KR20180003677A - Pharmaceutical compositions comprising mutant proteins of human growth hormone or transferrin fusion proteins thereof - Google Patents

Abstract

Human growth hormone modified proteins of the present invention having an amino acid sequence in which serines at positions 85^th and 144^th in a human growth hormone protein are substituted with hydrophobic amino acids or transferrin fusion proteins thereof, have significantly improved specific activity and stability in blood, and increased half-life, compared to pre-existing human growth hormone proteins, and thus may be beneficially used in the treatment of growth failure or growth retardation.

Description

A pharmaceutical composition comprising a human growth hormone mutant protein or a transferrin fusion protein thereof as an active ingredient,

The present invention relates to a human growth hormone mutation protein, a transferrin fusion protein thereof, or a pharmaceutical composition comprising the protein as an active ingredient.

Human growth hormone (hGH) (hGH) is a polypeptide hormone with a molecular weight of about 22,000 Da, which is expressed by 191 amino acids secreted from the pituitary gland. It stimulates chondrocyte differentiation of the bone growth plate And affects metabolism in adults, and plays a role in increasing protein synthesis and accumulation, thus regulating metabolism. When growth hormone deficiency occurs, many changes in body composition such as muscles, fat, and bones are caused, leading to diseases such as osteoporosis, arthritis, and obesity. In addition, deficiency symptoms due to natural reduction of growth hormone may cause diabetes or hypertension, and supplementation therapy of growth hormone is needed in such cases.

The market for human growth hormone in 2010 is 30% for norditropin (Novo Nordisk), 29% for genotropin (Pfizer), 15% for nutropin (Roche) and 15% for humatrope (Eli Lilly) %. In Korea, growth hormone sales are forecast to increase by more than 4% in 2011 and 2012, and will continue to grow. However, although growth hormone therapy is widely used, it causes physical and psychological burden on patients due to stability and convenience. Polypeptides such as hGH generally have low molecular weight and low stability. They are easy to metabolize. They are easily degraded by protein hydrolytic enzymes in the blood and are easily removed through the kidneys and liver. In order to maintain the human growth hormone concentration and activity, It needs to be administered frequently. These problems cause physical and psychological distress to the patient. To solve this problem, efforts have been made to increase the blood stability of the protein drug and to maintain the drug effect by maintaining the drug concentration in the blood.

Recently, a protein drug preparation capable of minimizing the increase of stability and degradation of activity has been proposed as a physiologically active polypeptide conjugate having increased persistence in vivo (Korean Patent Registration No. 10-0567902), a protein conjugate using immunoglobulin fragment and a method for producing the same (Korean Patent No. 10-0725315), an immunoglobulin Fc method such as a persistent human growth hormone preparation (Korean Patent No. 10-1535681), a human growth hormone chemically modified with polyethylene glycol, a method for producing the same, (Korean Patent No. 10-1104574) and a method using saccharification such as a hyperglycosylated sustained-type human growth hormone protein and a method for producing the same (Korean Patent No. 10-1504824) have been reported.

Transferrin is the third most abundant plasma protein and is responsible for the transport of iron ions in the blood to various tissues. It is shorter than albumin or immunoglobulin G (IgG), but has a relatively long half-life of 8 days. It enters the cell through the transferrin receptor on the cell surface to supply iron ions and binds to the receptor There is a characteristic of being circulated outside the cell. Using these features, they are used as fusion partners to increase the circulating half-life by combining proteins with short half-lives of the prior art.

In the present invention, serine, which is the 85th and 144th amino acids of human growth hormone, is substituted with cysteine and induced to make sulfide bond between helix and C-term loop. These sulphide bonds stabilize the structure of the protein and have resistance to proteolytic degradation. The name of the present invention is called GHC3-P00-Th.

Injection of this into the pituitary-derived Sprague-Dawley Rat model resulted in an increase in body weight compared to the control group, and the level of IGF-1 in the serum was higher than that of the control group .

