KR102599111B1 - Insulin-like growth factor-1 fusion protein and use thereof - Google Patents

Abstract

The present invention relates to a biologically active human insulin-like growth factor-1 fusion protein and its use, specifically to a human insulin-like growth factor-1 (hIGF-1) protein fused to the chaperone protein PDIb'a' or MBP. It has biological activity and is stable even without IGF binding protein (Insulin-like growth factor bidding protein, IGFBP), and stimulates the IGF receptor (Insulin-like growth factor 1 receptor, IGFR-1R), the receptor for insulin-like growth factor-1. Therefore, it can be usefully used to treat growth hormone deficiency.

Description

Insulin-like growth factor-1 fusion protein and use thereof {Insulin-like growth factor-1 fusion protein and use thereof}

The present invention relates to a biologically active human insulin-like growth factor-1 fusion protein and its use, specifically to human insulin-like growth factor-1 (hIGF) fused with a tag protein (chaperone protein PDIb'a' or MBP). -1) The protein has biological activity and is stable even without the IGF binding protein (Insulin-like growth factor bidding protein, IGFBP), and the IGF receptor (Insulin-like growth factor 1 receptor, IGFR-), which is the receptor for insulin-like growth factor-1. 1R), it can be useful for treating growth hormone deficiency.

Growth hormone is a single molecule polypeptide composed of 191 amino acids and is a hormone secreted from the pituitary bulb. It participates in cell growth and regeneration by combining with growth hormone receptors and expressing IGF-1 (Insulin like Growth Factor-1). Growth hormone is produced in the pituitary gland in a normal person's body, and its production is known to increase until puberty and gradually decrease with age.

Representative growth hormone deficiency symptoms include adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (PGHD). In the case of adult growth hormone deficiency, it occurs idiopathically or when the patient's pituitary gland is damaged by radiation or surgery during the treatment of brain tumor, cerebral hemorrhage, etc. If growth hormone is not secreted properly, symptoms such as weight loss, decreased bone mineral density, increased fat, decreased HDL, increased LDL, and decreased muscle strength appear, deteriorating the quality of life. Pediatric growth hormone deficiency occurs when the pituitary gland is damaged or has developmental disorders. If there is a growth hormone secretion disorder, short stature occurs. The height is in the bottom 3% of the growth curve of peers, or growth is less than 5 cm per year, and symptoms such as hypoglycemia, decreased stamina, depression, and mental immaturity may appear.

Meanwhile, human insulin-like growth factor 1 (hIGF1), also known as somatomedin-C, plays an important role in childhood growth and continues to have an anabolic effect in adults.

Insulin-like growth factor 1 (hIGF1) is a drug approved for the long-term treatment of growth failure and short stature in children with severe primary insulin-like growth factor deficiency or growth hormone insensitivity. Insulin-like growth factor is basically a growth factor that acts similar to insulin in the body, and can be used in all types of cells in the body, including skeletal muscle cells, bone cells, cartilage cells, liver cells, kidney cells, nerve cells, skin cells, and hematopoietic cells. It is known that it can promote the growth of cells. In addition, insulin-like growth factors maintain the self-renewal and pluripotency of human pluripotent stem cells (hPSCs) and transform hPSCs into beta cells and hair cells. can be differentiated into Insulin-like growth factor 1 (hIGF1) binds to the insulin-like growth factor 1 receptor (IGF1R) and has low affinity for the insulin receptor (INSR).

When mature insulin-like growth factor is used alone, it is unstable under physiological conditions, and in the case of denatured insulin-like growth factor, there is a limitation in that it does not bind to its receptor, the insulin-like growth factor receptor. Meanwhile, IGF binding protein (Insulin-like growth factor bidding protein, IGFBP) stabilizes the structure of human insulin-like growth factor (hIGF-1) in solution, but is separated before human insulin-like growth factor binds to the insulin-like growth factor receptor. It has to be.

Under this background, the inventors of the present invention proposed that stable insulin-like growth factor stimulates the insulin-like growth factor receptor even without IGF-binding protein (IGFBP), thereby preventing adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (pediatric growth hormone deficiency). We sought to develop a synthetic peptide that is excellent for treating growth hormone deficiencies such as deficiency (PGHD).

(0001) US Patent No. 7,355,018 (registered on April 8, 2008)

The inventors of the present invention proposed that stable insulin-like growth factor stimulates the insulin-like growth factor receptor even without IGF-binding protein (IGFBP), thereby treating adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (PGHD). In order to develop a synthetic peptide excellent for treating growth hormone deficiency, a fusion protein was prepared in which the chaperone protein PDIb'a' or MBP was fused to the N-terminus of human insulin-like growth factor-1 (hIGF-1). Its biological activity was confirmed. PDIb'a' has a longer shape than MBP, which reduces the burden of IGF1 binding to the receptor, and the b' and a' domains of PDIb'a' are connected by a flexible linker, which imposes less steric hindrance. It was confirmed that biological activity was superior. As a result, the inventors of the present invention found that PDIb'a'-hIGF1 or MBP-hIGF1 is a synthetic peptide that can replace hIGF1 or hIGF1/IGFBP complex and completed the present invention. Accordingly, the purpose of the present invention is to provide an insulin-like growth factor fusion protein in which the tag protein PDIb'a' or MBP is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1). I'm doing it.

