Fusion protein of Z-domain and calcein with improved reactivity, stability and antibody recovery rate and method for separating and purifying antibodies using the same

Abstract

The present invention relates to a fusion protein of a Z-domain and calcitonin with improved reactivity, stability and antibody recovery, and a method for separating and purifying antibodies using the same. Specifically, the present invention relates to a nucleic acid encoding a fusion protein of a Z-domain and calcitonin with improved reactivity and stability, a recombinant expression vector comprising the nucleic acid, a transgenic organism transformed by the recombinant expression vector, and a method for separating and purifying antibodies using the Z-domain and calcitonin fusion protein with improved reactivity, stability, antibody recovery and purity.

Description

Technical Field

[0001] The present invention relates to a fusion protein of a Z-domain and calcitonin with improved reactivity, stability and antibody recovery rate, and a method for separating and purifying antibodies using the same. Specifically, the present invention relates to a nucleic acid encoding a fusion protein of a Z-domain and calcitonin with improved reactivity, stability and antibody recovery rate, a recombinant expression vector comprising the nucleic acid, a transgenic organism transformed by the recombinant expression vector, and a method for separating and purifying antibodies using the fusion protein of the Z-domain and calcitonin with improved reactivity, stability, antibody recovery rate and purity. Background Art

[0002] The purification process of monoclonal antibodies generally includes four basic steps. These steps include: (1) harvesting - separation of host cells from the fermentation culture; (2) capture - separation of antibodies from the bulk of the purified harvest; (3) micro-purification - removal of residual host cell contaminants and aggregates; and (4) formulation - the step of placing the antibody in a suitable carrier for maximum stability and storage period. However, these steps will not necessarily produce an antibody composition with sufficient purity for use in a pharmaceutical setting. Therefore, it is of utmost importance to produce and purify the target antibody that is suitable for pharmaceutical use and in a purified form, from which impurities have been removed.

[0003] Since protein A chromatography can almost completely purify antibodies, generally IgG, from cell culture supernatants in a single step, it is widely used in industrial antibody preparation. However, protein A chromatography has the problem that the ligand of the chromatographic column must leak to a certain extent due to repeated use. Such protein A or protein A fragments have affinity for IgG, so they form complexes with antibodies to contaminate antibodies, and are difficult to remove from purified antibodies. In particular, since protein A is a bacterial protein, it can induce undesirable immune responses, and therefore, it is necessary to remove it from purified antibodies. Therefore, the antibody purification process using protein A chromatography has the problem of having to monitor and remove the residual amount of protein A in each process.

[0004] In this regard, the present inventors disclosed through Patent No. 10-1774354 (Title of the Invention: Method for Separating and Purifying Antibodies Using a Fusion Protein of a Z-Domain and Calcitonin) a method for purifying antibodies with high purity and high quality without using the expensive protein A chromatography method commonly used for antibody purification.

[0005] Previous Literature

[0006] 1. Korean registered patent No. 10-1774353 Summary of the invention

[0007] Technical issues

[0008] The present inventors have continued to study methods for separating and purifying antibodies and have confirmed that the reactivity with calcium, stability, antibody recovery rate and purity of a fusion protein of the Z-domain and calcitonin can be significantly improved by changing the amino acid sequence of the Z-domain consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of calcitonin consisting of the amino acid sequence of SEQ ID NO: 4 used in Patent No. 10-1774353, thereby completing the present invention.

[0009] Technical Solution

[0010] The present invention relates to a nucleic acid encoding a fusion protein of a Z-domain and calcineurin with improved reactivity, stability and antibody recovery rate, wherein the calcineurin consists of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0011] The present invention also relates to a recombinant expression vector comprising a nucleic acid encoding a fusion protein of a Z-domain and calcineurin having improved reactivity, stability and antibody recovery.

[0012] The present invention also relates to a transgenic organism transformed by a recombinant expression vector comprising a nucleic acid encoding a fusion protein of a Z-domain and calcineurin having improved reactivity, stability and antibody recovery.

[0013] The present invention also relates to a method for separating and purifying antibodies with improved reactivity, stability and antibody recovery, the method comprising:

[0014] A) preparing a recombinant vector comprising a nucleic acid encoding a fusion protein of a Z-domain and calcineurin;

[0015] B) transducing the recombinant vector into a host cell, thereby obtaining a transgenic organism;

[0016] C) expressing Z-calcein protein from said transgenic organism; and

[0017] D) isolating antibodies binding to the Z-calcein protein from the expressed transgenic organism using calcium, wherein the calcein consists of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0018] Beneficial Effects

[0019] Compared to the fusion protein disclosed in the existing registered patent No. 10-1774353, the fusion protein of the Z-domain and calcitonin according to the present invention is more stable, has an improved precipitation reaction according to calcium and antibody recovery rate, can easily and quickly separate and purify antibodies, and can separate a large amount of antibodies at a very high speed compared to the protein A chromatography method widely used in the existing industry, thus reducing purification costs and preventing antibody decomposition. In addition, since the fusion protein of the Z-domain and calcitonin can be reused by using a chelate, it is economical.

[0020] Brief description of the attached figure

[0021] Figure 1 The turbidity test results of ZZ-CSQ1 (all) and ZZ-CSQ2 (Δ) according to calcium concentration in Test Example 1 are shown.

[0022] Figure 2 The test results for confirming the stability of ZZ-CSQ1 (all) and ZZ-CSQ2 (Δ) according to Test Example 1 are shown.

[0023] Figure 3 The results of a test to confirm stability after changing the linker G4S of ZZ-CSQ1 (all) according to Test Example 1 to 4N, 8N, and LE8N are shown.

[0024] Figure 4 The test results of measuring the affinity of the ZZ-CSQ1 (all) fusion protein according to Test Example 1 for antibodies are shown.

[0025] Figure 5 The results of an experiment in which antibodies purified using the ZZ-CSQ1 (all) fusion protein according to Experimental Example 1 were confirmed in an antibody culture solution are shown.

[0026] Figure 6 The results of a test to confirm the antibody recovery rate (Recovery) of ZZ-CSQ1 (all) at each pH according to Test Example 1 are shown.

[0027] Figure 7 The results of an experiment for confirming the host cell protein (HCP) of the antibody after purifying the antibody using the ZZ-CSQ1 (all) fusion protein according to Experimental Example 1 are shown.

[0028] Figure 8 The results of an experiment for confirming the host cell DNA (HCD) of the antibody after purifying the antibody using the ZZ-CSQ1 (all) fusion protein according to Experimental Example 1 are shown.

