JP7353616B2 - Novel carbohydrate-binding protein and polynucleotide encoding it - Google Patents

Description

The present invention relates to a novel protein and a polynucleotide encoding the same, and particularly to a sugar chain-binding protein and a polynucleotide encoding the same.

The sugar chains of complex carbohydrates such as glycoproteins and glycolipids that exist on cell surfaces and in body fluids function as a type of information element and are deeply involved in important life phenomena such as development, immunity, cancer, and infection. . On the other hand, lectins, which are sugar chain-binding proteins, function as sugar chain recognition molecules and, like sugar chains, play a biologically important role.

To date, many types of lectins have been isolated from seaweeds or algae (freshwater blue-green algae), and their biochemical properties have been clarified. It is known that some lectins specifically bind to viruses such as human immunodeficiency virus (HIV) and influenza virus (Non-Patent Documents 1 to 11). In addition, sugar chains are related not only to viruses but also to cancer as mentioned above, and in recent years, it has been reported that sulfated glycosaminoglycans are the main cancer immune antigens in cancer tissues (Non-patent literature 12), it has been reported that it is deeply involved in the development and metastasis of cancer cells (Non-patent Documents 13 and 14).

Boyd, M. R. et al., Antimicrob. Agents Chemother.41, 1521-1530, 1997. O’Keefe, B. R. et al., Antimicrob. AgentsChemother. 47, 2518-2525, 2003. Helle, F., et al., J. Biol. Chem. 281, 25177-25183, 2006. Barrientos, L. G., et al., Antiviral. Res. 58, 47-56, 2003. Dey, B., et al., J. Virol. 74, 4562-4569, 2000. O’Keefe, B. R. et al.,J. Virol. 84, 2511-2521,2010. Hori, K. et al., Glycobiology, 17, 479-491, 2007. Sato, Y., Okuyama, S., and Hori, K., J. Biol. Chem. 282, 11021-11029, 2007. Sato, Y., Morimoto, K., Hirayama, M., and Hori, K. Biochem. Biophys. Res. commun. 405,291-296, 2011. Yuichiro Sato, Makoto Hirayama, Yoshifumi Fujiwara, Kinjiro Morimoto, Kanji Hori (2010) Abstracts of the 13th Marine Biotechnology Society Conference (presented on May 29, 2010) Sato, Y., Hirayama, M., Morimoto, K., Yamamoto, N., Okuyama, S., and Hori, K. J. Biol.Chem. 286, No.22, 19446-19458, 2011. Hiroto Kato et al., Cell Reports. 20,1073-1087, 2017. Adam Pudelko et al., The FEBS Journal(First published: 13 January2019, online: https://doi.org/10.1111/febs.14748) Shuji Mizumoto et al., J. Biol. Chem.287, 18985-18994, 2012.

However, at present, it cannot be said that the substances that specifically recognize such sulfated glycosaminoglycan sugar chains are sufficiently known, and the number of such substances is limited. We are not in a position to be able to supply this. Since such substances have the potential to be used for cancer treatment and diagnosis, it is necessary to discover more new substances and clarify their properties.

The present invention was made in view of the above-mentioned problems, and its purpose is to search for and provide a novel protein that recognizes sulfated sugar chains that can be used for the diagnosis and treatment of cancer as described above. The goal is to make it possible.

In order to achieve the above object, the present inventors, as a result of intensive research, synthesized cDNA from RNA obtained from algae of the genus Bryopsis, and expressed a new protein (lectin) from the cDNA. I succeeded in doing so.

Specifically, the novel polynucleotide according to the present invention encodes a sugar chain-binding protein and includes the nucleotide sequence of SEQ ID NO: 1 or 2, or the nucleotide sequence of SEQ ID NO: 1 or 2, or a sequence complementary thereto. It is characterized by comprising a nucleotide sequence that hybridizes with under stringent conditions.

Furthermore, the polynucleotide according to the present invention may be derived from the genus Bryopsis, such as Bryopsis corticulans, Bryopsis plumose, and Bryopsis maxima.

The novel protein according to the present invention has sugar chain binding properties, and has an amino acid sequence set forth in SEQ ID NO: 3 or 4, or a substitution of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 or 4. It is characterized by containing a deleted, inserted, or added amino acid sequence.

Furthermore, the novel protein according to the present invention is characterized in that it specifically recognizes sugar chains containing sulfate groups, and is suitable for use in Bryopsis corticulans such as Bryopsis corticulans, Bryopsis plumose, and Bryopsis maxima. It may be derived from the genus.

The pharmaceutical composition for treating or diagnosing cancer according to the present invention is characterized by containing the novel protein according to the present invention. The pharmaceutical composition according to the present invention contains the above-mentioned novel protein that can recognize sugar chains containing sulfate groups such as sulfated glycosaminoglycans, which are expressed in cancer cells and are known to be cancer antigens. , it can be used for cancer treatment and cancer diagnosis.

The polynucleotide according to the present invention encodes a novel sugar chain-binding protein, and the new protein can recognize sulfated sugar chains that are cancer antigens, so it has the potential to be used for cancer treatment and diagnosis. , extremely useful.

