Lactoferrin c-lobe-fused protein l single domain and use thereof

Abstract

The purpose of the present invention is to provide a new means for efficiently and / or simply detecting a target substance in an immunoassay. Provided is an LFC-fused PpL single domain in which lactoferrin C-lobe (LFC) and Protein L single domain (PpL single domain) are linked directly or via a linker.

Description

Lactoferrin C-lobe fusion protein L single domain and its use

[0001] It relates to a lactoferrin C-lobe fusion protein L single domain and its use.

[0002] Protein L is said to be a protein first isolated from Peptostreptococcus magnus, and it has been reported that it mainly binds to the κ light chain of immunoglobulins such as IgG, IgM, IgE, IgD, and IgA derived from mammals such as humans, rabbits, pigs, mice, and rats. The protein L single domain is known as a domain constituting a part of protein L.

[0003] Lactoferrin is a protein composed of a single-chain peptide. For example, in the case of cows, it is known to consist of 689 amino acid residues, and in the case of humans, it consists of 692 amino acid residues (Non-Patent Document 1). Lactoferrin is a glycoprotein contained in exocrine fluids such as mammalian milk and tears, has the ability to bind to trivalent iron, and has a structure in which two globular domains called the N-lobe (N-lobe) having amino acid residues on the N-terminal side and the C-lobe (C-lobe) having amino acid residues on the C-terminal side in the amino acid sequence constituting lactoferrin are connected by an α-helix polypeptide.

[0004] Conventionally, immunoassay has been used for detecting target substances such as antibodies, antigens, and enzymes by immunological methods. Among immunoassays, for example, according to dot blot, it is possible to detect antibodies and the like with a relatively simple operation. In addition, lateral flow is also used for POCT tests (Point of Care Testing; immediate clinical testing) known as pregnancy test kits, infectious disease diagnostic agents, etc. because the results of antigen-antibody reactions can be obtained relatively simply and quickly. Thus, immunoassay is used for the simple and rapid detection of target substances, but it is important to provide new means for efficiently and / or simply detecting target substances.

[0005] Yoshiharu Takayama, Oleo Science, Vol. 23, No. 8 (2023), pp. 423-429

[0006] The objective is to provide a new method for efficiently and / or simply detecting a target substance in an immunoassay.

[0007] As a result of diligent research, the inventors have found that LFC-fused PpL single domains, which are formed by linking the C lobe (LFC) of lactoferrin with a protein L single domain (PpL single domain), can be produced using E. coli according to a general recombinant protein expression procedure, and that these LFC-fused PpL single domains can be efficiently refolded, i.e., that they can be efficiently produced. Furthermore, the inventors have found that LFC-fused PpL single domains can be easily refolded while immobilized on a nitrocellulose membrane (in a denatured state) and refolded while immobilized on the membrane. The inventors have also found that LFC-fused PpL single domains have good antibody-binding activity. The present invention was completed based on these findings and further research, and this disclosure includes, for example, the inventions described below.

[0008] Item 1. An LFC-fused PpL single domain in which the C lobe (LFC) of lactoferrin and a protein L single domain (PpL single domain) are linked directly or via a linker. Item 2. The LFC-fused PpL single domain according to Item 1, wherein each LFC contains 1 to 10 PpL single domains. Item 3. The LFC-fused PpL single domain according to Item 1 or 2, wherein the amino acid sequence encoding a PpL single domain is directly or via a linker at the N-terminus of the amino acid sequence encoding the LFC. Item 4. The LFC-fused PpL single domain according to any one of Items 1 to 3, wherein the LFC consists of the amino acid sequence described in (1-1) or (1-2) below: (1-1) The amino acid sequence represented by Sequence ID No. 1 (1-2) The amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence described in (1-1). Item 5. A nitrocellulose membrane on which an LFC-fused PpL single domain as described in any one of items 1 to 4 is immobilized. Item 6. An antibody kit comprising an LFC-fused PpL single domain as described in any one of items 1 to 4, a nitrocellulose membrane, and an antibody. Item 7. The antibody is scFv, scAb, sdAb, Fab, F(ab) ２ 'Antibody kit as described in item 6, which is a full-length antibody.

[0009] This provides a novel method for efficiently and / or simply detecting target substances in immunoassays.

[0010] Schematic diagrams of each fusion protein constructed in Test Example 1 (Examples 1-3, Comparative Example 1, Reference Example 1) are shown. An outline of refolding by dialysis in Test Example 1 is shown. The results of Test Example 1 (electrophoresis results, refolding rate) are shown. The reaction principle and results (antibody binding activity) of Test Example 2 are shown. The reaction principle and results (antibody binding activity) of Test Example 3 are shown. Schematic diagrams of the LFC-fused PpL single domains constructed in Test Example 4 (Examples 4-6) are shown. The results of Test Example 4 (electrophoresis results, refolding rate) are shown. The reaction principle and results (antibody binding activity) of Test Example 5 are shown. The reaction principle and results (antibody binding activity after heat treatment) of Test Example 6 are shown. The results (antibody binding activity after heat treatment) of Test Example 7 are shown. The results of Test Example 8 (antibody binding activity using buffers of various pH values) are shown. The results of Test Example 9 (antibody binding activity using buffers of various pH values) are shown. The reaction principle of Test Example 10 is shown. The results of Test Example 10 (antigen-binding activity) are shown. The amino acid sequence used to construct the LFC-fused PpL single domain is shown. The amino acid sequence used to construct the LFC-fused PpL single domain is shown. The amino acid sequence used to construct the LFC-fused PpL single domain is shown.

[0011] The embodiments included in this disclosure are described in further detail below. In this disclosure, “contains” also means “substantially consists of” or “consists of.”

[0012] This disclosure includes LFC-fused PpL single domains, in which the C lobe (LFC) of lactoferrin and the protein L single domain (PpL single domain) are linked directly or via a linker.

[0013] Lactoferrin C-lobe (LFC) Lactoferrin (LF) is a protein composed of a single-chain peptide, and is known to consist of 689 amino acid residues in the case of cattle, for example, and 692 amino acid residues in the case of humans. Lactoferrin has a structure in which two spherical domains, called the N-lobe (hereinafter sometimes referred to as "LFN"), composed of amino acid residues at the N-terminus and the C-lobe (hereinafter sometimes referred to as "LFC"), composed of amino acid residues at the C-terminus, are linked by an α-helix polypeptide, as illustrated in Figure 1, for example, <Structure of LF>. The amino acid sequence of the LFC is publicly known and can be easily obtained from various amino acid sequence databases (e.g., the NCBI database). The origin of the LFC is not limited, and mammalian origins (e.g., cattle, human) are exemplified, with cattle origin being preferred. Although not limiting to this disclosure, the LFC encoded by the amino sequence represented by Sequence ID No. 1 is exemplified as the LFC. The known amino acid sequence encoding LFC may be wild-type (naturally occurring) or may be a mutated version of the wild-type.

[0014] In this disclosure, the LFC may be encoded by an amino acid sequence that is a mutation of a known amino acid sequence encoding the LFC. Examples of an amino acid sequence that is a mutation of a known amino acid sequence encoding the LFC include an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the known amino acid sequence.

[0015] Examples of mutations in known amino acid sequences encoding LFCs include 2 to 20, 2 to 17, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, and 2 or 3 mutations. In this disclosure, the mutated amino acids may be, for example, natural amino acids or artificial amino acids. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, and sulfur-containing amino acids. Examples of amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Techniques for deleting, substituting, inserting, and / or adding one or more amino acids to a specific amino acid sequence are well known.

[0016] Without limiting this disclosure, substitutions are preferredly exemplified as mutations, and conservative substitutions are preferredly exemplified as substitutions. In this disclosure, a conservative substitution means that an amino acid residue is replaced by an amino acid residue having a side chain of similar properties. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Similarly, substitutions between amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, are also considered conservative substitutions. Furthermore, although this disclosure is not limited, as illustrated in the test examples described later, substitution of cysteine ​​with serine is preferred in terms of suppressing complex formation of LFC-fused PpL single domains.

[0017] Furthermore, although not limiting to this disclosure, it is more preferable to illustrate an LFC that, when linked with a PpL single domain to form an LFC-fused PpL single domain, exhibits a refolding rate equivalent to or greater than that of an LFC encoded by the amino acid sequence represented by Sequence ID No. 1 (i.e., an LFC with a refolding ratio of 0.7 or more according to Formula 1 described later). Moreover, it is more preferable to illustrate an LFC that, when formed as an LFC-fused PpL single domain, exhibits an antibody-binding activity value equivalent to or greater than that of an LFC encoded by the amino acid sequence represented by Sequence ID No. 1 (i.e., an LFC with an antibody-binding activity ratio of 0.7 or more according to Formula 2 described later).

[0018] The length of the amino acid sequence encoding the LFC is not limited and should be similar to that of known LFCs. The number of amino acid residues is preferably around 340 to 370, more preferably around 345 to 365, and even more preferably around 350 to 360.

