Split-intein for affinity capture

Abstract

This specification discloses an affinity capture protein comprising a ligand moiety capable of affinity interaction with a target molecule, and a linker moiety capable of binding to the ligand moiety and coupling to a solid phase, wherein the ligand moiety does not contain lysine residues, and the linker moiety has at least two lysine residues located at the terminal of the protein, with at least one non-lysine residue located at the terminal relative to at least two lysine residues. Furthermore, this specification discloses an affinity capture medium containing the protein, a split-intane system for affinity capture of the target protein, and a method for purifying the target protein.

Description

Technical Field

[0001] The present invention relates to proteins for affinity capture. The present invention further relates to an affinity capture medium containing a protein, a split intein system for affinity capture containing a protein, and a method for purifying a target protein.

Background Art

[0002] The immobilization of proteins on a solid phase is important in many biotechnological applications, such as the development of biosensors, immunoassays, and bioseparation, where affinity-based separation is utilized. In affinity separation, it is desirable but difficult to bind a ligand in the form of a protein to a solid phase without causing denaturation of the protein or steric hindrance and blockage of the active site of the protein.

[0003] It has been proposed that immobilization of proteins on a solid phase in a preferred orientation can avoid protein denaturation, prevent steric hindrance, keep the active site exposed in solution, and thus preserve bioaffinity or biological activity (Wei Huang et al., Anal. Chem., 69 (22), pp. 4601-4607, 1997).

[0004] Furthermore, it has been proposed that problems associated with protein denaturation, steric hindrance or blockage of the active site can be reduced by inserting a linker between the protein and the site used for binding to the solid phase. Peptide linkers are present in nature in multi-domain proteins where they act as spacers, but they can also be used to immobilize proteins on a solid phase. However, it is difficult to achieve a high degree of immobilization of proteins on a solid phase.

[0005] Furthermore, it is difficult to achieve specific immobilization of proteins via a linker, i.e., to avoid direct linkage of the protein to the solid phase. Still further, it is difficult to achieve a high immobilization density on the surface of the solid phase.

[0006] It is highly desirable to provide ligands for affinity separation or capture in the form of proteins such as inteins, which can bind to a solid phase at a predictable and high surface density, thereby providing efficient and / or improved affinity separation or capture. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO 2021 / 099607 [Non-patent literature]

[0008] [Non-Patent Document 1] Wei Huang et al., Anal. Chem., 69 (22), pp. 4601-4607, 1997. [Non-Patent Document 2] Proteins-Structure and Molecular Properties 2nd Ed., TE Creighton, WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983) [Non-Patent Document 3] Merck Index (14th edition) [Non-Patent Document 4] the Physicians' Desk Reference (64th edition) [Non-Patent Document 5] The Pharmacological Basis of Therapeutics (1st edition) [Non-Patent Document 6] http: / / www.expasy.org / tools / pi_tool.html [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to overcome, at least partially, the problems or shortcomings of the prior art. [Means for solving the problem]

[0010] A protein for affinity capture is provided, comprising a ligand moiety capable of affinity interaction with a target molecule, and a linker moiety capable of binding to the ligand moiety and coupling to a solid phase, wherein the ligand moiety does not contain lysine residues, and the linker moiety is located at the terminal end of the protein and has at least two lysine residues such that at least one residue other than lysine is terminally positioned relative to at least two lysine residues.

[0011] A ligand moiety capable of affinity interaction with a target molecule enables affinity-based binding, purification, or separation of the target molecule, such as the protein of interest.

[0012] Proteins having a linker moiety that can bind to a ligand and couple to a solid phase enable the immobilization of the protein to a solid phase, thus providing a solid-phase material or medium for affinity chromatography. Furthermore, the linker moiety allows for a physical distance between the ligand moiety and the solid phase, thus reducing the risk of deformation such as denaturation of the ligand moiety when immobilized to a solid phase, thereby enabling improved affinity interactions and separation. Moreover, such a physical distance between the ligand moiety and the solid phase improves access to the active site of the ligand moiety, such as the affinity site, by reducing steric interference by the solid phase, thereby enabling improved affinity interaction and separation.

[0013] The ligand moiety without lysine residues provides improved binding to the solid phase via the linker moiety. Specific binding between the linker moiety and the solid phase can be achieved via a covalent bond between the solid phase and lysine while avoiding direct binding of the ligand moiety to the solid phase.

[0014] A linker moiety having at least two lysine residues enables the linking or binding of the linker moiety, and thereby the ligand moiety, to the solid phase via at least one lysine residue. Thereby, improved control of binding to the solid phase can be achieved.

[0015] Having at least two lysine residues in the linker moiety, with at least one residue other than lysine disposed terminally with respect to the at least two lysine residues, provides improved binding to the solid phase, as is evident from the experimental data provided herein.

[0016] The linker moiety can comprise, or consist of, an amino acid sequence having 5 to 100 amino acid residues.

[0017] The linker moiety may have an isoelectric point above 6.0, thereby enabling improved immobilization to the solid phase.

[0018] The linker moiety may have an isoelectric point above 8.0.

[0019] The linker moiety may have at least three lysine residues. Thereby, improved binding to the solid phase can be achieved.

[0020] When the linker moiety has three lysine residues, the linker moiety can have an isoelectric point above 8.

[0021] The ligand moiety may be a split N - intein or a split C - intein, and thus can respectively undergo affinity interaction with the corresponding split C - intein or split N - intein.

[0022] The ligand part may be a split N - intein, the linker part may be a linker part arranged at the C - terminus, and the split N - intein has the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLEDGSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (SEQ ID NO: 1), (wherein, X at positions 20, 35, 70, 73, and 95 are each independently selected from R or A; X at position 28 is C, A or S; X at position 36 is N, H or Q; X at position 25 is N or R; X at position 59 (X is position 59) is D or C; X at position 80 is E or Q; and X at position 90 is Q or R) is included.

[0023] It is beneficial for such a split N - intein not to contain lysine residues. When combined with a linker part having at least two lysine residues, especially when at least one residue other than lysine is arranged terminally with respect to the at least two lysine residues, specific binding to the solid phase via the linker part can be achieved. Thereby, the ligand can be immobilized on the solid phase while reducing the risk of deformation of the ligand part or reduction of affinity activity, and can improve affinity interaction and separation.

[0024] Among SEQ ID NO: 1, X at positions 20, 35, 70, 73, and 95 may be R; X at position 28 may be A; X at position 36 may be H; X at position 25 may be N; X at position 59 may be D; X in 80th place may also be E; and The 90th ranked X could also be Q. Thus, a split N-intane may contain, or may consist of, the sequence defined by sequence number 2.

[0025] The linker portion may contain an amino acid sequence selected from the list consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. All of these sequences (SEQ ID NOs: 3-12) contain at least two lysine residues, each having at least one non-lysine residue positioned at the terminal relative to at least two lysine residues, which provides improved immobilization to a solid phase.