1 is a diagram showing the nucleotide sequence and deduced amino acid sequence of the full-length cDNA of BcBry1-1 obtained in Example 1. FIG. 1 is a diagram showing the nucleotide sequence and deduced amino acid sequence of the full-length cDNA of BcBry1-2 obtained in Example 1. FIG. 2 is a photograph showing the results of SDS-PAGE for confirming the expression of His-rBcBry1-1 in Example 2. 2 is a photograph showing the results of SDS-PAGE for examining the enzyme digestion conditions for His-rBcBry1-1 with Factor Xa in Example 2. 3 is a photograph showing the results of SDS-PAGE and Western blotting for examining the detectability of His-rBcBry1-1 by an anti-His tag antibody in Example 2. FIG. 3 is a diagram showing sugar chains used in the sugar chain immobilized array in Example 4. 3 is a photograph showing the results of SDS-PAGE and Western blotting for examining the detectability of His-rBcBry1-1N and C by Anti-His-tag-Alexa Fluor 647 in Example 4. 3 is a graph showing the results of sugar chain immobilization array of His-rBcBry1-1C in Example 4.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated based on drawing. The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the invention, its application, or its uses.

One embodiment of the present invention is a novel protein (BcBry1) derived from Bryopsis corticulans, an alga of the genus Bryopsis. The novel protein according to the present invention has sugar chain binding properties and is encoded by a polynucleotide comprising, for example, the nucleotide sequence of SEQ ID NO: 1 or 2. The amino acid sequence of the protein expressed by the nucleotide sequence of SEQ ID NO: 1 or 2 is shown in SEQ ID NO: 3 or 4, respectively.

As used herein, the term "protein" is used interchangeably with "peptide" or "polypeptide." Moreover, the protein according to the present invention may be isolated from a natural source or may be chemically synthesized.

The term "isolated" polypeptide or protein is intended to refer to a polypeptide or protein that has been removed from its natural environment. However, in the case of recombinantly produced polypeptides and proteins expressed in host cells, those that have been substantially purified and isolated from the host cell by any suitable technique are used.

Proteins according to the invention may be natural purified products, products of chemical synthesis procedures, and derived from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Includes products produced by recombinant technology. Depending on the host used in the recombinant production procedure, proteins according to the invention may be glycosylated or non-glycosylated. Furthermore, proteins according to the invention may contain an initial modified methionine residue as a result of host-mediated processes.

In one embodiment, the protein according to the present invention is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, or a variant of the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or 4. The protein according to the present invention is characterized in that it is a novel protein with very low homology to the amino acid sequences of conventionally known lectins.

Variants include deletions, insertions, inversions, repeats, and type substitutions (e.g., substituting a hydrophilic residue for another, but usually replacing a strongly hydrophilic residue with a strongly hydrophobic residue). (without substitution). In particular, "neutral" amino acid substitutions in a polypeptide generally have little effect on the activity of that polypeptide.

It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is well known that there are not only artificially modified variants but also variants of natural proteins that do not significantly alter the structure or function of the protein.

Those skilled in the art can readily vary one or a few amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, any base of a polynucleotide encoding a polypeptide can be mutated according to known point mutation introduction methods. Furthermore, deletion mutants or addition mutants can be created by designing primers corresponding to any site of the polynucleotide encoding the polypeptide. Furthermore, by using the method described herein, it can be easily determined whether the produced mutant is a desired mutant having sugar chain binding properties.

The above expression "one or more amino acids have been substituted, deleted, inserted, or added" refers to the substitution, deletion, insertion, or addition of one or more amino acids by a known mutant polypeptide production method such as site-directed mutagenesis. Substitution, deletion, insertion or addition of as many amino acids as can be added (preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, particularly preferably 1 to 3) means that it has been As mentioned above, such mutant polypeptides are not limited to polypeptides having mutations artificially introduced by known mutant polypeptide production methods, but can be obtained by isolating and purifying naturally occurring polypeptides. It may be something that has been done.

In other embodiments, polypeptides of the invention may be expressed recombinantly in modified forms, such as fusion proteins. For example, regions of additional amino acids, in particular charged amino acids, of the polypeptides according to the invention may be used to improve stability and persistence within host cells during purification or subsequent manipulation and storage. It can be added to the N-terminus of a polypeptide.

Recombinant production can be performed using methods well known in the art, eg, using vectors and cells as detailed below.

The present inventors discovered that the protein according to the present invention recognizes a sugar chain containing a sulfate group. Here, "sugar chain" means a linear or branched oligosaccharide or polysaccharide. Furthermore, depending on the binding mode of the sugar chain with the protein, the N-glycoside-linked sugar chain (hereinafter referred to as "N-glycan" or "N-glycan") that binds to asparagine, and the O-glycoside-linked sugar chain that binds to serine, threonine, etc. It is broadly classified into glycoside-linked sugar chains (hereinafter referred to as "O-glycans" and "O-glycans"), and other types such as Lewis type, ABO type, glycosaminoglycan (GAG), and Lac series are known. .