[0019] No. is limited to the foregoing, but an example of an LFC is an LFC consisting of the amino acid sequence described in (1-1) or (1-2) below: (1-1) The amino acid sequence represented by Sequence ID No. 1 (1-2) The amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence described in (1-1) above

[0020] In (1-2) above, multiples are described in the same manner as described above, with examples of multiples including 2 to 20, 2 to 17, 2 to 15, etc. Furthermore, although not limiting to this disclosure, substitutions are preferably given as examples of mutations, conservative substitutions are preferably given as examples of substitutions, and conservative substitutions are described in the same manner as described above.

[0021] Mutations are not limited to this extent, but more preferably, the refolding rate in the liquid phase when using an LFC-fused PpL single domain (pre-mutation LFC-fused PpL single domain) obtained by linking an LFC encoded by the amino acid sequence represented by Sequence ID No. 1 (hereinafter sometimes referred to as "pre-mutation LFC") with a PpL single domain is compared with the refolding rate in the liquid phase when using an LFN-fused PpL single domain (post-mutation LFC-fused PpL single domain) obtained by linking an LFC encoded by the post-mutation amino acid sequence described in (1-2) above (hereinafter sometimes referred to as "post-mutation LFC") with a PpL single domain, and the value obtained by dividing the refolding rate of the latter by the refolding rate of the former according to Formula 1 below (refolding ratio) is preferably 0.7 or more. The range of this ratio is more preferably 0.7 to 3, even more preferably 0.8 to 2, and particularly preferably 0.9 to 1.5.

[0022] [Equation 1] Liquid-phase refolding ratio = Liquid-phase refolding rate when using the mutated LFC-fused PpL single domain / Liquid-phase refolding rate when using the pre-mutated LFC-fused PpL single domain

[0023] For example, in Test Example 1 described later, the refolding rate in the liquid phase when using the pre-mutation LFC-fused PpL single domain (Example 1) was 107.2%, and the refolding rate in the liquid phase when using the post-mutation LFC-fused PpL single domain (Example 2) was 99.1%. Applying these to Formula 1, the refolding ratio calculated was 0.94 (99.1 ÷ 107.2, rounded to the third decimal place). The LFC used in Example 2 is coded by the amino acid sequence represented by Sequence ID No. 2, which represents an amino acid sequence in which 17 cysteines are replaced with serine in the amino acid sequence represented by Sequence ID No. 1. Thus, although not limiting the present disclosure, a preferred embodiment of the amino acid sequence described in (1-2) above is preferably an amino acid sequence in which one or more cysteines are replaced with serine in the amino acid sequence represented by Sequence ID No. 1. Furthermore, an amino acid sequence in which 1 to 20 cysteines are replaced with serine in the amino acid sequence represented by Sequence ID No. 1 is more preferably exemplified. Furthermore, as mentioned above, examples of multiple units include, more preferably, 2 to 17 units, and even more preferably, 2 to 15 units. The refolding ratio is preferably satisfied when one PpL single domain is linked to one LFC, and more preferably, it is satisfied when a mutated LFC is used as the LFC in the LFC-fused PpL single domain shown in Example 1, as exemplified in Example 2.

[0024] The refolding rate in the liquid phase can be determined by confirming the refolding rate in the liquid phase under the same conditions except for whether the pre-mutation LFC or post-mutation LFC is fused to the PpL single domain, and calculating it according to Equation 1 above. The confirmation of the refolding rate in the liquid phase is preferably done according to the procedure of Test Example 1 described below. Specifically, the fusion protein (LFC-fused PpL single domain) is expressed according to a general protein expression procedure using E. coli, and the intracellular insoluble fraction containing the fusion protein is recovered. Next, following a general refolding procedure, the fraction is dialyzed overnight at 4°C using 8M urea (pH 8) as a denaturant, and then dialyzed for two nights at 4°C in phosphate-buffered saline (pH 7.2) in a dialysis membrane. Next, the solution in the dialysis membrane is centrifuged at 4°C, 10,000 g, for 15 minutes, aggregates are removed, and the supernatant is collected to obtain the refolded LFC-fused PpL single domain. The refolding rate (%) is calculated by comparing the concentration of LFC-fused PpL single domains in the supernatant after centrifugation with the concentration of LFC-fused PpL single domains in the solution before centrifugation within the dialysis membrane.

[0025] Furthermore, while mutations are not limited to this extent, when using the LFC-fused PpL single domain of this disclosure immobilized on a nitrocellulose (NC) membrane, for simple immobilization on the NC membrane, it is preferable to utilize the adsorption capacity to the NC membrane derived from the LFC constituting the LFC-fused PpL single domain of this disclosure. From this viewpoint, mutations to the known amino acid sequence of the LFC are more preferably exemplified in a range that does not interfere with the adsorption capacity to the NC membrane derived from the LFC. The adsorption capacity to the NC membrane derived from the LFC can be confirmed based on the antibody binding property of the LFC-fused PpL single domain after immobilization by contacting the LFC-fused PpL single domain of this disclosure with the NC membrane, as shown in Test Example 2. In this disclosure, the higher the antibody binding property, the higher the adsorption capacity. Therefore, as the mutation, it is preferable to compare the antibody binding activity value when the pre-mutation LFC-fusion PpL single domain is immobilized on the NC membrane with the antibody binding activity value when the post-mutation LFC-fusion PpL single domain is immobilized on the NC membrane, and preferably exemplified a mutation in which the value obtained by dividing the latter antibody binding activity value by the former antibody binding activity value according to the following formula 2 (antibody binding activity ratio) is 0.7 or more. The range of this ratio is more preferably 0.7 to 2, even more preferably 0.8 to 1.5, and particularly preferably 0.9 to 1.2.

[0026] [Equation 2] Antibody binding activity ratio = Antibody binding activity value when using the mutated LFC-fused PpL single domain / Antibody binding activity value when using the pre-mutated LFC-fused PpL single domain

[0027] For example, in Test Example 2 described later, the antibody binding activity value when using the pre-mutation LFC-fused PpL single domain (Example 1) was 225.0, and the solution antibody binding activity value when using the post-mutation LFC-fused PpL single domain (Example 2) was 158.1. The antibody binding activity ratio calculated by applying these values ​​to Formula 2 is 0.70 (158.1 ÷ 225.0, rounded to the third decimal place). The antibody binding activity ratio is preferably satisfied when one PpL single domain is linked to one LFC, and more preferably when the post-mutation LFC is used as the LFC in the LFC-fused PpL single domain shown in Example 1.

[0028] The antibody binding activity ratio can be determined by fixing the LFC-fused PpL single domain by adsorption onto an NC membrane, under the same conditions except for whether the pre-mutation LFC or post-mutation LFC is fused to the PpL single domain, confirming the antibody binding activity value, and calculating it according to formula 2 above. The confirmation of the antibody binding activity value is preferably done according to the procedure in Test Example 2 described below. Specifically, the LFC-fused PpL single domain is dropped onto an NC membrane (product name Hi-Flow Plus HFC13504 2.5 cm, manufactured by Merck KGaA) and air-dried (25°C). Next, following a general antigen-antibody reaction procedure, the NC membrane is blocked and reacted with an antibody appropriate for the target (e.g., Bt-Human IgG (biotin-labeled human IgG)). Then, SA-AP (streptavidin-conjugated alkaline phosphatase) is added, followed by BCIP / NBT (manufactured by Nacalai Tesque Co., Ltd.). The antibody binding activity value can then be confirmed based on the resulting signal.

[0029] Protein L Single Domain (PpL Single Domain) A PpL single domain is known as a domain that constitutes a part of protein L. Protein L is said to be the first protein isolated from Peptostreptococcus magnus. The amino acid sequence of the PpL single domain is publicly known and can be easily obtained from various databases on amino acid sequences. PpL single domains are broadly classified into four types: protein L C1 domain, protein L C2 domain, protein L C3 domain, and protein L C4 domain, and in this disclosure, the PpL single domain may be any of these single domains. Although not limiting to this disclosure, examples of PpL single domains include domains encoded by amino sequences represented by any of sequence numbers 3 to 9. For example, sequence numbers 3 and 4 encode the protein L C1 domain, sequence number 5 encodes the protein L C2 domain, sequence number 6 encodes the protein L C3 domain, and sequence number 7 encodes the protein L C5 domain, respectively. The origin of the PpL single domain is not limited, and, for example, bovine origin is preferred. Furthermore, the known amino acid sequence encoding the PpL single domain may be wild-type (naturally occurring) or may be a mutated version of the wild-type. Examples of PpL single domains include preferably the protein L C1 domain (PpL1) and the protein L C3 domain (PpL3), more preferably PpL1, and even more preferably PpL1 encoded by the amino acid sequence represented by Sequence ID No. 3.

