Recombinant Proteins and Anticancer Therapeutics

Abstract

To provide a recombinant protein and an anti-cancer therapeutic agent that can be used in the treatment of cancer. [Solution] The recombinant protein comprises a lectin site and a cytotoxic activity site. The cytotoxic activity site is, for example, the following (a) or (b): (a) a protein consisting of an amino acid sequence in which the 111th phenylalanine is replaced with tyrosine and the 163rd lysine is replaced with arginine in the amino acid sequence of the RIP site contained in bitter melon lectin. (b) a protein consisting of the amino acid sequence of (a) in which one or several amino acids are deleted, substituted or added, and having cytotoxic activity.

Description

[Technical field]

[0001] The present disclosure relates to recombinant proteins and anti-cancer therapeutics. [Background technology]

[0002] Bitter melon lectin has a lectin site and a RIP (Ribosome inactivating protein) site. The lectin site and the RIP site are linked by a disulfide bond. The lectin site has H antigen binding properties and binds to specific cells. As described in Patent Document 1, bitter melon lectin is used for various purposes. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 3849945 Summary of the Invention [Problem to be solved by the invention]

[0004] Proteins that have a lectin site that recognizes glycans specific to cancer cells and binds to cancer cells, and a cytotoxic activity site that has cytotoxic activity against cancer cells, can be used for cancer treatment. However, bitter melon lectin cannot be used as it is for cancer treatment because the RIP site in bitter melon lectin does not have cytotoxic activity.

[0005] In one aspect of the present disclosure, it is preferred to provide recombinant proteins and anti-cancer therapeutic agents that can be used in the treatment of cancer. [Means for solving the problem]

[0006] One aspect of the present disclosure is a recombinant protein comprising a lectin site and a cytotoxic activity site. The recombinant protein of one aspect of the present disclosure can be used for cancer treatment.

[0007] Another aspect of the present disclosure is an anti-cancer therapeutic agent comprising a recombinant protein comprising a lectin site and a cytotoxic activity site. The anti-cancer therapeutic agent that is another aspect of the present disclosure can be used for cancer treatment. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1A is an explanatory diagram showing the structure of wild-type bitter melon lectin. FIG. 1B is an explanatory diagram showing the structure of recombinant bitter melon lectin G72Y. FIG. 1C is an explanatory diagram showing the structure of recombinant bitter melon lectin F111Y. FIG. 1D is an explanatory diagram showing the structure of recombinant bitter melon lectin K163R. FIG. 1E is an explanatory diagram showing the structure of recombinant bitter melon lectin G72Y / F111Y. FIG. 1F is an explanatory diagram showing the structure of recombinant bitter melon lectin G72Y / K163R. FIG. 1G is an explanatory diagram showing the structure of recombinant bitter melon lectin F111Y / K163R. FIG. 1H is an explanatory diagram showing the structure of recombinant bitter melon lectin G72Y / F111Y / K163R. [Diagram 2] Figure 2A is a photograph showing the results of silver staining and Figure 2B is a photograph showing the results of Western blotting for wild-type bitter gourd lectin. [Diagram 3] Figure 3A is a photograph showing the results of silver staining for each recombinant bitter melon lectin and wild-type bitter melon lectin, and Figure 3B is a photograph showing the results of Western blotting for each recombinant bitter melon lectin and wild-type bitter melon lectin. [Figure 4] 1 is a graph showing the results of measuring wavelength absorbance for each recombinant bitter melon lectin and wild-type bitter melon lectin. [Diagram 5] FIG. 1 is an explanatory diagram showing the base sequences of SEQ ID NOs: 1 to 8. [Figure 6] FIG. 1 is an explanatory diagram showing the amino acid sequence of SEQ ID NO:9. [Figure 7] FIG. 1 is an explanatory diagram showing the amino acid sequence of SEQ ID NO:10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Exemplary embodiments of the present disclosure will now be described with reference to the drawings. 1. Recombinant Protein Construction The recombinant protein of the present disclosure comprises a lectin site and a cytotoxic activity site, the lectin site and the cytotoxic activity site being linked together, for example, by a disulfide bond.

[0010] The cytotoxic activity site is, for example, the following (a) or (b): (a) A protein consisting of an amino acid sequence in which the 111th phenylalanine is replaced by tyrosine and the 163rd lysine is replaced by arginine in the amino acid sequence of the RIP site contained in bitter melon lectin.

