JP5531290B2 - gene - Google Patents

Description

  The present invention relates to a gene encoding a protein or peptide having sugar recognition activity, and more particularly to a gene encoding a protein or peptide that specifically recognizes α1 → 6 fucose and a genetic engineering production method thereof.

  The fucose α1 → 6 transferase (α1 → 6FucT) gene, which transfers fucose as α1 → 6 linkage to N-acetylglucosamine at the reducing end of the N-linked sugar chain, may be expressed as hepatocytes become cancerous. Are known. The presence or absence of cancerous hepatocytes can be detected by lectin affinity electrophoresis using lentil lectin (LCA) having affinity for fucose.

  Conventionally, as a lectin having an affinity for fucose α1 → 6 sugar chain, in addition to the above LCA, pea lectin (PSA), white bamboo lectin (AAL), daffodils lectin (NPA), broad bean lectin (VFA), bacilli Lectin (AOL) etc. are known (patent documents 1 to 5).

  However, the above-mentioned known lectin has affinity for a glycolipid sugar chain having fucose other than α1 → 6 bond in addition to fucose α1 → 6 sugar chain, and fucose α1 → 6 having three or more chains. No affinity is shown for sugar chains. Specifically, AAL and AOL have affinity for fucose α1 → 2, fucose α1 → 3, and the like. LCA, PSA and VFA do not have affinity for fucose α1 → 6 sugar chain of 3 or more chains.

  The present inventors have discovered a novel lectin derived from basidiomycetes and specifically binding to fucose α1 → 6 sugar chain, and can be used for its production method and sugar chain detection and separation method using the lectin. (Patent Document 6).

WO2002 / 030954 WO2003 / 084569 Examples of JP-A-02-083337 Example 5 of JP-A-2002-112786 JP2007-161633 PCT / JP2009 / 003346

  If the α1 → 6 fucose-specific lectin obtained from natural products by the present inventors can be stably produced as a recombinant protein using genetic engineering techniques, the utilization of the α1 → 6 fucose-specific lectin can be improved. To do. Therefore, an object of the present invention is to provide a gene encoding an α1 → 6 fucose-specific lectin newly isolated from basidiomycete by the present inventors, and a protein or a protein that specifically recognizes α1 → 6 fucose based on the gene. It is to provide a method for producing a peptide by genetic engineering.

In order to solve the above-mentioned problems, the present inventors have developed a Rapid Amplification of cDNA end method (hereinafter referred to as RACE method) based on a partial amino acid sequence of a lectin derived from Tsutsugitake (hereinafter referred to as PTL). The full-length cDNA of the gene encoding the lectin was cloned and the nucleotide sequence was successfully determined. That is, the present invention relates to a gene encoding the following protein or peptide (1) or (2):
(1) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 A protein or peptide consisting of a sequence,
(2) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 Provided is a protein or peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence and having sugar recognition activity.

  The gene encoding the amino acid sequence of SEQ ID NO: 2 is represented by SEQ ID NO: 1, for example.

  The present invention also provides a recombinant vector containing the gene.

  The present invention also provides a transformant obtained by introducing the above recombinant vector into a host.

  The present invention also provides a recombinant protein or peptide expressed from the transformant.

  The present invention is also a method for producing a recombinant protein or peptide, characterized by culturing or growing the transformant.

  The present invention also provides a recombinant protein or peptide characterized in that the recombinant vector is subjected to a cell-free protein synthesis system to express the recombinant protein or peptide (ie, in vitro transcription and translation). Is the method.

  The present invention is also a method for producing a recombinant protein or peptide, which comprises subjecting the recombinant protein or peptide obtained above to affinity purification via a tag sequence and / or sugar recognition activity.

  According to the present invention, a gene encoding PTL is provided. If a gene is used, mass production of PTL becomes possible. Since PTL can specifically detect fucose α1 → 6 sugar chains, it includes tumor markers related to fucose α1 → 6 sugar chains, including fucose α1 → 6 sugar chain specific binding agents and sugar chain research reagents. It is also useful for accurate detection, prognosis determination, therapeutic effect determination, and search for new tumor markers. The present invention makes it possible to produce a recombinant protein or peptide having one or more sugar recognition sites. Since the aggregation of cells or glycoproteins can be controlled by changing the number of sugar recognition sites, it can be applied to detection techniques that have been difficult with conventional lectins.

A full-length sequence diagram of the PTL gene is shown. Agarose electrophoresis of PCR products is shown. The SDS electropherogram of rPTL7 is shown. The SDS electropherogram of rPTL8-10 is shown. The SDS electropherogram of rPTL1-6 is shown. The glycoprotein array figure of rPTL is shown. The glycoprotein array by Strep * Tag II removal rPTL is shown. The structural diagram of PA sugar chain is shown. The structural diagram of PA sugar chain is shown. SRL gene full-length sequence. 1 shows an SDS electropherogram of rSRL. Figure 2 shows a glycoprotein array of rSRL.

Hereinafter, an embodiment of the present invention will be described in detail.
(A. Tsutsugitake lectin)
Tsutsugitake is a type of mushroom belonging to the family Basidiomyceae Moegitake. Lectin is a general term for proteins that exist widely in the living world and specifically recognize and bind to sugars. Even when only one sugar recognition site is present in the intramolecular subdomain, many molecules form multimers and thus have the ability to form crosslinks via sugar chain molecules.