The inventors of the present invention have found that the specific activity and the stability in blood are remarkably increased as compared with the non-fused circular human growth hormone by fusing a mutant in which amino acids 85 and 114 of human growth hormone are substituted with cysteine with transferrin Thereby completing the invention.

It is an object of the present invention to provide a human growth hormone mutation protein comprising an amino acid sequence in which the 85th and 144th serines in the amino acid sequence of a human growth hormone protein are respectively substituted with hydrophobic amino acids, Or a pharmaceutical composition containing the fusion protein or the protein as an active ingredient.

In order to achieve the above object,

The present invention relates to a human growth hormone mutation protein consisting of an amino acid sequence in which the 85th and 144th serines in the amino acid sequence of human growth hormone protein are respectively substituted with a hydrophobic amino acid, An expression vector containing the gene, and a transformant into which the expression vector has been introduced into a host cell.

The present invention also relates to a fusion protein of a human growth hormone mutation protein and a transferrin in which a transferrin is bound to the N-terminal of the human growth hormone mutation protein, a gene encoding the protein, an expression vector containing the gene, Wherein the expression vector is introduced into the host cell.

The present invention also provides a method for producing a human growth hormone mutant protein comprising the human growth hormone mutation protein, the fusion protein, the expression vector comprising the respective genes encoding the protein, or the respective transformants into which the respective expression vectors are introduced into the host cell, A pharmaceutical composition for the treatment of growth failure or growth retardation.

In addition, the present invention relates to a human growth hormone mutant protein, a fusion protein, an expression vector comprising each gene coding for the protein, or a childhood organism containing as an active ingredient a transformant into which the respective expression vector is introduced into a host cell The present invention provides a pharmaceutical composition for the prevention or treatment of starvation deficiency or adult onset type deficiency.

The human growth hormone mutant protein comprising the amino acid sequence in which the 85th and 144th serines of the human growth hormone protein of the present invention are substituted with hydrophobic amino acids, respectively, or a transferrin fusion protein thereof Specific activity and blood stability of the human growth hormone protein compared to the conventional human growth hormone protein and has an effect of increasing the half life of the conventional human growth hormone protein and thereby being useful for the treatment of growth failure or growth retardation .

1 is a diagram showing a cleavage map of a pcDNA3.1 (+) / preTf vector.
2 is a diagram showing a cleavage map of the pcDNA3.1 (+) / preTf (B) vector.
3 is a diagram showing a cleavage map of a pcDNA3.1 (+) / Tf (B) vector.
4 is a diagram showing a cleavage map of the pcDNA3.1 (+) / GHC3-P00-Th vector.
5 (5a, 5b) is a graph showing the expression amount of GGHC3-P00-Th in the Expi293F cell line transformed with the GHC3-P00-Th plasmid.
FIG. 6 shows the results of Q-Sepharose chromatography in the purification process of the human growth hormone fusion protein. FIG.
FIG. 7 is a graph showing the results of Urea-page determination of transferrin fused to human growth hormone after binding to Q-Sepharose. FIG.
FIG. 8 is a graph showing the results of size exclusion chromatography to remove remaining free iron ions and to change buffers after iron-ion binding of GHC3-P00-Th protein.
FIG. 9 is a graph showing the result of measurement of purity using reverse phase-HPLC for a target protein elution interval during a size exclusion chromatography process.
FIG. 10 is a graph showing the results of measurement of protein target band, purity and impurity using SDS-PAGE, SEC-HPLC, and RP-HPLC for the final purified protein.
Fig. 11 shows the results of confirming the presence or absence of free cysteine residues in proteins. Fig.
12 is a diagram showing the results of confirming intracellular activity of GHC3-P00-Th expressed in an animal cell line through Nb2-11 cell proliferation experiments.
Fig. 13 is a diagram showing a comparison of degradation resistance of hGH and GHC3-P00-Th proteins by proteolytic enzymes.
14 shows a comparison of in vivo plasma half-life of hGH and GHC3-P00-Th proteins.
15 is a graph showing the results of in vivo activity analysis by administering hGH and GHC3-P00-Th to pituitary-removed rats.
FIG. 16 is a graph showing IGF-1 serum concentration in pituitary-extracted rats after SC injection of hGH and GHC3-P00-Th.
FIG. 17 shows the results of weight gain by administering hGH, GHC3-P00-Ta, and GHC3-P00-Th to pituitary-removed rats:
GHC3-P00-Ta: unconjugated form of iron ion;
GHC3-P00-Th: Iron ion-bound form, free iron ion present together; And
GHC3-P00-Th (free-iron free): Recombinant protein in the form of free iron ion removed.

