Recombinant protein and anticancer therapeutic

Abstract

To provide a recombinant protein and an anticancer therapeutic that are usable for cancer treatment.SOLUTION: A recombinant protein comprises a lectin domain and a cytotoxic domain. The cytotoxic domain is exemplified by (a) or (b) below. (a) A protein composed of an amino acid sequence, being the amino acid sequence of the RIP domain contained in goya lectin, wherein the phenylalanine at position 111 is substituted with tyrosine, and the lysine at position 163 is substituted with arginine. (b) A protein having cytotoxic activity and being composed of an amino acid sequence, wherein one or several amino acids are deleted, substituted, or added in the amino acid sequence of (a).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to recombinant proteins and anti-cancer therapeutic agents.

Background Art

[0002] Bitter gourd 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 gourd lectin is used for various applications.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] A protein having a lectin site that recognizes sugar chains specific to cancer cells and binds to cancer cells, and a cytotoxic activity site having cytotoxic activity against cancer cells can be used for cancer treatment. However, since the RIP site of bitter gourd lectin does not have cytotoxic activity, bitter gourd lectin cannot be used for cancer treatment as it is.

[0005] In one aspect of the present disclosure, it is preferable to provide a recombinant protein and an anti-cancer therapeutic agent that can be used for cancer treatment.

Means for Solving the Problems

[0006] One aspect of the present disclosure is a recombinant protein having a lectin site and a cytotoxic activity site. The recombinant protein which is 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 having a lectin site and a cytotoxic activity site. The anti-cancer therapeutic agent, which is another aspect of the present disclosure, can be used for cancer treatment.

Brief Description of the Drawings

[0008]

Figure 1

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BEST MODE FOR CARRYING OUT THE INVENTION

[0009] Exemplary embodiments of the present disclosure will be described with reference to the drawings. 1. Composition of the recombinant protein The recombinant protein of the present disclosure comprises a lectin site and a cytotoxic activity site. The lectin site and the cytotoxic activity site are linked by, for example, 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 substituted with tyrosine and the 163rd lysine is substituted with arginine in the amino acid sequence of the RIP site contained in Momordica lectin.

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

[0012] 2. Effects of the recombinant protein 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] Cancer cells to which the lectin moiety specifically binds are, for example, Capan-1 cells. In that 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, for cancer treatment. The anti-cancer therapeutic agent of the present disclosure contains the recombinant protein of the present disclosure. The recombinant protein of the present disclosure can be used, for example, for pancreatic cancer treatment. The anti-cancer therapeutic agent of the present disclosure is, for example, an anti-pancreatic cancer therapeutic agent.

[0015] 3. Examples (1) Isolation of bitter gourd lectin cDNA (1a) Commercially available bitter gourd seeds were germinated on a non-woven fabric containing water. Next, the germinated sprouts were collected. Next, the collected sprouts were frozen in liquid nitrogen. Next, the frozen sprouts were ground in a mortar.

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

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

[0018] (1e) The product obtained in (1d) was inserted into the XhoI / BamHI site of the pCAG-Hyg vector (FUJIFILM Wako Pure Chemical Corporation) to cause a ligation reaction. (2) Transformation into Escherichia coli (2a) 0.5 μL of the solution after the ligation reaction in (1e) was added to 10 μL of Escherichia coli DH5α strain. Next, it was left standing on ice for 20 minutes. Next, transformation was carried out by applying a heat shock in a 42 °C water bath for 90 seconds.

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

[0020] (3) Plasmid extraction (3a) Colonies of the Escherichia coli cultured in (2b) were inoculated into 3 mL of LB medium containing 50 μg / mL of Hygromycin. Next, shaking culture was carried out at 37 °C and 300 rpm for 8 hours.

[0021] (3b) After the shaking culture in (3a), using the Fast Gene plasmid mini kit (Nippon Genetics) and following its protocol, plasmids were extracted from E. coli. As a result, 50 μL of plasmid solution was obtained.