The present inventors extracted and purified PTL from the fruit body of Tsutsugitake, and revealed its physical properties and amino acid sequence at the N-terminal (Patent Document 6). According to this, PTL has a molecular weight of 4,000 to 40,000 by sodium dodecyl sulfate electrophoresis (hereinafter referred to as SDS electrophoresis), and has a binding constant of 1. It is 0 × 10 ⁴ M ⁻¹ or more. PTL also has high affinity for those having sialic acid at the non-reducing end of the fucose α1 → 6 sugar chain. Conventional fucose α1 → 6 specific lectins (eg, LCA, NPA and PSA) have low affinity for fucose α1 → 6 sugar chains having sialic acid at the non-reducing end. This means that PTL is superior to conventional lectins.

(B. Cloning of PTL gene)
A gene encoding PTL (hereinafter referred to as a PTL gene) can be cloned from Tsutsugitake cDNA or a cDNA library according to a well-known method based on the amino acid sequence of the N-terminal portion already specified. For example, a polymerase chain reaction (hereinafter referred to as PCR) is performed on cDNA using a primer prepared from the above partial amino acid sequence, and a target PTL gene is obtained from the Tsutsugitake cDNA library using the obtained amplified fragment. Cloning. Alternatively, the target PTL gene is cloned from cDNA using the RACE method without preparing a cDNA library. In general, it is often difficult to obtain a full-length cDNA from a cDNA library, and here, cloning by the RACE method will be described.

1. Extraction of mRNA First, mRNA is prepared from Tsutsugitake. The supply source of mRNA may be a part of the fruiting body such as an umbrella, a fungus, a mycelium, a fungus pattern, a footpad, a mycelium, or the like, but the whole fruiting body is preferable.

Extraction of mRNA can be performed by a commonly performed technique. For example, the whole fruit body is frozen with liquid nitrogen. Next, after the frozen fruit body is pulverized, a nucleic acid is extracted by adding a trisol reagent (manufactured by Invitrogen) or the like. Or you may extract using the MACS mRNA isolation kit (product made from Miltenyi Biotec) etc. which are commercially available kits. The extracted nucleic acid is treated with chloroform, a phenol reagent or the like to obtain total RNA. Then, using an oligo (dT) magnetic bead and magnet, an affinity column using oligo (dT) -cellulose or the like, or a poly (A) ⁺ isolation kit (made by NIPPON GENE) from total RNA, mRNA (Poly (A) ⁺ RNA) is prepared.

2. Synthesis of cDNA Using the thus obtained mRNA as a template, a commercially available kit such as Marathon (registered trademark) cDNA amplification kit (manufactured by Clontech) or the like is used to make a single strand with an oligo (dT) primer and reverse transcriptase. After the cDNA is synthesized, a double-stranded cDNA is synthesized from the single-stranded cDNA.

3. Acquisition of full-length cDNA by RACE method After adding an appropriate adapter to the obtained double-stranded cDNA, an adapter primer and a gene-specific primer as shown in Table 6 (Gene Specific Primer, hereinafter referred to as GSP) ) To perform PCR. The nucleotide sequences at the ends of the 5′-RACE product and 3′-RACE product obtained are determined, 5′-side and 3′-side GSPs are newly prepared from the sequences, PCR is performed, and the desired full length is obtained. Obtain cDNA.

  In the case of PTL, after a primary PCR reaction using a degenerate primer and an adapter primer prepared based on the amino acid sequence at the N-terminus, a second PCR using a nested degenerate primer and a nested-adapter primer prepared based on the sequence inside the degenerate primer is used. Next, a target PTL cDNA is amplified by performing a PCR reaction.

4). Determination of the base sequence The obtained PTL cDNA is commercially available, such as direct sequencing method, pGEM-T easy vector (Promega), pBlue Script SK (+) (Stratagene), pCR2.1 (Invitrogen). The nucleotide sequence is analyzed by the cycle sequencing method of subcloning into the plasmid vector. The base sequence can be determined by a known method such as a chemical modification method of Maxam-Gilbert and a dideoxynucleotide chain termination method using M13 phage. A method using an automatic base sequence analyzer (manufactured by BECKMAN COULTER: CEQ ^™ 8000 Genetic Analysis System, etc.) is simple and preferred.

(C. PTL gene)
The PTL gene (cDNA) isolated by the above method has a base sequence consisting of 695 bases shown in SEQ ID NO: 1. The base sequence of SEQ ID NO: 1 is converted into 180 amino acid residues shown in SEQ ID NO: 2 based on the codon table. FIG. 1 shows the characteristics of the amino acid sequence. In FIG. 1, the partial sequences of CRD1, CRD2, and CRD3 regarded as sugar recognition domains (Carbohydrate Recognition Domain) have very high homology with the analysis result of N-terminal amino acid sequence of natural PTL (40 amino acid residues) (80 %that's all). Furthermore, the homology between these peptides is also very high as shown in Table 1.

  As described above, CRD1, CRD2, and CRD3 are highly homologous to the N-terminal amino acid sequence of lectin derived from Tsutsugitake and other CRDs. Further, as shown in Examples 5 and 7, it has been found that all of CRD1, CRD1 + CRD2, CRD1 + CRD2 + CRD3, CRD2, CRD2 + CRD3, and CRD3 are proteins or peptides that are specifically recognized by α1 → 6 fucose.