[0011] (b) A protein having the amino acid sequence of (a) in which one or several amino acids have been deleted, substituted or added, and having cytotoxic activity. The lectin site is, for example, a lectin site contained in bitter melon lectin. The lectin site recognizes H antigen and specifically binds to cancer cells. The lectin site specifically binds, for example, to human pancreatic cancer cell line Capan-1 (hereinafter referred to as Capan-1 cells). The lectin site binds, for example, to a specific sugar chain of Capan-1 cells.

[0012] 2. Effects of recombinant proteins The recombinant protein of the present disclosure comprises a lectin site. The lectin site recognizes the H antigen and specifically binds to cancer cells. The recombinant protein of the present disclosure comprises a cytotoxic activity site. Therefore, the recombinant protein of the present disclosure has cytotoxic activity against cancer cells to which the lectin site binds. The recombinant protein of the present disclosure has low cytotoxic activity against cells to which the lectin site does not bind.

[0013] The cancer cells to which the lectin site specifically binds are, for example, Capan-1 cells, in which case the recombinant protein of the present disclosure has cytotoxic activity against Capan-1 cells.

[0014] The recombinant protein of the present disclosure can be used, for example, in cancer treatment. The anti-cancer therapeutic agent of the present disclosure includes the recombinant protein of the present disclosure. The recombinant protein of the present disclosure can be used, for example, in pancreatic cancer treatment. The anti-cancer therapeutic agent of the present disclosure is, for example, an anti-pancreatic cancer therapeutic agent.

[0015] 3. Working Example (1) Isolation of bitter gourd lectin cDNA (1a) Commercially available bitter melon seeds were germinated on a nonwoven cloth soaked in water. The germinated sprouts were then harvested. The harvested sprouts were then frozen in liquid nitrogen. The frozen sprouts were then crushed in a mortar.

[0016] (1b) RNA was extracted from the sprouts ground in (1a) above using an RNeasy mini kit (Qiagen) according to the protocol. (1c) cDNA was synthesized from the RNA extracted in (1b) above using an RT kit (Takara) according to the protocol. The synthesized cDNA was amplified by PCR using a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2. The base sequences of SEQ ID NOs: 1 and 2 are shown in FIG. 5.

[0017] (1d) The cDNA amplified in (1c) was subjected to agarose gel electrophoresis. Next, cDNA was recovered with 50 μL of ultrapure water using a Gel extraction kit (Qiagen) according to the protocol. The ultrapure water was produced by MilliQ (registered trademark), an ultrapure water production device. Next, the recovered cDNA was digested with restriction enzymes XhoI and BamHI. Digestion was performed at 37° C. for 15 hours. Next, the cDNA was subjected to agarose gel electrophoresis again. Next, cDNA was recovered with 50 μL of ultrapure water using a Gel extraction kit (Qiagen) according to the protocol. The ultrapure water was produced by MilliQ. As a result, 50 μL of product containing cDNA was obtained.

[0018] (1e) The product obtained in (1d) above was inserted into the XhoI / BamHI site of pCAG-Hyg vector (FUJIFILM Wako Pure Chemical Industries, Ltd.) to cause a ligation reaction. (2) Transformation into E. coli (2a) 0.5 μL of the solution obtained after the ligation reaction in (1e) was added to 10 μL of E. coli DH5α strain. The mixture was then left to stand on ice for 20 minutes. Then, the mixture was subjected to a heat shock in a water bath at 42° C. for 90 seconds, thereby carrying out transformation.

[0019] (2b) After the transformation in (2a), 200 μL of SOB medium was added to the E. coli. The E. coli was then cultured at 37° C. for 10 minutes. 20 μL of the medium containing the E. coli was then spread onto an LB agar medium. The LB agar medium contained 50 μg / L of hygromycin. The E. coli was then cultured overnight at 37° C.

[0020] (3) Plasmid extraction (3a) The E. coli colony cultured in (2b) above was inoculated into 3 mL of LB medium containing 50 μg / mL hygromycin, and then cultured with shaking at 37° C. and 300 rpm for 8 hours.

[0021] (3b) After the shaking culture in (3a), plasmids were extracted from E. coli using a Fast Gene plasmid mini kit (Nihon Genetics) according to the protocol, resulting in 50 μL of a plasmid solution.

[0022] (4) Transfection (4a) 7 μL of the plasmid solution obtained in (3b) was mixed with 247 μL of Opti-MEM (Gibco) and allowed to stand at room temperature for 5 minutes to prepare a first solution. The 7 μL of the plasmid solution obtained in (3b) contained about 4 μg of plasmid.