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

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

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

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

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

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

[0028] (4h) After the culture in (4g), the HEK293 cells were seeded in three 15-cm dishes. Next, the seeded HEK293 cells were subjected to an expansion culture for 1 week. Next, the steps of centrifuging at 4°C and 1300 rpm for 5 minutes and recovering the culture supernatant were alternately repeated, and 300 mL of the culture supernatant was recovered.

[0029] (5) Recovery of wild-type bitter gourd lectin (5a) 100 mL of the culture supernatant recovered in (4h) was adsorbed onto a column overnight. The column was filled 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. Next, the column was washed with 50 mL of PBS over 8 hours.

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

[0031] The eluate contains wild-type bitter gourd lectin. As shown in Figure 1A, wild-type bitter gourd lectin comprises 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 Figure 6.

[0032] (6) Preparation of recombinant bitter gourd lectin G72Y (6a) In wild-type bitter gourd lectin, a recombinant bitter gourd lectin in which glycine at the 72nd position in the RIP site 3A is substituted with tyrosine is designated as recombinant bitter gourd lectin G72Y. The recombinant bitter gourd lectin G72Y was prepared as follows by site-directed mutagenesis.

[0033] Using the plasmid extracted in (3) above as a template, inverse PCR was performed using the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4. The nucleotide sequences of SEQ ID NOs: 3 to 4 are shown in Figure 5.

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

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

[0036] (6e) After culturing as described in (6d), plasmids were extracted from the cultured colonies in the same manner as in (3). Next, sequence analysis was performed on the extracted plasmids to confirm mutagenesis. Next, recombinant bitter gourd lectin G72Y was recovered in the same manner as in (4) to (5).

[0037] As shown in Figure 1B, recombinant bitter gourd 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. The RIP site 3B has the 72nd glycine substituted with tyrosine when compared with the RIP site 3A.

[0038] (7) Preparation of recombinant bitter gourd lectin F111Y In wild-type bitter gourd lectin wild-type, a recombinant bitter gourd lectin in which the 111th phenylalanine at the RIP site 3A was substituted with tyrosine was designated as recombinant bitter gourd lectin F111Y.

[0039] Recombinant bitter gourd lectin F111Y could basically be prepared in the same manner as recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 were used. The nucleotide sequences of SEQ ID NOs: 5 to 6 are shown in Figure 5.

[0040] As shown in Figure 1C, recombinant bitter gourd 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. The RIP site 3C has the 111th phenylalanine substituted with tyrosine when compared with the RIP site 3A.

[0041] (8) Preparation of recombinant bitter gourd lectin K163R In the wild-type bitter gourd lectin, the recombinant bitter gourd lectin with the 163rd lysine at the RIP site 3A replaced by arginine was designated as recombinant bitter gourd lectin K163R.

[0042] Recombinant bitter gourd lectin K163R could basically be prepared in the same manner as recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 were used.

[0043] As shown in Figure 1D, recombinant bitter gourd lectin K163R comprises the RIP site 3D and the 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. When the RIP site 3D is compared with the RIP site 3A, the 163rd lysine is replaced by arginine.

[0044] (9) Preparation of recombinant bitter gourd lectin G72Y / F111Y In the wild-type bitter gourd lectin, the recombinant bitter gourd lectin with the 72nd glycine at the RIP site 3A replaced by tyrosine and the 111th phenylalanine replaced by tyrosine was designated as recombinant bitter gourd lectin G72Y / F111Y.

[0045] Recombinant bitter gourd lectin G72Y / F111Y could basically be prepared in the same manner as recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the plasmid used in the preparation of recombinant bitter gourd lectin G72Y was used as a template. Also, when performing inverse PCR, the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 were used.

[0046] As shown in FIG. 1E, the recombinant bitter gourd 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. The RIP site 3E has the 72nd glycine substituted with tyrosine and the 111th phenylalanine substituted with tyrosine when compared with the RIP site 3A.

[0047] (10) Preparation of recombinant bitter gourd lectin G72Y / K163R In the wild-type bitter gourd lectin wild-type, a recombinant bitter gourd lectin in which the 72nd glycine at the RIP site 3A was substituted with tyrosine and the 163rd lysine was substituted with arginine was designated as recombinant bitter gourd lectin G72Y / K163R.

