Fusion proteins containing proteins with phase behavior

Abstract

Provided herein is a fusion protein comprising a first polypeptide and a second polypeptide, the second polypeptide having a phase behavior. The first polypeptide can be a polypeptide having a desired biological activity, such as a polypeptide of therapeutic, cosmetic, and / or industrial importance. The fusion of the first polypeptide to a polypeptide having a phase behavior can also facilitate its purification and help stabilize the first polypeptide during expression, storage, or after exposure to various conditions known to unfold, degrade, or misfold proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 151,524, filed February 19, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] Instructions for electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Copy of Sequence Listing in Computer Readable Format (Filename: ISOL_006_00US_SeqList_ST25.txt, Recorded: February 19, 2021, File Size: 1 MB).

[0003] The present disclosure relates generally to compositions and methods for the purification of biologics. More specifically, the disclosure relates to purification matrices comprising adeno-associated virus binding polypeptides and methods of using the same. [Background technology]

[0004] The use of biologics in medicine and other fields is rapidly increasing. Biologics are often characterized by high affinity and specificity for a given target, as well as low toxicity and biodegradability. However, their production and purification can be very challenging. Biologics, including therapeutic enzymes, antibodies, gene delivery vectors, signaling molecules, hormones, and other proteins, are typically produced recombinantly in bacterial, yeast, or mammalian host cells. To meet acceptable purity standards (e.g., standards set by the FDA or other regulatory agencies), numerous downstream purification steps are required. Host cell proteins, nucleic acids, endotoxins, and viruses are often the main contaminants that must be removed from biological products.

[0005] In addition, biologics must maintain their structure to remain active. Several factors can cause loss of activity in biologics, including salt, temperature, lyophilization, oxidative conditions, pH, light, agitation, orientation of the biologic during storage, freeze-thawing, and the tendency of the biologic to aggregate.

[0006] There is a need in the art for improved compositions and methods for the production, purification, and stabilization of biologics. Summary of the Invention [Means for solving the problem]

[0007] In some embodiments, provided herein is a fusion protein comprising a first polypeptide and a second polypeptide, the second polypeptide having a phase behavior. In some embodiments, the first polypeptide comprises (i) an enzyme or a derivative or catalytic fragment thereof; (ii) an antibody or a derivative or an antigen-binding fragment thereof; (iii) a signaling molecule or a fragment or derivative thereof; (iv) a structural protein or a fragment or derivative thereof; or (v) a hormone or a fragment or derivative thereof.

[0008] In some embodiments, provided herein are methods of performing a multi-step enzymatic process on a substrate, the method comprising the steps of: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that enables the first enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate; and (iv) applying a second environmental factor that enables the second enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate.

[0009] In some embodiments, provided herein are methods of contacting, isolating, and / or purifying a substrate, the methods including the steps of: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that enables the first enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate; and (iv) applying a second environmental factor that enables the second enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate.

[0010] In some embodiments, provided herein is a method for purifying a first polypeptide, the method comprising the steps of: (i) providing a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior; (ii) applying a first environmental agent to the fusion protein; (iii) separating the fusion protein aggregates from at least one contaminant based on size and / or density; and (iv) applying a second environmental agent to disaggregate the fusion protein.

[0011] In some embodiments, provided herein are methods of performing a multi-step enzymatic process on a substrate, the method comprising the steps of: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that enables the first enzyme to contact the substrate; and (iv) applying a second environmental factor that enables the second enzyme to contact the substrate.

[0012] In some embodiments, provided herein are methods of improving the yield of a first polypeptide, the methods comprising: (i) expressing a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior; and (ii) separating the first polypeptide from the second polypeptide, wherein the yield of the first polypeptide is improved when expressed as a fusion protein compared to the yield of the first polypeptide when not expressed as a fusion protein.

[0013] In some embodiments, provided herein is a method of substantially preventing loss of activity of a first polypeptide following exposure to one or more conditions known to unfold, degrade, and / or misfold the first polypeptide, the method comprising expressing a fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide is a polypeptide having phase behavior.

[0014] In some embodiments, provided herein is a method of substantially preventing unfolding, degradation, and / or misfolding of a first polypeptide, the method comprising expressing a fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide is a polypeptide having phase behavior.

[0015] In some embodiments, provided herein are methods of stabilizing a first polypeptide, the method comprising expressing a fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide is a polypeptide having a phase behavior such that when the fusion protein is exposed to one or more conditions that destabilize the first polypeptide, the first polypeptide substantially retains its activity.

[0016] In embodiments, the method includes removing the fusion protein from the conditions and cleaving the first polypeptide from the second polypeptide, wherein the first polypeptide retains its activity relative to a control first polypeptide not exposed to the conditions.

[0017] In embodiments, the one or more conditions that unfold, degrade, misfold, or destabilize the first polypeptide include: exposure to an oxidizing agent, lyophilization, exposure to a non-physiological pH, exposure to a chaotropic agent, exposure to a temperature of at least 50° C., exposure to an organic solvent, exposure to urea, exposure to a detergent, exposure to autoclaving, freeze-thaw cycles, heat shock, or a combination thereof.

[0018] In embodiments, the one or more conditions that unfold, degrade, misfold, or destabilize the first polypeptide include exposure to a non-physiological pH. In embodiments, the exposure to a non-physiological pH is exposure to an acid. In embodiments, the acid is guanidine hydrochloride. In embodiments, the exposure to a non-physiological pH is exposure to a base. In embodiments, the base is sodium hydroxide or urea.

[0019] In an embodiment, the one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide are exposure to guanidine hydrochloride, exposure to urea, lyophilization, freeze-thaw cycles, autoclaving, exposure to sodium hydroxide, or exposure to a temperature of at least 90° C. In an embodiment, the one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide are exposure to 0.1 M NaOH. In an embodiment, the one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide are exposure to 0.1 M NaOH for 30 minutes. In an embodiment, the one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide are exposure to 6 M guanidine hydrochloride. In embodiments, the one or more conditions known to unfold, degrade, or destabilize the first polypeptide is exposure to 6M guanidine hydrochloride for 30 minutes. In embodiments, the one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide is heating to at least 95° C. In embodiments, the condition is heat shock, which comprises: heating a fusion protein comprising the first polypeptide at 95° C. for 30 minutes; placing a container containing the fusion protein on ice, followed by allowing the fusion protein to return to room temperature.

[0020] In embodiments, the fusion protein comprising the first polypeptide is exposed to one or more conditions for about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In embodiments, the exposure to the one or more conditions is for at least about 30 minutes. In embodiments, exposure to the one or more conditions occurs for about 30 minutes to about 12 hours, about 30 minutes to about 11 hours, about 30 minutes to about 10 hours, about 30 minutes to about 9 hours, about 30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30 minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, or about 30 minutes to about 1 hour.

[0021] In embodiments, the activity of the first polypeptide is its affinity for a binding partner of the first polypeptide. In embodiments, the first polypeptide is an enzyme and its activity is k cat It is.

[0022] In embodiments, the first polypeptide loses less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% of its activity after exposure to one or more conditions compared to a control, the control not being exposed to conditions known to unfold, degrade, or destabilize the first polypeptide. In embodiments, the first polypeptide retains 65%-100% of its activity after exposure to one or more conditions compared to a control. In embodiments, the first polypeptide retains at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of its activity after exposure to one or more conditions compared to a control. In embodiments, the first polypeptide loses less than 20% or less than 25% of its activity after exposure to one or more conditions compared to a control, the control being not exposed to conditions known to unfold, degrade, or destabilize the first polypeptide. In embodiments, the first polypeptide retains at least 80% of its activity after exposure to one or more conditions compared to a control. In embodiments, the first polypeptide retains its activity for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months at 4°C. In embodiments, the first polypeptide retains its activity at -20°C for about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years.

[0023] In embodiments, the yield of the first polypeptide is greater than 15 mg per liter, greater than 30 mg per liter, greater than 50 mg per liter, greater than 75 mg per liter, greater than 100 mg per liter, greater than 200 mg per liter, or greater than 300 mg per liter of host cell suspension.In embodiments, the yield of the first polypeptide in the fusion protein is at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 440%, at least about 460%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 610%, at least about 620%, at least about 630%, at least about 640%, at least about 650%, at least about 660%, at least about 670%, at least about 680%, at least about 690%, at least about 700%, at least about 710%, at least about 720%, at least about 750%, at least about 750%, at least about 760%, at least about 770%, at least about 780%, at least about 790%, at least about 800%, at least about 820%, at least about 840%, at least about 850%, at least about 860%, at least about 870%, at least about 880%, at least about 890%, at least about 900%, at least about 910%, at least about 920%, at least about 930%, at least about 940%, at least about 450%, at least about 475%, at least about 500%, at least about 525%, at least about 550%, at least about 575%, at least about 600%, at least about 625%, at least about 650%, at least about 675%, at least about 700%, at least about 725%, at least about 750%, at least about 775%, at least about 800%, at least about 825%, at least about 850%, at least about 875%, at least about 900%, at least about 925%, at least about 950%, at least about 975%, at least about 1, 000%, at least about 1,100%, at least about 1,125%, at least about 1,150%, at least about 1,175%, at least about 1,200%, at least about 1,225%, at least about 1,250%, at least about 1,275%, at least about 1,300%, at least about 1,325%, at least about 1,350%, at least about 1,375%, at least about 1,400%, at least about 1,425%, at least about 1,450%, at least about 1,475%, at least about 1,500%, at least about 1,525%, at least about 1,550%, at least about 1,575%, at least about 1,600%, at least about 1,625%, at least about 1,650%, at least about 1,675%, at least about 1,700%, at least about 1,725%, at least about 1,750%, at least about 1,775%, at least about 1,800%, at least about 1,825%, at least about 1,850%, at least about 1,875%, at least about 1,900%, at least about 1,925%, at least about 1,950%, at least about 1,975%, or at least about 2,000% higher.In embodiments, the yield of the first polypeptide is greater than 75 mg per liter, hi embodiments, the yield of the first polypeptide is about 300% greater than the yield of the first polypeptide not expressed as a fusion protein.

[0024] In embodiments, provided herein is a method of performing an enzymatic process on a nucleic acid substrate, the method comprising: (i) providing a first fusion protein comprising a first enzyme and a first polypeptide having phase behavior; and (ii) applying a first environmental factor that allows the first enzyme to contact the substrate. In some embodiments, the method comprises: (iii) providing a second fusion protein comprising a second enzyme and a second polypeptide having phase behavior; and (iv) applying a second environmental factor that allows the second enzyme to contact the substrate. In embodiments, the method comprises: (v) applying a third environmental factor to separate the first enzyme from the substrate; and (vi) applying a fourth environmental factor to separate the second enzyme from the substrate. In embodiments, the first enzyme, the second enzyme, or both comprise a nucleic acid binding protein (NBP).

[0025] These and other embodiments are further described in the detailed description, examples, and claims that follow. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a graph showing the percent activity of the fusion protein after treatment with various conditions known to unfold, degrade, or misfold proteins, including lyophilization, exposure to 0.1 M sodium hydroxide (NaOH), 6 M guanidine hydrochloride (GuHCl), and heating to 95° C. [Diagram 2] FIG. 2 shows the percent activity of the fusion proteins after lyophilization and resuspension in PBS (untreated) or after lyophilization, autoclaving, and resuspension in PBS (autoclaved). [Diagram 3]Figure 3 shows the percent loss of fusion protein after exposure to 0.1 M sodium hydroxide, heating to 95°C, and heating to 95°C at low pH (i.e., pH 4). After treatment, the fusion protein from each exposure group was neutralized and / or cooled, centrifuged to remove aggregates, and assayed for total protein using a commercial ELISA. Less than 2% of the starting protein material was lost in each group. Data represent the mean and standard error (n=3). [Figure 4A] , [Figure 4B] , [Figure 4C] Figures 4A-4C show that a fusion protein comprising the PKD2 domain of the AAV receptor (AAVR) and a polypeptide having phase behavior has superior expression to the expression of the PKD2 domain of the AAVR in the absence of the polypeptide having phase behavior. Figure 4A shows the concentration of PKD2 and purified PKD2-containing fusion protein per liter. Figure 4B shows the amount of PKD2 and purified PKD2-containing fusion protein per liter. Figure 4C shows the expression of PKD2 and PKD2-containing fusion protein on a gel. [Diagram 5] FIG. 5 shows that a fusion protein comprising a polypeptide having the CR3 domain and phase behavior of the LDL receptor (LDLR) retains its ability to capture lentivirus after exposure to conditions known to degrade, aggregate, or inactivate polypeptides (e.g., 95° C., 0.1 M NaOH incubation, or 6 M GuHCl incubation). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" refers to a single protein or a mixture of such proteins, reference to a "method" includes references to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0028] As used herein, the term "about" or "approximately" preceding a numerical value refers to the value plus or minus a range of 10%. For example, "about 100" includes both 90 and 110.

[0029] Also, as used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0030] Unless the context dictates otherwise, it is expressly intended that the various features described herein can be used in any combination.

[0031] Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. To further explain, for example, if the specification indicates that a particular amino acid may be selected from A, G, I, L, and / or V, this language also indicates that the amino acid may be selected from any subset of these amino acid(s), such as A, G, I, or L; A, G, I, or V; A or G; L only, etc., as if each such subcombination were expressly described herein. Furthermore, such language also indicates that one or more of the specified amino acids may be excluded. For example, in certain embodiments, the amino acid is not A, G, or I; is not A; is not G or V; etc., as if each such possible exclusion were expressly described herein.

[0032] "Adeno-associated virus" (AAV) is a small, replication-deficient parvovirus. As used herein, AAV may refer to wild-type or mutant AAV of any one of the following serotypes: AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAVAnc80, AAVPHP.B, and other AAVs now known or hereafter discovered. In some embodiments, the AAV can have a single-stranded genome or a double-stranded genome (e.g., a self-complementary AAV).

[0033] An "AAV particle" typically comprises a capsid and a nucleic acid (e.g., a nucleic acid containing a transgene) encapsidated by the protein capsid. A "capsid" is a near-spherical protein shell that comprises individual "capsid protein subunits" or "capsid proteins" (e.g., about 60 capsid protein subunits) assembled and arranged with T=1 icosahedral symmetry. Thus, the capsid of the AAV vector described herein comprises multiple capsid proteins. When an AAV particle is described as comprising a capsid protein, it will be understood that the AAV particle comprises a capsid, which in turn comprises one or more AAV capsid proteins. When an AAV particle is described as binding to a binding domain, it will be understood that the binding domain can bind to one or more capsid proteins within the capsid. The term "empty AAV particle" or "empty capsid" refers to an AAV particle or capsid that does not contain any vector genome or nucleic acid, including an expression cassette or transgene.

[0034] As used herein, the term "AAV sample", which is used interchangeably herein with "AAV composition", refers to a composition that contains AAV particles. In some embodiments, "AAV sample" refers to a composition that contains one or more serotypes of AAV. For example, "AAV8 sample" refers to a composition that contains AAV8 particles.

[0035] A "virus particle" typically includes a protein shell (eg, a capsid or envelope) and nucleic acid contained therein (eg, a nucleic acid containing a transgene).

[0036] As used herein, the term "fragment" referring to a polypeptide includes truncated forms of the polypeptide. The fragment has substantially the same activity as the full-length protein or polypeptide. For example, a fragment of a nucleic acid binding protein refers to a truncated form of the nucleic acid binding protein that substantially retains its binding affinity to nucleic acid. As a further example, a fragment may contain about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% of the amino acids of the full-length protein. As used herein, the term "substantially" with respect to activity such as binding affinity refers to a truncated form that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as active (e.g., binding affinity) as the full-length protein or polypeptide.

[0037] As used herein, the terms "contaminant" and "impurity" are used interchangeably. A contaminant may refer to any substance that is undesirable in a purified composition. In some embodiments, a contaminant is any substance other than the biologic that is desired to be purified. Non-limiting examples of contaminants include, but are not limited to, solvents, proteins, peptides, carbohydrates, nucleic acids, viruses, cells (e.g., bacterial, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides. In some embodiments, a contaminant is an endotoxin or a mycotoxin.

[0038] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up the sequence of a protein. The term "peptide" may refer to a short chain of amino acids, including, for example, natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. Proteins and peptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0039] A "polynucleotide" is a sequence of nucleotide bases, which may be RNA, DNA, or a DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides). In some embodiments, a polynucleotide is a single- or double-stranded DNA sequence.

[0040] As used herein, "isolated" or "purified" (or grammatical equivalents) a viral particle means that the viral particle has been at least partially separated from at least some of the other components in the starting material (e.g., a cell lysate) that contains the viral particle. In representative embodiments, the "isolated" or "purified" viral particle is enriched at least about 10-fold, about 100-fold, about 1,000-fold, about 10,000-fold, or more, as compared to the starting material.

[0041] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Naturally occurring levorotatory (L-) amino acids are shown in Table 1.

[0042] [Table 1]

[0043] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 2) and / or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0044] [Table 2]

[0045] [Table 3]

[0046] Furthermore, a non-naturally occurring amino acid may be a "non-natural" amino acid.

[0047] As used herein, the term "environmental factor" refers to any factor that, when applied to a composition comprising a protein-based purification matrix, changes one or more properties of the composition. Non-limiting examples of environmental factors include changes in one or more of temperature, pH, salt concentration, purification matrix concentration, biological agent concentration, or pressure; addition of one or more of detergents, cofactors, vitamins, molecular crowding agents, denaturants, reducing agents, or oxidizing agents; or application of electromagnetic waves.

[0048] As used herein, the term "polypeptide having phase behavior" refers to any polypeptide that can undergo a phase transition. In some embodiments, the polypeptide undergoes a phase transition upon application of an environmental factor. Examples of polypeptides having phase behavior include elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs).

[0049] As used herein, the term "fusion protein" refers to a polypeptide produced when two heterologous nucleotide sequences, or fragments thereof, encoding two (or more) different polypeptides that are not found fused together in nature are fused to each other in the correct translation reading frame.

[0050] The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses complete polyclonal or monoclonal antibodies and antigen-binding fragments thereof. For example, a natural immunoglobulin molecule is composed of two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides associates with a light chain polypeptide through an interchain disulfide bond between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein composed of two heterologous polypeptide chains). The two heterodimeric proteins then associate through an additional interchain disulfide bond between the heavy chain polypeptides to form an Ig molecule. The term "antibody" also includes multispecific antibodies (e.g., bispecific antibodies).

[0051] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one complementarity determining region (CDR) of an immunoglobulin heavy and / or light chain that binds to at least one epitope of an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein may contain one, two, three, four, five, or all six CDRs of the variable heavy (VH) and variable light (VL) chain sequences of the antibody that specifically bind to a target molecule. Antigen-binding fragments include proteins that contain a portion of a full-length antibody, generally an antigen-binding region or variable region thereof, such as Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single domain antibodies (dAbs), single chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, and other modified configurations of immunoglobulin molecules that contain an antigen-binding site or fragment of the required specificity.

[0052] As used herein, the term "complementarity determining region" or "CDR" refers to an immunoglobulin (antibody) molecule. There are three CDRs per variable domain: CDR1, CDR2, and CDR3 in the variable domain of the light chain, and CDR1, CDR2, and CDR3 in the variable domain of the heavy chain.

[0053] The term "F(ab')2" refers to a protein fragment of IgG generated by proteolytic cleavage with the enzyme pepsin. Each F(ab')2 fragment is a bivalent antigen-binding fragment because it contains two F(ab') fragments linked by disulfide bonds in the hinge region. The term "Fab'" refers to a fragment derived from F(ab')2 and may contain a small portion of Fc. Each Fab' fragment is a monovalent antigen-binding fragment.

[0054] The term "F(ab)" refers to the two protein fragments resulting from the proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) contains a covalent heterodimer of a VH and VL chain and contains a complete antigen-binding site. Each F(ab) is a monovalent antigen-binding fragment.

[0055] "Fv fragment" refers to a non-covalent VH::VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule, but lacks the CH1 and CL domains contained within the Fab. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0056] The binding affinity (Ka) is 1 / M or M -1Affinity refers to the equilibrium association of a particular interaction, expressed in units of M. Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, in units of M. Affinity can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0057] Fusion proteins In some embodiments, the present disclosure provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide has a phase behavior. In some embodiments, the first polypeptide is an enzyme. In some embodiments, the first polypeptide is any polypeptide that has therapeutic, cosmetic, or industrial purposes. Also provided herein are methods for stabilizing, purifying, and producing the first polypeptide.

[0058] First Polypeptide In some embodiments, the present disclosure provides a fusion protein comprising a first polypeptide. The first polypeptide can be, for example, any polypeptide having therapeutic, cosmetic, or industrial purpose. In some embodiments, the first polypeptide is (i) an enzyme or a catalytic fragment thereof; (ii) an antibody or an antigen-binding fragment thereof; (iii) a signaling molecule or a fragment thereof; (iv) a structural protein or a fragment thereof; (v) a hormone; (vi) a nucleic acid binding protein (NBP) or a fragment thereof; (vii) a therapeutic drug or a fragment thereof; (viii) a carrier protein or a fragment thereof; (ix) a cytokine or a fragment thereof; or (x) a toxin or a fragment thereof.

[0059] In some embodiments, the first polypeptide is a carrier protein. In embodiments, the carrier protein is selected from the group consisting of: human transcription factor TAF12 (TAF12), ketosteroid isomerase (KSI), maltose binding protein (MBP), β-galactosidase (β-Gal), glutathione-S-transferase (GST), thioredoxin (Trx), chitin binding domain (CBD), BMP-2 mutant (BMPM), SUMO, CAT, TrpE, staphylococcal protein A, streptococcal protein, starch binding protein, cellulose of endoglucanase A. The binding domain is selected from the cellulose binding domain of exoglucanase Cex, the biotin binding domain, recA, F1ag, poly(His), poly(Arg), poly(Asp), poly(G1n), poly(Phe), poly(Cys), green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, biotin, anti-biotin, streptavidin, an antibody epitope, albumin, bovine serum albumin, keyhole limpet hemocyanin, and variants and fragments thereof. In an embodiment, the carrier protein is albumin. In an embodiment, the carrier protein is bovine serum albumin.

[0060] In embodiments, the first polypeptide is a therapeutic agent. Non-limiting examples of therapeutic agents include antibodies, cytokines, lepirudin, cetuximab, dornase alfa, denileukin diftitox, etanercept, bivalirudin, leuprolide, alteplase, interferon alfa-n1, darbepoetin alfa, reteplase, epoetin alfa, salmon calcitonin, interferon alfa-n3, pegfilgrastim, sargramostim, secretin, peginterferon alfa-2b, asparaginase, thyrotropin alfa, antihemophilic factor, anakinra, gramicin, leuprolide ... Zin D, intravenous immunoglobulin, anistreplase, insulin (regular), tenecteplase, menotropin, interferon gamma-1b, interferon alfa-2a (recombinant), clotting factor Vila, oprelvekin, palifermin, glucagon (recombinant), aldesleukin, botulinum toxin type B, omalizumab, lutropin alfa, insulin lispro, insulin glargine, collagenase, rasburicase, adalimumab, imiglucerase, abciximab, alpha-1-proteinase inhibitors, pegasparga interferon beta-1a, bovine pegademase, human serum albumin, eptifibatide, iodized serum albumin, infliximab, follitropin beta, vasopressin, interferon beta-1b, hyaluronidase, rituximab, basiliximab, muromonab, digoxin immune Fab (sheep), ibritumomab, daptomycin, tositumomab, pegvisomant, botulinum toxin type A, pancrelipase, streptokinase, alemtuzumab, alglucerase, capromab, laronidase, urofolitol lopin, efalizumab, serum albumin, choriogonadotropin alfa, antithymocyte globulin, filgrastim, coagulation factor IX, becaplermin, agalsidase beta, interferon alfa-2b, oxytocin, enfuvirtide, palivizumab, daclizumab, bevacizumab, arcitumomab, eculizumab, panitumumab, ranibizumab, idursulfase, alglucosidase alfa, exenatide, mecasermin, pramlintide, galsulfase, abatacept, cosyntropin, corticotropin,Insulin aspart, insulin detemir, insulin glulisine, pegaptanib, nesiritide, thymalfasin, defibrotide, natural alpha interferon / multiferon, glatiramer acetate, preotact, teicoplanin, canakinumab, ipilimumab, sulodexide, tocilizumab, teriparatide, pertuzumab, rilonacept, denosumab, liraglutide, golimumab, belatacept, buserelin, velaglucerase alfa, tesamorelin, brentuximab vedotin, taliglucerase alfa, belimumab, aflibercept, asparaginase Erwinia chrysanthemi, ocriplasmin, glucarpidase, teduglutide, raxibacumab, certolizumab pegol, insulin isophane isophane), epoetin zeta, obinutuzumab, fibrinolysin aka plasmin, follitropin alfa, romiplostim, lucinactant, natalizumab, aliskiren, ragweed pollen extract, secukinumab, somatotropin (recombinant), drotrecogin alfa, alefacept, OspA lipoprotein, urokinase, abarelix, sermorelin, aprotinin, gemtuzumab ozogamicin, satumomab pendetide, albiglutide, antithrombin alfa, antithrombin III (human), asfotase alfa alfa), atezolizumab, autologous cultured chondrocytes, beractant, blinatumomab, C1 esterase inhibitor (human), coagulation factor XIIIA subunit (recombinant), conestat alfa, daratumumab, desirudin, dulaglutide, elosulfase alfa, evolocumab, fibrinogen concentrate (human), filgrastim-sndz, gastric intrinsic factor, hepatitis B immunoglobulin, human calcitonin, human Clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin, human Rho(D) immunoglobulin, human Rho(D) immunoglobulin, hyaluronidase (human, recombinant), idarucizumab, immunoglobulin (human), vedolizumab, ustekinumab, turoctocog alfa, tuberculin purified protein derivative, simoctocog alfa,Siltuximab, sebelipase alfa, saclocsidase, ramucirumab, prothrombin complex concentrate, poractant alfa, pembrolizumab, peginterferon beta-1a, ofatumumab, obiltoxaximab, nivolumab, necitumumab, metreleptin, methoxypolyethylene glycol-epoetin beta, mepolizumab, ixekizumab, insulin degludec, insulin (porcine), insulin (bovine), thyroglobulin, anthrax immunoglobulin (human), anti Inhibitor coagulation complex, brodalumab, C1 esterase inhibitor (recombinant), chorionic gonadotropin (human), chorionic gonadotropin (recombinant), coagulation factor X (human), dinutuximab, efmoloctocog alfa, factor IX complex (human), hepatitis A vaccine, human varicella zoster immune globulin, ibritumomab tiuxetan, lenograstim, pegloticase, protamine sulfate, protein S (human), sipuleucel T, somatropin (recombinant), susoctocog alfa, and thrombomodulin alpha.