Hereinafter, the present invention will be described in detail with reference to examples.

The present invention relates to a human growth hormone mutation protein consisting of an amino acid sequence in which the 85th and 144th serines in the amino acid sequence of human growth hormone protein are respectively substituted with a hydrophobic amino acid, An expression vector containing the gene, and a transformant into which the expression vector has been introduced into a host cell.

The term human growth hormone (hGH), as used herein, refers to human growth hormone and also analogs, fragments, homologs, derivatives or allelic variants of hGH having the same function as a naturally occurring polypeptide allelic variant. The growth hormone according to the present invention can be purified from human or animal sources, and can be produced chemically or recombinantly. hGH preparations are commercially available.

The amino acid sequence of the human growth hormone is preferably represented by SEQ ID NO: 1, but it is preferable that the amino acid sequence of the human growth hormone is the same as the protein or that the gene coding for the human growth hormone protein on the chromosome is at the same position, Deleted, or substituted.

The human growth hormone protein comprises at least 80% homology, more specifically at least 90% homology, most particularly at least 95%, 96%, 97%, 98%, 99% or 99.5% homology to the amino acid sequence of SEQ ID NO: But it is not limited thereto.

The hydrophobic amino acid is preferably an amino acid capable of sulfidation bonding, and the hydrophobic amino acid is preferably cysteine.

The present invention relates to a fusion protein of a human growth hormone mutation protein and transferrin in which a transferrin is bound to the end of the human growth hormone mutant protein, a gene encoding the protein, an expression vector comprising the gene, Thereby providing a transformant introduced into the cell.

The terminal to which the transferrin is connected may be either an amino terminal (5-terminal, N-terminal) or a carboxy terminal (3-terminal, C-terminal).

The transferrin is a protein represented by the sequence of SEQ ID NO: 2, or a protein having the same activity as the protein or having a gene position encoding the transferrin on the chromosome is equal to or more than that of one or several amino acids of the protein, Base sequence.

The transferrin protein has at least 80% homology, more specifically at least 90% homology, most specifically at least 95%, 96%, 97%, 98%, 99% or 99.5% homology to the amino acid sequence of SEQ ID NO: But it is not limited thereto.

The human growth hormone mutant protein is a protein represented by the sequence of SEQ ID NO: 3 or a protein having the same activity as the protein or a gene position encoding the human growth hormone mutant protein on a chromosome, Amino acid may be added, deleted, or substituted.

The fusion protein is preferably represented by SEQ ID NO: 4, but it is preferable that the fusion protein has the same activity as the protein or that the gene position encoding the fusion protein on the chromosome is the same, Sequence.

The human growth hormone protein or transferrin used in the present invention may be derived from an animal, a plant, or a microorganism and is preferably a human-derived growth hormone protein or transferrin, but is preferably derived from a human-derived growth hormone protein or a heterologous Protein.

Such proteins may additionally be modified such as phosphorylation, acetylation, methylation, glycosylation, etc., and may bind to other proteins, but they may be the same as the proteins before modification as long as they do not change to such an extent that the function of the protein is lost .