Note that oligosaccharides refer to those produced by dehydration bonding of 2 to 10 monosaccharides or substituted derivatives of monosaccharides. Carbohydrates that have many monosaccharides linked together are called polysaccharides. Polysaccharides differ depending on the type of constituent sugars, but carbohydrates containing a large amount of uronic acid and ester sulfate are called acid polysaccharides, and those containing only neutral sugars are called neutral polysaccharides. Among polysaccharides, a group of polysaccharides called mucopolysaccharides are mostly bound to proteins and are called proteoglycans. Monosaccharides are the constituent units of sugar chains, and are basic substances that cannot be made into simpler molecules through hydrolysis.

Furthermore, monosaccharides are broadly classified into three types: acidic sugars having acidic side chains such as carboxyl groups, amino sugars in which the hydroxyl group is substituted with an amino group, and other neutral sugars. Among the monosaccharides that exist in living organisms, acidic sugars include sialic acids such as N-acetylneuraminic acid and N-glycolylneuraminic acid (hereinafter referred to as "Neu5Gc"), and uronic acids, and amino sugars include Examples include N-acetylglucosamine (hereinafter referred to as "GlcNAc") and N-acetylgalactosamine, and examples of neutral sugars include glucose, mannose, galactose, and fucose.

The method shown in the example below can be used to determine whether a polypeptide binds to a sugar chain, but it is also possible to use a column on which a target sugar chain or a glycoprotein to which a sugar chain is bound is immobilized. , the polypeptide to be tested is passed through, and whether or not the polypeptide binds to the column is evaluated by the amount of polypeptide contained in the passed through solution or the amount of polypeptide eluted from the column with a specific eluent. be able to. In addition, Western blotting (Forensic Medicine Practice and Research, 37, 155, 1994), dot blot method (see AnalyticalBiochemistry, 204(1), 198, 1992), etc.

The present invention, as described above, provides polynucleotides encoding the polypeptides of the present invention. As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and contemplates a polymer of nucleotides. As used herein, the term "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence," and is a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). is shown as

Polynucleotides according to the invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).

As used herein, the term "oligonucleotide" is intended to have several to several dozen nucleotides linked together, and is used interchangeably with "polynucleotide." Short oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers), and long oligonucleotides are expressed by the number of polymerized nucleotides, such as 30-mer or 100-mer. Oligonucleotides may be produced as fragments of longer polynucleotides or chemically synthesized.

Furthermore, the polynucleotide according to the present invention can be fused to a polynucleotide encoding the above-mentioned tag label (tag sequence or marker sequence) on its 5' or 3' side.

The invention further relates to variants of polynucleotides encoding the polypeptides according to the invention. Variants can occur naturally, such as natural allelic variants. By "allelic variant" is intended one of several interchangeable forms of a gene that occupies a given locus on the chromosome of an organism. Non-naturally occurring variants can be generated using, for example, mutagenesis techniques well known in the art.

Such variants include variants in which one or several bases are deleted, substituted, or added in the base sequence of the polynucleotide encoding the polypeptide of the present invention. Variants may be mutated in the coding or non-coding regions, or both. Mutations in the coding region can produce conservative or non-conservative amino acid deletions, substitutions, or additions.

The invention further provides an isolated polynucleotide comprising a polynucleotide encoding or hybridizing to a polypeptide of the invention under stringent hybridization conditions.

The above-mentioned "stringent conditions" mean that hybridization occurs only when there is at least 90% identity, preferably at least 95% identity, and most preferably 97% or more identity between the sequences. means something that happens.

The above hybridization can be performed by well-known methods such as those described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (the more difficult hybridization becomes), and the more homologous polynucleotides can be obtained.

As conditions for hybridization, conventionally known conditions can be suitably used, and are not particularly limited. For example, 42°C, 6x SSPE, 50% formamide, 1% SDS, 100 μg/ml salmon sperm DNA, 5x Denhardt's solution (1x SSPE; 0.18M sodium chloride, 10mM sodium phosphate, pH 7.7, 1mM EDTA. 5x Denhardt's solution; 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone).

The polynucleotide or oligonucleotide according to the present invention includes not only double-stranded DNA but also single-stranded DNA and RNA such as the sense strand and antisense strand that constitute it. Furthermore, DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. Furthermore, the polynucleotide or oligonucleotide according to the present invention may contain sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences).

A method for obtaining the polynucleotide or oligonucleotide according to the present invention includes a method of isolating and cloning a DNA fragment containing the polynucleotide or oligonucleotide according to the present invention using a known technique. For example, a probe that specifically hybridizes with a portion of the base sequence of the polynucleotide of the present invention may be prepared and a genomic DNA library or cDNA library may be screened. Such a probe may be of any sequence and/or length as long as it specifically hybridizes to at least a portion of the base sequence of the polynucleotide according to the present invention or its complementary sequence. good.

Alternatively, as a method for obtaining the polynucleotide according to the present invention, a method using an amplification means such as PCR can be mentioned. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide of the present invention, and these primers are used to template genomic DNA (or cDNA), etc. By performing PCR or the like to amplify the DNA region sandwiched between both primers, a large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained.

