displaying labels (e.g., antibody fragments) that recognize specific cell surface markers can selectively associate with cells, thereby effectively reducing their density for separation using countercurrent centrifugal force as a more continuous process than using discrete batches of density media.

[0093] In some embodiments, during a second time period, the captured target protein-bubble composition redistributes within the sample mixture to accumulate at the sample mixture-air interface. In some embodiments, during a second time period, the captured target protein-bubble composition redistributes within the sample mixture but does not reach the sample mixture-air interface.

[0094] In some embodiments, any affinity tag of the labeled GV or the corresponding binding site of the affinity tag present on a target, target protein, or a cell having a target protein or target peptide on its surface may have a protease cleavage site or a self-splicing peptide sequence. The protease cleavage site or self-splicing sequence is contemplated in the present method for both native recognition sites and for recognition sites installed by one of ordinary skill in the art on a target, target protein, or a cell having a target protein or target peptide on its surface. A non-limiting example of a protease cleavage site contemplated by the present disclosure is the TEV protease cleavage site. A non-limiting example of a self-splicing peptide sequence contemplated by the method of the present disclosure is an intein. The method of the present disclosure optionally includes protease-mediated cleavage or self-splicing at the protease cleavage site or self-splicing sequence, respectively, to separate the target protein from the captured target protein-bubble composition. In some embodiments, internalization of GV by target cells may not require modification of the affinity tag of GV. In some embodiments, chemical conjugation of an antibody with GV will not form with a self-splicing peptide sequence. In some embodiments, the self-splicing peptide allows for increased purity of the target protein of interest in downstream purification. According to the present method, the presence or inclusion of a self-splicing sequence in any component of the labeled GV does not imply or require that self-splicing of the sequence will occur. In some embodiments, the captured target protein-bubble composition or the captured target cell-bubble composition will be separated intact and the target protein or target cell will not be separated from the bubble.

[0095] II. Affinity Tag

[0096] The present disclosure provides labeled GV which, in some embodiments, comprises an affinity tag that binds to a target located on the cell surface. In certain embodiments, the affinity tag is a peptide sequence engineered according to the present disclosure. Modification of the GV surface as described in the above section may include adding at least one affinity tag to the coat protein, such as adding an affinity tag to the C-terminus of gvpB ( Figure 1)。According to some embodiments, the affinity tag can be, for example, an antibody, a lipid, a carbohydrate, a polysaccharide, a growth factor, a hormone, a peptide, an aptamer, a small molecule such as a hormone, an imaging agent, or a cofactor, or a cytokine. In some embodiments, the affinity tag is a functional group associated with the cell membrane, a carbohydrate or polysaccharide that binds to one or more markers on the cell membrane, a lipid that binds to one or more markers on the cell membrane, a small molecule that binds to one or more markers on the cell membrane, an aptamer that binds to one or more markers on the cell membrane, or a peptide or antibody that binds to one or more markers on the cell membrane. In some embodiments, the affinity tag can target human cells, such as cancer cells or T cells. For example, the affinity tag according to some embodiments can bind to low-count circulating tumor cells (CTCs). It has been shown that the gp240 antigen is expressed in a variety of melanomas but not in normal tissues. Thus, in some embodiments, the compositions of the present disclosure can be conjugated with an antibody against a specific antigen that is expressed by cancer cells but not in normal tissues.

[0097] In certain embodiments, the affinity tag is a functional group, such as a positively charged group, such as an amine. The positively charged group can be used to associate with negatively charged groups on the cell membrane surface. It is contemplated that the group can be used to associate with other negatively charged groups (such as negatively charged proteins or nucleic acids).

[0098] In certain additional embodiments, it is contemplated that the affinity tag binds to multiple types of cancer cells. For example, the 8H9 monoclonal antibody and single-chain antibodies derived therefrom bind to glycoproteins expressed on breast cancer, sarcoma, and neuroblastoma (Onda, et al., 2004). Another example is the cell targeting agent described in U.S. Patent Publication No. 2004 / 005647 and Winthrop, et al. (2003), which binds to the antigen MUC-1 expressed on multiple cancer types. Thus, it should be understood that in certain embodiments, the affinity tag according to some embodiments can target multiple cancer or tumor types.

[0099] In addition, certain cell surface molecules are highly expressed in tumor cells, including hormone receptors such as human chorionic gonadotropin receptor and gonadotropin-releasing hormone receptor (Nechushtan et al., 1997). Thus, the corresponding hormones can be used as affinity tags in cancer therapy. In addition, affinity tags that can be used include cofactors, sugars, drug molecules, imaging agents, or fluorescent dyes. It is known that many cancer cells overexpress folate receptors, and thus folic acid or other folate derivatives can be used as affinity tags to allow specific binding of labeled GV of the present disclosure (Campbell, et al., 1991; Weitman, et al., 1992).

[0100] Since a large number of cell surface receptors have been identified in hematopoietic cells of multiple lineages, ligands or antibodies specific for these receptors can be used as affinity tags. IL-2 can also be used as an affinity tag in chimeric proteins to target IL-2R+ cells. Alternatively, other molecules such as B7-1, B7-2, and CD40 can be used to specifically target activated T cells (The Leucocyte Antigen Facts Book, 1993, Barclay, et al. (eds.), Academic Press). In addition, B cells express CD19, CD40, and IL-4 receptors and can be targeted by affinity tags that bind these receptors (such as CD40 ligand, IL-4, IL-5, IL-6, and CD28). Elimination of immune cells (such as T cells and B cells) is particularly useful in the treatment of lymphoid tumors.

[0101] Other cytokines that can be used to target specific cell subsets include interleukins (IL-1 to IL-15), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, leukemia inhibitory factor, tumor necrosis factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and / or fibroblast growth factor (Thompson (ed.), 1994, The Cytokine Handbook, Academic Press, San Diego). In some aspects, the targeting peptide is a cytokine that binds to the Fn14 receptor, such as TWEAK (see, e.g., Winkles, 2008, which is incorporated herein by reference).

[0102] Those skilled in the art recognize that there are a variety of known cytokines, including hematopoietins (four-helix bundles) [such as EPO (erythropoietin), IL-2 (T cell growth factor), IL-3 (multi-colony CSF), IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6IL-4 (IFN-b2, BSF-2, BCDF), IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T cell growth factor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM (OM, oncostatin M), and LIF (leukemia inhibitory factor)]; interferons [such as IFN-g, IFN-a, and IFN-b]; immunoglobulin superfamilies [such as B7.1 (CD80) and B7.2 (B70, CD86)]; TNF families [such as TNF-a (cachectin), TNF-b (lymphotoxin, LT, LT-a), LT-b, CD40 ligand (CD40L), Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and 4-1BBL]; and those not assigned to a specific family [such as TGF-b, IL 1a, IL-1b, IL-1RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NK cell stimulating factor), MIF, IL-16, IL-17 (mCTLA-8), and / or IL-18 (IGIF, interferon-g inducing factor)]. In addition, the Fc portion of an antibody heavy chain can be used to target Fc receptor-expressing cells, such as targeting mast cells and basophils using the Fc portion of an IgE antibody.

[0103] In addition, in some aspects, the affinity tag is a peptide sequence or a cyclic peptide as described above. Some examples of cell and tissue targeting peptides that can be used according to embodiments are provided in, for example, U.S. Patent Nos. 6,232,287; 6,528,481; 7,452,964; 7,671,010; 7,781,565; 8,507,445; and 8,450,278, each of which is incorporated herein by reference.

[0104] Thus, in some embodiments, the affinity tag is an antibody or an avimer. Thus, antibodies and avimers can be generated against almost any cell surface marker, providing a method for targeting labeled GVs to almost any population of target cells. Methods for generating antibodies that can be used as affinity tags are known in the art and are detailed below. Methods for generating avimers that bind to a given cell surface marker are detailed in U.S. Patent Publications Nos. 2006 / 0234299 and 2006 / 0223114, each of which is incorporated herein by reference. In certain embodiments of the present disclosure, any other antigen-specific biomolecule, some non-limiting examples of which include nanobodies and aptamers, is an affinity tag.

[0105] In addition, it is contemplated that the compositions described herein may comprise nanoparticles or other nanomaterials. Some non-limiting examples of nanoparticles include metallic nanoparticles such as gold or silver nanoparticles or polymeric nanoparticles such as poly-L-lactic acid or poly(ethylene) glycol polymers. Nanoparticles and nanomaterials that can be conjugated to the compounds of the invention include those described in U.S. Patent Publications Nos. 2006 / 0034925, 2006 / 0115537, 2007 / 0148095, 2012 / 0141550, 2013 / 0138032 and 2014 / 0024610 and PCT Publications Nos. 2008 / 121949, 2011 / 053435 and 2014 / 087413, each of which is incorporated herein by reference.

[0106] III. Kits

[0107] The techniques disclosed herein include kits for forming labeled GVs for purifying peptides, proteins, or cells. In some embodiments, the kits described herein may comprise preformed labeled GVs for purifying peptides, proteins, or cells. A "kit" refers to a combination of physical elements. For example, a kit may include, for example, one or more components such as specific primers, enzymes, reaction buffers, instructions, and other elements that can be used to practice the techniques described herein. These physical elements can be arranged in any manner suitable for carrying out the present disclosure.