[0029] Fig. 9 The following are test results of confirming the aggregation of antibodies or ZZ-CSQ1 (all) by size exclusion high performance liquid chromatography (SEC-HPLC) after purifying antibodies using the ZZ-CSQ1 (all) fusion protein according to Test Example 1.

[0030] Fig.10 Shown are the test results of measuring the antibody recovery rate after changing the ZZ domain according to Test Example 1 to Z6G and ZL4G.

[0031] Fig.11 The turbidity test results of ZZ-CSQ2 (all) and ZZ-CSQ2 (Δ) of Test Example 2 according to the calcium concentration are shown.

[0032] Fig.12 The results of a test to confirm the degree of antibody recovery of ZZ-CSQ2 (all) and ZZ-CSQ2 (Δ) according to Test Example 2 are shown.

[0033] Fig.13 The results of a test to confirm changes in stability after the linker G4S of ZZ-CSQ2 (all) according to Test Example 2 was changed to 4N, 8N, and LE8N are shown.

[0034] Fig.14 The results of an experiment confirming that only antibodies can be separated from an antibody culture solution by using the ZZ-CSQ2 (all) fusion protein according to Experimental Example 2 are shown.

[0035] Fig.15 The test results for measuring the affinity of the Z-CSQ1 (all) fusion protein according to Test Example 3 for antibodies are shown.

[0036] Fig.16 The test results of measuring the affinity of the (Z)5-CSQ1(all) fusion protein according to Test Example 4 for antibodies are shown.

[0037] Fig.17 The results of a test for confirming the binding ratio between (Z)5-CSQ1 (all) and antibodies according to Test Example 5 are shown.

[0038] Best Mode

[0039] According to a first specific embodiment,

[0040] The present invention provides a nucleic acid encoding a fusion protein of a Z-domain and calcineurin with improved reactivity, stability and antibody recovery.

[0041] The term "fusion protein" used in this specification refers to a protein prepared by combining (joining) two or more genes encoded independently of each other. The fusion protein can be artificially prepared by recombinant DNA technology. In addition, the fusion protein can be a physiologically active protein, or a peptide and an immunoglobulin Fc fragment in the absence of a joint, or a peptide and an immunoglobulin Fc fragment connected by a peptide linker. Here, a peptide linker refers to a peptide that connects the two proteins constituting the fusion protein to each other. The peptide linker can be included so that the two proteins as components of the fusion protein can be folded independently (folding) or maintain their respective functions even in the form of a fusion protein, but is not limited thereto.

[0042] The term "immunoglobulin Fc fragment" used in this specification refers to the heavy chain and light chain variable regions of an immunoglobulin, the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) excluding the heavy chain constant region 1 (CH1) and the light chain constant region 1 (CL1), and includes a hinge portion in the heavy chain constant region. In addition, as long as the immunoglobulin Fc fragment of the present invention has the same or improved effect as the natural type, it may be an enlarged Fc fragment including part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1) excluding only the heavy chain and light chain variable regions of the immunoglobulin. In addition, the immunoglobulin Fc fragment may also be a region in which some relatively long amino acid sequences corresponding to CH2 and / or CH3 have been removed. The immunoglobulin Fc fragment of the present invention includes a natural amino acid sequence and its sequence derivatives. An amino acid sequence derivative refers to a sequence having at least one amino acid residue different from a natural amino acid sequence, and may be naturally occurring or artificially occurring. The Fc fragment of an immunoglobulin includes derivatives formed by deletion, insertion, non-complementary or complementary substitution or a combination thereof. Insertions are generally composed of a continuous sequence of about 1 to 20 amino acids, but larger insertions are also possible. Deletions are generally composed of about 1 to 30 residues. The technology for preparing sequence derivatives of such immunoglobulin Fc fragments is disclosed in International Patent Publication No. WO97 / 34631, International Patent Publication No. WO96 / 32478, etc., which are included as references to this specification. Amino acid substitutions in proteins and peptides that do not completely change the activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions are substitutions between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, the immunoglobulin Fc fragment can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc. Such immunoglobulin Fc fragments can be obtained from natural forms isolated from animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, and can be recombinant or derivatives thereof obtained from transformed animal cells or microorganisms.Here, the method for obtaining immunoglobulin Fc fragment from natural type can include obtaining immunoglobulin Fc fragment by isolating whole immunoglobulin from living body of human or animal, and then treating with proteolytic enzyme. When treated with papain, immunoglobulin is cut into Fab and Fc, and when treated with pepsin, immunoglobulin is cut into pF'c and F(ab')2. Size exclusion chromatography can be used to separate Fc or pF'c. Preferably, immunoglobulin Fc fragment according to the present invention can include protein A, protein G, protein A / G or protein L derived from microorganisms.

[0043] The term "Z protein" used in this specification is obtained from protein A of Staphylococcus aureus, which refers to a protein used to defend against antibodies present in host cells. The Z protein is used to prevent the self-defense mechanism of phagocytosis by macrophages by binding to the Fc site of antibodies in host cells. The tandem repeat dimer made by operating only the site in protein A that binds to antibodies is called a "Z-domain".

[0044] The term "calsequestrin" used in this specification is a calcium binding protein of the sarcoplasmic reticulum, and although the calcium concentration in the sarcoplasmic reticulum is higher than that in the cytoplasm, calsequestrin binds to calcium after muscle contraction and allows calcium ions to be stored in the endoplasmic reticulum cleft of the sarcoplasmic reticulum. Since one calsequestrin molecule can bind multiple calcium (for example, each calsequestrin molecule has 40 to 50 calcium binding sites), its ability to store calcium is very good.

[0045] In the nucleic acid according to the invention, the Z-domain is characterized in that it comprises Z in the form of Z-domain repeats. n (n is an integer greater than or equal to 1). For example, the Z-domain may be in the form of 1 or more, 1 to 20, 1 to 10, or 1 to 5 Z-domain repeats. Preferably, the Z-domain may be in the form of 2 to 5 Z-domain repeats.

[0046] In the nucleic acid according to the present invention, the Z-domain is characterized by consisting of the amino acid sequence of SEQ ID NO: 13 and has a form of 1 to 5 repeats.

[0047] In the nucleic acid according to the present invention, the Z-domain is characterized by being a ZZ domain, a Z6G domain, or a ZL4G domain. The ZZ domain may include or consist of the amino acid sequence of SEQ ID NO: 1. The Z6G domain may include or consist of the amino acid sequence of SEQ ID NO: 2. The ZL4G domain may include or consist of the amino acid sequence of SEQ ID NO: 3.