Therefore, the gene of the present invention can be defined as a gene encoding the following protein or peptide (1) or (2).
(1) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 A protein or peptide consisting of a sequence,
(2) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 A protein or peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence and having sugar recognition activity.
Table 2 shows the meaning of the range of the amino acid sequence and the base sequence.

  The PTL gene of the present invention is any of SEQ ID NO: 1, 20 to 562, 113 to 232, 113 to 397, 113 to 541, 278 to 397, 278 to 541, and 422 to 541. As long as it encodes the PTL protein having the sugar recognizing activity of the present invention, even other polynucleotides capable of hybridizing under stringent conditions with the polynucleotide having the base sequence represented by Included in the gene. Here, the stringent condition refers to, for example, a condition where the sodium concentration is 30 mM and the temperature is 65 ° C., preferably the sodium concentration is 10 mM and the temperature is 65 ° C.

  In the present specification, the term “gene” includes not only DNA but also mRNA, cDNA and cRNA thereof. Therefore, the gene of the present invention always includes both the coding strand shown in the sequence listing and its complementary strand, and includes all of DNA, mRNA, cDNA, and cRNA having the base sequence. The base sequences in the sequence listing are all described as DNA sequences, but when the sequences represent RNA, the base symbol “t” in the sequence listing shall be read as “u”.

  Once the PTL gene and its base sequence are determined, then the PTL gene can be obtained by chemical synthesis, PCR using genomic DNA as a template, or by hybridizing a DNA fragment having the base sequence as a probe. It is easy for those skilled in the art.

(D. Production of recombinant PTL protein)
The present inventors have developed an effective purification method for this PTL (Patent Document 6). However, since the method uses the natural tsutsutake mushroom as a raw material, the problem of securing the raw material due to the weather and seasons was included.

  In addition to its application to sugar chain research reagents, PTL is used for the accurate diagnosis of tumor markers and the search for new tumor markers in cancer medicine from the knowledge that fucose α1 → 6 sugar chains increase with canceration. Is also expected to be applicable. Therefore, if PTL can be mass-produced by genetic engineering, it becomes an extremely efficient method for obtaining the lectin. Hereinafter, a method for producing a recombinant protein or peptide using the gene of the present invention will be described.

1. Production of Recombinant Vector A recombinant vector containing a PTL gene can be obtained by linking (inserting) a PTL gene into a known vector. The vector is not particularly limited as long as it can be replicated in a host, and for example, a plasmid vector, a phage vector, a virus vector and the like can be used.

  Examples of the plasmid vector include plasmids derived from E. coli (eg, pBR322, pUC18, pUC119, pTrcHis, pBlueBacHis, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.), and yeast-derived plasmids (eg, YEp13, YEp24, YCp50, pYE52). Etc.). Examples of the phage vector include λ phage and the like. Furthermore, animal virus vectors such as retrovirus and vaccinia virus, insect virus vectors such as baculovirus, plant virus vectors such as potato ex virus, and the like may be used.

  For the insertion of the PTL gene into the vector, first, a method is used in which the purified DNA is cleaved with an appropriate restriction enzyme and inserted into an appropriate restriction enzyme site or a multicloning site of the vector DNA and ligated to the vector. .

  The PTL gene needs to be incorporated into a vector so that the function of the gene can be suitably exerted. Therefore, in addition to the PTL gene, the vector can contain a promoter, a cis element such as an enhancer, if necessary, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like. . Examples of selection markers include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.

  The recombinant vector may be a PTL gene expressed directly from the start codon or may be expressed as a fusion protein. Furthermore, a DNA fragment containing the PTL gene may be expressed.

2. Production of Transformant A transformant introduced with a PTL gene can be obtained by introducing the above-described recombinant vector into a host in such a manner that the PTL gene can be expressed. The host is not particularly limited as long as it can express the PTL gene. For example, the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas putida such as Pseudomonas putida, and the genus Pseudomonas i Bacteria, yeasts such as Saccharomyces cerevisiae, Saccharomyces pombe (S. pombe), animal cells such as monkey cells (COS cells), Chinese hamster ovary cells (CHO cells), insect cells such as Sf19 and Sf21 , Insects such as Bombyx mori, soybean (Glycine max), duckweed (Spi) a plant such as rodella polyrhiza).

  When a bacterium such as Escherichia coli is used as a host, it is desirable that the recombinant vector is capable of autonomous replication in each bacterium and is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained. Examples of Escherichia coli include Escherichia coli K12, DH1, and TOP10F. Examples of Bacillus subtilis include B. subtilis MI114 and 207-21.

  The promoter is not particularly limited as long as it can be expressed in a host such as Escherichia coli. For example, E. coli-derived promoters such as trp promoter, lac promoter, PL promoter, PR promoter and the like can be used. An artificially designed promoter such as a tac promoter may be used.

  The method for introducing the recombinant vector into bacteria such as Escherichia coli is not particularly limited as long as it is a method capable of introducing DNA into bacteria. For example, a method using calcium ions (Cohen, SN et al .: Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)), an electroporation method, and the like can be given.

  When yeast is used as a host, for example, S. cervisiae, S. pombe, Pichia pastoris and the like are used. The promoter is not particularly limited as long as it can be expressed in yeast. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX promoter and the like can be mentioned.

  The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method capable of introducing DNA into yeast. For example, electroporation method (Becker, DM et al .: Methods. Enzymol., 194: 180 (1990)), spheroplast method (Hinnen, A. et al .: Proc. Natl. Acad. Sci. USA, 75: 1929 (1978)), the lithium acetate method (Itoh, H .: J. Bacteriol., 153: 163 (1983)), and the like.