[0108] The components of the kit can be packaged in an aqueous medium or in a lyophilized form. The container devices of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container device in which the components can be placed and, preferably, appropriately aliquoted (e.g., into the wells of a microtiter plate). In the case where there is more than one component in the kit, the kit will generally also contain a second, third, or other additional container in which the additional components can be placed separately. However, a single vial can contain multiple combinations of the components. The kits of the present disclosure will generally also include means for enclosing the containers that restrictively house the labeled GV and any other reagents for commercial sale. Such containers can include injection or blow molded plastic containers that retain the desired vials.

[0109] The kit will also include instructions for using the kit components, as well as the uses of any other reagents not included in the kit. The instructions can include variations that can be implemented. Such reagents are contemplated to be some embodiments of the kits of the present disclosure. However, such kits are not limited to the specific articles defined above.

[0110] IV. Process Scale-up

[0111] Using the principles and techniques of process chemistry, bacterial growth, or bioprocessing as applied by those skilled in the art, the methods described herein can be further modified and optimized for preparative, pilot, or large-scale production, whether batch or continuous. For example, such principles and techniques are taught in Practical Process Research & Development (2000), which is incorporated herein by reference. The methods described herein can be used to produce preparative-scale amounts of any of the compounds or compositions described herein.

[0112] V. Definitions

[0113] When used in the claims and / or the specification in conjunction with the term "comprising / including", the use of a term without a quantifier can mean "one / a", but it is also consistent with the meanings of "one / a or more / more than one", "at least one / a", and "one / a or more than one / a".

[0114] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variations of the device, method used to determine the value, or the variations that exist between subjects or patients. Unless otherwise specified, the term "about" is used to indicate a value that is ±10% of the reported value, preferably ±5% of the reported value. It is understood that whenever the term "about" is used, a specific reference to the exact numerical value is also included.

[0115] The terms "comprise", "have", and "include" are open-ended conjunctive verbs. Any form or tense of one or more of these verbs is also open-ended, e.g., "comprises / comprising", "has / having", or "includes / including". For example, any method that "comprises", "has", or "includes" one or more steps is not limited to merely having those one or more steps, but also encompasses other unlisted steps.

[0116] The above definitions supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be taken to indicate that any terms that are not defined are unclear. On the contrary, all terms used are considered to describe the invention in a sense that enables one of ordinary skill in the art to understand the scope and practice of the invention.

[0117] VI. Examples

[0118] The following examples are included to illustrate some preferred embodiments of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present disclosure and, thus, can be considered to constitute a preferred mode for the practice of the present disclosure. However, according to the present disclosure, those skilled in the art will understand that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0119] A. Cell sorting

[0120] a. SpyTag GV allows for modular functionalization with SpyCatcher fusion proteins

[0121] In some embodiments, the gas vesicles used in the methods of the invention are composed of repeating subunits of the major shell protein GvpA, which can be reinforced at the aqueous boundary by GvpC (Figure 4A). In some embodiments, GVs can be simply modified using known conjugation techniques (e.g., NHS ester chemical modification reagents) to conjugate to lysine residues or the N-terminal free primary amine (Figure 4B). By more structured protein engineering methods, the minor shell protein GvpC can be stripped (Figure 4C) or replaced with engineered variants. In some embodiments, SpyTag3 (hereinafter referred to as SpyTag) is fused to the C-terminus of GvpC such that it can be displayed on the outer surface of the GV body (Figure 4D) for modular functionalization with SpyCatcher fusions such as mWasabi-SpyCatcher or anti-CD3scFv-SpyCatcher (Figure 4E, Figure 4F). Incubation of SpyTag GV with the SpyCatcher-mWasabi fusion results in modification of the GV with the fluorescent protein, which can be detected by the formation of a ligation product between SpyTag and SpyCatcher, shown as a higher MW band on SDS-Page (Figure 4G). Ligation of mWasabi-SpyCatcher also results in enrichment of the fluorescent signature in the buoyant fraction containing GVs (Figure 4H). Additionally, the concentration of mWasabi-SpyCatcher on the GV can be regulated by controlling the amount of GvpC-SpyTag input during GvpC replacement on the GV.

[0122] b. Gas vesicles reduce cell density for density-based separation

[0123] Initially, unmodified WT GVs were tested by incubating them with RAW 264.7 macrophages, and a maximum reduction in cell density of 0.03 g / cm was measured 3 (Figure 5). Phagocytosis-dependent density-based cell separation strategies require extended incubation times at 37 °C for robust GV internalization.

[0124] GV for antigen - specific sorting (e.g., for sorting T cells) was developed by connecting anti - CD3 single - chain variable fragment (scFv) to GvpC using the SpyTag / SpyCatcher ligation system (Figure 6A). Before GV modification, the reference CD3 - scFv sequence was first validated by cell - binding studies. CD3scFv - mWasabi was cloned and expressed in Escherichia coli. The purified CD3 - scFv - mWasabi was applied to HEK cells (negative), pure patient - derived T - cell samples (positive), and freshly isolated peripheral blood mononuclear cells (PBMC; mixed samples), and showed a staining distribution comparable to that of the commercial anti - CD3 antibody (OKT3 clone) (Figure 7). Then, CD3 scFv with a C - terminal SpyCatcher fusion was cloned, expressed, and purified before ligation to SpyTag GV. By increasing the total binding of GV, the cell density on CD3+ T cells could be proportionally decreased (Figure 6B). Thus, in some embodiments, the present invention provides a method for cell sorting with improved predictability and control compared to methods known in the art. For example, common methods for preparing PBMC from blood include overlaying the buffy coat on Ficoll - Paque (a medium with a specific gravity of 1.08 g / mL) (Turner et al., 2020). While PBMC can be captured as a buoyant cell layer above the dense Ficoll medium, even denser red blood cells will form a distinct fraction below the Ficoll at the bottom of the container. In addition to separating PBMC, the ability of individual density media to sort specific cell types (e.g., T cells) is limited. According to the methods disclosed herein, GV binds to specific cell - surface markers, and then it can be separated using existing and relatively cost - effective density media.

[0125] One of the initial challenges when dealing with buoyant samples is the efficient and complete separation of the buoyant fraction. To overcome this obstacle, microcentrifuge spin columns were repurposed to rapidly separate the buoyant top fraction and the pelleted bottom fraction (Figure 6C). Instead of using filters or packed beads inside the column, it was kept open and filled with a medium modified to a specific density. After centrifugation, cells with an effective density lower than the modified medium remained in the top buoyant fraction, and these cells could be completely and easily separated by removing the column insert and transferring it to a second container. This system provides a device - based solution for separating buoyant fractions that are otherwise vulnerable to disruption and difficult to fully recover.

[0126] CD3-GV was incubated with freshly isolated PBMCs and fractionated at 1.06 g / mL to evaluate cell capture in the buoyant fraction. A robust increase in SSC signal was observed in the CD3-GV-treated samples, particularly in the top non-precipitating fraction. Without being bound by theory, the significant increase in SSC indicates a higher degree of GV binding. Gating on CD4+ and CD8+ cells in each fraction, the greatest enrichment of T cells was observed in the top fraction of PBMCs treated with CD3-GV: more than 80% of the isolated sample consisted of T cells compared to 50% of both the original input and the PBS-treated control. Based on the FSC and SSC profiles of the second cell population, the presence of a subpopulation with both high FSC and SSC indicates monocyte contamination when the sample is fractionated in a 1.06 g / mL density medium.

[0127] c. Density medium component reduces monocyte contamination

[0128] To reduce monocyte contamination, the density of the medium used for sorting was decreased. CD3-GV was applied to freshly isolated PBMCs and fractionated through a series of Ficoll-Paque dilutions with a density ranging from 1.005 g / mL (PBS) to 1.08 g / mL in 0.02 g / mL steps (Figure 8A). At the upper density range, samples treated with both PBS and CD3-GV showed almost complete depletion of the precipitating fraction at 1.08 g / mL as it was equivalent to the density initially used for PBMC isolation. A difference in buoyant fraction cell counts was first observed at 1.06 g / mL, where 20% of the original cell input no longer precipitated when treated with PBS, but 60% of the cells no longer precipitated when treated with CD3-GV. When the sample was treated with GV, further reducing the density to 1.04 g / mL reliably captured the buoyant cell fraction, although the total yield was much lower compared to 1.06 g / mL. Additionally, cell count events were only observed in the top fraction of the 1.02 g / mL and 1.005 g / mL samples when treated with CD3-GV, but not when treated with PBS alone.

[0129] The % distribution of monocytes in the isolated fractions was measured using flow cytometry. PBMCs not treated with CD3-GV showed a monocyte population % of 50.6% in the 1.06 g / mL density medium and 18.4% in the 1.04 g / mL density medium (Figure 8B). In contrast, for PBMCs treated with CD3-GV, reducing the density medium from 1.06 g / mL to 1.04 g / mL resulted in a decrease in monocytes from the top fraction from 11% to 0.18% (Figure 8C). The high SSC characteristics in the top fraction of GV-treated cells continued to be consistent with GV-cell interactions, driving the reduction in target T cell density. While the lower density medium increased the final purity of the isolated T cells, this corresponded to a decrease in the total cell yield. Without being bound by theory, it is reasonable to speculate that only cells with high-density surface antigens available for GV binding can achieve a greater reduction in cell density.