[0030] In this disclosure, a PpL single domain may be encoded by an amino acid sequence that is mutated from a known amino acid sequence encoding a PpL single domain. Examples of amino acid sequences that are mutated from a known amino acid sequence encoding a PpL single domain include amino acid sequences in which one or more amino acids are substituted, deleted, inserted, or added to the known amino acid sequence. Examples of multiple substitutions include 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2, or 3. Conservative substitutions are preferably exemplified as substitutions, and conservative substitutions are described in the same manner as described above. Although not limiting to this disclosure, preferred examples include the substitution of lysine with arginine and the substitution of cysteine ​​with serine.

[0031] The length of the amino acid sequence encoding the PpL single domain is not limited, but it should be similar to that of known amino acid sequences encoding PpL single domains. Preferably, the number of amino acid residues is about 55 to 95, more preferably about 65 to 90, and even more preferably about 70 to 88.

[0032] No. is limited to the foregoing, but as one embodiment of a PpL single domain, a PpL single domain consisting of the amino acid sequences described in (2-1) or (2-2) below is exemplified: (2-1) The amino acid sequence represented by Sequence ID No. 3 (2-2) The amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence described in (2-1) above

[0033] In (2-2) above, multiple instances are explained in the same manner as described above. Furthermore, substitutions are preferably given as examples of mutations, and conservative substitutions are preferably given as examples of substitutions, and conservative substitutions are explained in the same manner as described above.

[0034] Without limiting this disclosure, as one embodiment of the amino acid sequence described in (2-2) above, an amino acid sequence is provided in which one or more lysines are substituted with arginine in the amino acid sequence represented by Sequence ID No. 3, and all lysines present in the amino acid sequence may be substituted with arginine, or some of the lysines may be substituted with arginine. More preferably, an amino acid sequence is provided in which 1 to 9 lysines are substituted with arginine, and even more preferably, an amino acid sequence is provided in which at least 1, 2, 3, 4, 5, 6, 7, or 8 of the 8 lysines located at the 3rd, 17th, 23rd, 32nd, 39th, 58th, 77th, and 81st positions from the N-terminus of the amino acid sequence represented by Sequence ID No. 3 are substituted with arginine. Without limiting this disclosure, the amino acid sequence represented by Sequence ID No. 10 represents an amino acid sequence in which 8 lysines are substituted with arginine in the amino acid sequence represented by Sequence ID No. 3.

[0035] Mutations to known amino acid sequences of PpL single domains are not limited to this extent, but preferably examples of mutations are those in which the refolding rate in the liquid phase is 0.7 or higher when comparing the refolding rate in the liquid phase when using an LFC-fused PpL single domain (LFC-fused pre-mutation PpL single domain) obtained by linking an LFC with a PpL single domain encoded by a known amino acid sequence (hereinafter sometimes referred to as "pre-mutation PpL single domain"), and the refolding rate in the liquid phase when using an LFN-fused PpL single domain (LFC-fused post-mutation PpL single domain) obtained by linking an LFC with a PpL single domain encoded by a mutated amino acid sequence (hereinafter sometimes referred to as "post-mutation PpL single domain"), and the value obtained by dividing the refolding rate of the latter by the refolding rate of the former according to formula 3 below (refolding ratio). There is no upper limit to the ratio, but a more preferred range for the ratio is 0.7 to 2, even more preferably 0.8 to 1.5, and particularly preferably 0.9 to 1.

[0036] [Equation 3] Liquid-phase refolding ratio = Liquid-phase refolding rate when using the PpL single domain after LFC fusion mutation / Liquid-phase refolding rate when using the PpL single domain before LFC fusion mutation

[0037] The refolding rate in the liquid phase can be determined by confirming the refolding rate in the liquid phase under the same conditions except for whether a pre-mutation PpL single domain or a post-mutation PpL single domain is fused to the LFC, and calculating it according to formula 3 above. The confirmation of the refolding rate in the liquid phase can preferably be performed according to the procedure of Test Example 1 described later, that is, it will be explained in the same way as above. The refolding rate is more preferably satisfied when a post-mutation PpL single domain is used as the PpL single domain in the LFC-fused PpL single domain shown in Example 1 or Example 5.

[0038] Mutations are not limited to this extent, but for the simple detection of the target substance, mutations that do not interfere with the antibody binding ability derived from the PpL single domain are preferably exemplified, given that the antibody binding ability derived from the PpL single domain constituting the LFC-fused PpL single domain of this disclosure is utilized. The antibody binding ability derived from the PpL single domain can be confirmed based on the antibody binding properties of the LFC-fused PpL single domain after immobilizing it by contacting the LFC-fused PpL single domain of this disclosure with an NC membrane, as shown in Test Examples 2 and 5 (liquid-phase refolding). In this disclosure, a higher antibody binding property indicates higher antibody binding ability. Therefore, as the mutation, the antibody binding activity value when the PpL single domain before the LFC fusion mutation is immobilized on the NC membrane is compared with the antibody binding activity value when the PpL single domain after the LFC fusion mutation is immobilized on the NC membrane (more preferably, the antibody binding activity value when the LFC fusion PpL single domain (PpL single domain before the LFC fusion mutation), which is formed by linking the PpL single domain encoded by the amino acid sequence represented by SEQ ID NO: 3 (hereinafter sometimes referred to as the "pre-mutation PpL single domain") with the LFC, is immobilized on the NC membrane is compared with the antibody binding activity value when the mutated amino acid sequence described in (2-2) above is compared with the antibody binding activity value when the LFC fusion PpL single domain (pre-mutation PpL single domain) is immobilized on the NC membrane A mutation is preferably exemplified in which the antibody binding activity value obtained by dividing the antibody binding activity value of the latter by the antibody binding activity value of the former, according to the following formula 4, is 0.7 or higher, when the PpL single domain encoded by the column (hereinafter, it may be referred to as the "mutated PpL single domain" or, in some cases, the LFC-fused PpL single domain (LFC-fused mutant PpL single domain) linked with the LFC is immobilized on an NC membrane, and the mutation is preferably exemplified in which the antibody binding activity value of the latter is 0.7 or higher. There is no upper limit to this ratio, but a more preferably given range for this ratio is 0.7 to 3, even more preferably 0.8 to 2, and particularly preferably 0.9 to 1.5.

[0039] [Equation 4] Antibody binding activity ratio = Antibody binding activity value when using the post-LFC fusion mutation PpL single domain / Antibody binding activity value when using the pre-LFC fusion mutation PpL single domain

[0040] For example, in Test Example 5 described below, the antibody binding activity value when using the PpL single domain before LFC fusion mutation (Example 5: PpL1T-LFC-PpL1T-v1) was 45.3, and the liquid antibody binding activity value when using the PpL single domain after LFC fusion mutation (Example 6: PpL1T-LFC-PpL1T-v2) was 58.6. The antibody binding activity ratio calculated by applying these to the above formula 4 is 1.29 (58.6÷45.3, rounding off the third decimal place). Note that the antibody binding activity ratio preferably suffices when using the mutated PpL single domain as the PpL single domain in the LFC fusion PpL single domain shown in Example 1 or Example 5.

[0041] The antibody binding activity ratio can be determined by adsorbing the LFC fusion PpL single domain onto a solid phase under the same conditions except for whether the unmutated PpL single domain or the mutated PpL single domain is fused to LFC, confirming the antibody binding activity value, and calculating according to the above formula 4. The confirmation of the antibody binding activity value can preferably be performed according to the procedures of Test Examples 2 and 5 (liquid phase refolding) described below. That is, similar to the above, it is explained in the same way as the procedure according to Test Example 2.

[0042] In the LFC fusion PpL single domain of the present disclosure, LFC and the PpL single domain are linked directly or via a linker. The PpL single domain may be linked to the N-terminal side of LFC, may be linked to the C-terminal side of LFC, or may be linked to both the N-terminal side and the C-terminal side. Preferably, it is exemplified that the PpL single domain is at least linked to the N-terminal side of LFC.

[0043] The LFC-fused PpL single domain of this disclosure is linked directly or via a linker between the LFC and the PpL single domain, preferably via a linker. The linker is not limited to the extent that it does not impede the effects of this disclosure, and examples include a linker consisting only of alanine (A), a linker consisting only of glycine (G), a linker consisting only of serine (S), a GS linker, an EAAAK linker, etc. The length of the linker is not particularly limited, and examples of the number of amino acid residues of the linker include preferably 2 to 10, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2, 3, or 4. As a GS linker, (G4S)n is preferably exemplified, where n is an integer of 1 or 2. As an EAAAK linker, (EAAAK)n is exemplified, where n is an integer of 1 or 2 (EAAAK is shown in Sequence ID No. 11). As a linker, a linker consisting only of alanine (A) is preferably exemplified. The linker may be used alone or in combination of two or more types. Furthermore, the LFC-fused PpL single domain of this disclosure may have amino acid sequences other than the linker, such as restriction enzyme sites, tag sequences (His tag, Myc tag, etc.), and signal peptides, to the extent that it does not interfere with the effects of this disclosure.