[0048] In the nucleic acid according to the present invention, the calcineurin is characterized by comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or is composed of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0049] In the nucleic acid according to the present invention, the Z-domain and the calcitonin are characterized in that they are fused via a linker, which comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 7 to 10 or consists of said amino acid sequences.

[0050] In the nucleic acid according to the present invention, the antibody is characterized by comprising an antibody, a fragment, a derivative or an analogue thereof that can specifically bind to protein Z. For example, the antibody may be IgG.

[0051] According to a second specific embodiment, there is provided a method for separating and purifying antibodies with improved reactivity and stability, the method comprising:

[0052] (A) preparing a recombinant vector comprising a nucleic acid encoding a fusion protein of a Z-domain and calcineurin;

[0053] (B) transducing the recombinant vector into a host cell to obtain a transgenic organism;

[0054] (C) expressing a fusion protein of the Z-domain and calcein from the transgenic organism; and

[0055] (D) Antibodies binding to the fusion protein of the Z-domain and calcein were isolated from the expressed transgenic organisms using calcium.

[0056] The term "vector" used in this specification refers to an expression vector capable of expressing a target protein in a suitable host cell, which refers to a nucleic acid construct comprising operably linked necessary regulatory elements so that a nucleic acid insert is expressed. The vector can be prepared by operating a plasmid commonly used in the art (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series, pACYC184 and pUC19, etc.), a phage (e.g., λ-gt4, λB, λ-Charon, λΔz1 and M13, etc.) or a virus (e.g., SV40, etc.), but is not limited thereto.

[0057] The term "transformation" used in this specification refers to the introduction of DNA into a host so that the DNA can be replicated as a factor of a chromosome or can be replicated by completing chromosome fusion, and refers to the phenomenon of artificially causing genetic changes by introducing external DNA into cells. The transformation methods include CaCl2 precipitation method, Hanahan method that improves efficiency by using a reducing substance called dimethyl sulfoxide (DMSO) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using polyethylene glycol (PEG), dextran sulfate, liposome and drying / inhibition-mediated transformation method, but are not limited thereto.

[0058] The term "host cell" used in this specification refers to a cell that provides nutrition by allowing the parasitism of other microorganisms or genes, and refers to a cell that causes various genetic or molecular effects in the host cell by vector transformation. The host cell may include bacteria of the genus Escherichia such as Escherichia coli; bacteria of the genus Bacillus such as Bacillus subtilis; bacteria of the genus Pseudomonas such as Pseudomonas putida; lactobacilli such as lactobacilli and enterococci; yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; animal cells and insect cells, but are not limited thereto.

[0059] In the method for separating and purifying antibodies according to the present invention, the Z-domain is characterized by comprising Z in the form of Z-domain repeats. n(n is an integer greater than or equal to 1). For example, the Z-domain may be in the form of 1 or more, 1 to 20, 1 to 10, or 1 to 5 Z-domain repeats. Preferably, the Z-domain may be in the form of 2 to 5 Z-domain repeats.

[0060] In the nucleic acid according to the present invention, the Z-domain is characterized by consisting of the amino acid sequence of SEQ ID NO: 13 and has a form of 1 to 5 repeats.

[0061] In the method for separating and purifying antibodies according to the present invention, the Z-domain is characterized by being a ZZ domain, a Z6G domain or a ZL4G domain. The ZZ domain may include the amino acid sequence of SEQ ID NO: 1 or consist of the amino acid sequence of SEQ ID NO: 1. The Z6G domain may include the amino acid sequence of SEQ ID NO: 2 or consist of the amino acid sequence of SEQ ID NO: 2. The ZL4G domain may include the amino acid sequence of SEQ ID NO: 3 or consist of the amino acid sequence of SEQ ID NO: 3.

[0062] In the method for separating and purifying antibodies according to the present invention, the calcineurin is characterized by comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or is composed of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0063] In the method for separating and purifying antibodies according to the present invention, the Z-domain and the calcitonin are characterized in that they are fused via a linker, and the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 7 to 10 or consists of the amino acid sequences.

[0064] In the method for separating and purifying antibodies according to the present invention, the antibody is characterized in that it is an antibody, a fragment, a derivative or an analogue thereof that can specifically bind to the Fc, fragments thereof, derivatives thereof / analogues thereof, of the antibody. For example, the antibody can be IgG.

[0065] In the method for separating and purifying antibodies according to the present invention, the step (D) is characterized in that it is carried out under the condition of pH 3.0 to pH 4.5. Preferably, the step (D) can be carried out under the condition of pH 3.0 to pH 3.5.

[0066] In the method for separating and purifying antibodies according to the present invention, the step of (D) separating the antibody bound to the fusion protein of the Z-domain and calcineurin using calcium is characterized by comprising:

[0067] (d-1) using calcium precipitation to bind to an antibody that binds to a fusion protein of the Z-domain and calcein; and

[0068] (d-2) Remove the precipitate by centrifugation or filtration. DETAILED DESCRIPTION

[0069] Unless otherwise defined in this specification, all technical and scientific terms used are synonymous with the meanings commonly understood by those of ordinary skill in the art, and the present invention is described in more detail by the following examples and with reference to the accompanying drawings of the present invention. However, these examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0070] <Example>

[0071] Example 1. Preparation of Z domain-calcein fusion protein

[0072] A Z-domain-calcein fusion protein including a Z-domain consisting of the amino acid sequence shown in Table 1 below, a linker, and calcein was designed. On the one hand, as a comparative example, a fusion protein of registered patent No. 10-1774353 was used.

[0073]

Table 1

[0074]

[0075]

[0076] Example 2. Gene cloning of Z-domain-calcein fusion protein

[0077] In order to clone the nucleic acid encoding the Z-domain-calcitonin fusion protein into pET28b as an expression vector, polymerase chain reaction (PCR) was used for amplification. For this purpose, a forward primer (AATAAAA CATATG GTAGACAACAAATTCAAC, sequence number 11) and a reverse primer (ATT CTCGAG TTAATCATCATCATCATCATCTTC, sequence number 12) (Bioneer) were designed. The PCR product was purified using a DNA purification kit (Ginol) and treated with NdeI and XhoI restriction enzymes (the underlined parts in the primers, NEB). In addition, the pET28b vector (Novagen) was also treated with NdeI and XhoI restriction enzymes. The DNA fragment cut with NdeI and XhoI and the pET28b vector were connected using a ligase (NEB) at 16°C for 12 hours. The ligated product was transfected into DH5a Escherichia coli and cultured in an agar medium containing kanamycin for one day. The vector was then extracted from the grown colonies and sequenced (biooneer).

