Fucose-binding protein with improved thermal stability and method for producing same

Abstract

To provide a fucose-binding protein having binding affinity with any carbohydrate chain including a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3) GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4) GalNAc, and having improved heat stability and a method for producing the same.SOLUTION: The present invention discloses a method for producing a fucose-binding protein by replacing a specific amino acid residue with an another amino acid residue in a specific amino acid sequence constituting the fucose-binding protein, represented by a specific sequence, and also using Escherichia coli.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a fucose-binding protein with improved thermostability and a method for producing the same. [Background technology]

[0002] BC2LCN, derived from the N-terminal domain of BC2L-C lectin produced by the Gram-negative bacterium Burkholderia cenocepacia, is a lectin with binding affinity for glycans containing fucose residues. It is known to have high binding affinity for several types of glycans containing fucose residues, including H-type 1 glycans (Fucα1-2Galβ1-3GlcNAc), H-type 3 glycans (Fucα1-2Galβ1-3GalNAc), Lewis Y glycans (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), Lewis b glycans (Fucα1-2Galβ1-3(Fucα1-4)GalNAc), and Lewis X glycans (Galβ1-4(Fucα1-3)GlcNAc) (Non-Patent Document 1). Because of its binding affinity for these glycans, BC2LCN can be used to detect and separate cells and proteins that overexpress these glycans. For example, Non-Patent Document 2, Patent Document 1, and Patent Document 2 disclose a method for detecting undifferentiated cells such as human ES cells and iPS cells using BC2LCN, based on the fact that undifferentiated human ES cells and iPS cells highly express H type 1 glycans (Fucα1-2Galβ1-3GlcNAc) and H type 3 glycans (Fucα1-2Galβ1-3GalNAc). Furthermore, H type 1 glycans and Lewis Y glycans are known to be highly expressed in certain cancer cells (H1: Non-Patent Document 3, Lewis Y: Non-Patent Document 4). Lewis b glycans are blood group antigens on red blood cells, and Helicobacter pylori is known to use Lewis b glycans on host cells for infection (Non-Patent Document 5).

[0003] One known method for improving protein function is to introduce amino acid mutations into the protein using protein engineering techniques to improve the desired function. Patent Document 3 discloses that replacing specific amino acid residues in BC2LCN with other amino acid residues improves its heat stability; however, there is a demand for BC2LCN with even higher heat stability. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 065302 [Patent Document 2] International Publication No. 2013 / 128914 [Patent Document 3] Japanese Patent Publication No. 2020-025535 [Non-patent literature]

[0005] [Non-Patent Document 1] Sulak, O. et al., Structure.2010, 18(1):59-72. [Non-patent document 2] Tateno, H. et al., Stem Cells Transl. Med.2013, 2(4):265-273. [Non-patent document 3] Tang, C. et al., Nat. Biotechnol. 2011, 29(9):829-835. [Non-patent document 4] Westwood JA et al., J. Immunother.2009,32(3):292-301. [Non-Patent Document 5] Hage, N. et al., Sci. Adv.2015,14;1(7):e1500315. Summary of the Invention [Problem to be solved by the invention]

[0006] BC2LCN has high binding affinity to various glycans and is therefore useful for detecting specific cells; however, BC2LCN is heat-labile, and suffers from inactivation and denaturation due to heat during production, storage, and use. Therefore, there is a need for a highly stable BC2LCN that can guarantee long-term binding affinity to glycans. In particular, for industrial production and provision of BC2LCN, there is a need for a BC2LCN that is easy to manufacture, process, and store and has improved stability. The objective of the present invention is to provide a fucose-binding protein with high thermal stability. [Means for solving the problem]

[0007] As a result of extensive research by the present inventors to achieve the above object, it has been found that the amino acid sequence constituting BC2LCN, which consists of 155 amino acid residues as shown in SEQ ID NO: 1, can be substituted by substituting the 83rd threonine residue with one type of amino acid residue selected from a proline residue, an alanine residue, a tyrosine residue, a tryptophan residue, and a serine residue, and / or by substituting the 86th methionine residue with an isoleucine residue or a valine residue, and / or by substituting the 1st proline residue, the 37th lysine residue, the 39th glutamine residue, and the 4 ... The present inventors have found that by substituting one amino acid residue selected from the group consisting of the threonine residue at position 47, the proline residue at position 48, and the serine residue at position 122 with an alanine residue, a fucose-binding protein can be obtained that maintains its binding affinity to any of glycans containing the structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc, while improving its heat stability, thereby completing the present invention.

[0008] That is, the present invention includes the inventions described in [1] to

[11] below. [1] A fucose-binding protein according to any one of (a) to (c) below: (a) A fucose-binding protein containing one or more substitutions in the amino acid sequence shown in SEQ ID NO: 1 with any one of the amino acid sequences (1) to (3) below: (1) Substitution of the threonine residue at position 83 of the amino acid sequence shown in SEQ ID NO: 1 with any amino acid residue other than threonine (2) Substitution of the methionine residue at position 86 of the amino acid sequence shown in SEQ ID NO: 1 with any amino acid residue other than methionine. (3) Substitution of any one amino acid residue other than an alanine residue in the amino acid sequence shown in SEQ ID NO: 1 with an alanine residue (b) The amino acid sequence of the fucose-binding protein described in (a) above includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added at positions 83 and 86 of SEQ ID NO: 1, and in a region not containing an alanine amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, and the fucose-binding protein contains Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or F A fucose-binding protein that has binding affinity for either glycans containing the structure ucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc. (c) A fucose-binding protein having an amino acid sequence having 90% or more homology to the amino acid sequence having one or more substitutions described in (1) to (3) above, and having binding affinity for any of sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc. [2] 2. The fucose-binding protein according to claim 1, wherein in (1), any amino acid residue other than a threonine residue is substituted with one amino acid residue selected from a proline residue, an alanine residue, a tyrosine residue, a tryptophan residue, and a serine residue; and / or in (2), any amino acid residue other than a methionine residue is substituted with an isoleucine residue or a valine residue; and / or in (3), any one amino acid residue substituted with an alanine residue is one amino acid residue selected from a proline residue at position 1, a lysine residue at position 37, a glutamine residue at position 39, a threonine residue at position 46, a proline residue at position 47, or a serine residue at position 122 in the amino acid sequence shown in SEQ ID NO: 1. [3] The fucose-binding protein according to [1] or [2] above, which comprises an amino acid sequence shown in any one of SEQ ID NOs: 2 to 24. [4] The fucose-binding protein according to any one of [1] to [3] above, which has an additional amino acid sequence at the N-terminus and / or C-terminus. [5] The fucose-binding protein according to any one of [1] to [4] above, wherein the amino acid sequence added to the C-terminus is an oligopeptide containing a cysteine ​​residue. [6] The fucose-binding protein according to any one of [1] to [5] above, wherein the amino acid sequence added to the N-terminus is an oligopeptide containing a polyhistidine sequence. [7] A DNA encoding the fucose-binding protein according to any one of [1] to [6] above. [8] An expression vector containing the DNA described in [7] above. [9] A transformant obtained by transforming a host with the expression vector according to [8] above, which is capable of producing the fucose-binding protein according to any one of [1] to [6].

[10] The transformant according to [9] above, wherein the host is Escherichia coli.

[11] A method for producing the fucose-binding protein according to any one of [1] to [6], comprising two steps: a step of producing the fucose-binding protein by culturing the transformant according to [9] or

[10] above; and a step of recovering the fucose-binding protein produced from the obtained culture.

[0009] The present invention will be described in further detail below.