  When animal cells are used as hosts, monkey cells (COS-7), Vero, Chinese hamster ovary cells, mouse L cells, rat GH3 cells, human FL cells, and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like are used.

  Examples of methods for introducing the recombinant vector into animal cells include electroporation, calcium phosphate, and lipofection.

  When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As the promoter, polyhedrin promoter, p10 promoter or the like is used.

  As a method for introducing the recombinant vector into insect cells, for example, an electroporation method, a calcium phosphate method, a lipofection method, a baculovirus method, or the like is used.

  Silkworms are used when insects are used as lodging species. As the promoter, a polyhedrin promoter, an artificially designed E-vp39pu romotor, or the like is used.

  As a method for introducing a recombinant vector into an insect, for example, a baculovirus method, microinjection into an egg, or the like is used.

  When a plant is used as the seed, soy, duckweed, corn and the like are used. As the promoter, 35S promoter, NOS promoter and the like are used.

  As a method for introducing a recombinant vector into a plant, for example, an Agrobacterium method, a particle gun method, or the like is used.

3. Production of Recombinant PTL Protein The PTL protein can be obtained by culturing or growing the transformant described in the previous section (2. Production of transformant) and collecting the protein from the culture. The method of culturing the transformant is performed according to a usual method used for culturing a host. Alternatively, it can be obtained by subjecting the recombinant vector described in the previous section (1. Preparation of recombinant vector) to a cell-free protein synthesis system and expressing the protein or peptide in vitro.

  As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the microorganisms, and the transformants can be cultured efficiently. As long as it is a medium, either a natural medium or a synthetic medium may be used.

  As the carbon source, carbohydrates such as glucose, fructose, sucrose, starch, maltose and dextrin, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates such as ammonium salts of organic acids or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, casamino acid, NZ amine Etc. are used. Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, calcium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, zinc sulfate, and cobalt chloride.

  The culture is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture, at a temperature of about 15 ° C. to 40 ° C. and a culture time of 3 to 24 hours. During the culture period, the pH is maintained at 5.0 to 8.0. The pH is adjusted with an inorganic or organic acid or alkaline solution.

  During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when a microorganism transformed with a recombinant vector using the Lac promoter is cultured, a microorganism transformed with isopropyl-β-thiogalactopyranoside (IPTG) or the like with a recombinant vector using the trp promoter is cultured. In this case, indoleacrylic acid (IAA) or the like may be added to the medium.

Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPM11640 medium, DMEM medium, and the like, such as a medium obtained by adding fetal calf serum or the like to these mediums. The culture is usually performed at 20 to 37 ° C. for 1 to 7 days in the presence of 5% CO ₂ . During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  As a medium for culturing a transformant obtained by using insect cells as a host, fetal calf serum or the like is added to Grace's Insect Medium (Grace, TCC: Nature, 195: 788 (1962)) Medium. The culture is usually performed at 25 ° C. for 1 to 7 days. During the culture period, the pH is maintained at 6.0 to 7.0, and aeration and agitation are added as necessary.

  After culturing, when the protein of the present invention is produced in cells or cells, the cells or cells are disrupted. On the other hand, when the protein of the present invention is secreted outside the cells or cells, the culture solution is used as it is or after removing the cells or cells by centrifugation or the like, a supernatant is obtained. For protein isolation / purification, generally, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, or the like is used alone or in appropriate combination to obtain the above culture (cell disruption solution). The recombinant PTL protein of the present invention can be isolated and purified from the culture medium or the supernatant thereof.

  As the cell-free protein synthesis system, Escherichia coli extract, wheat germ extract, rabbit reticulocyte extract, insect cell extract, mammalian cell extract, in vitro protein synthesis kit and the like are used.

  As described above, by using the PTL gene, PTL can be produced by genetic engineering and can be obtained stably and efficiently.

  In addition, it is easy to fuse a known protein having a function other than a lectin to a recombinant PTL protein or peptide by genetic engineering techniques, such as an Fc chain of an antibody or a green fluorescent protein (Green Fluorescent Protein, GFP). It can be done. This makes it possible to produce a PTL having a useful function not found in natural products.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
[Example 1] (Cloning of lectin gene derived from Tsutsugitake mushroom)
The present inventors used the Peptide Sequencer PPSQ-21 System (manufactured by Shimadzu Corporation) with the amino acid sequence of PTL in the previous PCT / JP2009 / 003346 (invention name: α1 → 6 fucose specific lectin). The analysis results are shown. Based on the obtained N-terminal amino acid sequence, cloning of the lectin gene was attempted by the following procedure by 5 ′ RACE method and 3 ′ RACE method.

(MRNA extraction)
Tsutsugitake fruit bodies were crushed in liquid nitrogen with a mortar and pestle. 1 mL of a trizol reagent (manufactured by Invitrogen) was added to a 100 mg crushed sample to extract total RNA. For purification of mRNA from total RNA, Poly (A) ⁺ Isolation Kit from Total RNA (manufactured by NIPPON GENE) was used to purify mRNA according to the instructions of the relevant kit.