[0078] Example 3. Expression and purification of Z-domain-calcein fusion protein

[0079] The plasmid prepared in Example 2 was transformed into Escherichia coli strain BL21 (DE3) (Novagen, Northumberland, UK). The bacteria were treated with 1 mM isopropyl β-D-thiogalactoside (IPTG) (Sigma, St. Louis, Missouri) for 6 hours at 37°C to induce transgenic organisms. The bacteria were then harvested and resuspended in lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl and 5 mM imidazole) and lysed by sonication. The lysate was centrifuged at 1,550 g for 1 hour at 4°C. The supernatant was loaded onto a gravity flow column (gravity-flow column; BioRad, Hercules, California) equipped with a Ni-NTA affinity resin (Peptron, Daejeon, South Korea) pre-equilibrated with lysis buffer (3 mL per 1 liter of culture fluid). After washing the column and matrix with 10 bead volumes of lysis buffer, calcineurin was eluted and harvested. The protein was further purified by gel filtration using a Superdex 200 column (Amersham P Harmacia, Bucks, UK) pre-equilibrated with phosphate buffer solution (PBS, pH 7.4).

[0080] <Test example>

[0081] Test Example 1: Confirmation of the reaction of a fusion protein comprising calcein consisting of the amino acid sequence of SEQ ID NO: 5 Sex and stability

[0082] 1-1. Comparison between ZZ-CSQ1 and ZZ-CSQ2(Δ)

[0083] (1) Confirmation of calcium responsiveness

[0084] In order to confirm the turbidity of the ZZ-CSQ1 (all) fusion protein according to Preparation Example 1 and the ZZ-CSQ2 (Δ) fusion protein according to Comparative Example, the concentration of each protein was adjusted to 1 mg / ml to measure the turbidity. As a buffer for the fusion protein, 20 mM Tris-HCl (pH 7.0) was used, and calcium adjusted to 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM and 20 mM was added to the fusion protein, and then mixed and reacted at 4°C for 1 hour to allow the fusion protein to react with calcium. The absorbance value of each concentration was measured in UV-Vis (350 nm) using Nano-drop (Thermo). As a result, it was confirmed that ZZ-CSQ1(all) and ZZ-CSQ2(Δ) reacted at calcium concentrations of 2 mM and 4 mM, respectively, and that ZZ-CSQ1(all) according to Preparation Example 1 could react with calcium at a concentration lower than that of ZZ-CSQ2(Δ) to precipitate ( Figure 1 ).

[0085] (2) Confirm stability

[0086] In order to confirm the stability of the fusion protein of ZZ-CSQ1 (all) and ZZ-CSQ2 (Δ), the fusion protein of ZZ-CSQ1 (all) and ZZ-CSQ2 (Δ) at a concentration of 1 mg / ml was treated in a medium at 37°C for 1 day and 2 days. In order to confirm the change of the stability of the fusion protein over time, 4X sample buffer was added, and after heat treatment at 80°C for 10 minutes, it was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). As a result, it was confirmed that the degradation of ZZ-CSQ2 (Δ) increased over time compared with ZZ-CSQ1 (all) ( Figure 2 ).

[0087] (3) Check the stability of the connector type

[0088] After G4S (SEQ ID NO: 7) as the linker portion of the ZZ-CSQ1 (all) fusion protein was changed to 4N (SEQ ID NO: 8), 8N (SEQ ID NO: 9) and LE8N (SEQ ID NO: 10), respectively, each of the fusion proteins ZZ-CSQ1 (all) and ZZ-CSQ2 (Δ) according to Preparation Examples 1 to 4 was treated at a concentration of 1 mg / ml in a culture medium at 25° C. and 37° C. for 12 hours, 24 hours, 36 hours and 48 hours in order to confirm the stability in 1M NaOH and CHO cell culture supernatant (Cell Culture Sup). In order to confirm the change in the stability of the fusion protein over time, 4X sample buffer was added, and after heat treatment at 80° C. for 10 minutes, it was confirmed by SDS-PAGE. As a result, the ZZ-CSQ2(Δ) fusion protein was rapidly or completely decomposed after 24 hours, whereas in the case of ZZ-CSQ1(all) according to Preparation Examples 2 to 4 in which the linker was changed to 4N, 8N, and LE8N, respectively, it had a stability of 50% or more in the CHO cell culture supernatant and a stability of 70% or more in 0.1 M NaOH within 24 hours, thus confirming that the reuse rate was improved ( Figure 3 ).

[0089] 1-2. Measurement of affinity of ZZ-CSQ1 (all) fusion protein for antibodies

[0090] In order to measure the affinity of the ZZ-CSQ1 (all) fusion protein according to Preparation Example 1, surface plasmon resonance (SPR) analysis was performed. ZZ-CSQ1 (all) was immobilized on a CM5 chip, and various concentrations (7.5nM, 10nM, 12.5nM, 15nM and 25nM) of bevacizumab were injected into a 50mM Tris and pH 7.4 buffer solution at a flow rate of 30μl / min. Kinetic data were analyzed using BIA Evaluation 2.1 software (GE Healthcare). As a result, it was confirmed that the divalent Z-domain of ZZ-CSQ1 (all) according to Preparation Example 1 was able to strongly bind to the antibody with an affinity of 1.39nM ( Figure 4 )

[0091] 1-3. Purification of antibodies using ZZ-CSQ1 (all) fusion protein

[0092] Confirm the process of purifying only antibodies in antibody culture medium using ZZ-CSQ1 (all) fusion protein according to Preparation Example 1. ZZ-CSQ1 (all) fusion protein was added to the antibody culture medium at a ratio of 1:1 and reacted at 4°C for 30 minutes. After adding 10mM calcium, reacted at 4°C for 30 minutes, and then centrifuged at 4000g for 30 minutes to separate the supernatant (Supernatant) and the precipitate (Pellet). In order to elute the antibody in ZZ-CSQ1 (all) + antibody, 50mM citrate (Citrate) (pH 3.5) was used to elute the antibody. In order to confirm the degree of antibody recovery, 4X sample buffer (sampleBuffer) was added, and after heat treatment at 80°C for 10 minutes, it was confirmed by SDS-PAGE. As a result, it was confirmed that only the antibody (IgG) ( Figure 5 ).