[0026] The linker portion may contain an amino acid sequence that is at least 80%, for example at least 90%, or 90-100% identical to any one of sequence numbers 3-12, insofar as the linker portion has at least two lysine residues, with at least one residue other than lysine being located at the terminal of at least two lysine residues.

[0027] In a second aspect, an affinity capture medium is provided, comprising a protein for affinity capture according to the first aspect and a solid phase, wherein the ligand portion of the protein is covalently bonded to the solid phase via a linker portion.

[0028] The linker portion of the protein may be covalently bonded to the solid phase via at least one lysine residue in the amino acid sequence of the linker portion.

[0029] The solid phase may be in the form of beads, films, particles, chips, fibrous or nanofiber-like structures, or monolithic structures, or a combination thereof.

[0030] The solid phase may contain natural or synthetic polymer materials, preferably polysaccharides such as cellulose or agarose, or may be composed of them.

[0031] A split-intane system for affinity capture of a POI is provided, comprising a protein for affinity capture according to the first embodiment, wherein the ligand is a split N-intane, and a target molecule which is a protein of interest (POI) fused to a split C-intane.

[0032] The protein for affinity capture may be contained in the affinity capture medium according to the second embodiment.

[0033] A method for purifying a target protein (POI) is provided according to a fourth aspect. The method includes loading a sample liquid containing the POI onto an affinity capture medium containing an affinity capture protein that is covalently bonded to a solid phase via a linker portion and has affinity for the POI or a target molecule containing the POI, wherein the affinity capture protein is a protein according to the first aspect; capturing the POI or a target molecule containing the POI by binding to the protein of the affinity capture medium; washing the affinity capture medium with a washing solution to remove compounds other than the POI or the target molecule containing the POI; and eluting the POI or the target molecule containing the POI from the affinity capture medium.

[0034] The affinity capturing medium may also be a separation medium.

[0035] Target molecules containing POIs, which are eluted from the affinity capture medium, are released from the proteins of the affinity capture medium. The release of POIs or target molecules containing POIs may occur with elution, initiated by the eluate, and / or independently of elution.

[0036] The method may further include releasing a POI or a target molecule containing a POI from the affinity capture medium protein, the release of which may occur before or simultaneously with the elution of the POI or the target molecule containing a POI from the affinity capture medium, or a combination thereof. The release and / or elution of the POI or the target molecule containing a POI from the protein may occur via different mechanisms, for example, depending on the type of ligand, POI, and / or target molecule. For example, the release or elution may occur spontaneously by using an additive or reagent, or by a change in the properties of the present buffer or liquid. Such properties may be, for example, pH, ionic concentration, or salt concentration, or hydrophobicity or hydrophilicity, or a combination of different such properties. For example, spontaneous release may be performed using a split intein that enables spontaneous cleavage or self-cleavage, thereby detaching the POI from the split intein to which it is fused.

[0037] The affinity capturing medium may be an affinity capturing medium according to the second embodiment.

[0038] Features of one embodiment may be similarly applicable to any of the other embodiments. To avoid unnecessary repetition, refer to the features between embodiments where applicable.

[0039] Further scope of the applicability of this disclosure will become apparent from the detailed description below. However, while the detailed description and specific examples illustrate preferred variations of the concept of the invention, it should be understood that various changes and modifications within the scope of the concept of the invention will become apparent to those skilled in the art from this detailed description and are therefore shown merely as illustrations. Thus, it should be understood that the concept of the invention is not limited to specific steps of the described method or component parts of the described system, as the described method and system may vary. Similarly, it should be understood that the terms used herein are intended solely to illustrate specific embodiments and are not intended to be limiting. It should be noted that, as used herein and in the appended claims, the articles “a,” “an,” “the,” and “the foregoing” are intended to mean that there is one or more elements unless it expressly indicates otherwise. Furthermore, “comprising,” “including,” “containing,” and similar terms do not exclude other elements or steps.

[0040] Aspects of the present disclosure will be described in more detail below with reference to the accompanying drawings illustrating variations of the present invention. The drawings should not be considered to limit the concepts of the present invention to any particular variation; rather, they are used to illustrate and understand the concepts of the present invention. As illustrated in the drawings, for example, the sizes of multiple parts or solid phases are approximate and may be exaggerated for illustrative purposes, and are thus provided to illustrate the general structure of variations of the concepts of the present invention. Similar reference numerals refer to similar elements throughout. [Brief explanation of the drawing]

[0041] [Figure 1] This diagram illustrates proteins involved in affinity capture. [Figure 2] This figure illustrates the deformation morphology of affinity-capturing proteins bound to a solid phase. [Figure 3] This is a diagram illustrating an affinity capture medium. [Figure 4] [Figure 4a] This figure illustrates a split-intane system. [Figure 4b] This figure illustrates a split-intane system. [Figure 5] This diagram illustrates a method for purifying the target protein. [Figure 6] This diagram illustrates a modified form of a method for purifying a target protein. [Figure 7] This diagram illustrates the level of fixation. [Figure 8] This is a diagram illustrating the level of connection. [Figure 9] This is a UV chromatogram obtained from the experiment. [Figure 10] This is a photograph of an SDS-PAGE gel from an experiment. [Modes for carrying out the invention]

[0042] The present invention will be described in more detail below. However, the present invention may be embodied in many different forms and should be construed as not being limited to the embodiments described herein; rather, these embodiments are provided for thoroughness and completeness, and to adequately convey the scope of the invention to those skilled in the art. Individual features may be included in different embodiments, but they may also be combined in other ways, and their inclusion in different embodiments does not imply that combinations of features are not feasible.

[0043] The concept of the present invention is based on the recognition that a protein for affinity capture, at least in part, comprising a ligand moiety and a linker moiety, for example, a protein capable of affinity interaction with a target moiety, wherein the ligand moiety does not contain lysine residues, but the linker moiety contains at least two lysine residues, with at least one non-lysine residue positioned terminally relative to at least two lysine residues in the linker moiety, thereby enabling improved protein immobilization to a solid phase.

[0044] Affinity capture is understood to include, and refer to, any preferred form of capture based on affinity recognition or interaction, in which a target molecule can be captured by or interact with a ligand moiety based on affinity recognition. Thus, as used herein, affinity capture may refer to, or include, affinity separation such as affinity chromatography or affinity-based purification, but may also refer to, or include, affinity sensing or affinity-based assays. Proteins for affinity capture may thus, and for example, be proteins for affinity separation.