[0130] d. Streptavidin-modified GV can be combined with commercially available biotinylated antibodies

[0131] The method of the present invention may also involve using streptavidin-modified GV and commercially available biotinylated antibodies. Lysine residues and termini on the GV capsid protein were targeted with an NHS-streptavidin modification kit and the conjugation was confirmed by SDS-Page (Figure 9A). Without being bound by theory, after chemical reactions and multiple buoyancy purifications, the retention of streptavidin monomer and multimer bands in the buoyancy fractions indicated successful modification of GV. The functionality of streptavidin GV was further confirmed in a clustering assay, where streptavidin GV was mixed with biotinylated GV and the GV-GV interaction behavior was observed using TEM imaging (Figure 9B).

[0132] Functionally validated streptavidin-GV was used in a two-part cell labeling process, where samples were first stained with biotinylated anti-CD3 antibody (clone: OKT3), and then secondary labeled with streptavidin-GV (Figure 10A, Figure 10B). The biotin-streptavidin-based system for GV cell labeling resulted in an increase in SSC within subsets of PBMC samples, similar to scFv-GV assembled using the Spy system (Figure 10C). Untreated PBMC showed 50.6% CD14+ monocyte loading in the top fraction of a density medium at 1.06 g / mL and 18.4% monocyte loading in a density medium at 1.04 g / mL (Figure 10D). In contrast, PBMC treated with biotinylated anti-CD3 antibody and streptavidin-GV showed only 6.9% monocyte burden in the 1.06 g / mL medium and only 8.71% monocyte burden in the 1.04 g / mL medium. Based on previous CD4+ / CD8+ assessments, the FSC characteristics of the isolated cells, and the concentration of the low-density medium used, the remaining fraction was presumed to be positively isolated CD3+ T cells.

[0133] e. Method

[0134] i Air vesicle harvesting and buoyancy purification

[0135] As described above, according to the procedures described in previous publications, gas vesicles (GVs) are naturally produced in Anabaena flos-aquae or Haloarchae salinarum, or heterologously produced in Escherichia coli. Anabaena flos-aquae is cultured for the native cyanobacterial expression of GVs. The Anabaena flos-aquae culture is inoculated in BG11 freshwater medium and incubated in a 1% CO2 incubator modified with LED strip lights (2000 lumens) with a 12-hour on / off light / dark cycle. The Anabaena flos-aquae is cultured for about one month and then the GVs are harvested. GV harvesting is performed by first allowing the Anabaena flos-aquae to float in a separatory funnel and then draining the clarified supernatant. The concentrated Anabaena flos-aquae is lysed by mixing with an equal volume of 2× homemade lysis buffer (1M sorbitol, 100 mM Tris-HCl, and 1 wt% Tween-80) and shaking overnight at 4°C. Complete lysis is indicated by the optical transparency of the solution (i.e., no particles). The lysis buffer is removed by first centrifuging the lysed solution at 200 rcf for 24 hours at 4°C. The released gas vesicles accumulate at the air-water interface and form a white film on the surface. After lysis, the released GVs are washed by multiple rounds of buoyancy purification, where each round involves overnight centrifugation (200×g, 4°C) and then removal of the supernatant using a syringe immersed under the floating GV layer. The dehydrated GV sample is then resuspended in fresh TBS and the buoyancy purification cycle is repeated. The GVs undergo a final concentration cycle to bring the stock solution OD 500 = 30 (about 1 mg / mL of protein in PBS). The number of cycles required depends on the percentage of supernatant removed and the dilution factor per wash. At least 1000×DF was found to be required for complete removal of soluble impurities.

[0136] ii Cloning

[0137] Plasmids for the recombinant expression of GvpC-SpyTag and SpyCatcher fusions were constructed using Gibson assembly of the amplified PCR products. Using the Gibson Assembly Master Mix (New England Biolabs), PCR of the insert and pET28a vector was performed with amplified primers with designed overhangs for assembly. The recombinant GvpC was designed with a C-terminal SpyTag followed by a C-terminal his-tag. The SpyCatcher fusion was designed to include a C-terminal his-tag and an N-terminal fusion with the protein of interest (mWasabi or anti-CD3 scFv). The plasmid assembly was transformed into Turbo Competent Escherichia coli, cultured overnight in LB containing 50 μg / mL kanamycin, and used The Miniprep plasmid DNA extraction kit was used to isolate plasmids for sequencing verification by Sanger sequencing using forward and reverse sequencing primers for the T7 promoter and T7 terminator.

[0138] In some embodiments, the major capsid protein gvpB was modified by adding the amino acid sequence GSGKLGDIEFIKVNK (SEQ ID NO: 1) to the C-terminus. Note that KLGDIEFIKVNK (SEQ ID NO: 2) is the sequence of the previously described SnoopTag (Veggiani et al, 2016). In a separate experiment, the major capsid protein gvpB was modified by adding the amino acid sequence GSGWSHPQFEK (SEQ ID NO: 3) to the C-terminus, where WSHPQFEK (SEQ ID NO: 4) is the previously described StrepTag II sequence (Korndofer, 2002). The constructed plasmids were transformed into BL21-Star TM (DE3)pLysS-One Shot TM chemically competent Escherichia coli. The cultures were induced with 20 μM IPTG at OD 600 = 0.4 and incubated overnight for 22 hours at 30 °C. GVs were harvested by adding SoluLyse TM bacterial protein extraction reagent and DNase I and purified by repeated buoyant density purification with low-speed centrifugation (400 × g) and 1× PBS washes at 4 °C. The purified high-density surface-labeled GVs were stored at 4 °C.

[0139] In some embodiments, the surface of GVs can be modified by chemical conjugation using a variety of click chemistry kits, antibody-specific engineered peptides, or other techniques known in the art.

[0140] iii Expression and purification of recombinant proteins

[0141] The plasmid for expressing the anti-CD3 scFv fusion protein was heat-shock transformed into Shuffle T7 Express, or into BL21(DE3) chemically competent cells for all other protein expressions. After overnight culture with kanamycin antibiotic selection (50 μg / mL), 100 mL of fresh LB containing kanamycin was inoculated to an OD600 = 0.05 and incubated until reaching an OD600 = 0.4 to 0.5, at which point protein expression was induced using 400 mM IPTG and then expressed at 30 °C for 20 hours. The cells were collected by centrifugation at 2000 × g for 20 minutes at 4 °C and subsequently resuspended in Solulyse lysis reagent supplemented with lysozyme (250 μg / mL), DNase I (10 μg / mL), and MgCl2 (2 mM). To further facilitate lysis, the cell suspension was sonicated on ice for 5 rounds of 20-second on / off cycles using a Branson Sonifier 250 with the output control set to 6. For soluble proteins, the lysed cells were centrifuged at 20,000 × g for 20 minutes at 4 °C to pellet the insoluble fraction. The soluble supernatant was incubated with Ni-NTA agarose beads pre-equilibrated with wash buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole). After 1 hour, the flow-through was collected and the beads were washed with wash buffer and then eluted with elution buffer (20 mM Tris, 500 mM NaCl, 250 mM imidazole) (Figure 11B, Figure 11C). For GvpC purification, the lysed cell solution was centrifuged at 20,000 × g for 20 minutes at 4 °C. The soluble supernatant was discarded and the pellet was resuspended in wash buffer supplemented with 6 M urea. After shaking at 4 °C for 30 minutes, the solution was centrifuged at 20,000 × g for 20 minutes at 4 °C. The supernatant was transferred to incubate with Ni-NTA agarose beads pre-equilibrated with wash buffer supplemented with 6 M urea. The elution buffer was also supplemented with 6 M urea (Figure 11A). For all protein purifications, the eluates were combined and concentrated in a 10,000 kDa MWCO Amicon filter insert and buffer exchanged by three rounds of concentration and resuspension with fresh TBS (for soluble proteins) and TBS containing 6 M urea (for GvpC-SpyTag) to remove imidazole.

[0142] In some embodiments, the soluble protein of interest (POI) is fused to the C-terminus of the capture protein SnoopCatcher / monomeric streptavidin (mSA), which is described in (Keeble et al, 2019; DeMonte et al., 2013). In bench-scale experiments, Superfolder GFP (sfGFP) was used as the POI. The constructed plasmid was transformed into BL21-Star TM (DE3)pLysS-One Shot TM chemically competent Escherichia coli. The culture was induced with 400 μM IPTG at OD600 = 0.8 and incubated overnight for 20 hours at 18 °C. The culture was then centrifuged at 3000×g for 30 minutes to remove the liquid medium, and the cells were resuspended in the prepared lysis buffer. The cells in the lysis buffer were rapidly frozen in liquid nitrogen and thawed in a 42 °C water bath, repeating the freeze-thaw cycle five times. The cell lysate was then sonicated for 10 seconds five times. DNase I activated by the addition of MgCl2 was added to the cell lysate and incubated at room temperature for 30 minutes. The cell lysate was spun at 20000×g for 20 minutes to separate the supernatant and pellet.