[0093] 1-4. Confirmation of antibody recovery of ZZ-CSQ1 (all) fusion protein at each pH

[0094] In order to confirm the degree of antibody recovery according to pH change, ZZ-CSQ1 (whole) and antibody according to Preparation Example 1 were added in a ratio of 1:1 and reacted at 4°C for 30 minutes, and then centrifuged at 180000g for 30 minutes to separate the supernatant and the precipitate. 100mM Citrate Buffer (pH 3.0, 3.5, 4.0 and 4.5) was added to the ZZ-CSQ1 (whole) + antibody (IgG) complex and the antibody was eluted. After elution, the supernatant and the precipitate were separated by centrifugation at 4000g for 30 minutes, and in order to confirm the degree of antibody elution, 4X sample buffer was placed and heated at 80°C for 10 minutes, and then confirmed by SDS-PAGE. As a result, it was confirmed that the antibody was eluted better when the pH of the recovery buffer was more acidic (pH 3.0 and pH 3.5) than when the pH was 4.0 and 4.5. Figure 6 ).

[0095] 1-5. Measurement of host cell protein (HCP) of antibodies after antibody purification using ZZ-CSQ1 (all) fusion protein

[0096] After the antibody recovery of the ZZ-CSQ1 (all) fusion protein according to Preparation Example 1, measurement was performed to confirm HCP. In order to measure the HCP of the purified antibody, a CHO HCP enzyme-linked immunosorbent assay (ELISA; enzyme linked immunosorbent assay) kit (CANOPY) was used. 100ul of the antibody sample eluted by using ZZ-CSQ1 (all) was added to a 96-well plate attached with Anti-CHO antibody and reacted at room temperature for 1.5 hours. After washing 3 times with 250ul each time with 1X PBS-T, 100ul of the reporting antibody (Reporting antibody) was added, and the lid was covered and reacted at room temperature for 45 minutes. After washing 3 times with 250ul each time with 1X PBS-T, 100ul of streptavidin (Streptavidin)-horseradish peroxidase (HRP; horseradish peroxidase) at a concentration of 0.1ug / ml was added and reacted at room temperature for 30 minutes. After washing 3 times with 1X PBS-T with 250ul each time, the cells were treated with tetramethylbenzidine (TMB) and reacted for 10 minutes. Stop Solution was added, and the absorbance was measured at 450nm using a microplate reader. As a result, when ZZ-CSQ1 (all) according to Preparation Example 1 was used, it was confirmed that the HCP value was reduced by 1 / 4 (0.05) compared with the protein A chromatography method. Figure 7 ).

[0097] 1-6. Measurement of host cell DNA (HCD) of antibodies after antibody purification using ZZ-CSQ1 (all) fusion protein

[0098] After the antibody recovery of the ZZ-CSQ1 (all) fusion protein according to Preparation Example 1, HCP was confirmed. To measure the HCP of the purified antibody, the Quant-iT ds DNA High-Sensitivity Assay Kit (Invitrogen) was used. The provided working solution (Working Solution) was kept in a light-proof state, 200ul of the working solution was added to the microplate (Microplate), and stored at room temperature for 3 hours. The purified antibody sample was placed in each well and mixed, and the fluorescence (Fluorescence) (excitation / emission maximum: 480 / 530nm) was measured using a microplate reader (PerkinElmer). Confirmation was performed by repeating the measurement 3 times. As a result, it was confirmed that when using ZZ-CSQ1 (all) according to Preparation Example 1, the HCD value was reduced by 1 / 332 (compared with the protein A chromatography method). Figure 8 ).

[0099] 1-7. Measurement of size-exclusion high-performance liquid chromatography (SEC-HPLC) after antibody purification of ZZ-CSQ1 (all) fusion protein

[0100] After antibody purification using the ZZ-CSQ1 (all) fusion protein according to Preparation Example 1, in order to confirm the aggregation of the antibody or ZZ-CSQ1 (all), SEC-HPLC was used for confirmation. For analysis, a Superdex200 size exclusion chromatography column (SEC; Size Exclusion Chromatography Column) was used. 50mM citrate was used as an elution buffer, and 1M Tris (pH9.0) was immediately added for neutralization. 20mM Tris (pH 7.5) + 150mM NaCl was flowed at a flow rate of 0.2ml / min and analyzed. As a control group, an antibody sample purified with a protein A chromatography column was confirmed under the same conditions. As a result, it was confirmed that an antibody quality equivalent to that of protein A chromatography could be obtained ( Fig. 9 ).

[0101] 1-8. Confirmation of recovery of Z6G-CSQ1 (all) and L4G-CSQ1 (all) fusion proteins during antibody elution

[0102] In order to improve the antibody recovery rate, the fusion proteins of Z6G-CSQ1 (all) according to Preparation Example 5 and ZL4G-CSQ1 (all) according to Preparation Example 6 were prepared by changing the ZZ domain position in ZZ-CSQ1 (all) according to Preparation Example 1 to Z6G (SEQ ID NO: 2) and ZL4G (SEQ ID NO: 3). In order to confirm the antibody recovery rate of the fusion protein having the new domain, Z6G-CSQ1 (all) and ZL4G-CSQ1 (all) were added to the antibody culture solution at a ratio of 1:1, respectively, and reacted at 4°C for 30 minutes. 10mM calcium was added and reacted at 4°C for 30 minutes, and then centrifuged at 4000g for 30 minutes to separate the supernatant (Supernatant) and the precipitate (Pellet). 50mM citrate (pH 3.5) and 50mM citrate (pH 4.0) buffer were added to the precipitate and eluted for 20 minutes. After elution, the supernatant and the precipitate were separated by centrifugation at 4000 g for 30 minutes, and in order to confirm the degree of elution of the antibody, 4X sample buffer was placed and heat-treated at 80°C for 10 minutes, and then confirmed by SDS-PAGE. As a result, it was confirmed that the degree of antibody elution increased by 10% after the linker was changed to Z6G and ZL4G compared with ZZCSQ1 (all). In addition, it was confirmed that after changing to Z6G-CSQ1 (all) according to Preparation Example 5 and ZL4G-CSQ1 (all) according to Preparation Example 6, the recovery rate of the antibody was improved compared with ZZCSQ1 (all) according to Preparation Example 1 ( Fig.10 ).

[0103] Test Example 2: Confirmation of the reaction of a fusion protein comprising calcein consisting of the amino acid sequence of SEQ ID NO: 6 Sex and stability

[0104] 2-1. Comparison between ZZ-CSQ2(all) and ZZ-CSQ2(Δ)

[0105] (1) Confirmation of calcium responsiveness

[0106] The turbidity and antibody elution of the ZZ-CSQ1 (all) fusion protein according to Preparation Example 7 and the ZZ-CSQ2 (Δ) fusion protein according to Comparative Example were measured in the same manner as in Experimental Example 1, and the results are shown in Fig.11 .