[0010] The fucose-binding protein of the present invention is a lectin that has binding affinity to fucose-containing glycans such as H-type 1 glycans having a structure consisting of Fucα1-2Galβ1-3GlcNAc, H-type 3 glycans having a structure consisting of Fucα1-2Galβ1-3GalNAc, Lewis Y glycans having a structure consisting of Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, and Lewis b glycans having a structure consisting of Fucα1-2Galβ1-3(Fucα1-4)GalNAc. The amino acid sequence of BC2LCN (which is an amino acid sequence consisting of 155 amino acid residues shown in SEQ ID NO: 1 and corresponds to the amino acid sequence from the 2nd to the 156th amino acid residues of the amino acid sequence registered with GenPept under registration number WP_006490828) has been mutated into amino acid residues at specific positions and expressed as a recombinant protein in transformed Escherichia coli. Specifically, the 83rd threonine residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with one type of amino acid residue selected from proline residue, alanine residue, tyrosine residue, tryptophan residue, serine residue, and cysteine ​​residue, and / or the 86th methionine residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an isoleucine residue or a valine residue, and / or one amino acid residue selected from the 1st proline residue, the 37th lysine residue, the 39th glutamine residue, the 46th threonine residue, the 47th proline residue, and the 122nd serine residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an alanine residue, and the resulting protein is expressed as a recombinant protein in transformed Escherichia coli.By substituting the threonine residue at position 83 in the amino acid sequence shown in SEQ ID NO: 1 with a proline residue, an alanine residue, a tyrosine residue, a tryptophan residue, or a serine residue, and / or substituting the methionine residue at position 86 with an isoleucine residue or a valine residue, and / or substituting the proline residue at position 1, the lysine residue at position 37, the glutamine residue at position 39, the threonine residue at position 46, the proline residue at position 47, or the serine residue at position 122 with an alanine residue, the thermal stability of the recombinant protein can be improved while maintaining its binding affinity to sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc. Among these amino acid residue substitutions, substitution of the 83rd threonine residue with a proline residue and the 86th methionine residue with an isoleucine residue or a valine residue is preferred in terms of high thermal stability, and substitution of the 83rd threonine residue with a proline residue, or substitution of the 83rd threonine residue with a tyrosine residue or a serine residue, and substitution of the 86th methionine residue with an isoleucine residue or a valine residue is preferred in terms of significantly improving thermal stability by 10°C or more higher midpoint temperature than the recombinant protein without amino acid substitutions while maintaining binding affinity to all of the four types of sugar chains. As shown in the Examples and Comparative Examples described below, by substituting amino acid residues at specific positions in the amino acid sequence shown in SEQ ID NO: 1 with specific amino acid residues, rather than substituting amino acid residues at any position with other amino acid residues, it is possible to improve thermal stability while maintaining binding affinity to glycans containing structures consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc.

[0011] Specific examples of the fucose-binding protein of the present invention include SEQ ID NO: 2 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a proline residue), SEQ ID NO: 3 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with an alanine residue), SEQ ID NO: 4 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue), SEQ ID NO: 5 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tryptophan residue), SEQ ID NO: 6 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a serine residue), sequence), SEQ ID NO:7 (amino acid sequence in which the 86th methionine residue of SEQ ID NO:1 is substituted with an isoleucine residue), SEQ ID NO:8 (amino acid sequence in which the 86th methionine residue of SEQ ID NO:1 is substituted with a valine residue), SEQ ID NO:9 (amino acid sequence in which the 83rd threonine residue of SEQ ID NO:1 is substituted with a proline residue and the 86th methionine residue of SEQ ID NO:1 is substituted with an isoleucine residue), SEQ ID NO:10 (amino acid sequence in which the 83rd threonine residue of SEQ ID NO:1 is substituted with an alanine residue and the 86th methionine residue of SEQ ID NO:1 is substituted with an isoleucine residue), SEQ ID NO:11 (amino acid sequence in which the 83rd threonine residue of SEQ ID NO:1 is substituted with an alanine residue and the 86th methionine residue of SEQ ID NO:1 is substituted with an isoleucine residue), SEQ ID NO: 12 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tryptophan residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue), SEQ ID NO: 13 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a serine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue), SEQ ID NO: 14 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a proline residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue), 1), SEQ ID NO: 15 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with an alanine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue), SEQ ID NO: 16 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue), SEQ ID NO: 17 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tryptophan residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue), SEQ ID NO: 18 (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a serine residue,Examples of fucose-binding proteins include those comprising the amino acid sequence shown in any one of SEQ ID NO: 1 (the amino acid sequence in which the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue), SEQ ID NO: 19 (the amino acid sequence in which the first proline residue of SEQ ID NO: 1 is substituted with an alanine residue), SEQ ID NO: 20 (the amino acid sequence in which the lysine residue at position 37 of SEQ ID NO: 1 is substituted with an alanine residue), SEQ ID NO: 21 (the amino acid sequence in which the glutamine residue at position 39 of SEQ ID NO: 1 is substituted with an alanine residue), SEQ ID NO: 22 (the amino acid sequence in which the threonine residue at position 46 of SEQ ID NO: 1 is substituted with an alanine residue), SEQ ID NO: 23 (the amino acid sequence in which the proline residue at position 47 of SEQ ID NO: 1 is substituted with an alanine residue), and SEQ ID NO: 24 (the amino acid sequence in which the serine residue at position 122 of SEQ ID NO: 1 is substituted with an alanine residue). Among these fucose-binding proteins, the fucose-binding proteins containing the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 14 are preferred because they have higher thermal stability than recombinant BC2LCNcys containing the amino acid sequence shown in SEQ ID NO: 1, as will be shown in the Examples below. The fucose-binding proteins containing the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18 are preferred because they maintain binding affinity to all of the H-type 1 glycan, H-type 3 glycan, Lewis Y glycan and Lewis b glycan, and have a denaturation midpoint temperature that is 10°C or more higher than that of the recombinant BC2LCNcys, resulting in significantly improved thermal stability.

[0012] The fucose-binding protein of the present invention may be a variant of the amino acid sequence shown in SEQ ID NO: 1 having one or more of the amino acid substitutions listed in (1) to (3) below, as long as it has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc. (1) Substitution of the threonine residue at position 83 of the amino acid sequence shown in SEQ ID NO: 1 with any amino acid residue other than threonine (2) Substitution of the methionine residue at position 86 of the amino acid sequence shown in SEQ ID NO: 1 with any amino acid residue other than methionine. (3) An amino acid sequence in which any one amino acid residue other than an alanine residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an alanine residue. Variant sequences include those amino acid sequences of SEQ ID NO: 1 in which one or more amino acid residues are deleted, substituted, inserted, or added at positions 83 and 86 and in a region not containing an alanine residue. The term "one or several" may vary depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but may mean, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. An example of the amino acid residue substitution is a conservative substitution between amino acids with similar physical and / or chemical properties. Examples of amino acid residues with similar physical and / or chemical properties include amino acid residues with similar side chain properties. Examples of amino acid residues with similar side chain properties are listed below.

[0013] The variant sequence includes the amino acid sequence shown in SEQ ID NO: 1, which has the above (1) and (2). Also included are amino acid sequences that are highly homologous to the amino acid sequences having the amino acid substitutions described in (2) and / or (3). "High homology" may mean, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more homology. "Homology" may mean identity or similarity. In this specification, "identity" refers to the proportion of identical amino acid residues in the amino acid sequences being compared, and "similarity" refers to the proportion of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). Here, examples of amino acid residues with similar side chain properties include amino acid residues with hydrophobic side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Furthermore, examples of amino acids with hydrophilic acidic side chains include aspartic acid and glutamic acid; examples of amino acids with hydrophilic basic side chains include lysine, arginine, and histidine; and examples of amino acids with hydrophilic uncharged side chains include asparagine, glutamine, serine, threonine, cysteine, and tyrosine. Amino acid sequence homology can be determined using alignment programs such as BLAST. For example, amino acid sequence identity may refer to the identity between amino acid sequences calculated using BLASTp, specifically, the identity between amino acid sequences calculated using BLASTp with default parameters. Amino acid sequences with 90% or more homology are preferred, as this will result in high thermal stability of the resulting fucose-binding protein.

[0014] Furthermore, as long as the fucose-binding protein of the present invention has binding affinity for glycans containing the structure Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc, it may have an additional amino acid sequence at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1 that is useful for separating the protein from a solution containing contaminants. Examples of such additional amino acid sequences include oligopeptides containing a polyhistidine sequence, glutathione S-transferase, maltose-binding protein, cellulose-binding domains, myc tags, and FLAG tags. Among these additional amino acid sequences, oligopeptides containing a polyhistidine sequence are preferred because they facilitate purification by nickel chelate affinity chromatography. The number of histidine repeats in the oligopeptide containing the polyhistidine sequence is not particularly limited, but a short histidine repeat makes purification by nickel chelate affinity chromatography difficult, while a long histidine repeat may impair the binding affinity of the fucose-binding protein of the present invention to the sugar chain. Therefore, the number of histidine repeats in the oligopeptide containing the polyhistidine sequence is preferably a repeat sequence consisting of 5 to 15 histidine residues, and more preferably a repeat sequence consisting of 5 to 10 histidine residues. The length of the oligopeptide containing the polyhistidine sequence is not particularly limited as long as it contains the histidine repeat sequence, and is preferably an oligopeptide of 20 or less residues containing a repeat sequence consisting of 5 to 15 histidine residues, and more preferably an oligopeptide of 15 or less residues containing a repeat sequence consisting of 5 to 10 histidine residues.There are no particular limitations on the position at which the oligopeptide containing the polyhistidine sequence is added to the fucose-binding protein; it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus. However, in terms of efficient purification by nickel chelate affinity chromatography, it is preferable that the oligopeptide containing the polyhistidine sequence be added to the N-terminus of the fucose-binding protein.