[0061] In some embodiments, the first polypeptide is a growth factor such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor (IGF1), sonic hedgehog (SHH), bone morphogenetic protein 2 (BMP2), glial cell line-derived neurotrophic factor (GDNF), or noggin.

[0062] In some embodiments, the first polypeptide is a cytokine. In some embodiments, the first polypeptide is a cytokine selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-12), interleukin-12 (IL-13), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-29), interleukin-31 (IL-29), interleukin-32 (IL-29), interleukin-33 (IL-29), interleukin-34 (IL-29), interleukin-35 (IL-29), interleukin-36 (IL-29), interleukin-37 (IL-29), interleukin-38 (IL-29), interleukin-39 (IL-39), interleukin-39 ( ), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), Interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), In some embodiments, the cytokine is an interleukin, such as IL-31, interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38), interleukin-39 (IL-39), or interleukin-40 (IL-40). In some embodiments, the cytokine is tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-g), granulocyte-macrophage colony stimulating factor (GM-CSF), or transforming growth factor beta (TGV-B).

[0063] In some embodiments, the first polypeptide is a receptor or a receptor fragment. For example, the first polypeptide can be a cell surface receptor or its extracellular portion (e.g., extracellular domain). In some embodiments, the receptor is an ion channel-associated receptor, an enzyme-linked receptor, or a G protein-coupled receptor. In some embodiments, the receptor can be a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine-threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.

[0064] In some embodiments, the first polypeptide is an enzyme or a derivative or catalytic fragment thereof. As used herein, the term "enzyme" includes a protein or a derivative or fragment thereof that is capable of catalyzing a chemical change in another substance without itself being altered. Non-limiting examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, hemicellulases, peroxidases, proteases, gluco-amylases, amylases, alkaline / acid phosphatases, isomerases, oxidases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, β-glucosidases, lamarinases, kinases, oxidorectuases, ligases, lysozymes, pentosanases, malanases, glucanases, arabinosidases, hyaluronidases, chondroitinases, dehydrogenases, decarboxylases, kinases, phytases, laccases, and derivatives thereof.

[0065] In some embodiments, the enzyme or a derivative or catalytic fragment thereof is isolated or derived from a bacterium or a fungus, hi some embodiments, the enzyme or a derivative or catalytic fragment thereof is isolated or derived from a mammal.

[0066] In some embodiments, the enzyme or derivative or catalytic fragment thereof is a naturally occurring enzyme. In some embodiments, the enzyme or derivative or catalytic fragment thereof has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations compared to the naturally occurring enzyme. In some embodiments, the enzyme or derivative or catalytic fragment thereof comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to the naturally occurring enzyme.

[0067] Unless otherwise indicated, sequence identity is determined using the National Center for Biotechnology Information (NCBI)'s Basic Local Alignment Search Tool (BLAST®), available at blast.ncbi.nlm.nih.gov / Blast.cgi. In some embodiments, sequence identity is calculated over the entire length of the sequences being compared. In some embodiments, sequence identity is calculated over a 20 amino acid, 50 amino acid, 75 amino acid, 100 amino acid, 250 amino acid, 500 amino acid, 750 amino acid, or 1000 amino acid fragment of each comparison sequence.

[0068] In some embodiments, the first polypeptide is an antibody or a derivative or antigen-binding fragment thereof. In some embodiments, the antibody is selected from the group consisting of rituximab, trastuzumab, retifanlimab, amivantamab, ublituximab, anifrolumab, loncastuximab tesirin, valstilimab, bimekizumab, tralokinumab, evinacumab, stimulimab, aducanumab, teplizumab, dostarlimab, tanezumab, inolimomab, oportuzumab monatox, narsoplimab, ansuvimab, margetuximab, naxitamab, atortivimab, maftivimab, and odesivimab-ebgn, belantamab mafode. Chin, tafasitamab, satralizumab, inebilizumab, sacituzumab govitecan, teprotumumab, isatuximab, eptinezumab, [fam]-trastuzumab deruxtecan, enfortumab vedotin, crizanlizumab, brolucizumab, polatuzumab vedotin, risankizumab, romosozumab, caplacizumab, ravulizumab, emapalumab, cemiplimab, fremanezumab, moxetumomab passudotox, galcanezumab, lanadelumab, mogamuizumab, erenumab, til Drakizumab, Ibalizumab, Burosumab, Durvalumab, Emicizumab, Benralizumab, Ocrelizumab, Guselkumab, Inotuzumab, Ozogamicin, Sarilumab, Dupilumab, Avelumab, Brodalumab, Atezolizumab, Bezlotoxumab, Olaratumab, Reslizumab, Oviltoxaximab, Ikekizumab, Daratumumab, Elotuzumab, Necitumumab, Idarucizumab, Alirocumab, Mepolizumab, Evolocumab, Dinutuximab, Secukinumab, Nivolumab, Blinatumomab, Pembrolizumab, La Musilumab, Vedolizumab, Siltuximab, Obinutuzumab, Ad-trastuzumab emtansine, Raxibacumab, Pertuzumab, Brentuximab vedotin, Belimumab, Ipilimumab, Denosumab, Tocilizumab, Ofatumumab, Canakinumab, Golimumab, Ustekinumab, Certolizumab pegol, Catumaxomab, Eculizumab, Ranibizumab, Panitumumab, Natalizumab, Bevacizumab, Cetuximab, Efalizumab, Omalizumab, Tositumomab-i131, Ibritumomab tiuxetan, Adalimumab,alemtuzumab, gemtuzumab, ozogamicin, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, or a derivative or antigen-binding fragment thereof having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of the aforementioned antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or derivative or antigen-binding fragment thereof has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations compared to any one of the aforementioned antibodies or antigen-binding fragments thereof.

[0069] In some embodiments, the first polypeptide is a signaling molecule or a fragment or derivative thereof. The term "signaling molecule" refers to a protein that causes a cell to undergo a process that involves a defined series of biochemical reactions within the cell. Non-limiting examples of signaling molecules include receptor tyrosine kinases (e.g., G protein-coupled receptors), nuclear hormone receptors, extracellular signal-regulated kinases (ERKs), vaccinia virus (VHR) H1-associated proteins, members of the mitogen-activated protein kinase (MKP) family of phosphatases, interleukins, cytokines, transcriptional activators, or transcription factors. In some embodiments, the signaling molecule or a fragment or derivative thereof is a naturally occurring signaling molecule. In some embodiments, the signaling molecule or fragment or derivative thereof has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more amino acid mutations compared to a naturally occurring signaling molecule or fragment or derivative thereof. In some embodiments, the signaling molecule or fragment or derivative thereof comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to a naturally occurring signaling molecule or fragment or derivative thereof.

[0070] In some embodiments, the first polypeptide is a structural protein or a fragment or derivative thereof. The term "structural protein" refers to a class of non-catalytic proteins that can function as biological structural supports. Proteins can function as biological structural supports by themselves, in combination with other proteins, or as matrices or supports for other materials. Non-limiting examples of structural proteins include spider silk, porin (e.g., outer membrane porin F precursor), keratin, collagen, actin, actinin, aggrecan, biglycan, cadherin, clathrin, decorin, elastin, fibrinogen, fibrin, heparin, laminin, mucin, myelin-associated glycoprotein, myelin basic protein, myosin, spectrin, tropomyosin, troponin, tubulin, vimentin, vitronectin, and recognin. In some embodiments, the structural proteins or fragments or derivatives thereof have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more amino acid mutations compared to a naturally occurring structural protein or fragment or derivative thereof. In some embodiments, the structural protein or fragment or derivative thereof comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to a naturally occurring structural protein or fragment or derivative thereof.

[0071] In some embodiments, the first polypeptide is a hormone or a fragment or derivative thereof. The term "hormone" refers to a chemical released by cells, glands, or organs in one part of an organism that sends a message that affects cells in another part of the organism. Non-limiting examples of hormones include thyrotropin releasing hormone, corticotropin releasing hormone, growth hormone releasing hormone (GHRH), dopamine, somatostatin, vasopressin, growth hormone, thyroid stimulating hormone, adrenocorticotropic hormone (ACTH), follicle stimulating hormone (FSH), melanocyte stimulating hormone (MSH), luteinizing hormone (LH), prolactin, oxytocin, thymopoietin, IGF, THPO, androgens, glucocorticoids, aldosterone, adrenaline, noradrenaline, estrogen, progesterone, prolactin, relaxin, melatonin, calcitonin, PTH, gastrin, ghrelin, histamine, neuropeptide Y, insulin, glucagon, calcitriol, renin, erythropoietin, inhibin, and calcitonin. In some embodiments, the hormone is a growth factor.

[0072] In some embodiments, the first polypeptide is a mammalian polypeptide. The mammalian polypeptide can be a human polypeptide, a goat polypeptide, a rabbit polypeptide, a mouse polypeptide, a rat polypeptide, a primate polypeptide, or a baboon polypeptide.

[0073] In some embodiments, the first polypeptide is a viral polypeptide. Non-limiting examples of viral polypeptides include gag protein, DNA polymerase, protease, capsid protein, envelope polypeptide, fusion polypeptide, and spike protein. In some embodiments, the viral polypeptide is a capsid protein. In some embodiments, the viral polypeptide is an envelope protein. In some embodiments, the viral polypeptide is a spike protein. In some embodiments, the capsid protein is an AAV capsid protein. In some embodiments, the viral polypeptide is a bacteriophage polypeptide, such as T7 polymerase.

[0074] In some embodiments, the first polypeptide is a bacterial polypeptide or a derivative or fragment thereof. The term "bacterial polypeptide" refers to a polypeptide or protein that is naturally produced by bacteria or other microorganisms. In some embodiments, the bacterial polypeptide is botulinum toxin, diphtheria toxin, anthrax toxin, pseudomonas exotoxin A, or shiga toxin. In some embodiments, the bacterial polypeptide is Staphylococcus protein A (SPA) or protein L.

[0075] In some embodiments, the first polypeptide is a toxin. Non-limiting examples of toxins include heat-labile toxins (LT), heat-stable toxins (ST), verotoxins, Shiga-like toxins (Stxs), cytotoxins, endotoxins (e.g., lipopolysaccharide (LPS)), enteroaggregative ST toxins (EAST), Shigella enterotoxin 1 (ShET1), Shigella enterotoxin 2 (ShET2), neurotoxins, cytolethal distending toxins (Cdt), AvrA toxins, cytotoxic necrotizing factors, mouse toxins, cytolethal ... lethal distending toxin, AvrA toxin, toxin complex, cytotoxin necrotizing factor, Yst toxin, heat-stable toxin, Shiga-like toxin II, leukotoxin, enterotoxin, heat-stable-like enterotoxin, exotoxin complex, hemolysin, pore-forming toxin, alpha hemolysin, heat-stable-like toxin, exotoxin complex (ETC), alpha toxin, beta toxin, neurotoxin, C2 toxin, tetanus neurotoxin, epsilon toxin, tetanospasmin, lecithinase, cholera toxin, accessory cholera enterotoxin, RTX toxin, C3 toxin, iota toxin, theta toxin, delta hemolysin, delta hemolysin, gamma hemolysin, Panton-Valentine leukocidin, staphylococcal enterotoxin, toxic shock syndrome toxin-1, Zot toxin (zona occludens toxin), bile toxin, cholera toxin (CTx), paracholera enterotoxin (Ace), RTX toxin, zonula occludens toxin (Zot), Cholix toxin, β-hemolysin / cytolysin, CAMP factor, streptolysin O, streptolysin S, pneumolysin, exotoxin, vacuolating lethal toxin A (VacA), cytolytic toxin, exotoxin A, exotoxin S, exotoxin T, exotoxin U, exotoxin Y, phospholipase C, Cry toxins, anthrax toxin, cytolethal distending toxin A, cytolethal distending toxin B, cytolethal distending toxin C, cholera-like enterotoxin, adenylate cyclase toxin, pertussis toxin, tracheal cytotoxin, dermonecrotic toxin, diphtheria toxin, Bacteroides fragilis toxin, and listeriolysin O. In some embodiments, the toxin is a botulinum toxin, a diphtheria toxin, anthrax toxin, Pseudomonas exotoxin A, or a shiga toxin.

[0076] In some embodiments, the toxin is from a bacteria, such as, for example, Yersinia, Salmonella, Shigella, Escherichia, Enterobacter, Klebsiella, Serratia, Proteus, Citrobacter, Clostridium, Vibrio, Staphylococcus, Streptococcus, Helicobacter, The bacterium is isolated from a bacterium selected from any one of the genera: Bacillus, Pseudomonas, Pasteurella, Bacillus, Campylobacter, Aeromonas, Neiserria, Bordetella, Haemophilus, Chlamydia, Corynebacteria, Bacteroides, Corynebacteria, and Listeria.

[0077] In some embodiments, the first polypeptide is an antigenic polypeptide. The term "antigenic polypeptide" refers to any polypeptide that elicits an immune response in an organism. In some embodiments, the antigenic polypeptide triggers the development of a humoral and / or cellular immune response against the antigenic polypeptide. In some embodiments, the antigenic polypeptide is a component of a vaccine. In some embodiments, the antigenic polypeptide is a hemagglutinin, spike protein, neuraminidase, Hepatitis B surface antigen (HBsAg), a fusion protein, or a capsid protein. In some embodiments, the antigenic polypeptide is a spike protein from SARS-CoV or a spike protein from SARS-CoV-2.

[0078] In some embodiments, the first polypeptide is an enzyme capable of carrying out one or more steps involved in protein synthesis. Non-limiting examples of enzymes involved in protein synthesis include ribosomal proteins, such as proteins encoded by any one of the following genes: RPSA, RPS3, RPS3A, RPS4X, RPS4Y, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS 15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3. RPS14, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL12, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27A, RPL30, RPL31, RPL32, RPL34, RPL25, RPL26, RPL36A, RPL37, RPL39, RPL50, RPP0, RPP1, RPP1. In some embodiments, the first polypeptide is involved in folding a protein, such as a chaperone protein. Non-limiting examples of chaperone proteins include heat shock proteins such as Hsp70 (cpn60, GroEL), Hsp60 (DNAK, BiP), Hsp25, HSP90 (Clp), calnexin, calreticulin, PDI, PPI, alpha-lytic protease, or subtilisin.

[0079] In some embodiments, the first polypeptide is an enzyme capable of performing one or more steps involved in protein modification. For example, the first polypeptide can be a kinase, a phosphatase, a methylase, a glycosyltransferase, an enzyme that adds or removes lipids, a capping enzyme, or a tailing enzyme. In some embodiments, the first polypeptide is involved in post-translational modification, such as an enzyme involved in phosphorylation, glycosylation, S-nitrosylation, methylation, n-acetylation, palmitoylation, n-myristoylation, prenylation, sumoylation, or ubiquitination. In some embodiments, the first polypeptide is involved in phosphorylation, methylation, lipidation, capping, or tailing. In some embodiments, the first polypeptide is a kinase or a phosphorylase. In some embodiments, the enzyme is AMAN1, MGAT / GNT1, AMAN II, MGAT2 / GNT II, ​​MGAT3 / GNT III, MGAT4A / GNT IV, MGAT5 / GNT V, FUT8, B4GALT1, ST3GAL3, ST3GAL1, FUT11, XYLT, POMT1, POMT2, GALNT1, POFUT1, XYLT1, HPAT1, HPAT3, GALT2, SERGT1, or RRA1.

[0080] In some embodiments, the first polypeptide is an enzyme capable of performing one or more steps involved in DNA or RNA synthesis. For example, the first polypeptide can be a polymerase, such as a DNA polymerase or an RNA polymerase. In some embodiments, the first polypeptide can be a helicase.

[0081] In some embodiments, the first polypeptide is an enzyme capable of performing one or more steps involved in DNA or RNA modification. For example, the first polypeptide can be a Cas enzyme, such as Cas9 or Cas12. In some embodiments, the first polypeptide can be a Zn finger nuclease. In some embodiments, the first polypeptide can be a TALEN. In some embodiments, the first polypeptide can be a meganuclease. In some embodiments, the first polypeptide can be a deaminase.

[0082] In some embodiments, the first polypeptide is a nucleic acid binding protein (NBP). In some embodiments, the nucleic acid binding protein is an RNA binding protein (RBP). In some embodiments, the nucleic acid binding protein is a DNA binding protein (DBP). RBPs bind RNA, while DBPs bind DNA. In embodiments, NBPs bind DNA, microRNA, capped RNA, DNA, double-stranded RNA, transfer RNA, ribosomal RNA, small nuclear RNA, regulatory RNA, ribozyme, transfer RNA, or messenger RNA.

[0083] In embodiments, the NBP binds to a polyA tail, a double-stranded RNA, an AU-rich element (ARE), a positively charged intrinsically disordered region (IDR) of a nucleic acid, or an mRNA cap.

[0084] In embodiments, the RBP binds to an AU-rich element. An AU-rich element is referred to herein as an "ARE." ARE refers to an adenylate-uridylate-rich element in the 5' or 3' untranslated region of an mRNA. An ARE contains the core sequence AUUUA (SEQ ID NO:X). AREs are determinants of RNA stability and often occur in the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Proteins that bind to AREs are referred to as ARE-binding proteins (ARE-BPs). In embodiments, ARE-BPs stabilize mRNAs. Non-limiting examples of ARE-BPs include human antigen R (huR, also referred to as "ELAV"), tristetrapolin (TTP), AU-rich element RNA-binding protein (AUF), and fragile X mental retardation syndrome-related protein 1 (FXR1).The following papers describe ARE-BPs and are incorporated by reference in their entirety: Otsuka et al. Front. Genet., 02 May 2019; Brennan and Steitz. Cell Mol Life Sci. 2001 Feb; 58(2): 266-77; Carballo et al. 1998. Science, 281, 1001-1005; Mazan-Mamczarz et al. Oncogene volume 27, pages 6151-6163 (2008); Vasudevan and Steitz. Cell. 2007 Mar 23; 128(6): 1105-18; Curr Cancer Drug Targets. 2019; 19(5): 382-399; J Biol Chem. 2017 Apr 28;292(17):6869-6881.doi:10.1074 / jbc.M116.772947.Epub 2017 Mar 16;Wiley Interdiscip Rev RNA.Jul-Aug 2014;5(4):549-64.doi:10.1002 / wrna.1230;and Elife.2017 Aug 2;6:e26129.doi:10.7554 / eLife.26129;Mazan-Mamczarz et al.2008.Nucleic Acids Research,37,204-214. In embodiments, the NBP binds to an ARE. In embodiments, the NBP that binds to an ARE incorporates a binding element for huR, TTP, AUF, or FXR1.

[0085] In embodiments, the NBP binds to double-stranded RNA. In embodiments, the NBP comprises a dsRNA binding protein (dsRBD) or a fragment thereof. dsRBD is described in the following references, the entire contents of which are incorporated herein by reference: Banerjee et al. RNA Biol. 2014 Oct; 11(10): 1226-1232.

[0086] In embodiments, the NBP binds to a capped mRNA. In embodiments, the NBP that binds to a capped mRNA comprises eukaryotic translation initiation factor 4E (eIF4E), eukaryotic translation initiation factor 3 subunit D (eIF3D), or a combination thereof.

[0087] In embodiments, the NBP binds to the groove of DNA or RNA. Non-limiting examples of nucleic acid binding proteins that bind to the groove of DNA or RNA include the transactivator of transcription (Tat) protein of human immunodeficiency virus-1 (HIV-1), the REV protein of HIV-1, and the RSG-1.2 peptide. The RSG-1.2 peptide is a synthetic peptide that binds to the Rev response element present in the env gene of the HIV-1 genome. The RSG-1.2 peptide is described in the following paper, which is incorporated herein by reference in its entirety: Kumar et al. PLoS One. 2011; 6(8): e23300.

[0088] In an embodiment, the NBP binds to mRNA. In an embodiment, the NBP that binds to mRNA is a ribosomal protein. In an embodiment, the ribosomal protein is a 70S ribosome or an 80S ribosome. In an embodiment, the ribosomal protein is from the 40S or 60S subunit of the 80S ribosome. In an embodiment, the ribosomal protein is from the 30S or 50S subunit of the 70S ribosome. In an embodiment, the ribosomal protein is selected from the group consisting of L3 ribosomal protein, L4 ribosomal protein, L13 ribosomal protein, L20 ribosomal protein, L22 ribosomal protein, L24 ribosomal protein, L24e ribosomal protein, S12 ribosomal protein, S14 ribosomal protein, and eukaryotic initiation factor 4E binding protein 1 (4EBP1).

[0089] In an embodiment, the NBP that binds to the mRNA is part of the spliceosome. In an embodiment, the NBP that is part of the spliceosome is a splicing factor. In an embodiment, the splicing factor is selected from ASF / SF2 splicing factor, serine / arginine-rich splicing factor 4 (SRp75), and serine and arginine-rich splicing factor 1 (SRSF1).

[0090] In an embodiment, the NBP that binds to the mRNA is a protein that is localized in P granules. In an embodiment, the protein that is localized in P granules is selected from the group consisting of LAF-1, MEG-1, and MEG-3. LAF-1, MEG-1, and MEG-3 are described in the following references, which are incorporated herein by reference in their entirety: Leacock et al. Genetics, Volume 178, Issue 1, 1 January 2008, Pages 295-306; Wu et al. Mol Biol Cell. 2019 Feb 1; 30(3): 333-345; Elbaum-Garfinkle et al. Proc Natl Acad Sci USA. 2015 Jun 9; 112(23): 7189-94.

[0091] In embodiments, the NBP that binds to mRNA is a protein that removes or facilitates the removal of the 5' cap of the mRNA, referred to herein as a "decapping protein." In embodiments, the protein that removes or facilitates the removal of the 5' cap of the mRNA is Dcp1, Dcp2, or a combination thereof. Dcp1 and Dcp2 are described in the following references, which are incorporated herein by reference in their entirety: Valkov et al. Nature Structural & Molecular Biology volume 23, pages 574-579 (2016).

[0092] In embodiments, the NBP that binds to mRNA is a component of a processing body (p-body). In embodiments, the component of the p-body is Edc3, DHX9, or Xrn1. The components of p-bodies are described in the following references, which are incorporated herein by reference in their entirety: Luo et al. Biochemistry 2018, 57, 17, 2424-2431.

[0093] In an embodiment, the NBP that binds to mRNA is a stem-loop binding protein (SLBP). SLBP binds to the histone 3' untranslated region (UTR) stem-loop structure of replication-dependent histone mRNA. In an embodiment, the NBP that binds to mRNA is a heterogeneous nuclear ribonucleoprotein (hnRNP). hnRNP is described in the following references, the entire contents of which are incorporated herein by reference: Geuens et al. Hum Genet. 2016; 135: 851-867.

[0094] In an embodiment, the NBP that binds to mRNA is GroEL.

[0095] In an embodiment, the NBP is a protein involved in in vitro transcription.Non-limiting examples of NBPs involved in in vitro transcription include T7 RNA polymerase, RNase inhibitor, 2'-O-methyltransferase, inorganic pyrophosphatase, poly(A) polymerase, DNase I, calf intestinal phosphatase, Antarctic phosphatase, D1 subunit of vaccinia virus mRNA capping enzyme, guanine-7-methyltransferase (present in D1 subunit of vaccinia virus mRNA capping enzyme), guanylyltransferase (present in D1 subunit of vaccinia virus mRNA capping enzyme), RNA triphosphatase (present in D1 subunit of vaccinia virus mRNA capping enzyme), and D12 subunit of vaccinia virus mRNA capping enzyme. The following references describe the aforementioned proteins and are incorporated by reference in their entirety: Dickson et al. Prog Nucleic Acid Res Mol Biol. 2005; 80:349-374; Shuman et al. J Biol Chem. 1980 Dec 10; 255(23):11588-11598; Luo et al. J Virol. 1995 Jun; 69(6):3852-3856; Kobori et al. PNAS November 1, 1984 81(21)6691-6695.

[0096] In embodiments, the NBP is selected from the group consisting of poly(A) binding protein (PABP), eukaryotic translation initiation factor 4E (eIF4E), eukaryotic translation initiation factor 3 subunit D (eIF3D), heterogeneous nuclear ribonucleoprotein (hnRNP), RNA-specific adenosine deaminase 1 (ADAR1), RNA-specific adenosine deaminase 2 (ADAR2), CspB from Bacillus subtilis (Bscscp), Y-box protein 1 cold shock domain (YB1-CSD), Fox-1 protein (FOX1), poly(A) binding protein (PABP), Staufen protein, TIS11d, zinc finger protein (ZNF), Z-DNA binding protein 1 (ZBP1), retinoic acid inducible gene-I (RIG-I)-like protein, Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TL R3), Toll-like receptor 8 (TLR8), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), interferon-inducible protein with tetratricopeptide repeats 1 (IFIT1), protein kinase R (PKR), 2'-5'-oligoadenylate synthetase, oligoadenylate synthase-like (OASL) proteins (e.g., OAS1, OAS2, OAS3, or OASL), ribonuclease E (RNASE E), gamma interferon-inducible protein Ifi-16 (IF116), and cyclic GMP-AMP synthase (cGAS).The following references describe selected aforementioned proteins and are incorporated by reference in their entirety: Kuroyanagi. Cell Mol Life Sci. 2009;66(24):3895-3907; Baou et al. J Biomed Biotechnol. 2009;2009:634520; and Brisse and Ly. Front. Immunol.,17 July 2019;10:1586; Rehwinkel et al. Nature Reviews Immunology volume 20, pages537-551(2020); and Brisse et al. Front Immunol. 2019;10:1586; Luo et al. Cell. 2011 Oct 14;147(2):409-422.

[0097] In embodiments, the NBP comprises one or more RNA binding domains (RBDs) and one or more intrinsically disordered regions (IDRs). In embodiments, the IDRs comprise RG[G] repeats, RS / RG-rich domains, K / R patches, molecular recognition functions, low complexity sequences, pentatricopeptide domains, or combinations thereof.