[0107] (2) Confirmation of improvement in antibody recovery and purity

[0108] In order to confirm the antibody elution recovery rate of the fusion protein of ZZ-CSQ2 (Δ) and ZZ-CSQ2 (all), the fusion protein of ZZ-CSQ2 (Δ) and ZZ-CSQ2 (all) was added to the antibody culture solution at a ratio of 1:1 and reacted at 4°C for 30 minutes. 20mM Cacl2 was added to the antibody + fusion protein and reacted at 4°C for 30 minutes, and then centrifuged at 4000g for 30 minutes to separate the supernatant and the precipitate. 50mM citrate (pH 3.5) was added to the precipitate and eluted for 20 minutes. After elution, centrifuged at 4000g for 30 minutes, and the supernatant and the precipitate were separated. In order to confirm the degree of antibody elution in the fusion protein + antibody, 4X sample buffer was added and heat treated at 80°C for 10 minutes, and then confirmed by SDS-PAGE. As a result, it was confirmed that approximately 60% of the antibodies were recovered using ZZ-CSQ2(Δ), and that greater than or equal to 80% of the antibodies were recovered in ZZ-CSQ2(all). In addition, it was confirmed that approximately 10% of the antibodies recovered using ZZ-CSQ2(Δ) were recovered together with the ZZ-CSQ2(Δ) fusion protein, but that the ZZ-CSQ2(all) fusion protein was less than 1% of the antibodies recovered using ZZ-CSQ2(all), so that highly pure antibodies could be recovered. Fig.12 )

[0109] (3) Check the stability of the connector type

[0110] After G4S (SEQ ID NO: 7) as the linker portion of the ZZ-CSQ2 (all) fusion protein was changed to 4N (SEQ ID NO: 8), 8N (SEQ ID NO: 9) and LE8N (SEQ ID NO: 10), respectively, each of ZZ-CSQ2 (all) and ZZ-CSQ2 (Δ) was treated at a concentration of 1 mg / ml in a culture medium at 25° C. and 37° C. for 12 hours, 24 hours, 36 hours and 48 hours in order to confirm the stability in 1 M NaOH and CHO cell culture supernatant. In order to confirm the change in the stability of the fusion protein over time, 4X sample buffer was added, and after heat treatment at 80° C. for 10 minutes, it was confirmed by SDS-PAGE. As a result, in the case of ZZ-CSQ2 (all) according to Preparation Examples 8 to 10 in which the linker in ZZ-CSQ2 (all) was changed to 4N, 8N and LE8N, respectively, the stability was improved by at least 25% when in the CHO cell culture supernatant for 24 hours, and the stability of 50% or more was maintained in 0.1 M NaOH for up to 36 hours, thus confirming that the reuse rate can be improved ( Fig.13 ).

[0111] 2-2. Antibody purification of ZZ-CSQ2 (all) fusion protein

[0112] Confirm the process of purifying only antibodies in antibody culture medium using ZZ-CSQ2 (whole) fusion protein. ZZ-CSQ2 (whole) fusion protein was added to the antibody culture medium at a ratio of 1:1 and reacted at 4°C for 30 minutes. After adding 20mM calcium, reacted at 4°C for 30 minutes, and then centrifuged at 4000g for 30 minutes to separate the supernatant (Supernatant) and the precipitate (Pellet). In order to elute the antibody (IgG) in ZZ-CSQ2 (whole) + antibody (IgG), the antibody was eluted using 50mM citrate (pH3.5). In order to confirm the degree of antibody recovery, 4X sample buffer was added and after heat treatment at 80°C for 10 minutes, confirmed by SDS-PAGE. As a result, it was confirmed that only the antibody (IgG) was eluted in ZZ-CSQ2 (whole) + antibody (IgG) ( Fig.14 ).

[0113] Experimental Example 3. Measurement of the affinity of Z-CSQ1 (all) fusion protein for antibodies

[0114] Z-CSQ1 (all) was prepared using the Z-domain consisting of SEQ ID NO: 13 instead of the ZZ domain consisting of SEQ ID NO: 1 in the preparation example 1, and SPR (BIACORE 3000) analysis was performed to measure the affinity of the Z-CSQ1 (all) fusion protein. Herceptin was immobilized on a CM5 chip, and various concentrations (30nM, 60nM, and 90nM) of Z-CSQ1 (all) were injected into a PBS buffer solution at a flow rate of 30μl / min. The kinetic data were analyzed using BIA evaluation 2.1 software (GE Healthcare). As a result, it was confirmed that the Z-domain of Z-CSQ1 can also strongly bind to the antibody ( Fig.15 ).

[0115] Experimental Example 4. Measurement of affinity of (Z)5-CSQ1(all) fusion protein for antibodies

[0116] In order to measure the affinity of (Z)5-CSQ(all) with 5 Z-domains, SPR (BIACORE 3000) analysis was performed. Herceptin was immobilized on a CM5 chip, and various concentrations (0.96nM, 1.96nM and 3.84nM (Z)5-CSQ(all)) were injected into a PBS buffer solution at a flow rate of 30μl / min. The kinetic data were analyzed using BIA Evaluation 2.1 software (GE Healthcare). As a result, it was confirmed that the Z-domain of (Z)5-CSQ(all) can also strongly bind to the antibody with an affinity of 1.14nM ( Fig.16 ).

[0117] Test Example 5: Confirmation of the antibody binding ratio of (Z)5-CSQ (all) fusion protein to antibody

[0118] The (Z)5-CSQ(total) fusion protein and the antibody were added at a ratio of 0.5:1, 1:1, and 2:1, and reacted at 4°C for 30 minutes to allow binding. Calcium was added to the reaction solution of the fusion protein and the antibody, and then centrifuged at 4000g for 30 minutes at 4°C. The supernatant and the precipitate were separated, and the precipitate was placed in a 20mM Tris-HCl (pH 7.0) buffer and dissolved. In order to confirm the antibody binding ratio of the fusion protein, 4X sample buffer was added and heat-treated at 80°C for 10 minutes, and then confirmed by SDS-PAGE. As a result, it was confirmed that (Z)5-CSQ(total) was completely precipitated with the antibody even at a ratio of 2:1 and 4:1 ( Fig.17 ).