[0023] (4b) 10 μL of Lipofectamine 2000 Transfection Reagent (Thermo Fisher) was mixed with 240 μL of Opti-MEM and allowed to stand at room temperature for 5 minutes to prepare a second solution. (4c) The first solution and the second solution were mixed and allowed to stand at room temperature for 15 minutes to prepare a transfection mixture.

[0024] (4d) HEK293 cells were cultured overnight at 37°C in a 6-well plate (Iwaki) using D10 medium. Next, the D10 medium was removed from the 6-well plate using an aspirator. Next, 1.5 mL of fresh D10 medium and 500 μL of the transfection mixture prepared in (4c) above were added to the 6-well plate. Next, HEK293 cells were cultured at 37°C in the presence of 5% CO2 for 48 hours.

[0025] (4e) Trypsin-ethylenediaminetetraacetic acid (EDTA) solution was added to HEK293 cells and treated for 5 minutes. Next, the HEK293 cells were detached from the 6-well plate by pipetting. Next, the cell suspension containing HEK293 cells was collected.

[0026] (4f) The cell suspension collected in (4e) above was centrifuged at 4°C and 1000 rpm for 5 minutes. The supernatant was then discarded. The pellet of HEK293 cells was then suspended in 10 mL of D10 medium containing 50 μg / mL hygromycin. As a result, a suspension was obtained.

[0027] (4g) The suspension obtained in (4f) was seeded on a 10 cm dish. Next, the HEK293 cells contained in the suspension were cultured at 37°C for 2 weeks. The culture was passaged twice.

[0028] (4h) After the culture in (4g), HEK293 cells were seeded on three 15 cm dishes. The seeded HEK293 cells were then expanded for one week. Next, a process of centrifuging the cells at 4°C and 1300 rpm for 5 minutes and a process of recovering the culture supernatant were alternately repeated, and 300 mL of the culture supernatant was recovered.

[0029] (5) Recovery of wild-type bitter melon lectin (5a) 100 mL of the culture supernatant collected in (4h) was allowed to adsorb onto the column overnight. The column was packed with 5 mL of Lactose-Sepharose 4B equilibrated with 10 mM phosphate buffer (PBS). The pH of the 10 mM PBS was 7.4. The 10 mM PBS contained 0.15 M NaCl. The column was then washed with 50 mL of PBS for 8 hours.

[0030] (5b) After washing the column in (5a), 0.5M lactose-PBS was injected into the column and elution was performed quickly. At this time, 1mL of the eluate was collected at each time. As a result, 10 elution fractions were obtained. The total volume of the 10 elution fractions was 10mL. Each of the 10 elution fractions was assigned a fraction number from 1 to 10. The fraction number is smaller the earlier the fraction is eluted. For example, the fraction number of the first eluted fraction is 1, and the fraction number of the second eluted fraction is 2.

[0031] The eluate contains wild-type bitter melon lectin. As shown in FIG. 1A, the wild-type bitter melon lectin contains a RIP site 3A and a lectin site 5. The RIP site 3A and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3A contains a signal sequence 9 and a PA tag 11. The amino acid sequence of the RIP site 3A is as shown in SEQ ID NO: 9 and FIG. 6.

[0032] (6) Construction of recombinant bitter melon lectin G72Y (6a) The recombinant bitter melon lectin G72Y is a wild-type bitter melon lectin in which the 72nd glycine at RIP site 3A is replaced with tyrosine. The recombinant bitter melon lectin G72Y was prepared by site-directed mutagenesis as follows.

[0033] Using the plasmid extracted in (3) above as a template, inverse PCR was performed using a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4. The base sequences of SEQ ID NOs: 3 to 4 are shown in FIG.

[0034] (6b) 1 μL of DpnII (9 U / μL) was added to 25 μL of the PCR reaction solution obtained in (6a) above, followed by restriction enzyme treatment at 37° C. for 2 hours. (6c) Using 2 μL of the product after the treatment in (6b) above, self-ligation was carried out at 16° C. for 1 hour.

[0035] (6d) The product after the treatment in (6c) was transformed into Escherichia coli DH5α strain in the same manner as in (2) above. Next, the Escherichia coli was cultured overnight in LB medium supplemented with 50 μg / mL hygromicine.

[0036] (6e) After the cultivation in (6d), a plasmid was extracted from the cultured colony in the same manner as in (3). Next, the extracted plasmid was subjected to sequence analysis to confirm the introduction of the mutation. Next, the recombinant bitter melon lectin G72Y was recovered in the same manner as in (4) to (5).