[0143] iv Protein quantification

[0144] The concentration of the purified and buffer-exchanged protein was determined using Bradford reagent. A 1 mg / mL BSA standard was prepared in 1×TBS (pH 7.4) to determine the soluble protein concentration, or a 1 mg / mL BSA standard was prepared in 6 M urea in 1×TBS to determine the concentration of recombinant GvpC. 5 μL of the sample or standard was mixed with 250 μL of Bradford reagent, then mixed and the absorbance was read at a wavelength of 595 nm. A standard curve of BSA was prepared from 0 to 1 mg / mL and compared with the diluted protein sample to infer the original unknown sample protein concentration.

[0145] In some embodiments, the SnoopTag-labeled GV is incubated with the lysate supernatant of Snoopcatcher-POI for a given time to allow binding. For example, for SnoopCatcher-sfGFP, incubation at 4 °C for 2 hours enables efficient binding. Next, buoyancy-assisted protein purification is carried out using gravity flotation or centrifugation-assisted flotation. For gravity flotation, the solution is left to stand for 5.3 hours. Theoretical estimates based on the hydrodynamic radius of the GV complex (approximately 1402.2 nm) suggest that the particles should rise by approximately 4E-5 mm per hour. The gravity flotation protocol results in particle aggregation on the top surface of 2 cm of water after 2 hours. For centrifugation-assisted flotation, the solution is centrifuged at 400×g for 15 minutes, and the lower supernatant is removed by syringe. The top fraction of the remaining GV-POI is resuspended in 1×PBS. The centrifugation and resuspension processes are repeated three times to increase purity. The enrichment of GV-POI in each repeated centrifugation run is calculated as the ratio of the remaining top fraction to the total volume of the solution. The purity of the sfGFP sample is evaluated by fluorescence intensity to indicate the POI (sfGFP) bound to the GV, and the results show purities of 81.9%, 99.3%, and 99.6% after 3 repeats.

[0146] GvpC replacement on v WT GV

[0147] Recombinant GvpC is replaced onto wild-type GV by dissolution of native GvpC and re-annealing of GvpC-SpyTag. GvpC-SpyTag at a molar ratio increased relative to the WT GvpC present on the GV (0.1:1 to 5:1. GvpC-SpyTag:WT GvpC) is reconstituted with WT GV in the presence of 6 M urea. 6 M urea effectively dissolves GvpC and its derivatives from the primary GV bodies ( Figure 12 ), and the sample is rapidly diluted with TBS to a final 3 M urea, resulting in re-annealing of GvpC and GvpC-SpyTag GV. Excess and un-annealed GvpC, as well as residual urea, can be removed by two rounds of buoyancy purification.

[0148] vi SpyTag-SpyCatcher ligation assay and fluorescence quantification

[0149] The SpyTag-modified GV resuspended in 1×TBS (pH 7.4) was incubated with the SpyCatcher fusion for overnight at 4 °C. During the ligation reaction, SpyTag-GvpC was present at approximately 1 μM and the SpyCatcher fusion was added at a 5- to 20-fold molar excess. The excess and unreacted SpyCatcher were removed by multiple rounds of buoyant purification. After buoyant purification, the relative amount of mWasabi-SpyCatcher ligated to the prepared GV was determined by measuring the fluorescence intensity using 485 / 529 nm (ex / em) wavelengths, with the gain automatically adjusted and normalized to the final OD of the treated GV sample.

[0150] In some embodiments, the SnoopTag-labeled GV was incubated with the lysate supernatant of Snoopcatcher-POI for a given time to allow binding. For example, for SnoopCatcher-sfGFP, 2 hours at 4 °C allowed efficient binding. Next, buoyancy-assisted protein purification was performed with gravity flotation or centrifugation-assisted flotation. For gravity flotation, the solution was allowed to stand for 5.3 hours. Theoretical estimates based on the hydrodynamic radius of the GV complex (≈1402.2 nm) suggest that the particles should rise approximately 4E-5 mm per hour. The gravity flotation protocol resulted in particle aggregation on the top surface of 2 cm of water after 2 hours. For centrifugation-assisted flotation, the solution was centrifuged at 400×g for 15 minutes and the lower supernatant was removed by syringe. The remaining top fraction of GV-POI was resuspended in 1×PBS. The centrifugation and resuspension process was repeated three times to increase purity. The enrichment of GV-POI in each repeated centrifugation run was calculated as the ratio of the remaining top fraction to the total volume of the solution. The purity of the sfGFP sample was evaluated by fluorescence intensity to indicate the POI (sfGFP) bound on the GV, and the results showed purities of 81.9%, 99.3%, and 99.6% after 3 repeats.

[0151] vii SDS-Page

[0152] SDS-Page was performed using a 4%-15% Mini-PROTEAN TGX unstained protein gel. The samples were mixed with an equal volume of 2× Laemmli sample buffer containing 2-mercaptoethanol as a reducing agent. The gel was run at 100 V using a running buffer consisting of 25 mM Tris, 192 mM glycine, 0.1% SDS (pH 8.3). The running gel was stained with Coomassie blue solution, then de-stained and imaged. A larger MW ligation band was observed in the gel, indicating successful covalent ligation of the SpyTag and SpyCatcher fusion proteins.

[0153] viii NHS modification of GV

[0154] The NHS modification was completed using the Lightning Link chemistry modification kit according to the supplier's instructions for the modification of both biotin and streptavidin on GV. The purified and concentrated GV (OD 500 = 50) was directly added to the lyophilized NHS modification reagent and allowed to react overnight at 4 °C. Unreacted and soluble biotin or streptavidin was depleted by two rounds of buoyant density purification.

[0155] ix Transmission electron microscopy

[0156] For negative staining TEM of purified GV, the sample was diluted to OD 500 = 0.2 in 1×PBS and loaded onto 200-mesh carbon-coated copper grids (Ted Pella, Redding, CA) for 3 minutes. Excess liquid was carefully blotted away with filter paper. The sample was then stained with 2% (w / v) uranyl acetate solution (Electron Microscopy Sciences, Hatfield, PA). High-resolution TEM images were acquired using a JEOL JEM-2010 TEM and a JEOL JEM-2100 field emission gun TEM.

[0157] x Preparation of density gradient buffers

[0158] Density gradient buffers of 1.02 g / mL, 1.04 g / mL, 1.06 g / mL, and 1.08 g / mL were obtained by mixing the running buffer (1×PBS with 2% BSA) with Cytiva Ficoll-Paque TM (Ficoll, Fisher Scientific) in the correct amounts for each condition. These mixtures were stored at 4 °C when not in use and tested against controls to ensure correct density values. xi Isolation of CD3+ T cells from PBMCs by scFv-CD3-GV

[0159] For all cell studies, GV was washed and concentrated to a final OD500 = 10. GV was formulated in TBS and 2% BSA and stored at 4 °C for up to 1 month. Buffy coats from donors were obtained from the Gulf Coast Consortium.

[0160] Standard buffy coat isolation using an industry-standard Ficoll procedure was performed to obtain peripheral blood mononuclear cells (PBMCs). After isolation, the PBMCs were counted and the required number of PBMCs was added to Eppendorf tubes (2 × 10 in 50 μL of running buffer 6cells). Incubate the cell suspension with 350 μL of scFv-CD3-GV solution (5 OD) at 4 °C with vigorous rotation for 1 hour.

[0161] Use a rotating column with a collection tube (Fisher Scientific) to establish a buoyancy-activated cell sorting system. The filter in the rotating column has been pre-removed. Prepare 1 mL of density buffer as described previously and place it in each collection tube. Gently add the cell suspension and GV mixture to the rotating column. Then centrifuge the collection tube and the rotating column at 400 g for 30 minutes. Obtain the top fraction of the mixture by collecting the solution in the rotating column, while the bottom fraction is the solution remaining in the collection tube. Use a Countess TM cell counting chamber slide (Invitrogen) to obtain the cell numbers of the top and bottom fractions.

[0162] xii Isolate CD3+ T cells from PBMCs by streptavidin-GV

[0163] As described previously, obtain PBMCs and establish a buoyancy-activated cell sorting system. After isolation, count the PBMCs and add the required number of PBMCs to an Eppendorf tube (2 × 10 6 cells in 50 μL of flow buffer). Incubate the cell suspension with 5 μL of biotinylated anti-human CD3 (Thermo Fisher Scientific) at 4 °C for 45 minutes. Then, centrifuge the cell solution at 400 g for 10 minutes to remove the excess antibody. Add 400 μL of streptavidin-GV solution (5 OD) to the cell solution and incubate at 4 °C with vigorous rotation for 1 hour. Gently add the cell suspension and GV mixture to the rotating column. Then centrifuge the collection tube and the rotating column at 400 g for 30 minutes.