[0098] In embodiments, the NBP comprises one or more of the following domains: short linear motif (SLiM), RG repeats, RGG repeats, RS / RG-rich domains, K / R basic patches, molecular recognition functions, low complexity sequences, RNA recognition motifs, double-stranded RNA binding domains, K homology domains, zinc finger domains (e.g., CCHH ZF domains, CCCC (Ran-BP2) domains, CCCH ZF domains), RGG domains, Pumillo family domains, pentatricopeptide domains, cold shock domains, helicase domains, La motifs, Piwi-Argonaute-Zwille (PAZ) domains, P-element induced wimpy testis (PIWI), pseudouridine synthase, and archaeosine kinase (PUA). transglycosylate), Pumillo-like repeats (PUM), ribosomal S1-like (S1), Sm and Like-Sm (Sm / Lsm) repeats, thiuridine synthase and RNA methylase and pseudouridine synthase (THUMP), and a domain with YT521-B homology. The following references describe many of these domains and are incorporated by reference in their entirety: Balcerak et al. Open Biol. 2019 Jun;9(6)190096; Jarvelin et al. Cell Commun Signal, 2016:14,9; Corley et al. Mol. Cell. 2020 Apr 2;78(1):9-29; De Franco et al. Sci Rep:2019:9,2484; Shotwell et al. 2020. Wiley Interdiscip Rev RNA, 11, e1573; Simon et al. 2019. Molecular Cell, 75, 66-75.e5; Varadi et al, 2015, PLoS One, 10, e0139731; Zeke et al, 2020, WIREs RNA, n / a, e1714.

[0099] In embodiments, NBPs contain short sequence motifs (SLiMs). SLiMs are composed of up to 10 amino acid residue motifs that are primarily located outside of protein domains. SLiMs bind non-specifically to RNA with low affinity. SLiMs are often repeated multiple times throughout the protein.

[0100] In an embodiment, the NBP comprises an RS / RG rich domain. The RS / RG rich domain comprises an arginine-serine repeat (RS), an arginine-glycine repeat (RG), or a combination thereof. The RS / RG rich domain mediates specific or non-specific interaction with RNA. Examples of proteins that comprise an RS / RG rich domain include SR proteins and SR-like proteins such as serine / arginine-rich splicing factor 1 (SRSF1) and RNA helicase DDX23.

[0101] In an embodiment, the NBP comprises an RG[G] repeat. The RG[G] repeat is known to have extensive degenerate binding. The RG[G] repeat is an arginine and glycine rich motif consisting of at least three RG / RGG repeats (e.g., 3-500) separated by 10 amino acid residues. The RG / RGG motif comprises RGG and / or RG repeats of various lengths interspersed with spacers of different amino acids. In an embodiment, the NBP comprises a di-RGG motif. The di-RGG motif comprises two repeated RGG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a di-RG motif. The di-RG motif comprises two repeated RG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a tri-RGG motif. The tri-RGG motif comprises three repeated RGG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a tri-RGG motif. The tri-RG motif contains three repeated RG sequences separated by 0-4 amino acids. These motifs are described in the following papers, which are incorporated herein by reference in their entirety: Thandapani et al. (2013). Molecular Cell, 50, 613-623.

[0102] In embodiments, the amino acid sequence of the NBP comprises one or more of RG, RGG, RGGR, or RGGGR, or combinations thereof. In embodiments, NBPs comprising RG, RGG, RGGR, RGGGR, or combinations thereof mediate hydrogen bonding and base stacking with DNA and RNA via arginine moieties. In embodiments, NBPs comprising RG, RGG, RGGR, RGGGR, or combinations thereof bind to DNA G-quadruplexes. An exemplary protein comprising repeats of RGG, RGGR, or RGGGR is the RNA binding protein FUS. In embodiments, the NBP comprises FUS. In embodiments, the NBP sequence comprises consecutive repeats of RGG, RGGR, RGGGR, or combinations thereof. An exemplary NBP comprising a combination of RGG, RGGR, or RGGGR repeats may comprise the sequence RGGRGGRGGRRGGRRGGRRGGGRRGG. In embodiments, the NBP may comprise one or more of RGG, RGGR, or RGGGR interspersed throughout its sequence. In embodiments, the NBP comprises 1-100 RG, RGG, RGGR, or RGGGR sequences. The RGG, RGGR, and RGGGR may be interspersed (separated by one or more amino acids) or may be contiguous throughout the sequence. In embodiments, the NBP is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 RG, RGG, RGGR, or RGGGR repeats.The RG, RGG, RGGR, and RGGGR repeats may be contiguous or interspersed throughout the sequence. The following paper describes exemplary RGG sequences and is incorporated by reference in its entirety: Simon et al. Molecular Cell (2019), 75, 66-75.e5.

[0103] In an embodiment, the NBP comprises an RG domain. The RG domain comprises about 2 to about 500 RG (arginine-glycine) repeats. In an embodiment, the NBP comprises an RGG domain. The RGG domain comprises about 2 to about 500 RGG (arginine-glycine-glycine) repeats. In an embodiment, the NBP comprises an RGGR domain. The RGGR domain comprises about 2 to about 500 RGGR (arginine-glycine-glycine-arginine) repeats. In an embodiment, the NBP comprises an RGGGR domain. The RGGGR domain comprises about 2 to about 500 RGG (arginine-glycine-glycine-glycine-arginine) repeats. In an embodiment, the NBP comprises an RG mixed domain. The RG mixed domain comprises 2 to 500 simultaneous repeats of RG, RGG, RGGR, and / or RGGGR. For example, the RG mixed domain may comprise RGG, followed by RG, followed by RGGR, followed by RG, followed by RGGGR.

[0104] In an embodiment, the NBP comprises a K / R basic patch. The K / R basic patch comprises 4-8 consecutive lysines, arginines, or a combination thereof. The K / R basic patch forms a strong positive exposed interface that binds to RNA. K / R basic patches are often found in multiple clusters on the same protein.

[0105] In embodiments, the NBP comprises a molecular recognition function (MoRF). In embodiments, the MoRF is up to 25 amino acids long, 50 or more amino acids long, or 25-50 amino acids long. The MoRF dynamically transitions from disordered to ordered upon ligand binding.

[0106] In an embodiment, the NBP comprises a low complexity (LC) sequence. In an embodiment, the LC sequence comprises up to 100 amino acids and is composed of multiple repeats of the same amino acid or several amino acids. The LC sequence can polymerize into amyloid-like fibers and undergo a reversible phase transition to a hydrogel-like state. Examples of proteins that contain LC sequences include FUS and hnRNPA2.

[0107] In an embodiment, the NBP comprises an RNA recognition motif (RRM). The RRM binds to RNA. Typically, the binding is sequence specific. In an embodiment, the RRM has a length of about 75 to about 125 amino acids, e.g., about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, or about 125 amino acids. In an embodiment, the RRM comprises about 85 amino acids. The RRM typically adopts a β1α1β2β3α2β4 topology that forms two α-helices on an antiparallel β-sheet and accommodates the RNA-binding RNP1 and RNP2 motifs conserved in the central β1 and β3 strands.

[0108] In embodiments, the NBP comprises a double-stranded RNA binding domain (dsRBD). In embodiments, the dsRBD comprises about 55 to about 80 amino acids or about 65 to about 70 amino acids. In embodiments, the dsRBD comprises 68 amino acids. The dsRBD typically adopts an αβββα structure. In embodiments, the dsRBD exists as a tandem repeat or in combination with other RNA binding domains. There are two subclasses of dsRBD, type B and type A. Type A binds better to dsRNA than type B. dsRBD typically binds in a shape-dependent manner rather than sequence-specifically. However, ADAR2 is a rare example of a dsRBD that exhibits sequence-specific binding.

[0109] In an embodiment, the NBP comprises a K homology domain. In an embodiment, the K homology domain comprises 60 to 80 amino acids. In an embodiment, the K homology domain comprises 70 amino acids. There are two types of K homology domains: type I and inverted type II. Type I K homology domains have a β1α1α2β2β'α' topology. Inverted type II K homology domains have an α'β'β1α1α2β2 topology. K homology domains do not use aromatic amino acids for binding, but instead use hydrogen bonds. NBPs containing K homology domains are difficult to design due to their strict sequence specificity.

[0110] In embodiments, the NBP comprises one or more zinc finger (ZF) domains. In embodiments, the NBP comprises 1-100 ZF domains, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 ZF domains. In embodiments, the zinc finger domain is selected from one of the following subtypes: CCHC (zinc knuckle), CCCH, CCCC (RanBP2), and CCHH. C and H refer to interspersed cysteine ​​and histidine residues that coordinate the zinc atom. In embodiments, the zinc finger domain comprises about 20 to about 40 amino acids, e.g., about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, or about 40 amino acids. The CCHH ZF domain comprises two conserved cysteine ​​residues and two conserved histidine residues. The CCHH ZF domain recognizes both structure-specific and sequence-specific elements. To date, there are no engineered versions of the CCHH ZF domain. The CCHH ZF domain binds both single-stranded and double-stranded DNA and RNA. CCCC ZFs may not require a specific RNA conformation for binding. Typically, CCCC ZFs recognize short triplet nucleotide repeats.An engineered version of the CCCC ZF is described in the following reference, the entire contents of which are incorporated herein by reference: De Franco et al. Sci Rep: 2019:9, 2484.

[0111] In embodiments, the NBP comprises pentatricopeptide repeats (PPRs). The PPRs comprise 20-50 amino acids, e.g., about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 amino acids. In embodiments, the PPRs comprise about 35 amino acids. In embodiments, the NBP comprises a PPR that is repeated about 2-30 times within the NBP sequence. In embodiments, the NBP comprises a PPR that is repeated about 10-30 times within the NBP sequence. In embodiments, the PPR may be repeated about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 21 times, about 22 times, about 23 times, about 24 times, about 25 times, about 26 times, about 27 times, about 28 times, about 29 times, or about 30 times in the NBP. In embodiments, the PPR is repeated at least 10 times. The PPR repeats may be contiguous or separated by one or more amino acids. The PPR repeats form two antiparallel α-helices. In embodiments, the NBP containing the PPR forms a solenoid structure. In embodiments, the NBP containing the PPR binds to single-stranded RNA, single-stranded DNA, or mRNA. In embodiments, the NBP containing a PPR binds to the 5' cap of an mRNA. In embodiments, the NBP containing a PPR binds to the 3' poly A tail of an mRNA.

[0112] In embodiments, the NBP comprises a Pumilio homology domain (also called Pumilio-like repeats, abbreviated as "PUF"). The PUF domain comprises eight alpha-helical repeats of a conserved 36 amino acid sequence that form a concave RNA-binding surface. In embodiments, the NBP comprises one to eight alpha-helical repeats of the PUF domain, e.g., one, two, three, four, five, six, seven, or eight alpha-helical repeats. In embodiments, the NBP comprising the PUF binds to a poly-A tail. In embodiments, the NBP comprising the PUF binds to an mRNA. The following papers describe PUF domains and are incorporated herein by reference in their entirety: Zhao et al. Nucleic Acids Res. 2018 May 18;46(9):4771-4782. In embodiments, the NBP comprising the PUF domain binds to the 3' untranslated region of an mRNA.

[0113] In an embodiment, the NBP comprises a cold shock domain (CSD). The CSD comprises five antiparallel β-strands that form a β-barrel structure known as the oligosaccharide / oligonucleotide binding fold. The CSD binds single-stranded RNA and single-stranded DNA. In an embodiment, the CSD is comprised of 60-80 amino acids, e.g., about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 amino acids. In an embodiment, the CSD comprises about 70 amino acids. In an embodiment, the CSD is a bacterial CSD. In an embodiment, the bacterial CSD prefers ssDNA over ssRNA up to 10-fold.

[0114] In an embodiment, the NBP comprises a helicase domain. Helicases include six superfamilies (SFs), including SF1, SF2, SF3, SF4, SF5, and SF6. In an embodiment, the helicase domain is a eukaryotic RNA and DNA helicase from the SF1 or SF2 superfamily. Non-limiting examples of families within the SF1 and SF2 superfamily include the Upf1-like family, the DEAD-box family, the DEAH family, the RIG-I-like family, the Ski2-like family, and the NS3 family. In an embodiment, the helicase domain is a bacterial or viral helicase from the SF3, SF4, SF5, or SF6 superfamily. ATP binding to the helicase promotes a higher affinity of the helicase domain for RNA. ATP hydrolysis promotes a conformational change that causes the helicase to unwind the substrate and / or displace one nucleotide.

[0115] In an embodiment, the NBP comprises an La motif. The La motif consists of five α-helices and three β-strands that form a small antiparallel β-sheet to a modified "winged helix" fold. In an embodiment, the La motif binds to a 3'-terminal UUU-OH element on a polymerase III transcribed small RNA. The La motif comprises 80-100 amino acids, e.g., about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 amino acids. In an embodiment, the La motif comprises about 90 amino acids.

[0116] In embodiments, the NBP comprises a Piwi-Argonautre-Zwille (PAZ) domain. In embodiments, the PAZ domain promotes the binding of small interfering mRNAs and / or microRNA guides to mRNA targets. In embodiments, the PAZ domain is derived from a Dicer protein or an Argonaute protein. The PAZ domain displays a six-stranded beta barrel with two alpha helices on top and a special appendage on the other side that includes a beta hairpin and a short alpha helix.

[0117] In embodiments, the NBP comprises a P-element induced wimpy testis (PIWI) domain. In embodiments, the PIWI domain promotes binding of small interfering mRNAs and / or microRNA guides to their mRNA targets. In embodiments, the PIWI domain is found on the Argonaute protein. The tertiary structure of the PIWI domain forms an RNase H-like fold consisting of a five-stranded beta sheet flanked by alpha helices on both sides.

[0118] In an embodiment, the NBP comprises a PAZ domain and a PIWI domain.

[0119] In embodiments, the NBP comprises a pseudouridine synthase and an archaeosine transglycosylase (PUA) domain. The PUA domain ranges from 67-94 amino acids in length and comprises a β1α1β2β3β4β5α2β6 structure that forms a pseudo-barrel wrapped by two α-helices. In embodiments, the PUA-containing NBP binds to double-stranded RNA.

[0120] In an embodiment, the NBP comprises an S1 RNA binding domain. In an embodiment, the NBP comprising an S1 RNA binding domain interacts with single-stranded RNA, double-stranded RNA, or mRNA. In an embodiment, the S1 RNA binding domain comprises about 60 to about 80 amino acids, for example, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 amino acids. In an embodiment, the S1 RNA binding domain comprises about 70 amino acids.

[0121] In an embodiment, the NBP comprises an Sm RNA-binding motif. The Sm RNA-binding motif is found in eukaryotic and archaeal Sm and Like-sm (Lsm) proteins, and in prokaryotic Hfq proteins. The Sm motif consists of approximately 70 residues with an α1β1β2β3β4β5 topology that forms a curved antiparallel β-sheet. Sm-containing proteins readily multimerize through interactions between strands β4 and β5 of two Sm motifs. In an embodiment, the NBP comprises one, two, three, four, five, six, seven, eight, nine, or ten Sm motifs. In an embodiment, the NBP comprises two Sm motifs. In an embodiment, the Sm-binding motif binds to RNA through hydrogen bonds and base-stacking interactions.

[0122] In an embodiment, the NBP comprises a thiouridine synthase and RNA methylase and pseudouridine synthase (THUMP) domain. THUMP domains are found in many tRNA modifying enzymes. THUMP domains are found adjacent to RNA modifying domains and in some cases, adjacent to N-terminal ferredoxin-like domains. THUMP domains exhibit an α1α2β1α3β2β2 topology that forms parallel α-helices adjacent to β-sheets. In an embodiment, NBPs containing a THUMP domain bind to tRNA.

[0123] In an embodiment, the NBP comprises a YT521-B homology domain. In an embodiment, the YT521-B homology domain is a 100-150 amino acid sequence, for example, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 1 about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, or about 150 amino acids. In embodiments, NBPs comprising a YT521-B homology domain bind to methylated adenosines.

[0124] In some embodiments, the first polypeptide is selected from cluster of differentiation 4 (CD4), the Z domain of Staphylococcus protein A (SpA Z domain), low density lipoprotein receptor (LDLR), albumin binding polypeptide (ABD), coxsackievirus and adenovirus receptor (CAR), fibronectin type III (FN3), poly(A) binding protein (PABP), Z-DNA binding protein 1 (ZBP1), or a fragment or derivative thereof.

[0125] In some embodiments, the first polypeptide of the fusion protein comprises cluster of differentiation 4 (CD4) or a fragment or derivative thereof. CD4 binds to glycoprotein 120 (gp120) of lentiviral particles, retroviral particles, and human immunodeficiency viruses.

[0126] CD4 (see, e.g., Uniprot Accession No. P01730) is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. The amino acid sequence of CD4 from humans is: [ka] It is.

[0127] In some embodiments, the first polypeptide comprises CD4 having the amino acid sequence of SEQ ID NO:78 and having at least one, at least two, at least three, or at least four mutations at amino acids 112, 113, 116, and 117 to glycine, alanine, lysine, arginine, histidine.

[0128] In some embodiments, the first polypeptide comprises the extracellular domain of CD4. In some embodiments, the first polypeptide comprises the extracellular domain of CD4 having the amino acid sequence of SEQ ID NO:79.

[0129] In some embodiments, the first polypeptide comprises a fragment of the extracellular domain of CD4 having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the first polypeptide comprises domain 1 of CD4 having the amino acid sequence of SEQ ID NO: 81. In some embodiments, the first polypeptide comprises the extracellular domain of CD4 or a fragment or derivative thereof (SEQ ID NOs: 79-81) with at least one, at least two, at least three, or at least four mutations at amino acids 88, 89, 92, and 93 to glycine, alanine, lysine, arginine, or histidine.

[0130] In some embodiments, the first polypeptide comprising CD4 or a fragment or derivative thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 78-81. In some embodiments, the first polypeptide comprising CD4 or a fragment or derivative thereof comprises an amino acid sequence of any one of SEQ ID NOs: 78-81 and has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising CD4 or a fragment thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 78-81.

[0131] In some embodiments, the first polypeptide comprises Protein A or a derivative or fragment thereof. Protein A (see, e.g., Uniprot Accession No. Q70AB8) binds to the Fc region of most immunoglobulins. In some embodiments, Protein A is Staphylococcal Protein A. The amino acid sequence of Protein A is: [ka] It is.

[0132] In some embodiments, the first polypeptide comprises protein A having the amino acid sequence of SEQ ID NO: 136 with an A117G mutation. In some embodiments, the first polypeptide comprises the B domain of protein A, or a fragment or derivative thereof, with the amino acid sequence of SEQ ID NO: 137. In some embodiments, the first polypeptide comprises the B domain of protein A, or a fragment or derivative thereof, with the amino acid sequence of SEQ ID NO: 137 with an A2G amino acid mutation. In some embodiments, the first polypeptide comprises the C domain of protein A, or a fragment or derivative thereof, with the amino acid sequence of SEQ ID NO: 138. In some embodiments, the first polypeptide comprises the Z domain of protein A, or a fragment or derivative thereof, with the amino acid sequence of SEQ ID NO: 180.

[0133] In some embodiments, the first polypeptide comprising protein G or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 136-138 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising an albumin binding polypeptide or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NO: 136-138.

[0134] In some embodiments, the first polypeptide comprises Protein G or a derivative or fragment thereof. Protein G (see, e.g., Uniprot Accession No. P19909) binds to the Fc region of most immunoglobulins. The amino acid sequence of Protein G is: [ka] It is.

[0135] In some embodiments, the first polypeptide comprises a G domain of Protein G having the amino acid sequence of SEQ ID NO: 155, or a fragment or derivative thereof. In some embodiments, the first polypeptide comprises a fragment of Protein G having the amino acid sequence of SEQ ID NO: 156.

[0136] In some embodiments, the first polypeptide comprising protein G or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 154-156 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising an albumin binding polypeptide or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NO: 154-156.

[0137] In some embodiments, the first polypeptide binds to a kappa light chain. In some embodiments, the kappa light chain is part of an antibody or an antigen-binding portion of a monoclonal antibody thereof. In some embodiments, the first polypeptide comprises Protein L. Protein L binds to an antibody or an antibody-binding fragment thereof via interaction with a kappa light chain. The amino acid sequence of Protein L is: [ka] It is.

[0138] In some embodiments, the first polypeptide comprises a fragment of protein L having the amino acid sequence of SEQ ID NO:140.

[0139] In some embodiments, the first polypeptide comprising protein L or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 139-140 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising an albumin binding polypeptide or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 139 or 140.

[0140] In some embodiments, the first polypeptide of the purification matrix provided herein comprises LDLR or a fragment or derivative thereof. The low density lipoprotein receptor LDLR (see, e.g., Uniprot Accession No. P01130) is a cell surface receptor that mediates endocytosis of cholesterol-rich low density lipoproteins. The amino acid sequence of LDLR from humans is: [ka] It is.

[0141] In some embodiments, the first polypeptide comprises an extracellular domain of LDLR having the amino acid sequence of SEQ ID NO: 127. In some embodiments, the first polypeptide comprises a fragment of the extracellular domain of LDLR having the amino acid sequence of SEQ ID NO: 130. In some embodiments, the first polypeptide comprises a CR2 domain of LDLR having the amino acid sequence of SEQ ID NO: 128. In some embodiments, the first polypeptide comprises a CR3 domain of LDLR having the amino acid sequence of SEQ ID NO: 129.

[0142] In some embodiments, the first polypeptide comprising LDLR or a fragment or derivative thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 126-129. In some embodiments, the first polypeptide comprising LDLR or a fragment or derivative thereof comprises an amino acid sequence of any one of SEQ ID NOs: 126-129 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least six, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising LDLR or a fragment thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 126-129.

[0143] In some embodiments, the first polypeptide binds to albumin or a derivative or fusion thereof. In some embodiments, the albumin is human serum albumin (HSA), bovine serum albumin (BSA), or ovalbumin. In some embodiments, the first polypeptide that binds to albumin comprises an albumin binding polypeptide (ABP) or a fragment or derivative thereof. In some embodiments, the albumin binding polypeptide comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the first polypeptide comprising an albumin binding polypeptide or a fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 135 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising an albumin binding polypeptide or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO:135.

[0144] In some embodiments, the first polypeptide is the Coxsackievirus and Adenovirus Receptor (CAR) or a fragment or derivative thereof. The Coxsackievirus and Adenovirus Receptor (see, e.g., Uniprot Accession No. P78310) is expressed on cardiac, brain epithelial, and endothelial cells and binds to the fiber protein of the adenovirus capsid. The amino acid sequence of the Coxsackievirus and Adenovirus Receptor from humans is: [ka] It is.

[0145] In some embodiments, the first polypeptide of the purification matrix provided herein comprises a coxsackievirus and adenovirus receptor, or a fragment or derivative thereof.

[0146] In some embodiments, the first polypeptide comprising a coxsackievirus and adenovirus receptor or fragment thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 117-125, 24, and 25. In some embodiments, the first polypeptide comprising a coxsackievirus and adenovirus receptor or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 117-125, 24, and 25 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising a coxsackievirus and adenovirus receptor or a fragment thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 117-125, 24, and 25.

[0147] In some embodiments, the first polypeptide comprises an extracellular domain of a coxsackievirus and adenovirus receptor or a fragment thereof. In some embodiments, the first polypeptide comprising an extracellular domain of a coxsackievirus and adenovirus receptor has an amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the first polypeptide comprises domain 1 of a coxsackievirus and adenovirus receptor or a fragment thereof. In some embodiments, the first polypeptide comprising domain 1 of a coxsackievirus and adenovirus receptor has an amino acid sequence of SEQ ID NO: 120. In some embodiments, the first polypeptide comprises domain 2 of a coxsackievirus and adenovirus receptor or a fragment thereof. In some embodiments, the first polypeptide comprising domain 2 of a coxsackievirus and adenovirus receptor has an amino acid sequence of SEQ ID NO: 121. In some embodiments, the first polypeptide comprises isoform 3 of a coxsackievirus and adenovirus receptor or a fragment thereof. In some embodiments, the first polypeptide comprising coxsackievirus and adenovirus receptor isoform 3 has the amino acid sequence of SEQ ID NO: 122. In some embodiments, the first polypeptide comprises coxsackievirus and adenovirus receptor isoform 4 or a fragment thereof. In some embodiments, the first polypeptide comprising coxsackievirus and adenovirus receptor isoform 4 has the amino acid sequence of SEQ ID NO: 123. In some embodiments, the first polypeptide comprises coxsackievirus and adenovirus receptor isoform 5 or a fragment thereof. In some embodiments, the first polypeptide comprises coxsackievirus and adenovirus receptor isoform 5 has the amino acid sequence of SEQ ID NO: 124. In some embodiments, the first polypeptide comprises coxsackievirus and adenovirus receptor isoform 7 or a fragment thereof. In some embodiments, the first polypeptide comprises coxsackievirus and adenovirus receptor isoform 7 has the amino acid sequence of SEQ ID NO: 125.

[0148] In some embodiments, the first polypeptide is M(RAIVFRVQWLRRYFVNGSRSGGG) n where n is an integer from 1 to 8 (SEQ ID NO: 24), e.g., n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the first polypeptide comprises an amino acid sequence of (RAIVFRVQWLRRYFVNGSRSGGG) n where n is an integer from 1 to 8 (SEQ ID NO: 25); for example, n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0149] In some embodiments, the first polypeptide comprises the mRNA decay activator protein ZFP36L2 (Tis11d) or a fragment or derivative thereof. Tis11d G (see, e.g., Uniprot Accession No. P47974) binds to adenosine and uridine rich elements (AREs). The amino acid sequence of Tis11d is: [ka] The bracketed methionine (M) in the above sequence or any other sequence described herein is the initiator methionine. The presence of the initiator methionine is optional.

[0150] In some embodiments, the first polypeptide comprises a Tis11d fragment having the amino acid sequence of SEQ ID NO: 170. In some embodiments, the first polypeptide comprises the RNA-binding domain of Tis11d having the amino acid sequence of SEQ ID NO:171.

[0151] In some embodiments, the first polypeptide comprises Tis11d having the amino acid sequence of SEQ ID NO: 169 with at least one mutation selected from E195D, E195H, E195G, E195A, E195R, and E195K. In some embodiments, the first polypeptide comprises a Tis11d fragment having the amino acid sequence of SEQ ID NO: 170 with at least one mutation selected from E46D, E46H, E46G, E46A, E46R, and E46K. In some embodiments, the first polypeptide comprises an RNA-binding domain of Tis11d having the amino acid sequence of SEQ ID NO: 171 with at least one mutation selected from E27D, E27H, E27G, E27A, E27R, and E27K.