Another object of the present invention is to provide a pharmaceutical composition for treating growth hormone deficiency containing the insulin-like factor fusion protein.

In order to achieve the above object, the present invention is an insulin-like growth factor fusion in which the tag protein PDIb'a' or MBP is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1). Provides protein.

In addition, the present invention provides a pharmaceutical composition for treating growth hormone deficiency, which contains the insulin-like factor fusion protein, a gene encoding the protein, or an expression vector containing the gene encoding the protein as an active ingredient. do.

The fusion protein according to the present invention is stable, improves solubility, and exhibits biological activity due to the attachment of the tag protein PDIb'a' or MBP. Accordingly, stable insulin-like growth factor stimulates the insulin-like growth factor receptor even without IGF-binding protein (IGFBP), thereby promoting the growth of adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (PGHD). Excellent for treating hormone deficiency.

In particular, PDIb'a' has a longer shape than MBP, which reduces the burden of IGF1 binding to the receptor, and the b' and a' domains of PDIb'a' are connected by a flexible linker, imposing less steric hindrance. Therefore, it is more effective in treating growth hormone deficiency.

The fusion protein attached to PDIb'a' or MBP according to the present invention can be used as a replacement for hIGF1 or hIGF1/IGFBP complex for the treatment of growth hormone deficiency.

Figure 1 shows the crystal structure of the hIGF1 and IGFBP complex (hIGF1: magenta, IGFBP1: yellow, and IGFBP4: gray; PDB code is 2DSQ).
Figure 2(A) shows a schematic diagram of the production process of the Tag-hIGF 1 expression vector and the structure of the fusion protein with TEVrs.
Figure 3 shows the results of analyzing the expression and solubility of hIGF 1 fused with 7 different tag proteins using SDS-PAGE: M - molecular weight size marker, C - control, T - total after sonication. , S - soluble fraction, I - insoluble fraction. (A) Seven tags are displayed on BL21(DE3) induced at 18°C. (B) The tag of BL21 (DE3) induced at 37°C is displayed. (C) and (D) show Origami 2 strains induced at 18°C and 37°C, respectively. (E) and (F) show Shuffle strains induced at 18°C and 37°C, respectively. The red arrow indicates the expected protein size band.
Figure 4 shows the results of purification, (A) the affinity chromatography analysis result of the PDIb'a'-hIGF 1 purification, and (B) the cation exchange chromatography analysis result of the PDIb'a'-hIGF 1 purification. and (C) the results of SDS-PAGE gel analysis from all purification processes of PDIb'a'-hIGF 1 purification; M - molecular weight marker, 1 - total after sonication (obtained from cultures previously induced with 0.5 mM IPTG), 2 - soluble fraction from sonicated, 3 - solubilized fraction from affinity column, 4 - cation exchange. Product eluted from the column, 5 - final product after dialysis and endotoxin removal (reduced state), 6 - final product after dialysis and endotoxin removal (non-reducing state), (D) Affinity chromatography of MBP-hIGF 1 purification. (affinity chromatography) is the result of analysis; M - molecular weight marker, 1 - solubilized fraction after sonication (obtained from cultures previously induced with 0.5 mM IPTG), 2 - solubilized fraction from affinity column.
Figure 5 is a flow chart showing the purification process. (A) PDIb'a'-IGF 1 purification flow chart is shown. (B) shows the flow chart of MBP-IGF 1 purification.
Figure 6 shows the results showing dose-dependent activation of the hIGF1 fusion protein of MCF-7, a human breast cancer cell line: A - PDIb'a'-hIGF1. The EC ₅₀ is 348.0 pg/mL, which corresponds to 88.0 pg/mL hIGF1. B - MBP-hIGF1. The EC ₅₀ is 6.96 ng/mL, which corresponds to 1.46 ng/mL of hIGF1.
Figure 7 shows the atomic structures of IGF1, IGF1R complex, PDIb'a', and MBP. (A) Shows the IGF1 and IGF1R complex structure, indicating the N-terminus and C-terminus of IGF1. Gray is IFG1R. The PDB code is 6PYH. (B) PDIb'a' structure. The PDB code is 6PYH. (C) MBP structure. The PDB code is 1ANF.
Figure 8 shows the results of measuring the protein stability of PDIb'a'-hIGF1 using SYPRO Orange Fluorescent Dye.

Hereinafter, the present invention will be described in detail.

The present invention provides treatment for adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (PGHD) by stimulating the insulin-like growth factor receptor with stable insulin-like growth factor without IGF-binding protein (IGFBP). This relates to a synthetic peptide that is excellent for treating growth hormone deficiency. In detail, it involves manufacturing a fusion protein in which the chaperone protein PDIb'a' or MBP is fused to the N-terminus of human insulin-like growth factor-1 (hIGF-1). Its biological activity was confirmed. PDIb'a' has a longer shape than MBP, which reduces the burden of IGF1 binding to the receptor, and the b' and a' domains of PDIb'a' are connected by a flexible linker, which imposes less steric hindrance. It was confirmed that biological activity was superior.