[0164] In some embodiments, buoyancy-assisted cell sorting is accomplished with cells expressing a protein of interest (e.g., CD3, CD28). First, cells expressing the protein of interest are incubated with the corresponding biotinylated antibody at room temperature for 10 minutes. After incubation, streptavidin-modified air vesicles are mixed into the sample and further incubated for 30 minutes to promote biotin-streptavidin interaction on the cell surface. Then, the labeled cell sample is either processed as is or loaded on top of a Percoll density medium, the final solution density of which is adjusted to about 1.01 g / mL, about 1.02 g / mL, about 1.03 g / mL, about 1.04 g / mL, about 1.05 g / mL, about 1.06 g / mL, about 1.07 g / mL, or about 1.08 g / mL to assist in separating the air vesicle-modified cell population. The sample is then centrifuged at 400×g for 10 minutes, yielding a top fraction containing air vesicle-labeled cells and a bottom fraction containing unmodified cells. The top fraction is a floating cell layer that can be manually separated or, in one embodiment, can be captured in an insert that retains the liquid sample by surface tension, removal of which results in complete recovery of the top fraction.

[0165] xiii Cell Staining

[0166] Collect cell samples and stain for flow cytometry. Mix the cell sample with 10 μL of Fc Block (BD Bioscience) and pre-incubate at 4°C for 15 minutes. Subsequently, add 5 μL of the desired antibody to the mixture and incubate at 4°C in the dark for 45 minutes. Then, spin the cell solution at 400 g for 10 minutes and discard the supernatant. Then add flow buffer to the cell sample for flow cytometry analysis.

[0167] Stain the cell sample with CD3 (Alexa 700 mouse anti-human CD3, BD Bioscience), CD4 (Alexa 647 mouse anti-human CD4, BioLegend), CD8 (CD8 monoclonal antibody (3B5), PE Texas Red, Invitrogen), CD14 (CD14 monoclonal antibody (61D3), PE-Cyanine5, Invitrogen), and 7-AAD (7-aminoactinomycin D, Invitrogen).

[0168] xiv Cell Type Analysis by Flow Cytometry

[0169] All flow cytometry analyses were performed on a Sony MA3800 cell sorter. Before the experiment, different fluorescence libraries were established using UltraComp eBeads TM (Invitrogen). The desired antibodies were incubated with the beads at 4°C for 45 minutes. Excess antibodies were removed by centrifuging the solution at 400 g for 10 minutes. Then the stained beads were fed into the instrument and the fluorescence signals were measured.

[0170] Before running, the instrument was set up and standards were run to ensure proper working conditions. For each run, fluorescence controls and / or biological controls were added to ensure proper gating. As for the gating strategy, generally FSC vs SSC was gated to target the population of interest. After this parent gate, FSH vs FSA was gated to exclude any doublets / triplets and only the single-cell population was shown. After this gating, all viable single-cell populations were gated using a viability control (7-AAD). All flow cytometry analyses were completed using FlowJo software and compared to the controls of the same run.

[0171] B. Protein purification for industrial enzyme production

[0172] a. Creating high-density affinity tags on the GV surface

[0173] Previous studies have shown that the A. flos-aquae scaffolding protein GvpC from the genetically engineered GV construct Acoustic reporter gene 1 (ARG1) (Bourdeau et al., 2018; Hurt et al., 2023) can be appended with affinity tags at the N-terminus or C-terminus (Lakshmanan et al., 2016). Additionally, GvpC from other diverse species can accommodate fusions with bacterial and viral polypeptides. However, GvpC is only sparsely present on the GV surface, with an estimated ratio to the major shell protein of 1:20 (Huber et al., 2023; Dutka et al., 2023). Consequently, by adding affinity tags to GvpC, the availability of binding sites for the protein of interest (POI) on each GV particle surface becomes insufficient. Given this limited binding capacity, a large number of GVs are required to bind and purify substantial amounts of the desired protein, which undermines the goal of developing an economical and scalable method. Therefore, GVs encoded by the pNL29 operon, initially cloned from Bacillus megaterium, were selected (Figure 13A) because they can be robustly expressed in E. coli and purified without GvpC (Figure 13B) (Li & Cannon, 1998). Specifically, compositions containing affinity tags appended to the primary shell protein GvpB were designed and studied.

[0174] Recent studies by Huber et al. have shown that the cylindrical shell of GV in Bacillus megaterium (B. megaterium) is composed of GvpB monomers (Huber et al., 2023). These monomers are densely packed in a helix with a lateral spacing of 1.2 nm, a helical pitch of 4.9 nm, and approximately 93 monomers per turn of the helix (Figure 13C) (Huber et al., 2023). Since the shell proteins are tightly packed in this highly crystalline manner, there are considerable challenges in altering the protein sequence of the major shell protein. Mutating any part of the shell protein risks both distorting the tight crystal packing and interfering with their interaction with the assembly factor proteins. However, they also showed that the C-terminus of GvpB has a more limited role in stabilizing the assembly compared to the N-terminus (Huber et al., 2023). Additionally, the C-terminus is relatively flexible within the assembled GV shell and is also exposed on the outer surface of GV (Huber et al., 2023). Therefore, without being bound by theory, the C-terminus of GvpB can be genetically modified without disrupting GV assembly (Figure 13D). If affinity tags are genetically appended to the C-terminus of GvpB, after expression and assembly, the outer surface of GV can be densely covered with these affinity tags (Figure 13E).

[0175] A series of fusions to the C-terminus of Mega-GV (Figure 13F) were screened, including a variety of protein tags and linkers in between that further enhanced the flexibility of the C-terminus of GvpB. After expression optimization and centrifugation-assisted purification, most designs failed to produce fully assembled GVs, as expected. However, two candidate tags emerged: SnoopTag (KLGDIEFIKVNK (SEQ ID NO: 2)) and Strep-tag II (WSHPQFEK (SEQ ID NO: 4)). After the purification process, these two tagged GV variants were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), with wild-type pNL29GV as a control (Figures 13G to 13J). Notably, like their wild-type counterparts, the tagged GVs remained clustered after purification from Escherichia coli. However, the difference that emerged from the DLS measurements (Figures 13I, 13J) was that the clusters formed by the tagged GVs were larger in size than wild-type GVs. The established protocol (Figures 14A, 14B) involving treatment of the wild-type pNL29 GV that was de-clustered with 6 M urea (Lakshmanan et al., 2017) was found to be ineffective for the tagged GVs. Unexpectedly, contrary to a one-order-of-magnitude decrease in the hydrodynamic diameter of the GV, the size of the clusters increased after treatment with 6 M urea (Figures 14C to 14F).

[0176] After successful expression of the two tagged GVs in Escherichia coli, the functional efficacy of the high-density affinity tags on the GV surface was evaluated. The aim was to effectively bind these GVs to the target protein using the corresponding binding partners of the tags and then perform buoyancy-driven separation of the target protein (Figure 13K).

[0177] b. Buoyant separation of fluorescent proteins

[0178] To establish the process design and obtain quantitative results of its performance, fluorescent proteins were selected as the POI to enhance the convenience of visualization and quantification of protein levels based on fluorescence readings (Figures 15A, 16).

[0179] The process began with the tagged GV being combined with a fluorescently labeled binding partner (purified His6-tag::SnoopCatcher::superfolder green fluorescent protein (sfGFP) for SnoopTag GV and Alexa Fluor for Strep-tag II GV) TM488 streptavidin) was mixed in a tube and incubated for one hour at room temperature. The fluorescence intensity after incubation was measured (Figure 15A). After centrifugation, the floating GVs bound to the labeled ligand were separated by removing the lower liquid with a syringe (Figure 15A). These GVs were resuspended in 1×PBS and their fluorescence was measured again ( Figure 2 a). This cycle was continued until there was no significant change in fluorescence, and at least three rounds of buoyancy separation were performed to confirm that the POI was tightly bound to the GV surface, as shown by the fluorescence readout (Figure 15B, Figure 15D). In addition, direct fluorescence imaging of the plastic tube showed that the POI was separated to the top of the tube as expected (Figure 15C, Figure 15E). In contrast, WT GVs without an affinity tag pulled out the least amount of non-specific binding in the first round, and this binding was almost completely removed starting from the second round of buoyancy separation (Figure 15B, Figure 15D). Direct fluorescence imaging also showed that no POI bound to the WT GVs after multiple rounds of buoyancy-driven separation. The fluorescence readout of the removed lower liquid further confirmed that most of the POI bound to the labeled GVs rather than the WT GVs (Figure 17).

[0180] The evidence provided above supports that GVs featuring SnoopTag or Strep-tag II surface modification can effectively interact with their respective binding ligands. Therefore, SnoopTag and SnoopCatcher were selected for further development.

[0181] c. Performance comparison with common affinity chromatography methods

[0182] The purified labeled GVs were evaluated as affinity substrate analogs of "resin" or "beads", which are commonly used for the affinity chromatography purification of the POI. For this purpose, a fusion protein used as the POI was designed by linking miniSOG (a flavin-binding protein with a dual ability for fluorescence and singlet oxygen photo-generation of 12.3 kDa) to the C-terminus of SnoopCatcher (Ruiz-González et al., 2013; Qi et al., 2012), and the SnoopCatzer had been engineered with a His6-tag at its N-terminus (Figure 18A). Given the irreversible covalent bond between SnoopTag and SnoopCatcher, a TEV protease cleavage site (ENLYFQ|S (SEQ ID NO: 5)) was strategically introduced between SnoopCatcher and miniSOG. This modification allowed miniSOG to be separated from the GV surface upon incubation with TEV protease (Figure 18A, Figure 18B). In addition, the presence of the His6-tag enabled a comparative analysis of buoyancy-assisted affinity chromatography (BAAC) of the labeled GVs with conventional His-tag / Ni-NTA immobilized metal affinity chromatography (IMAC) (Figure 13C).