[0119] So far, for the present invention, its preferred embodiments have been described. It will be appreciated by those skilled in the art that the present invention may be implemented in modified form without departing from the basic features of the present invention. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is represented by the claims rather than the above description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention. <110> Korea Ceramic Technology Institute <120> Fusion protein of Z-domain and calcein with improved reactivity, stability and antibody recovery rate and method for separating and purifying antibodies using the same <130> DP-2020-0054 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 116 <212> PRT <213> Artificial sequence <220> <223> ZZ <400> 1 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn 50 55 60 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro 85 90 95 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala 100 105 110 Gln Ala Pro Lys 115 <210> 2 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Z6G <400> 2 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Gly Gly Gly Gly Gly Gly Ser Ala 35 40 45 Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 60 Val Asp Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 65 70 75 80 Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe 85 90 95 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Gly Gly Gly Gly Gly Gly 100 105 110 Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 115 120 125 Pro Lys Val Asp 130 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> ZL4G <400> 3 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Gly Gly Gly Gly Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Val Asp Asn Lys 50 55 60 Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro 65 70 75 80 Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp 85 90 95 Gly Gly Gly Gly Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn 100 105 110 Asp Ala Gln Ala Pro Lys Val Asp 115 120 <210> 4 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> CSQ2(delta) <400> 4 Glu Glu Gly Leu Asn Phe Pro Thr Tyr Asp Gly Lys Asp Arg Val Val 1 5 10 15 Ser Leu Ser Glu Lys Asn Phe Lys Gln Val Leu Lys Lys Tyr Asp Leu 20 25 30 Leu Cys Leu Tyr Tyr His Glu Pro Val Ser Ser Asp Lys Val Thr Pro 35 40 45 Lys Gln Phe Gln Leu Lys Glu Ile Val Leu Glu Leu Val Ala Gln Val 50 55 60 Leu Glu His Lys Ala Ile Gly Phe Val Met Val Asp Ala Lys Lys Glu 65 70 75 80 Ala Lys Leu Ala Lys Lys Leu Gly Phe Asp Glu Glu Gly Ser Leu Tyr 85 90 95 Ile Leu Lys Gly Asp Arg Thr Ile Glu Phe Asp Gly Glu Phe Ala Ala 100 105 110 Asp Val Leu Val Glu Phe Leu Leu Asp Leu Ile Glu Asp Pro Val Glu 115 120 125 Island Island Ser Ser Lys Leu Glu Val Gln Ala Phe Glu Arg Ile Glu Asp 130 135 140 Tyr Ile Lys Leu Ile Gly Phe Phe Lys Ser Glu Asp Ser Glu Tyr Tyr 145 150 155 160 Lys Ala Phe Glu Glu Ala Ala Glu His Phe Gln Pro Tyr Ile Lys Phe 165 170 175 Phe Ala Thr Phe Asp Lys Gly Val Ala Lys Lys Leu Ser Leu Lys Met 180 185 190 Asn Glu Val Asp Phe Tyr Glu Pro Phe Met Asp Glu Pro Ile Ala Ile 195 200 205 Pro Asn Lys Pro Tyr Thr Glu Glu Glu Leu Val Glu Phe Val Lys Glu 210 215 220 His Gln Arg Pro Thr Leu Arg Arg Leu Arg Pro Glu Glu Met Phe Glu 225 230 235 240 Thr Trp Glu Asp Asp Leu Asn Gly Ile His Ile Val Ala Phe Ala Glu 245 250 255 Lys Ser Asp Pro Asp Gly Tyr Glu Phe Leu Glu Ile Leu Lys Gln Val 260 265 270 Ala Arg Asp Asn Thr Asp Asn Pro Asp Leu Ser Ile Leu Trp Ile Asp 275 280 285 Pro Asp Asp Phe Pro Leu Leu Val Ala Tyr Trp Glu Lys Thr Phe Lys 290 295 300 Ile Asp Leu Phe Arg Pro Gln Ile Gly Val Val Asn Val Thr Asp Ala 305 310 315 320 Asp Ser Val Trp Met Glu Ile Pro Asp Asp Asp Asp Leu Pro Thr Ala 325 330 335 Glu Glu Leu Glu Asp Trp Ile Glu Asp Val Leu Ser Gly Lys Ile Asn 340 345 350 Thr Glu Asp Asp Asp Glu Asp Asp Asp Asp Asp Asp 355 360 <210> 5 <211> 362 <212> PRT <213> Artificial Sequence <220> <223> CSQ1 (All) <400> 5 Gln Glu Gly Leu Asp Phe Pro Glu Tyr Asp Gly Val Asp Arg Val Ile 1 5 10 15 Asn Val Asn Ala Lys Asn Tyr Lys Asn Val Phe Lys Lys Tyr Glu Val 20 25 30 Leu Ala Leu Leu Tyr His Glu Pro Pro Glu Asp Asp Lys Ala Ser Gln 35 40 45 Arg Gln Phe Glu Met Glu Glu Leu Ile Leu Glu Leu Ala Ala Gln Val 50 55 60 Leu Glu Asp Lys Gly Val Gly Phe Gly Leu Val Asp Ser Glu Lys Asp 65 70 75 80 Ala Ala Val Ala Lys Lys Leu Gly Leu Thr Glu Val Asp Ser Met Tyr 85 90 95 Val Phe Lys Gly Asp Glu Val Ile Glu Tyr Asp Gly Glu Phe Ser Ala 100 105 110 Asp Thr Ile Val Glu Phe Leu Leu Asp Val Leu Glu Asp Pro Val Glu 115 120 125 Leu Ile Glu Gly Glu Arg Glu Leu Gln Ala Phe Glu Asn Ile Glu Asp 130 135 140 Glu Ile Lys Leu Ile Gly Tyr Phe Lys Ser Lys Asp Ser Glu His Tyr 145 150 155 160 Lys Ala Phe Glu Asp Ala Ala Glu Glu Phe His Pro Tyr Ile Pro Phe 165 170 175 Phe Ala Thr Phe Asp Ser Lys Val Ala Lys Lys Leu Thr Leu Lys Leu 180 185 190 Asn Glu Ile Asp Phe Tyr Glu Ala Phe Met Glu Glu Pro Val Thr Ile 195 200 205 Pro Asp Lys Pro Asn Ser Glu Glu Glu Ile Val Asn Phe Val Glu Glu 210 215 220 His Arg Arg Ser Thr Leu Arg Lys Leu Lys Pro Glu Ser Met Tyr Glu 225 230 235 240 Thr Trp Glu Asp Asp Met Asp Gly Ile His Ile Val Ala Phe Ala Glu 245 250 255 Glu Ala Asp Pro Asp Gly Phe Glu Phe Leu Glu Thr Leu Lys Ala Val 260 265 270 Ala Gln Asp Asn Thr Glu Asn Pro Asp Leu Ser Ile Ile Trp Ile Asp 275 280 285 Pro Asp Asp Phe Pro Leu Leu Val Pro Tyr Trp Glu Lys Thr Phe Asp 290 295 300 Ile Asp Leu Ser