Therefore, the present invention provides a tagged protein, PDIb'a' or MBP, at the N-terminus of human insulin-like growth factor 1 (hIGF-1), which can replace hIGF1 or hIGF1/IGFBP complex. Provided is a fused, biologically active insulin-like growth factor fusion protein.

In detail, the tag protein, PDIb'a' protein, may be SEQ ID NO: 1, and the MBP protein may be SEQ ID NO: 2. Figure 1 shows the crystal structure of the hIGF1 and IGFBP complex (hIGF1: magenta, IGFBP1: yellow, and IGFBP4: gray; PDB code is 2DSQ). Figure 2 also shows a schematic diagram of the production process of the Tag-hIGF 1 expression vector and the structure of the fusion protein with TEVrs.

As an example, when the tag protein PDIb'a' is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1), it can represent SEQ ID NO: 3. When MBP is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1), it can represent SEQ ID NO: 4 (Table 1).

division No. Sequence PDIb’a’tag One MKHHHHHHHHEGGGLIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANE VEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAV MBP tag 2 MGSSHHHHHHGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKA GLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT PDIb’a’-IGF1 3 MKHHHHHHHHEGGGLIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANE VEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVGTGSYITSLYKKAGFENLYFQGGTGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM* MBP-IGF1 4 MGSSHHHHHHGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKA GLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGTGSYITSLYKKAGFENLYFQGGTGPETLC GAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM*

In the present invention, “vector” refers to a self-replicating DNA molecule used to transport a clonal gene (or other fragment of clonal DNA). “Expression vector” means a recombinant DNA molecule containing a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the operably linked coding sequence in a specific host cell. The expression vector may preferably contain one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicilin, kanamycin, G418, Bleomycin, hygromycin, and chloramphenicol, and may be identified by those skilled in the art. You can select appropriately. The host cell may be any one selected from the group consisting of E. coli, yeast, mold, plant cell, and animal cell, but is not limited thereto, and any host cell used in the art for producing recombinant proteins can be used.

Accordingly, the present invention can provide a gene encoding the fusion protein or an expression vector containing a gene encoding the fusion protein.

In addition, the Gateway Vector system used in the present invention consists of an entry vector and a destination vector, where the entry vector is a vector having attL1 and attL2 at both ends of the target gene, and the destination vector is a vector containing attR1 and attR2. The entry vector and the destination vector cause an LR reaction by a recombinase, and attR1 and attR2 of the destination vector are replaced with attL1 and attL2 of the entry vector. In this process, the target gene contained in the entry vector is transferred to the target vector.

The fusion protein according to the present invention has biological activity even without IGF binding protein (IGFBP) and can stimulate IGFRs (insulin-like growth factors). In addition, the tag proteins, PDIb'a' protein and MBP protein, improve solubility and thermal stability. In particular, the b' domain and a' domain of PDIb'a' are connected by a flexible linker, which imposes less steric hindrance, so its biological activity is better than that of MBP protein.

In addition, the present invention provides a biologically active insulin-like growth factor fusion protein in which the tag protein PDIb'a' or MBP is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1). , or a gene encoding the protein, or an expression vector containing the gene encoding the protein as an active ingredient. It provides a pharmaceutical composition for treating growth hormone deficiency.

Representative growth hormone deficiency symptoms include adult growth hormone deficiency (AGHD) and pediatric growth hormone deficiency (PGHD). In the case of adult growth hormone deficiency, it occurs idiopathically or when the patient's pituitary gland is damaged by radiation or surgery during the treatment of brain tumor, cerebral hemorrhage, etc. If the secretion of growth hormone is not normal, symptoms such as weight loss, decreased bone mineral density, increased fat, decreased HDL, increased LDL, and decreased muscle strength appear, deteriorating the quality of life. Pediatric growth hormone deficiency occurs when the pituitary gland is damaged or has developmental disorders. If there is a growth hormone secretion disorder, short stature occurs. The height is in the bottom 3% of the growth curve of peers, or growth is less than 5 cm per year, and symptoms such as hypoglycemia, decreased stamina, depression, and mental immaturity may appear. If the height is more than 3 SD lower than the average for the same age group, if the height is more than 1.5 SD lower than the average height of the parents, if the height is more than 2 SD lower than the average height, if the height is more than 1 SD lower than the average height of peers for more than 1 year, or if the height is more than 2 SD lower than the average height of the parents If the SD value is lower than 0.5, or if no symptoms of short stature appear, but remains below 2 SD for more than 1 year or 1.5 for more than 2 years, it can be diagnosed as pediatric growth hormone deficiency.

In addition, when the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the active ingredients. Such pharmaceutically acceptable carriers are commonly used in drug preparation, such as lactose and dextrose. , sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, It may include, but is not limited to, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the pharmaceutical composition may further include fillers, weighting agents, binders, lubricants, wetting agents, disintegrants, sweeteners, flavoring agents, emulsifiers, suspending agents, fragrances, preservatives, etc. as additives and auxiliaries.