[0183] The amount of His6-tagged::SnoopCatcher::miniSOG was calculated based on fluorescence readout (Figure 16B) to precisely match the theoretical binding capacity of Ni-NTA beads or labeled GVs. Following the workflow outlined above (Figure 18A), after four rounds of buoyant separation, the fluorescence readout of the POI on SnoopTag GVs remained consistently stable (Figure 19D), indicating tight binding of the miniSOG version of the POI to the GV surface. After incubation with TEV protease and subsequent buoyant separation to release miniSOG from SnoopTag GV, approximately 60% of the initial fluorescence was observed to remain on the GV (Figure 19D). This result confirmed the successful partial release of miniSOG. Direct fluorescence imaging after TEV protease treatment and buoyant separation showed that a greater amount of miniSOG remained on the GV surface compared to that released into the supernatant (Figure 19E). Without being bound by theory, a reasonable explanation for this observation could be that the tight clustering of GVs limits the accessibility of TEV protease to the GV surface. Despite partial release, the amount of miniSOG purified from clarified cell lysates using BAAC was comparable to that obtained using Ni-NTA beads (Figure 19F). Additionally, purification using labeled GVs produced miniSOG with a higher purity level (79.8 ± 0.4%) compared to the Ni-NTA-based purification method (66.8 ± 1.9%), as shown by densitometry of elution fractions after removal of TEV protease (Figure 19G, Figure 19H). Similarly, the consistency of these findings was also observed in the purification of sfGFP, further confirming the general applicability of our method (Figure 20). Additionally, loss of the POI was observed during the buffer exchange process used to remove imidazole in His-tag / Ni-NTA IMAC ( Figure 21 ); however, this could have been avoided since a consistent buffer was used in the labeled GV BAAC procedure.

[0184] d. Simple one-step purification workflow

[0185] A key advantage of the GV-based buoyant separation method disclosed herein is the elimination of the need to prepare affinity beads themselves. When users scale up their POI, they can simply culture a bacterial culture to produce the "beads", i.e., engineered GVs. Then, one can mix the two types of cells together, lyse them in a single vessel, and proceed with protein purification. Described below is the use of a fluorescent protein to test this simple one-step purification workflow.

[0186] The one-step protein purification workflow begins with the simultaneous lysis of E. coli cultures of SnoopTag GV and POI, His6-tagged::SnoopCatcher::sfGFP. This step is followed by a one-hour incubation at room temperature to allow the labeled GV to bind to the POI. After centrifugation for 30 minutes, the SnoopTag GV with the bound POI is separated from the unbound POI, impurities, and inclusion bodies by buoyancy separation (Figure 19A). The remaining steps, including multiple rounds of buoyancy purification and incubation with TEV protease, follow the protocol described above. Due to the similar molecular weights of sfGFP and the commercially available TEV protease, which is characterized by His6-tag, Western blotting was used as an additional analytical tool to distinguish between them. When comparing the results of SDS-PAGE and Western blotting, the bands present in lanes 2 and 3 in the SDS-PAGE analysis were absent in the Western blotting, confirming the complete removal of TEV protease or capsid protein from the labeled GV, which was shown as a smear in lanes 1 and 4 of the Western blotting results (Figure 19B, Figure 19C). Using this one-step buoyancy separation method, a yield of nearly 5 mg of protein per 50 mL of E. coli culture was achieved (Figure 19D).

[0187] e. Purification without equipment

[0188] Next, an attempt was made to completely bypass centrifugation and rely solely on normal gravity for protein separation.

[0189] Wild-type GV is a nanoscale object with a diameter of several hundred nanometers and thus typically requires centrifugation for at least several hours to separate the GV (Lakshmanan et al., 2017). Although buoyancy is proportional to the volume of the object and thus to the diameter, increasing the overall particle size would be the most feasible method to increase the flotation rate. Fortunately, both types of engineered GV were observed in a clustered form (Figure 13G to 13J, Figure 16).

[0190] To test the flotation rate of GV, a time-lapse photography of tubes was set up for a certain time frame (Figure 19E). In both labeled GV without POI and GV bound to a fluorescent protein, most GV particles took more than about 5.3 hours to rise by buoyancy to the surface of 1×PBS (Figure 19F). Since the opacity of the solution can indicate the position of GV, the intensity within the region of interest (ROI) at the center of the tube was measured every hour to quantify the flotation rate. The ROI grayscale intensity decreased over time, following a single-phase decay non-linear model with a half-life of 0.87 hours (Figure 19G). This indicates that, without being bound by theory, in a fixed-volume container, most clustered GV particles can rise approximately 5 cm within one hour.

[0191] f. Discussion

[0192] The air vesicles used in the currently disclosed methods are preliminary examples, which demonstrate that the major capsid protein of GV can be genetically modified without compromising the efficient expression and assembly of GV in Escherichia coli, highlighting the potential of the genetically engineered GV surface with high density as a versatile platform for various applications. In addition, by demonstrating that GV with an affinity tag on its surface can function similarly to an affinity resin for purifying POI, the potential use of GV has been extended from biomedical engineering to biomanufacturing applications, especially the purification of protein biologics.

[0193] The successful attachment of two tag systems, Strep-tag II and SnoopTag, to GV is of great significance in protein tag research. Their complementary properties allow for wide applications in biotechnology and related disciplines. Specifically, the non-covalent nature of Strep-tag II provides flexibility in the release of traditional protein cargos via competitive binding with small molecules, which is highly valuable when fine-tuning or targeted release of proteins is required (Voss, S. & Skerra, 1997; Schmidt et al., 2013). On the other hand, the covalent bond of SnoopTag (Veggiani et al., 2016) paves the way for innovative applications, opening the door to the potential of directly utilizing GV-binding proteins for enzyme catalysis, thus breaking through the boundaries of traditional enzyme applications. In addition, this covalency provides opportunities for designing controllable, self-cleaving protein or tag modules (Levitin et al., 2005; Li, 2011), with fine-tuned cleavage and activation using stimulus-responsive mechanisms of inteins induced by, for example, pH or temperature (Wood et al., 1999; Zeidler et al., 2004).

[0194] Meanwhile, the method disclosed herein provides a potential alternative to traditional affinity chromatography in the field of protein purification. Specifically, one-step buoyancy separation using labeled GV provides a "gentle" technique. Traditional methods typically subject POI to precipitation and changes in buffer conditions, leading to potential inconsistencies, such as incomplete removal of unwanted chemicals or loss of the target protein (Block et al., 2009; Burgess, 2018; Charcosset, 1998). In contrast, the method disclosed herein ensures that the protein remains under consistent buffer conditions, enhancing the reproducibility and integrity of the results, highlighting its promise for a wider range of biotechnological applications. In addition, the results from device-free purification suggest that the method disclosed herein has the potential to be used in low-resource settings, especially in the fields of biomanufacturing and diagnostics.

[0195] In summary, the present disclosure provides new compositions and methods for the industrial-scale purification of recombinant proteins, which reduce the reliance on affinity resins and columns. Additionally, the high-density surface modification of gas vesicles provides potential use as an assembly platform for a variety of nanotechnology and biomaterial applications( et al., 2002; Farooq et al., 2020; Sato et al., 2016).

[0196] g. Method

[0197] Cloning, expression, and purification of iGV

[0198] The plasmid pST39-pNL29 encoding the GV gene cluster from Bacillus megaterium was obtained from Addgene (#91696). Oligonucleotides for molecular cloning were synthesized (Integrated DNA Technologies (IDT), Coralville, IA). All GV variants with an affinity tag attached to the C-terminus of gvpB were cloned into the pST39 vector backbone using site-directed mutagenesis (SDM) (New England Biolabs (NEB), Ipswich, MA). For GV expression, the plasmid was transformed into the BL21 Star TM (DE3)pLysS One Shot TM E. coli strain (Thermo Fisher Scientific, Waltham, MA), and then it was cultured in a 250 mL flask (Thermo Fisher Scientific, Waldham, MA) in 50 mL of LB Miller Broth (Thermo Fisher Scientific, MA) containing 25 μg / ml chloramphenicol (MilliporeSigma, Burlington, MA), 100 μg / ml carbenicillin (Gold Biotechnology, Olivette, MO), and 0.2% glucose (MilliporeSigma, Burlington, MA). The cells were grown at 30 °C until the optical density (OD) at 600 nm 600)Reached 0.4 and was then induced with 20 μM Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Teknova, Hollister, CA) at 30 °C for 22 h. Cells were harvested and pelleted by centrifugation at 400 × g for 6 h in 50 mL conical tubes with a liquid depth ≤10 cm. The middle layer between the buoyant and pelleted cells was removed, and the remaining cells were mixed with 4 mL SoloLyse-Tris (Genlantis, San Diego, CA), 250 μL / mL lysozyme (MilliporeSigma, Burlington, MA), and 10 μL / mL RNase-free deoxyribonuclease I (DNase I, MilliporeSigma, Burlington, MA) per 50 mL of culture. After rotation at 4 °C for 30 min, the lysate was transferred to 2 mL tubes and centrifuged at 400 g for 2 h at 4 °C. The floating gas vesicles (GVs) located at the top of the liquid surface were separated by removing the liquid and the cell pellet beneath it using a syringe. The separated GVs were then washed by resuspending them in 1× PBS (Teknova, Hollister, CA) followed by three cycles of centrifugation-assisted flotation.