(CDNA synthesis)
Double-stranded cDNA synthesis from the purified mRNA was performed using a Marathon (registered trademark) cDNA amplification kit (Clontech). That is, 0.5 μL of the attached cDNA Synthesis primer solution (10 μM) was added to 2.5 μL of the mRNA solution (460 ng). The solution was heated at 70 ° C. for 2 minutes and then left on ice for 2 minutes. Subsequently, the reaction composition shown in Table 3 was used for reaction for 1 hour at 42 ° C. to prepare a single-stranded cDNA reaction solution.

  This single-stranded cDNA reaction solution was reacted at 16 ° C. for 1.5 hours with the reaction composition shown in Table 4.

  Further, 1 μL of T4 DNA polymerase (3 units) was added and reacted at 16 ° C. for 45 minutes. Subsequently, 2 μL of EDTA / glycogen solution was added to stop the double-stranded cDNA synthesis reaction. To this reaction solution, 50 μL of a phenol: chloroform: isoamyl alcohol (25: 24: 1) solution was added and mixed. After centrifugation (14,000 rpm, 10 min, 25 ° C.), the aqueous layer was recovered. To the recovered aqueous layer, 50 μL of a chloroform: isoamyl alcohol (24: 1) solution was added and mixed. After centrifugation (14,000 rpm, 10 min, 25 ° C.), the aqueous layer was recovered. To the collected solution, 1/2 volume of 4M ammonium acetate solution and 2.5 volume of 95% ethanol were added and mixed. Centrifugation (14,000 rpm, 20 min, 25 ° C.) was performed to precipitate cDNA. To the precipitate excluding the supernatant, 150 μL of 80% ethanol was added to wash the precipitate. After centrifugation (14,000 rpm, 10 min, 25 ° C.), the supernatant was removed and the precipitate was dried. A double-stranded cDNA solution was prepared by dissolving the dried precipitate with 5 μL of sterile water.

(Addition of adapter sequence to double-stranded cDNA)
The adapter sequence was added to the prepared double-stranded cDNA using an adapter attached to a Marathon (registered trademark) cDNA amplification kit (Clontech). That is, a 2.5 μL double-stranded cDNA solution was reacted overnight at 16 ° C. with the reaction composition shown in Table 5, and an adapter sequence added double-stranded cDNA (adapter sequence-added double-stranded cDNA solution) Was prepared.

(Degenerate primer design)
Peptide sequencer: A degenerate primer was prepared based on the N-terminal partial amino acid sequence of PTL analyzed by Protein Peptide Sequencer PPSQ-21 System (manufactured by Shimadzu Corporation). Four types of primers shown in Table 6 were prepared. NGSP_sense_PTL and NGSP_antisense_PTL were created based on sequences inside GSP_sense_PTL and GSP_antisense_PTL, respectively.

(Primary PCR)
PCR was performed using Takara Ex-Taq (registered trademark) (manufactured by Takara Bio Inc.). First, using the previously obtained cDNA as a template, adapter primer and GSP_sense_PTL (5′RACE) or GSP_antisense_PTL (3′RACE) attached to Marathon (registered trademark) cDNA amplification kit (manufactured by Clontech) are used. The primary PCR was carried out under the reaction composition described in Table 7 and the reaction conditions described in Table 8. The primary PCR product was stored at 4 ° C. until analysis. It was confirmed by agarose electrophoresis of the PCR product and ethidium bromide staining that the DNA fragment was amplified by primary PCR.

(Secondary PCR)
Next, using the amplified DNA fragment as a template, the nested-adapter primer and NGSP_sense_PTL (5′RACE) or NGSP_antisense_PTL (3′RACE) attached to the Marathon (registered trademark) cDNA amplification kit (Clontech) are used. Using the reaction composition described in Table 9 and the reaction conditions described in Table 10, secondary PCR (nested-PCR) was performed. Secondary PCR products were stored at 4 ° C. until analysis.

  The amplification of the DNA fragment by secondary PCR was confirmed by agarose electrophoresis and ethidium bromide staining of the PCR product. The results are shown in FIG. In order to determine the base sequence of the obtained DNA fragment, subcloning was performed according to the following procedure.

(Subcloning)
The obtained DNA fragment was TA-cloned using a pGEM-T easy vector (manufactured by Promega). First, reaction solutions shown in Table 11 were prepared and reacted at room temperature for 1 hour.

  Next, 1 μL of the ligation reaction solution was thawed on ice. Electroporation was performed by mixing with 40 μL of E. coli DH5α Electro-Cells (manufactured by Takara Bio Inc.). Thereafter, 500 μL of SOC medium (manufactured by Takara Bio Inc.) was added and cultured with shaking at 37 ° C. for 1 hour. The culture solution after shaking culture was developed on an LB agar medium (containing ampicillin, isopropyl-β-thiogalactopyranoside (IPTG) and X-gal). The cells were cultured at 37 ° C. for 16 hours to form transformed colonies.

  PCR was performed using a primer set (M13 forward and M13 reverse) (manufactured by Sigma-Aldrich) designed to amplify the multiple cloning site using 2 × PCR Master Mix (manufactured by Promega). First, the transformed colony was scraped with a sterilized toothpick and suspended in the reaction solution described in Table 12. PCR was performed under the reaction conditions described in Table 13. PCR products were stored at 4 ° C. until analysis. From the PCR product, a clone in which a DNA fragment of 1000 bp or less was incorporated was selected by agarose electrophoresis and ethidium bromide staining.