[0152] In some embodiments, a first polypeptide comprising Tis11d or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 169-171 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more amino acid mutations. In some embodiments, a first polypeptide comprising Tis11d or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 169-171.

[0153] In some embodiments, the first polypeptide comprises eukaryotic translation initiation factor 4E (eIF4E) or a fragment or derivative thereof. eIF4E (see, e.g., Uniprot Accession No. P06730) binds to the mRNA cap. The amino acid sequence of eIF4E is: [ka] It is.

[0154] In some embodiments, the first polypeptide comprises an eIF4E fragment having the amino acid sequence of SEQ ID NO:173.

[0155] In some embodiments, the first polypeptide comprising eIF4E or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 172 or 173 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising eIF4E or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NO: 172 or 173.

[0156] In some embodiments, the first polypeptide comprises a poly(A) binding protein (PABP) or a fragment or derivative thereof. PABP (see, e.g., Uniprot Accession No. P11940) binds to the poly(A) tail of mRNA. The amino acid sequence of PABP is: [ka] It is.

[0157] In some embodiments, the first polypeptide comprises a PABP fragment having the amino acid sequence of SEQ ID NO: 175. In some embodiments, the first polypeptide comprises an RNA recognition motif (RRM) 1 domain of PABP having the amino acid sequence of SEQ ID NO: 176. In some embodiments, the first polypeptide comprises an RRM2 domain of PABP having the amino acid sequence of SEQ ID NO: 177. In some embodiments, the first polypeptide comprises an RRM3 domain of PABP having the amino acid sequence of SEQ ID NO: 178. In some embodiments, the first polypeptide comprises an RRM4 domain of PABP having the amino acid sequence of SEQ ID NO: 179.

[0158] In some embodiments, the first polypeptide comprising PABP or a fragment or derivative thereof comprises an amino acid sequence of any one of SEQ ID NOs: 174-179 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising PABP or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 174-179.

[0159] In some embodiments, the first polypeptide comprises Z-DNA binding protein 1 (ZBP1) or a fragment or derivative thereof. ZBP1 (see, e.g., Uniprot Accession No. Q9H171) binds to double-stranded DNA. The amino acid sequence of ZBP1 is: [ka] It is.

[0160] In some embodiments, the first polypeptide comprises Z-binding domain 1 of ZBP1 having the amino acid sequence of SEQ ID NO: 182. In some embodiments, the first polypeptide comprises Z-binding domain 2 of ZBP1 having the amino acid sequence of SEQ ID NO: 183.

[0161] In some embodiments, the first polypeptide comprising ZBP1 or a fragment or derivative thereof comprises an amino acid sequence of any one of SEQ ID NOs: 181-183 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising ZBP1 or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 181-183.

[0162] In some embodiments, the first polypeptide comprises a PUM-HD domain-containing protein (PUM-HD) or a fragment or derivative thereof. PUM-HD (see, e.g., Uniprot Accession No. B2BXX4) binds to mRNA. The amino acid sequence of PUM-HD is: [ka] It is.

[0163] In some embodiments, the first polypeptide comprising PUM-HD or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 184 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising PUM-HD or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 184.

[0164] In some embodiments, the first polypeptide is fibronectin (FN). In some embodiments, the fibronectin is a fibronectin type III (FN3) repeat. The FN3 repeat is the largest and most common of the subdomains of fibronectin. Domains homologous to the FN3 repeat are found in a variety of animal protein families, including other extracellular matrix molecules, cell surface receptors, enzymes, and muscle proteins. The FN3 domain has a conserved beta sandwich fold, with one beta sheet containing four chains and another sheet containing three chains. The amino acid sequence of an exemplary fibronectin (see, e.g., UniProt Accession No. P02751) is: [ka] [ka] It is.

[0165] In some embodiments, the first polypeptide comprising fibronectin or a fragment or derivative thereof comprises an amino acid sequence of SEQ ID NO: 26 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations. In some embodiments, the first polypeptide comprising fibronectin or a fragment or derivative thereof comprises a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 26.

[0166] In some embodiments, the first polypeptide is an adeno-associated virus receptor (AAVR) or a fragment or derivative thereof. AAVR, also referred to as KIAA0319L (see, e.g., Uniprot Accession No. Q8IZA0), is a 150 kDa glycoprotein that binds to the capsid of multiple AAV serotypes, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. The following references describe AAVR and are incorporated by reference in their entireties: Zhang et al. Nat Microbiol. 2019 Apr;4(4):675-682; Pillay et al. Nature. 2016 Feb 4;530(7588):108-12; Zhang et al. Nat Commun. 2019 Aug 21;10(1):3760; Summerford et al. Mol Ther. 2016 Apr;24(4):663-6.

[0167] The extracellular domain of AAVR contains an N-terminal motif with eight cysteines (MANEC) and five immunoglobulin domains known as polycystic kidney disease (PKD) domains. AAV particles bind to the PKD domains to facilitate transduction. Thus, in some embodiments, the AAVR fragment or derivative thereof contains one, two, three, four, or five PKD domains or fragments or derivatives thereof. In some embodiments, the AAVR is human AAVR (SEQ ID NO: 35), mouse AAVR (SEQ ID NO: 36), or orangutan AAVR (SEQ ID NO: 42), or a fragment or derivative thereof. In some embodiments, the AAVR contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to any one of SEQ ID NOs: 35, 36, or 42. Unless otherwise indicated, sequence identity is determined using the Basic Local Alignment Search Tool (BLAST®) from the National Center for Biotechnology Information (NCBI), available at blast.ncbi.nlm.nih.gov / Blast.cgi. In some embodiments, sequence identity is calculated over the entire length of the sequences being compared. In some embodiments, sequence identity is calculated over a 20 amino acid fragment, a 50 amino acid fragment, a 75 amino acid fragment, a 100 amino acid fragment, a 250 amino acid fragment, a 500 amino acid fragment, a 750 amino acid fragment, or a 1000 amino acid fragment of each sequence being compared.

[0168] In some embodiments, the AAVR fragment or derivative thereof comprises the extracellular domain of AAVR or a fragment or derivative thereof. In some embodiments, the AAVR comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, or a sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identical thereto. In some embodiments, the AAV binding polypeptide comprises the sequence of SEQ ID NO: 33 or SEQ ID NO: 34 having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, or more amino acid mutations.

[0169] In some embodiments, the AAVR or a fragment or derivative thereof comprises one or more PKDs, e.g., two, three, four, five, or more PKDs. In some embodiments, the PKDs are each individually selected from the PKDs listed in Table 3. For example, in some embodiments, the PKDs are each individually selected from SEQ ID NOs: 28-32, 37-41, and 43-47. In some embodiments, the AAVR or a fragment or derivative thereof comprises multiple PKDs, and the PKDs are connected to each other by a linker. Non-limiting examples of linkers are described throughout this disclosure. In some embodiments, the AAVR or a fragment or derivative thereof comprises multiple PKD domains, and each PKD domain has the same or substantially the same sequence. In some embodiments, the AAVR or a fragment or derivative thereof comprises multiple PKD domains, and each PKD has a different sequence.

[0170] [Table 4]

[0171] In some embodiments, the AAVR, or a fragment or derivative thereof, comprises a polycystic kidney disease 1 (PKD1) domain, a polycystic kidney disease 2 (PKD2) domain, a polycystic kidney disease 3 (PKD3) domain, a polycystic kidney disease 4 (PKD4) domain, a polycystic kidney disease 5 (PKD5) domain, or a combination thereof.

[0172] In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD1 and PKD2 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD1 and PKD2 domains having the amino acid sequence of SEQ ID NO: 92. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD1 and PKD3 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD1 and PKD4 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD1 and PKD5 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD2 and PKD3 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD2 and PKD4 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD2 and PKD5 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD3 and PKD4 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises PKD3 and PKD5 domains. In some embodiments, the AAVR or a fragment or derivative thereof comprises a PKD4 and a PKD5 domain. In some embodiments, the AAVR or a fragment or derivative thereof comprises three PKD domains, each PKD domain being independently selected from any one of PKD1-5. In some embodiments, the AAVR or a fragment or derivative thereof comprises four PKD domains, each PKD domain being independently selected from any one of PKD1-5. In some embodiments, the AAVR or a fragment or derivative thereof comprises five PKD domains, each PKD domain being independently selected from any one of PKD1-5. In some embodiments, the AAVR or a fragment or derivative thereof comprises six or more PKD domains, each PKD domain being independently selected from any one of PKD1-5.

[0173] In any of the embodiments of the previous paragraph, each of the PKD domains may be independently selected from wild-type or mutant PKD domains. In some embodiments, each PKD may have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to wild-type PKD. In some embodiments, the AAVR or fragment or derivative thereof disclosed herein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to wild-type PKD. In some embodiments, the AAVR or fragment or derivative thereof binds to AAV using one or more PKDs.

[0174] In some embodiments, the AAVR or fragment or derivative thereof described herein comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 amino acid mutations compared to wild-type AAVR or its PKD. In some embodiments, the AAVR or fragment or derivative thereof comprises up to about 25 or more amino acid mutations compared to wild-type AAVR or its PKD. For example, the AAV binding polypeptide may comprise about 25-35, about 35-45, about 45-55, about 55-65, or about 65-75 amino acid mutations compared to wild-type AAVR.

[0175] In some embodiments, the AAVR or fragment or derivative thereof described herein comprises at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about eleven, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 amino acid mutations compared to wild-type AAVR or its PKD, each mutation comprising a change of a native amino acid residue to histidine.

[0176] In some embodiments, the AAVR, or a fragment or derivative thereof, comprises the sequence of SEQ ID NO: 29. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises the sequence of SEQ ID NO: 29, where at least one amino acid (i.e., a non-histidine amino acid) is mutated to histidine.

[0177] In some embodiments, the AAVR, or a fragment or derivative thereof, comprises SEQ ID NO: 35 with at least one, at least two, at least three, at least four, or at least five mutations. In some embodiments, each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H.

[0178] In some embodiments, the AAVR, or a fragment or derivative thereof, comprises amino acids 411-499 of SEQ ID NO: 35 having at least one, at least two, at least three, at least four, or at least five mutations, each mutation individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H.

[0179] In some embodiments, the AAVR having the amino acid sequence of SEQ ID NO: 35, or a fragment or derivative thereof, has one or more of the mutation combinations in Table 4. Each row in Table 4 represents a mutation combination.

[0180] [Table 5]

[0181] [Table 6]

[0182] [Table 7]

[0183] [Table 8]

[0184] [Table 9]

[0185] [Table 10]

[0186] [Table 11]

[0187] [Table 12]

[0188] [Table 13]

[0189]

Table 14

[0190]

Table 15

[0191]

Table 16

[0192]

Table 17

[0193]

Table 18

[0194]

Table 19

[0195]

Table 20

[0196]

Table 21

[0197]

Table 22

[0198]

Table 23

[0199]

Table 24

[0200]

Table 25

[0201]

Table 26

[0202]

Table 27

[0203]

Table 28

[0204]

Table 29

[0205]

Table 30

[0206]

Table 31

[0207] In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations, each mutation being individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with V32H and V34H mutations. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with S23H and Q24H mutations. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations, each mutation being individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 76-86.

[0208] In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29, an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus, and an additional five amino acids having the amino acid sequence of VDYPG (SEQ ID NO:90) at the C-terminus. In some embodiments, the AAVR or fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations (each mutation individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H), an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus, and an additional five amino acids having the amino acid sequence of VDYPG (SEQ ID NO:90) at the C-terminus. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises SEQ ID NO:52 having at least one, at least two, at least three, at least four, or at least five mutations, each mutation individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H.

[0209] In some embodiments, the AAVR or fragment or derivative thereof is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 93-116. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO: 29 with V32H and V34H mutations, an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and an additional five amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO: 29 with S23H and Q24H mutations, and an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and an additional five amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations (each mutation individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H), and five additional amino acids at the N-terminus with the amino acid sequence of GNRPP (SEQ ID NO:89), and five additional amino acids at the C-terminus with the amino acid sequence of VDYPG (SEQ ID NO:90). In some embodiments, the AAVR or fragment or derivative thereof comprises the amino acid sequence of any one of SEQ ID NOs:53-63.

[0210] In some embodiments, the AAVR or fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO:29, plus an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus and an additional four amino acids having the amino acid sequence of VDYP (SEQ ID NO:91) at the C-terminus. In some embodiments, the AAVR or fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO:64. In some embodiments, the AAVR or fragment or derivative thereof comprises SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations (each mutation individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H), and an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus and an additional four amino acids having the amino acid sequence of VDYP (SEQ ID NO:91) at the C-terminus. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises SEQ ID NO:29, in addition to V32H and V34H mutations, and an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus, and an additional four amino acids having the amino acid sequence of VDYP (SEQ ID NO:91) at the C-terminus. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises SEQ ID NO:29, with S23H and Q24H mutations, and an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus, and an additional four amino acids having the amino acid sequence of VDYP (SEQ ID NO:91) at the C-terminus. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO:29 with at least one, at least two, at least three, at least four, or at least five mutations (each mutation individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H), and an additional five amino acids having the amino acid sequence of GNRPP (SEQ ID NO:89) at the N-terminus and an additional four amino acids having the amino acid sequence of VDYP (SEQ ID NO:91) at the C-terminus.In some embodiments, the AAVR, or a fragment or derivative thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 65-75.

[0211] In some embodiments, the AAVR or fragment or derivative thereof described herein comprises one or more MANEC motifs (see, e.g., SEQ ID NOs: 49-51). In some embodiments, the AAVR or fragment or derivative thereof described herein comprises one or more recombinant MANEC motifs. In some embodiments, the AAVR or fragment or derivative thereof described herein comprises an amino acid sequence having at least about 80%, at least about 90%, at least about 95, or at least about 95% identity to a wild-type MANEC motif. In some embodiments, the AAVR or fragment or derivative thereof comprises a MANEC motif having at least about 80%, at least about 90%, or at least about 95% identity to any one of SEQ ID NOs: 49-51. In some embodiments, the AAVR or fragment or derivative thereof binds to an AAV particle via one or more MANEC motifs.

[0212] In some embodiments, the AAVR or fragment or derivative thereof described herein comprises an N-terminal methionine. In some embodiments, the N-terminal methionine initiates translation of the AAVR or fragment or derivative thereof described herein. In some embodiments, the AAVR or fragment or derivative thereof described herein lacks an N-terminal methionine.

[0213] In some embodiments, the AAVR or a fragment or derivative thereof described herein binds to AAV particles of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, and / or AAVrh74. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV particles of one or more of the following serotypes: AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV1 particles. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV2 particles. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV3B particles. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV5 particles. In some embodiments, the AAVR or a fragment or derivative thereof binds to AAV6 particles. In some embodiments, the AAVR or a fragment or derivative thereof binds to an AAV8 particle. In some embodiments, the AAVR or a fragment or derivative thereof binds to an AAV9 particle.

[0214] In some embodiments, the AAVR is human AAVR. In some embodiments, the AAVR is primate AAVR. In some embodiments, the AAVR is wild-type AAVR. In some embodiments, the AAVR is mutant AAVR.

[0215] In some embodiments, the AAVR is a glycoprotein. In some embodiments, the AAVR, or a fragment or derivative thereof described herein, comprises one or more glycosylation sites. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises an O-linked glycosylation site. In some embodiments, the AAVR, or a fragment or derivative thereof, comprises an N-linked glycosylation site. In some embodiments, the AAVR, or a fragment or derivative thereof, is glycosylated at one or more asparagine and / or glutamine residues.

[0216] In some embodiments, the fusion protein comprises about 1 to about 100 AAVRs or fragments or derivatives thereof. In some embodiments, the number of AAVRs or fragments or derivatives thereof in the fusion protein is about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100. In some embodiments, a single second polypeptide having phase behavior may be bound to multiple AAVRs or fragments or derivatives thereof, for example, about 1 to 100 AAVRs or fragments or derivatives thereof.

[0217] In some embodiments, the AAVR or fragment or derivative thereof comprises the sequence of any one of SEQ ID NOs: 28-34, 37-41, 43-47, and 52-86. In some embodiments in which a single polypeptide having phase behavior is linked to multiple AAVRs or fragments or derivatives thereof, each AAVR or fragment or derivative thereof may be independently selected from SEQ ID NOs: 28-34, 37-41, 43-47, and 52-86.

[0218] In some embodiments, the first polypeptide is a polypeptide isolated or derived from SARS-CoV-2. In some embodiments, the polypeptide isolated or derived from SARS-CoV-2 is a spike protein, a membrane protein, an envelope protein, or a nucleocapsid protein. In some embodiments, the first polypeptide is a SARS-CoV-2 spike protein or a fragment or derivative thereof. The SARS-CoV-2 spike protein (see, e.g., Uniprot Accession No. P0DTC2) mediates fusion of the virion to the host cell membrane via human angiotensin-converting enzyme 2 (ACE2). An exemplary sequence of the SARS-CoV-2 S protein is: [ka] In some embodiments, the first polypeptide is a SARS-CoV-2 S protein having an amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:27.

[0219] Second Polypeptide In some embodiments, the disclosure provides a fusion protein comprising a polypeptide having phase behavior.

[0220] In some embodiments, the polypeptide having phase behavior is a resilin-like polypeptide (RLP). A resilin-like polypeptide is an elastic polypeptide having mechanical properties including desirable elasticity, compressive modulus, tensile modulus, shear modulus, elongation to break, ultimate tensile strength, hardness, rebound, and compression set. In some embodiments, the resilin-like polypeptide described herein is a polymer comprising one or more repeats. In some embodiments, the polymer repeat may have an amino acid sequence selected from any one of SEQ ID NOs: 1-9.

[0221] In some embodiments, a resilin-like polypeptide comprises two or more types of repeats, for example, a repeat of SEQ ID NO:1 and a repeat of SEQ ID NO:3.

[0222] In some embodiments, the resilin-like polypeptides described herein comprise repeats that occur up to 500 times within a given RLP. In some embodiments, the repeats are about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or more. , about 190 times, about 200 times, about 210 times, about 220 times, about 230 times, about 240 times, about 250 times, about 260 times, about 270 times, about 280 times, about 290 times, about 300 times, about 310 times, about 320 times, about 330 times, about 340 times, about 350 times, about 360 times, about 370 times, about 380 times, about 390 times, about 400 times, about 450 times, or about 500 times.

[0223] In some embodiments, the RLP comprises one or more partial repeats. In some embodiments, the partial repeat is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the RLP comprises one or more additional amino acids at the N-terminus or C-terminus of the RLP that are not part of the repeat.

[0224] In some embodiments, one or more of the RLP repeats are scrambled, i.e., they comprise different amino acid sequences but retain the same amino acid composition. For example, a repeat may have an amino acid sequence that is different from SEQ ID NO:8 but retain the same amino acid composition.

[0225] In some embodiments, the polypeptide having phase behavior is an elastin-like polypeptide. Elastin-like polypeptides (ELPs) are biopolymers derived from tropoelastin. In some embodiments, the elastin-like polypeptides described herein have the sequence (Val-Pro-Gly-Xaa-Gly). n (SEQ ID NO:10), where Xaa is defined herein.

[0226] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 08, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 (including all values ​​and ranges therebetween).

[0227] In some embodiments, the pentapeptide repeats are scrambled, e.g., comprise different amino acid sequences but maintain the same amino acid composition. For example, an ELP may comprise a different amino acid sequence than SEQ ID NO: 10, but maintain the same amino acid composition, e.g., 40% of the sequence is glycine, 20% of the sequence is Xaa (e.g., any amino acid except proline), 20% of the sequence is proline, and 20% of the sequence is valine.

[0228] In some embodiments, the ELP comprises one or more partial repeats. In some embodiments, the partial repeat is one, two, three, or four amino acids in length. In some embodiments, the ELP comprises one or more additional amino acids at the N-terminus or C-terminus of the ELP that are not part of the repeat.

[0229] In some embodiments, the ELP or RLP comprises 30 to about 150 amino acids. In some embodiments, the ELP or RLP comprises about 50 to about 100 amino acids. In embodiments, the ELP or RLP comprises about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60 , about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, About 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124 , about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, or about 150 amino acids. In embodiments, the ELP or RLP is at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67,at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least about 110, at least about at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, at least about 141, at least about 142, at least about 143, at least about 144, at least about 145, at least about 146, at least about 147, at least about 148, at least about 149, or at least about 150 amino acids. In embodiments, the polypeptide having phase behavior may include any 30-150 amino acid fragment or any 50-100 amino acid fragment of an ELP or RLP described herein.

[0230] ELPs and RLPs undergo a phase transition in response to environmental factors. ELPs and RLPs retain the ability to undergo a phase transition when conjugated to one or more polypeptides (such as one or more AAV-binding polypeptides) or when expressed as a fusion protein with one or more other polypeptides (such as one or more AAV-binding polypeptides). Polymers such as ELPs and RLPs have a cloud point temperature (T c ) also called the transition temperature (T t In some embodiments, the ELP and RLP are t The ELP undergoes a reversible phase transition from the soluble phase to the insoluble phase at a temperature called the lower critical solution temperature (LCST), T t The RLP that transitions from the soluble phase to the insoluble phase by cooling or decreasing the salt concentration has a temperature called the lower critical solution temperature (UCST), T t In some embodiments, the phase transition occurs due to a change in the secondary structure of the ELP and / or RLP. For example, the phase transition of the ELP is a random coil (T t The change in secondary structure occurs due to a change in the secondary structure from a type II β-turn (less than 100 nm) to a type II β-turn. In some embodiments, the change in secondary structure is characterized by a method selected from circular dichroism spectropolarimetry, small angle X-ray scattering, UV-visible spectrophotometry, static light scattering, dynamic light scattering, nuclear magnetic resonance spectroscopy, solid-state nuclear magnetic resonance spectroscopy, infrared spectroscopy, Fourier transform infrared spectroscopy (FTIR), small angle neutron scattering, microscopy, and cryo-electron microscopy. In some embodiments, the phase transition of the ELP is not due to a change in secondary structure.

[0231] In some embodiments, the RLPs and ELPs described herein have a transition temperature of about 0° C. to about 100° C. In some embodiments, the RLPs and / or ELPs described herein have a transition temperature of about 10° C. to about 50° C. In some embodiments, the transition temperature is about 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, About 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C or about 100°C. In some embodiments, the transition temperature is at least about 0° C., at least about 1° C., at least about 2° C., at least about 3° C., at least about 4° C., at least about 5° C., at least about 6° C., at least about 7° C., at least about 8° C., at least about 9° C., at least about 10° C., at least about 11° C., at least about 12° C., at least about 13° C., at least about 14° C., at least about 15° C., at least about 16° C., at least about 17° C., at least about 18° C., at least about 19° C., at least about 20° C., at least about 21° C., at least about 22° C., at least about 23° C., at least about 24° C., at least about 25° C., at least about 26° C., at least about 27° C., at least about 28° C., at least about 29° C., at least about 30° C., at least about 31° C., at least about 32° C., at least about 33° C., at least about 34° C., at least about 35° C., at least about 36° C.,at least about 37°C, at least about 38°C, at least about 39°C, at least about 40°C, at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, at least about 45°C, at least about 46°C, at least about 47°C, at least about 48°C, at least about 49°C, at least about 50°C, at least about 51°C, at least about 52°C, at least about 53°C, at least about 54°C, at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, At least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, at least about 80°C, at least about 81°C, at least about 82°C, at least about 83°C, at least about 84°C, at least about 85°C, at least about 86°C, at least about 87°C, at least about 88°C, at least about 89°C, at least about 90°C, at least about 91°C, at least about 92°C, at least about 93°C, at least about 94°C, at least about 95°C, at least about 96°C, at least about 97°C, at least about 98°C, at least about 99°C, or at least about 100°C. In some embodiments, the RLPs described herein have a transition temperature of about 10°C to about 100°C.

[0232] In some embodiments, the T of the RLPs and ELPs described herein t can be regulated by manipulating the primary structure (e.g., amino acid sequence) of RLPs and ELPs. 。 In some embodiments, the hydrophobicity of the ELP or RLP is modulated. In some embodiments, the hydrophobicity of the ELP is modified by altering the nature of the guest residue Xaa. In some embodiments, the hydrophobicity of the ELP or RLP is increased, resulting in a higher T tIn some embodiments, the hydrophobicity of the ELP or RLP is reduced, such that the T t In some embodiments, the polarity of the ELP or RLP is modulated. In some embodiments, the polarity of the ELP is modulated by altering the nature of the guest residue Xaa. In some embodiments, the polarity of the ELP or RLP is increased, resulting in an increase in T t In some embodiments, the polarity of the ELP or RLP is decreased, resulting in an increase in T t decreases.

[0233] In some embodiments, the number of ELP pentapeptide repeats (n) is adjusted to increase the T t In some embodiments, the pentapeptide repeat (Val-Pro-Gly-Xaa-Gly) is nIn (SEQ ID NO: 10), n is an integer from 1 to 500, inclusive. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 08, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 (including all values ​​and ranges therebetween).

[0234] In some embodiments, Xaa is a "guest residue", i.e., any amino acid that does not eliminate the phase behavior of the ELP. In some embodiments, Xaa is any amino acid except proline. In some embodiments, Xaa is selected independently for each repeat. For example, a given ELP may contain the guest residues alanine, glycine, and valine in a ratio of 8:7:1. In some embodiments, Xaa is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa is a non-classical amino acid selected from the group consisting of Table 2, 2,4-diaminobutyric acid, α-aminoisobutyric acid, alloisoleucine, 4-aminobutyric acid, 2-aminobutyric acid (Abu), ε-Ahx, 6-aminohexanoic acid, 2-aminoisobutyric acid (Aib), 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. In some embodiments, Xaa is the D-isomer of a natural or non-classical amino acid.