[0199] ii Transmission electron microscopy

[0200] For negative staining TEM of purified GVs, the sample was diluted to OD 500 = 0.2 in 1× PBS and loaded onto 200-mesh carbon-coated copper grids (Ted Pella, Redding, CA) for 3 min. The excess liquid was carefully blotted away with filter paper. The sample was then stained with 2% (w / v) uranyl acetate solution (Electron Microscopy Sciences, Hatfield, PA). High-resolution TEM images were captured using a JEOL JEM-2010 TEM and a JEOL JEM-2100 field emission gun TEM.

[0201] iii Hydrodynamic size measurement

[0202] The hydrodynamic diameter of all GV variants was measured using a Malvern Zen 3600 Zetasizer (Malvern, UK). The purified GV samples were diluted to OD 500= 0.2. Then, 500 μL of each sample was transferred to a cuvette (Thermo Fisher Scientific, Waltham, MA). At least three reads were obtained for each sample, and each GV variant had at least three biological replicates.

[0203] iv Cloning, expression, and purification of the target protein

[0204] The open reading frames of SnoopCatcher::sfGFP and SnoopCatcher::miniSOG were cloned into the pST39 vector with an N-terminal His6-tag. The transformed cells were grown at 30 °C until the OD 600 reached 0.6 and were subsequently induced with 400 mM IPTG at 20 °C for 20 h. The cells were harvested and resuspended in lysis buffer containing 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, and pH 7.5. The cells were lysed by 5 cycles of freeze-thaw and then 5 cycles of probe sonication. DNase I, nuclease (MilliporeSigma, Burlington, MA), and dithiothreitol (DTT) were added at 20 μg / mL, 5000 units / mL, and 1 mM, respectively, and then incubated at 37 °C for 30 min. Next, the samples were centrifuged at 24,000 × g for 30 min, and the supernatant was used for downstream experiments.

[0205] Next, to obtain the purified fluorescent protein, the supernatant was loaded onto a Ni-NTA affinity column (Qiagen USA, Germantown, MD). After washing with 20 mM imidazole, the target protein was eluted with 300 mM imidazole. The fractions containing the target protein were buffer-exchanged with 1× PBS buffer and stored at -80 °C.

[0206] v SDS-PAGE and protein purity quantification

[0207] SDS-PAGE was performed using 4% to 15% Mini-PROTEAN TM TGX Stain-free TMPerformed using a protein gel (Bio-Rad Laboratories, Hercules, CA). The sample was mixed with 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) at a final concentration and 10 mM dithiothreitol (DTT) was added to reduce the sample. The SDS-PAGE gel was run at 120 V in 25 mM Tris, 192 mM glycine, 0.1% (w / v) SDS, pH 8.3. The gel was stained with Coomassie Brilliant Blue G-250 stain (Bio-Rad Laboratories, Hercules, CA), decolorized with a homemade decolorizing buffer (1:1:8 volume % methanol, glacial acetic acid, H2O), and imaged using a FluorChem M imager. The gel band intensity profile was analyzed using Fiji (ImageJ2) by plotting the profile of the selected lane (Wash in Figure 19). The percentage purity was defined as 100×(intensity of the target protein band in the lane / intensity of all protein bands in the lane)

[0208] vi Conversion standard for protein fluorescence intensity and concentration

[0209] The purified SnoopCatcher fusion fluorescent protein was serially diluted with 1× PBS. The fluorescence intensity of 150 μL of the diluted solution in a 96-well plate ( 96Well microplate, Corning) was measured using a microplate reader (Agilent BioTek Synergy H4 Hybrid, Agilent) (for His6-tag::SnoopCatcher::sfGFP: excitation = 485, emission = 510; His6-tag::SnoopCatcher::miniSOG: excitation = 447, emission = 501).

[0210] Measured according to the protocol provided by Pierce TM Modified Lowry Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) for the concentration of those same dilutions. vii Functional test of affinity tags on GV

[0211] Both the purified WT Mega GV and SnoopTag GV were diluted to 500 nm (OD in a 2.0 mL microcentrifuge tube by adding a total volume of 800 μL of 1× PBS 500) The optical density under is equal to 0.5. These GVs were loaded into 200 μL of a purified His6-tag::SnoopCatcher::sfGFP solution at a concentration of 345 μg / mL and then mixed with a pipette. Then these mixture samples were subjected to rotary incubation for 1 hour at room temperature. After incubation, the fluorescence intensity readout of the samples was obtained in the same manner as above. Subsequently, the samples were centrifuged at 400×g for 10 minutes to separate the GVs with bound target protein and the supernatant with unbound target protein. The buoyant GV-target protein complex at the top of the liquid surface was separated by removing the liquid below it using a syringe. Then the complex samples were washed by resuspending in 1000 μL of 1×PBS and then the fluorescence intensity was measured. As shown in Figure 18A, the centrifugation-resuspension cycle was repeated a total of 5 times.

[0212] The purified Strep-tag II GVs and WT Mega GVs were diluted in 1×PBS with a total volume of 1000 μL in a 1.5 mL microcentrifuge tube to an OD 500 = 1.0. 10 μL of 2 mg / mL Alexa Fluor TM 488 streptavidin (ThermoFisher Scientific, Waltham, MA) was introduced into each GV sample. The sample incubation, fluorescence intensity measurement, and centrifugation-resuspension cycle were treated in a manner similar to that of SnoopTag GVs. For Alexa Fluor TM 488, the fluorescence intensity measurement was adjusted to excitation = 495, emission = 519. The centrifugation-resuspension cycle was repeated three times. The percentage of the fluorescence intensity retained on the GVs after each wash was calculated as 100×(fluorescence intensity after each resuspension wash / fluorescence brightness after incubation). The FluorChem M imager was used to visualize the fluorescence between samples by setting two channels with the same exposure time (2.4 seconds): Channel 1 with epi-illuminated white light and no filter (no filter), and Channel 2 with a blue and green filter (green filter).

[0213] viii Parallel protein purification comparison

[0214] 50 μL of a Ni-NTA magnetic bead slurry with a binding capacity of 7.5 mg / mL (Biolabs, 2020) (New England Biolabs (NEB), Ipswich, MA) was used as the OD 500Counterparts for parallel protein purification of SnoopTag GV with 12. The fluorescence intensity of the His6-tag::SnoopCatcher::miniSOG lysate supernatant was measured using the aforementioned method to estimate the target protein concentration. Lysate supernatants containing an amount of target protein sufficient to saturate the binding capacity of both SnoopTag-GV (500 μL) and Ni-NTA magnetic beads (700 μL) were loaded into each sample accordingly. For Ni-NTA magnetic beads, the provided protocol was followed, including a 1-hour incubation at 4 °C, collection of the flow-through, three washing steps, and three elution steps, and the fluorescence intensity was measured at all steps. By using MilliporeSigma TM Amicon TM Ultra-0.5 centrifugal filter units (MilliporeSigma, Burlington, MA) and a molecular weight cut-off membrane of 10 kDa, the samples from the elution step were buffer-exchanged with 1×PBS. The purified His6-tag::SnoopCatcher::miniSOG was incubated with 30 μL of His6-tagged TEV protease (New England Biolabs (NEB), Ipswich, MA) at 30 °C for 12 hours, and then additional Ni-NTA magnetic bead slurry was added to remove His6-tag::SnoopCatcher and TEV protease.

[0215] For SnoopTag GV, after incubation with the lysate supernatant at 4 °C for 1 hour, the fluorescence intensity and OD of the sample were measured 500 . Subsequently, the samples were centrifuged and washed three times with 1×PBS following the same procedure as described in the previous section. The TEV protease procedure for GV with bound His6-tag::SnoopCatcher::miniSOG was the same as in Ni-NTA magnetic bead purification. Fluorescence intensity and OD 500 measurements were obtained after each procedure. Samples collected from each step of the two purification methods were subjected to SDS-PAGE and analyzed as described in the previous section.

[0216] ix Co-lysis of SnoopTag GV and SnoopCatcher::sfGFP E. coli cultures

[0217] Two cultures, each with a volume of 50 mL, were grown and harvested according to the same procedure as described previously. The GV culture was centrifuged at 400×g for 6 hours, and then the middle layer between the buoyant cells and the pellet cells was removed, resulting in a final volume of 10 mL. The SnoopCatcher::sfGFP culture was centrifuged at 3000×g for 15 minutes, then the supernatant was discarded, and the pelleted cells were transferred to the 10 mL GV culture. The cells from both cultures were co-lysed with 4 mL SoluLyse-Tris (Genlantis, San Diego, CA), 90 U / mL endonuclease containing 1 mM Mg 2+ of (MilliporeSigma, Burlington, MA), 0.5 mg / mL lysozyme (MilliporeSigma, Burlington, MA), and 100 μL Halt TM Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). After incubation at 37 °C for 30 minutes, the whole lysed culture was centrifuged at 400×g for 10 minutes to separate the GV with the bound target protein, and then the underlying liquid and inclusion bodies were removed. The GV was resuspended in 1×PBS and subjected to three buoyant separation washes as before. The TEV protease procedure was also carried out as shown in the previous section, along with the fluorescence intensity and OD 500 . Samples after the TEV protease procedure were collected and subjected to SDS-PAGE and Western blotting. His6-tag monoclonal antibody (HIS.H8), HRP (Thermo Fisher Scientific, Waltham, MA) was used to label proteins in Western blotting; and the bands were visualized by applying Pierce TM ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA).