[0045] The terms “peptide,” “polypeptide,” and “protein” are used herein and include proteins and fragments thereof. Peptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right, from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named using either three-letter or one-letter notation, as shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Peptides include any oligopeptides, polypeptides, gene products, expression products, or proteins. Peptides are composed of a sequence of amino acids and include naturally occurring or synthetic molecules. In addition, as used herein, the term “peptide” refers to amino acids linked to one another by peptide bonds or modified peptide bonds, such as peptide isosteas, and may contain modified amino acids other than the 20 gene-coding amino acids. Peptides can be modified by natural processes, such as post-translational processing or chemical modification techniques well known in the art. Modifications can occur at any site on the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. The same type of modification may be present to the same or different degrees at several sites of a given polypeptide. Similarly, a given peptide may have many types of modifications.Modifications include, but are not limited to, linking of separate domains or motifs, acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodation, methylation, myristoylation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer of amino acids to proteins - RNA-mediated addition, including, for example, arginylation (Proteins-Structure and Molecular Properties 2nd Ed., TE Creighton, WH Freeman and Company, New York (1993); Posttranslational Covalent See Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

[0046] As used herein, the term “Protein of Interest (POI)” includes any synthetic or naturally occurring protein or peptide. Therefore, this term encompasses molecules such as proteins, peptides, and similars, including those compounds conventionally considered as drugs, vaccines, and biologics. Examples of therapeutic agents are found in the Merck Index (14th edition) and the Physicians' Desk Reference (64). thThese are described in well-known literature such as (edition) and The Pharmacological Basis of Therapeutics (1st edition), and include, but are not limited to, medicines; substances used for the treatment, prevention, diagnosis, cure, or relief of diseases or illnesses; substances that affect the structure or function of the body; or prodrugs that become or become more bioactive after being placed in a physiological environment.

[0047] "Intein" refers to an in-frame intervening sequence in a protein. Inteins can catalyze their own excision from a protein through the post-translational protein splicing process, yielding free intein and mature protein. Inteins can also catalyze the cleavage of intein-extrain bonds at either the intein N-terminus, the intein C-terminus, or both the intein-extrainus. As used herein, "intine" includes mini-intine, modified or mutant inteins, and split inteins. As used herein, the term "split intein" refers to any intein in which one or more peptide bond breaks are present between the N-terminal intein segment and the C-terminal intein segment, thereby forming separate molecules in which the N-terminal and C-terminal intein segments can be reassigned non-covalently to an intein functional for splicing or cleavage reactions. Split inteins for use in the proteins, systems, and methods disclosed herein may be derived using any catalytically active intein or a fragment thereof. For example, in one variant, a split intein may be derived from a eukaryotic intein. According to another variant, a split intein may be derived from a bacterial intein. According to yet another variant, a split intein may be derived from an archaeal intein. The split intein thus derived may possess only the amino acid sequence essential for catalyzing the splicing reaction. As used herein, “split N-intine,” “N-terminal intein segment,” or “N-intine” refers to any intein sequence containing an N-terminal amino acid sequence that, when combined with the corresponding C-terminal intein segment, is functional for splicing and / or cleavage reactions. The N-terminal intein segment or split N-intine thus also includes the sequence that is spliced ​​and removed when splicing occurs. Split N-intines may contain sequences that are modifications to the N-terminal portion of naturally occurring (natural) intein sequences.Non-intane residues can also be gene-fused to the intein segment to provide additional functionality, such as the ability to be affinity-purified or immobilized by covalent bonding. As used herein, “split C-intane,” “C-terminal intein segment,” or “C-intane” refers to any intein sequence containing a C-terminal amino acid sequence that is functional for splicing or cleavage reactions when combined with the corresponding N-terminal intein segment. Depending on the variant, the C-terminal intein segment may contain a sequence that is spliced ​​and removed when splicing occurs. In another variant, the split C-intane may be cleaved from the peptide sequence fused to its C-terminus. The sequence cleaved from the C-terminus of the C-terminal intein may be referred herein to as the “target protein POI.” The C-terminal intein segment may contain a sequence that is a modification of the C-terminal portion of a naturally occurring (natural) intein sequence. For example, the C-terminal intein segment may contain additional amino acid residues and / or mutated residues, provided that such additional residues and / or mutated residues do not render the C-terminal intein segment non-functional for splicing or cleavage.

[0048] Deformed forms of N-intene proteins A variant of the embodiment relates to affinity separation, such as affinity chromatography. A single-step affinity-tagged cleavage mechanism using a split intein system that cleaves with broad amino acid tolerance to produce an untagged target protein (POI) as the final product may be used according to a variant of the embodiment herein. The two halves of the intein may be a ligand moiety (e.g., split N-intine) and an affinity tag fused to the target molecule (e.g., split C-intine), which associate rapidly. Immobilizing one half (split N-intine) on a solid phase, such as a chromatography resin, allows for the capture of the other half (split C-intine) fused to or coupled to the POI from the sample liquid or sample solution. For some inteins, e.g., Zn 2+The presence of ions can inhibit the cleavage reaction, for example, while washing away impurities, thereby enabling the formation of a stable complex. For the other intein, cleavage can be inhibited or promoted by altering other properties, such as pH or ionic strength. After impurities have been excluded or removed, a chelating agent or reducing agent may be added to allow the cleavage reaction to proceed, enabling the collection of POIs with the intein tag remaining non-covalently bound to a covalent intein linked to the solid phase.

[0049] Natural inteins are known in the art. A list of inteins can be found in Table 1 of WO 2021 / 099607, which is incorporated herein by reference. All inteins have the potential to become split inteins, although some inteins exist naturally in split form.

[0050] The variant forms of the split inteins described herein may be modified or mutated inteins. The modified inteins may include modifications to the N-terminal intein segment, the C-terminal intein segment, or both. The modifications may include additional amino acids at the N-terminus or C-terminus of any portion of the split intein, or within any portion of the split intein.

[0051] Disclosure of sequences used in this specification The following list of arrays is disclosed and used herein.

[0052] [Table 1]

[0053] Referring to Figure 1, we will now consider protein 1 for affinity capture. Protein 1 may be protein 1 according to the first embodiment or a variant thereof. Figure 1 is a schematic diagram representing an attempt to improve understanding of protein 1 as it may be used according to embodiments of this specification. Protein 1 comprises a ligand moiety 2 capable of affinity interaction with a target molecule, and a linker moiety 4 capable of binding to ligand moiety 2 and coupling to a solid phase (not shown). Furthermore, ligand moiety 2 does not contain lysine residues, and linker moiety 4 is located at the terminal of protein 1 and has at least two lysine residues in the linker moiety, wherein at least one non-lysine residue is terminally positioned relative to at least two lysine residues. With respect to the at least one non-lysine residue that is terminally positioned relative to at least two lysine residues, it should be understood that "terminally positioned" means that it is located distal to the ligand moiety and is positioned in such a manner.

[0054] The ligand moiety may contain a polypeptide. In particular, the ligand moiety may consist of a polypeptide.