[0037] As shown in Fig. 1B, the recombinant bitter melon lectin G72Y comprises a RIP site 3B and a lectin site 5. The RIP site 3B and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3B contains a signal sequence 9 and a PA tag 11. In the RIP site 3B, the 72nd glycine is replaced with tyrosine when compared with the RIP site 3A.

[0038] (7) Construction of recombinant bitter melon lectin F111Y The recombinant bitter melon lectin, in which the 111th phenylalanine in the RIP site 3A of the wild-type bitter melon lectin was replaced with tyrosine, was named recombinant bitter melon lectin F111Y.

[0039] The recombinant bitter melon lectin F111Y was basically prepared in the same manner as the recombinant bitter melon lectin G72Y. However, when performing the inverse PCR method, a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6 were used. The base sequences of SEQ ID NOs: 5 to 6 are shown in FIG. 5.

[0040] As shown in Fig. 1C, the recombinant bitter melon lectin F111Y comprises a RIP site 3C and a lectin site 5. The RIP site 3C and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3C contains a signal sequence 9 and a PA tag 11. In the RIP site 3C, the 111th phenylalanine is replaced with tyrosine when compared with the RIP site 3A.

[0041] (8) Construction of recombinant bitter melon lectin K163R The recombinant bitter melon lectin was obtained by substituting lysine at position 163 of the wild-type bitter melon lectin with arginine at RIP site 3A, and named the recombinant bitter melon lectin K163R.

[0042] The recombinant bitter melon lectin K163R was basically prepared in the same manner as the recombinant bitter melon lectin G72Y, except that the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 were used in the inverse PCR.

[0043] As shown in Fig. 1D, the recombinant bitter melon lectin K163R comprises a RIP site 3D and a lectin site 5. The RIP site 3D and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3D contains a signal sequence 9 and a PA tag 11. In the RIP site 3D, the 163rd lysine is replaced with arginine when compared with the RIP site 3A.

[0044] (9) Construction of recombinant bitter melon lectin G72Y / F111Y The recombinant bitter melon lectin was obtained by substituting glycine at position 72 with tyrosine and phenylalanine at position 111 with tyrosine in the RIP site 3A of the wild-type bitter melon lectin, and named recombinant bitter melon lectin G72Y / F111Y.

[0045] The recombinant bitter melon lectin G72Y / F111Y was basically produced in the same manner as the recombinant bitter melon lectin G72Y. However, when performing the inverse PCR method, the plasmid used in the production of the recombinant bitter melon lectin G72Y was used as a template. In addition, when performing the inverse PCR method, a forward primer of SEQ ID NO:5 and a reverse primer of SEQ ID NO:6 were used.

[0046] As shown in Fig. 1E, the recombinant bitter melon lectin G72Y / F111Y comprises a RIP site 3E and a lectin site 5. The RIP site 3E and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3E contains a signal sequence 9 and a PA tag 11. Compared with the RIP site 3A, the RIP site 3E has a substitution of tyrosine for glycine at position 72 and a substitution of tyrosine for phenylalanine at position 111.

[0047] (10) Construction of recombinant bitter melon lectin G72Y / K163R The recombinant bitter melon lectin was obtained by substituting glycine at position 72 with tyrosine and lysine at position 163 with arginine in the RIP site 3A of the wild-type bitter melon lectin, and named recombinant bitter melon lectin G72Y / K163R.

[0048] The recombinant bitter melon lectin G72Y / K163R was basically produced in the same manner as the recombinant bitter melon lectin G72Y. However, when performing the inverse PCR method, the plasmid used in the production of the recombinant bitter melon lectin G72Y was used as a template. In addition, when performing the inverse PCR method, a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8 were used.

[0049] As shown in FIG. 1F, the recombinant bitter melon lectin G72Y / K163R comprises a RIP site 3F and a lectin site 5. The RIP site 3F and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3F contains a signal sequence 9 and a PA tag 11. Compared with the RIP site 3A, the RIP site 3F has a substitution of glycine at position 72 with tyrosine and a substitution of lysine at position 163 with arginine.

[0050] (11) Construction of recombinant bitter melon lectin F111Y / K163R The recombinant bitter melon lectin was obtained by substituting 111th phenylalanine with tyrosine and 163rd lysine with arginine in the RIP site 3A of the wild-type bitter melon lectin, and named recombinant bitter melon lectin F111Y / K163R.