Examples are shown below to explain in detail the novel protein of the present invention and the polynucleotide encoding it.

[Example 1: cDNA cloning of BcBry-1 derived from Nezashihanemo]
<Method>
(Sample for RNA collection)
As a sample for RNA collection, Bryopsis corticulans algae collected in Ujina, Hiroshima City, Hiroshima Prefecture were shredded in RNAlater (Ambion) and stored at -30°C until use.

(Extraction of total RNA, purification of mRNA and synthesis of cDNA)
Total RNA was extracted from the above sample using PureLink Plant RNA Reagent (Invitrogen), and using the obtained total RNA as a sample, mRNA was purified using NucleoTrap mRNA (Macherey-Nagel). Furthermore, full-length cDNA was synthesized from the same mRNA using GeneRacer Kit (Invitrogen). All operations were performed according to the manual included with the kit.

(cDNA cloning of BcBry-1)
The Rapid Amplification of cDNA Ends (RACE) method targeting Bry-1 (BcBry-1) derived from Bryopsis corticulans was performed using GeneRacer KitTM (Invitrogen) and Blend Taq (registered trademark) (TOYOBO) according to the manual. First, degenerate primers Bry1-F1 and Bry1-F2 were designed with reference to the known N-terminal partial amino acid sequence of Bry-1 derived from Bryopsis sp., and 3' RACE was performed. First, a PCR product (annealing temperature 52°C) amplified using a primer pair of Bry1-F1 (5'- GGIGGITAYGTIATHAA-3'; SEQ ID NO: 5) and GeneRacer 3' primer was excised and purified after agarose gel electrophoresis. . Here, I is an IUB code that represents inosine, Y represents C or T, and H represents A, C, or T, respectively. Using this as a template, PCR amplification was further performed (annealing temperature: 64° C.) using the primer pair of Bry1-F2 (5'-ACITTYGAYGAYGCIACITAYGA-3'; SEQ ID NO: 6) and GeneRacer 3' nested primer as a nested primer. The obtained PCR product was inserted into the pGEM-T Easy vector, and then subjected to nucleotide sequencing using the BigDye Terminator v3.1 Cycle Sequencing kit in a conventional manner. As a result, two types of BcBry-1 with clearly different sequences, including the 3'-terminal sequence and the polyA tail, were obtained, so one was designated as BcBry1-1 and the other as BcBry1-2. In order to further determine the full-length nucleotide sequences of these two genes, primers BcBry1-1-3'endR (5'-GCCGCACAATGGAGAAAGCGATTAC-3'; SEQ ID NO: 7) were used from the 3' end sequences of BcBry1-1 and BcBry1-2. ) and BcBry1-2-3'endR (5'- CGCACGATGGAGAAACCAGTTAC-3'; SEQ ID NO: 8), and BcBry1-common-R1 (5'-ACTTTGTAAGCCGTGCACTTCTC-3'; SEQ ID NO: 9) from the sequence common to both BcBry1s. Designed. Perform 5'RACE using BcBry1-1-3'endR or BcBry-1-2-3'endR in 1st PCR and BcBry1-common-R1 in nested PCR, and perform 5'RACE using the nucleotide sequence as described above for the obtained PCR product. Submitted for decision. As a result, the 5' end sequences of BcBry1-1 and BcBry1-2 were determined. Primers BcBry1-1-5'endF (5'-ACACACTTTGCGAGCTCGTGTG-3'; SEQ ID NO: 10) and BcBry1-2-5'endF (5'-ACAGTGTCAACATGAAGCTGACAGCC-3) were prepared from the 5' end sequences of BcBry1-1 and BcBry1-2, respectively. '; SEQ ID NO: 11) was designed using the primer pair of BcBry1-1-5'endF and BcBr1-1-3'endR, and the primer pair of BcBry1-1-5'endF and BcBr1-1-3'endR. Then, PCR was performed using a high-accuracy DNA polymerase, KOD plus Neo (TOYOBO), using Bryopsis corticulans-derived cDNA as a template. As a result, DNA fragments encoding the full length cDNAs of BcBry1-1 and BcBry1-2 were obtained, and their nucleotide sequences were confirmed using conventional methods.

<Results>
(cDNA sequence and amino acid sequence of BcBry-1)
Sequence information of the obtained full-length BcBry1-1 and BcBry1-2 cDNAs are shown in FIGS. 1 and 2, respectively. Furthermore, the mature protein region was estimated from the comparison with known N-terminal amino acids of Bryopsis sp.-derived Bry-1 and from the signal peptide sequence prediction results using SignalPprogram. The amino acid sequences obtained from the above BcBry1-1 and BcBry1-2 cDNAs are also shown in FIGS. 1 and 2, respectively. As shown in Figure 1, the full-length cDNA of BcBry1-1 each has a length of 575 nucleotides (the full-length cDNA sequence is SEQ ID NO: 12), and the 81st to 83rd nucleotides are the start codons, and 438 The ~440th nucleotide becomes the stop codon. The 81st to 440th nucleotide sequence of the cDNA is designated as SEQ ID NO: 1, and the amino acid sequence of the protein obtained from this nucleotide sequence is designated as SEQ ID NO: 3. On the other hand, as shown in Figure 2, the full-length cDNAs of BcBry1-2 each have a length of 495 nucleotides (the full-length cDNA sequence is SEQ ID NO: 13), and the 12th to 15th nucleotides are the start codons. , 369th to 371st nucleotides become the stop codon. The 12th to 371st nucleotide sequence of the cDNA is designated as SEQ ID NO: 2, and the amino acid sequence of the protein obtained from this nucleotide sequence is designated as SEQ ID NO: 4.