[0055] The linker portion may contain polypeptides. In particular, the linker portion may consist of polypeptides.

[0056] A protein 1 having a linker moiety 4 that can bind to a ligand moiety 2 and couple to a solid phase 8 allows for the immobilization of protein 1 to the solid phase 8, as illustrated in Figure 2. Such a solid phase 8 to which protein 1 is bound may be used as a solid phase 8 material or affinity capture medium in affinity chromatography applications. Thus, Figure 2 can also illustrate an affinity capture medium or a modified form thereof according to a second embodiment. Referring further to Figure 2, the linker moiety 4 provides distance between the ligand moiety 2 and the solid phase 8, thus reducing the risk of deformation of the ligand moiety 2, e.g., denaturation, and further reducing the risk of steric interference or other obscuration of the interaction or binding of the active site of the ligand moiety 2 with a target molecule such as a POI or a target sample containing a POI. Although the solid phase 8 is schematically illustrated as a surface, it is recognized that the solid phase can be provided in any preferred form, such as beads, chips, fibrous or nanofiber-like structures, or monolithic structures. Furthermore, the solid phase 8 may be provided with magnetic properties, thereby facilitating, for example, the control or manipulation of the solid phase 8. For example, the solid phase 8 may be in the form of magnetic beads. Any suitable solid phase 8 that can be used as a medium for chromatography or separation purposes and is suitable for binding protein 1 may be used as described herein. When the solid phase takes the form of a convex surface or structure, such as beads or spheres, it is recognized that the linker portion 4 can link a greater number of ligand portions 2 to the solid phase compared to when the ligand portions 2 are directly bound to the solid phase 8 or bound by a fairly short linker portion 4. Porous media can also allow a greater number of ligand portions 2 to link to the solid phase.

[0057] The linker portion may contain, or may consist of, an amino acid sequence having 5 to 100 amino acid residues.

[0058] The linker portion may contain, or consist of, an amino acid sequence having 5 to 75 amino acid residues, for example, 5 to 50, 5 to 30, 9 to 30, or 12 to 20 amino acid residues.

[0059] The ligand moiety may or may consist of a peptide or protein. Thus, the ligand moiety may or may consist of an amino acid sequence.

[0060] The linker portion may have an isoelectric point above 6.0. This can improve bonding to the solid phase.

[0061] The linker portion may have at least three lysine residues. This can improve binding to the solid phase.

[0062] The linker portion may have an isoelectric point above 8.0. In particular, if the linker portion has three lysine residues, it can have an isoelectric point above 8, thereby improving the immobilization of the protein to the solid phase.

[0063] The ligand portion may be a split N-intane or a split C-intane, and in this way, affinity interactions with the corresponding split C-intane or split N-intane are possible.

[0064] The target molecule, for example POI, may be fused with a split intein capable of interacting with the ligand portion of the split intein, and may be expressed together with it, for example.

[0065] Split N-intanes and split C-intanes may be derived from naturally occurring split inteins or engineered split inteins.

[0066] The ligand portion may be a split N-intane, and the linker portion may be a linker portion located at the C-terminus. The split N-intane has the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLEDGSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (Sequence ID 1), (In the formula, The X at positions 20, 35, 70, 73, and 95 are each independently selected from R or A; The 28th ranked X is C, A, or S; The 36th ranked X is N, H, or Q; The 25th ranked X is either N or R; The 59th ranked X is either D or C; The 80th X is either E or Q; and (X in 90th place is either Q or R) Includes.

[0067] Such split N-intanes are beneficial because they do not contain lysine residues. When combined with a linker moiety having at least two lysine residues, specific binding to the solid phase via the linker moiety can be achieved, particularly if at least one non-lysine residue is terminal to at least two lysine residues. This allows the ligand to be immobilized on the solid phase while reducing the risk of ligand moiety deformation, potentially enabling improved affinity interactions and separation. In addition, certain sequences have been selected with respect to improved alkaline stability, providing resistance to increases in pH values ​​during chromatographic procedures, such as regeneration of the solid phase using a highly alkaline solution. This can enable the reuse of the solid phase or the column used for affinity interactions.

[0068] In sequence number 1, The X values ​​in positions 20, 35, 70, 73, and 95 may also be R; X, which is ranked 28th, could also be A; The 36th ranked X could also be H; The 25th ranked X could also be N; X, which is ranked 59th, could also be D; X in 80th place may also be E; and The 90th ranked X could also be Q.

[0069] Thus, the split N-intane has the following sequence: ALSYDTEILTVEYGFLPIGRIVEENIEATVYSVDRHGFVYTQPIAQWHNRGEQEVFEYDLEDGSIIRATRDHRFMTTDGEMLPIDEIFEQGLDLRQV (Sequence ID 2) It may include, or may consist of.

[0070] Furthermore, an N-intane protein variant of the natural N-intane domain of Nostoc punctiforme (Npu) is provided, wherein the natural N-intane domain has the sequence defined by SEQ ID NO: 21.

[0071] With respect to an affinity capture protein following a variant of the first embodiment, wherein the ligand portion is a split N-intane and the linker portion is a linker portion located at the C-terminus, the split N-intane may contain or consist of a sequence having at least 70% or 80% identity, e.g., at least 90% or 95% identity, with SEQ ID NO: 21. With respect to such a split N-intane, position 36 of SEQ ID NO: 21 may preferably be replaced with histidine, H, and any present lysine may be replaced with an amino acid other than lysine, thereby enabling the provision of a ligand portion that does not contain lysine residues. Lysine may preferably be replaced with arginine, which retains the charge and charge properties of the split N-intane.

[0072] The linker portion may contain an amino acid sequence selected from the list consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0073] These particular sequences (SEQ ID NOs: 3-12) have been selected from a larger number of sequences to provide equivalent improvements in immobilization to the solid phase. It should be noted that each of these sequences contains at least two lysine residues, each having at least one non-lysine residue terminally relative to at least two lysine residues, thus providing improved immobilization to the solid phase. In other words, at least two lysine residues must have at least one non-lysine amino acid residue, i.e., a non-lysine amino acid residue, terminally. In particular, the linker portion may be described by SEQ ID NO: 3.

[0074] An example of a linker moiety directly bound to a ligand moiety can be illustrated, for example, by using SEQ ID NO: 3 fused to the N-intane described in SEQ ID NO: 2, which together forms an affinity capture protein described in SEQ ID NO: 20.

[0075] Referring to Figure 3, an affinity capture medium 100 described in the second embodiment is schematically illustrated. The affinity capture medium 100 comprises an affinity capture protein 101 according to the first embodiment and a solid phase 108. The ligand portion 102 of the protein is covalently bound to the solid phase 108 via a linker portion 104. Figure 3 illustrates an example in which the solid phase 108 is illustrated as a sphere, but it will be recognized that other preferred shapes are possible. The ligand portion 102 is illustrated to have an interaction site 110 capable of affinity interaction with a target molecule. Figure 7 schematically illustrates with only one affinity capture protein 101 bound to the solid phase 108, but it will be understood that having several proteins 101 bound to the solid phase 108 structure is achievable and beneficial.