Ala Pro Gln Ile Gly Val Val Asn Val Thr Asp Ala 305 310 315 320 Asp Ser Val Trp Met Glu Met Asp Asp Glu Glu Asp Leu Pro Ser Ala 325 330 335 Glu Glu Leu Glu Asp Trp Leu Glu Asp Val Leu Glu Gly Glu Ile Asn 340 345 350 Thr Glu Asp Asp Asp Asp Asp Asp Asp Asp 355 360 <210> 6 <211> 380 <212> PRT <213> Artificial Sequence <220> <223> CSQ2 (Full) <400> 6 Glu Glu Gly Leu Asn Phe Pro Thr Tyr Asp Gly Lys Asp Arg Val Val 1 5 10 15 Ser Leu Ser Glu Lys Asn Phe Lys Gln Val Leu Lys Lys Tyr Asp Leu 20 25 30 Leu Cys Leu Tyr Tyr His Glu Pro Val Ser Ser Asp Lys Val Thr Pro 35 40 45 Lys Gln Phe Gln Leu Lys Glu Ile Val Leu Glu Leu Val Ala Gln Val 50 55 60 Leu Glu His Lys Ala Ile Gly Phe Val Met Val Asp Ala Lys Lys Glu 65 70 75 80 Ala Lys Leu Ala Lys Lys Leu Gly Phe Asp Glu Glu Gly Ser Leu Tyr 85 90 95 Ile Leu Lys Gly Asp Arg Thr Ile Glu Phe Asp Gly Glu Phe Ala Ala 100 105 110 Asp Val Leu Val Glu Phe Leu Leu Asp Leu Ile Glu Asp Pro Val Glu 115 120 125 Ile Ile Ser Ser Lys Leu Glu Val Gln Ala Phe Glu Arg Ile Glu Asp 130 135 140 Tyr Ile Lys Leu Ile Gly Phe Phe Lys Ser Glu Asp Ser Glu Tyr Tyr 145 150 155 160 Lys Ala Phe Glu Glu Ala Ala Glu His Phe Gln Pro Tyr Ile Lys Phe 165 170 175 Phe Ala Thr Phe Asp Lys Gly Val Ala Lys Lys Leu Ser Leu Lys Met 180 185 190 Asn Glu Val Asp Phe Tyr Glu Pro Phe Met Asp Glu Pro Ile Ala Ile 195 200 205 Pro Asn Lys Pro Tyr Thr Glu Glu Glu Leu Val Glu Phe Val Lys Glu 210 215 220 His Gln Arg Pro Thr Leu Arg Arg Leu Arg Pro Glu Glu Met Phe Glu 225 230 235 240 Thr Trp Glu Asp Asp Leu Asn Gly Ile His Ile Val Ala Phe Ala Glu 245 250 255 Lys Ser Asp Pro Asp Gly Tyr Glu Phe Leu Glu Ile Leu Lys Gln Val 260 265 270 Ala Arg Asp Asn Thr Asp Asn Pro Asp Leu Ser Ile Leu Trp Ile Asp 275 280 285 Pro Asp Asp Phe Pro Leu Leu Val Ala Tyr Trp Glu Lys Thr Phe Lys 290 295 300 Ile Asp Leu Phe Arg Pro Gln Ile Gly Val Val Asn Val Thr Asp Ala 305 310 315 320 Asp Ser Val Trp Met Glu Ile Pro Asp Asp Asp Asp Leu Pro Thr Ala 325 330 335 Glu Glu Leu Glu Asp Trp Ile Glu Asp Val Leu Ser Gly Lys Ile Asn 340 345 350 Thr Glu Asp Asp Asp Glu Asp Asp Asp Asp Asp Asp Asn Ser Asp Glu 355 360 365 Glu Asp Asn Asp Asp Ser Asp Asp Asp Asp Asp Glu 370 375 380 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> G4S <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 4N <400> 8 Gly Ser Asn Asn Asn Asn 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 8N <400> 9 Gly Ser Asn Asn Asn Asn Asn Asn Asn Asn 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> LE8N <400> 10 Leu Glu Asn Asn Asn Asn Asn Asn Asn Asn 1 5 10 <210> 11 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Primers <400> 11 Ala Ala Thr Ala Ala Ala Ala Cys Ala Thr Ala Thr Gly Gly Thr Ala 1 5 10 15 Gly Ala Cys Ala Ala Cys Ala Ala Ala Thr Thr Cys Ala Ala Cys 20 25 30 <210> 12 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Primer <400> 12 Ala Thr Thr Cys Thr Cys Gly Ala Gly Thr Thr Ala Ala Thr Cys Ala 1 5 10 15 Thr Cys Ala Thr Cys Ala Thr Cys Ala Thr Cys Ala Thr Cys Thr Thr 20 25 30 Cys <210> 13 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Z <400> 13 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 14 <211> 290 <212> PRT <213> artificial sequence <220> <223> (Z)5 <400> 14 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn 50 55 60 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro 85 90 95 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala 100 105 110 Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala 115 120 125 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn 130 135 140 Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu 145 150 155 160 Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp 165 170 175 Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His 180 185 190 Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu 195 200 205 Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys 210 215 220 Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu 225 230 235 240 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu 245 250 255 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln 260 265 270 Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 275 280 285 Pro Lys 290

Claims

1. A fusion protein of a Z-domain and calcineurin, wherein: The Z-domain and calcitonin are connected via a linker, The calcineurin consists of the amino acid sequence of SEQ ID NO: 5, The Z-domain consists of the amino acid sequence of any one of SEQ ID NOs: 1-3, 13 and 14, The linker consists of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 10.

2. The fusion protein according to claim 1, wherein The Z-domain consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 3.

3. The fusion protein according to claim 1, wherein The fusion protein comprises a Z-domain consisting of the amino acid sequence of SEQ ID NO: 1, a linker consisting of any one of the amino acid sequences of SEQ ID NO: 8 to 10, and a calcineurin consisting of the amino acid sequence of SEQ ID NO:

5.

4. Use of the fusion protein according to any one of claims 1 to 3 for purifying antibodies.

5. The use according to claim 4, wherein The fusion protein comprises a Z-domain consisting of the amino acid sequence of SEQ ID NO: 1, a linker consisting of any one of the amino acid sequences of SEQ ID NO: 8 to 10, and a calcineurin consisting of the amino acid sequence of SEQ ID NO: 5.

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