[0044] In the LFC-fused PpL single domains of this disclosure, the number of PpL single domains and the number of LFCs may be the same or different. Preferably, the number of PpL single domains and LFCs are equal, or the number of PpL single domains is greater than the number of LFCs. Without limiting this disclosure, examples of LFC-fused PpL single domains of this disclosure include those containing preferably 1 to 10 PpL single domains (1 to 10 decamers) per LFC (monomer), more preferably 1 to 8, even more preferably 1 to 7, and particularly preferably 1, 2, 3, 4, 5, or 6. When the number of LFCs is greater than the number of PpL single domains, examples include those containing preferably 2 to 5 LFCs, more preferably 2 to 4, and even more preferably 2 or 3 LFCs per PpL single domain. If the LFC-fused PpL single domain of this disclosure includes two or more LFCs and / or two or more PpL single domains, the LFCs and PpL single domains may each be used individually or in combination of two or more types.

[0045] For the purpose of facilitating understanding, an example in the case where the number of PpL single domains is the same as or greater than the number of LFCs is shown in FIG. 6. In FIG. 6, Example 1 (PpL1-LFC) is an example in the case of the same number (one PpL single domain (monomer) and one LFC (monomer) are linked), and Examples 4 (PpL1T-LFC), Example 5 (PpL1T-LFC-PpL1T-v1), and Example 6 (PpL1T-LFC-PpL1T-v2) are all examples in the case where the number of PpL single domains is greater than the number of LFCs (in Example 4, three PpL single domains (trimer) and one LFC (monomer) are linked, and in Examples 5 and 6, six PpL single domains (hexamer) and one LFC (monomer) are linked). In the LFC fusion PpL single domain of the present disclosure, when two or more PpL single domains and / or two or more LFCs are fused, it is not necessary for all of the PpL single domains to be present continuously. As illustrated in Examples 5 and 6 of FIG. 6, among a total of six PpL single domains, three consecutive PpL single domains (PpL single domain trimer) may be present on both the N-terminal side and the C-terminal side of the LFC. That is, one or a plurality of consecutive PpL single domains may be present only on the N-terminal side of the LFC, only on the C-terminal side of the LFC, or on both the N-terminal side and the C-terminal side of the LFC. The same explanation applies to LFC. Although not limiting the present disclosure, when two or more PpL single domains are present continuously, it is preferably exemplified that 2 to 5 PpL single domains are present continuously, and more preferably, it is exemplified that 2, 3, or 4 PpL single domains are present continuously. Although not limiting the present disclosure, when two or more LFCs are present continuously, it is preferably exemplified that 2 to 5 LFCs are present continuously, and more preferably, it is exemplified that 2, 3, or 4 LFCs are present continuously.

[0046] In the LFC-fused PpL single domain of this disclosure, if there are multiple consecutive PpL single domains, the PpL single domains are linked to each other directly or via a linker, preferably directly (by peptide bonds). In the LFC-fused PpL single domain of this disclosure, if there are multiple consecutive LFCs, the LFCs are linked to each other directly or via a linker, preferably directly. The linker is described in the same manner as described above.

[0047] Furthermore, in the LFC-fused PpL single domain of this disclosure, when the LFC and the PpL single domain are linked via a linker, the number of amino acid residues (amino acid length) between the LFC and the PpL single domain is preferably exemplified as 2 to 10. For example, in Examples 1 and 4-6 shown in Figure 6, the number of amino acid residues is 3 (AAA).

[0048] In the LFC-fused PpL single domain of this disclosure, as long as the LFC and the PpL single domain are linked directly or via a linker, this does not prevent the LFN from being further linked; however, preferably, the LFN is not linked in the LFC-fused PpL single domain of this disclosure. From this viewpoint, a more preferable example of the LFC-fused PpL single domain of this disclosure is one in which the LF itself (i.e., the LF having the LFC and LCN) is not linked.

[0049] Without limiting this disclosure, examples of molecular weights for LFC-fused PpL single domains include 40 to 150 (40 to 150 kDa), more preferably 45 to 120 (45 to 120 kDa), and even more preferably 50 to 100 (50 to 100 kDa). The molecular weight of an LFC-fused PpL single domain is calculated using the Expasy protparam tool (https: / / web.expasy.org / protparam / ). The Expasy protparam tool is a protein analysis tool operated by the Swiss Institute of Bioinformatics (SIB), and is a well-known tool that can calculate the molecular weight of a protein by inputting an amino acid sequence.

[0050] The LFC-fused PpL single domain of this disclosure can be manufactured according to procedures known in the field of genetic engineering, etc. Without limiting this disclosure, for example, it can be manufactured by constructing a polynucleotide containing a nucleotide sequence encoding the PpL single domain, a nucleotide sequence encoding the LFC, and optionally a linker, transforming host cells using a vector, and then culturing and purifying the cells. Thus, the LFC-fused PpL single domain of this disclosure may further contain any sequences other than the PpL single domain, LFC, and linker, as long as they do not interfere with the effects of this disclosure. Therefore, sequences of any substance such as restriction enzyme sites, tags, or proteins may be introduced into the nucleotides as needed.

[0051] As shown in Test Example 1 below, the LFC-fused PpL single domain of this disclosure can be easily produced according to conventionally known protein expression principles using E. coli and the like. Furthermore, this disclosure is useful because it allows for the production of LFC-fused PpL single domains with a relatively high refolding rate, meaning that LFC-fused PpL single domains can be produced efficiently.

[0052] Furthermore, among immunoassays, methods such as dot plots and POCT (point-of-care testing) using lateral flow are means of detecting a target substance by loading an antibody onto a solid phase and applying a sample to the solid phase, with nitrocellulose (NC) membranes mainly used as the solid phase. In these methods, it is important to be able to easily load the antibody onto the NC membrane while maintaining the antigen recognition ability of the antibody. According to this disclosure, the LFC-fused PpL single domain of this disclosure can be easily immobilized on the NC membrane by contacting it with the NC membrane, and by applying an antibody to the NC membrane, the antibody can be easily loaded onto the NC membrane via the LFC-fused PpL single domain of this disclosure. Furthermore, according to this disclosure, not only can the LFC-fused PpL single domain after refolding be immobilized on the NC membrane to support antibodies, but even when the LFC-fused PpL single domain before refolding (denatured state) is immobilized on the NC membrane and then refolded on the NC membrane, antibodies can be easily supported on the NC membrane via the LFC-fused PpL single domain of this disclosure.

[0053] Furthermore, antibodies supported on an NC membrane via the LFC-fused PpL single domain of this disclosure have binding properties to a target substance (e.g., an antigen). Thus, the LFC-fused PpL single domain of this disclosure allows antibodies to be easily supported on an NC membrane via the LFC-fused PpL single domain of this disclosure while maintaining the desired antigen recognition ability.

[0054] Therefore, it can be said that this disclosure also encompasses NC membranes in which the LFC-fused PpL single domain of this disclosure is immobilized.

[0055] NC membranes are conventionally known as immobilization carriers for antibodies, enzymes, etc., in various immunoassays such as immunochromatography, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blotting, and are not limited as long as they can be used in immunoassays. In this respect, the thickness and shape of the NC membrane are also not limited. Although not limiting to this disclosure, examples of NC membrane thicknesses include approximately 80 to 500 μm, preferably approximately 80 to 400 μm. Also, although not limiting to this disclosure, examples of NC membrane average pore size include approximately 0.1 to 5 μm, preferably approximately 0.45 μm to 5 μm. In this disclosure, the average pore size is the value according to the product catalog. NC membranes are commercially available, and examples include High-Flow plus HF180, Hi-Flow Plus HFC13504 (both manufactured by Merck KGaA), Amersham Protran (manufactured by Cytiva), etc.

[0056] The LFC-fused PpL single domains of this disclosure, immobilized on the NC membrane, may be in a pre-refolding state (denatured state) or a post-refolding state (undenatured state). Refolding is in the common sense of this art, that is, generally, it refers to solubilizing and denaturing inclusion bodies (aggregates) or misfolded proteins formed when producing proteins using an expression system such as Escherichia coli, using a denaturing agent (denatured state), and then unfolding them back into their native structure (undenatured state). Thus, in this disclosure, the NC membrane may be immobilized with LFC-fused PpL single domains in a pre-refolding state (denatured state) or with LFC-fused PpL single domains in a post-refolding state (undenatured state).

[0057] The method for immobilizing the LFC-fused PpL single domains of this disclosure onto an NC membrane is not limited as long as such immobilization is possible. As a simple example, the LFC-fused PpL single domains of this disclosure can be immobilized by contacting the NC membrane with a solution containing the LFC-fused PpL single domains of this disclosure and then drying it.