[0076] The linker moiety 104 of protein 101 can be covalently bonded to the solid phase via at least one lysine residue in the amino acid sequence of the linker moiety. The linker moiety 104 can be covalently bonded to the solid phase 108 by a chemical reaction involving contact between the lysine residue and the solid phase 108. Examples of suitable chemicals or reagents for the reaction with the lysine residue include, for example, cyanide bromide, epoxide, or N-hydroxysuccinimide esters.

[0077] The solid phase may be in the form of beads, films, particles, chips, fibrous or nanofiber-like structures, or monolithic structures, or a combination thereof.

[0078] In particular, the solid phase may be in the form of beads.

[0079] The solid phase may contain natural or synthetic polymer materials, preferably polysaccharides such as cellulose or agarose, or may be composed of them.

[0080] The solid phase may contain, or be composed of, one or more compounds selected from the group consisting of agarose, polystyrene, methacrylate, and cellulose, or combinations thereof.

[0081] Referring to Figures 4a and 4b, we consider a split intein system 200 for affinity capture of a target protein 220 (POI) according to a variant of the third embodiment. The split intein system 200 comprises a protein 201 for affinity capture according to the first embodiment, where the ligand portion 202 is a split N-intane. The split intein system 200 further comprises a target molecule which may be a POI fused to, for example, a split C-intane 222. The split C-intane 222 may be expressed, for example, as a fusion with POI 220. The split N-intane and split C-intane 222 are sequences of split inteins. Protein 201 can be coupled to solid phase 208, for example, via covalent bonds or binding, which is illustrated using the dashed lines of solid phase 208. For example, covalent bonds or binding can be achieved using the chemistry described above. Figure 4a illustrates the split intein system 200, in which the target molecule, a POI fused to split C-intane 222, has not yet interacted with protein 201. Figure 4b illustrates the split intein system 200, in which the target molecule, a POI fused to split C-intane 222, has come into contact with and is interacting with protein 201 via split C-intane 222.

[0082] The use of the split intein described above offers several advantages. The affinity recognition and interaction between the split N-intane and split C-intane of the split intein provides high selectivity in separation. Furthermore, the intrinsic properties of the intein allow for spontaneous or autocleavage, thereby detaching the POI from the split intein to which it is fused, thereby enabling efficient elution of the POI in separation or chromatographic methods. The split C-intane can dissociate from the split N-intane upon regeneration of protein 201 or an affinity capture medium containing protein 201.

[0083] The protein for affinity capture may be contained in the affinity capture medium according to the second embodiment.

[0084] Referring to Figure 5, we consider a method 300 for purifying a target protein (POI) according to a further modified form. Method 300 comprises loading a sample liquid containing a POI onto an affinity capture medium containing an affinity capture protein that is covalently bonded to a solid phase via a linker portion and has affinity for the POI or a target molecule containing a POI, wherein the affinity capture protein is a protein according to the first embodiment; capturing the POI or a target molecule containing a POI by binding of the affinity capture medium to the protein; washing the affinity capture medium with a washing solution to remove compounds other than the POI or a target molecule containing a POI; and eluting the POI or a target molecule containing a POI from the affinity capture medium.

[0085] POIs or target molecules containing POIs can be released from proteins by the split-intane system and the intein's inherent ability to cleave fusion proteins, such as POIs.

[0086] The affinity capturing medium may be an affinity capturing medium according to the second embodiment.

[0087] The target molecule containing the POI may be, for example, a target molecule in which the POI is fused to a peptide, or the target molecule containing the POI may be the POI itself, in which case a ligand portion suitable for affinity interaction with the POI may be selected with respect to the protein for affinity capture.

[0088] The affinity capturing medium according to the fourth embodiment may be the affinity capturing medium according to the second embodiment.

[0089] The affinity capture protein may be a protein according to the first embodiment, wherein the ligand is a split N-intane and the target molecule is a POI fused to a split C-intane.

[0090] While variations of Method 300 and the fourth embodiment describe the purification of POIs, it is recognized that alternative variations may relate to the purification of other molecules of interest that are suitable for purification by such methods.

[0091] Referring to Figure 6, a method 400 for purifying POIs following a modified form of the method discussed with reference to Figure 5 will be described in more detail. Method 400 involves the use of an affinity capture protein 401 following a modified form of the first embodiment, in other words, the use of an affinity capture protein 401 that includes a ligand moiety 402 capable of affinity interaction with a target molecule 421, in this example a POI 420 fused to a target molecule moiety 422, or other target molecules may be usable for method 400 if suitable. With respect to the example of a target molecule 421 containing a POI 420, the target molecule 421 may include a target molecule moiety 422 capable of affinity interaction with the ligand moiety 402. The affinity capture protein 401 further includes a linker moiety 404 capable of binding to the ligand moiety 402 and coupling with the solid phase 408. In this example, the ligand moiety 402 does not contain a lysine residue, and the linker moiety 404 is located at the terminus of protein 401. Furthermore, the linker portion contains an amino acid sequence having 5 to 100 amino acid residues, of which at least two are lysine residues. In addition, at least one residue other than lysine is located at the terminal of at least two lysine residues.