The pharmaceutical composition can be formulated into tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, emulsions, freeze-dried preparations, suppositories, and sterile injectable solutions. The pharmaceutical composition of the present invention can be administered by intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, intrasternal administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, topical administration, oral administration and inhalation. It can be administered in a conventional manner.

The method of administration of the pharmaceutical composition is determined depending on the severity of symptoms, and topical administration is usually preferable. In addition, the effective amount of the active ingredient of the pharmaceutical composition refers to the amount required for the prevention or treatment of disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It is stated that it may be adjusted according to various factors including secretion rate, treatment period, and concurrently used drugs, but is not limited thereto.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response regulators.

Therefore, PDIb'a'-hIGF1 and MBP-hIGF1 according to the present invention are stable without IGFBP and can be activated by stimulating tagged IGFR and show the effect of increasing the existing half-life, thereby producing hIGF1 or hIGF1/IGFBP complex. Since it can be replaced, it can be useful in treating growth hormone deficiency.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited to these examples.

<Materials and Methods>

1. Materials

Dithiothreitol (DTT) and 1-thio-β-d-galactopyranoside (IPTG) were purchased from AnaSpec (Fremont, CA) and Coomassie brilliant blue R-250. and Tris were obtained from Amresco (Solon, OH). Imidazole and ampicillin were obtained from Daejeong Chemical (Siheung, Korea) and DuchefaBiochemie (Haarlem, Netherlands), respectively. Lambda integrase and excision enzyme were purchased from Elpis Biotech (Daejeon, Korea). Sodium chloride (NaCl) and glycerol were purchased from Samcheon Chemicals (Pyeongtaek, Korea), and 2-mercaptoethanol was purchased from Yakuri Pure Chemicals (Kyoto, Japan). All purification columns were obtained from GE healthcare (Piscataway, NJ), and the Limulus Amebocyte Lysate test kit was obtained from Lonza (Basel, Switzerland). E. coli BL21 (DE3) and Origami 2 (DE3) cells were obtained from Novagen (Waddison, Madison). Amicon Ultra-15 centrifugal filter devices were purchased from Millipore (Billerica, MA), and commercial HSA and l -arginine were purchased from Sigma-Aldrich (St. Louis, MO). Guanidine-HCl was supplied by Biosesang (Seongnam, Korea), and BODIP FL l-cystine and native polyacrylamide gel electrophoresis (PAGE) gel were purchased from Thermo Fisher Scientific Korea Ltd (Seoul, Korea) and Koma Biotech (Seoul, Korea), respectively. did. All chemicals purchased above are of analytical grade.

2. Construction and expression design of pDestH8G4-Pb'a'-TEVrs-IGF1 clone

Genes were obtained from a commercially obtained plasmid (21C Frontier Human Gene Bank, clone ID: KU029721, full, gene bank: NM_000618). In detail, the gene is first inserted into the "pOTB7" vector, and then through polymerase chain reaction (PCR), there are restriction enzyme sites (KpnI: GGTACC and XbaI: TCTAGA) at the ends, and primers (forward primer: ATA GGTACC GGA CCG) GAG ACG CTC TGC and reverse primer: CGC TCTAGA CTA CAT CCT GTA GTT CTT) were inserted linearly. The pENTR-His-ccdb vector was digested with the same enzymes. The gene and digested vector were linked by ligase, and the new clone was called ‘His Tagged IGF 1’.

The second clone was prepared by the same enzymes and plasmids: His Tagged IGF 1 and pDONR 207 were ligated using ligase enzyme. The subclone generated from the His Tagged IGF 1-pDONR 207 reaction was named pENTR-IGF 1 and was submitted through LR reaction (Gateway Clone, Invitrogen, CA, USA) to generate the desired target vector pDestH8G4-Pb'a'. . The expression vector called hPDIb'a'-hIGF1 generated the structure of the PDIb'a' tag, the hIGF1 gene, and the tobacco etch virus recognition site (TEVrs) to secure free hIGF1 (Figure 2).

3. Expression and solubility of recombinant fusion protein

E. coli BL21 strain was transformed by heat shock method, and the overnight culture was incubated with Luria Bertani (LB medium) and 50 ug/mL ampicillin at 37°C, shaking at 180 rpm/min until OD 0.5 was reached at a 1:100 ratio. Subculture was performed. After dividing into three fractions, two fractions were induced with 0.5mM IPTG (the third was used as a non-inducted control). One fraction and the control group were incubated at 37°C for 4 hours with shaking. The other fraction was incubated at 17°C overnight at the same rate. Fractions were collected and the pellet was sonicated to obtain total soluble and insoluble fractions. In the control group, only the total portion was observed. Fractions were visualized through SDS-Page gel 10% Tricine and percentages were obtained by gel analysis in GelAnalyzer software (Copyright 2010, Istvan Lazar and Dr Istvan Lazar).