[0015] Furthermore, the fucose-binding protein of the present invention may have an additional amino acid sequence consisting of an oligopeptide containing cysteine ​​or lysine residues (hereinafter referred to as a carrier immobilization tag) at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, which is useful for immobilizing the fucose-binding protein of the present invention to a carrier such as a chromatographic support. There are no particular limitations on the length of the carrier immobilization tag, as long as the fucose-binding protein of the present invention has binding affinity to sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc and / or Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Fucα1-2Galβ1-3(Fucα1-4)GalNAc. As the tag for carrier immobilization, an oligopeptide consisting of 2 to 10 amino acid residues and containing one or more cysteine ​​residues is preferred, in that it allows for highly selective and efficient immobilization to an insoluble carrier. Specific examples include an oligopeptide consisting of the three amino acid residues "Gly-Gly-Cys," an oligopeptide consisting of the five amino acid residues "Ala-Ser-Gly-Gly-Cys," and an oligopeptide consisting of the seven amino acid residues "Gly-Gly-Gly-Ser-Gly-Gly-Cys." There are no particular restrictions on the position at which the oligopeptide containing one or more cysteine ​​residues is added to the fucose-binding protein, and it may be added to both the N-terminus and C-terminus, or to either the N-terminus or the C-terminus. However, in that it allows for efficient immobilization of the fucose-binding protein to a carrier, it is preferred that the oligopeptide containing one or more cysteine ​​residues be added to the C-terminus of the fucose-binding protein.

[0016] In addition, a signal peptide may be added to the N-terminus of the fucose-binding protein of the present invention to promote efficient expression in a host. When the host is Escherichia coli, examples of the signal peptide include signal peptides that direct protein secretion into the periplasm, such as PelB, DsbA, MalE, and TorT.

[0017] Next, the DNA encoding the fucose-binding protein of the present invention (hereinafter referred to as the DNA of the present invention) and the expression vector containing the DNA of the present invention (hereinafter referred to as the expression vector of the present invention) will be described.

[0018] The DNA of the present invention can be obtained by, for example, using a DNA amplification method such as polymerase chain reaction (PCR) to prepare the DNA by modifying the BC2L-C gene region of the genomic DNA of Burkholderia cenocepacia; by converting the amino acid sequence of recombinant BC2LCN (SEQ ID NO: 1) into a nucleotide sequence, artificially preparing DNA containing that nucleotide sequence, and then further modifying the resulting DNA using a DNA amplification method; or by converting the amino acid sequences set forth in SEQ ID NOs: 2 to 24 into nucleotide sequences, artificially preparing DNA containing that nucleotide sequence. In these methods, when converting an amino acid sequence into a nucleotide sequence, it is preferable to consider the codon usage frequency in the microorganism or cell (host) used to produce the fucose-binding protein of the present invention. For example, when Escherichia coli is used as a host, AGA, AGG, CGG, or CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are rare codons, so it is preferable to select and convert codons other than these codons. Analysis of codon usage can also be performed using public databases (e.g., the Codon Usage Database on the Kazusa DNA Research Institute website, http: / / www.kazusa.or.jp / codon / , accessed February 1, 2018). Furthermore, when constructing the DNA of the present invention, an appropriate restriction enzyme recognition sequence may be introduced without changing the amino acid sequence surrounding the amino acid residue substitution site, simplifying the amino acid residue substitution procedure.

[0019] Specific examples of the DNA of the present invention include DNA consisting of the nucleotide sequence of SEQ ID NO: 25 which encodes the amino acid sequence of SEQ ID NO: 2, DNA consisting of the nucleotide sequence of SEQ ID NO: 26 which encodes the amino acid sequence of SEQ ID NO: 3, DNA consisting of the nucleotide sequence of SEQ ID NO: 27 which encodes the amino acid sequence of SEQ ID NO: 4, DNA consisting of the nucleotide sequence of SEQ ID NO: 28 which encodes the amino acid sequence of SEQ ID NO: 5, DNA consisting of the nucleotide sequence of SEQ ID NO: 29 which encodes the amino acid sequence of SEQ ID NO: 6, DNA consisting of the nucleotide sequence of SEQ ID NO: 30 which encodes the amino acid sequence of SEQ ID NO: 7, DNA consisting of the nucleotide sequence of SEQ ID NO: 31 which encodes the amino acid sequence of SEQ ID NO: 8, DNA consisting of the nucleotide sequence of SEQ ID NO: 32 which encodes the amino acid sequence of SEQ ID NO: 9, DNA consisting of the nucleotide sequence of SEQ ID NO: 33 which encodes the amino acid sequence of SEQ ID NO: 1, DNA consisting of the nucleotide sequence of SEQ ID NO: 34 which encodes the amino acid sequence of SEQ ID NO: 11, DNA consisting of the nucleotide sequence of SEQ ID NO: 35 which encodes the amino acid sequence of SEQ ID NO: 12, and DNA consisting of the nucleotide sequence of SEQ ID NO: 13. Examples of the DNA encoding the amino acid sequence of SEQ ID NO: 36 include DNA encoding the amino acid sequence of SEQ ID NO: 14, DNA encoding the amino acid sequence of SEQ ID NO: 37, DNA encoding the amino acid sequence of SEQ ID NO: 14, DNA encoding the amino acid sequence of SEQ ID NO: 38, DNA encoding the amino acid sequence of SEQ ID NO: 15, DNA encoding the amino acid sequence of SEQ ID NO: 39, DNA encoding the amino acid sequence of SEQ ID NO: 16, DNA encoding the amino acid sequence of SEQ ID NO: 40, DNA encoding the amino acid sequence of SEQ ID NO: 17, DNA encoding the amino acid sequence of SEQ ID NO: 41, DNA encoding the amino acid sequence of SEQ ID NO: 18, DNA encoding the amino acid sequence of SEQ ID NO: 42, DNA encoding the amino acid sequence of SEQ ID NO: 19, DNA encoding the amino acid sequence of SEQ ID NO: 43, DNA encoding the amino acid sequence of SEQ ID NO: 20, DNA encoding the amino acid sequence of SEQ ID NO: 44, DNA encoding the amino acid sequence of SEQ ID NO: 21, DNA encoding the amino acid sequence of SEQ ID NO: 45, DNA encoding the amino acid sequence of SEQ ID NO: 22, DNA encoding the amino acid sequence of SEQ ID NO: 46, and DNA encoding the amino acid sequence of SEQ ID NO: 24.

[0020] To transform a host using the DNA of the present invention, the DNA of the present invention itself may be used for transformation. However, it is preferable to use an expression vector of the present invention, for example, by inserting the DNA of the present invention into a suitable position in a vector based on a bacteriophage, cosmid, or plasmid, which is commonly used for transforming prokaryotic and eukaryotic cells, to thereby obtain the expression vector, which can then be used for transformation, in order to achieve stable transformation. Here, "suitable position" means a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, and the region involved in transferability. Furthermore, when inserting the DNA of the present invention into a vector, it is preferable to insert it into the vector in a state where it is linked to functional DNA, such as a promoter, required for expression.

[0021] The expression vector of the present invention is not particularly limited as long as it is a vector that can stably exist and replicate in a host, and examples thereof include, when Escherichia coli is used as a host, pET vectors, pUC vectors, pTrc vectors, pCDF vectors, and pBBR vectors. Furthermore, examples of promoters used in the present invention when Escherichia coli is used as a host include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and the λPL promoter and λPR promoter of λ phage.

[0022] A transformant capable of producing the fucose-binding protein of the present invention (hereinafter referred to as the transformant of the present invention) can be obtained by transforming a host with the expression vector of the present invention. There are no particular limitations on the host used as the transformant of the present invention; however, Escherichia coli is preferred because it facilitates genetic engineering experiments. Transforming a host with the expression vector of the present invention can be carried out using methods commonly used by those skilled in the art. For example, when Escherichia coli strains such as JM109, BL21(DE3), or W3110 are selected as hosts, methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used. The expression vector of the present invention can be extracted from the transformant of the present invention using an appropriate extraction method or a commercially available kit. For example, when the host is Escherichia coli, alkaline extraction or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (trade name, manufactured by Qiagen) can be used.

[0023] Next, a method for producing the fucose-binding protein of the present invention (hereinafter referred to as the production method of the present invention) will be described. The production method of the present invention comprises two steps: a step of producing the fucose-binding protein of the present invention by culturing the transformant of the present invention (hereinafter referred to as step 1), and a step of recovering the fucose-binding protein of the present invention from the obtained culture (hereinafter referred to as step 2). In this specification, the culture includes not only the cultured transformant cells themselves and cell secretions, but also the medium used for culture.