As shown in FIGS. 1 and 2, the mature protein regions of BcBry1-1 and BcBry1-2 both consisted of 100 amino acid residues, and their amino acid identity was 83%. As a result of a BLAST search, both BcBry1s showed no homology not only with known lectins but also with known proteins. In other words, it was revealed that the protein has an extremely novel primary structure.

[Example 2: Construction of E. coli expression system for BcBry1-1]
<Method>
(Preparation of expression construct pCold-rBcBry1-1)
A DNA encoding BcBry1-1 optimized for E. coli codons was designed and synthesized, and a primer BcBry1-1_pCold_F (5'-ATCATCATCATCATATATCGAAGGTAGGTCCCGCACAATTACTGTGTTC -3'; PCR was performed using BcBry1-1_pCold_R (5'-AGATTACCTATCTAGCTAGCATGTGTAGGCCCATG-3'; SEQ ID NO: 14) and Platinum Pfx DNA polymerase (Invitrogen) as a DNA polymerase. The obtained amplification product was inserted into pCold II DNA (Takara Bio), which had been linearized by restriction enzymes XbaI and NdeI, using the In-FusionAdvantage PCR Cloning Kit (Clontech), and the nucleotide sequence was confirmed. The construct pCold-rBcBry1-1 was constructed.

(Preparation of His-rBcBry1-1)
E. coli strain SHuffle Express was transformed using the above expression construct pCold-rBcBry1-1 to obtain a recombinant BcBry1-1 expression strain (SHuffle Express/pCold-BcBry1-1). For the same expression strain, 1/20 volume was added to 3 ml of LB/Amp ⁺ liquid medium, and cultured with shaking at 37°C until the mid-logarithmic growth phase. When the OD ₆₀₀ reached 0.5, the culture solution was allowed to stand at 15° C. for 30 minutes, and then IPTG was added to a final concentration of 0.1 mM, followed by shaking culture at 15° C. for 24 hours to induce expression. After expression induction, the culture solution was centrifuged (20,400 x g, 5 minutes, 4°C), the bacterial cells were collected, and 1/20 volume of the culture solution was added with an ultrasonic disruption buffer (50mM Tris-HCl (pH 8.0), 150 mM NaCl) and sonicated. This was centrifuged again (20400×g, 20 minutes, 4° C.), and the supernatant was classified as a soluble fraction and the residue as an insoluble fraction. Regarding the soluble fraction, His-rBcBry1-1 was purified using nickel chelate beads MagExtractor (registered trademark) His-tag (Toyobo), and this was used as a His-tag purified specimen.

(Study of enzymatic digestion conditions using Factor Xa)
To prepare rBcBry1-1 from the His-tagged fusion protein His-rBcBry1-1, enzymatic digestion with Factor Xa was performed. First, the purified specimen His-rBcBry1-1 was sufficiently dialyzed against a dialysis buffer (50mM Tris-HCl (pH 8.0), 100mM NaCl). This dialyzed fluid was centrifuged (20,400 xg, 20 minutes, 4°C), and the resulting supernatant His-rBcBry1-1 was collected. In order to determine the optimal conditions for the enzyme reaction from this supernatant, the amount of enzyme added and the reaction time were investigated. Prepare 50 μl of enzyme reaction solution (10 μg of His-rBcBry1-1, 5 μl of 10× Factor Xa treatment buffer, 1 μl of Factor Xa (0.1, 0.2, 0.5 U), 24 μl of sterile water), Enzyme treatment was carried out at 20°C and 37°C, and at 37°C the enzyme addition amount was Factor Xa (0.1U, 0.2U). In addition, a sample without the addition of Factor Xa was prepared under both temperature conditions and subjected to the same treatment to serve as a negative control. From this reaction solution, 10 μl each was collected over time (2, 4, 8, and 16 hours) and subjected to SDS-PAGE.