[0235] In some embodiments, the T of the RLPs and ELPs described herein t is modulated by introducing one or more environmental factors into a composition comprising RLPs and / or ELPs. 。 In some embodiments, the T of the ELP and / or the RLP tis adjusted by adjusting the ionic strength of the solvent. In some embodiments, the ionic strength of the solvent is adjusted by adding salt. In some embodiments, the ELP and / or RLP have a lower T in a solvent that contains an anion classified as a kosmotrope. t The anion, which is a kosmotrope, is highly hydrated and affects the water shield of the ELP and / or RLP. In some embodiments, the T t can be adjusted by the addition of anions that are chaotropes. At low concentrations, the addition of chaotropes reduces the T of ELP and / or RLP. t At high concentrations, the addition of chaotropes increases the T t In some embodiments, the T of ELP and / or RLP is reduced. t can be adjusted by introducing one or more reagents that disrupt hydrogen bonds. Non-limiting examples of reagents that disrupt hydrogen bonds include sodium dodecyl sulfate (SDS) and urea. In some embodiments, reagents that promote the formation of hydrogen bonds are utilized to adjust the T t In some embodiments, a reagent that promotes hydrophobic interactions is used to modulate T t Trifluoroethanol promotes both hydrophobic interactions and hydrogen bond formation, modulating T t It is a reagent that causes a decrease in

[0236] In some embodiments, the concentrations of ELP and / or RLP are adjusted to provide a T t In some embodiments, high ELP and / or RLP concentrations can modulate T t In some embodiments, lower ELP and / or RLP concentrations reduce T t increases.

[0237] In addition, adjustments of pH, light, and ion concentration can be used to increase T t can be adjusted.

[0238] In some embodiments, adjusting the number (e.g., addition or removal) and nature (e.g., positively or negatively charged) of charged amino acids (e.g., histidine, lysine, arginine, glutamic acid, aspartic acid, ornithine, or other non-naturally charged amino acids) can be used to increase the T t This allows for adjustment.

[0239] In some embodiments, the ELPs and / or RLPs described herein are block copolymers. Block copolymers contain two or more sequence domains or blocks, where the two or more blocks have different properties. Non-limiting examples of properties that can be tailored include hydrophilicity, hydrophobicity, polarity, and secondary structure. In some embodiments, the block copolymers are amphiphilic, e.g., contain at least one hydrophobic block and at least one hydrophilic block.

[0240] In some embodiments, the ELPs and / or RLPs described herein are formed into various forms. Non-limiting examples of forms include globulomers, micelles, vesicles, fibrils, nanofibrils, nanotubes, and hydrogels. In some embodiments, the RLPs and / or ELPs described herein are formed into various forms after the addition of an environmental factor. In some embodiments, the RLPs and / or ELPs described herein change from one form to another form after the addition of an environmental factor. In some embodiments, the RLPs and / or ELPs described herein change from one form to another form after the addition of an AAV particle.

[0241] In some embodiments, the addition of an environmental factor causes the RLP and / or ELP to undergo a phase transition, hi some embodiments, in the phase transition of the RLP and / or ELP, the RLP and / or ELP are converted from one form to another.

[0242] In some embodiments, the phase transition of the RLP and / or ELP results in the formation of dense droplets.

[0243] In some embodiments, the polypeptide has the phase behavior: (a)(GRGDSPY) n (SEQ ID NO:1) (b)(GRGDSPH) n (SEQ ID NO:2) (c)(GRGDSPV) n (SEQ ID NO:3) (d)(GRGDSPYG) n (SEQ ID NO:4) (e)(RPLGYDS) n (SEQ ID NO:5) (f)(RPAGYDS) n (SEQ ID NO:6) (g)(GRGDSYP) n (SEQ ID NO:7) (h)(GRGDSPYQ) n (SEQ ID NO:8) (i) (GRGNSPYG) n (SEQ ID NO:9) (j)(GVGVP) n (SEQ ID NO:11); (k)(GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO:12); (l)(GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO:13); (m)(GVGVPGWGVPGVGVPGWGVPGVGVP) m (SEQ ID NO:14); (n)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP) m (SEQ ID NO:15); (o)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m (SEQ ID NO:16); and (p)(GAGVPGVGVPGAGVPGVGVPGAGVP) m (SEQ ID NO: 17) or a randomized, scrambled analog thereof; During the ceremony; n is an integer from 1 to 500, inclusive; and m is an integer from 4 to 25, inclusive.

[0244] In some embodiments, the polypeptide having the phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 12), where m is 16. In some embodiments, the polypeptide having the phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 12), where m is 16 and there are up to 10 additional amino acids at the N-terminus or C-terminus. In some embodiments, the polypeptide having the phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 12), where m is 16 and there is an additional C-terminal glycine. In some embodiments, the polypeptide having the phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 12), where m is 16 and there is an additional N-terminal methionine. In some embodiments, the polypeptide having the phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO:12), where m is 16 and there is an additional C-terminal glycine and an additional N-terminal methionine.

[0245] In some embodiments, the polypeptide having the phase behavior has the amino acid sequence of SEQ ID NO:88.

[0246] In some embodiments, the polypeptide having the phase behavior is (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 144) or (GVGVPGVGVPGLGVPGVGVPGVGVP) m(SEQ ID NO: 146), where m is an integer from 2 to 32, inclusive. In some embodiments, the polypeptide having the phase behavior is (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 144), where m is 8 or 16. In some embodiments, the polypeptide having the phase behavior is (GVGVPGAGVP) m (SEQ ID NO: 145), where m is an integer from 5 to 80, inclusive. In some embodiments, the polypeptide having the phase behavior is (GXGVP) m (SEQ ID NO:147), where m is an integer from 10 to 160, inclusive, and X of each repeat is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine.

[0247] In some embodiments, the polypeptide having the phase behavior is (a)(GVGVP) m (SEQ ID NO:143); (b)(ZZPXXXXGZ) m (SEQ ID NO:148); (c)(ZZPXGZ) m (SEQ ID NO:149); (d)(ZZPXXGZ) m (SEQ ID NO: 150); or (e)(ZZPXXXGZ) m (SEQ ID NO: 151), where m is an integer from 10 to 160, inclusive; X, if present, is any amino acid except proline and glycine; and Z, if present, is any amino acid.

[0248] In some embodiments, the polypeptide having the phase behavior is (GVGVP) m(SEQ ID NO: 143), where m is 20, 40, or 80. In some embodiments, the polypeptide having the phase behavior is (GRGDXPZX) m (SEQ ID NO: 152) or (XZPXDGRG) m (SEQ ID NO: 153), wherein X is glutamine or serine, Z is tyrosine or valine, and m is an integer from 10 to 160, inclusive.

[0249] In some embodiments, the polypeptide with phase behavior comprises a first set of repeat sequences and a second set of repeat sequences. The first set of repeat sequences and the second set of repeat sequences may each individually comprise a sequence that is repeated one or more times. In some embodiments, the first set of repeat sequences and / or the second set of repeat sequences comprise a repeat sequence comprising any one of SEQ ID NOs: 1-17 and 143-153. In some embodiments, the polypeptide with phase behavior comprises a first set of repeat sequences and a second set of repeat sequences, wherein the first set of repeat sequences comprises (GRGDXPZX) 40 (SEQ ID NO: 185), and the second set of repeats comprises the amino acid sequence (GVGVP) 80(SEQ ID NO: 186), where X is glutamine and Z is tyrosine. In some embodiments, the first set of repeat sequences comprises the sequence of SEQ ID NO: 187. In some embodiments, the polypeptide having phase behavior comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different sets of repeat sequences. In some embodiments, each set of repeat sequences in a polypeptide having phase behavior is about 5 to about 400 times, e.g., about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, about 100 times, about 105 times, about 110 times, about 115 times, about 120 times, about 125 times, about 130 times, about 135 times, about 140 times, about 145 times, about 150 times, about 155 times, about 160 times, about 165 times, about 170 times, about 175 times, about 180 times, about 185 times, about 190 times, about 1 95 times, about 200 times, about 205 times, about 210 times, about 215 times, about 220 times, about 225 times, about 230 times, about 235 times, about 240 times, about 245 times, about 250 times, about 255 times, about 260 times, about 265 times, about 270 times, about 275 times, about 280 times, about 285 times, about 290 times, about 295 times, about 300 times, The present invention includes a sequence that is repeated about 305 times, about 310 times, about 315 times, about 320 times, about 325 times, about 330 times, about 335 times, about 340 times, about 345 times, about 350 times, about 355 times, about 360 times, about 365 times, about 370 times, about 375 times, about 380 times, about 385 times, about 390 times, about 395 times, or about 400 times.

[0250] In some embodiments, a polypeptide having phase behavior comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises up to 10 additional N-terminal and / or C-terminal amino acids. In some embodiments, a polypeptide having phase behavior comprising an amino acid sequence of any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises an additional N-terminal methionine. In some embodiments, a polypeptide having phase behavior comprising an amino acid sequence of any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises an additional C-terminal glycine.

[0251] In some embodiments, the polypeptide with phase behavior has the same amino acid composition as the ELP and / or RLP, but does not include repeats. In some embodiments, the polypeptide with phase behavior comprises an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the ELP and / or RLP. In some embodiments, the polypeptide with phase behavior has an amino acid composition that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the ELP and / or RLP. In some embodiments, the polypeptide with phase behavior has a hydrophobic amino acid composition that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the ELP and / or RLP.

[0252] In some embodiments, the polypeptide having phase behavior comprises a non-repetitive, unstructured polypeptide. In some embodiments, the non-repetitive, unstructured polypeptide has an amino acid sequence that comprises at least 50 amino acids. In some embodiments, the non-repetitive, unstructured polypeptide has an amino acid sequence that comprises at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In some embodiments, the sequence of the non-repetitive, unstructured polypeptide is at least about 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%) proline and at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) glycine. In some embodiments, the non-repetitive, unstructured polypeptide has a sequence that comprises at least about 40% amino acids selected from the group consisting of valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0253] In some embodiments, the polypeptide with phase behavior comprises a sequence that does not contain three consecutive identical amino acids, no 5-10 amino acid subsequence occurs more than once in the polypeptide with phase behavior, and the polypeptide with phase behavior comprises a subsequence that begins and ends with a proline, the subsequence further comprising at least one glycine.

[0254] The ELP and / or RLP described herein are expressed as a fusion protein, e.g., as a component of a second polypeptide, where the second polypeptide has a phase behavior. In some embodiments, the fusion protein comprises a first polypeptide. Examples of the first polypeptide are provided throughout this example. In some embodiments, the first polypeptide is (i) an enzyme or a derivative or catalytic fragment thereof; (ii) an antibody or a derivative or an antigen-binding fragment thereof; (iii) a signaling molecule or a fragment or derivative thereof; (iv) a structural protein or a fragment or derivative thereof; or (v) a hormone or a fragment or derivative thereof.

[0255] In some embodiments, the fusion protein is expressed in bacterial cells or mammalian cells.

[0256] In some embodiments, the fusion protein is expressed in Escherichia coli. In some embodiments, the fusion protein is expressed in insect cells. In some embodiments, the sequence of the non-repetitive, unstructured polypeptide is at least about 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%) proline, at least 20% (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) glycine, and at least 40% (e.g., at least 40%, at least 50%, at least 60%, or at least 70%) amino acids selected from the group consisting of valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0257] In some embodiments, the polypeptide with phase behavior does not contain three consecutive identical amino acids. In some embodiments, the polypeptide with phase behavior contains a subsequence that occurs only once in the amino acid sequence of the polypeptide with phase behavior (e.g., a fragment of a polypeptide with phase behavior). In some embodiments, the polypeptide with phase behavior contains a subsequence that begins and ends with a proline. In some embodiments, the polypeptide with phase behavior contains a subsequence that includes at least one glycine.

[0258] In some embodiments, the polypeptide with phase behavior comprises a signal peptide. In some embodiments, the polypeptide with phase behavior comprises an N-terminal methionine. In some embodiments, the polypeptide with phase behavior lacks an N-terminal methionine. As used herein, "(M)" refers to an optional N-terminal methionine.

[0259] Linker In some embodiments, a linker separates the first and second polypeptides of the fusion protein. In some embodiments, any linker that does not interfere with the function of the fusion protein can be utilized. In some embodiments, the linker can be flexible. In some embodiments, the linker can be rigid.

[0260] In some embodiments, the linker maintains the phase behavior of a polypeptide having a phase behavior. In some embodiments, the linker maintains the T of a polypeptide having a phase behavior. tIn some embodiments, the linker preserves the structure of the first polypeptide. In some embodiments, the linker comprises 1-50 amino acids. In some embodiments, the linker comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0261] In some embodiments, the rigidity of the linker is increased by including prolines in the amino acid sequence of the linker.

[0262] In some embodiments, the flexibility of the linker is increased by including small polar amino acids, including threonine, serine, and glycine.

[0263] In some embodiments, the linker may adopt a variety of secondary structures, including, but not limited to, alpha helices, beta strands, and random coils. In some embodiments, the linker adopts an alpha helix structure, (EAAAK) n (SEQ ID NO: 18), where n is the number of repeats, from 1 to 20.

[0264] In some embodiments, the linker is (G4S) n (SEQ ID NO: 19), where n can be a repeat number between 1 and 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In an embodiment, the linker has repeats of (SGGG)n (SEQ ID NO: 20), where n is an integer between 1 and 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In an embodiment, the linker has repeats of (GGGS)n (SEQ ID NO: 20), where n is an integer between 1 and 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). n(SEQ ID NO:21), where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker has repeats of (G x S) n (SEQ ID NO: 141), where x is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) and n is an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). x G) n (SEQ ID NO: 142), where x is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) and n is an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

[0265] In some embodiments, the linker has an amino acid sequence of KESGSVSSEQLAQFRSLD (SEQ ID NO: 22). In some embodiments, the linker has an amino acid sequence of EGKSSGSGSESKST (SEQ ID NO: 23). In some embodiments, the linker contains only glycines.

[0266] In some embodiments, the linker is cleavable. For example, the linker can be cleaved by a protease. In some embodiments, the peptide linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a furin cleavage site.

[0267] In some embodiments, the linker is poly-(Gly) nand n is a linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (SEQ ID NO: 48). In other embodiments, the linker is selected from the group consisting of a dipeptide, a tripeptide, and a quadripeptide. In embodiments, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0268] In some embodiments, the linker is selected from GKSSGSGSESKS (SEQ ID NO: 157), GSTGSSGKSSEGKG (SEQ ID NO: 158), GSTGSSGKSSEGSGSTKG (SEQ ID NO: 159), GSTGSSGKPGSGEGSTKG (SEQ ID NO: 160), EGKSSGSGSESKEF (SEQ ID NO: 161), SRSSG (SEQ ID NO: 162), and SGSSC (SEQ ID NO: 163).

[0269] In some embodiments, the linker is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A peptide. 2A peptides are a class of 18-22 amino acid long peptides that induce ribosomal skipping during translation of proteins in cells. In some embodiments, the 2A peptide is a T2A peptide having an amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 164), a P2A peptide having an amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 165), an E2A peptide having an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 166), or an F2A peptide having an amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 167). In some embodiments, the 2A peptide has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NOs: 164-167. In some embodiments, the 2A peptide further comprises GSG (SEQ ID NO: 168) at the N-terminus.

[0270] In some embodiments, a fusion protein comprises, from N-terminus to C-terminus, a first polypeptide, a linker, and a second polypeptide having phase behavior. In some embodiments, a fusion protein comprises, from N-terminus to C-terminus, a second polypeptide having phase behavior, a linker, and a first polypeptide.

[0271] Methods for stabilizing a first polypeptide of a fusion protein, improving its yield, and preventing its loss of activity, unfolding, degradation, and / or misfolding - Patents.com The inventors have unexpectedly found that expression of a first polypeptide as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior can be useful for stabilizing the first polypeptide during production, purification, and / or storage of the first polypeptide. As used herein with respect to a first polypeptide, the term "stabilize" or "stabilizing" refers to the ability of a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior to reduce degradation or aggregation of a sample comprising a plurality of first polypeptides, to prevent the first polypeptide from binding to other proteins or to itself, to promote synthesis of the first polypeptide by a producing cell, to prevent unfolding of the first polypeptide, and to prevent misfolding of the first polypeptide.

[0272] Thus, in some embodiments, a method of stabilizing a first polypeptide is provided herein, comprising expressing the first polypeptide as a fusion protein comprising the first polypeptide and a second polypeptide, wherein the second polypeptide is a polypeptide having phase behavior. In embodiments, the first polypeptide substantially retains its activity after exposure of the fusion protein to one or more conditions that would destabilize the first polypeptide. In some embodiments, a method of substantially preventing unfolding, degradation, and / or misfolding of a first polypeptide is provided herein, comprising expressing the first polypeptide as a fusion protein of the first polypeptide and a second polypeptide having phase behavior, wherein the fusion protein is exposed to one or more conditions that would cause unfolding, degradation, and / or misfolding. In some embodiments, provided herein are methods of substantially preventing loss of activity of a first polypeptide after exposure to one or more conditions known to unfold, degrade, and / or misfold the first polypeptide, the methods comprising expressing a fusion protein comprising a first polypeptide and a second polypeptide, the second polypeptide being a polypeptide having phase behavior. In embodiments, any of the foregoing methods comprise excluding the fusion protein from conditions known to destabilize the first polypeptide or conditions known to unfold, degrade, and / or misfold the first polypeptide, wherein the first polypeptide retains its activity compared to a control first polypeptide. A "control first polypeptide" refers to a first polypeptide that is not fused to a polypeptide having phase behavior and that has not been exposed to conditions that cause loss of activity, misfolding, unfolding, or degradation.

[0273] The term "activity" may refer to the binding affinity of a first polypeptide to its binding partner. For example, if the first polypeptide is PKD2 of AAVR, the binding partner may be the capsid of an AAV viral particle. If the first polypeptide is protein A, the binding partner may be the Fc region of an immunoglobulin. When referring to an enzyme, the term "activity" refers to the k, also referred to herein as the "turnover number." cat It can refer to k cat is expressed by the formula V max / E t where V max is the maximum reaction rate when all enzyme catalytic sites are saturated with substrate, and E t is the total enzyme concentration or the concentration of all enzyme catalytic sites. cat " and "V max To determine enzyme kinetic parameters such as ", several techniques are available, such as surface plasmon resonance, Forster resonance energy transfer (FRET), isothermal titration calorimetry, colorimetry, or fluorimetric techniques.

[0274] Conditions known to destabilize, unfold, degrade, or misfold the first polypeptide include the introduction of one or more of the following: salt, exposure to base (e.g., Bronsted-Lowry or Lewis base), exposure to acid (e.g., Bronsted-Lowry or Lewis acid), exposure to a temperature of at least 50° C., lyophilization, freeze-thaw cycles, autoclaving, oxidizing agents, reducing agents, chaotropic agents, detergents, organic solvents, urea, guanidine hydrochloride. In some embodiments, autoclaving, heat shock, change in pH, exposure to light, agitation, mixing, temperature change (e.g., freeze-thaw), storage of the first polypeptide in a non-ideal orientation, or tendency of the first polypeptide to aggregate causes the first polypeptide to unfold, degrade, or misfold. In embodiments, any of the environmental factors described herein can be conditions that destabilize, unfold, degrade, or misfold the first polypeptide.

[0275] In embodiments, the conditions known to unfold, degrade, misfold, or destabilize the first polypeptide are exposure to guanidine hydrochloride, lyophilization, freeze-thaw cycles, exposure to sodium hydroxide, autoclaving, exposure to a temperature of at least 50° C., or a combination thereof.

[0276] In embodiments, the fusion protein is exposed to one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide for about 5 minutes to about 1 day. In embodiments, the first polypeptide is exposed to one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide for about 10 minutes to about 30 minutes, about 15 minutes to about 30 minutes, about 15 minutes to 1 hour, about 30 minutes to about 12 hours, about 30 minutes to about 11 hours, about 30 minutes to about 10 hours, about 30 minutes to about 9 hours, about 30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30 minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, or about 45 minutes to 1 hour. In embodiments, the first polypeptide is administered for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 61 minutes, about 62 minutes, about 63 minutes, about 64 minutes, about 65 minutes, about 66 minutes, about 67 minutes, about 68 minutes, about 69 minutes, about 70 minutes, about 71 minutes, about 72 minutes, about 73 minutes, about 74 minutes, about 75 minutes, about 76 minutes, about 77 minutes, about 78 minutes, about 79 minutes, about 80 minutes, about 81 minutes, about 82 minutes, about 83 minutes, about 84 minutes, about 85 minutes, about In some embodiments, the method further comprises exposing the sample to the conditions for about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours (including all subranges and values ​​therebetween).In embodiments, the first polypeptide is stimulated for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, at least about 31 minutes, at least about 32 minutes, at least about 33 minutes, at least about 34 minutes, at least about 35 minutes, at least about 36 minutes, at least about 37 minutes, at least about 38 minutes, at least about 39 minutes, at least about 40 minutes, at least about 41 minutes, at least about 42 minutes, at least about 43 minutes, at least about 44 minutes, at least about 45 minutes, at least about 46 minutes, at least about 47 minutes, at least about 48 minutes, at least about 49 minutes, at least about 50 minutes, at least about 51 minutes, at least about 52 minutes, at least about 53 minutes, at least about 54 minutes, at least about 55 minutes, at least about 56 minutes, at least about 57 minutes, at least about 58 minutes, at least about 59 minutes, at least about 60 minutes, at least about 61 minutes, at least about 62 minutes, at least about 63 minutes, at least about 64 at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, at least about 31 minutes, at least about 32 minutes, at least about 33 minutes, at least about 34 minutes, at least about 35 minutes, at least about 36 minutes, at least about 37 minutes, at least about 38 minutes, at least about 39 minutes, at least about 40 minutes, at least about 41 minutes, at least about 42 minutes, at least about 43 minutes, at least about 44 minutes, at least about 45 minutes, at least about 46 minutes, at least about 47 minutes, at least about 48 minutes, at least about 49 minutes, at least about 50 minutes, at least about 51 minutes, at least about 52 minutes, at least about 53 minutes, at least about 54 minutes, at least about 55 minutes, at least about 56 minutes, at least about 57 minutes, at least about 58 minutes, at least about 59 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours.In an embodiment, the first polypeptide is exposed to one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide for about 30 minutes. In an embodiment, the first polypeptide is exposed to one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide for at least about 30 minutes. In an embodiment, the first polypeptide is exposed to one or more conditions known to unfold, degrade, misfold, or destabilize the first polypeptide for about 10 minutes to about 30 minutes.

[0277] In embodiments, the fusion protein is exposed to a temperature ranging from about 50° C. to about 99° C., e.g., about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C., about 90° C., about 91° C., about 92° C., about 93° C., about 94° C., about 95° C., about 96° C., about 97° C., about 98° C., or about 99° C., including all subranges and values ​​therebetween. In embodiments, the fusion protein is exposed to at least about 50° C., at least about 55° C., at least about 60° C., at least about 65° C., at least about 70° C., at least about 75° C., at least about 80° C., at least about 85° C., at least about 90° C., at least about 91° C., at least about 92° C., at least about 93° C., at least about 94° C., at least about 95° C., at least about 96° C., at least about 97° C., at least about 98° C., or at least about 99° C.

[0278] In embodiments, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is exposure to guanidine hydrochloride. In embodiments, the first polypeptide is exposed to 1 M to about 10 M guanidine hydrochloride. In embodiments, the first polypeptide is exposed to about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, about 3.1 M, about 3.2M, approximately 3.3M, approximately 3.4M, approximately 3.5M, approximately 3.6M, approximately 3.7M, approximately 3.8M, approximately 3.9M, approximately 4M, approximately 4.1M, approximately 4.2M, approximately 4.3M, approximately 4.4 M, about 4.5M, about 4.6M, about 4.7M, about 4.8M, about 4.9M, about 5M, about 5.1M, about 5.2M, about 5.3M, about 5.4M, about 5.5M, about 5.6M, about 5.7M, approximately 5.8M, approximately 5.9M, approximately 6M, approximately 6.1M, approximately 6.2M, approximately 6.3M, approximately 6.4M, approximately 6.5M, approximately 6.6M, approximately 6.7M, approximately 6.8M, approximately 6. 9M, about 7M, about 7.1M, about 7.2M, about 7.3M, about 7.4M, about 7.5M, about 7.6M, about 7.7M, about 7.8M, about 7.9M, about 8M, about 8.1M, about 8 0.2M, about 8.3M, about 8.4M, about 8.5M, about 8.6M, about 8.7M, about 8.8M, about 8.9M, about 9M, about 9.1M, about 9.2M, about 9.3M, about 9.4M, about 9.5M, about 9.6M, about 9.7M, about 9.8M, about 9.9M, or about 10M (including all subranges and values ​​therebetween) guanidine hydrochloride. In embodiments, the first polypeptide is at least about 1M, at least about 1.1M, at least about 1.2M, at least about 1.3M, at least about 1.4M, at least about 1.5M, at least about 1.6M, at least about 1.7M, at least about 1.8M, at least about 1.9M, at least about 2M, at least about 2.1M, at least about 2.2M, at least about 2.3M, at least about 2.4M, at least about 2.5M, at least about 2.6M, at least about 2.7M, at least about 2.8M, at least about 2.9M, at least about 3M, at least about 3.1M, at least about 3.2M, at least about 3.3M, at least about 3.4M, at least about 3.5M, at least about 3.6M, at least about 3.7M, at least about 3.8M, at least about 3.9M, at least about 4.5M, at least about 3.6M, at least about 3.7M, at least about 3.8M, at least about 3.9M, at least about 4M, at least about 4.1M, at least about 4.2M, at least about 4.3M, at least about 4.4M, at least about 4.5M, at least about 4.6M, at least about 4.7M, at least about 4.8M, at least about 4.9M, at least about 5M, at least about 5.1M, at least about 5.2M, at least about 5.3M, at least about 5.4M, at least about 5.5M, at least about 5.6M, at least about 5.7M, at least about 5.8M, at least about 5.9M, at least about 6M, at least about 6.1M, at least about 6.2M, at least about 6.3M, at least about 6.4M, at least about 6.5M, at least about 6.6M, at least about 6. 7M, at least about 6.8M, at least about 6.9M, at least about 7M, at least about 7.1M, at least about 7.2M, at least about 7.3M, at least about 7.4M, at least about 7.5M, at least about 7.6M, at least about 7.7M, at least about 7.8M, at least about 7.9M, at least about 8M, at least about 8.1M, at least about 8.2M, at least about 8.3M, at least about 8.4M, at least about 8.5M, at least about 8.6M, at least about 8.7M, at least about 8.8M, at least about 8.9M, at least about 9M, at least about 9.1M, at least about 9.2M, at least about 9.3M, at least about 9.4M, at least about 9.5M, at least about 9.6M, at least about 9.7M, at least about 9.8M, at least about 9.In an embodiment, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposed to 6M guanidine hydrochloride. In an embodiment, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposed to 6M guanidine hydrochloride for 30 minutes. In an embodiment, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposed to 6M guanidine hydrochloride for at least 30 minutes. In an embodiment, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposed to 6M guanidine hydrochloride for 10 to 30 minutes.