[0051] The recombinant bitter melon lectin F111Y / K163R was basically produced in the same manner as the recombinant bitter melon lectin G72Y. However, when performing the inverse PCR method, the plasmid used in the production of the recombinant bitter melon lectin F111Y was used as a template. In addition, when performing the inverse PCR method, a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8 were used.

[0052] As shown in FIG. 1G, the recombinant bitter melon lectin F111Y / K163R comprises a RIP site 3G and a lectin site 5. The RIP site 3G and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3G comprises a signal sequence 9 and a PA tag 11. In the RIP site 3G, the 111th phenylalanine is replaced with a tyrosine, and the 163rd lysine is replaced with an arginine, when compared with the RIP site 3A. The RIP site 3G corresponds to the hindered active site. The amino acid sequence of the RIP site 3G is as shown in SEQ ID NO: 10 and FIG. 7.

[0053] (12) Construction of recombinant bitter melon lectin G72Y / F111Y / K163R The recombinant bitter melon lectin was designed by substituting glycine at position 72 with tyrosine, phenylalanine at position 111 with tyrosine, and lysine at position 163 with arginine in the RIP site 3A of the wild-type bitter melon lectin, and named G72Y / F111Y / K163R.

[0054] The recombinant bitter melon lectin G72Y / F111Y / K163R was basically produced in the same manner as the recombinant bitter melon lectin G72Y. However, when performing the inverse PCR method, the plasmid used in the production of the recombinant bitter melon lectin F111Y / K163R was used as a template. In addition, when performing the inverse PCR method, a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 were used.

[0055] As shown in FIG. 1H, the recombinant bitter melon lectin G72Y / F111Y / K163R comprises a RIP site 3H and a lectin site 5. The RIP site 3H and the lectin site 5 are linked by a disulfide bond 7. The RIP site 3H contains a signal sequence 9 and a PA tag 11. Compared with the RIP site 3A, the RIP site 3H has a substitution of tyrosine for glycine at position 72, a substitution of tyrosine for phenylalanine at position 111, and a substitution of arginine for lysine at position 163.

[0056] (13) Expression confirmation (13a) Expression of wild-type bitter gourd lectin The eluate of the wild-type bitter melon lectin recovered in (5) above was subjected to SDS-PAGE using a 10% polyacrylamide gel under reducing conditions. SDS-PAGE is sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The elution of the wild-type bitter melon lectin was confirmed by Western blotting using silver staining and anti-PA-tag antibody (NZ-1, Wako) staining.

[0057] The results of SDS-PAGE are shown in Figures 2A and 2B. Figure 2A shows the results of silver staining. Figure 2B shows the results of Western blotting. In Figures 2A and 2B, lane M indicates a molecular weight marker. In Figures 2A and 2B, 1 to 10 arranged horizontally indicate the fraction numbers of the eluted fractions. It was confirmed that there were peaks in the eluted fractions with fraction numbers 3 to 4.

[0058] (13b) Expression of each recombinant bitter melon lectin The expression of each of the recombinant bitter melon lectins G72Y, F111Y, K163R, G72Y / F111Y, G72Y / K163R, F111Y / K163R, and G72Y / F111Y / K163R (hereinafter referred to as each recombinant bitter melon lectin) was confirmed in the same manner as described above in (13a). Expression of wild-type bitter melon lectin was also confirmed. The eluted fraction with fraction number 4 was used for expression confirmation. The results are shown in Figures 3A and 3B.

[0059] It was confirmed that each recombinant bitter gourd lectin and the wild-type bitter gourd lectin had a band at approximately 80 kDa. (14) Concentration of bitter melon lectin (14a) Enrichment of wild-type bitter melon lectin Among the eluted fractions collected in (5) above, fractions with fraction numbers 3 to 6 were transferred to an Amicon Ultra-4 centrifugal (Merck). Next, (i) the fractions were centrifuged at 3000 rpm for 30 minutes. Next, (ii) 2 mL of PBS solution was added and centrifuged. Furthermore, the above (i) and (ii) treatments were alternately repeated to finally obtain 300 μL of a concentrated solution. This concentrated solution is a concentrate of wild-type bitter melon lectin.

[0060] (14b) Concentration of each recombinant bitter melon lectin Concentrates were obtained for each recombinant bitter melon lectin in the same manner as described above in (14a). (15) Measurement of cytotoxicity (15a) Cytotoxicity of wild-type bitter melon lectin The cytotoxic activity of the wild-type bitter melon lectin obtained in (14a) above was measured against Capan-1 cells by MTT assay.