(Detection of His-rBcBry1-1 by Western blotting using anti-His tag antibody)
The prepared His-rBcBry1-1 was subjected to a sugar chain immobilization array to comprehensively investigate the binding with sugar chains.An anti-His tag antibody, Anti-His-tag-Alexa Fluor, was used as a labeled antibody to detect the recombinant. (Registered Trademark) 647 (Medical and Biological Research Institute) was tested by Western blotting. That is, purified His-rBcBry1-1, recombinant Kappaphycus alvarezii agglutinin 1 (His-rKAA1) with a His tag on the N-terminal side, which was preserved in our laboratory as a positive control, and His-rKAA1 with a His tag as a negative control. Using the removed rKAA, each sample was prepared in an amount equivalent to 5 μg and subjected to SDS-PAGE under non-reducing conditions. Proteins were transferred from a different electrophoresis gel than that stained with CBB to a PVDF membrane using a wet transfer device Mini Trans-Brot Cell Module (Bio-Rad). After transfer, the membrane was soaked in 5% blocking buffer (5% skim milk, TBS-T (20mM Tris-HCl (pH 8.0), 0.15M NaCl, 0.05% Tween20)) and gently incubated at room temperature for 1 hour. I shook it. Next, the plate was rinsed twice with TBS-T, and washed once with TBS-T for 15 minutes and twice for 5 minutes. After washing, add 3 ml of Anti-His-tag-Alexa Fluor 647 diluted to 1 μg/ml with 2.5% blocking buffer (2.5% skim milk, TBS-T), shake at room temperature for 1 hour, Antibody was reacted. After the reaction was completed, the plate was washed with TBS-T by shaking for 10 minutes three times. After the membrane was air-dried, the presence or absence of detection by the anti-His tag antibody was examined using a detector Typhoon FLA 7000 (GE Healthcare) at an excitation wavelength of 633 nm.

<Results>
(Expression and preparation of His-rBcBry1-1)
A liquid volume of approximately 450 μl was obtained for the soluble fraction of the E. coli strain and approximately 200 μl for the purified sample, and clear hemagglutination activity was detected in the soluble fraction and the His-tagged purified sample. For evaluation of hemagglutination activity, 25 μL of each fraction was serially diluted 2-fold with physiological saline to each well of a 96-well plate, and an equal volume of 2% red blood cell suspension was added to each fraction. After addition, the mixture was gently shaken, incubated at room temperature for 60 minutes, and hemagglutination activity was visually observed. Furthermore, the His-tagged purified sample gave a single band at the expected molecular weight position in non-reduced and reduced SDS-PAGE, confirming the expression of His-rBcBry1-1 (FIG. 3). In FIG. 3, lane M is a molecular weight marker, lane 1 is a cell lysate before expression induction, lane 2 is a cell lysate after expression induction, lane 3 is a soluble fraction, lane 4 is an insoluble fraction, and lane 5 is a cell lysate. is the non-adsorbed fraction, lanes 6 and 7 are the washing solution, lane 8 is the His-tag purified sample (reduced), and lane 9 is the His-tag purified sample (non-reduced). Moreover, the yield of His-rBcBry1-1 was 40 μg per 9 ml of the medium, and the minimum aggregation concentration was 1.55 μg/ml. In addition, when expression was induced by scaling up separately, 240 μg of His-rBcBry1-1 was obtained per 200 ml of culture solution.

(Study of enzymatic digestion conditions using Factor Xa)
In order to remove the His tag portion of His-rBcBry1-1, we tried treating it with Factor Xa, but it was not cleaved at all at 20°C under any conditions ((a) to (d) in FIG. 4). In Figure 4, (a) is untreated with Factor Xa at 20°C, (b) is treated with 0.1U/50μl of Factor Treatment with Factor Xa, (d) treatment with 0.5 U/50 μl of Factor Xa at 20°C, (e) treatment with 0.2 U/50 μl of Factor Xa at 37°C, (f) treatment with 0.5 U/50 μl of Factor Xa at 37°C. These are the results under conditions of treatment with 5U/50 μl of Factor Xa. Furthermore, although the enzyme treatment was carried out at a reaction temperature of 37°C, it was found that the fusion protein portion was not cleaved as in the case of 20°C ((e) and (f) in FIG. 4). The reason why the fusion protein portion was not cleaved was considered to be that the Factor Xa recognition sequence present in His-rBcBry1-1 could not be recognized or contacted due to steric interference.

(Detection of His-rBcBry1-1 by Western blotting using anti-His tag antibody)
Since His-rBcBry1-1 has a Factor Xa recognition sequence fused to the N-terminus of the lectin coding region and a His tag further upstream, it is expected that the recombinant can be detected using a fluorescently labeled anti-His tag antibody. Ta. Therefore, we investigated the reactivity between His-rBry1-1 and anti-His tag antibody by Western blotting, but although a clear signal was obtained with His-rKAA1 used as a positive control, similar to rKAA1 used as a negative control, No signal was obtained for His-rBcBry1-1 (Fig. 5). In FIG. 5, lane M is a molecular weight marker, lane 1 is His-rBcBry1-1, lane 2 is rKAA1, and lane 3 is His-rKAA1. Combined with the above-mentioned result that tag cleavage by Factor Xa was not possible, it was considered that there may be difficulty in contacting the enzyme/antibody with their respective recognition sites.