[0279] In embodiments, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is exposure to sodium hydroxide. In embodiments, the first polypeptide is exposed to 0.001M to about 10M sodium hydroxide. In embodiments, the first polypeptide is exposed to about 0.001M, about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, Approximately 1.6M, approximately 1.7M, approximately 1.8M, approximately 1.9M, approximately 2M, approximately 2.1M, approximately 2.2M, approximately 2.3M, approximately 2.4M, approximately 2.5M, approximately 2.6M, approximately 2.7M, approximately 2.8M, approximately 2.9M, approximately 3M, approximately 3 .1M, about 3.2M, about 3.3M, about 3.4M, about 3.5M, about 3.6M, about 3.7M, about 3.8M, about 3.9M, about 4M, about 4.1M, about 4.2M, about 4.3M, about 4.4M, about 4.5M, about 4. 6M, approximately 4.7M, approximately 4.8M, approximately 4.9M, approximately 5M, approximately 5.1M, approximately 5.2M, approximately 5.3M, approximately 5.4M, approximately 5.5M, approximately 5.6M, approximately 5.7M, approximately 5.8M, approximately 5.9M, approximately 6M, approximately 6.1M , about 6.2M, about 6.3M, about 6.4M, about 6.5M, about 6.6M, about 6.7M, about 6.8M, about 6.9M, about 7M, about 7.1M, about 7.2M, about 7.3M, about 7.4M, about 7.5M, about 7.6M, The method is exposed to about 7.7M, about 7.8M, about 7.9M, about 8M, about 8.1M, about 8.2M, about 8.3M, about 8.4M, about 8.5M, about 8.6M, about 8.7M, about 8.8M, about 8.9M, about 9M, about 9.1M, about 9.2M, about 9.3M, about 9.4M, about 9.5M, about 9.6M, about 9.7M, about 9.8M, about 9.9M, or about 10M (including all subranges and values ​​therebetween) sodium hydroxide. In embodiments, the first polypeptide has a concentration of at least about 0.001M, at least about 0.01M, at least about 0.02M, at least about 0.03M, at least about 0.04M, at least about 0.05M, at least about 0.06M, at least about 0.07M, at least about 0.08M, at least about 0.09M, at least about 0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M, at least about 0.5M, at least about 0.6M, at least about 0.7M, at least about 0.8M, at least about 0.9M, at least about 1M, at least about 1.1M, at least about 1.2M, at least about 1.3M, at least about 1.4M, at least about 1.5M, at least about 1.6M, at least about 1.7M, at least about 1.8M, at least about 1.9M, at least about 2M, at least about 2.1M, at least about 2.2M, at least about 2.3M, at least about 2.4M, at least about 2.5M, .5M, at least about 2.6M, at least about 2.7M, at least about 2.8M, at least about 2.9M, at least about 3M, at least about 3.1M, at least about 3.2M, at least about 3.3M, at least about 3.4M, at least about 3.5M, at least about 3.6M, at least about 3.7M, at least about 3.8M, at least about 3.9M, at least about 4M, at least about 4.1M, at least about 4.2M, at least about 4.3M, at least about 4.4M, at least about 4.5M, at least about 4.6M, at least about 4.7M, at least about 4. .8M, at least about 4.9M, at least about 5M, at least about 5.1M, at least about 5.2M, at least about 5.3M, at least about 5.4M, at least about 5.5M, at least about 5.6M, at least about 5.7M, at least about 5.8M, at least about 5.9M, at least about 6M, at least about 6.1M, at least about 6.2M, at least about 6.3M, at least about 6.4M, at least about 6.5M, at least about 6.6M, at least about 6.7M, at least about 6.8M, at least about 6.9M, at least about 7M, at least about 7. 1M, at least about 7.2M, at least about 7.3M, at least about 7.4M, at least about 7.5M, at least about 7.6M, at least about 7.7M, at least about 7.8M, at least about 7.9M, at least about 8M, at least about 8.1M, at least about 8.2M, at least about 8.3M, at least about 8.4M, at least about 8.5M, at least about 8.6M, at least about 8.7M, at least about 8.8M, at least about 8.9M, at least about 9M, at least about 9.1M, at least about 9.2M, at least about 9.3M, at least about 9.In an embodiment, the condition known to destabilize, unfold, degrade or misfold the first polypeptide is exposure to 0.1 M sodium hydroxide. In an embodiment, the condition known to destabilize, unfold, degrade or misfold the first polypeptide is exposure to 0.1 M sodium hydroxide for about 30 minutes. In an embodiment, the condition known to destabilize, unfold, degrade or misfold the first polypeptide is exposure to 0.1 M sodium hydroxide for at least about 30 minutes. In an embodiment, the condition known to destabilize, unfold, degrade or misfold the first polypeptide is exposure to 0.1 M sodium hydroxide for 10 to 30 minutes.

[0280] In an embodiment, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is a freeze-thaw cycle. In an embodiment, the freeze-thaw cycle comprises freezing a solution containing the first polypeptide and then bringing the solution to a temperature above freezing. In an embodiment, the first polypeptide is subjected to multiple freeze-thaw cycles. In an embodiment, the first polypeptide is subjected to 2-10, 2-20, 2-30, 2-40, or 2-50 freeze-thaw cycles. In embodiments, the first polypeptide is fused at least once, at least twice, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once, at least once 5 times, about 26 times, about 27 times, about 28 times, about 29 times, about 30 times, about 31 times, about 32 times, about 33 times, about 34 times, about 35 times, about 36 times, about 37 times, about 38 times, about 39 times, about 40 times, about 41 times, about 42 times, about 43 times, about 44 times, about 45 times, about 46 times, about 47 times, about 48 times, about 49 times, about 50 times, about 51 times , about 52 times, about 53 times, about 54 times, about 55 times, about 56 times, about 57 times, about 58 times, about 59 times, about 60 times, about 61 times, about 62 times, about 63 times, about 64 times, about 65 times, about 66 times, about 67 times, about 68 times, about 69 times, about 70 times, about 71 times, about 72 times, about 73 times, about 74 times, about 75 times, about 76 times, about 77 times, about The polymer may be subjected to 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 freeze-thaw cycles. In embodiments, the first polypeptide is ligated at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 21 times, at least about 22 times,at least about 23 times, at least about 24 times, at least about 25 times, at least about 26 times, at least about 27 times, at least about 28 times, at least about 29 times, at least about 30 times, at least about 31 times, at least about 32 times, at least about 33 times, at least about 34 times, at least about 35 times, at least about 36 times, at least about 37 times, at least about 38 times, at least about 39 times, at least about 40 times, at least about 41 times, at least about 42 times, at least about 43 times, at least about 44 times, at least about 45 times, at least about 46 times, at least about 47 times, at least about 48 times, at least about 49 times, at least about 50 times, at least about 51 times, at least about 52 times, at least about 53 times, at least about 54 times, at least about 55 times, at least about 56 times, at least about 57 times, at least about 58 times, at least about 59 times, at least about 60 times, at least about 61 times, at least about 62 times , at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, or at least about 100 freeze-thaw cycles.

[0281] In an embodiment, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is heat shock. In an embodiment, the first polypeptide is heat shocked by heating the first polypeptide to at least 90°C, at least 91°C, at least 92°C, at least 93°C, at least 94°C, or at least 95°C for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, or more; placing the container containing the first polypeptide on ice and then returning the fusion protein to room temperature. In embodiments, the first polypeptide is heat shocked by heating the first polypeptide to at least 90°C, at least 91°C, at least 92°C, at least 93°C, at least 94°C, or at least 95°C for at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, or more; placing the container containing the first polypeptide on ice and then allowing the fusion protein to return to room temperature.

[0282] In an embodiment, heating to at least 90°C, at least 91°C, at least 92°C, at least 93°C, at least 94°C, or at least 95°C is known to destabilize, unfold, degrade, or misfold the first polypeptide. In an embodiment, the one or more conditions known to destabilize, unfold, degrade, or misfold the first polypeptide is heating to at least 90°C. In an embodiment, the one or more conditions known to destabilize, unfold, degrade, or misfold the first polypeptide is heating to at least 95°C. In an embodiment, the one or more conditions known to destabilize, unfold, degrade, or misfold the first polypeptide is heating to at least 90°C for at least 30 minutes. In an embodiment, the one or more conditions known to destabilize, unfold, degrade, or misfold the first polypeptide is heating to at least 95°C for at least 30 minutes. In embodiments, the one or more conditions known to destabilize, unfold, degrade, or misfold the first polypeptide is heating to at least 95° C. for 10-30 minutes.

[0283] In an embodiment, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is exposure to a non-physiological pH. The term non-physiological pH refers to a pH that is not 7.2 to about 7.4. In an embodiment, the non-physiological pH is an acidic pH. In an embodiment, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is exposure to an acidic pH. In an embodiment, the condition known to destabilize, unfold, degrade, or misfold the first polypeptide is exposure to an acidic pH of about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, or about 0.5 to about 1. In embodiments, the acidic pH is about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4. 2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, or about 7.1 (including all subranges and ranges therebetween). In embodiments, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposure to a pH of about 4. In embodiments, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposure to a pH of about 4 for about 10 to 30 minutes. In embodiments, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposure to a pH of about 4 for about 30 minutes.

[0284] In embodiments, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposure to a pH of about 4 and a temperature of at least 95° C. for at least 30 minutes. In embodiments, the conditions known to destabilize, unfold, degrade, or misfold the first polypeptide are exposure to a pH of about 4 and a temperature of at least 95° C. for between 10 and 30 minutes.

[0285] In an embodiment, the non-physiological pH is a basic pH, which is about 7.5 to about 14, about 7.5 to about 13, about 7.5 to about 12, about 7.5 to about 11, about 7.5 to about 10, about 7.5 to about 9, or about 7.5 to about 8.5. In embodiments, the basic pH is about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9 ...5, about 11.6, about 11.7, about 11.8, about 11.9, about 11.1, about 11.2, about 11.3, 0.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12, about 12.1, about 12.2, about 12.3, about 12.4, about 12.5, about 12.6, about 12.7, about 12.8, about 12.9, about 13, about 13.1, about 13.2, about 13.3, about 13.4, about 13.5, about 13.6, about 13.7, about 13.8, about 13.9, or about 14 (including all subranges and ranges therebetween).

[0286] In some embodiments, the activity of the first polypeptide is lost less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 2%, or less than 1% compared to a control polypeptide after exposure to one or more conditions. In some embodiments, the activity of the first polypeptide is lost less than about 25% compared to a control polypeptide after exposure to one or more conditions. In some embodiments, the activity of the first polypeptide is lost less than about 20% compared to a control polypeptide after exposure to one or more conditions.

[0287] In some embodiments, a first polypeptide expressed as a fusion protein comprising a first polypeptide and a second polypeptide having phase behavior retains about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of its activity compared to a control polypeptide after exposure to one or more conditions. In some embodiments, the first polypeptide retains at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of its activity compared to a control after exposure to one or more conditions. In some embodiments, the first polypeptide retains at least 80% of its activity after exposure to one or more conditions compared to a control. The activity of the first polypeptide can be measured using a functional assay, an enzyme-linked immunosorbent assay, or flow cytometry.

[0288] In some embodiments, the first polypeptide is maintained at a temperature between about −20° C. and about 35° C. (e.g., about −20° C., about −19° C., about −18° C., about −17° C., about −16° C., about −15° C., about −14° C., about −13° C., about −12° C., about −11° C., about −10° C., about −9° C., about −8° C., about −7° C., about −6° C., about −5° C., about −4° C., about −3° C., about −2° C., about −4° C., about −5 ... 1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C 31°C, about 32°C, about 33°C, about 34°C, about 35°C (including any values ​​and subranges therebetween) for about 1 week to about 10 years (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years (including all subranges and values ​​therebetween)).In embodiments, the first polypeptide is expressed for a period of between about 1 week and 10 weeks (e.g., at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, At least about 9 years, or for at least about 10 years (including any value or subrange therebetween), at -20°C to about 35°C (e.g., about -20°C, about -19°C, about -18°C, about -17°C, about -16°C, about -15°C, about -14°C, about -13°C, about -12°C, about -11°C, about -10°C, about -9°C, about -8°C, about -7°C, about -6°C, about -5°C, about -4°C, about -3°C, about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 12°C, about 14°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 39°C, about 40°C, about 40°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, C., about 6.0°C, about 7.0°C, about 8.0°C, about 9.0°C, about 10.0°C, about 11.0°C, about 12.0°C, about 13.0°C, about 14.0°C, about 15.0°C, about 16.0°C, about 17.0°C, about 18.0°C, about 19.0°C, about 20.0°C, about 21.0°C, about 22.0°C, about 23.0°C, about 24.0°C, about 25.0°C, about 26.0°C, about 27.0°C, about 28.0°C, about 29.0°C, about 30.0°C, about 31.0°C, about 32.0°C, about 33.0°C, about 34.0°C, about 35.0°C (including all subranges and values ​​therebetween).

[0289] In some embodiments, the first polypeptide retains its activity after exposure for about 1 week to about 10 years at about 4° C. In some embodiments, the first polypeptide retains its activity at about 4° C. for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more (including all subranges and values ​​therebetween). In some embodiments, the first polypeptide retains its activity at least about 4° C. for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, or more (including all subranges and values ​​therebetween).

[0290] In some embodiments, the first polypeptide retains its activity after exposure for about 6 months to about 10 years at −20° C. In some embodiments, the first polypeptide retains its activity at about 4° C. for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more (including all subranges and values ​​therebetween). In some embodiments, the first polypeptide retains its activity at least about 4° C. for at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, or more (including all subranges and values ​​therebetween).

[0291] In some embodiments, one or more of the following techniques are used to assess the ability of the methods of the disclosure to stabilize a first polypeptide: size exclusion chromatography, ion exchange and reverse-phase high performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, capillary electrophoresis, potency assays, dynamic light scattering, spectroscopy, microscopy, and physicochemical measurements of appearance, pH, and particle size.

[0292] In some embodiments, expression of the first polypeptide as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior protects the first polypeptide from degradation during storage (e.g., freezing). In some embodiments, the methods described herein protect the first polypeptide from degradation, particularly during multiple freeze-thaw cycles. Aggregation of the first polypeptide can be observed visually by microscopy and / or by a technique selected from the group consisting of X-ray scattering, laser diffraction, analytical ultracentrifugation, dynamic light scattering, nanoparticle tracking analysis, resonance mass spectrometry, size exclusion chromatography, gel permeation chromatography, light obscuration methods, and combinations thereof. In some embodiments, the first polypeptide can be frozen and stored as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior at a temperature of about -80°C to about 40°C, e.g., about -80°C, about -75°C, about -70°C, about -65°C, about -60°C, about -55°C, about -50°C, about -45°C, about -40°C, about -35°C, about -30°C, about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, or about 40°C.

[0293] In some embodiments, when a first polypeptide is stored as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior, the shelf life of the first polypeptide is at least about 10% longer than the shelf life of a first polypeptide not expressed as a fusion protein comprising a second polypeptide having phase behavior. For example, in some embodiments, the shelf life is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% longer than the shelf life of a first polypeptide not expressed as a fusion protein comprising a second polypeptide having phase behavior stored at the same temperature. In some embodiments, when a first polypeptide is stored as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior, the shelf life of the first polypeptide is at least one month longer than a first polypeptide that is not expressed as a fusion protein comprising a second polypeptide having phase behavior. For example, in some embodiments, the shelf life is at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months longer than the shelf life of a first polypeptide that is not expressed as a fusion protein comprising a second polypeptide having phase behavior stored at the same temperature.

[0294] In some embodiments, the fusion polypeptide comprising the first polypeptide and the second polypeptide having phase behavior is stored at about -80°C. In some embodiments, the fusion polypeptide comprising the first polypeptide and the second polypeptide having phase behavior is stored at about -20°C. In some embodiments, the fusion polypeptide comprising the first polypeptide and the second polypeptide having phase behavior is stored at about 4°C. In some embodiments, the fusion polypeptide comprising the first polypeptide and the second polypeptide having phase behavior is stored at about 37°C. In some embodiments, when the fusion polypeptide comprising the first polypeptide and the second polypeptide having phase behavior is stored at about -80°C, about -20°C, about 4°C, or about 37°C, the shelf life of the first polypeptide is at least about 10% longer than when the first polypeptide is not expressed as a fusion protein and stored under the same conditions. For example, the shelf life of the first polypeptide will be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% longer than the shelf life of the first polypeptide not expressed as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior at the same temperature. As used herein, an increase in shelf life may refer to an increase in the time that the first polypeptide is stored and still retains its function. For example, a first polypeptide comprising a monoclonal antibody that binds to programmed cell death protein 1 (PD1) that retains its function retains the same affinity for PD1.

[0295] Methods for improving production of a first polypeptide In some embodiments, provided herein are methods of improving the yield of a first polypeptide, the methods comprising: (i) expressing a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior; and (ii) separating the first polypeptide from the second polypeptide, wherein the yield of the first polypeptide when expressed as a fusion protein is improved compared to the yield of the first polypeptide when the first polypeptide is not expressed as a fusion protein.

[0296] In some embodiments, the first polypeptide is separated from the second polypeptide by protease cleavage. In some embodiments, the protease is furin.

[0297] In some embodiments, the fusion protein is expressed in a host cell selected from a mammalian cell, a bacterial cell, a fungal cell, a yeast cell, and a plant cell. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a CHO cell. In some embodiments, the host cell is a HEK293 cell.

[0298] In some embodiments, methods are provided herein for increasing the yield of a first polypeptide during production of the first polypeptide. In some embodiments, the yield of a first polypeptide produced as a fusion protein with a second polypeptide having phase behavior is greater than the yield of the first polypeptide not expressed as a fusion protein (i.e., not fused to a second polypeptide having phase behavior). In embodiments, the yield of the first polypeptide when expressed as a fusion protein with a second polypeptide having phase behavior is about 1 mg / liter to about 1,000 mg / liter. In embodiments, the yield of the first polypeptide when expressed as a fusion protein with a second polypeptide having phase behavior is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 10 ... g, about 21mg, about 22mg, about 23mg, about 24mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30mg, about 31mg, about 32mg, about 33mg, about 34mg, about 35mg , about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, About 51mg, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg, about 61mg, about 62mg, about 63mg, about 64mg, about 65mg, about 66mg, about 67mg, about 68mg, about 69mg, about 70mg, about 71mg, about 72mg, about 73mg, about 74mg, about 75mg, about 76mg, about 77mg, about 78mg, about 79mg, about 80mg, about 8 1 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,About 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 40 0mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1,000 mg. In embodiments, the yield of a first polypeptide when expressed as a fusion protein with a second polypeptide having a phase behavior is at least about 1 mg, at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, at least about 6 mg, at least about 7 mg, at least about 8 mg, at least about 9 mg, at least about 10 mg, at least about 11 mg, at least about 12 mg, at least about 13 mg, at least about 14 mg, at least about 15 mg, at least about 16 mg, at least about 17 mg, at least about 18 mg, at least about 19 mg, at least about 20 mg, at least about 21 mg, at least about 22 mg, at least about 23 mg, at least about 24 mg, at least about 25 mg, at least about 26 mg, at least about 27 mg, at least about 28 mg, at least about 29 mg, at least about 30 mg, at least about 31 mg, at least about 32 mg, at least about 33 mg,at least about 34 mg, at least about 35 mg, at least about 36 mg, at least about 37 mg, at least about 38 mg, at least about 39 mg, at least about 40 mg, at least about 41 mg, at least about 42 mg, at least about 43 mg, at least about 44 mg, at least about 45 mg, at least about 46 mg, at least about 47 mg, at least about 48 mg, at least about 49 mg, at least about 50 mg, at least about 51 mg, at least about 52 mg, at least about 53 mg, at least about 54 mg, at least about 55 mg, about 56 mg, at least about 57 mg, at least about 58 mg, at least about 59 mg, at least about 60 mg, at least about 61 mg, at least about 62 mg, at least about 63 mg, at least about 64 mg, at least about 65 mg, at least about 66 mg, at least about 67 mg, at least about 68 mg, at least about 69 mg, at least about 70 mg, at least about 71 mg, at least about 72 mg, at least about 73 mg, at least about 74 mg, at least about 75 mg, at least about 76 mg, at least about 77 mg, at least about 78 mg, at least about 79 mg, at least about 80 mg, at least about 81 mg, at least about 82 mg, at least about 83 mg, at least about 84 mg, at least about 85 mg, at least about 86 mg, at least about 87 mg, at least about 88 mg, at least about 89 mg, at least about 90 mg, at least about 91 mg, at least about 92 mg, at least about 93 mg, at least about 94 mg, at least about 95 mg, at least about 96 mg, at least about 97 mg, at least about 98 mg, at least about 99 mg, at least about 100 mg, at least Also about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg,at least about 320 mg, at least about 330 mg, at least about 340 mg, at least about 350 mg, at least about 360 mg, at least about 370 mg, at least about 380 mg, at least about 390 mg, at least about 400 mg, at least about 410 mg, at least about 420 mg, at least about 430 mg, at least about 440 mg, at least about 450 mg, at least about 460 mg, at least about 470 mg, at least about 480 mg, at least about 490 mg, at least about 500 mg, at least about 510 mg, at least about 520 mg, at least about 530 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 570 mg, at least about 580 mg, at least about 590 mg, at least about 600 mg, at least about 610 mg, at least about 620 mg, at least about 630 mg, at least about 640 mg, at least about 650 mg, at least about 660 mg, mg, at least about 670 mg, at least about 680 mg, at least about 690 mg, at least about 700 mg, at least about 710 mg, at least about 720 mg, at least about 730 mg, at least about 740 mg, at least about 750 mg, at least about 760 mg, at least about 770 mg, at least about 780 mg, at least about 790 mg, at least about 800 mg, at least about 810 mg, at least about 820 mg, at least about 830 mg, at least about 840 mg, at least about 850 mg, at least about 860 mg, at least about 870 mg, at least about 880 mg, at least about 890 mg, at least about 900 mg, at least about 910 mg, at least about 920 mg, at least about 930 mg, at least about 940 mg, at least about 950 mg, at least about 960 mg, at least about 970 mg, at least about 980 mg, at least about 990 mg, or at least about 1,000 mg. In some embodiments, the yield of the first polypeptide is greater than 15 mg per liter of host cell suspension, greater than 30 mg per liter, greater than 50 mg per liter, greater than 100 mg per liter, greater than 200 mg per liter, or greater than 200 mg per liter. In embodiments, the yield of the first polypeptide is greater thanMore than 75 mg per liter of host cell suspension. The cell suspension may be referred to interchangeably as a cell culture.

[0299] In some embodiments, the yield of a first polypeptide produced as a fusion protein with a second polypeptide having phase behavior is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, than the yield of the first polypeptide not expressed as a fusion protein (i.e., expressed not fused to a second polypeptide having phase behavior). In embodiments, the yield of a first polypeptide produced as a fusion protein with a second polypeptide having phase behavior is about 3-fold greater than the yield of the first polypeptide not expressed as a fusion protein.

[0300] In some embodiments, the yield of a first polypeptide produced as a fusion protein with a second polypeptide having phase behavior is at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 380%, at least about 400%, at least about 425%, at least about 440%, at least about 460%, at least about 485%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 585%, at least about 590%, at least about 600%, at least about 610%, at least about 620%, at least about 630%, at least about 640%, at least about 650%, at least about 660%, at least about 670%, at least about 685%, at least about 690%, at least about 700%, at least about 710%, at least about 720%, at least about 750%, at least about 750%, at least about 760%, at least about 770%, at least about 785%, at least about 790%, at least about 800%, at least about 810%, at least about 820%, at least about 830%, at least about 840%, at least about 850%, at least about 860%, at least about 870%, at least about 885%, at least about 890%, at least about 900%, at at least about 400%, at least about 425%, at least about 450%, at least about 475%, at least about 500%, at least about 525%, at least about 550%, at least about 575%, at least about 600%, at least about 625%, at least about 650%, at least about 675%, at least about 700%, at least about 725%, at least about 750%, at least about 775%, at least about 800%, at least about 825%, at least about 850%, at least about 875%, at least about 900%, at least about 925%, at least about 950%, at least about 975%, at least about 1,000%, at least about 1,100%, at least about 1,125%, at least about 1,150%, at least about 1,175%, at least about 1,200%, at least about 1,225%, at least about 1,250%, at least about 1,275%, at least about 1,300%, at least about 1,325%, at least about 1,350%, at least about 1,375%, at least about 1,400%, at least about 1,425%, at least about 1,450%, at least about 1,475%, at least about 1,500%, at least about 1,525%, at least about 1,550%, at least about 1,575%, at least about 1,600%, at least about 1,625%, at least about 1,650%, at least about 1,675%, at least about 1,700%, at least about 1,725%, at least about 1,750%, at least about 1,775%, at least about 1,800%, at least about 1,825%, at least about 1,850%, at least about 1,875%, at least about 1,900%, at least about 1,925%, at least about 1,950%, at least about 1,975%, or at least about 2,000% greater (including all ranges and values ​​therebetween). In embodiments, the yield of the first polypeptide is about 300% greater than the yield of the first polypeptide not expressed as a fusion protein.

[0301] Method for purifying a first polypeptide of a fusion protein Also provided herein is a method of purifying a first polypeptide, the method comprising the steps of: (i) providing a fusion protein comprising the first polypeptide and a second polypeptide having a phase behavior; (ii) applying a first environmental agent to reversibly aggregate the fusion protein; (iii) separating the fusion protein aggregates from at least one contaminant; and (iv) applying a second environmental agent to disaggregate the fusion protein. In some embodiments, the fusion protein is any fusion protein described herein.

[0302] In some embodiments, the at least one contaminant is a solvent, a protein, a peptide, a carbohydrate, a nucleic acid, a virus, a cell (e.g., a bacterial, yeast, or mammalian cell), a carbohydrate, a lipid, or a lipopolysaccharide. In some embodiments, the contaminant is an endotoxin or a mycotoxin.