The gene encoding the protein may be obtained by substituting the base sequence of the restriction enzyme recognition site in the gene with another base sequence encoding the same amino acid as the amino acid encoding the base sequence, Or may be added with a restriction enzyme recognition site inserted. For example, the protein may be a protein obtained by replacing the Thymine of the BamH I restriction enzyme recognition site (GGATCC) in the protein gene with cytosine.

The type of the restriction enzyme is EcoR I, BamH I, Hind Ⅲ , kpn Ⅰ, Not Ⅰ, Pst Ⅰ, Sma Ⅰ, Xho Ⅰ, Fok Ⅰ, Alw26 Ⅰ, Bbv Ⅰ, Bsr Ⅰ, Ear Ⅰ, Hph Ⅰ, Mbo Ⅰ used, and the like SfaN ⅰ, Tth111 ⅰ, Nae ⅰ, Nhe I, NgoM ⅳ, Nhe I, Eco57 ⅰ, Bcg ⅰ, Bp ⅰ, Bsp24 ⅰ, Bae ⅰ, Cje ⅰ, EcoP ⅰ, Hint ⅲ, StyLT ⅰ, Any restriction enzyme used in the art may be used without limitation, depending on the gene, expression vector or genetic engineering environment in question.

The expression vector may be prepared from a vector represented by any one of the cleavage maps selected from the group consisting of FIGS. 1, 2, 3 and 4.

The expression vector of the present invention includes various forms of plasmid, cosmid, BAC, viral nucleic acid and the like as a means for introducing a nucleic acid sequence encoding a target protein into a host cell. The vector generally includes an optional marker such as an antibiotic resistance gene that can confirm that the target gene has been successfully introduced into the host cell. The vector includes a promoter, an operator, an initiation codon, a termination codon, A denylated sequence, an enhancer, a Kozak sequence, a Shine-Dalgano sequence, and the like. For replicable expression vectors, it includes the origin of replication.

The host cell may be any one selected from the group consisting of E. coli, yeast, fungi, plant cells, and animal cells, and is not limited thereto. Any host cell used in the art for producing recombinant proteins may be used.

In a specific embodiment of the present invention, the human growth hormone mutant protein (GHC3-P00-Th) of the present invention is substituted with cysteine at the 85th serine and the 144th serine in the amino acid sequence of the native growth hormone (GH) Lt; / RTI > Free cysteine was confirmed and the disulfide bonds of cysteines 85 and 144 were confirmed indirectly.

The present inventors also found that GHC3-P00-Th had lower cell proliferation activity than hGH as a result of intracellular activity analysis of human growth hormone (hGH) and human growth hormone mutant protein (GHC3-P00-Th) of the present invention. Based on the above results, the value of EC ⁵⁰ was calculated. The calculated EC ⁵⁰ values were 0.07 ng / ml of hGH and 0.47 ng / ml of GHC3-P00-Th, respectively (see FIG. 12). In the case of GHC3-P00-Th, it was confirmed that the biological efficacy of the hGH-domain was preserved even with increasing molecular weight.

In order to measure the degree of degradation resistance of the GHC3-P00-Th protein of the present invention to the proteolytic enzyme in blood, the inventors of the present invention found that the GHC3-P00- The amount was measured. In the case of natural type GH, the amount of residual GHC3-P00-Th protein was found to be 30% at 15 hours, as the reaction time with serum increased. (See Fig. 13). It was confirmed that disulfide bond formation between cysteine 85 and 144 helps increase the resistance of protein to protease.

In addition, when the GHC3-P00-Th fusion protein of the present invention was administered to a living body, the present inventors measured the amount of growth hormone present in the serum after administration of the protein in mice to determine the biological half-life of the protein. As a result, the GHC3-P00-Th fusion protein showed a higher blood half-life than the native human Growth Hormone (see Table 1). This is a result of slower filtration rate in the kidney, as it has a relatively larger protein radius than Growth Hormone. In addition, fusion of Growth Hormone mutant protein with increased resistance to serum protease in transferrin resulted in a decrease in renal filtration rate and improvement in blood stability, resulting in maximization of blood half-life in the living body (see FIG. 14).