[0024] In the first step of the production method of the present invention, the transformant of the present invention may be cultured in a medium suitable for its culture. For example, when Escherichia coli is used as the host, it is preferable to use Terrific Broth (TB) medium, Luria-Bertani (LB) medium, or the like supplemented with necessary nutrients. When the expression vector of the present invention contains a drug resistance gene, selective growth of the transformant can be achieved by adding a drug corresponding to the gene to the medium and performing the first step. For example, when the expression vector contains a kanamycin resistance gene, it is preferable to add kanamycin to the medium. The culture temperature may be any temperature generally known for the host used. For example, when the host is Escherichia coli, it is 10°C to 40°C, preferably 20°C to 37°C, and may be appropriately determined taking into consideration the production amount of the fucose-binding protein of the present invention, etc. The pH of the medium may be within a generally known pH range for the host used. For example, when the host is Escherichia coli, the pH range is from 6.8 to 7.4, preferably around 7.0, and may be appropriately determined taking into consideration the amount of the fucose-binding protein of the present invention to be produced, etc.

[0025] When an inducible promoter is introduced into the expression vector of the present invention, expression can be induced by adding an inducer to the medium under conditions that allow good production of the fucose-binding protein of the present invention. A preferred inducer is isopropyl-β-D-thiogalactopyranoside (IPTG), and its concentration ranges from 0.005 to 1.0 mM, preferably from 0.01 to 0.5 mM. Expression induction by adding IPTG can be carried out under conditions generally known for the host used. Even when an inducible promoter is introduced into the expression vector of the present invention, it is not necessary to add an inducer as long as good production of the fucose-binding protein of the present invention is possible.

[0026] In the second step of the production method of the present invention, the fucose-binding protein of the present invention is recovered by a commonly known recovery method from the culture obtained in the first step. For example, when the fucose-binding protein of the present invention is secreted and produced in the culture medium, the cells can be separated by centrifugation and the fucose-binding protein of the present invention can be recovered from the resulting culture supernatant; when the fucose-binding protein of the present invention is expressed intracellularly (including the periplasm in prokaryotes), the cells can be collected by centrifugation, disrupted by adding an enzyme treating agent, a surfactant, or the like, and the fucose-binding protein of the present invention can be recovered from the cell lysate.

[0027] To improve the purity of the fucose-binding protein of the present invention recovered by the production method of the present invention, methods known in the art can be used, such as separation and purification using liquid chromatography. Liquid chromatography preferably includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and more preferably a combination of these chromatographic methods. Furthermore, the purity of the fucose-binding protein of the present invention purified by the above chromatography can be determined using methods known in the art, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography.

[0028] The binding affinity of the fucose-binding protein of the present invention to glycans can be evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, or the like. Surface plasmon resonance will be described as an example. Binding affinity evaluation by surface plasmon resonance can be performed, for example, using a Biacore 8K instrument (GE Healthcare) with a recombinant protein as the analyte and a glycan as the solid phase. A sensor chip with immobilized glycans can be prepared using a streptavidin-coated sensor chip (Sensor Chip SA, GE Healthcare) or a dextran-coated sensor chip (Sensor Chip CM5, GE Healthcare) with streptavidin immobilized in advance, using biotin-labeled glycans. Furthermore, binding affinity evaluation can be performed using the kinetics analysis program provided with the instrument.

[0029] The thermal stability of the fucose-binding protein of the present invention can be evaluated by thermal shift assay or differential scanning calorimetry, and the binding affinity to glycans before and after heat treatment can be evaluated by comparing the binding affinity using enzyme-linked immunosorbent assay or surface plasmon resonance. As examples, thermal shift assay and differential scanning calorimetry are described below. Both thermal shift assay and differential scanning calorimetry involve increasing the temperature of a protein solution at a constant rate and measuring the denaturation midpoint temperature at which half of the protein is denatured. In the thermal shift assay, a fluorescent dye that binds to the hydrophobic surface of the protein, such as SYPRO Orange, is added to the protein solution in advance, and the structural changes of the protein are measured by measuring the fluorescence intensity of the fluorescent dye bound to the hydrophobic surface of the protein whose higher-order structure has been disrupted by heat. On the other hand, differential scanning calorimetry measures the change in heat produced by the thermal denaturation of the protein. [Effects of the Invention]

[0030] The present invention provides fucose-binding proteins that have improved heat stability compared to fucose-binding proteins before amino acid residue substitution and that maintain binding affinity to H-type 1 glycans having a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or H-type 3 glycans having a structure consisting of Fucα1-2Galβ1-3GalNAc and / or Lewis Y glycans having a structure consisting of Fucα1-2Galβ1-4(Fucα1-3)GlcNAc and / or Lewis b glycans having a structure consisting of Fucα1-2Galβ1-3(Fucα1-4)GalNAc, as well as methods for producing them.

[0031] H-type 1 and H-type 3 glycans are known as undifferentiated markers specifically present in undifferentiated cells such as human iPS cells and ES cells. Furthermore, H-type 1 and Lewis Y glycans are known to be highly expressed in certain cancer cells. Lewis b glycans are blood group antigens on red blood cells and are known to be involved in Helicobacter pylori infection. Therefore, because the fucose-binding protein of the present invention has improved thermal stability, it is expected to stably bind to glycans on the surface of specific cells, such as undifferentiated cells and cancer cells, while suppressing functional degradation due to temperature changes. For example, fluorescent labeling of the fucose-binding protein of the present invention enables highly sensitive detection of undifferentiated cells, cancer cells, and other cells. Furthermore, immobilizing the fucose-binding protein of the present invention on a water-insoluble carrier allows the preparation of a cell adsorbent capable of selectively adsorbing specific cells, such as undifferentiated cells and cancer cells. [Example]

[0032] The present invention will be explained in more detail below by giving Preparation Examples, Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these.

[0033] Preparation example 1 (1-1) Construction of expression vector pET-BC2LCNcys and recombinant Escherichia coli BL21(DE3) / pET-BC2LCNcys The expression vector pET-BC2LCNcys is an expression vector for expressing recombinant BC2LCNcys. The amino acid sequence of recombinant BC2LCNcys is SEQ ID NO: 48. Specifically, positions 5 to 10 of SEQ ID NO: 48 correspond to a polyhistidine sequence, positions 15 to 169 correspond to the amino acid sequence of SEQ ID NO: 1 (the amino acid sequence of positions 2 to 156 of GenPept registration number WP_006490828), and positions 170 to 174 correspond to an oligopeptide sequence containing a cysteine ​​residue. The expression vector pET-BC2LCNcys has the same nucleotide sequence as the plasmid pET-BC2LCNcys disclosed in Japanese Patent Application Laid-Open No. 2018-000038 and was prepared using the method disclosed in that publication. Next, Escherichia coli BL21(DE3) was transformed with the expression vector pET-BC2LCNcys to generate recombinant Escherichia coli BL21(DE3) / pET-BC2LCNcys. (1-2) Introduction of a mutation into the threonine residue identified as the 83rd threonine residue of SEQ ID NO: 1 into the recombinant BC2LCNcys Mutation was introduced into the recombinant BC2LCNcys at the threonine residue identified as the 83rd threonine residue in SEQ ID NO: 1. That is, mutation was introduced to replace the 97th threonine residue in the amino acid sequence of the recombinant BC2LCNcys (SEQ ID NO: 48) with another amino acid residue.

[0034] PCR was performed using the expression vector pET-BC2LCNcys described in Preparation Example (1-1) above as a template and the oligonucleotides consisting of the sequences set forth in SEQ ID NO: 49 and SEQ ID NO: 50 as PCR primers according to the method disclosed in JP 2018-000038 A. The PCR primer consisting of the sequence set forth in SEQ ID NO: 49 had the degenerate sequence NNB (N = A, C, G, or T, B = C, G, or T), and was designed so that the 97th threonine residue in SEQ ID NO: 48 (corresponding to the 83rd threonine residue in SEQ ID NO: 1) was randomly substituted with other amino acid residues. The resulting PCR product was digested with the restriction enzymes KpnI and XhoI and ligated with the expression vector pET-BC2LCNcys described in (1-1) above that had been similarly digested with the restriction enzymes. This ligation product was used to transform Escherichia coli BL21(DE3) to obtain multiple transformants. The expression vector was extracted from each transformant and the nucleotide sequence was analyzed. As a result, 19 types of expression vectors and transformants harboring them were obtained, as shown in Table 1. The recombinant proteins in Table 1 were produced as described in Preparation Example (1-3) below.

[0035] [Table 1]

[0036] (1-3) Production of recombinant proteins The recombinant Escherichia coli BL21(DE3) / pET-BC2LCNcys prepared in Example 1-1 and the 19 transformants (Table 1) prepared in Example 1-2 were inoculated into LB medium (10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl) supplemented with 30 μg / mL kanamycin, and precultured overnight with shaking at 37°C. The precultures were then inoculated into 1 L of TB medium (24 g / L yeast extract, 12 g / L tryptone, 9.4 g / L KHPO, 2.2 g / L KHPO, and 4 mL / L glycerol) supplemented with 30 μg / mL kanamycin, and cultured with shaking at 37°C. When the turbidity (OD600) of the culture medium reached approximately 0.6, the culture temperature was changed to 30°C and cultured overnight to express each recombinant protein (recombinant BC2LCNcys and the 19 recombinant proteins listed in Table 1). Soluble protein extracts were collected from each bacterial cell using ultrasonic disruption or a BugBuster Protein extraction kit (Merck). The recombinant proteins were purified from the soluble protein extracts by nickel chelate affinity chromatography and then transferred to storage buffer (50 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 20% glycerol) using an ultrafiltration filter.