[0303] Without being bound by theory, application of the first environmental agent causes aggregation of the fusion protein through intermolecular interactions between the polypeptides having phase behavior, resulting in fusion protein aggregates. In some embodiments, the environmental agent causes the formation of fusion protein aggregates that are larger than the size of the fusion protein before the introduction of the environmental agent. As used herein, the phrase "increased size" can refer to an increase in the diameter of the fusion protein or an increase in the mass of the fusion protein. In some embodiments, the increase in size is an increase in the molar mass of the fusion protein. In some embodiments, the increase in size is an increase in the hydrodynamic radius of the fusion protein.

[0304] In some embodiments, the increase in size is stabilized by non-covalent interactions between the polypeptides having phase behavior, hi some embodiments, the non-covalent interactions are dipole-dipole forces, van der Waals forces, London dispersion forces, hydrogen bonds, hydrophobic interactions, and / or electrostatic interactions.

[0305] In some embodiments, the size of the fusion protein aggregates is at least about 2 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least about 100 times larger than the size of the fusion protein before the introduction of the environmental factor. In some embodiments, the size of the fusion protein aggregates is at least 2 times larger than the size of the fusion protein before the introduction of the environmental factor. In some embodiments, the size of the fusion protein aggregates is at least 5 times larger than the size of the fusion protein before the introduction of the environmental factor. In some embodiments, the size of the fusion protein aggregates is at least 10 times larger than the size of the fusion protein before the introduction of the environmental factor. In some embodiments, the size of the fusion protein aggregates is at least 25 times larger than the size of the fusion protein before the introduction of the environmental factor.

[0306] In some embodiments, the increase in size of the fusion protein aggregates compared to the fusion protein can be visually observed with the naked eye, for example, the increase in size can cause a change in color, clarity, viscosity of a composition containing the complex, and / or the solubility of the complex can change (e.g., precipitate out of solution), and such changes are observable by humans without the use of any special equipment.

[0307] In some embodiments, one of skill in the art can measure the increase in size of the fusion protein aggregates according to methods known in the art, hi some embodiments, the increase in size can be measured using a technique selected from the group consisting of X-ray scattering, small angle X-ray scattering, wide angle X-ray scattering, dynamic light scattering, analytical ultracentrifugation, size exclusion chromatography, and photon correlation spectroscopy.

[0308] In some embodiments, the environmental factor is an environmental factor described herein. For example, in some embodiments, the environmental factor is (a) one or more changes in temperature, pH, salt concentration, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, enzymes, denaturants; or (c) the application of electromagnetic waves.

[0309] In some embodiments, the fusion protein aggregates are separated from one or more impurities by washing the fusion protein aggregates. In some embodiments, washing the fusion protein aggregates does not prevent aggregation of the fusion protein. In some embodiments, the fusion protein aggregates are washed with a buffer. Non-limiting examples of buffers include sodium acetate, saline, glycine-HCL, cacodylate buffer, Tris-HCl, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), citrate, phosphate buffer, tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), and tris(hydroxymethyl)aminomethane (Tris). In some embodiments, the buffer comprises one or more of arginine, histidine, urea, pluronic acid, and Triton-X-100. In some embodiments, the AAV-purified matrix complexes are washed with a solvent. Non-limiting examples of solvents include acetone, acetonitrile, dimethylformamide, water, ethanol, toluene, methyl acetate, and ethyl acetate.

[0310] In some embodiments, the fusion protein aggregates are separated from at least one impurity based on size. In some embodiments, the fusion protein aggregates are separated from at least one impurity based on diameter. In some embodiments, the fusion protein aggregates are separated from at least one impurity based on radius. In some embodiments, the fusion protein aggregates are separated from at least one impurity based on mass. In some embodiments, the fusion protein aggregates are separated from at least one impurity based on molar mass. In some embodiments, the AAV purification matrix is ​​a fusion protein aggregate separated from at least one impurity based on size by using a technique selected from the group consisting of centrifugation, tangential flow filtration, analytical ultracentrifugation, membrane chromatography, high performance liquid chromatography, size exclusion chromatography, dead-end filtration, sonication, centrifugation, counter-current centrifugation, and fast protein liquid chromatography.

[0311] In some embodiments, separation is achieved using sonic separation. In some embodiments, the sonic waves have a frequency of about 1 Hz to 2,000 kHz. In some embodiments, the sonic waves have a frequency of about 1 Hz, about 5 Hz, about 10 Hz, about 20 Hz, about 30 Hz, about 40 Hz, about 50 Hz, about 60 Hz, about 70 Hz, about 80 Hz, about 90 Hz, about 100 Hz, about 200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900 Hz, about 1 kHz, about 100 kHz, about 200 kHz, about 300 kHz ... Hz, about 400 kHz, about 500 kHz, about 600 kHz, about 700 kHz, about 800 kHz, about 900 kHz, about 1,000 kHz, about 1,100 kHz, about 1,200 kHz, about 1,300 kHz, about 1,400 kHz, about 1,500 kHz, about 1,600 kHz, about 1,700 kHz, about 1,800 kHz, about 1,900 kHz, or about 2,000 kHz.

[0312] In some embodiments, the fusion protein aggregates are separated from at least one impurity based on size using centrifugation. In some embodiments, a relative centrifugal force (RCF) of about 100 to about 16,000 RCF, e.g., about 500 to about 16,000 RCF, about 1,000 RCF to 16,000 RCF, is applied to separate the fusion protein aggregates from at least one impurity. In some embodiments, a relative centrifugal force (RCF) of at least 500, e.g., at least about 500 RCF, at least about 600 RCF, at least about 700 RCF, at least about 800 RCF, at least about 900 RCF, at least about 1,000 RCF, at least about 2,000 RCF, at least about 3,000 RCF, at least about 3,500 RCF, at least about 4,000 RCF, at least about 5,000 RCF, at least about 6,000 RCF, at least about 7,000 RCF, at least about 8,000 RCF, is applied to separate the fusion protein aggregates from at least one impurity. In some embodiments, a RCF of at least about 9,000 RCF, at least about 10,000 RCF, at least about 11,000 RCF, at least about 12,000 RCF, at least about 13,000 RCF, at least about 14,000 RCF, at least about 15,000 RCF, at least about 16,000 RCF, at least about 17,000 RCF, at least about 18,000 RCF, at least about 19,000 RCF, or at least about 20,000 RCF is applied to separate the fusion protein aggregates from the at least one impurity.

[0313] In some embodiments, the fusion protein aggregates are separated from at least one impurity based on size by using TFF. In some embodiments, the fusion protein aggregates can be separated from at least one impurity based on size using TFF, a process also referred to herein as "diafiltration". Diafiltration includes both a washing step and an elution step. Washing removes impurities contained in the composition comprising the fusion protein aggregates. Elution separates the purified first polypeptide from the second polypeptide. In some embodiments, the fusion protein aggregates are concentrated using TFF. In some embodiments, TFF can be used to increase the concentration of fusion protein aggregates in the composition, a process also referred to herein as "concentration".

[0314] Tangential flow filtration uses both microfiltration and ultrafiltration membranes to separate and / or concentrate molecules. Microfiltration membranes typically have pore sizes between 0.1 μm and 10 μm. Ultrafiltration membranes typically have smaller pore sizes than microfiltration membranes, between 0.001 μm and 0.1 μm. In some embodiments, membranes with pore sizes between about 0.001 μm and about 10 μm are utilized in the methods of the present disclosure. In some embodiments, the membrane has a pore size of about 0.001 μm, about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm (including all values ​​and ranges therebetween). In some embodiments, the membrane has a pore size of about 0.1 μm. In some embodiments, the membrane has a pore size of about 0.2 μm.

[0315] In some embodiments, the membrane is formed from hydrophilized poly(vinylidene fluoride) (PVDF), polyethersulfone (PES), cellulose phosphate, diethylaminoethyl cellulose, polysulfone, regenerated cellulose, nylon, cellulose nitrate, cellulose acetate, PEGylated PES, and sulfonated PES.

[0316] In TFF, a membrane is placed tangentially to the flow of a fluid mixture, causing the fluid mixture to flow tangentially across a first side of the membrane. At the same time, a fluid medium is placed in contact with the second surface of the membrane. Transmembrane pressure is the force that forces the fluid through the membrane, carrying the permeable molecules with it.

[0317] In some embodiments, size-based separation of fusion protein aggregates from one or more contaminants or impurities is performed using TFF at a transmembrane pressure of about 0.1 bar to about 3 bar. In some embodiments, the transmembrane pressure is about 0.1 bar, about 0.2 bar, about 0.3 bar, about 0.4 bar, about 0.5 bar, about 0.6 bar, about 0.7 bar, about 0.8 bar, about 0.9 bar, about 1.0 bar, about 1.1 bar, about 1.2 bar, about 1.3 bar, about 1.4 bar, about 1.5 bar, about 1.6 bar, about 1.7 bar, about 1.8 bar, about 1.9 bar, about 2.0 bar, about 2.1 bar, about 2.2 bar, about 2.3 bar, about 2.4 bar, about 2.5 bar, about 2.6 bar, about 2.7 bar, about 2.8 bar, about 2.9 bar, or about 3.0 bar (including all values ​​and ranges therebetween). In some embodiments, the transmembrane pressure is about 1.5 bar.

[0318] In some embodiments, the cross-flow flow rate is adjusted to improve separation of the fusion protein aggregates herein from one or more contaminants. The cross-flow flow rate is the flow rate of the solution passing through the membrane through the feed channel. This provides a force to sweep out molecules that can limit the flow of the filtrate. In some embodiments, the cross-flow flow rate is about 500 L / m2 / h to about 2,000 L / m2 / h. In some embodiments, the cross flow rate is about 500 L / m2 / h, about 600 L / m2 / h, about 700 L / m2 / h, about 800 L / m2 / h, about 900 L / m2 / h, about 1,000 L / m2 / h, about 1,100 L / m2 / h, about 1,200 L / m2 / h, about 1,300 L / m2 / h, about 1,400 L / m2 / h, about 1,500 L / m2 / h, about 1,600 L / m2 / h, about 1,700 L / m2 / h, about 1,800 L / m2 / h, about 1,900 L / m2 / h, or about 2,000 L / m2 / h (including all values ​​and ranges therebetween). In some embodiments, the cross flow rate is about 960 L / m2 / h. In some embodiments, TFF separation is performed by using a membrane that retains fusion protein aggregates while allowing contaminants to pass through.

[0319] In some embodiments, after separation of the fusion protein aggregate from at least one contaminant, the first polypeptide is separated from the second polypeptide.In some embodiments, the first polypeptide is separated from the second polypeptide by introducing an enzyme that breaks amide bond to separate the first polypeptide from the second polypeptide.In some embodiments, the enzyme is a protease.

[0320] In some embodiments, after the separation of the fusion protein aggregates from at least one contaminant, an environmental factor is applied to disaggregate the fusion protein aggregates. The environmental factor applied to disaggregate the fusion protein aggregates can be any environmental factor described herein.

[0321] In some embodiments, after the fusion protein aggregates are separated from at least one impurity, the first polypeptide is eluted as a fusion protein comprising the first polypeptide and a second polypeptide having phase behavior, in some embodiments, the first polypeptide is eluted as a disaggregated fusion protein comprising the first polypeptide and a second polypeptide having phase behavior.

[0322] In some embodiments, the environmental factor comprises altering the pH of the composition comprising the first polypeptide. In some embodiments, the pH is about 0.1 units, about 0.2 units, about 0.3 units, about 0.4 units, about 0.5 units, about 0.6 units, about 0.7 units, about 0.8 units, about 0.9 units, about 1.0 units, about 1.1 units, about 1.2 units, about 1.3 units, about 1.4 units, about 1.5 units, about 1.6 units, about 1.7 units, about 1.8 units, about 1.9 units, about 2.0 units, about 2.1 units, about 2.2 units, about 2.3 units, about 2.4 units, about 2.5 units, about 2.6 units, about 2.7 units, about 2.8 units, about 2.9 units, about 3.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, about 6.0 units, about 6.1 units, about 6.2 units, about 6.3 units, about 6.4 units, about 6.5 units, about 6.6 units, about 6.7 units, about 6.8 units, about 6.9 units, about The amount can be increased by about 0.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, or about 6.0 units. In some embodiments, the pH is about 0.1 units, about 0.2 units, about 0.3 units, about 0.4 units, about 0.5 units, about 0.6 units, about 0.7 units, about 0.8 units, about 0.9 units, about 1.0 units, about 1.1 units, about 1.2 units, about 1.3 units, about 1.4 units, about 1.5 units, about 1.6 units, about 1.7 units, about 1.8 units, about 1.9 units, about 2.0 units, about 2.1 units, about 2.2 units, about 2.3 units, about 2.4 units, about 2.5 units, about 2.6 units, about 2.7 units, about 2.8 units, about 2.9 units, about 3.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, about 6.0 units, about 6.1 units, about 6.2 units, about 6.3 units, about 6.4 units, about 6.5 units, about 6.6 units, about 6.7 units, about 6.8 units, about 6.9 units, about 7.0 units, about 7.1 units, about 7.2 units, In some embodiments, the purified fusion protein is reduced by about 0.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, or about 6.0 units. In some embodiments, the purified fusion protein is aggregated at a pH of about 2.In some embodiments, the fusion protein is purified at a pH of about 3.

[0323] In some embodiments, the environmental factor comprises altering the temperature of the composition comprising the fusion protein, in some embodiments, the temperature is increased by 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the temperature is reduced by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0324] In some embodiments, the environmental factor comprises altering the ionic strength of the composition comprising the fusion protein. In some embodiments, the alteration of the ionic strength is effected by increasing the concentration of a salt. In some embodiments, the alteration of the ionic strength is effected by decreasing the concentration of a salt. Non-limiting examples of salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium citrate, glycine, arginine, copper sulfate, sodium iodide, ammonium sulfate, and sodium sulfate. In some embodiments, dialysis is used to alter the concentration of salts of the composition comprising the fusion protein, contaminants, and / or molecules.

[0325] In some embodiments, the environmental factor comprises the addition of a reducing agent to the composition comprising the fusion protein, in some embodiments, the one or more reducing agents are selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), hydrazine, borohydride, amine boranes, lower alkyl substituted amine boranes, triethanolamine, and N,N,N',N'-tetramethylethylenediamine (TEMED).

[0326] In some embodiments, the method for purifying a fusion protein comprising a first polypeptide described herein is completed in about 30 minutes to about 24 hours. In some embodiments, the method described herein is completed in about 30 minutes to about 24 hours. In some embodiments, the method is completed in about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In some embodiments, the method for purifying a fusion protein and / or a first polypeptide described herein is completed in about 2 hours to about 10 hours.

[0327] In some embodiments, after purification of the fusion protein, the first polypeptide is separated from the second polypeptide. In some embodiments, the first polypeptide is separated from the second polypeptide by introducing an enzyme that cleaves amide bonds to separate the first polypeptide from the second polypeptide. In some embodiments, the enzyme is a protease.

[0328] In some embodiments, the purification yield of the fusion protein and / or first polypeptide is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0329] In some embodiments, the fusion protein and / or first polypeptide is purified to a purity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0330] In some embodiments, the methods described herein allow for the purification of at least 0.1 kg, at least about 0.2 kg, at least about 0.3 kg, at least about 0.4 kg, at least about 0.5 kg, at least about 0.6 kg, at least about 0.7 kg, at least about 0.8 kg, at least about 0.9 kg, at least about 1 kg, at least about 2 kg, at least about 3 kg, at least about 4 kg, at least about 5 kg, at least about 6 kg, at least about 7 kg, at least about 8 kg, at least about 9 kg, at least about 10 kg, or more (including all values ​​and ranges therebetween) of the fusion protein and / or first polypeptide per day.

[0331] Methods for carrying out enzymatic processes on nucleic acid substrates The fusion protein can be used to perform an enzymatic process on a nucleic acid substrate in a controlled manner. In some embodiments, the first protein of the fusion protein is an enzyme or a catalytic fragment or derivative thereof. For example, the process can be performed in a two-phase composition. In such a composition, the substrate can be present in a first phase and the fusion protein can be present in a second phase. If necessary, an environmental factor can be applied that causes the fusion protein to enter the first phase and thereby contact the substrate. In some embodiments, a second environmental factor can be applied that causes the fusion protein to exit the first phase and the fusion protein can no longer contact the substrate. This process can be used to perform a multi-step enzymatic process on a substrate in a controlled manner. For example, a two-phase composition can be provided in which the substrate can be present in a first phase and multiple fusion proteins can be present in a second phase. The multiple fusion proteins include fusion proteins that include different phase behaviors. If necessary, a first environmental factor can be applied that causes one or more fusion proteins to contact and / or separate from the substrate. In addition, a second environmental factor can be applied that brings one or more additional fusion proteins into contact with and / or separates them from the substrate. Such a process can aid in the synthesis of a biologic of interest, such as in vitro transcription of RNA.

[0332] In an embodiment, the nucleic acid substrate is DNA or RNA. In an embodiment, the DNA or RNA is single stranded. In an embodiment, the DNA or RNA is double stranded. In an embodiment, the nucleic acid is RNA, and the RNA is selected from messenger RNA (mRNA), transfer RNA (tRNA), microRNA, or ribosomal RNA (rRNA). In an embodiment, the mRNA is a small RNA. The small RNA comprises about 18 to about 30 nucleotides, for example, about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0333] In embodiments, the enzyme is any one of the NBPs described herein. In embodiments, the NBP is selected from the group consisting of T7 RNA polymerase, RNase inhibitor, 2'-O-methyltransferase, inorganic pyrophosphatase, poly(A) polymerase, DNase I, calf intestinal phosphatase, Antarctic phosphatase, the D1 subunit of vaccinia virus mRNA capping enzyme, guanine-7-methyltransferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), guanylyltransferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), RNA triphosphatase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), the D12 subunit of vaccinia virus mRNA capping enzyme, stem-loop binding protein (SBP), and the like. protein, heterogeneous nuclear ribonucleoprotein (hnRNP), GroEL, Edc3, DHX9, Xrn1, Dcp1, Dcp2, LAF-1, MEG-1, MEG-3, ASF / SF2 splicing factor, serine / arginine-rich splicing factor 4 (SRp75), serine and arginine-rich splicing factor 1 (SRSF1), L3 ribosomal protein, L4 ribosomal protein, L13 ribosomal protein, L20 ribosomal protein, L22 ribosomal protein, L24 ribosomal protein, L24e ribosomal protein, S12 ribosomal protein, S14 ribosomal protein, and eukaryotic initiation factor 4E-binding protein 1 (4EBP1), Tat, Rev, and RSG-1.2 peptide, poly(A) binding protein (PABP), eukaryotic translation initiation factor 4E (eIF4E), eukaryotic translation initiation factor 3 subunit D (eIF3D), heterogeneous nuclear ribonucleoprotein (hnRNP), RNA-specific adenosine deaminase 1 (ADAR1), RNA-specific adenosine deaminase 2 (ADAR2), Bacillus subtilis subtilis (Bscscp), Y-box protein 1 cold shock domain (YB1-CSD), Fox-1 protein (FOX1), poly(A)-binding protein (PABP), Staufen protein, TIS11d, zinc finger protein (ZNF), Z-DNA binding protein 1 (ZBP1), retinoic acid-inducible gene-I (RIG-I)-like protein, Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR3), Toll-like receptor 8 (TLR8), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), interferon-inducible protein with tetratricopeptide repeats 1 (IFIT1), protein kinase R (PKR), oligoadenylate synthase-like (OASL) proteins (e.g., OAS1, OAS2, OAS3, or OASL), ribonuclease E (RNASE E), gamma interferon-inducible protein Ifi-16 (IF116), cyclic GMP-AMP synthase (cGAS), or catalytic fragments thereof.

[0334] In embodiments, the NBP is a short sequence motif (SLiM), RG[G] repeat, RGG repeat, RS / RG rich domain, K / R basic patch, molecular recognition function, low complexity sequence, RNA recognition motif, double stranded RNA binding domain, K homology domain, zinc finger domain (e.g., CCHH ZF domain, CCCC (Ran-BP2) domain, CCCH ZF domain), RGG domain, Pumillo family domain, pentatricopeptide domain, cold shock domain, helicase domain, La motif, Piwi-Argonaute-Zwille (PAZ) domain, PIWI (P-element induced wimpy testis), pseudouridine synthase, and PUA (archaeosine transglycosylate), Pumillo-like repeats (PUM), ribosomal S1-like (S1), Sm and Like-Sm (Sm / Lsm) repeats, thiuridine synthase and RNA methylase and pseudouridine synthase (THUMP), or domains with YT521-B homology.

[0335] In embodiments, the polypeptide having the phase behavior is any polypeptide having the phase behavior described herein.

[0336] In some embodiments, a method of performing an enzymatic process on a substrate comprises the steps of: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; and (ii) applying a first environmental factor that allows the first enzyme to contact the substrate. In some embodiments, the method further comprises an additional environmental factor that separates the first enzyme from the substrate.

[0337] In some embodiments, a method for performing a multi-step enzymatic process on a substrate includes the steps of (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that allows the first enzyme to contact the substrate; and (iv) applying a second environmental factor that allows the second enzyme to contact the substrate. In some embodiments, the method further includes the steps of (v) applying a third environmental factor to separate the first enzyme from the substrate. In some embodiments, the method further includes the steps of (vi) applying a fourth environmental factor to separate the second enzyme from the substrate.

[0338] In some embodiments, the method further comprises providing additional fusion proteins, e.g., a third fusion protein, a fourth fusion protein, a fifth fusion protein, etc. Each additional fusion protein comprises an additional enzyme and a polypeptide having a phase behavior. For example, the third fusion protein comprises a third enzyme and a polypeptide having a third phase behavior. In some embodiments, a plurality of fusion proteins are provided, each fusion protein comprising a different enzyme, but at least two of the fusion proteins comprise a polypeptide having the same phase behavior.

[0339] In some embodiments, provided herein are methods of performing a multi-step enzymatic process on a substrate, the method comprising: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that enables the first enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate; and (iv) applying a second environmental factor that enables the second enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate. In some embodiments, provided herein are methods of contacting, isolating, and / or purifying a substrate, the methods including: (i) providing a first fusion protein comprising a first enzyme and a polypeptide having a first phase behavior; (ii) providing a second fusion protein comprising a second enzyme and a polypeptide having a second phase behavior; (iii) applying a first environmental factor that allows the first enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate; and (iv) applying a second environmental factor that allows the second enzyme to contact the substrate, isolate the substrate, and / or concentrate the substrate. In some embodiments, the methods described herein allow for the formation of concentrated droplets comprising the fusion protein and the substrate. For example, in some embodiments, the concentrated droplets are formed by the addition of salt to a composition comprising (i) a fusion protein comprising an enzyme polyadenylate polymerase and a polypeptide having a phase behavior, (ii) an mRNA substrate, and (iii) poly(adenylate) nucleotides. In some embodiments, polyadenylate polymerase adds poly(adenylate) nucleotides to mRNA substrates in the concentrated droplets.

[0340] In some embodiments, the method includes incubating a fusion protein comprising an enzyme and a polypeptide having phase behavior with a substrate. In some embodiments, the fusion protein is incubated with the substrate for about 10 minutes to about 24 hours, e.g., 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, or about 60 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours, including all ranges therebetween. In some embodiments, the fusion protein is incubated with the substrate for about 1 hour to about 4 hours. In some embodiments, the fusion protein is incubated with the substrate for about 10 minutes to about 1 hour.

[0341] In some embodiments, incubation is carried out at a temperature between about 10°C and about 80°C, e.g., about 10°C, 15°C, 16°C, 20°C, 25°C, 30°C, 35°C, 37°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C.

[0342] In some embodiments, the introduction of an environmental factor allows access to the substrate of an enzyme of a fusion protein comprising a polypeptide having phase behavior. For example, the introduction of an environmental factor can solubilize the fusion protein and allow a reaction between the enzyme and the substrate to occur. In some embodiments, the introduction of an environmental factor prevents access to the substrate of the enzyme. For example, the introduction of an environmental factor can precipitate the fusion protein.

[0343] In some embodiments, the fusion protein is separated from the substrate. In some embodiments, the separation is based on size and / or density, e.g., based on molecular weight or diameter. In some embodiments, any of the techniques described herein for separating molecules based on size can be applied to separate the fusion protein from the substrate. In some embodiments, continuous centrifugation, tangential flow filtration, or centrifugation are used to separate the fusion protein from the substrate based on size.

[0344] In some embodiments, the multi-step enzymatic process is in vitro transcription and the substrate is DNA. In some embodiments, the DNA is linear or circular DNA. In some embodiments, the first enzyme of the first fusion protein comprises an RNA polymerase. In some embodiments, the RNA polymerase is T7, T3, or SP6 RNA polymerase. In some embodiments, the first fusion protein is added to a composition comprising a DNA substrate. In some embodiments, the composition also comprises ribonucleotide triphosphates and / or a buffer (e.g., a buffer comprising dithiothreitol and magnesium). In some embodiments, an environmental factor is added to allow the first enzyme (e.g., RNA polymerase) access to the DNA substrate. In some embodiments, the fusion protein and DNA substrate are incubated for about 10 minutes to about 24 hours. In some embodiments, incubation of the RNA polymerase with the DNA substrate produces RNA. In some embodiments, the first fusion protein is separated from the substrate by application of an environmental factor. In some embodiments, the RNA is separated from the first fusion protein using any method described herein for separating molecules based on size.

[0345] In some embodiments, the RNA produced after reaction of the first fusion protein with the first enzyme is used as a substrate for further enzymatic reactions. For example, in some embodiments, a second fusion protein comprising a second enzyme mRNA Cap2'-O-methyltransferase and a polypeptide having phase behavior is added to the RNA. This reaction produces RNA that contains a methyl group at the 5' end. In some embodiments, the second fusion protein and the RNA substrate are incubated for about 10 minutes to about 24 hours. In some embodiments, the second fusion protein is separated from the substrate by application of an environmental factor. In some embodiments, the capped mRNA is separated from the second fusion protein using any method described herein for separating molecules based on size.