[0061] (a) A dilution series of wild-type bitter melon lectin was prepared in a first collagen-coated 96-well plate (Iwaki) using PBS. The dilution series consisted of a solution obtained by diluting the concentrated solution of wild-type bitter melon lectin at 1 / 10, 1 / 40, and 1 / 160.

[0062] (b) A second collagen-coated 96-well plate (Iwaki) was seeded with 100 μL of a solution containing Capan-1 cells per well. The solution containing Capan-1 cells was Iscove's modified medium containing 20% ​​fetal bovine serum (FBS), and the concentration of Capan-1 cells was 5.0 × 10 4 The solution was adjusted to give a concentration of 1000 cells / mL. Next, the solution was cultured at 37° C. for 12 hours to allow Capan-1 cells to adhere to the wells.

[0063] (c) On the day after (b), 90 μL of medium containing the Capan-1 cells cultured in (b) and 10 μL of one of the dilution series of wild-type bitter melon lectin were added to the wells of a third collagen-coated 96-well plate. Then, the wells were cultured at 37°C in the presence of 5% CO2 for 5 days. Then, the medium containing the wild-type bitter melon lectin was removed from the wells using an aspirator.

[0064] (ii) After removing the medium in (iii) above, 100 μL of solution was added to the well. The added solution was a 5 mg / mL 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution diluted 10-fold with Iscove's modified medium. The wells were then incubated at 37°C in a CO2 incubator in the presence of 5% CO2 for 4 hours.

[0065] (e) After the reaction in (ii) was completed, the medium was removed from the wells using an aspirator. Next, washing was performed once with 200 μL of PBS solution. Next, 50 μL of dimethyl sulfoxide (DMSO) solution was added to the wells to dissolve the Capan-1 cells, and the wells were shaken at room temperature for 15 minutes until a homogenous solution was obtained.

[0066] (f) Next, the solution containing Capan-1 cells was measured for absorbance at a wavelength of 570 nm using a microplate reader (Thermo Bioanalysis Japan). The measurement results are shown in FIG. (15b) Cytotoxicity of each recombinant bitter melon lectin Instead of wild-type bitter melon lectin, the concentrates of each recombinant bitter melon lectin obtained in (14b) above were used to measure the cytotoxicity against Capan-1 cells in the same manner as in (15a) above. The measurement results are shown in Figure 4. When a solution with a dilution ratio of 1 / 10 was used in the dilution series of recombinant bitter melon lectin F111Y / K163R, the absorbance decreased. A decrease in absorbance means that the product has cytotoxicity against Capan-1 cells.

[0067] The reason why the more toxic activity there is against Capan-1 cells, the lower the absorbance is is as follows. Capan-1 cells contain an oxidoreductase that reduces MTT to purple formazan crystals. Purple formazan crystals are a pigment. The fewer the surviving Capan-1 cells, the less likely MTT is to be converted to a pigment, and the lower the absorbance. Therefore, the more toxic activity there is against Capan-1 cells, the lower the absorbance. 4. Other embodiments Although the embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments and can be implemented in various modified forms.

[0068] (4-1) The function of one component in each of the above embodiments may be shared by multiple components, or the functions of multiple components may be performed by one component. Also, part of the configuration of each of the above embodiments may be omitted. Also, at least part of the configuration of each of the above embodiments may be added to or substituted for the configuration of another of the above embodiments.

[0069] (4-2) In addition to the recombinant proteins described above, the present disclosure can be realized in various forms, such as a system including the recombinant proteins as a component, a method for producing recombinant proteins, and genes encoding recombinant proteins. [Explanation of symbols]

[0070] wild-type...wild-type bitter gourd lectin, G72Y, F111Y, K163R, G72Y / F111Y, G72Y / K163R, F111Y / K163R, G72Y / F111Y / K163R...recombinant bitter gourd lectin, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H...RIP site, 5...lectin site, 7...disulfide bond, 9...signal sequence, 11...PA tag

Claims

1. The antibody has a lectin site and a cytotoxic activity site, A recombinant protein, wherein the cytotoxic activity site is either (a) or (b) below: (a) a protein having an amino acid sequence in which the 111th phenylalanine is replaced by tyrosine and the 163rd lysine is replaced by arginine in the amino acid sequence represented by SEQ ID NO: 9 of the RIP site contained in bitter melon lectin; (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence in (a) above, and having cytotoxic activity;

2. An anti-cancer therapeutic agent comprising the recombinant protein of claim 1.

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