[0092] Method 400 includes loading a sample liquid containing a target molecule 421 including (I)POI 420, in this example an aqueous solution, onto an affinity capture medium 100 containing an affinity capture protein 401 having affinity to the target molecule including the POI, which is covalently bonded to a solid phase 208 via a linker portion 404. Loading 450 may, for example, be injected into a chromatography column or chromatography membrane (the column or membrane is not illustrated in Figure 6). The sample liquid may, for example, be a cell culture, or may be obtained from a cell culture, for example, from the cell culture supernatant or lysate. Such a sample liquid may typically contain a considerable amount of contaminants or molecules other than the target molecule. The sample liquid may be subjected to cleaning or purification of the sample before loading the sample. A suitable binding buffer may exist for the purpose of loading and binding the sample, for example, the sample liquid may be a sample mixed with the sample buffer. The sample buffer or sample liquid may contain buffers and / or salts. The sample liquid or binding buffer may have a pH in the range of 6 to 9. The salt concentration may be in the range of 0 to 1 M, for example, 1 mM to 1 M. The binding buffer or sample liquid may contain NaCl and / or phosphate. The phosphate may be used to adjust or set the pH value. NaCl may preferably be used to adjust the salt concentration. For example, the binding buffer may contain or consist of phosphate-buffered saline (PBS) at pH 7.4. Method 400 further comprises (II) capturing a target molecule 421 containing POI 420 by binding to a protein 401 of the affinity capture medium 100 452. The binding of the target molecule 421 containing POI 420 to the protein 401 may also be achieved by enabling contact between the target molecule 421 containing POI 420 and the protein 401, for example, by the diffusion of the target molecule 421 containing POI in the sample liquid or by the diffusion of the target molecule 421 containing POI in the sample liquid.For example, if the target molecule 421 is a POI fused to a split C-intine, for example, a POI expressed in a fusion with a split C-intine, and the ligand portion contains the corresponding split N-intine, then capture 452 may involve affinity interactions between the split C-intine and the split N-intine. Subsequently, (III) the affinity capture medium is washed with a washing solution 354 to remove compounds other than the target molecule containing POI 420 from the sample liquid. Compounds other than POI 420 424 are schematically represented by triangles in Figure 6 and may be any compounds present in the sample liquid other than the target molecule, e.g., POI, such as other proteins, DNA, RNA, salts, or reagents. Therefore, compounds other than the target molecule 424 may be considered contaminants or impurities. Due to the specificity of protein 401 to target molecule 421 containing POI 420, compound 424 does not bind to protein 401 via affinity interactions during or after loading 405. However, contaminants or compounds may interact with the solid phase 408 or affinity capture medium 100 using other mechanisms, such as one or more mechanisms based on hydrogen bonding, ionic interactions, hydrophobicity, or hydrophilic interactions. Washing 354 can remove at least a large portion of other compounds 424 from the affinity capture medium 100. Washing 354 can be carried out using a suitable buffer, which may be water-soluble and may contain salts and / or detergents. Washing may be carried out using a high pH, ​​which can provide efficient removal of compounds other than POI, by selecting a ligand moiety 402 that can handle high pH solutions or alkaline solutions. When using split-intane, Zn is used to prevent elution of target molecules containing POI during washing. 2+ Ions may be present in the washing buffer.

[0093] Following washing 454, the POI or target molecule containing the POI is eluted from the affinity capture medium 456. Elution 456 can be at least partially achieved or proceed by waiting for a retention time, for example, if a self-cleaving split-intane is used as the ligand portion and the corresponding split-intane is expressed in the fusion with the POI 420. The retention time may be, for example, between 1 and 30 hours, for example between 1 and 25 hours, or between 2 and 6 hours, or between 15 and 25 hours, for example about 4 hours or about 20 hours. Next, the POI or target molecule containing the POI is eluted from the affinity capture medium, thereby obtaining the POI purified from the compound in the sample liquid. For example, elution may be facilitated or performed by changing the properties of the buffer or liquid present. For example, pH, ionic concentration, or salt concentration, or hydrophobicity or hydrophilicity, or different combinations of such properties may be changed to facilitate elution.

[0094] For example, elution may be carried out using imidazole in PBS, for example, a solution containing or consisting of 250 mM imidazole in PBS adjusted to pH 7.4.

[0095] After elution, the solid phase or the column containing the solid phase may be regenerated. Regeneration may include washing with a regeneration solution, which may be an alkaline solution. For example, regeneration of the solid phase contained in the column may be used to prepare the solid phase or column for further purification of the target protein (POI).

[0096] Thus, the method 400 for purifying the target protein (POI) may further include solid phase regeneration, including washing with a regeneration solution.

[0097] Regeneration can be carried out using a regeneration solution containing, for example, an aqueous solution of sodium hydroxide and urea, such as an aqueous solution of 100 mM sodium hydroxide and 4 M urea.

[0098] The ligand portion of this variant of the fourth embodiment may be a split N-intane, the linker portion may be a C-terminal linker portion, and the split N-intane includes the sequence defined by SEQ ID NO: 2.

[0099] The C-terminal linker portion may contain an amino acid sequence selected from the list consisting of SEQ ID NOs: 3 to 12.

[0100] Thus, it is recognized that methods 300 and 400 may also use a split-intane system for affinity capture of a POI, comprising a protein for affinity capture according to the first embodiment and a variant thereof, wherein the ligand is a split N-intane, and a split C-intane expressed in the fusion with the POI.

[0101] The ligand moieties of these modified forms of the fourth embodiment, as illustrated with reference to Figures 5 and 6, can be covalently bonded to solid phase 408 via lysine residues of the linker moiety, thus forming an affinity capture medium.

[0102] The sample liquid may be any suitable liquid containing POI. For example, the sample liquid may be a cell culture medium in which POI is expressed, or may be obtained therefrom. The sample may be an aqueous sample liquid or an aqueous solution. [Examples]

[0103] Experiments were conducted to evaluate protein immobilization for affinity capture in a solid phase. The objective was to achieve high surface concentrations of proteins in the solid phase, as high surface concentrations provide desirable separation or chromatographic properties. A further objective was to achieve efficient binding of target molecules using a solid phase with immobilized proteins.

[0104] The affinity capture protein used in the experiment consisted of a ligand moiety capable of affinity interaction with the target molecule, and a linker moiety bound to the ligand moiety. Immobilization to the solid phase was achieved by binding the linker moiety to the solid phase via covalent linkage of lysine residues to the solid phase.

[0105] The experimental setup was based on the understanding that a ligand moiety that does not contain lysine residues, having a linker moiety with an amino acid sequence containing lysine, could lead to efficient binding to a solid phase via the linker moiety.

[0106] Ligand portion Regarding the experiment, the following sequence: ALSYDTEILTVEYGFLPIGRIVEENIEATVYSVDRHGFVYTQPIAQWHNRGEQEVFEYDLEDGSIIRATRDHRFMTTDGEMLPIDEIFEQGLDLRQV (Sequence ID 2) The ligand moiety containing [the specified characteristic] was used. The ligand moiety used in the experiment may further be described as an N-intei.

[0107] Linker section For the experiment, we designed a ligand moiety that is an N-intane, and provided it with different linker moies at the C-terminus in the form of amino acid sequences of various lengths, including variations in the number and position of lysine residues.

[0108] Therefore, the ligand portion provided with the linker portion is a protein containing the ligand portion and the linker portion bound to the ligand portion. The ligand portion can be considered to contain a ligand protein or ligand peptide that does not contain lysine residues, and the linker portion is located at the end of the protein and contains an amino acid sequence.

[0109] The amino acid sequence of the linker portion used in the experiment is provided in Table 1 (Table 2).