  The colonies selected above were inoculated into 2 mL of LB liquid medium containing 50 μg / mL of ampicillin, and cultured with shaking at 37 ° C. for 16 hours. After culturing, the cells were collected by centrifugation (14,000 rpm, 1 min, 4 ° C.). Plasmid extraction from the obtained cells was performed using a commercially available QIAprep Spin Miniprep kit (manufactured by QIAGEN). The operation followed the instructions.

(Determination of nucleotide sequence)
Using the thus purified plasmid, the nucleotide sequences of the inserted 5 ′ RACE and 3 ′ RACE DNA fragments were determined. A DNA sequencer was used to determine the base sequence. That is, the base sequence was determined using CEQ ^™ 8000 Genetic Analysis System (manufactured by BECKMAN COULTER) using the Dye Terminator method and capillary sequence.

  The resulting plasmid was prepared to be 100-200 ng / μL. Cycle sequencing was performed using a DTCS quick start master mix kit (manufactured by BECKMAN COULTER). The operation followed the instructions. The PCR reaction was performed under the conditions described in Table 14 and Table 15. As the primer, M13 forward and M13 reverse were appropriately used.

  After completion of the cycle sequence, 5 μL of the reaction stop solution described in Table 16 was added. Subsequently, 60 μL of 100% ethanol was added and stirred.

  The reaction product was centrifuged (14,000 rpm, 20 min, 4 ° C.). The supernatant was removed and 200 μL of 70% ethanol was added. The precipitate was washed again by centrifugation (14,000 rpm, 20 min, 4 ° C.). This operation was repeated twice. The precipitate was air-dried, and 40 μL of a sample loading solution (manufactured by BECKMAN COULTER) attached to the kit was added to dissolve the precipitate. The lysed sample was transferred to the entire CEQ sample plate. One drop of mineral oil was dropped into each well, and a capillary sequence was performed.

  The full-length sequence of PTL cDNA (FIG. 1, SEQ ID NO: 1) was determined from the results of fluorescence patterns obtained by analyzing 5′RACE and 3′RACE.

  The amino acid sequence obtained by converting the full-length sequence of cDNA based on the codon table (FIG. 1, SEQ ID NO: 2) contained a base sequence encoding 180 amino acids from the start codon (ATG) to the stop codon (TAA). . This amino acid sequence was found to have three highly homologous sequences to the previously purified PTL N-terminal amino acid sequence analysis results (40 amino acid residues). These highly homologous sequences were named CRD1, CRD2 and CRD3 in order from the 5 'side.

  When the obtained 180-residue amino acid sequence was compared with a known amino acid sequence using BLAST, no protein showing significant homology was confirmed.

[Example 2] (Expression 1 of recombinant PTL in E. coli)
A histidine tag (His tag) was added to the coding region of the full-length sequence obtained above for expression. First, a restriction enzyme sequence was added to the gene fragment of the base sequence of rPTL0 shown in Table 2 and inserted upstream of the His tag sequence of the pET27b vector (Novagen).

  The prepared pET27b / PTL0 plasmid was transformed into E. coli One Shot BL21 Star (DE3) (manufactured by Invitrogen). The transformed E. coli was developed on LB agar medium (containing kanamycin) and cultured at 37 ° C. for 16 hours. The obtained single colony was inoculated into LB liquid medium (containing kanamycin) and cultured with shaking at 37 ° C. for 16 hours. The whole amount of the preculture solution was added to the LB liquid medium (containing kanamycin). The cells were cultured at 37 ° C. until the absorbance at a wavelength of 600 nm reached 0.6 to 1.0. IPTG was added to a final concentration of 1 mM. Furthermore, after culturing at 25 ° C. for 24 hours, the cells were collected by centrifugation (5000 g, 20 min, 4 ° C.). To the cell pellet, 10 mM phosphate buffered saline (pH 7.4, hereinafter referred to as PBS) was added and suspended. The bacterial cells were crushed by ultrasonic crushing treatment. The supernatant was recovered by centrifuging the cell disruption solution (8500 g, 30 min, 4 ° C.).

(Purification with His tag)
The obtained supernatant was subjected to affinity purification of rPTL7 shown in Table 2 using HiTrap Chelating HP (GE Healthcare Bioscience). The purification result is shown in FIG. The expected 22 kDa band was detected. From the above, the expression of rPTL7 was confirmed from the pET27b / PTL0 plasmid, and rPTL7 could be purified.

[Example 3] (Expression of recombinant PTL in E. coli 2)
Of the full-length sequence obtained above, a region having high homology with the N-terminal amino acid sequence of PTL was expressed. First, a stop codon (TAA) and a restriction enzyme sequence were added to gene fragments of three types of base sequences rPTL1, rPTL2 and rPTL3 shown in Table 2. Three types of recombinant vectors were prepared by inserting the pET51b vector (manufactured by Novagen) downstream of the Strep Tag (registered trademark) II sequence.

  The three kinds of prepared pET51b / PTL plasmids were each transformed into E. coli BL21-codon plus (registered trademark) (DE3) -RIL by electroporation. The transformed E. coli was developed on LB agar medium (containing ampicillin) and cultured at 37 ° C. for 16 hours. The obtained single colony was inoculated into LB liquid medium (containing ampicillin) and cultured with shaking at 37 ° C. for 16 hours. The whole amount of the preculture solution was added to the LB liquid medium (containing ampicillin). The cells were cultured at 37 ° C. until the absorbance at a wavelength of 600 nm reached 0.6 to 1.0. IPTG was added to a final concentration of 1 mM. Furthermore, after culturing at 25 ° C. for 24 hours, the cells were collected by centrifugation (5000 g, 20 min, 4 ° C.). PBS was added to the cell pellet and suspended. The bacterial cells were crushed by ultrasonic crushing treatment. The supernatant was recovered by centrifuging the cell disruption solution (8500 g, 30 min, 4 ° C.).