4. Cell disruption

Induced cell cultures were harvested at 3000 rpm for 20 min at 4°C and the pellet was incubated in Lysis buffer [20 mM Tris-HCl, pH 8, 5% glycerol, 250 mM NaCl, 1X Protease Inhibitor Cocktail, Sigma P2714-IBTL. ] was resuspended. They were then sonicated on ice with 60 cycles of 3 second burst and 27 second rest. The sonicated product was centrifuged at 13000 rpm for 20 min at 4°C. The supernatant was filtered through a hydrophilic membrane filter (0.45 ㎛, polyethersulfone, Hyundai Micro Co., Ltd.).

5.1. Purification of recombinant PDIb'a'-hIGF1 fusion protein.

a) Affinity column

The soluble fraction of the cell lysate was applied to a HiTrap HisTrap (GE Healthcare, Bucks, UK) column previously equilibrated with 5 column volumes of buffer A (20 mM Tris-HCl, pH8, 5% glycerol, 250 mM NaCl). Once the sample was bound, the column was washed with 2 column volumes of buffer A and 5 volumes of 50 mM imidazole in buffer A and then eluted isotropically with 500 mM imidazole in buffer A (Figure 5).

b) Desalting step and cation exchange column

The product eluted from the affinity column was desalted by dialysis against A1 buffer (20 mM Tris-HCl, pH 8, 5% glycerol) using a 10 KDa dialysis membrane, and the ions were previously equilibrated with 5 column volumes of A1 buffer. It was applied to an exchanged HiTrap SP HP column (GE Healthcare, Bucks, UK). Once the sample was applied, the unbound sample was washed with 5 column volumes of Buffer A1, followed by elution with a gradient of 0-1 M NaCl in Buffer A1 per 10 column volumes. Once again, the fractions were confirmed through electrophoresis using 10% tricine gel SDS-PAGE (Figure 5).

5.2. Purification of recombinant MBP-hIGF1 fusion protein

The soluble fraction of the cell lysate was applied to a HiTrap MBP HP (GE Healthcare, Bucks, UK) column previously equilibrated with 5 column volumes of Buffer A (20 mM Tris-HCl, pH8, 5% glycerol, 250 mM NaCl). .

Once the sample was bound, the column was washed with 10 column volumes of Buffer A and eluted isotropically with 10 mM maltose in Buffer A. The eluted fractions were confirmed through electrophoresis on 10% tricine gel SDS-PAGE.

6. Dialysis of final buffer

The fusion protein was concentrated using an AMICON ultracentrifugation filter 10 KDa (Millipore, MA, USA) and dialyzed against phosphate-buffered saline, pH 7.4.

7. Endotoxin removal and quantification

Endotoxins were removed by adding Triton ^® did. The supernatant was then carefully collected, filtered through a 0.2 μm, 13 mm, polyethersulfone (PES), sterile syringe filter (Hyundai Micro, Korea), and stored at -20 for further analysis. Concentration was measured by Bradford Assay. For endotoxin quantification, the ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript, USA) was used according to the manufacturing instructions (Figure 5).

8. Activity Assay

MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. MCF-7 cells were seeded in 24-well plates at a density of 5 x 10 ⁴ cells per well. After 24 hours of incubation, the medium was removed and each well was washed twice with 0.5 mL PBS. Then, the medium was changed to serum-free medium, and the cells were treated with different concentrations of PDIb'a'-IGF. After 72 hours of culture with protein, the medium was removed, 0.5 mL of MTT solution (0.04 mg/mL in serum-free medium) was added, and cultured for 2 hours. Afterwards, the MTT solution was removed and 0.5 mL DMSO was added. After incubation for 2 hours, absorbance was measured at 595 nm. Cell proliferation was calculated using the following equation and Microsoft Excel.

Re = Bl + ( Max Bl )/(1 + (EC ₅₀ / conc ) ^Hs ) ( Max Bh )/(1 + (IC ₅₀ / conc ) ^Hi )

Here, Re means the response of the cells, Bl is the baseline at low concentration, Max is the maximum response, and conc is the concentration of the protein. , Hs means Hill coefficient of stimulation, Bh means baseline at high concentration, and Hi means Hill coefficient of inhibition.

9. Statistics

All data are expressed as mean ± standard error (SE). The experiment was performed independently three times.

<Example 1> Construction of recombinant protein

To arrive at the final expression vector used to obtain the fusion protein, a PCR reaction was first performed on the commercially obtained gene by adding restriction enzyme sites to each end. After treating the vector pENTR-His-ccdb with the same restriction enzyme and ligating the plasmid gene with ligase enzyme, the His Tagged IGF 1 clone was successfully obtained. Then, cloning was performed once more through a restriction reaction using the same site. At this time, clone pENTR-IGF 1 was inserted into the pDONR 207 vector.

LR reaction was performed with the pDESTH8G4 pb'a' vector using the pENTR-IGF 1 clone to generate the expression clone (gateway clone, Invitrogen, CA, USA) pDESTH8G4 pb'a' (Figure 2a).

Once the expression clone was obtained, overexpression was induced through IPTG induction on the T7 promoter, and the fusion protein shown in Figure 2b was obtained as the final product (hPDIb'a' tag, tobacco etch virus recognition site (TEVrs), and hIGF 1 in that order). configured).