[0346] In some embodiments, a second fusion protein comprising a second enzyme polyadenylate polymerase is added to the RNA substrate. In some embodiments, the composition comprising the RNA substrate comprises poly(adenylate) nucleotides. In some embodiments, the composition comprising the RNA comprises polyadenylate polymerase. The polyadenylate polymerase catalyzes the addition of a poly(A) tail to the RNA. In some embodiments, the second fusion protein and the RNA substrate are incubated for about 10 minutes to about 24 hours. In some embodiments, the second fusion protein is separated from the substrate by application of an environmental factor. In some embodiments, the RNA comprising the poly(A) tail is separated from the second fusion protein using any method described herein for separating molecules based on size.

[0347] In some embodiments, a second fusion protein comprising a second enzyme, PABP, is added to the RNA substrate. In some embodiments, the composition comprising the RNA substrate comprises poly(adenylate) nucleotides. In some embodiments, the composition comprising the RNA comprises polyadenylate polymerase. PABP aids in the addition of a poly(A) tail to the RNA. In some embodiments, the second fusion protein and the RNA substrate are incubated for about 10 minutes to about 24 hours. In some embodiments, the second fusion protein is separated from the substrate by application of an environmental factor. In some embodiments, the RNA comprising the poly(A) tail is separated from the second fusion protein using any method described herein for separating molecules based on size.

[0348] environmental factors In some embodiments, the method of the disclosure provides one or more environmental factors to a composition comprising a fusion protein of the present disclosure. In some embodiments, the one or more environmental factors are applied to reversibly aggregate the fusion protein. The application of the environmental factors causes a change in the composition comprising the fusion protein. In some embodiments, the environmental factors are used to reversibly aggregate the fusion protein. In some embodiments, the environmental factors are used to reversibly disaggregate the fusion protein. In some embodiments, the environmental factors are used to separate the fusion protein from one or more impurities in the composition comprising the fusion protein. In some embodiments, the one or more environmental factors increase the size of the fusion protein aggregates. In some embodiments, the one or more environmental factors allow the first polypeptide of the fusion protein to retain its structure, function, and activity. In some embodiments, the one or more environmental factors allow the first polypeptide of the fusion protein to enhance its native structure, function, and activity.

[0349] In some embodiments, the methods of the disclosure include applying at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten environmental factors to a composition comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten fusion proteins.

[0350] In some embodiments, the environmental factor is a change in temperature. In some embodiments, the temperature is increased by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the temperature is reduced by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0351] In some embodiments, the environmental factor is a change in pH. In some embodiments, the pH is about 0.1 units, about 0.2 units, about 0.3 units, about 0.4 units, about 0.5 units, about 0.6 units, about 0.7 units, about 0.8 units, about 0.9 units, about 1.0 units, about 1.1 units, about 1.2 units, about 1.3 units, about 1.4 units, about 1.5 units, about 1.6 units, about 1.7 units, about 1.8 units, about 1.9 units, about 2.0 units, about 2.1 units, about 2.2 units, about 2.3 units, about 2.4 units, about 2.5 units, about 2.6 units, about 2.7 units, about 2.8 units, about 2.9 units, about 3.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, about 6.0 units, about 6.1 units, about 6.2 units, about 6.3 units, about 6.4 units, about 6.5 units, about 6.6 units, about 6.7 units, about 6.8 units, about 6.9 units, about 7.0 units, about The amount can be increased by about 0.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, or about 6.0 units.

[0352] In some embodiments, the pH is about 0.1 units, about 0.2 units, about 0.3 units, about 0.4 units, about 0.5 units, about 0.6 units, about 0.7 units, about 0.8 units, about 0.9 units, about 1.0 units, about 1.1 units, about 1.2 units, about 1.3 units, about 1.4 units, about 1.5 units, about 1.6 units, about 1.7 units, about 1.8 units, about 1.9 units, about 2.0 units, about 2.1 units, about 2.2 units, about 2.3 units, about 2.4 units, about 2.5 units, about 2.6 units, about 2.7 units, about 2.8 units, about 2.9 units, about 3.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, about 6.0 units, about 6.1 units, about 6.2 units, about 6.3 units, about 6.4 units, about 6.5 units, about 6.6 units, about 6.7 units, about 6.8 units, about 6.9 units, about 7.0 units, about 7.1 units, about 7.2 units, The amount of the drug can be reduced by 0.0 units, about 3.1 units, about 3.2 units, about 3.3 units, about 3.4 units, about 3.5 units, about 3.6 units, about 3.7 units, about 3.8 units, about 3.9 units, about 4.0 units, about 4.1 units, about 4.2 units, about 4.3 units, about 4.4 units, about 4.5 units, about 4.6 units, about 4.7 units, about 4.8 units, about 4.9 units, about 5.0 units, about 5.1 units, about 5.2 units, about 5.3 units, about 5.4 units, about 5.5 units, about 5.6 units, about 5.7 units, about 5.8 units, about 5.9 units, or about 6.0 units.

[0353] In some embodiments, the environmental factor is a change in ionic strength. In some embodiments, the change in ionic strength is caused by increasing the concentration of a salt. In some embodiments, the change in ionic strength is caused by decreasing the concentration of a salt. Non-limiting examples of salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium acetate, sodium citrate, copper sulfate, sodium iodide, and sodium sulfate. In some embodiments, the salt is about 0.1M to about 5M, for example, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.1M, about 2.2M, about 2.3M, about The salt has a concentration of about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, about 4 M, about 4.1 M, about 4.2 M, about 4.3 M, about 4.4 M, about 4.5 M, about 4.6 M, about 4.7 M, about 4.8 M, about 4.9 M, or about 5 M. In some embodiments, the salt has a concentration of 0.6 M. In some embodiments, dialysis is used to alter the salt concentration of the composition comprising the protein-based purification matrix and the biologic, the contaminant, and / or the molecule.

[0354] In some embodiments, the environmental factor is the addition of a cofactor. Non-limiting examples of cofactors include calcium, magnesium, cobalt, copper, zinc, iron, manganese, selenium, molybdenum, potassium, coenzyme A (CoA), nucleoside triphosphates, and vitamins (e.g., vitamins A, B, C, D, or F). In some embodiments, the cofactor is calcium. In some embodiments, the nucleoside triphosphate is adenosine triphosphate, uridine triphosphate, guanosine triphosphate, cytidine triphosphate, or thymidine triphosphate. In some embodiments, the vitamin is fat-soluble. In some embodiments, the vitamin is water-soluble. Non-limiting examples of vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12, vitamin C, vitamin D, vitamin E, vitamins K, K1, and K2, folic acid, and biotin.

[0355] In some embodiments, the environmental factor is a change in concentration of a protein-based purification matrix, hi some embodiments, the environmental factor is a change in concentration of a biologic, a contaminant, and / or a molecule.

[0356] In some embodiments, the environmental factor is a change in pressure of the composition, contaminant, and / or molecule comprising the protein-based purification matrix and the biologic, hi some embodiments, the change in pressure can be brought about by increasing or decreasing the volume of the composition.

[0357] In some embodiments, the environmental factor is the addition of one or more surfactants. In some embodiments, the one or more surfactants are free fatty acid salts, soaps, fatty acid sulfonates, such as sodium lauryl sulfate, ethoxylated compounds, such as ethoxylated propylene glycol, lecithin, polygluconates, quaternary ammonium salts, lignin sulfonates, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sugars, including sucrose and glucose, Triton X-100, and NP-40. In some embodiments, the surfactant is anionic, nonionic, or amphoteric.

[0358] In some embodiments, the environmental factor is the addition of one or more molecular crowding agents. Non-limiting examples of molecular crowding agents include polyethylene glycol, dextran, and ficoll. PEGs can include PEG400, PEG1450, PEG3000, PEG8000, and PEG10000.

[0359] In some embodiments, the environmental factor is the addition of one or more oxidizing agents, non-limiting examples of which include hydrogen peroxide, hydrophilic or hydrophobically activated hydrogen peroxide, preformed peracid, monopersulfate, or hypochlorite.

[0360] In some embodiments, the environmental factor is the addition of one or more reducing agents. In some embodiments, the one or more reducing agents are selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), hydrazine, borohydride, amine borane, lower alkyl substituted amine borane, triethanolamine, and N,N,N',N'-tetramethylethylenediamine (TEMED). In some embodiments, the environmental factor is the addition of one or more denaturing agents. Non-limiting examples of denaturing agents include urea, guanidine hydrochloride, guanidine, sodium salicylate, dimethyl sulfoxide, and propylene glycol.

[0361] In some embodiments, the environmental factor is the addition of one or more enzymes, non-limiting examples of which include proteases, kinases, phosphatases, synthetases, transferases, nucleases, such as restriction endonucleases, lyases, isomerases, dehydrogenases, decarboxylases, and lipases.

[0362] In some embodiments, the environmental factor is the application of electromagnetic waves. In some embodiments, the environmental factor is the application of light. In some embodiments, the electromagnetic waves have a wavelength of about 0.0001 nm to about 100 m. In some embodiments, the electromagnetic waves are selected from the group consisting of gamma rays, X-rays, ultraviolet rays, visible light, infrared rays, and radio waves. In some embodiments, the electromagnetic waves are gamma rays. In some embodiments, the gamma rays have a wavelength of about 0.0001 nm to about 0.01 nm, for example, 0.0001 nm, 0.0005 nm, 0.001 nm, 0.002 nm, 0.003 nm, 0.004 nm, 0.005 nm, 0.006 nm, 0.007 nm, 0.008 nm, 0.009 nm, and 0.01 nm. In some embodiments, the X-rays have a wavelength of about 0.01 nm to about 10 nm, e.g., about 0.01 nm, 0.02 nm, 0.03 nm, 0.04 nm, 0.05 nm, 0.06 nm, 0.07 nm, 0.08 nm, 0.09 nm, 0.10 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or about 10 nm. In some embodiments, the ultraviolet light has a wavelength of about 10 nm to about 400 nm, for example, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 280 nm, about 300 nm, about 350 nm, or about 400 nm. In some embodiments, the visible light has a wavelength of about 400 nm to about 800 nm, for example, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm.In some embodiments, the infrared radiation has a wavelength of about 800 nm to about 0.1 cm, e.g., about 800 nm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 0.1 cm. In some embodiments, the radio waves have a wavelength of about 0.1 cm to 100 m, e.g., about 0.1 cm, about 1 cm, about 10 cm, about 100 cm, about 1,000 cm, about 2,000 cm, about 3,000 cm, about 4,000 cm, about 5,000 cm, about 6,000 cm, about 7,000 cm, about 8,000 cm, about 9,000 cm, or about 100 m.

[0363] All patents, patent applications, references, and journal articles cited in this disclosure are expressly incorporated herein by reference in their entirety for all purposes. EXAMPLES

[0364] Example 1. Expression of a fusion protein comprising a binding domain of an AAV receptor and a second polypeptide having phase behavior The nucleic acid encoding the extracellular domain of the AAV receptor (AAVR) is synthesized as a polypeptide having the phase behavior ((GVGVPGLGVPGVGVPGLGVPGVGVP) 16 (SEQ ID NO:87) and cloned into the pET24 plasmid. The plasmid was transformed into BL21 E. coli cells, which were maintained under conditions allowing expression of the fusion protein.

[0365] The fusion proteins were purified, aliquoted and formulated in PBS (as a control) or subjected to the following conditions known to affect protein function: (1) lyophilization and resuspension in PBS, (2) incubation in 0.1M NaOH for 30 minutes followed by neutralization and buffer exchange into PBS, (3) incubation in 6M guanidine hydrochloride (GuHCl) for 30 minutes followed by buffer exchange into PBS, (4) incubation in PBS at 95°C for 30 minutes followed by cooling on ice and returning to room temperature. The activity of each sample (i.e., the ability of AAVR to bind to its target) was then measured by assaying for AAV8 capture. Briefly, activity was defined as the percentage of captured AAV8 and quantified using the Progen AAV8 total capsid ELLISA.

[0366] The results are shown in Figure 1. In all treatment conditions, the activity of the fusion protein remained within 80% of the untreated control.

[0367] Example 2: Expression of a fusion protein containing the Z domain of Staphylococcus aureus protein A and a second polypeptide having phase behavior A nucleic acid encoding the Z domain from Staphylococcus aureus protein A, known to bind to antibodies (SEQ ID NO: 180), was synthesized using a polypeptide having the phase behavior ((GVGVPGLGVPGVGVPGLGVPGVGVP) 16 (SEQ ID NO: 87)) and cloned into the pET24 plasmid. The plasmid was transformed into BL21 E. coli cells, which were maintained under conditions allowing expression of the fusion protein. The fusion protein was then purified and lyophilized.

[0368] The fusion protein was autoclaved on a gravity cycle and resuspended in PBS. Protein A activity was then tested. Specifically, activity was measured using an assay for antibody capture. The results are shown in Figure 2. Fusion to a polypeptide with phase behavior helped Protein A retain at least 85% activity after autoclaving / resuspension in PBS compared to the non-autoclaved control.

[0369] Proteins were also tested to determine whether fusion to a polypeptide with phase behavior could prevent the loss of soluble folded Protein A following treatment with 0.1 M NaOH, heating to 95°C in PBS, or heating in acidic buffer at pH 4. For a given treatment condition, samples were incubated for 10-30 minutes and then neutralized or cooled and centrifuged to remove any aggregated protein. To measure the percent protein loss due to treatment, total protein was measured using a Protein A ELISA assay and compared to untreated controls. As shown in Figure 3, exposure to these conditions resulted in less than 2% loss.

[0370] Example 3. Expression of a fusion protein comprising a binding domain of an AAV receptor and a second polypeptide having phase behavior The nucleic acid (Cys-His-PKD2) encoding the AAV receptor PKD2 was fused to the nucleic acid encoding the polypeptide with phase behavior (ELP) and cloned into the pET24 plasmid. As a control, the nucleic acid encoding the AAV receptor PKD2 was cloned into the pET24 plasmid. The nucleic acid sequence and protein sequence of the AAV receptor PKD2 and the polypeptide with phase behavior are included in Table 5 below.

[0371] [Table 32]

[0372] [Table 33]

[0373] [Table 34]

[0374] [Table 35]

[0375] 5 mL overnight cultures of BL21 E. coli cells were grown to express His-tagged PKD2 protein alone or as a fusion with ELP. 250 μL of this culture was used to inoculate 50 mL cultures, which were induced with 0.5 mM IPTG after 9 hours. After 24 hours of incubation, cells from each culture were centrifuged, resuspended in 10 mL PBS buffer, and sonicated. Lysates were clarified by centrifugation. 1.5 mL of each clarified lysate was purified using the HisLink Protein Purification Kit (Promega) and 200 μL of the elution fraction. Eluted material was quantified by measuring absorbance at 280 nm. Beer's law was then used to calculate grams and moles of purified protein and fusion protein per liter of E. coli cell culture. Purity and correct molecular weight were also qualitatively assessed using SDS-PAGE and Coomassie staining.

[0376] Expression of a fusion protein containing PKD2 was compared to expression of PKD2 in the absence of a polypeptide having the phase behavior.

[0377] Figure 4A shows the concentration of purified PKD2 and fusion protein containing PKD2 per liter. Figure 4B shows the amount of purified PKD2 and fusion protein containing PKD2 per liter. Figure 4C shows the expression of PKD2 and fusion protein containing PKD2 on a gel. These results show that when the PKD2 domain is fused to a polypeptide having phase behavior, 10 times more mass of soluble protein (fusion protein) and more than 3 times more PKD2 (first polypeptide) can be extracted compared to when PKD2 is expressed without a polypeptide having phase behavior.

[0378] Example 4. Expression of a fusion protein comprising a binding domain of an AAV receptor and a second polypeptide having phase behavior A nucleic acid encoding the CR3 domain of the LDL receptor (LDLR) was fused to a nucleic acid encoding a polypeptide having phase behavior and cloned into the pET24 plasmid. The plasmid was transformed into BL21 E. coli cells, which were maintained under conditions that allowed expression of the fusion protein. The fusion protein is referred to in this example as IsoTag-LV. The amino acid sequence of IsoTag-LV is shown in Table 6 below.

[0379] [Table 36]

[0380] The stability of IsoTag-LV was assessed by measuring the lentivirus (LV) capture activity of a fusion protein control versus a fusion protein exposed to extreme conditions known to degrade, aggregate, or inactivate polypeptides.

[0381] 20 μM IsoTag-LV was incubated in 6 M guanidine-HCL (6M GuHCl) or 0.1 M NaOH (0.1 M NaOH) for 30 min, followed by six washes with 500 μL PBS in a 30 kDa Amicon spin filter. 20 μM IsoTag-LV was incubated at 95° C. for 30 min, followed by incubation on ice for 30 min.

[0382] 10 μM IsoTag and untreated IsoTag (Control IsoTag-LV) from the described incubation were added to 500 μL of lentivirus and incubated on ice for 5 min, then incubated at 37° C. for 5 min before centrifugation at 10,000 rpm for 5 min to capture and pellet lentiviral targets. Harvested supernatants (representing uncaptured LV targets) were analyzed using a p24 assay (VPK-107, Cell Biolabs) to measure LV envelope p24 protein as a surrogate measure of lentiviral titer. As a control for no capture activity, lentivirus without added IsoTag was also measured in duplicate (Control (No IsoTag-LV)). Absorbance values ​​were plotted using GraphPad Prism V9. Error bars represent standard error of the mean. There was no statistically significant difference in the LV capture activity of the control fusion protein compared to the fusions treated by heating to 95°C and incubated with 0.1 M NaOH or 6 M GuHCl, indicating that the CR3 domain of the LDLR is protected from degradation when expressed as a fusion protein with a polypeptide having phase behavior. The results are shown in Figure 5.

[0383] Example 5: Estimated yield in E. coli of additional fusion proteins comprising a first polypeptide and a second polypeptide with phase behavior Nucleic acids encoding various proteins having biological functions (referred to herein as target proteins) are synthesized as polypeptides having the phase behavior ((GVGVPGLGVPGVGVPGLGVPGVGVP) 16(SEQ ID NO: 87) and expressed as a recombinant fusion protein in E. coli BL21 cells. More specifically, the nucleic acid encoding the fusion protein was cloned into a pET24 plasmid, which was transfected into E. coli cells. Expression of the fusion protein was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a shake flask containing the cells. The fusion protein was purified using phase separation and shown to have a yield of at least 10 mg / L. Table 7 shows a list of target proteins used for fusion. It is estimated that the yield of each purified protein can be at least 15 mg per liter, with some yields reaching as high as 300 mg per liter.

[0384] [Table 37]

[0385] Example 6: Use of fusion proteins in the modification of nucleic acids This example evaluates the ability of the fusion proteins herein to promote DNA and RNA modification. A fusion protein comprising a nucleic acid binding protein (NBP) and a polypeptide having phase behavior is incubated with a nucleic acid substrate (e.g., DNA or RNA) for a sufficient time for either soluble or phase-separated NBP to produce a product or to produce an affinity binding complex. An environmental factor (e.g., 0.6 M NaCl) is then added to the solution to phase separate the fusion protein. This phase separation helps to enhance the enzymatic reaction, concentration, and / or purity of the nucleic acid target. The fusion protein is separated from the product by tangential flow filtration, depth filtration, or centrifugation. The product is incubated with additional fusion protein and the same procedure as described for the first fusion protein is followed to perform additional modifications to the nucleic acid. At each of the above steps, additional reagents (e.g., cofactors, ribonucleotide triphosphates, deoxynucleoside triphosphates, salts, etc.) can be added to promote the function of the fusion protein.

[0386] At any point, the sample can be centrifuged or filtered to separate the fusion protein and any bound affinity material from the soluble phase components. At any point, a second environmental factor (e.g., low pH, cofactor, salt) can be added to disrupt the binding of the product or substrate to the soluble or phase-separated NBP fusion protein.

[0387] An exemplary process for transcribing DNA into mRNA is described below. A nucleic acid encoding a polypeptide having NBP T7 RNA polymerase and phase behavior (e.g., GVGVPGLGVPGVGVPGLGVPGVGVP). 16 (SEQ ID NO: 87) is expressed as a recombinant fusion protein in E. coli BL21 cells and purified. The purified fusion protein is incubated with a composition containing a DNA substrate and rNTPs for about 1 hour at 37° C. under conditions in which the fusion protein is soluble to form RNA. Salt (0.6 M NaCl) is added to separate the fusion protein bound to the RNA product from the composition. The RNA product is separated from the fusion protein by successive centrifugations.

[0388] Subsequent reactions are performed to add a poly(A) tail to the 3' end of the RNA product and a methyl group to the 5' end of the RNA product. The poly(A) tail is added via a fusion protein comprising a PABP and a polypeptide with phase behavior. The methyl group is added to the 5' end of the RNA product via a fusion protein comprising an mRNA Cap2'-O-methyltransferase and a polypeptide with phase behavior. The final product is an mRNA containing a 5' methyl group and a 3' poly(A) tail.

[0389] Additional modifications to DNA and RNA can be performed in a similar manner using any of the NBPs described herein. For example, the NBP can be any of the following proteins: T7 RNA polymerase, RNase inhibitor, 2'-O-methyltransferase, inorganic pyrophosphatase, poly(A) polymerase, DNase I, calf intestinal phosphatase, Antarctic phosphatase, D1 subunit of vaccinia virus mRNA capping enzyme, guanine-7-methyltransferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), guanylyltransferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), RNA triphosphatase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), and D12 subunit of vaccinia virus mRNA capping enzyme, stem-loop binding protein. protein, heterogeneous nuclear ribonucleoprotein (hnRNP), GroEL, Edc3, DHX9, Xrn1, Dcp1, Dcp2, LAF-1, MEG-1, MEG-3, ASF / SF2 splicing factor, serine / arginine-rich splicing factor 4 (SRp75), serine and arginine-rich splicing factor 1 (SRSF1), L3 ribosomal protein, L4 ribosomal protein, L13 ribosomal protein, L20 ribosomal protein, L22 ribosomal protein, L24 ribosomal protein, L24e ribosomal protein, S12 ribosomal protein, S14 ribosomal protein, and eukaryotic initiation factor 4E-binding protein 1 (4EBP1), Tat, Rev, and RSG-1.2 peptide, poly(A) binding protein (PABP), eukaryotic translation ini...

Claims

1. 1. A method for stabilizing a first polypeptide, comprising expressing a fusion protein comprising the first polypeptide and a second polypeptide, wherein the second polypeptide is a polypeptide having a phase behavior such that the first polypeptide substantially retains its activity when the fusion protein is exposed to one or more conditions that would destabilize the first polypeptide.

2. 1. A method for enhancing yield of a first polypeptide, comprising: (i) expressing a fusion protein comprising the first polypeptide and a second polypeptide having a phase behavior; and (ii) separating the first polypeptide from the second polypeptide; The method, wherein the yield of the first polypeptide is increased when expressed as a fusion protein compared to the yield of the first polypeptide when not expressed as a fusion protein.

3. 3. The method of claim 1 or 2, wherein the first polypeptide comprises cluster of differentiation 4 (CD4), the Z domain of Staphylococcus protein A (SpA), low density lipoprotein receptor (LDLR), albumin binding polypeptide (ABD), coxsackievirus and adenovirus receptor (CAR), fibronectin type III (FN3), poly(A) binding protein (PABP), Z-DNA binding protein 1 (ZBP1), the extracellular domain of the adeno-associated virus receptor (AAVR), polycystic kidney disease 2 (PKD2) domain of AAVR, complement receptor 3 (CR3) domain of the LDL receptor (LDLR), the B domain of SpA, the C domain of SpA, the binding domain of AAVR, AAVR, LDLR domain, ZBP, or a fragment or derivative thereof.

4. The method of any one of claims 1 to 3, wherein the second polypeptide having a phase behavior is an elastin-like polypeptide (ELP).

5. The second polypeptide having a phase behavior of: (a) (GVGVPGLGVPGVGVPGLGVPGVGVP) 16 (SEQ ID NO: 87); (b) (GVGVP) 80; (c) (GVGVPGAGVP) 40; (d) (GVGVP) n (SEQ ID NO:11), where n is 20 to 360 (inclusive); (e) (GVGVP) m (SEQ ID NO:143), where n is 10 to 180 (inclusive); (f) (GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO:12), where m is 4 to 25 (inclusive); (g) (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO:13), where m is 4 to 25 (inclusive); (h) (GVGVPGWGVPGVGVPGWGVPGVGVGVP) m (SEQ ID NO:14), where m is 4 to 25 (inclusive); (i) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVGVGEGVPGFGVPGVGVP) m (SEQ ID NO:15), where m is 4 to 25 (inclusive); (j) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m (SEQ ID NO:16), where m is 4 to 25 (inclusive); (k) (GAGVPGVGVPGAGVPGVGVPGAGVP) m (SEQ ID NO:17), where m is 4 to 25 (inclusive); (l) (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO:144), where m is 2 to 32 (inclusive); (m) (GVGVPGAGVP) m (SEQ ID NO:145), where m is 5 to 80 (inclusive); (n) (GVGVPGVGVPGLGVPGVGVPGVGVP) m (SEQ ID NO:146), where m is 2 to 32, inclusive; 5. The method of any one of claims 1 to 4, comprising an amino acid sequence that is the amino acid sequence of claim 1, or a randomized, scrambled analog thereof.

6. 6. The method of any one of claims 1 to 5, wherein the one or more conditions that will destabilize the first polypeptide are selected from any one of the following: lyophilization, exposure to sodium hydroxide (NaOH), exposure to guanidine hydrochloride, exposure to a temperature of at least 95°C, exposure to a non-physiological pH, exposure to autoclaving, freeze-thaw cycles, heat shock, or a combination thereof.

7. The one or more conditions that result in destabilization of the first polypeptide include: (i) exposure to 0.1 M NaOH; (ii) exposure to 6M guanidine hydrochloride; (iii) heating the first polypeptide to at least 95° C.; or (iv) heat shock, heating the fusion protein comprising the first polypeptide to 95° C. for 30 minutes; placing a container containing the fusion protein on ice; and allowing the fusion protein to return to room temperature. The method of claim 6, wherein the ion exchange is selected from any one of the following:

8. The method of claim 6 or 7, wherein the first polypeptide is exposed to the one or more conditions for about 10 to about 30 minutes.

9. 9. The method of any one of claims 1 to 8, wherein the first polypeptide retains at least 80% of its activity after exposure to the one or more conditions compared to a control that is not exposed to the one or more conditions.

10. The method of any one of claims 1 to 8, wherein the yield of the first polypeptide is at least three times the yield of the first polypeptide not expressed as a fusion protein.

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