[Example 3: Construction of an E. coli expression system for BcBry1-1 with different His tag fusion positions]
<Method>
(Preparation of expression constructs pET28a-rBcBry1-1N and pET28a-rBcBry1-1C)
Based on the above results, we created His-rBcBry1-1N with a linker inserted between the N-terminal His tag and the lectin region, and the His tag fusion position so that the tag-cleaving enzyme and the tag-recognizing antibody can contact their respective recognition sites. An attempt was made to prepare His-rBcBry1-1C in which Bry1-1C was placed on the C-terminal side. First, the rBcBry1-1 coding region was amplified using the BcBry1-1 coding synthetic DNA optimized for E. coli codons as a template and the following primer pair. That is, for His-rBcBry1-1N expression, pET_rBcBry1-1_F (5'- CGCGCGGCAGCCATATGAGCCGTACCATTACCGTGTT -3'; SEQ ID NO: 16) and pET_rBcBry1-1_R (5'-GGTGGTGGTGCTCGATTAG CAGGTATATGCCCACG-3'; SEQ ID NO: 17), His-rBcBry1- For 1C expression, pET_rBcBry1-1_CF (5'-AGGAGATATACCATGAGCCGTACCATTACCGTGTT-3'; SEQ ID NO: 18) and pET_rBcBry1-1_CR (5'-GGTGGTGGTGCTCCAGGCTGCCGCGCGGCACCA Amplification was performed by PCR using GACCACCACCACCGCTGCAGGTATATGCCCACGGAT -3'; SEQ ID NO: 19). Next, the expression vector pET28a (Novagen) was treated with restriction enzymes NdeI and XhoI for the His-rBcBry1-1N expression construct, and with restriction enzymes NcoI and XhoI for the His-rBcBry1-1C expression construct. Expression constructs pET28a-rBcBry1-1N and pET28a were created using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) using linearized pET28a and the PCR amplification product containing the coding region of each BcBry1-1 described above. -rBcBry1-1C was created.

(Preparation of E. coli strain expressing His-rBcBry1-1N and His-rBcBry1-1C)
E. coli strain SHuffle T7 Express was transformed using the constructed expression construct. An appropriate amount of the transformant was spread on an LB/Kan ⁺ agar medium and cultured overnight at 37°C. Correct insertion of the insert was confirmed by insert checking and sequencing of the obtained colonies. The corresponding clones were cultured in LB/Kan ⁺ liquid medium to obtain His-rBcBry1-1 expression strains SHuffle T7Express/pET28a-rBcBry1-1N and SHuffle T7 Express/pET28a-rBcBry1-1C, and 1/2 volume of 40% glycerol was added ( final concentration 20%) and stored at -80°C until use.

(Large expression of His-rBcBry1-1N and His-rBcBry1-1C)
The above-mentioned SHuffle T7 Express/pET28a-rBcBry1-1N and SHuffleT7 Express/pET28a-rBcBry1-1C expression strains were inoculated into 1 L of LB/Kan ⁺ liquid medium and cultured with shaking at 37°C until the mid-logarithmic growth phase was reached. The cells were cultured until the OD ₆₀₀ was between 0.5 and 0.8, and expression induction was started by adding IPTG to a final concentration of 0.5 mM, followed by shaking culture at 20° C. for 12 hours. After culturing, collect the bacteria by centrifugation (10,000 x g, 4°C, 10 minutes), and add 1/20 volume of ultrasonic disruption buffer (20mM phosphate buffer (pH 7.4), 500mM NaCl, After suspending in 20mM imidazole), ultrasonic disruption was performed. After sonication, the mixture was centrifuged (10,000×g, 4° C., 30 minutes), and the supernatant was collected as a soluble fraction and the residue as an insoluble fraction.

(Purification using nickel chelate column)
The His-tagged recombinant contained in the previously prepared soluble fraction was purified using a nickel chelate column His Gravi Trap (GE Healthcare Bioscience). After fully equilibrating the column with an equilibration buffer (20mM phosphate buffer (pH 7.4), 500mM NaCl, 20mM imidazole), the soluble fraction was added, and the column carrier and His-rBcBry1-1N and His-rBcBry1 were separated. -1C was combined. After thoroughly washing the column with an equilibration buffer, an elution buffer (20mM phosphate buffer (pH 7.4), 500mM NaCl, 500mM imidazole) was added to elute the recombinant. The eluted fraction was thoroughly dialyzed against a buffer solution (300 mM arginine hydrochloride, 50 mM Tris (pH 8.0), 100 mM NaCl) to obtain a purified sample.

<Results>
(Yield of purified drift product and its hemagglutination activity)
Expression of SHuffleT7 Express/pET28a-rBcBry1-1N and SHuffle T7Express/pET28a-rBcBry1-1C is each induced with 1 L of LB/Kan ⁺ , and after ultrasonic disruption, the soluble fraction is subjected to a nickel chelate column for purification and dialysis. As a result, a refined specimen was obtained. When the protein yield and hemagglutination activity of the purified sample were measured, 0.96 mg and 512 HA activities were confirmed for rBcBry1-1N, and 2.52 mg and 128 HA activities were confirmed for rBcBry1-1C (Table 1). . In addition, protein yield was determined by measuring absorbance to UV 280 nm. Specifically, if the absorbance at 280 nm is 1.0, the protein concentration is estimated to be 1 mg/mL, or using bovine serum albumin (BSA) as a standard, the Pierce BCA Protein Assay Kit (Thermo FisherScientific, IL, USA) is used. ), and the hemagglutination activity was measured in the same manner as above. When this result was compared with the yield of His-rBcBry1-1 obtained by expressing the SHuffle Express/pCold-rBcBry1-1 strain described above (240 μg per 200 ml of culture solution), it was found that using the pET system and E. coli strain SHuffle T7 Express. No dramatic increase in yield was observed.