(Purification by Strep / Tactin superflow column)
The resulting supernatant was subjected to affinity purification of rPTL8, rPTL9 and rPTL10 shown in Table 2 using Strep Tactin (registered trademark) superflow column (manufactured by Novagen). The purification results are shown in FIG. As expected, bands of 7.3 kDa, 13.2 kDa, and 18.3 kDa were detected. From the above, the expression of rPTL8, rPTL9, and rPTL10 was confirmed, and rPTL8, rPTL9, and rPTL10 could be purified.

[Example 4] (Expression of recombinant PTL in E. coli 3)
Of the full-length sequence obtained above, only the region having high homology with the N-terminal amino acid sequence of PTL was expressed. First, a start codon (ATG), a stop codon (TAA), and a restriction enzyme sequence were added before and after the gene fragments of six types of base sequences rPTL1 to 6 shown in Table 2. By inserting into the pET51b vector (Novagen) excluding the tag sequence, six types of recombinant vectors were prepared.

  The same operation as in Example 3 was performed, and the supernatant was collected.

(Purification by affinity chromatography)
The obtained supernatant was subjected to thyroglobulin-immobilized agarose equilibrated with PBS. The column was washed with PBS and eluted with 0.2 M ammonia. The eluted fractions were analyzed by SDS-PAGE and CBB staining. The result is shown in FIG. As expected, bands of 4.6 kDa, 10.5 kDa, 15.6 kDa, 4.6 kDa, 9.7 kDa, and 4.5 kDa were detected, respectively. From the above, the expression of rPTL1-6 was confirmed, and rPTL1-6 could be purified.

[Example 5] (Glycoprotein array)
Thyroglobulin was prepared as a glycoprotein having fucose α1 → 6 sugar chain, and transferrin was prepared as a glycoprotein having no fucose α1 → 6 sugar chain. These glycoproteins were immobilized on a glass substrate, and glycoprotein arrays were performed using GlycoStation ^™ Reader 1200 (manufactured by GP Bioscience). First, purified recombinant PTL was labeled with Cy3 according to the instructions of Cy3 Mono-Reactive Dye Pack (manufactured by GE Healthcare Bioscience). Suspended labeled recombinant PTL in Tris buffer (pH 7.4, hereinafter referred to as Probing buffer) containing 500 mM glycine, 1% Triton X-100, 1 mM CaCl ₂ , 1 mM ZnCl _2, 0.8% NaCl The labeled recombinant PTL was provided on a glass substrate on which the glycoprotein was immobilized. After reacting overnight at 20 ° C., the glass substrate was washed with a Probing buffer.

The washed glass substrate was subjected to GlycoStation ^™ Reader 1200, and the binding between the immobilized glycoprotein and Cy3-labeled recombinant PTL was analyzed. The result is shown in FIG.

  FIG. 6 shows that all recombinant PTLs bind to glycoproteins having fucose α1 → 6 sugar chains and not to glycoproteins having no fucose α1 → 6 sugar chains.

[Example 6] (Removal of Strep • Tag (registered trademark) II sequence and analysis of specificity to glycoprotein)
The Strep Tag (registered trademark) II sequence added to the N-terminus of rPTL8 was removed by Recombinant Enterokinase (manufactured by Novagen). First, purified rPTL8 was reacted overnight at room temperature with the reaction composition shown in Table 17. The solution after the reaction was subjected to the same operation as in Example 5, and the specificity was analyzed with a glycoprotein array. The result is shown in FIG.

  From FIG. 7, rPTL from which the Strep • Tag (registered trademark) II sequence has been removed also binds to a glycoprotein having fucose α1 → 6 sugar chain and not to a glycoprotein having no fucose α1 → 6 sugar chain. I understand that. In this way, by removing the tag sequence after affinity purification with the tag sequence, it is possible to produce rPTL that does not contain an extra sequence such as a tag sequence.

  Furthermore, rPTL8 from which the Strep • Tag (registered trademark) II sequence was removed from rPTL8 showed increased fluorescence intensity to glycoprotein having fucose α1 → 6 sugar chain. This means that it may be preferable not to add an extra sequence such as a tag sequence in order to maximize the activity of rPTL.

[Example 7] (Frontal affinity chromatography)
A pyridylamino (PA) sugar chain having the structure shown in FIG. 8 was prepared. Frontal affinity chromatography was performed using FAC-1 (manufactured by Shimadzu Corporation). First, rPTL-Sepharose was produced from rPTL1-3 according to the instructions of NHS-activated Sepharose (manufactured by GE Healthcare Bioscience). RPTL-Sepharose was suspended in 10 mM Tris buffer (pH 7.4, hereinafter referred to as TBS) containing 0.8% NaCl, and packed in a miniature column (φ2 mm × 10 mm, 31.4 μL). The prepared column was inserted into a stainless steel holder and connected to the FAC-1 apparatus. The flow rate and column temperature were kept at 0.125 ml / min and 25 ° C., respectively. After equilibrating the miniature column with the TBS, an excessive volume (0.5 ml to 4 ml) of a PA sugar chain (3.75 nM or 7.5 nM) was injected into the column using an automatic sampling device or a manual injection device.