[0037] (1-4) Evaluation of glycan binding affinity of recombinant proteins The binding affinity of the recombinant proteins produced in (1-3) above (recombinant BC2LCNcys and the recombinant proteins in Table 1) to H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans was evaluated by surface plasmon resonance. Specifically, kinetic analysis was performed using a Biacore 8K (GE Healthcare) with each recombinant protein as the analyte and H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, or Lewis b glycans as the solid phase. The sensor chip used was a dextran-coated Sensor Chip CM5 (GE Healthcare). Streptavidin was immobilized to the dextran by the amine coupling method, followed by the addition of biotin-labeled H-type 1 glycans (Glycotech), H-type 3 glycans (Glycotech), Lewis Y glycans (Glycotech), or Lewis b glycans (Glycotech). Each glycan was immobilized on the sensor chip via a biotin-streptavidin reaction, producing a sensor chip with immobilized H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, or Lewis b glycans. The amount of immobilized streptavidin was approximately 400 RU, and the amount of immobilized glycans was approximately 35 RU.

[0038] The glycan binding affinity was measured using HBS-EP+ (10 mM HEPES, 150 mM sodium chloride, 3 mM EDTA, 0.05% (v / v) Tween 20, pH 7.4) as the buffer, with a flow rate of 30 μL / min, a binding time of 3 minutes, and a dissociation time of 6 minutes. The sensor chip was regenerated using 5 mM sodium hydroxide at a flow rate of 30 μL / min and a regeneration time of 10 seconds. When using a sensor chip with a large amount of immobilized glycans, 20 mM sodium hydroxide was used. Analysis was performed using the analysis software (Biacore 8K Evaluation Software) provided with the Biacore 8K instrument, and the dissociation constant (KD) was calculated by 1:1 binding fitting.

[0039] Tables 2 to 4 show the dissociation constants for H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans for the 19 recombinant proteins prepared using the recombinant BC2LCNcys (Comparative Example 1) produced in (1-3) above and the 19 transformants listed in Table 1, as Examples 1 to 5 and Comparative Examples 1 to 15. A smaller dissociation constant indicates higher binding affinity. For mutants with clearly reduced glycan binding (those whose binding was confirmed from the sensorgram shape but whose maximum binding amount to the sensor chip, Rmax, was approximately 10 RU or less, approximately one-third that of recombinant BC2LCNcys), we determined that most of the recombinant proteins had lost glycan-binding activity and thus had reduced glycan-binding activity. Therefore, the dissociation constants were not calculated and the results were recorded as "reduced." Furthermore, mutants without glycan-binding activity (where glycan binding could not be confirmed from the sensorgram shape) were recorded as "x."

[0040] Table 2 shows the results for recombinant BC2LCNcys (Comparative Example 1) described in Preparation Example (1-5) below, and for fucose-binding proteins in Examples 1 to 5 that retain affinity for any of the four types of glycans measured and have improved thermal stability compared to recombinant BC2LCNcys (Comparative Example 1) in the thermal stability evaluation described in Preparation Example (1-5) below. The thermal stability measurements in Table 2 were performed as described in Preparation Example (1-5) below. The denaturation midpoint temperatures in Table 2 were measured as described in Preparation Example (1-6) below. As shown in Table 2, the fucose-binding protein T83P described as Example 1 (the threonine specified as the 83rd threonine residue in SEQ ID NO: 1 is replaced with proline), the fucose-binding protein T83A described as Example 2 (the threonine specified as the 83rd threonine residue in SEQ ID NO: 1 is replaced with alanine), the fucose-binding protein T83Y described as Example 3 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 4 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 5 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 6 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 7 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 8 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 9 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 10 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with tyrosine), the fucose-binding protein T83Y described as Example 11 (the 83rd threonine residue specified as SEQ ID NO: 1 is replaced with 3W (in which the threonine specified as the 83rd threonine residue in SEQ ID NO: 1 is replaced with tryptophan) and the fucose-binding protein T83S described in Example 5 (in which the 83rd threonine residue in SEQ ID NO: 1 is replaced with serine) have binding affinity to all of the H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans, similar to the recombinant BC2LCNcys described in Comparative Example 1 (in which the 83rd threonine specified as the 83rd threonine in SEQ ID NO: 1 is not replaced).

[0041] [Table 2]

[0042] The amino acid sequence of the fucose-binding protein T83P described in Example 1 is SEQ ID NO: 51, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 2 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a proline residue). Sequence analysis of the expression vector pET-83P confirmed that the expression vector pET-83P contains the nucleotide sequence of SEQ ID NO: 25, which encodes the amino acid sequence of SEQ ID NO: 2.

[0043] The amino acid sequence of the fucose-binding protein T83A described in Example 2 is SEQ ID NO: 52, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 3 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with an alanine residue). Sequence analysis of the expression vector pET-83A confirmed that the expression vector pET-83A contains the nucleotide sequence of SEQ ID NO: 26, which encodes the amino acid sequence of SEQ ID NO: 3.

[0044] The amino acid sequence of the fucose-binding protein T83Y described in Example 3 is SEQ ID NO: 53, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 4 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tyrosine residue). As a result of sequence analysis of the expression vector pET-83Y, it was confirmed that the expression vector pET-83Y contains the nucleotide sequence of SEQ ID NO: 27 that encodes the amino acid sequence of SEQ ID NO: 4.

[0045] The amino acid sequence of the fucose-binding protein T83W described in Example 4 is SEQ ID NO: 54, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 5 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tryptophan residue). Sequence analysis of the expression vector pET-83W confirmed that the expression vector pET-83W contains the nucleotide sequence of SEQ ID NO: 28, which encodes the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the fucose-binding protein T83S described in Example 5 is SEQ ID NO: 55, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 6 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a serine residue). Sequence analysis of the expression vector pET-83S confirmed that the expression vector pET-83S contains the nucleotide sequence of SEQ ID NO: 29, which encodes the amino acid sequence of SEQ ID NO: 6.

[0046] Table 3 shows the results of Comparative Examples 2 to 11 for fucose-binding proteins that retained affinity for any of the four types of sugar chains measured and had the same or lower thermal stability compared to Comparative Example 1. The thermal stability measurements in Table 3 were performed as described in Preparation Examples (1-5) below.

[0047] [Table 3]

[0048] Table 4 shows the results of recombinant proteins in which the binding affinity to all four types of glycans measured was significantly reduced or lost as Comparative Examples 12 and 15. The thermal stability measurements in Table 4 were performed as described in Preparation Examples (1-5) below.

[0049] [Table 4]

[0050] (1-5) Evaluation of the thermal stability of recombinant proteins The thermal stability of the recombinant proteins (recombinant BC2LCNcys and the recombinant proteins in Table 1) produced in (1-3) above was evaluated by Thermal Shift Assay (TSA). Specifically, a real-time PCR system, QuantStudio3 (Thermo Fisher Scientific), was used. The reaction solution consisted of 0.25 mg / mL recombinant protein, 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, and 0.5% (v / v) SYPRO Orange. The reaction conditions were a temperature ramp from 30°C to 98°C at a rate of 0.5°C / s. The denaturation midpoint temperature was determined by simultaneously measuring the recombinant BC2LCNcys from Comparative Example 1 and comparing the peak fluorescence intensity temperatures.

[0051] Tables 2 to 4 show the results of the thermal stability evaluation by the TSA method. In the tables, "Improved" indicates fucose-binding proteins whose peak showing the denaturation midpoint temperature was shifted to a higher temperature compared to recombinant BC2LCNcys (Comparative Example 1) and whose thermal stability was improved, or fucose-binding proteins whose denaturation midpoint temperature was higher than the measurement range and no peak was observed. In the tables, "Equivalent" indicates fucose-binding proteins whose denaturation midpoint temperature was similar to that of recombinant BC2LCNcys (Comparative Example 1) and whose thermal stability was equivalent, and "Decreased" indicates fucose-binding proteins whose denaturation midpoint temperature was lower than that of recombinant BC2LCNcys (Comparative Example 1) and whose thermal stability was reduced.

[0052] Table 2 shows the results for fucose-binding proteins that retained their affinity for one of the four types of sugar chains measured according to the method in (1-4) above and had improved thermal stability compared to recombinant BC2LCNcys (Comparative Example 1). Table 3 shows the results for fucose-binding proteins that retained their affinity for one of the four types of sugar chains measured according to the method in (1-4) above and had thermal stability equivalent to or lower than that of Comparative Example 1. Table 4 shows the results for fucose-binding proteins that lost their affinity for all four types of sugar chains in the sugar chain binding affinity measured in (1-4) above.