[0110] [Table 2]

[0111] Evaluation of ligand immobilization in the solid phase Proteins for different affinity capture were evaluated. All evaluated proteins contained the ligand moiety described by Sequence ID No. 2, as discussed above, and one of the linker moieties in Table 1 (Table 2). Evaluation was performed using a Biacore T-200 instrument (Cytiva, Sweden) with surface plasmon resonance (SPR) for detection and analysis. Proteins containing the ligand moiety and their respective linker moieties were expressed in Escherichia coli (E. coli) BL21 (DE3) and purified to >95% purity before SPR analysis using a combination of hydrophobic interaction chromatography and ion exchange chromatography (HiTrap Phenyl Sepharose HP and Capto Q, Cytiva, respectively). Purity was determined using CBB-stained, i.e., Coomassie brilliant blue-stained SDS-PAGE polyacrylamide gel (SERVA Electrophoresis GmbH) and size exclusion chromatography analysis on Superdex 75 Increase (Cytiva). The identification of each protein, including the ligand and linker moieties, was confirmed by comparing the theoretical molecular weight from the amino acid sequence with full-length LC-MS analysis data (BioAccord System, Waters). C-intane-tagged proteins used for experimental isolation were purified with Strep-Tactin XT Sepharose and analyzed using the same method as the proteins containing the ligand and linker moieties. The Biacore method was performed at 25°C using HBS-EP+, pH 7.4 (Cytiva AB) as the running buffer.

[0112] All reagents and buffers were purchased from Cytiva and used according to the user's instructions and standard settings. Immobilization of proteins, including the ligand and linker moieties, was performed using a Series-S sensor chip CM5 as the solid phase. This involved activating the chip surface using EDC and NHS from an amine coupling kit, followed by injecting the purified ligand moiety coupled to the linker moiety, diluted to 20 μg / ml with 10 mM sodium acetate pH 4.0, to bring the activated surface into contact with the chip. Sample injection was performed at 10 μl / min for 420 seconds. Finally, any remaining unreacted NHS groups were blocked by injecting 1 M ethanolamine. The response, RU, or resonance unit was directly proportional to the protein concentration on the surface. The immobilization level equal to the relative response was determined by subtracting the absolute response before immobilization from the absolute response after immobilization.

[0113] The relative responses from the immobilization of different linker-ligand pairs of proteins are shown in Figure 7 and similarly in Table 2 (Table 3). From the experimental data illustrated in Figure 7, along with the sequences in Table 1 (Table 2), it can be concluded that a higher level of immobilization was achieved when the linker moiety had at least two lysine residues, with at least one non-lysine residue positioned terminally relative to at least two lysine residues.

[0114] It can be further concluded that linker portions having an isoelectric point (pI) above approximately 6, and especially those having a pI above approximately 8, can provide a beneficial level of immobilization.

[0115] Evaluation of the binding ability of immobilized ligand-linker pairs The relative binding ability of each ligand-linker pair (Table 1 (Table 2)) was tested after immobilizing the ligand portion on a sensor chip. Binding ability was evaluated using test protein B64 (interleukin 1-b, IL-1b) tagged with a mutant non-cleavable split C-intane. Self-cleavage functionality was inactivated by point mutation of the catalytic C-terminal Asn in the C-intane sequence to Ala(N36A). Purified test protein B64 was diluted to 20 μg / ml with HBS-EP+, pH 7.4 buffer, and then injected into the sensor chip at 30 μl / min for 1 minute. The relative response was measured 10 seconds after sample injection. Binding levels are shown as relative responses in Table 2 (Table 3), which also shows the immobilization levels from Table 1 (Table 2).

[0116] [Table 3]

[0117] In Table 2 (Table 3), linker moieties containing at least two lysine residues, where at least one non-lysine residue is terminally positioned relative to at least two lysine residues, are marked with an asterisk along with their identifying names. In particular, these linker moieties associated at the highest level of immobilization.

[0118] Figure 8 illustrates the join level compared to the immobilization level using data from Table 2 (Table 3).

[0119] From Table 2 (Table 3) and Figure 8, we can conclude that there is a clear and strong correlation between the immobilization level and the binding level. Furthermore, we can conclude that the linker region that yields the highest immobilization level also has the highest, and thus most desirable, binding level. In Figure 8, linker regions having at least two lysine residues, i.e., linker regions with the highest immobilization level, are marked with dashed boxes, where at least one non-lysine residue is terminally positioned relative to at least two lysine residues.

[0120] Experiment - Column Application An N-intane protein (identification name C25), consisting of a ligand moiety according to SEQ ID NO: 2 and a linker moiety according to SEQ ID NO: 7, was expressed in Escherichia coli BL21 (DE3) and purified to >95% purity using a combination of hydrophobic interaction chromatography and ion exchange chromatography (HiTrap Phenyl Sepharose HP and Capto Q, Cytiva, respectively). Purity was determined by size exclusion chromatography analysis using a Coomassie brilliant blue stained SDS-PAGE gel (SERVA Electrophoresis GmbH) and a Superdex 75 Increase column (Cytiva). The identification of the N-intane protein was confirmed by comparing the experimental molecular weight obtained from mass spectrometry data (BioAccord System, Waters) with the theoretical molecular weight calculated from the amino acid sequence. The N-intane protein was further concentrated to a concentration of 41 mg / ml by ultrafiltration in a buffer containing 50 mM sodium phosphate and 0.5 M sodium chloride adjusted to pH 7.0. Before coupling the epoxy-activated solid phase in the form of agarose beads, the pH was adjusted to pH 10-11. 8 grams of epoxy-activated agarose beads were added to a small 50 ml reactor flask. 3.7 ml of ligand solution was added. The pH of the resulting reaction mixture was adjusted to pH 11 with 2 M sodium hydroxide. The reaction mixture was mixed for 3 hours. The resulting agarose bead slurry was transferred to a glass filter and washed five times with 10 ml of distilled water.

[0121] After washing, the gel resin was transferred to a small 50 ml reactor flask, and 8 ml of 0.1 M Tris buffer pH 10 and 0.6 ml of thioglycerol were added. The pH was adjusted to pH 8.5. The reaction mixture was stirred for 2 hours. The resulting agarose bead slurry was transferred to a glass filter and washed with 0.1 M Tris buffer, followed by washing with 0.5 M acetic acid. This base / acid washing was repeated two more times. The gel resin was then washed with distilled water. The washed and drained gel resin was finally maintained in 20% ethanol and then packed into a 1 ml column.

[0122] The receptor-binding domain (RBD) from the SARS-CoV-2 spike protein was used as the test protein. Test protein B58, possessing a C-terminal polyhistidine tag, an N-terminal signal sequence, and a C-intane tag, was expressed in Expi293 cells and secreted into cell culture medium. Six days after transfection, the cell culture medium was collected, and cells and particles were removed by tangential flow filtration. The final sample was stored at -80°C until purified.