[0058] The solution containing the LFC-fused PpL single domain is not limited as long as immobilization is possible, and may be a conventionally known buffer (Tris buffer, phosphate buffer, Good's buffer, carbonate buffer, acetate buffer, glycine-HCl buffer, citrate buffer, borate buffer, etc.). Furthermore, as mentioned above, the immobilized LFC-fused PpL single domain may be in either a denatured or undenatured state. Therefore, the solution may be a solution obtained after solubilization treatment (before adding the refolding solution) according to a conventionally known refolding procedure, a solution obtained after refolding is completed by adding the refolding solution, or any mixture thereof. The pH of the solution containing the LFC-fused PpL single domain is also not limited as long as the effects of this disclosure are obtained, with pH 1 to 14 being an example, preferably pH 2 to 13.5, more preferably pH 6 to 13.5, and even more preferably pH 10.5 to 13. The pH is a value measured at 25°C using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.) and represents the pH of the solution when it is brought into contact with the NC film. The contact temperature and time between the solution and the NC film are not limited, but preferably 20 to 60°C and 10 seconds to 1 hour are given as examples. Examples of contact include dropping the solution onto the NC film and immersing the NC film in the solution, and the amount of the solution applied to the NC film can be determined as appropriate.

[0059] The drying may be done naturally, or it may be done using a device that allows adjustment of temperature, airflow speed, etc., such as a constant temperature bath. The drying temperature is not limited, but is exemplified as 25 to 80°C, preferably 25 to 70°C, and more preferably 30 to 50°C.

[0060] In this way, an NC film on which the LFC-fused PpL single domain of the present disclosure is immobilized can be manufactured. In the NC film, the LFC-fused PpL single domain of the present disclosure is, simply put, immobilized on the NC film via the LFC constituting the LFC-fused PpL single domain, and can be said to be directly immobilized (adsorbed) by the adsorption characteristics of the LFC to the NC film.

[0061] Furthermore, in this disclosure, the denatured LFC-fusion PpL single domain can also be refolded on an NC membrane by contacting the NC membrane, on which the denatured LFC-fusion PpL single domain is immobilized, with a solution that does not contain a denaturing agent (such as a refolding solution or buffer). As mentioned above, in protein refolding, refolding is performed using a refolding solution after solubilization, and a buffer is generally used as the refolding solution. Typically, this buffer does not contain a denaturing agent (such as guanidine hydrochloride or urea used during denaturation and solubilization). The solution that does not contain a denaturing agent may be a solution containing an antibody or the like for binding to the LFC-fusion PpL single domain of this disclosure, as exemplified in the test examples described later.

[0062] Therefore, this disclosure also encompasses an antibody kit comprising the LFC-fused PpL single domain, NC membrane, and antibody of this disclosure. Hereinafter, this antibody kit may be referred to as the antibody kit of this disclosure.

[0063] In the antibody kit of this disclosure, the LFC-fused PpL single domain and the NC membrane are described in the same manner as described above, and the LFC-fused PpL single domain may or may not be immobilized on the NC membrane.

[0064] In this disclosure, antibodies are not limited as long as they can bind to a PpL single domain; however, since PpL single domains are known to primarily bind to the κ light chain of immunoglobulins, antibodies having a κ light chain are preferred examples. Examples of antibodies include full-length antibodies (whole antibodies; IgG, IgM, IgE, IgD, IgA, etc.), single-chain antibodies (single-chain Fv; scFv), scAb (single-chain antibody), sdAb ((single-domain antibody), VHH, etc.), Fab, F(ab). ２Examples include the following. Furthermore, monoclonal antibodies and polyclonal antibodies can be selected according to the purpose, and monoclonal antibodies are preferred. The origin of the antibody is also not limited, and examples include those derived from rabbits, humans, mice, rats, pigs, guinea pigs, chickens, camels, and sharks.

[0065] Furthermore, examples of antibodies include, but are not limited to this disclosure, antibodies against viruses and bacteria such as anti-influenza antibodies (type A, type B, etc.), anti-norovirus antibodies, anti-human chorionic gonadotropin antibodies, anti-Helicobacter pylori antibodies, anti-SARS antibodies (CoV-2 antibodies, etc.), anti-E. coli O-157 antibodies, anti-rotavirus antibodies, anti-adenovirus antibodies, anti-cytomegalovirus antibodies, and anti-Legionella antibodies. Examples of antibodies include antibodies against troponin T, troponin I, CK-MB (creatine kinase-MB), myoglobin, LH (luteinizing hormone), and E. coli verotoxin.

[0066] Antibodies may be used individually or in combination of two or more types.

[0067] As described above, according to this disclosure, antibodies can be easily immobilized on an NC membrane via the LFC-fused PpL single domain of this disclosure. In the antibody kit of this disclosure, the antibody may or may not be immobilized on the NC membrane.

[0068] Furthermore, in immunoassays, an antigen targeted by the antibody is further reacted with the antibody supported on the NC membrane to produce an antigen-antibody reaction between the antibody and the antigen, or a substance capable of binding to the antibody is further reacted with the antibody to detect the antigen or target substance through a binding reaction between the antibody and the substance. For this reason, the antibody kit of this disclosure may further contain any components as needed. Examples of any components include antigens, enzymes, fluorescent dyes, fluorescent labels, enzyme labels, metal labels (metal colloids, etc.), substrates, biotin, avidin, streptavidin, secondary antibodies, latex (colored latex, etc.), and other substances capable of directly or indirectly binding to the antibody, as well as refolding solutions, various buffers, etc. Furthermore, the antibody may or may not be modified with the said substances as needed. Furthermore, the antibody kit disclosed herein may include instructions for use, for example, instructions that include a URL or scannable code for a webpage, and instructions for use may be obtainable via the URL or scannable code. In this disclosure, detection also includes measurement.

[0069] Thus, the LFC-fused PpL single domain of this disclosure, the NC membrane immobilized with the LFC-fused PpL single domain of this disclosure, and the antibody kit of this disclosure are useful in the detection of target substances such as antibodies, antigens, and enzymes using immunoassays, as well as in immunological tests.

[0070] Furthermore, as mentioned above, since the LFC-fused PpL single domain of this disclosure can recognize (bind) antibodies, for example, by contacting a complex containing an antibody and an antigen obtained separately by an antibody-antigen reaction with the LFC-fused PpL single domain of this disclosure immobilized on an NC membrane, the antigen (complex) can be easily supported on the NC membrane through binding between the LFC-fused PpL single domain of this disclosure and the antibody. In this way, by using the LFC-fused PpL single domain of this disclosure, target substances such as antigens can be easily detected. Moreover, according to this disclosure, since it is not essential to pre-support the antibody on the NC membrane, antibody detachment due to washing of the NC membrane can also be suppressed.

[0071] Furthermore, as described above, the LFC-fused PpL single domain of this disclosure can be refolded well on the NC membrane even when the LFC-fused PpL single domain is immobilized on the NC membrane before refolding. Therefore, the NC membrane can be stored for a long period of time after the LFC-fused PpL single domain of this disclosure is immobilized on the NC membrane before refolding and dried. By applying a solution containing antibodies or the like (dropping, immersion, etc.) to the dried NC membrane at the time of use, refolding can be performed and the membrane can be used for the detection of the target substance.

[0072] Furthermore, while proteins are susceptible to functional degradation due to heat denaturation, the LFC-fused PpL single domain of this disclosure, as shown in Test Example 6 below, possesses heat resistance and is therefore useful in suppressing heat denaturation, thus offering ease of use. In addition, as mentioned above, the LFC-fused PpL single domain of this disclosure can be immobilized on an NC membrane due to its adsorption properties, and as mentioned above, it can be easily produced. For this reason, the membrane on which the LFC-fused PpL single domain of this disclosure is immobilized is not limited to an NC membrane, but may be any membrane usable for immunoassays. The immobilization can be carried out according to immobilization methods known in the art.

[0073] The various characteristics (properties, structure, function, etc.) described for each embodiment in this disclosure may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein.

[0074] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0075] Test Example 1 <Test Procedure> 1-1) Production of LFC-fused PpL single domains Fusion proteins (Examples 1-3, Comparative Example 1, Reference Example 1) were produced. Schematic diagrams of each fusion protein of Examples 1-3, Comparative Example 1, and Reference Example 1 are shown in Figure 1.

[0076] In Examples 1 and 2, the C lobe (LFC) of lactoferrin encoded by the amino acid sequences represented by SEQ ID NOs: 1 and 2, respectively, was used. SEQ ID NOs: 2 represents an amino acid sequence in which 17 cysteine ​​molecules are replaced with serine molecules in the amino acid sequence represented by SEQ ID NOs: 1. In Example 3, lactoferrin (LF) encoded by the amino acid sequence represented by SEQ ID NOs: 12 was used.

[0077] In Comparative Example 1, the N-lobe (LFN) of lactoferrin encoded by the amino acid sequence represented by Sequence ID No. 13 was used.

[0078] In Reference Example 1, concanavalin A (ConA), encoded by the amino acid sequence represented by Sequence ID No. 14, was used. ConA was used as a reference example because, like LF, it is a protein that can adsorb to nitrocellulose (NC) membranes.

[0079] In all of Examples 1-3, Comparative Example 1, and Reference Example 1, a PpL single domain encoded by the amino acid sequence represented by Sequence ID No. 3 was used. Since the amino acid sequence represented by Sequence ID No. 3 encodes the protein L C1 domain (PpL1) as a PpL single domain, it will be referred to as "PpL1" below.