[0053] (1-6) Measurement of the denaturation midpoint temperature of recombinant proteins The denaturation midpoint temperatures of the fucose-binding proteins and recombinant BC2LCNcys that exhibited higher thermal stability than the recombinant BC2LCNcys of Comparative Example 1 in (1-5) and retained affinity for one of the four types of glycans in (1-4) were measured using a differential scanning calorimeter. Specifically, the recombinant proteins produced in (1-3) were buffer-exchanged in dialysis buffer (50 mM sodium acetate, 150 mM sodium chloride, pH 5.5) using a regenerated cellulose membrane (Thermo Fisher Scientific, molecular weight cutoff 3500). The concentration of the recombinant protein in the dialyzed solution was measured by ultraviolet absorption spectroscopy, and the dialyzed solution was diluted to 500 μg / mL with the dialysis buffer. The denaturation midpoint temperatures were measured using a differential scanning calorimeter (Malvern Panathelial, MicroCal VP-Capillary DSC). The conditions for measuring the denaturation midpoint temperature were as follows: the solution volume of each recombinant protein was 400 μL, the heating rate was 60°C / h, and the heating temperature was 40°C-120°C.

[0054] Table 2 shows the denaturation midpoint temperatures of the recombinant proteins produced in (1-3) above as Examples 1 to 5. The denaturation midpoint temperature is the temperature at which half of the protein is denatured. A higher denaturation midpoint temperature indicates higher thermal stability. As shown in Table 2, fucose-binding protein T83P (Example 1), fucose-binding protein T83A (Example 2), fucose-binding protein T83Y (Example 3), fucose-binding protein T83W (Example 4), and fucose-binding protein T83S (Example 5) have higher denaturation midpoint temperatures than recombinant BC2LCNcys (in which the threonine residue identified as the 83rd threonine residue in SEQ ID NO: 1 is not substituted) described in Comparative Example 1, demonstrating improved thermal stability. Furthermore, it can be seen that these proteins retain their affinity for all four types of sugar chains measured, demonstrating their function as fucose-binding proteins.

[0055] Preparation example 2 (2-1) Introduction of a mutation into the methionine residue identified as the 86th methionine residue of SEQ ID NO: 1 into the recombinant BC2LCNcys Mutation was introduced into the recombinant BC2LCNcys at the methionine residue identified as the 86th methionine residue in SEQ ID NO: 1. That is, mutation was introduced to replace the 100th methionine residue in the amino acid sequence of the recombinant BC2LCNcys (SEQ ID NO: 48) with another amino acid residue.

[0056] PCR was performed using the expression vector pET-BC2LCNcys described in Preparation Example (1-1) above as a template and the oligonucleotides consisting of the sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 50 as PCR primers according to the method disclosed in JP 2018-000038 A. The PCR primer consisting of the sequence set forth in SEQ ID NO: 56 had the degenerate sequence NNB (N = A, C, G, or T, B = C, G, or T) and was designed so that the methionine residue at position 86 of SEQ ID NO: 48 (corresponding to the methionine residue at position 86 of SEQ ID NO: 1) was randomly replaced with another amino acid residue. The resulting PCR product was digested with the restriction enzymes KpnI and XhoI and ligated with the expression vector pET-BC2LCNcys described in (1-2) above, which had been similarly digested with the restriction enzymes. This ligation product was used to transform Escherichia coli BL21(DE3) to obtain multiple transformants. The expression vector was extracted from each transformant and the nucleotide sequence was analyzed. As a result, 19 types of expression vectors and transformants harboring them were obtained, as shown in Table 5. The recombinant proteins in Table 5 were produced as described in Preparation Example (2-2) below.

[0057] [Table 5]

[0058] (2-2) Production of recombinant proteins The recombinant BC2LCNcys and the 19 types of recombinant fucose-binding proteins (Table 5) described in (2-1) above were produced according to the method described in Preparation Example (1-3) above, using the recombinant Escherichia coli BL21(DE3) / pET-BC2LCNcys produced in Preparation Example (1-1) above and the 19 types of transformants (Table 5) produced in (2-1) above.

[0059] (2-3) Evaluation of glycan binding affinity of recombinant proteins The binding affinity of the recombinant proteins produced in (2-2) above (recombinant BC2LCNcys and the recombinant proteins in Table 5) to H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans was evaluated according to the method described in the above Preparation Example (1-4).

[0060] Tables 6 to 8 show the dissociation constants for H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans of 19 types of recombinant proteins prepared using the recombinant BC2LCNcys (Comparative Example 1) produced in the above Preparation Example (1-3) and the 19 types of transformants listed in Table 5, as Examples 6 and 7, Comparative Example 1, and Comparative Examples 16 to 32.

[0061] Table 6 shows the results for recombinant BC2LCNcys (Comparative Example 1) described in Preparation Example (1-5) and for fucose-binding proteins in Examples 6 and 7, which retain affinity for any of the four types of glycans measured and have improved thermal stability compared to recombinant BC2LCNcys (Comparative Example 1) in the thermal stability evaluation described in Preparation Example (2-4) below. The thermal stability measurements in Table 6 were performed as described in Preparation Example (2-4) below. The denaturation midpoint temperatures in Table 6 were measured as described in Preparation Example (2-5) below. As shown in Table 6, the fucose-binding protein M86I described in Example 6 (the methionine specified as the 86th methionine residue in SEQ ID NO: 1 is replaced with isoleucine) and the fucose-binding protein M86V described in Example 7 (the methionine specified as the 86th methionine residue in SEQ ID NO: 1 is replaced with valine) have binding affinity to all of the H-type 1 glycan, H-type 3 glycan, Lewis Y glycan, and Lewis b glycan, similar to the recombinant BC2LCNcys described in Comparative Example 1 (the methionine specified as the 86th methionine in SEQ ID NO: 1 is not replaced).

[0062] [Table 6]

[0063] The amino acid sequence of the fucose-binding protein M86I described in Example 6 is SEQ ID NO: 57, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 7 (the amino acid sequence in which the methionine residue at position 86 of SEQ ID NO: 1 is substituted with an isoleucine residue). Sequence analysis of the expression vector pET-86I confirmed that the expression vector pET-86I contains the nucleotide sequence of SEQ ID NO: 30 that encodes the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of the fucose-binding protein M86V described in Example 7 is SEQ ID NO: 58, and the amino acid sequence from positions 15 to 169 corresponds to the amino acid sequence of SEQ ID NO: 8 (the amino acid sequence in which the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue). Sequence analysis of the expression vector pET-86V confirmed that the expression vector pET-86V contains the nucleotide sequence of SEQ ID NO: 31, which encodes the amino acid sequence of SEQ ID NO: 8.

[0064] Table 7 shows the results of Comparative Examples 16 to 29 for fucose-binding proteins that retained affinity for any of the four types of sugar chains measured and had the same or lower thermal stability compared to Comparative Example 1. The thermal stability measurements in Table 7 were performed as described in Preparation Example (2-4) below.

[0065] [Table 7]

[0066] Table 8 shows the results of comparative examples 30 to 32, which show recombinant proteins that showed significantly reduced or no binding affinity to all four types of glycans measured. The thermal stability measurements in Table 8 were performed as described in Preparation Example (2-4) below.

[0067] [Table 8]

[0068] (2-4) Evaluation of the thermal stability of recombinant proteins The heat stability of the recombinant proteins produced in (2-3) above (recombinant BC2LCNcys and the recombinant proteins in Table 5) was evaluated according to the TSA method described in Preparation Example (1-5) above.

[0069] Tables 6 to 8 show the results of thermal stability evaluation by the TSA method. Table 6 shows the results for fucose-binding proteins with improved thermal stability compared to recombinant BC2LCNcys (Comparative Example 1). Table 7 shows the results for fucose-binding proteins that retained affinity for any of the four types of glycans measured according to the method in (2-3) above and had thermal stability equivalent to or lower than that in Comparative Example 1. Table 8 shows the results for fucose-binding proteins that lost affinity for all four types of glycans in the glycan-binding affinity measured in (2-3) above.

[0070] (2-5) Measurement of the denaturation midpoint of recombinant proteins In the above (2-4), the fucose-binding protein and recombinant BC2LCNcys, which showed higher thermal stability than the recombinant BC2LCNcys of Comparative Example 1 and retained affinity for any of the four types of glycans in the above (2-3), were subjected to measurement of the denaturation midpoint temperature using a differential scanning calorimeter as described in the above Preparation Example (1-6). Table 6 shows the denaturation midpoint temperatures of the recombinant proteins produced in (2-2) above as Examples 6 and 7. As shown in Table 6, the fucose-binding protein M86I (Example 6) and the fucose-binding protein M86V (Example 7) have higher denaturation midpoint temperatures than the recombinant BC2LCNcys described in Comparative Example 1 (the methionine residue identified as the 86th methionine residue in SEQ ID NO: 1 is not substituted), indicating improved thermal stability. It is also clear that they retain their affinity for the four types of sugar chains measured, and thus retain their function as fucose-binding proteins.