For the in vivo activity test of the GHC3-P00-Th fusion protein of the present invention, a 5-week-old male rat (hypophysectomized Sprague Dawley rat, SLC, Japan) in which pituitary gland was removed was used as an experimental animal. (HGH) and human growth hormone mutation protein (GHC3-P00-Th) in the day after the administration of the human control hormone mutant protein (GHC3-P00-Th) was higher than that of human growth hormone (hGH) at the 10th day after the administration for 10 days. The body weight gain of the human growth hormone mutant protein (GHC3-P00-Th) % (See Fig. 15). When the human growth hormone mutant protein (GHC3-P00-Th) was administered once, body weight was continuously increased until the third day after administration, and up to day 7, the activity was superior to that of human growth hormone (hGH) . When human growth hormone (GHC3-P00-Th) was administered in a single dose, the efficacy was remarkably high, and even when administered once a day, it was found to have an efficacy equal to or higher than that of human growth hormone (hGH). In addition, in rats that received a single dose of human growth hormone mutation protein (GHC3-P00-Th) compared with rats injected daily with human growth hormone (hGH) at a rate of weight gain, Respectively. Therefore, the GHC3-P00-Th of the present invention is expected to be able to solve the limitation of human growth hormone which has an excellent continuous activity as compared with hGH and has to be injected every day.

In addition, the present inventors measured the amount of IGF-1 protein present in the serum when the transferrin fusion protein of Growth hormone of the present invention was administered to a living body. As a result of ELISA analysis, test substance GHC3-P00-Th 30 ug / rat (Daily) and hGH 30 ug / rat (Daily) were significantly higher than vehicle (p <0.001 or p <0.01). GHC3-P00-Th 200 ug / rat (Single) was significantly higher up to day 5 than vehicle (p <0.001 or p <0.05). In addition, the GHC3-P00-Th 30 ug / rat (Daily) was significantly higher than the hGH 30 ug / rat (Daily) until the 10th day (p <0.001). GHC3-P00-Th 200 ug / rat (Single) was significantly higher up to day 3 than hGH 30 ug / rat (Daily) (p <0.001) (see FIG. 16).

Accordingly, the transspan fusion protein (GHC3-P00-Th) of the human growth hormone mutation protein of the present invention has a marked increase in specific activity and blood stability as compared with the conventional human growth hormone protein, And may be useful for the treatment of growth failure or growth retardation.

In addition, the present invention relates to a method for inhibiting growth of a human growth hormone mutant protein, its transferrin fusion protein, an expression vector comprising a gene encoding said protein, or a transformant into which said expression vector has been introduced into a host cell, failure or growth retardation in a mammal.

The growth failure or growth retardation may be caused by, but not limited to, pituitary gland growth hormone secretion disorder, chronic renal disease, Turner's syndrome, cachexia or AIDS wasting .

The present invention also relates to a method for the treatment and / or prevention of childhood-onset deficiency syndrome, which comprises as an active ingredient a human growth hormone mutation protein, a transferrin fusion protein thereof, an expression vector comprising a gene encoding said protein, or a transformant into which said expression vector has been introduced into a host cell Or a pharmaceutical composition for the prevention or treatment of adult onset type deficiency.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a commonly used filler, an extender, a binder, a wetting agent, a disintegrant or a surfactant may be used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally according to the intended method and may be administered orally, parenterally, rectally, intravenously, muscularly, subcutaneously, intracavitally, It is preferable to select it.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, But is not limited to. The individual dosages will specifically contain amounts in which the active drug is administered in one go.