[0080] The fusion protein was constructed by linking the following: • Example 1: PpL1-LFC (SEQ ID NO: 15, Figure 15) The amino acid sequence encoding LFC (SEQ ID NO: 1) was linked to the C-terminus of the amino acid sequence encoding PpL1 (SEQ ID NO: 3) via a linker sequence (AAA).

[0081] Example 2: PpL1-LFCS (SEQ ID NO: 16, Figure 15) The amino acid sequence encoding LFC (SEQ ID NO: 2) was linked to the C-terminus of the amino acid sequence encoding PpL1 via a linker sequence (same as in Example 1).

[0082] Example 3: PpL1-LF The amino acid sequence encoding LF (SEQ ID NO: 12) was linked to the C-terminus of the amino acid sequence encoding PpL1 via a linker sequence (same as in Example 1).

[0083] Comparative Example 1: PpL1-LFN The amino acid sequence encoding LFN (SEQ ID NO: 13) was linked to the C-terminus of the amino acid sequence encoding PpL1 via a linker sequence (the same as in Example 1).

[0084] • Reference Example 1: PpL1-ConA The amino acid sequence encoding ConA (SEQ ID NO: 14) was concatenated to the C-terminus of the amino acid sequence encoding PpL1 via a linker sequence (same as in Example 1).

[0085] 1-2) Production and Purification of Each Fusion Protein Each fusion protein was produced and purified using E. coli according to the conventionally known procedure described below, and various treatments and measurements were performed. The vector (vector for fusion protein expression) was synthesized by contract.

[0086] [Agar Culture] (1) 50 μL of Rosetta2(DE3) Competent Cell was thawed on ice, and 1 μL of each fusion protein expression vector solution was added according to general procedure, and incubated on ice for 15 min. (2) The solution obtained in (1) was heat-shocked at 42°C for 45 sec using a block incubator and immediately cooled on ice. (3) The solution obtained in (2) was plated onto LB agar medium (Amp: final concentration 50 μg / mL, Cm: final concentration 34 μg / mL) and incubated overnight at 37°C.

[0087] [Pre-culture] (1) Add 10 mL of autoclaved 2×YT medium (Amp: final concentration 50 μg / mL, Cm: final concentration 34 μg / mL) to a 50 mL Falcon tube. (2) Inoculate the single colonies on the agar plate obtained in (3) of [Agar culture] above, and culture overnight at 30°C and 200 rpm to obtain the pre-culture solution.

[0088] [Main Culture] (1) 50 mL of filter-sterilized Overnight Express medium (Amp: final concentration 50 μg / mL, Cm: final concentration 34 μg / mL) was added to an autoclaved 500 mL baffled flask. (2) The aforementioned pre-culture solution was OD 600(3) The culture solution was added to a ratio of 0.1 and incubated at 30°C, 200 rpm, and 24 hours. (4) The obtained culture solution was collected in a 50 mL Falcon tube and centrifuged at 4°C, 13420 g, and 20 min to separate and collect the culture supernatant and bacterial cells.

[0089] [Cell Disruption] (1) 5 mL of 1% triton x-100-PBS (PBS containing 1% Triton X-100, pH 7.2) was added to the cells obtained in (3) of [Main Culture] (50 mL Culture), vortexed, and then sonicated under the conditions Output 3, Duty 30, 10 min × 3 while cooling with ice. (2) The cells were centrifuged at 4°C, 13420 g, 20 min, and the supernatant was collected as the intracellular soluble fraction.

[0090] [Acquisition of Insoluble Fraction] (1) Add 1×PBS (pH 7.2) to the precipitate (bacterial cells) obtained in (2) of [Bacterial Cell Disruption], vortex, and centrifuge at 4°C, 13420g, and 20min to wash. This was repeated twice. (2) Add 1×PBS containing 8 M Urea, centrifuge at 4°C, 13420g, and 20min, and collect the supernatant as the insoluble fraction within the bacterial cells.

[0091] [Purification] (1) An open column packed with 1 mL of Ni-NTA agarose was supplied with Milli Q 5 CV (Column volume) x 3 times, and binding buffer (20 mM Imidazole, 8 M Urea-2 x PBS, pH 8.0) 5 CV x 3 times to equilibrate the column. (2) 5 mL of intracellular insoluble fraction was supplied to the column and the FT (pass-through fraction) was collected. (3) Subsequently, binding buffer 5 CV x 5 times was added and Wash 1-5 was collected. (4) Elution buffer (250 mM Imidazole, 8 M Urea-2 x PBS, pH 8.0) 1 CV x 5 times was added and Elutions 1-5 were collected. (5) A was collected using Nanodrop. 280 The absorption peak was measured, and the fractions in which the absorption peak was confirmed were collected and dialyzed overnight at 4°C in 8 M Urea-1 × PBS, pH 8.0.

[0092] [Refolding by Dialysis] (Figure 2) (1) The fusion protein dissolved in 8M Urea-PBS recovered in the [Purification] step above (before refolding (denatured state), unreduced, this was designated as Sample 1) was placed in an 18 / 32 cellulose tube (dialysis membrane) and dialyzed for 2 nights in 2 L of 1 × PBS (pH 7.2) (solution temperature 4°C). (2) After dialysis, the solution in the dialysis membrane was centrifuged at 4°C, 10,000 g, and 15 min to remove aggregates and collect the supernatant to obtain the fusion protein after refolding (unreduced) (this was designated as Sample 2).

[0093] [Protein Quantification] BIO-RAD DC for protein quantification TM A protein assay kit was used. The concentration of the fusion protein was quantified and the refolding rate was calculated according to the following microassay procedure. Microassay procedure (1) 20 μL of Protein Assay Reagent S was added to 1 ml of Protein Assay Reagent. (2) 25 μL of the solution from (1) was added to each of the 50 μL samples (the solution before centrifugation in the dialysis membrane and the supernatant after centrifugation in (2) of [Refolding by Dialysis] above) and 0-200 μg / ml BSA solutions. (3) 200 μL of Protein Assay Reagent B was added to (2) and incubated at 25°C for 15 min. (4) Absorbance at a wavelength of 750 nm was measured using a microplate. (5) A calibration curve was created from the measurement results of the BSA solution and the protein (fusion protein) concentration was quantified. (6) The protein concentration of the supernatant after centrifugation relative to the total protein concentration before centrifugation was calculated as the refolding rate (%).

[0094] [Reduction of Fusion Protein] (1) The fusion protein (before refolding (denatured state), unreduced) was mixed with 2×SDS Sample buffer containing 1 mM DTT in a 1:1 ratio and incubated at 98°C for 10 minutes to obtain the reduced fusion protein before refolding (this was designated as Sample 3). (2) The fusion protein (after refolding, unreduced) was also mixed with 2×SDS Sample buffer in a 1:1 ratio and incubated at 98°C for 10 minutes to obtain the reduced fusion protein after refolding (this was designated as Sample 4).

[0095] LFC has a thiol group, and during this experiment, it was found that both LFC and LF readily form a complex through thiol bonding by this thiol group (cysteine), that is, readily form the complex of the fusion protein (for example, lane 2 of "PpL1-LFC" in the electrophoresis results in Figure 3). Furthermore, it was found that the thiol bond forming the complex is cleaved by the reduction treatment, thereby obtaining monomers of the fusion protein (for example, lane 3 of "PpL1-LFC" in the electrophoresis results in Figure 3). The reduction treatment is a process that converts the fusion protein having a complex structure into a monomeric fusion protein.

[0096] [SDS-PAGE] (1) 50 μL of APS and 5 μL of TEMED were added to a 12% separation gel solution, poured onto a glass plate, and 200 μL of butanol was added to remove bubbles. The mixture was incubated at room temperature (25°C) for 40 minutes until the 12% separation gel solution polymerized. (2) After polymerization, the added butanol was removed, and 25 μL of APS and 5 μL of TEMED were added to a 4% stacking gel solution. The mixture was poured up to the top of the glass plate, a comb was inserted to prevent air bubbles, and the mixture was incubated at room temperature for 40 minutes until the 4% stacking gel solution polymerized. (3) 5 μL (15-well comb) of the molecular weight marker and samples 2-3 were applied to the wells, and electrophoresis was performed in 1× running buffer under conditions of 200 V, 2.00 A, and V constant. (4) After electrophoresis was completed, the gel was peeled off the glass plate. (5) The gel was washed with deionized water and incubated in approximately 30 mL of fixation solution at room temperature for 30 minutes. (6) The gel was washed with deionized water and incubated in approximately 30 mL of CBB staining solution at room temperature for 1 hour. (7) The gel was washed with deionized water and incubated overnight at room temperature in approximately 30 mL of decolorizing solution. (8) The gel after staining and decolorization was imaged with a Typhoon FLA 9000. Lane 1 shows the results for sample 3, Lane 2 shows the results for sample 2, and Lane 3 shows the results for sample 4.