[0071] Preparation example 3 (3-1) Introduction of mutations into the recombinant BC2LCNcys at the amino acid residues specified as the 83rd threonine residue and the 86th methionine residue of SEQ ID NO: 1 For recombinant BC2LCNcys, the threonine residue at position 83 of SEQ ID NO: 1 was substituted with one amino acid residue selected from proline, alanine, tyrosine, tryptophan, and serine, and the methionine residue at position 86 of SEQ ID NO: 1 was substituted with an isoleucine or valine residue. That is, mutations were introduced to substitute the methionine residues at positions 97 and 100 of the amino acid sequence of recombinant BC2LCNcys (SEQ ID NO: 48) with other amino acid residues.

[0072] PCR was performed using the expression vector pET-86I or expression vector pET-86V described in Preparation Example (2-1) above as a template, and the oligonucleotides consisting of the sequences set forth in SEQ ID NOs: 50 and 59 to 51 as PCR primers, in the combinations of template DNA and PCR primers shown in Table 9, according to the method described in Preparation Example (1-1) above. The resulting PCR product was digested with the restriction enzymes KpnI and XhoI and ligated with the expression vector pET-BC2LCNcys described in (1-1) above, which had been similarly treated with the restriction enzymes. Transformants were obtained according to the method described in Preparation Example (1-2) above. Expression vectors were extracted from each transformant and sequenced to obtain the 10 expression vectors shown in Table 10 and transformants containing them. The recombinant proteins in Table 10 were produced as described in Preparation Example (3-2) below.

[0073] [Table 9]

[0074] [Table 10]

[0075] Sequence analysis revealed that the expression vector pET-83P / 86I contains the nucleotide sequence of SEQ ID NO: 32, which encodes the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of the fucose-binding protein T83P / M86I is SEQ ID NO: 64, with the 15th to 169th amino acid sequences corresponding to the amino acid sequence of SEQ ID NO: 9 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a proline residue and the methionine residue at position 86 with an isoleucine residue). The expression vector pET-83A / 86I contains the nucleotide sequence of SEQ ID NO: 33, which encodes the amino acid sequence of SEQ ID NO: 10, and the amino acid sequence of the fucose-binding protein T83A / M86I is SEQ ID NO: 65, with the 15th to 169th amino acid sequences corresponding to the amino acid sequence of SEQ ID NO: 10 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with an alanine residue and the methionine residue at position 86 with an isoleucine residue). The expression vector pET-83Y / 86I contains the nucleotide sequence of SEQ ID NO: 34, which encodes the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of the fucose-binding protein T83Y / M86I is SEQ ID NO: 66, with the 15th to 169th sequences corresponding to the amino acid sequence of SEQ ID NO: 11 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tyrosine residue and the methionine residue at position 86 with an isoleucine residue).The expression vector pET-83W / 86I contains the nucleotide sequence of SEQ ID NO: 35, which encodes the amino acid sequence of SEQ ID NO: 12, and the amino acid sequence of the fucose-binding protein T83W / M86I is SEQ ID NO: 67, with the 15th to 169th sequences corresponding to the amino acid sequence of SEQ ID NO: 12 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tryptophan residue and the methionine residue at position 86 with an isoleucine residue). The expression vector pET-83S / 86I contains the nucleotide sequence of SEQ ID NO: 36, which encodes the amino acid sequence of SEQ ID NO: 13. The amino acid sequence of the fucose-binding protein T83S / M86I is SEQ ID NO: 68, the 15th to 169th amino acids of which correspond to the amino acid sequence of SEQ ID NO: 13 (the amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is replaced with a serine residue and the 86th methionine residue is replaced with an isoleucine residue).The expression vector pET-83P / 86V contains the nucleotide sequence of SEQ ID NO: 37, which encodes the amino acid sequence of SEQ ID NO: 14, and the amino acid sequence of the fucose-binding protein T83P / M86V is SEQ ID NO: 69, with the 15th to 169th sequences corresponding to the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a proline residue and the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue). The expression vector pET-83A / 86V contains the nucleotide sequence of SEQ ID NO: 38, which encodes the amino acid sequence of SEQ ID NO: 15, and the amino acid sequence of the fucose-binding protein T83A / M86V is SEQ ID NO: 70, with the 15th to 169th sequences corresponding to the amino acid sequence of SEQ ID NO: 15 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with an alanine residue and the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue). The expression vector pET-83Y / 86V contains the nucleotide sequence of SEQ ID NO: 39, which encodes the amino acid sequence of SEQ ID NO: 16, and the amino acid sequence of the fucose-binding protein T83Y / M86V is SEQ ID NO: 71, with the 15th to 169th sequences corresponding to the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tyrosine residue and the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue). The expression vector pET-83W / 86V contains the nucleotide sequence of SEQ ID NO: 40, which encodes the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of the fucose-binding protein T83W / M86V is SEQ ID NO: 72, with the 15th to 169 sequences corresponding to the amino acid sequence of SEQ ID NO: 17 (the amino acid sequence in which the threonine residue at position 83 of SEQ ID NO: 1 is substituted with a tryptophan residue and the methionine residue at position 86 of SEQ ID NO: 1 is substituted with a valine residue). The expression vector pET-83S / 86V contains the nucleotide sequence of SEQ ID NO: 41, which encodes the amino acid sequence of SEQ ID NO: 18. The amino acid sequence of the fucose-binding protein T83S / M86V is SEQ ID NO: 73, the 15th to 169th amino acids of which correspond to the amino acid sequence of SEQ ID NO: 18 (the amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is replaced with a serine residue and the 86th methionine residue is replaced with a valine residue).

[0076] (3-2) Production of recombinant proteins The 10 types of recombinant fucose-binding proteins (Table 10) described in (3-1) above were produced using the 10 types of transformants (Table 10) prepared in (3-1) above, according to the method described in the preparation example (1-3) above.

[0077] (3-3) Evaluation of glycan binding affinity of recombinant proteins The binding affinity of the 10 recombinant fucose-binding proteins (Table 10) produced in (3-2) above to H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans was evaluated according to the method described in the Preparation Example (1-4) above.

[0078] Table 11 shows the dissociation constants of the H type 1 sugar chain, H type 3 sugar chain, Lewis Y sugar chain, and Lewis b sugar chain for the 12 types of recombinant proteins produced in the above (3-2) as Examples 8 to 17. Also shown are the dissociation constants of the fucose-binding proteins of Examples 1 to 5 measured in the above Preparation Example (1-4) and the fucose-binding proteins of Examples 6 and 7 measured in the above Preparation Example (2-3).

[0079] The measurement of the denaturation midpoint temperature in Table 11 was performed as described in Preparation Example (3-4) described later. As shown in Table 11, the recombinant BC2LCNcys (Comparative Example 1) described in Preparation Example (1-5) and Examples 8 to 17 retained affinity for any of the four types of sugar chains measured.

[0080] [Table 11]

[0081] (3-4) Measurement of the denaturation midpoint of recombinant proteins The denaturation midpoint temperatures of the 10 recombinant proteins (Table 10) produced in (3-2) above were measured according to the differential scanning calorimeter measurement method described in Preparation Example (1-6) above.