<Example 2> Solubility and expression

Expression levels and solubilities were compared for two different tags (PDIb'a' and MBP) and at two different temperatures (37°C and 18°C). Both tags showed low overexpression but high solubility (Tables 2 to 4). This was especially noticeable in PDIb'a'. In detail, MBP was overexpressed in about 31.57% at 37°C and 26.6% at 18°C, and PDIb'a' was overexpressed in 38.85% at 37°C and 20.1% at 18°C. In the BL21 strain, MBP was 94.3% soluble at 18°C and PDIb'a' was 99.4% soluble at 18°C (Figures 2a and 2b, Table 2). For other strains tested, the values may show slight changes, but high solubility and low overexpression remain the same (Tables 3 and 4).

Expression level and solubility of hIGF in BL21 strain. hIGF1
(11.7 kDa) tag tag size
(kDa) Fusion protein size (kDa) Expression level (%) Solubility (%) 18℃ 37℃ 18 ^o C 37℃ MBP 40.2 55.79 26.6 31.57 94.3 92.2 PDIb'a' 30.6 46.66 20.1 38.85 99.4 53.8 His 6 3.77 15.46 - 16.37 - 89.41 Sumo 15.42 22.11 - - - - Trx 15.58 27.27 16.37 25.5 29.54 40.86 GST 29.47 41.16 13.44 14.82 61.36 38.62 NusA 58.64 70.33 51.73 21.49 97.81 71.21

Expression level and solubility of hIGF 1 in Origami 2 strain hIGF1
(11.7 kDa) tag tag size
(kDa) Fusion protein size (kDa) Expression level (%) Solubility (%) 18℃ 37℃ 18℃ 37℃ MBP 40.2 55.79 43.18 40.91 97.46 90.2 PDIb'a' 30.6 46.66 32.78 23.63 99.43 43.81 His 6 3.77 15.46 20.79 27.98 100 97.1 Sumo 15.42 22.11 - - - - Trx 15.58 27.27 41.36 48.62 93.66 52.68 GST 29.47 41.16 13.73 41.38 40.56 22.01 NusA 58.64 70.33 56.95 51.45 97.7 60.65

Expression level and solubility of hIGF 1 in Shuffle strain. hIGF1
(11.7 kDa) tag tag size
(kDa) Fusion protein size (kDa) Expression level (%) Solubility (%) 18℃ 37℃ 18℃ 37℃ MBP 40.2 55.79 24.45 19.35 97.84 72.01 PDIb'a' 30.6 46.66 33.85 36.96 99.05 26.96 His 6 3.77 15.46 26.98 41.42 80.32 15.23 Sumo 15.42 22.11 - - - - Trx 15.58 27.27 44.1 54.43 93.11 63.71 GST 29.47 41.16 19.36 31.39 73.42 14.42 NusA 58.64 70.33 39.63 53.13 87.9 94.56

<Example 3> PDIb'a'-hIGF 1 purification

Cells were induced with IPTG and the pellet was obtained, lysed by sonication, and the soluble fraction was applied to a His Trap FF column (GE Healthcare, Bucks, UK).

The final product eluted from the affinity column was desalted by dialysis to remove most impurities and then applied to a HiTrap SP HP column (GE Healthcare, Bucks, UK).

The product from the cation exchange column was dialyzed against PBS, endotoxin was removed with Triton ^® X-114 (Sigma - Aldrich), and filtered through a 0.2 um syringe filter. For endotoxin, ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript, USA) was used. As a result of the measurement, the final purified product was 0.13 EU/mL.

When the tag was removed, rhIGF1 aggregated. For NaCl sensitivity, NaCl was substituted for NMDG, but aggregation still occurred even after tag cleavage. Accordingly, the present inventors additionally measured activity.

<Example 4> MBP-hIGF 1 purification

The sonicated soluble fraction was applied to a HiTrap MBP HP column (GE Healthcare, Bucks, UK) in the same manner as PDIb'a'-hIGF 1, and the eluted product was dialyzed against PBS and purified against Triton ^® X-114 (Sigma). -Aldrich) and filtered through a 0.2um syringe filter. The fusion of hIGF1 with the MBP tag showed the same behavior, including aggregation after cleavage, in PDIb'a'-hIGF1 (Figure 4).

<Example 5> Biological Activity Measurement

In vitro activity for PDIb'a'-hIGF1 and MBP-hIGF1 was measured using MCF-7, a human breast carcinoma cell line (FIG. 6). Figure 6 is a result showing the dose-dependent activation of the hIGF1 fusion protein of the human breast cancer cell line MCF-7. The EC ₅₀ of PDIb'a'-hIGF1 is 348.0 pg/mL, which corresponds to 88.0 pg/mL hIGF1. The EC ₅₀ of MBP-hIGF1 is 6.96 ng/mL, which corresponds to 1.46 ng/mL of hIGF1.