[0123] Figure 9 shows the UV chromatogram, including the steps from column equilibration to regeneration. The sample containing test protein B58 was thawed and applied to a tandem column setup, with a 5 ml HisTrap Excel (Cytiva) connected in succession to a 1 ml column containing immobilized N-intane protein. Equilibration was performed using equilibration buffer in the form of phosphate-buffered saline, i.e., PBS, pH 7.4, with a flow rate of 1 ml / min during loading of 80 ml of sample and 2 ml / min during washing and elution. After elution with 12 column volumes of elution buffer containing 250 mM imidazole in pH 7.4 PBS, the flow rate was stopped, as indicated by "pause" in Figure 9, to allow for cleavage incubation during a 20-hour pause. The cleaved proteins eluted as sharp peaks in the UV chromatogram after incubation, as indicated by the arrows in Figure 9, and were collected for further analysis. Finally, the column was regenerated with 100 mM sodium hydroxide containing 4 M urea, which resulted in a sharp peak at the end of the chromatogram. The purification results are shown in Figure 9 (chromatogram) and Figure 10 (SDS-PAGE analysis). The estimated elution amount of RBD from UV absorption at 280 nm was 10.6 mg, and 67% of the fusion protein was recovered, with 33% found in the regenerated fraction.

[0124] The experiment was repeated, but the cleavage incubation time, washing, and elution time after sample application were changed from 20 hours to 4 hours. The amount of RBD cleaved and eluted was lower, as expected, compared to longer incubation times. The amount of RBD eluted, estimated from UV absorption at 280 nm, was 7.7 mg, and 46% of the fusion protein was recovered, but 54% was found in the regenerated fraction. The purity of the eluted RBD from both purifies was close to 100% according to SDS-PAGE. A photograph of the SDS-PAGE gel is shown in Figure 10. The data obtained from the SDS-PAGE gel is shown in Table 3 (Table 4).

[0125] [Table 4] [Explanation of Symbols]

[0126] 1. Protein 2. Ligand portion 4 Linker section 8 Solid phase 100 Affinity Capture Media 101 Proteins 102 Ligand portion 104 Linker section 108 Solid phase 110 Interaction site 200 Split-Intein System 201 Protein 202 Ligand portion 208 Solid phase 220 Target Protein (POI) 222 Split C-Intein 300 ways 350 Road 352 capture 354 Cleaning 358 Elution 400 ways 401 Protein 404 Linker section 408 Solid phase 420 POI 421 Target molecule 422 Target molecule moiety 424 Compound 450 Road 452 Capture 454 Cleaning

Claims

1. A ligand moiety capable of affinity interaction with the target molecule, and A linker portion capable of binding to the ligand portion and coupling to a solid phase, A protein for affinity capture, which includes The ligand portion does not contain lysine residues. The linker portion is located at the terminal end of the protein and has at least two lysine residues, wherein at least one residue other than lysine is located at the terminal end of at least two lysine residues. Proteins for affinity capture.

2. The affinity capture protein according to claim 1, wherein the linker portion comprises an amino acid sequence having 5 to 100, preferably 5 to 30 amino acid residues.

3. The affinity capture protein according to claim 1 or 2, wherein the linker portion has an isoelectric point above 6.

0.

4. The affinity capture protein according to any one of claims 1 to 3, wherein the linker portion has at least three lysine residues.

5. A protein for affinity capture according to any one of claims 1 to 4, wherein the ligand portion is a split N-intane or a split C-intane, and is thus capable of affinity interaction with the corresponding split C-intane or split N-intane, respectively.

6. The ligand portion is a split N-intane, and the linker portion is a linker located at the C-terminus, and the split N-intane has the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLEDGSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (Sequence ID 1), (In the formula, The X at positions 20, 35, 70, 73, and 95 are each independently selected from R or A; The 28th ranked X is C, A, or S; The 36th ranked X is N, H, or Q; The 25th ranked X is either N or R; The 59th ranked X is either D or C; The 80th X is either E or Q; and (X in 90th place is either Q or R) A protein for affinity capture according to any one of claims 1 to 5, comprising

7. X at positions 20, 35, 70, 73, and 95 is R; X, ranked 28th, is A; X, ranked 36th, is H; The 25th ranked X is N; X, ranked 59th, is D; X, ranked 80th, is E; and X, which is ranked 90th, is Q. The affinity capture protein according to claim 5.

8. The linker part, DADSGGSGKDDAKKKDDTKK (Sequence ID 3), GGSGKDDAKKKDDTKK (Sequence ID 4), DADSKDDAKKKDDTKK (Sequence ID 5), KDDAKKKDDTKK (Sequence ID 6), ARTKQTARKSTGGK (Sequence ID 7), ARTKQTARKSTGGKAPRK (Sequence ID 8), SGRGKGGKGLGKGGAK (Sequence ID 9), AKSAPAPKKGSKK (Sequence ID 10), PKKTESSKSKSK (Sequence ID 11), and ARTKQTARKSTGGR (Sequence ID 12) A protein for affinity capture according to any one of claims 1 to 7, comprising an amino acid sequence selected from a list consisting of the following.

9. An affinity capture medium comprising a protein for affinity capture according to any one of claims 1 to 8, and a solid phase, wherein the ligand portion of the protein is covalently bonded to the solid phase via a linker portion.

10. The affinity capture medium according to claim 9, wherein the linker portion of the protein is covalently bonded to the solid phase via at least one lysine residue in the amino acid sequence of the linker portion.

11. The affinity capturing medium according to claim 9 or 10, wherein the solid phase is in the form of beads, films, particles, chips, fibrous or nanofiber-like structures, or monolithic structures, or a combination thereof.

12. The affinity capturing medium according to any one of claims 9 to 11, wherein the solid phase comprises or is composed of natural or synthetic polymer materials, preferably polysaccharides such as cellulose or agarose.

13. A split-intine system for affinity capture of a target protein (POI), A protein for affinity capture according to any one of claims 1 to 8, wherein the ligand is split N-intei, and Target molecule that is a POI fused to split C-intei. A split-intein system, including [a specific feature / feature].

14. The split-intine system according to claim 11, wherein the protein for affinity capture is contained in the affinity capture medium according to any one of claims 9 to 12.

15. A method for purifying a target protein (POI), A step of loading a sample liquid containing a POI into an affinity capture medium containing an affinity capture protein that has affinity to the POI or a target molecule containing a POI, which is covalently bonded to a solid phase via a linker portion, wherein the affinity capture protein is the protein described in any one of claims 1 to 8. A step of capturing a POI or a target molecule containing a POI by binding to a protein of an affinity capture medium. The process involves washing the affinity capture medium with a washing solution to remove POIs or compounds other than target molecules containing POIs, and A step of eluting a POI or a target molecule containing a POI from an affinity capture medium. A method that includes this.

16. A method for purifying a target protein (POI) according to claim 12, wherein the affinity capture medium is the affinity capture medium described in any one of claims 7 to 10.

17. The protein for affinity capture is the protein described in any one of claims 1 to 8, and the ligand is split N-intei, and The target molecule is a POI fused to a split C-intei. A method for purifying the target protein (POI) according to claim 15 or 16.

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