[0218] x Time-lapse photography for GV buoyant flotation velocity analysis

[0219] Two initial OD 500 = 46 SnoopTag GV samples were prepared in 1×PBS with a total volume of 1 mL. One GV sample was loaded with 96 μL of 32 mg / mL His6-tag::SnoopCatcher::sfGFP, while the other sample was loaded with 96 μL of 1×PBS. The two samples were incubated with rotation at room temperature for 1 hour and then transferred to a transparent In a 14 mL round-bottom polystyrene test tube (Corning Inc., Corning, NY), and filled with 1×PBS to reach a final volume of 12 mL. Together with another test tube containing 12 mL of 1×PBS, the samples were placed in a self-made 3D printed holder. For unclustered GVs, sample photos were taken every 20 seconds, and for clustered GVs, sample photos were taken every 30 seconds, continuing until the end of the photography period. The photos of the samples were converted to 8-bit, and the grayscale of the selected region of interest (ROI) was analyzed using ImageJ.

[0220] xi Quantification and statistical analysis

[0221] The statistical information of the experiments, including the sample size (n) and P values, can be located in the figures, figure captions, and method sections. Statistical analysis was performed using GraphPad Prism software and presented as mean ± standard deviation (STD). For all multiple comparisons, Welch and Brown-Forsythe ANOVA tests were performed, which are a form of one-way ANOVA that does not assume that all groups are from populations with equal variances. p < 0.05 was set as the significant difference.

[0222] References

[0223] The following references, to some extent, provide supplements to the exemplary procedures or other details described herein and are specifically incorporated herein by reference:

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Claims

1. A method for purifying a target protein from a sample, comprising: (A) obtaining a collection of labeled gas vesicle compositions, wherein the gas vesicles comprise one or more proteins on the surface of the gas vesicles, and wherein the one or more proteins have been modified with one or more affinity tags to form the labeled gas vesicle compositions; and (B) contacting the collection of labeled gas vesicle compositions with a sample comprising the target protein for a first period of time; wherein the first period of time is sufficient for the labeled gas vesicle compositions and the target protein to bind to form a captured target protein-gas vesicle composition; and (C) allowing the components of the sample comprising the captured target protein-gas vesicle composition to equilibrate for a second period of time; and (D) separating the captured target protein-gas vesicle composition from the sample mixture.

2. The method of claim 1, wherein the captured target protein-gas vesicle composition forms a floating layer in the sample mixture.

3. The method of claim 2, wherein the floating layer forms during the first period of time or the second period of time.

4. The method according to any one of claims 1 to 3, wherein the target protein is on the surface of a cell type in the sample mixture.

5. The method of claim 4, wherein the cell is a lymphocyte.

6. The method of claim 4 or claim 5, wherein the cell is a T cell.

7. The method according to any one of claims 4 to 6, wherein the cell density of the captured target protein-gas vesicle composition is lower than the cell density of unbound cells.

8. The method according to any one of claims 4 to 7, wherein the cell density of the captured target protein-biovescence composition is about 0.01 g / cm lower than the cell density of the unbound cells 3 to about 0.04 g / cm 3 .

9. The method according to any one of claims 4 to 8, wherein the cell density of the captured target protein-biovescence composition is about 0.04 g / cm lower than the cell density of the unbound cells 3 .

10. The method according to any one of claims 4 to 9, wherein the cells are derived from a patient.

11. The method according to any one of claims 1 to 10, wherein the target protein is present on a single cell type in the sample mixture.

12. The method according to any one of claims 1 to 10, wherein the target protein is present on two or more cell types in the sample mixture.

13. The method according to any one of claims 1 to 12, wherein the collection of labeled gas vesicle compositions comprises two different sets of labeled gas vesicle compositions; wherein (i) the first set of labeled gas vesicle compositions comprises a first affinity tag; (ii) the second set of labeled gas vesicle compositions comprises a second affinity tag; and wherein the first affinity tag and the second affinity tag are complementary.

14. The method of claim 13, wherein the labeled gas vesicle compositions further comprise cell-specific markers.

15. The method of claim 14, wherein the cell-specific marker is linked to an affinity tag that is the same as the first affinity tag of the first set of labeled gas vesicle compositions.

16. The method according to any one of claims 1 to 15, wherein contacting the labeled gas vesicle compositions with the sample mixture is carried out in two steps, wherein (i) the first step comprises contacting the first set of labeled gas vesicle compositions with the sample mixture; and (ii) The second step comprises contacting the second group of labeled air vesicle compositions with the sample mixture.

17. The method according to claim 16, wherein the second group of labeled air vesicle compositions is present in excess relative to the first group of labeled air vesicle compositions.

18. The method according to any one of claims 1 to 17, wherein at least one of the affinity tags is a peptide or a protein.

19. The method according to claim 18, wherein at least one of the affinity tags is a peptide.

20. The method according to claim 19, wherein at least one of the affinity tags is a protein.

21. The method according to any one of claims 1 to 20, wherein at least one of the affinity tags is an antibody.

22. The method according to claim 21, wherein the antibody is an antibody against the target protein.

23. The method according to claim 22, wherein the target protein is expressed on the cell surface.

24. The method according to any one of claims 21 to 23, wherein the antibody is an anti-CD3 antibody.

25. The method according to any one of claims 21 to 23, wherein the antibody is an anti-CD28 antibody.

26. The method according to any one of claims 1 to 25, wherein at least one of the affinity tags is streptavidin.

27. The method according to any one of claims 1 to 26, wherein at least one of the affinity tags is biotin.

28. The method according to any one of claims 1 to 27, wherein at least one of the affinity tags is a SnoopTag.

29. The method according to any one of claims 1 to 28, wherein at least one of the affinity tags is a SnoopCatcher.

30. The method according to any one of claims 1 to 29, wherein at least one of the affinity tags is a SpyTag.

31. The method according to any one of claims 1 to 30, wherein the labeled air vesicle composition and the target protein bind to form a covalent bond.

32. The method according to any one of claims 1 to 30, wherein the labeled air vesicle composition and the target protein bind to form a non-covalent bond.

33. The method according to any one of claims 1 to 32, wherein the first time period is from about 15 minutes to about 5 hours.

34. The method according to claim 33, wherein the first time period is about 1 hour.

35. The method according to any one of claims 1 to 34, wherein the second time period is from about 15 minutes to about 5 hours.

36. The method according to claim 35, wherein the second time period is about 1 hour or about 2 hours.

37. The method according to claim 35, wherein the second time period is about 5 hours.

38. The method according to any one of claims 1 to 37, wherein the method does not include centrifugation.

39. The method according to any one of claims 1 to 37, wherein separating the captured target protein-air vesicle composition further includes centrifugation.

40. The method according to claim 39, wherein centrifugation forms a concentrated layer of the captured target protein - air vesicle composition.

41. The method according to claim 39 or claim 40, wherein the centrifugation is conventional centrifugation.

42. The method according to claim 39 or claim 40, wherein the centrifugation is density gradient centrifugation.

43. The method according to any one of claims 39 to 42, wherein the centrifugation is at 400×g.

44. The method according to any one of claims 1 to 43, wherein separating the captured target protein - air vesicle composition further comprises elutriation.

45. The method according to any one of claims 1 to 44, wherein the method further comprises separating the target protein from the captured target protein - air vesicle composition after separation from the sample mixture.

46. The method according to claim 45, wherein the separation is carried out by cleavage with a protease.

47. The method according to claim 46, wherein the protease is TEV protease.

48. The method according to claim 45, wherein the separation is carried out by intein self - splicing.

49. The method according to claim 48, wherein the intein is the ΔI - CM mini - intein.

50. The method according to claim 49, wherein the intein self - splices upon a change in pH.

51. The method according to claim 50, wherein the intein self - splices when the pH changes from about pH 4 to about pH 7.

52. The method according to claim 49, wherein the intein self - splices upon a change in temperature.

53. The method according to any one of claims 1 to 52, wherein the method further comprises detecting the presence of the target protein.

54. The method according to claim 53, wherein the detection is detecting the presence of cells.

55. The method according to any one of claims 1 to 54, wherein the volume of the sample containing the target protein is about 10 L or greater.

56. The method according to any one of claims 1 to 55, wherein the volume of the sample containing the target protein is about 100 L or greater.

57. The method according to any one of claims 1 to 56, wherein the volume of the sample containing the target protein is about 1000 L or greater.

58. The method according to any one of claims 1 to 57, wherein the volume of the sample containing the target protein is about 10,000 L.

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