PDIb'a'-hIGF1 and MBP-hIGF1 showed different dose responsiveness. PDIb'a'-hIGF1 showed a bell-shaped biphasic dose-response curve with a maximum response of 437% at 3 nM (Figure 6), and EC ₅₀ and IC ₅₀ values were calculated to be 0.35 nM and 30 nM, respectively. The Hill excitation coefficient and Hill inhibition coefficient were 2.3 and 1.6, respectively.

On the other hand, MBP-hIGF1 showed a monophasic sigmoid dose-response curve with a maximum response of 305% (Figure 6). EC ₅₀ and Hill coefficient were 6.5 nM and 1.15, respectively.

The EC ₅₀ of PDIb'a'-hIGF1 is 348 pM, which corresponds to 87.2 pg/mL of hIGF1. In other studies using hIGF1, the EC ₅₀ was reported to be 300 to 1,500 pg/mL. The ED ₅₀ of rhIGF1 produced in E. coli was less than 5 ng/ml as measured by cell proliferation assay using FDC-P1 cells, which corresponds to an activity of more than 2.0 x 10 ⁵ units/mg (Cancer Res. 1988 Jul 15;48(14):4083-92.).

Considering that the large size of the tag is still at the N-terminus of hIGF1, it is quite remarkable that PDIb'a'-hIGF1 still shows good activity. This is especially true considering that MBP-hIGF1 is not as active as PDIb'a'-hIGF1 or hIGF1. The cryo-EM structure of the full-length IGF1R-IGF1 complex in the active state shows that the N- and C-termini of IGF1 move away from IGF1R, allowing space for some of the tagged proteins (Figure 7). This suggests that PDIb'a' is a better tag for IGF1. First, PDIb'a' and MBP are 227-residue and 370-residue proteins, respectively, and PDIb'a' provides less steric hindrance to IGF1 than MBP. Second, PDIb'a' has an elongated shape, whereas MBP has a spherical shape, which may relieve PDIb'a's burden on IGF1 binding to the receptor. Third, the b'domain and a'domain of PDIb'a' are connected by a flexible linker. PDIb'a'-hIGF1 showed a bell-shaped biphasic dose-response curve with a peak at 3 nM (Figure 6). A similar biphasic dose-dependent response was observed in another study of hIGF1.

Meanwhile, MBP-hIGF1 showed a monophasic sigmoid dose-response curve rather than a bell-shaped dose-response curve. Considering that the EC ₅₀ of PDIb'a'-hIGF1 is lower than that of MBP-hIGF1, it can be seen that MBP-hIGF1 exhibits an inhibitory effect at a higher concentration.

<Example 6> Thermal stability

The thermal stability of the protein was measured using SYPRO Orange Fluorescent Dye. As a result of Figure 8, the melting temperature was about 61.4°C for PDIb'a'-IGF1 and about 40°C for hIGF1. This suggests that the thermal stability of IGF1 protein increased when tagged.

Taken together, mature hIGF1 alone is not stable and is denatured under physiological conditions. Denatured hIGF1 has no binding affinity for the receptor. Therefore, mature hIGF1 must bind to IBP4, IBP1, or their receptors for stabilization. According to the present invention, when the tag was removed, mature rhIGF1 aggregated and could not go any further. Therefore, the present inventors confirmed the effect of tagged protein fusion, and interestingly, both PDIb'a'-hIGF1 and MBP-hIGF1 showed biological activity in vitro. Therefore, it was confirmed that the PDIb'a' or MBP tag not only stabilizes rhIGF1, but also does not prevent the fusion protein from binding to the receptor and does not induce a cellular response.

That is, the fusion proteins PDIb'a'-hIGF1 and MBP-hIGF1 according to the present invention are stable without IGFBP and stimulate tagged IGFR. In particular, PDIb'a'-hIGF1 can replace hIGF1 or hIGF1/IGFBP complex for experiments and treatments.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the practical scope of the present invention will be defined by the appended claims and their equivalents.