[0097] 1-3) Molecular weight measurement of fusion proteins The molecular weight of the fusion proteins was calculated by inputting the amino acid sequence using the Expasy protparam tool (Swiss Institute of Bioinformatics (SIB); https: / / web.expasy.org / protparam / ), a tool for analyzing publicly known proteins, according to the instructions.

[0098] <Results> The results are shown in Figure 3. As shown in Figure 3, the refolding rates were high in Example 1 (PpL1-LFC) and Example 2 (PpL1-LFC-S), at 107.2% and 99.1%, respectively. Although inferior to Examples 1 and 2, Example 3 (PpL1-LF), which included LFN along with LFC, also had a refolding rate of 50.3%. On the other hand, in Comparative Example 1 (PpL1-LFN), where the LFC was not linked but LFN was linked, the refolding rate was low, at only 20.4%.

[0099] These findings indicate that LFC-fused PpL single domains, which are formed by fusing a PpL single domain with an LFC, exhibit higher refolding efficiency than LFN-fused PpL single domains in conventional protein expression procedures using E. coli, and therefore can be produced with greater efficiency.

[0100] Furthermore, the electrophoresis results shown in Figure 3 revealed that LFCs readily form complexes due to thiol bonding by the thiol group (cysteine) present in LFCs, and therefore readily form fusion protein complexes (for example, lane 2 of PpL1-LFC in Figure 3). In PpL1-LFCS, the formation of this complex was suppressed by reducing the number of thiol groups in LFCs by substituting cysteine ​​with serine (for example, lane 2 of PpL1-LFCS in Figure 3). From this, it was found that mutations that reduce the number of thiol groups in LFCs are preferable from the viewpoint of efficiently obtaining monomeric fusion proteins without reduction treatment. Moreover, since this mutation suppresses complex formation by disulfide bonds, and as a result can be efficiently produced as monomeric fusion proteins, it can be said that it has the advantages of improving the uniformity of the resulting molecules and eliminating the need for low molecular weight treatment.

[0101] Test Example 2 The fusion protein produced in Test Example 1 (reduced after refolding) was brought into contact with a nitrocellulose (NC) membrane to adsorb (immobilize) the fusion protein onto the NC membrane, and the antibody binding activity of the fusion protein was evaluated. Since the PpL single domain constituting the fusion protein is known to have binding activity to human IgG antibodies, human IgG antibodies were used as the antibody in this test example. PpL1-His was used as a control that does not adsorb to the NC membrane. Specifically, the evaluation was performed according to the following procedure.

[0102] <Test Procedure (Dot Blot Assay)> (Figure 4) (1) The fusion protein was prepared to 1.0 μM using 1 × PBS (pH 7.2), and 2 μL was dropped onto an NC membrane (product name Hi-Flow Plus HFC13504 2.5 cm, manufactured by Merck KGaA) and air-dried (room temperature). (2) The NC membrane was immersed in 2% BSA-TBS (TBS containing 2% BSA (Tris-buffered saline, pH 8.0)) and incubated at room temperature for 1 hour to block it. (3) The NC membrane blocked in (2) above was washed three times with TBST (TBS containing 0.1% Tween20, pH 8.0), immersed in 1 μg / mL Bt-Human IgG (biotin-labeled human IgG) in 0.2% BSA-TBST (TBST containing 0.2% BSA), and incubated at room temperature for 1 hour. (4) Next, the NC membrane was washed three times with 0.1% TBST, immersed in SA-AP (streptavidin-conjugated alkaline phosphatase) diluted 5000 times with 0.2% BSA-TBST, and incubated at room temperature for 1 hour. (5) Next, the NC membrane was washed three times with 0.1% TBST, 1 mL of BCIP / NBT (manufactured by Nacalai Tesque Co., Ltd.) was added, and incubated at room temperature for 10 minutes. (6) Next, the NC membrane was washed three times with 0.1% TBST, air-dried, placed on the measurement stage, and the signal was scanned at room temperature (25°C) using a scanner (GT-X830, manufactured by Seiko Epson Corporation) according to the instructions for use, and the antibody binding activity value was obtained based on the signal intensity.

[0103] <Results> The results are shown in Figure 4. As shown in Figure 4, compared to Comparative Example 1 (PpL1-LFN), good antibody binding activity was observed in Example 1 (PpL1-LFC), Example 2 (PpL1-LFCS), and Example 3 (PpL1-LF). In particular, high antibody binding activity was observed in Example 1 (PpL1-LFC).

[0104] Here, the antibody binding activity ratio obtained by dividing the antibody binding activity value in Comparative Example 1 by the antibody binding activity value in Test Example 1 was 0.63. Also, the antibody binding activity ratio obtained by dividing the antibody binding activity value in Example 2 by the antibody binding activity value in Example 1 was 0.70. Furthermore, the antibody binding activity ratio obtained by dividing the antibody binding activity value in Example 3 by the antibody binding activity value in Example 1 was 0.88.

[0105] Higher antibody binding activity was observed in the LFC-fused PpL single domain compared to when PpL1-His (control) was in contact with the NC membrane. This confirmed that the LFC-fused PpL single domain is directly immobilized (adsorbed) to the NC membrane due to the adsorption properties of the LFCs that constitute the LFC-fused PpL single domain. Furthermore, this confirmed that antibody binding activity based on the PpL single domain is exhibited even when the PpL single domain is immobilized on the NC membrane while fused with LFCs. In particular, according to Example 1, even higher antibody binding activity was obtained when immobilized on the NC membrane, indicating that antibody binding activity and, furthermore, the detection sensitivity of signals based on antibody binding activity can be further improved.

[0106] Test Example 3: The fusion protein prepared in Test Example 1 (denatured state, reduced state, Sample 3) was adsorbed (immobilized) onto an NC membrane, and then refolded while immobilized on the NC membrane. Antibody binding activity was then evaluated. The same human IgG antibody as in Test Example 2 was used. Specifically, the evaluation was performed according to the following procedure.

[0107] <Test Procedure (Solid-Phase Refolding and Antibody Binding Activity Evaluation)> (Figure 5) (1) The denatured fusion protein (Sample 3) was prepared to 50 μg / mL in 1×PBS in the same manner as described above, 2 μL was dropped onto the NC membrane, and air-dried in the same manner as in Test Example 2. (2) The NC membrane was immersed in 2% BSA-TBS and incubated for 1 hour to block it. (3) In the same manner as in Test Example 2, the NC membrane blocked in (2) was washed three times with TBST, immersed in 1 μg / mL Bt-Human IgG in 0.2% BSA-TBST and incubated for 1 hour, then (4) to (6) were performed in the same manner as in Test Example 2, and the signal was scanned.

[0108] <Results> The results are shown in Figure 5. As shown in Figure 5, compared to Reference Example 1 (PpL1-ConA), good antibody binding activity was observed in Example 1 (PpL1-LFC), Example 2 (PpL1-LFCS), and Example 3 (PpL1-LF). In particular, high antibody binding activity was observed in Example 1 (PpL1-LFC).

[0109] From this, it was found that, unlike when using ConA, the fusion protein was well refolded on the NC membrane in Examples 1 to 3. In particular, in Examples 1 to 3, it was found that good refolding was possible even when the fusion protein before refolding was immobilized on the NC membrane and refolding was performed while it was immobilized on the NC membrane, and good antibody binding activity was obtained. Furthermore, it was found that with the PpL single domain of Example 1, even when the fusion protein before refolding was immobilized on the NC membrane and refolding was performed while it was immobilized on the NC membrane, antibody binding activity and, furthermore, the detection sensitivity of signals based on antibody binding activity could be improved.

[0110] Test Example 4 <Test Procedure> An LCF-fused PpL single domain was created in the same manner as in Test Example 1, except that the PpL single domain and LFC were linked in the order shown in Figure 6 (Examples 1 and 4-6). In Figure 6, the PpL1-LFC is the same as in Example 1 of Test Example 1. Example 4 shows the case where three PpL1 and one LFC are linked, and Examples 5 and 6 show the cases where six PpL1 and one LFC are fused. Examples 5 and 6 are examples of LCF-fused PpL single domains in which three PpL1 single domains are linked to both the N-terminus and C-terminus of the LFC via a linker (the same as in Example 1). In Example 6, seven or eight lysine (K) molecules are replaced with arginine (R) molecules in the PpL1 that constitutes Example 5. The amino acid sequences of each fused PpL single domain in Examples 4-6 are shown in Figures 16-17.

[0111] For the LCF-fused PpL single domains of Examples 1, 4, and 6, the refolding rate was calculated using the same procedure as in Test Example 1, except that in step (3) of the [purification] of Test Example 1, 5 CV of binding buffer was added three times and Washes 1-3 were collected. This was followed by [agar culture] and [SDS-PAGE]. The molecular weight of the fusion protein was also measured in the same manner as in Test Example 1.

[0112] <Results> The results are shown in Figure 7. As shown in Figure 7, the refolding rate was high for all LFC-fused PpL1 single domains in Examples 1, 4, and 6. From these results, it was found that LFC-fused PpL1 single domains can be produced with high efficiency using the conventional protein expression procedure using E. coli, regardless of the number of PpL1 single domains constituting the LFC-fused PpL1 single domain.