The fluorescence intensity (excitation wavelength: 310 nm and fluorescence wavelength: 380 nm) of each PA sugar chain eluate was monitored, and the interaction intensity [difference in front eluate relative to standard oligosaccharide (sugar chain No. 003): V−V ₀ ] Was measured. The binding constant Ka was determined from the interaction strength and the effective ligand amount. The results are shown in Tables 18, 19 and 20A-20E.

The following can be understood from Tables 18-20. It can be seen that the binding constant for the fucose α1 → 6 sugar chain is Ka = 1.0 × 10 ⁵ M ⁻¹ or more, and the three types of rPTL are strongly bound. In addition, a binding constant for a non-fucose α1 → 6 sugar chain or a sugar chain not having fucose is Ka = 5.15 × 10 ³ M ⁻¹ or less means that rPTL does not bind to these sugar chains. Yes. In addition, fucose α1 → 6 sugar chain (fucose α1 → 6 sugar chain (sugar chain No. 410) or four chain (sugar chain No. 418) that is strongly bound to sialic acid is added. The binding constant of sugar chain No. 602) is not lowered. This means that it is superior to a lectin that binds to a conventional fucose α1 → 6 sugar chain.

  In addition, since any of CRD1, CRD2 and CRD3 can bind to fucose α1 → 6 sugar chain, a recombinant protein having high affinity for fucose α1 → 6 sugar chain can be obtained by arbitrarily combining these sequences. Or it can be estimated easily that a peptide can be manufactured.

[Example 8] (Cloning of Salmonella lectin gene)
Using the salmon bamboo fruit body as a raw material, the same procedure as in Example 1 was performed to determine the base sequence. From the analysis results, a full-length sequence of salmonella lectin-derived lectin (hereinafter referred to as SRL) cDNA (FIG. 9, SEQ ID NO: 7) was determined. The amino acid sequence obtained by converting the full-length sequence of SRL based on the codon table (FIG. 9, SEQ ID NO: 8) encoded 179 amino acids from the start codon (ATG) to the stop codon (TAA). In addition, when compared with the amino acid sequence from PTL cDNA, two amino acid deletions, one amino acid addition, and 59 amino acid substitutions were confirmed.

  Moreover, the site | part corresponded to CRD1-3 of PRL of SRL was identified from the homology of the amino acid sequence. Table 21 shows the homology between each SRL and PTL CRD. There was more than 64% homology between any CRD. Furthermore, the homology between the same CRD of PTL and SRL was 74% or more.

From Table 21, similarly to PTL, the gene encoding the following protein or peptide (1) or (2) is also predicted to be a gene having sugar-binding activity.
(1) Amino acids represented by any one of SEQ ID NO: 1 to 179, 30 to 68, 30 to 123, 30 to 172, 85 to 123, 85 to 172, and 134 to 172 A protein or peptide consisting of a sequence,
(2) Amino acids represented by any one of SEQ ID NOs: 1 to 179, 30 to 68, 30 to 123, 30 to 172, 85 to 123, 85 to 172, and 134 to 172 A protein or peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence and having sugar recognition activity.
Table 22 shows the meaning of the range of the above seven amino acid sequences.

[Example 9] (Expression and purification of recombinant SRL (rSRL) in E. coli)
The same operation as in Example 2 was performed to obtain rSRL0 (full-length cDNA). The result is shown in FIG. The expected 15.6 kDa band was detected. From the above, rSRL expression was confirmed and rSRL could be purified.

[Example 10] (Glycoprotein array)
The same operation as in Example 5 was performed to analyze the specificity of rSRL. The result is shown in FIG. FIG. 11 shows that rSRL binds to a glycoprotein having fucose α1 → 6 sugar chain and does not bind to a glycoprotein having no fucose α1 → 6 sugar chain.

  In the previous PCT / JP2009 / 003346 (title of the invention: α1 → 6 fucose specific lectin), the present inventors have shown that PTL and SRL are α1 → 6 fucose specific lectins. Also in the present invention, it was shown that rPTL and rSRL specifically recognize α1 → 6 fucose. From the above, the gene encoding rSRL can be regarded as an example of the gene of the present invention encoding amino acid deletion and amino acid addition and amino acid substitution of protein or peptide.

Claims (7)

Genes encoding the following proteins or peptides (1) or (2):
(1) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 A protein or peptide consisting of a sequence,
(2) Amino acids represented by any one of SEQ ID NO: 1 to 180, 32 to 71, 32 to 126, 32 to 174, 87 to 126, 87 to 174, and 135 to 174 A protein or peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence and having sugar recognition activity.   The gene according to claim 1, wherein the gene encoding the amino acid sequence of SEQ ID NO: 2 is represented by SEQ ID NO: 1.   A recombinant vector containing the gene according to claim 1 or 2.   A transformant obtained by introducing the recombinant vector according to claim 3 into a host.   A method for producing a recombinant protein or peptide, comprising culturing or growing the transformant according to claim 4.   A method for producing a recombinant protein or peptide, comprising subjecting the recombinant vector according to claim 3 to a cell-free protein synthesis system to express the recombinant protein or peptide. A method for producing a recombinant protein or peptide, wherein the recombinant protein or peptide expressed from the transformant according to claim 4 is subjected to affinity purification via a tag sequence and / or a sugar recognition activity.