[0048] The recombinant bitter gourd lectin G72Y / K163R could basically be prepared in the same manner as the recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the plasmid used in the preparation of the recombinant bitter gourd lectin G72Y was used as a template. Also, when performing inverse PCR, the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 were used.

[0049] As shown in FIG. 1F, the recombinant bitter gourd 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. The RIP site 3F has the 72nd glycine substituted with tyrosine and the 163rd lysine substituted with arginine when compared with the RIP site 3A.

[0050] (11) Preparation of recombinant bitter gourd lectin F111Y / K163R In the wild-type bitter gourd lectin, a recombinant bitter gourd lectin in which the 111th phenylalanine at the RIP site 3A was replaced with tyrosine and the 163rd lysine was replaced with arginine was designated as recombinant bitter gourd lectin F111Y / K163R.

[0051] Recombinant bitter gourd lectin F111Y / K163R could basically be prepared in the same manner as recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the plasmid used for the preparation of recombinant bitter gourd lectin F111Y was used as the template. Also, when performing inverse PCR, the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 were used.

[0052] As shown in FIG. 1G, recombinant bitter gourd 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 contains a signal sequence 9 and a PA tag 11. When the RIP site 3G is compared with the RIP site 3A, the 111th phenylalanine is replaced with tyrosine and the 163rd lysine is replaced with arginine. The RIP site 3G corresponds to the cytotoxic activity site. The amino acid sequence of the RIP site 3G is as shown in SEQ ID NO: 10 and FIG. 7.

[0053] (12) Preparation of recombinant bitter gourd lectin G72Y / F111Y / K163R In the wild-type bitter gourd lectin, a recombinant bitter gourd lectin in which the 72nd glycine at the RIP site 3A was replaced with tyrosine, the 111th phenylalanine was replaced with tyrosine, and the 163rd lysine was replaced with arginine was designated as recombinant bitter gourd lectin G72Y / F111Y / K163R.

[0054] The recombinant bitter gourd lectin G72Y / F111Y / K163R could basically be prepared in the same way as the recombinant bitter gourd lectin G72Y. However, when performing inverse PCR, the plasmid used for the preparation of the recombinant bitter gourd lectin F111Y / K163R was used as a template. Also, when performing inverse PCR, the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 were used.

[0055] As shown in FIG. 1H, the recombinant bitter gourd 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. When the RIP site 3H is compared with the RIP site 3A, the 72nd glycine is substituted with tyrosine, the 111th phenylalanine is substituted with tyrosine, and the 163rd lysine is substituted with arginine.

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

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

[0058] (13b) Confirmation of expression of each recombinant bitter gourd lectin For each of the recombinant bitter gourd lectins G72Y, F111Y, K163R, G72Y / F111Y, G72Y / K163R, F111Y / K163R, G72Y / F111Y / K163R (hereinafter referred to as each recombinant bitter gourd lectin), expression confirmation was performed in the same manner as in the above (13a). In addition, expression confirmation was also performed for wild-type bitter gourd lectin wild-type. For the expression confirmation, the elution fraction with a fraction number of 4 was used. The results are shown in FIGS. 3A and 3B.

[0059] It was confirmed that there was a band around about 80 kDa in each recombinant bitter gourd lectin and wild-type bitter gourd lectin wild-type. (14) Concentration of bitter gourd lectin (14a) Concentration of wild-type bitter gourd lectin wild-type Among the elution fractions collected in the above (5), those with a fraction number of 3 to 6 were transferred to an amicon Ultra-4 centrifugal (Merck). Next, (i) centrifugation was performed at 3000 rpm for 30 minutes. Next, (ii) 2 mL of PBS solution was added and centrifuged. Furthermore, by repeating the treatments of the above (i) and the above (ii) alternately, a final concentrated solution of 300 μL was obtained. This concentrated solution is a concentrated solution of wild-type bitter gourd lectin wild-type.