[0113] In Test Example 5, the LFC-fused PpL single domains were adsorbed (immobilized) onto an NC membrane in the same manner as in Test Example 2 (liquid-phase refolding) and Test Example 3 (solid-phase refolding), except that the LFC-fused PpL1 single domains from Examples 1 and 4-6 were used, and the antibody binding activity was evaluated. Specifically, the evaluation was performed according to the following procedure.

[0114] <Test Procedure> (1) The fusion protein after refolding or before refolding (denatured state) was prepared to 0.4 μM in 1 × PBS (pH 7.2), 2 μL of each was added dropwise to the NC membrane, and air-dried in the same manner as described above. (2) The NC membrane air-dried in (1) was immersed in the following solution and incubated for 30 minutes: 200 μL of a mixed solution of Bt-Human IgG (final concentration 1 μg / mL) and SA-AP (final concentration 5000-fold dilution). (3) Only the NC membrane portion was cut, washed three times with 0.1% TBST, 1 mL of BCIP / NBT was added, and incubated for 10 minutes. (4) The NC membrane was then washed three times with 0.1% TBST, air-dried, and the signal was scanned in the same manner as described above, following the usual procedure.

[0115] <Results> The results are shown in Figure 8. As shown in Figure 8, antibody binding activity was observed in all examples in both liquid-phase and solid-phase refolding. In particular, in solid-phase refolding, an improvement in the refolding rate was observed with increasing PpL single domains. In liquid-phase refolding, the antibody binding activity value when using Example 5 was 45.3, and the liquid antibody binding activity value when using Example 6 was 58.6. The value obtained by dividing the former value by the latter value (antibody binding activity ratio) was 1.29.

[0116] These results confirm that the LFC-fused PpL single domain is directly immobilized on the NC membrane due to the adsorption properties of the LFCs that constitute the LFC-fused PpL single domain. Furthermore, it was confirmed that antibody binding activity based on the PpL single domain is exhibited even when the PpL single domain is immobilized on the NC membrane while fused with LFCs. In addition, it was found that whether the PpL single domain is immobilized on the NC membrane after refolding or immobilized on the NC membrane before refolding and then refolded, the LFC-fused PpL single domain can further improve antibody binding activity and, moreover, the detection sensitivity of signals based on antibody binding activity.

[0117] In Test Example 6, the thermal stability of LFC-fused PpL1 single-domain immobilized NC films was verified by using the LFC-fused PpL1 single-domains from Examples 1 and 4-6, immobilizing them on NC films after refolding, and then performing heat treatment. Specifically, the evaluation was performed according to the following procedure.

[0118] <Test Procedure (Lateral Flow)> (Figure 9) (1) The LFC-fused PpL1 single domains of Examples 1 and 4-6 after refolding were prepared to 0.4 μM in the same manner as in Test Example 5, 2 μL was dropped onto the NC film, and it was air-dried. (2) The air-dried NC film was incubated at 37°C for 1 week (heat treatment 1) or at 80°C for 1 hour (heat treatment 2). (3) The NC films that underwent heat treatment (heat treatment 1 or 2) in (2) were incubated in the same procedure as in steps (2) to (5) of Test Example 5, and the signal was scanned. Similarly, the NC films that were not heat-treated in (2) were incubated in the same procedure as in steps (2) to (5) of Test Example 5, and the signal was scanned.

[0119] <Results> The results are shown in Figure 9. In Figure 9, "1" shows the result without heat treatment, "2" shows the result after heat treatment at 80°C for 1 hour, and "3" shows the result after heat treatment at 37°C for 1 week. From Figure 9, a similar level of signal was observed regardless of whether heat treatment was performed or not. From this, it was confirmed that antibody binding activity of the LFC-fused PpL1 single domain can be obtained even after such heat treatment. Furthermore, from this, it can be said that the LFC-fused PpL1 single domain has heat resistance.

[0120] In Test Example 7, the LFC-fused PpL1 single domains from Examples 1, 4, and 6 were immobilized on NC membranes before or after refolding, and the concentration-dependent antibody binding activity of the LFC-fused PpL1 single domains was verified. Specifically, the evaluation was performed according to the following procedure.

[0121] <Test Procedure (Dot Blot Assay)> (Figure 10) (1) The LFC-fused PpL1 single domains of Examples 1, 4, and 6 before or after refolding were prepared in 1×PBS (pH 7.2) at a concentration of 10 to 1000 μg / mL, respectively, and 2 μL was dropped onto each NC membrane and air-dried. (2) The samples were incubated in the same procedure as steps (2) to (6) of Test Example 2, and the signal was scanned.

[0122] <Results> The results are shown in Figure 10. As shown in Figure 10, it was confirmed that antibody binding activity tends to be obtained in a concentration-dependent manner for LFC-fused PpL1 single domains in both liquid-phase and solid-phase refolding.

[0123] Test Example 8: The antibody binding activity of LFC-fused PpL1 single domains immobilized on NC membranes under various pH conditions was evaluated. Specifically, the evaluation was performed according to the following procedure.

[0124] <Test Procedure (Dot Blot Assay)> (Figure 11) (1) The LFC-fused PpL1 single domain from Example 6 (after refolding) and each pH buffer shown in Figure 11 were mixed in a volume ratio of 4:1 to prepare a final concentration of 200 μg / mL of LFC-fused PpL1 single domain, and 2 μL was spotted and air-dried. (2) The sample was incubated in the same procedure as in steps (2) to (6) of Test Example 3, and the signal was scanned.

[0125] <Results> The results are shown in Figure 11. As shown in Figure 11, good antibody binding activity was observed when using any of the buffers with pH 1 to 14. In particular, higher antibody binding activity was observed when using buffers with pH 11 and 13.

[0126] Test Example 9: The antibody binding activity of LFC-fused PpL1 single domains immobilized on NC membranes under various pH conditions was evaluated. Specifically, the evaluation was performed according to the following procedure.

[0127] <Test Procedure (Lateral Flow)> (Figure 12) (1) The LFC-fused PpL1 single domain from Example 6 (after refolding) and each pH buffer shown in Figure 12 were mixed in a volume ratio of 4:1 to prepare a final concentration of 200 μg / mL of LFC-fused PpL1 single domain, and 2 μL was spotted and air-dried. (2) The sample was incubated in the same procedure as in steps (2) to (4) of Test Example 5, and the signal was scanned.

[0128] <Results> The results are shown in Figure 12. As shown in Figure 12, good antibody binding activity was observed in all cases using the buffer. In particular, higher antibody binding activity was observed when using the buffer with a pH of 11-13 compared to when using the buffer with a pH of 7.

[0129] In the 10th example, the antigen-binding ability of the antibody (C2R-v1) was evaluated using both indirect and direct methods, following the principle shown in Figure 13. Specifically, the evaluation was performed according to the following procedure: (1) 2 μL of the LFC-fused PpL1 single domain from Example 6 (after refolding, buffer pH 13) was spotted onto an NC membrane (strip) and air-dried, except that it was prepared to 500 μg / mL (5 μM) (Indirect). Separately, 2 μL of C2R-v1 (buffer pH 7) prepared to 50 μg / mL or 5 μM was spotted onto an NC membrane and air-dried (Direct). (2) Each strip was immersed in the following solution (total 200 μL, pH 7) and incubated for 30 minutes: Indirect: Mixed solution of C2R-v1 final concentration 0.5 μg / mL, Bt-CRP (antigen for C2R-v1) final concentration 1-0 μg / mL, and SA-AP final concentration diluted 5000 times. Direct: Mixed solution of Bt-CRP final concentration 1-0 μg / mL, and SA-AP final concentration diluted 5000 times. (3) The NC membrane was cut and washed three times with 0.1% TBST, 1 mL of BCIP / NBT was added and incubated for 10 minutes. (4) After incubation, the strips were washed three times with 0.1% TBST, air-dried, and scanned.

[0130] <Results> The results are shown in Figure 14. As shown in Figure 14, compared to when the antibody was directly immobilized on the NC membrane, higher antigen binding was observed when the antibody was indirectly immobilized on the NC membrane via the LFC-fused PpL1 single domain.

Claims

1. An LFC-fused PpL single domain in which the C lobe (LFC) of lactoferrin and the protein L single domain (PpL single domain) are linked directly or via a linker.

2. The LFC-fused PpL single domain according to claim 1, wherein each LFC contains 1 to 10 PpL single domains.

3. The LFC-fused PpL single domain according to claim 1, wherein the LFC consists of the amino acid sequence described in (1-1) or (1-2) below: (1-1) The amino acid sequence represented by Sequence ID No. 1 (1-2) The amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence described in (1-1).

4. A nitrocellulose membrane on which an LFC-fused PpL single domain as described in any one of claims 1 to 3 is immobilized.

5. An antibody kit comprising an LFC-fused PpL single domain as described in any one of claims 1 to 3, a nitrocellulose membrane, and an antibody.

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