[Example 4: Analysis of sugar chain binding specificity of BcBry1-1 using sugar chain immobilized array]
<Method>
(Detection by Western blotting using anti-His tag antibody)
The prepared His-rBcBry1-1N or His-rBry1-1C was subjected to a sugar chain immobilization array to comprehensively investigate the binding with sugar chains.Anti-His tag antibody Anti- Western blotting was used to test whether His-tag-Alexa Fluor 647 (Medical and Biological Research Institute) could be used. That is, His-rBcBry1-1N and His-rBcBry1-1C were targeted, His-rKAA was used as a positive control, and rKAA was used as a negative control. Each sample was prepared in an amount equivalent to 5 μg and subjected to SDS-PAGE under non-reducing conditions. Western blotting was performed in the same manner as described above.

(Glycan immobilized array)
His-rBcBry1-1C contains 7 types of N-glycan, 2 types of O-glycan, 5 types of glycosaminoglycan (GAG), 4 types of Lewis type, 7 types of Lac, and 3 types of ABO type, total 28 types. The oligosaccharide immobilized array (Fig. 6, Sumitomo Bakelite) was used to immobilize the type of sugar chain, and the anti-His tag antibody Anti-His-tag-Alexa Fluor 647 (Medical and Biological Research Institute) was used as a detection reagent. That is, rBcBry1-1C was diluted with a reaction buffer (50mM Tris-HCl (pH 7.5), 100mM NaCl, 1mM CaCl ₂ , 1mM MnCl ₂ , 1mM MgCl ₂ , 0.05% (v/v) Tween 20) (final concentration 83.9 μg/ml) was dropped onto the detection plate. After covering with a cover and incubating for 2 hours at room temperature, the cover was shaken off in ultrapure water, and then washed with washing buffer (50mM Tris-HCl (pH 7.5), 100mM NaCl, 1mM CaCl ₂ , 1mM MnCl ₂ , 1mM Wash gently in MgCl ₂ ) for 1 minute. Furthermore, after repeating the operation of gently washing for 1 minute with ultrapure water twice, the array slide was dried with air blow. Anti-His-tag-Alexa Fluor 647 was diluted with reaction buffer to 1 μg/ml, and an appropriate amount was dropped onto a detection plate and incubated at room temperature for 1 hour to react with the antibody. After the reaction was completed, the cover was shaken off in ultrapure water, and then gently washed in a washing buffer for 1 minute. Furthermore, after repeating the operation of gently washing for 1 minute with ultrapure water twice, the array slide was dried with air blow. Fluorescence detection and data analysis were outsourced to Sumitomo Bakelite.

<Results>
(Detection of His-rBcBry1-1N and C by Western blotting using anti-His tag antibody)

Western blotting was used to confirm whether Anti-His-tag-AlexaFluor 647 could be used as a detection antibody when His-rBcBry1-1N or C was applied to a sugar chain immobilized array. The detection results are shown in FIG. In FIG. 7, lane M is a molecular weight marker, lane 1 is His-rBcBry1-1N, lane 2 is His-rBcBry1-1C, lane 3 is His-rKAA, and lane 4 is rKAA. As shown in FIG. 7, detection of rKAA, which was used as a negative control, was not confirmed, but detection of His-rKAA, His-rBcBry1-1N, and His-rBcBry1-1C, which were used as positive controls, was confirmed. Therefore, it was shown that Anti-His-tag-Alexa Fluor 647 can be used to detect His-rBcBry1-1N and C. Furthermore, although the same amount of protein was added, His-rBcBry1-1C was detected more strongly than His-rBcBry1-1N, and His-rBcBry1-1 (pCold vector Considering that it could not be detected using the expression method (expressed using a glycoprotein), we decided to use His-rBcBry1-1C for the sugar chain immobilization array test.

(Glycan immobilization array test)
His-rBcBry1-1C was applied to a carbohydrate immobilized array (Sumitomo Bakelite) using Anti-His-tag-Alexa Fluor 647 as a detection reagent. The results are shown in FIG. As shown in FIG. 8, a significant signal was observed for heparin, and weak signals were also observed for De2S Hep, De6S Hep, and DeNS/AcHep. That is, binding was observed only in GAGs, and the signal particularly showed a tendency to decrease due to elimination of sulfate groups, suggesting that rBcBry1-1 recognizes the sulfate group portion of GAGs.

Claims (6)

Encodes a sulfated glycosaminoglycan sugar chain-binding protein,
A polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 1 . The polynucleotide according to claim 1, which is derived from the genus Bryopsis. The polynucleotide according to claim 2, which is derived from Bryopsis corticulans. Has sulfated glycosaminoglycan sugar chain binding properties,
A novel protein characterized by comprising the amino acid sequence set forth in SEQ ID NO: 3 , or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 3. . The novel protein according to claim 4, which is derived from the genus Bryopsis. The novel protein according to claim 5, which is derived from Bryopsis corticulans.