[0082] Table 11 shows the denaturation midpoint temperatures of the 10 types of recombinant proteins produced in (3-2) above as Examples 8 to 17. Also shown are the denaturation midpoint temperatures of the fucose-binding proteins of Examples 1 to 5 measured in Preparation Example (1-6) above and the fucose-binding proteins of Examples 6 and 7 measured in Preparation Example (2-5) above. As shown in Table 11, in Examples 8 to 17, the fucose-binding proteins in which the threonine residue at position 83 of SEQ ID NO: 1 was substituted with one amino acid residue selected from proline, alanine, tyrosine, tryptophan, and serine, and the methionine residue at position 86 of SEQ ID NO: 1 was substituted with an isoleucine or valine residue, showed improved denaturation midpoint temperatures compared to fucose-binding proteins in which either the threonine residue at position 83 or the methionine residue at position 86 of SEQ ID NO: 1 was substituted. Specifically, the fucose-binding protein T83P / M86I (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a proline residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) described in Example 8 is similar to the fucose-binding protein T83P (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a proline residue) of Example 1 and the fucose-binding protein M86I (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) of Example 6, and the fucose-binding protein T83A / M86I described in Example 9. M86I (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with an alanine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) is a fucose-binding protein T83A (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with an alanine residue) of Example 2, the fucose-binding protein M86I (an amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) of Example 6, and the fucose-binding protein T83Y / M86I (an amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue) described as Example 10.The fucose-binding protein T83Y (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue) of Example 3, the fucose-binding protein M86I (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) of Example 6, and the fucose-binding protein T83W / M86I (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tryptophan residue) of Example 11 are also included. The fucose-binding protein T83W (amino acid sequence of SEQ ID NO: 1 in which the 83rd threonine residue is substituted with a tryptophan residue) of Example 4, the fucose-binding protein M86I (amino acid sequence of SEQ ID NO: 1 in which the 86th methionine residue is substituted with an isoleucine residue) of Example 6, and the fucose-binding protein T83S / M86I (amino acid sequence of SEQ ID NO: 1 in which the 83rd threonine residue is substituted with an isoleucine residue) of Example 12 are also included. The fucose-binding protein T83S (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a serine residue) of Example 5, the fucose-binding protein M86I (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue) of Example 6, and the fucose-binding protein T83P / M86V (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a protease inhibitor) described as Example 13 are also included. The fucose-binding protein T83P (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a proline residue) of Example 1, the fucose-binding protein M86V (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue) of Example 7, and the fucose-binding protein T83A / M86V (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with an alanine residue) described as Example 14 are also included.The fucose-binding protein T83Y / M86V (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue) described as Example 15 is the fucose-binding protein T83Y (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tyrosine residue) described as Example 3 and the fucose-binding protein M86V (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue) described as Example 36. It can be seen that the fucose-binding protein T83W (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a tryptophan residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a tryptophan residue) of Example 4 and the fucose-binding protein M86V (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue) of Example 7 have an improved denaturation midpoint temperature, as compared with the fucose-binding protein T83S (amino acid sequence in which the 83rd threonine residue of SEQ ID NO: 1 is substituted with a serine residue and the 86th methionine residue of SEQ ID NO: 1 is substituted with a serine residue) of Example 5 and the fucose-binding protein M86V (amino acid sequence in which the 86th methionine residue of SEQ ID NO: 1 is substituted with a valine residue) of Example 7.

[0083] Preparation example 4 (4-1) Mutagenesis of recombinant BC2LCNcys by substituting amino acid residues with alanine In the recombinant BC2LCNcys, one amino acid residue selected from the first proline residue of SEQ ID NO: 1 (the 15th residue of SEQ ID NO: 48, which is the amino acid sequence of recombinant BC2LCNcys), the 37th lysine residue (the 51st residue of SEQ ID NO: 48), the 39th glutamine residue (the 53rd residue of SEQ ID NO: 48), the 46th threonine residue (the 60th residue of SEQ ID NO: 48), the 47th proline residue (the 61st residue of SEQ ID NO: 48), and the 122nd serine residue (the 136th residue of SEQ ID NO: 48) was substituted with an alanine residue.

[0084] The expression vectors shown in Table 13 were prepared by general methods similar to those described in Preparation Example (3-1) above. The expression vectors shown in Table 13 were prepared by PCR using appropriate PCR primers and the expression vector pET-BC2LCNcys from Preparation Example (1-1) above as a template, or by artificially preparing DNA encoding the desired amino acid sequence. Transformants containing each expression vector were obtained by the method described in Preparation Example (1-2) above. Expression vectors were extracted from each transformant and subjected to sequence analysis, yielding the six types of expression vectors shown in Table 13 and transformants containing them. The recombinant proteins in Table 13 were produced as described in Preparation Example (4-2) below.

[0085] [Table 12]

[0086] [Table 13]

[0087] As a result of confirming the nucleotide sequence by sequence analysis, the expression vector pET-P1A contains the nucleotide sequence of SEQ ID NO: 42, which encodes the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of fucose-binding protein P1A is SEQ ID NO: 74, with the 15th to 169th amino acid sequences corresponding to the amino acid sequence of SEQ ID NO: 19 (the amino acid sequence in which the first proline residue of SEQ ID NO: 1 is substituted with an alanine residue). The expression vector pET-K37A contains the nucleotide sequence of SEQ ID NO: 43, which encodes the amino acid sequence of SEQ ID NO: 20, and the amino acid sequence of fucose-binding protein K37A is SEQ ID NO: 75, with the 15th to 169th amino acid sequences corresponding to the amino acid sequence of SEQ ID NO: 20 (the amino acid sequence in which the 37th lysine residue of SEQ ID NO: 1 is substituted with an alanine residue). The expression vector pET-Q39A contains the nucleotide sequence of SEQ ID NO: 44, which encodes the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of fucose-binding protein Q39A is SEQ ID NO: 76, with the 15th to 169th residues corresponding to the amino acid sequence of SEQ ID NO: 21 (the amino acid sequence in which the glutamine residue at position 39 of SEQ ID NO: 1 is substituted with an alanine residue). The expression vector pET-T46A contains the nucleotide sequence of SEQ ID NO: 45, which encodes the amino acid sequence of SEQ ID NO: 22, and the 15th to 169th residues corresponding to the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence in which the threonine residue at position 46 of SEQ ID NO: 1 is substituted with an alanine residue). The expression vector pET-P47A contains the nucleotide sequence of SEQ ID NO: 46, which encodes the amino acid sequence of SEQ ID NO: 23, and the amino acid sequence of fucose-binding protein P47A is SEQ ID NO: 78, with the 15th to 169th residues corresponding to the amino acid sequence of SEQ ID NO: 23 (the amino acid sequence in which the proline residue at position 47 of SEQ ID NO: 1 is substituted with an alanine residue). The expression vector pET-S122A contains the nucleotide sequence of SEQ ID NO: 47, which encodes the amino acid sequence of SEQ ID NO: 24, and the 15th to 169th residues corresponding to the amino acid sequence of SEQ ID NO: 24 (the amino acid sequence in which the serine residue at position 122 of SEQ ID NO: 1 is substituted with an alanine residue).

[0088] (4-2) Production of recombinant proteins The six types of recombinant fucose-binding proteins (Table 13) described in (4-1) above were produced using the six types of transformants (Table 13) prepared in (4-1) above, according to the method described in Preparation Example (1-3) above. (4-3) Evaluation of glycan binding affinity of recombinant proteins The binding affinity of the six recombinant fucose-binding proteins (Table 13) produced in (4-2) above to H-type 1 glycans, H-type 3 glycans, Lewis Y glycans, and Lewis b glycans was evaluated according to the method described in the Preparation Example (1-4) above.

[0089] Table 14 shows the dissociation constants of the H type 1 sugar chain, H type 3 sugar chain, Lewis Y sugar chain, and Lewis b sugar chain for the six types of recombinant proteins produced in (4-2) above, as Examples 18 to 23.

[0090] The measurement of the denaturation midpoint temperature in Table 14 was performed as described in Preparation Example (4-4) described later. As shown in Table 14, the recombinant BC2LCNcys (Comparative Example 1) described in Preparation Example (1-5) and Examples 18 to 23 retained affinity for any of the four types of sugar chains measured.

[0091] [Table 14]

[0092] (4-4) Measurement of the denaturation midpoint of recombinant proteins The denaturation midpoint temperatures of the six recombinant proteins (Table 14) produced in (4-2) above were measured according to the differential scanning calorimeter measurement method described in Preparation Example (1-6) above.

[0093] Table 14 shows the denaturation midpoint temperatures of the six types of recombinant proteins produced in (4-2) above as Examples 18 to 23. As shown in Table 14, fucose-binding protein P1A (Example 18), fucose-binding protein K37A (Example 19), fucose-binding protein Q39A (Example 20), fucose-binding protein T46A (Example 21), fucose-binding protein P47A (Example 22), and fucose-binding protein S122A (Example 23) have higher denaturation midpoint temperatures than recombinant BC2LCNcys (in which none of the amino acid residues in SEQ ID NO: 1 are substituted with alanine residues) described in Comparative Example 1, demonstrating improved thermal stability.

Claims

1. A fucose-binding protein comprising an amino acid sequence shown in any of SEQ ID NOs: 2 to 6 and 9 to 18.

2. The fucose-binding protein according to claim 1, which has an additional amino acid sequence at the N-terminus and / or C-terminus.

3. The fucose-binding protein according to claim 1 or 2, wherein the amino acid sequence added to the C-terminus is an oligopeptide containing a cysteine ​​residue.

4. The fucose-binding protein according to any one of claims 1 to 3, wherein the amino acid sequence added to the N-terminus is an oligopeptide containing a polyhistidine sequence.

5. A DNA encoding the fucose-binding protein according to any one of claims 1 to 4.

6. An expression vector containing the DNA of claim 5.

7. A transformant obtained by transforming a host with the expression vector according to claim 6.

8. The transformant according to claim 7, wherein the host is Escherichia coli.

9. A method for producing a fucose-binding protein, comprising two steps: a step of producing the fucose-binding protein by culturing the transformant according to claim 7 or 8; and a step of recovering the produced fucose-binding protein from the obtained culture.

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