[0060] (14b) Concentration of each recombinant bitter gourd lectin For each recombinant bitter gourd lectin, a concentrated solution was obtained in the same manner as in the above (14a). (15) Measurement of cytotoxic activity (15a) Measurement of cytotoxic activity of wild-type bitter gourd lectin wild-type Using the concentrated solution of wild-type bitter gourd lectin wild-type obtained in the above (14a), the cytotoxic activity against Capan-1 cells was measured by the MTT assay method. Specifically, the following operations were performed.

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

[0062] (b) On a second collagen-coated 96-well plate (Iwaki), 100 μL of a solution containing Capan-1 cells was seeded per well. The solution containing Capan-1 cells was an Iscove's modified medium containing 20% fetal bovine serum (FBS), and was a solution prepared so that the concentration of Capan-1 cells was 5.0×10 4 cells / mL. Next, by culturing at 37°C for 12 hours, the Capan-1 cells were adhered to the wells.

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

[0064] (d) After removing the medium in (c) above, 100 μL of a solution was added to the wells. The added solution was a solution obtained by diluting a 5 mg / mL 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution 10-fold using Iscove's modified medium. Next, it was reacted at 37°C in the presence of 5% CO2 for 4 hours in a CO2 incubator.

[0065] (d) After completion of the reaction in (b) above, the medium was removed from the wells using an aspirator. Next, the wells were washed once with 200 μL of PBS solution. Next, 50 μL of dimethyl sulfoxide (DMSO) solution was added to the wells to lyse the Capan-1 cells, and the mixture was shaken at room temperature for 15 minutes until a uniform solution was obtained.

[0066] (f) Next, using a microplate reader (Thermo Bioanalysis Japan), the absorbance at a wavelength of 570 nm was measured for the solution containing Capan-1 cells. The measurement results are shown in FIG. 4. (15b) Measurement of the cytotoxic activity of each recombinant bitter gourd lectin Instead of wild-type bitter gourd lectin wild-type, the concentrated solutions of each recombinant bitter gourd lectin obtained in (14b) above were used, and in other respects, in the same manner as in (15a), the cytotoxic activity against Capan-1 cells was measured. The measurement results are shown in FIG. 4. Among the dilution series of recombinant bitter gourd lectin F111Y / K163R, when a solution with a dilution factor of 1 / 10 was used, the absorbance decreased. A decrease in absorbance means having cytotoxic activity against Capan-1 cells.

[0067] The reason why the absorbance decreases as the cytotoxic activity against Capan-1 cells increases is as follows. Capan-1 cells contain a redox enzyme that reduces MTT to purple formazan crystals. Purple formazan crystals are pigments. The fewer the number of viable Capan-1 cells, the more difficult it is for MTT to be converted into a pigment, and the lower the absorbance. Therefore, the more cytotoxic activity against Capan-1 cells, the lower the absorbance. 4. Other embodiments As described above, the embodiments of the present disclosure have been described, but the present disclosure is not limited to the above-described embodiments and can be implemented in various modifications.

[0068] (4-1) In each of the above embodiments, the functions of one component may be shared among a plurality of components, or the functions of a plurality of components may be exerted by one component. Also, a part of the configuration of each of the above embodiments may be omitted. Further, at least a part of the configuration of each of the above embodiments may be added to, replaced with, etc., the configuration of other above embodiments.

[0069] (4-2) In addition to the recombinant proteins described above, the present disclosure can also be realized in various forms such as a system having the recombinant protein as a component, a method for producing a recombinant protein, a gene encoding the recombinant protein, and the like.

Explanation of Reference Numerals

[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

Claim 1 A recombinant protein comprising a lectin site and a cytotoxic activity site. Claim 2 The recombinant protein according to Claim 1, wherein the cytotoxic activity site is the recombinant protein of the following (a) or (b). (a) A protein consisting of an amino acid sequence in which the 111th phenylalanine is substituted with tyrosine and the 163rd lysine is substituted with arginine in the amino acid sequence of the RIP site contained in Momordica charantia lectin (b) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in (a) above and having cytotoxic activity Claim 3 An anticancer therapeutic agent containing the recombinant protein according to Claim 1 or 2.

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