<110> University of Ulsan Foundation For Industry Cooperation THE ASAN FOUNDATION <120> Insulin-like growth factor-1 fusion protein and use thereof <130> ADP-2021-0011 <150> KR10-2020-0011469 <151> 2020-01-31 <160> 4 <170>CopatentIn 1.71 <210> 1 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> PDIb'a'tag <400> 1 Met Lys His His His His His His His His His His Glu Gly Gly Gly Gly Leu 1 5 10 15 Ile Glu Phe Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile 20 25 30 Lys Thr His Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp 35 40 45 Gly Lys Leu Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys 50 55 60 Ile Leu Phe Ile Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile 65 70 75 80 Leu Glu Phe Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu 85 90 95 Ile Thr Leu Glu Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu 100 105 110 Leu Thr Ala Glu Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly 115 120 125 Lys Ile Lys Pro His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp 130 135 140 Lys Gln Pro Val Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala 145 150 155 160 Phe Asp Glu Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys 165 170 175 Gly His Cys Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr 180 185 190 Tyr Lys Asp His Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala 195 200 205 Asn Glu Val Glu Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe 210 215 220 Phe Pro Ala Ser Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg 225 230 235 240 Thr Leu Asp Gly Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly 245 250 255 Ala Gly Asp Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro 260 265 270 Asp Met Glu Glu Asp Asp Asp Gln Lys Ala Val 275 280 <210> 2 <211> 378 <212> PRT <213> Artificial Sequence <220> <223> MBP tag <400> 2 Met Gly Ser Ser His His His His His His Gly Thr Lys Thr Glu Glu 1 5 10 15 Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu 20 25 30 Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr 35 40 45 Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala 50 55 60 Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly 65 70 75 80 Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala 85 90 95 Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn 100 105 110 Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile 115 120 125 Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile 130 135 140 Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met 145 150 155 160 Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp 165 170 175 Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp 180 185 190 Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val 195 200 205 Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile 210 215 220 Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly 225 230 235 240 Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val 245 250 255 Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly 260 265 270 Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala 275 280 285 Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala 290 295 300 Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu 305 310 315 320 Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala 325 330 335 Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp 340 345 350 Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr 355 360 365 Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 370 375 <210> 3 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> PDIb'a'-IGF1 <400> 3 Met Lys His His His His His His His His His His Glu Gly Gly Gly Gly Leu 1 5 10 15 Ile Glu Phe Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile 20 25 30 Lys Thr His Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp 35 40 45 Gly Lys Leu Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys 50 55 60 Ile Leu Phe Ile Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile 65 70 75 80 Leu Glu Phe Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu 85 90 95 Ile Thr Leu Glu Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu 100 105 110 Leu Thr Ala Glu Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly 115 120 125 Lys Ile Lys Pro His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp 130 135 140 Lys Gln Pro Val Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala 145 150 155 160 Phe Asp Glu Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys 165 170 175 Gly His Cys Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr 180 185 190 Tyr Lys Asp His Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala 195 200 205 Asn Glu Val Glu Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe 210 215 220 Phe Pro Ala Ser Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg 225 230 235 240 Thr Leu Asp Gly Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly 245 250 255 Ala Gly Asp Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro 260 265 270 Asp Met Glu Glu Asp Asp Asp Gln Lys Ala Val Gly Thr Gly Ser Tyr 275 280 285 Ile Thr Ser Leu Tyr Lys Lys Ala Gly Phe Glu Asn Leu Tyr Phe Gln 290 295 300 Gly Gly Thr Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala 305 310 315 320 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr 325 330 335 Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp 340 345 350 Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys 355 360 365 Ala Pro Leu Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg 370 375 380 His Thr Asp Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala 385 390 395 400 Ser Arg Gly Ser Ala Gly Asn Lys Asn Tyr Arg Met *** 405 410 <210> 4 <211> 508 <212> PRT <213> Artificial Sequence <220> <223>MBP-IGF1 <400> 4 Met Gly Ser Ser His His His His His His Gly Thr Lys Thr Glu Glu 1 5 10 15 Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu 20 25 30 Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr 35 40 45 Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala 50 55 60 Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly 65 70 75 80 Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala 85 90 95 Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn 100 105 110 Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile 115 120 125 Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile 130 135 140 Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met 145 150 155 160 Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp 165 170 175 Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp 180 185 190 Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val 195 200 205 Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile 210 215 220 Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly 225 230 235 240 Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val 245 250 255 Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly 260 265 270 Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala 275 280 285 Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala 290 295 300 Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu 305 310 315 320 Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala 325 330 335 Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp 340 345 350 Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr 355 360 365 Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly Thr Gly Ser Tyr Ile 370 375 380 Thr Ser Leu Tyr Lys Lys Ala Gly Phe Glu Asn Leu Tyr Phe Gln Gly 385 390 395 400 Gly Thr Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu 405 410 415 Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly 420 425 430 Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu 435 440 445 Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala 450 455 460 Pro Leu Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His 465 470 475 480 Thr Asp Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser 485 490 495 Arg Gly Ser Ala Gly Asn Lys Asn Tyr Arg Met *** 500 505

Claims (8)

It is an insulin-like growth factor fusion protein in which the tag protein PDIb'a' is bound to the N-terminus of human insulin-like growth factor 1 (hIGF-1),
The insulin-like growth factor fusion protein is an insulin-like growth factor fusion protein characterized in that it has the amino acid sequence of SEQ ID NO: 3.
According to claim 1,
The fusion protein is an insulin-like growth factor fusion protein, characterized in that it has biological activity even without IGF binding protein (IGFBP) and stimulates IGFRs (insulin-like growth factor).
According to claim 1,
The tag protein is an insulin-like growth factor fusion protein, characterized in that it improves solubility.
According to claim 1,
The PDIb'a' protein is an insulin-like growth factor fusion protein, characterized in that it has SEQ ID NO: 1.
A gene encoding the protein of claim 1.
An expression vector containing a gene encoding the protein of claim 1.
Treatment of growth hormone deficiency containing as an active ingredient the insulin-like factor fusion protein of any one of claims 1 to 4, or a gene encoding the protein, or an expression vector containing the gene encoding the protein. A pharmaceutical composition for:
The pharmaceutical composition for treating growth hormone deficiency according to claim 7, wherein the growth hormone deficiency is adult growth hormone deficiency (AGHD) or pediatric growth hormone deficiency (PGHD).