A single dose is 1 to 100 milligrams, preferably 3 to 50 milligrams, more preferably 6 to 30 milligrams, based on the amount of protein, and may be administered once or several times a day, . &Lt; / RTI &gt;

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

Accordingly, the transspan fusion protein (GHC3-P00-Th) of the human growth hormone mutation protein of the present invention has a marked increase in specific activity and blood stability as compared with the conventional human growth hormone protein, And may be useful for the treatment of growth failure or growth retardation.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Plasmid production

<1-1> Transferrin  Production of expression plasmids

HepG2, a human liver cancer cell, was used to obtain transferrin protein. HepG2 cells were subcultured in DMEM (HyClone) containing 10% fetal bovine serum (WelGNE) and 1% penicillin-streptomycin (Gibco) at 37 ° C in a 5% CO 2 incubator. After the subculture, the medium for HepG2 cells cultured for two days was removed, washed with phosphate buffer (HyClone), and the cell pellet was collected, centrifuged, and the medium was removed and stored at -70 ° C. Cells stored at -70 ° C were thawed and thawed, followed by RNA extraction kit (LeGene) using standard conditions as described in the guidelines to obtain total mRNA. The total mRNA was synthesized by using oligo (dT) as a cDNA synthesis system (LeGene).

Polymerase chain reaction (PCR) was performed using DNA polymerase (Phusion) as a template. The primers used for the reaction were custom-made in Cosmos Tech. The Nhe I restriction site, the Kozak sequence, the Xho I restriction site, and the Stop codon TAA). 10 pmol of the sense primer (5'-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3 ', SEQ ID NO: 10) and the antisense primer (5'-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3', SEQ ID NO: 11) were used. Distilled water was added to the DNA polymerase and buffer together with the cDNA template The total volume was 50 μl. The reaction was carried out at 98 ° C for 30 seconds using a PCR instrument. The circulation program was repeated 30 times at 98 ° C for 10 seconds, at 53 ° C for 30 seconds, at 72 ° C for 1 minute, and finally at 72 ° C for 10 minutes. The amplified transferrin gene was electrophoresed on a 1% agarose gel to confirm its size. The desired portion of the agarose gel was cut and the DNA was purified using a DNA purification kit (iNtRoN).

The Nhe I (Enzynomics) and Xho I (Enzynomics) restriction enzymes were treated with the transferrin gene and pcDNA3.1 (+) plasmid (Invitrogen) obtained through purification. 10 units of restriction enzyme and buffer solution (10 mM Tris-HCL pH 7.9, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, 100 μg / ml BSA) were added and reacted at 37 ° C for 2 to 3 hours. After the reaction was completed, the size was confirmed by electrophoresis on a 1% agarose gel, the agarose gel was cut out, and the DNA was purified using a DNA purification kit (iNtRoN). The purified transferrin gene and pcDNA3.1 (+) vector were reacted with T4 DNA-binding enzyme (Takara) at 16 ° C for 16 hours. After the reaction, E. coli DH10B was transformed and applied to an agar plate containing ampicillin antibiotics, followed by culturing at 37 DEG C for 16 hours to select E. coli colonies.

To confirm the successful insertion of the transferrin gene into the pcDNA3.1 (+) plasmid, DNA polymerase was used to confirm the polymerase chain reaction (PCR). (5'-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3 ', SEQ ID NO: 10) and an antisense primer (5'-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3, SEQ ID NO: 11) used for the amplification of transferrin gene were used as a template and the E. coli colonies were diluted in distilled water The reaction was carried out at 98 ° C for 30 seconds. The cycle was repeated 30 times at 98 ° C for 10 seconds, at 53 ° C for 30 seconds, and at 72 ° C for 1 minute, and finally at 72 ° C for 10 minutes. After PCR, the genes were confirmed by electrophoresis on 1% agarose gel. The colonies were inoculated into LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl) And cultured in a 37 ° C shaking incubator. The cultured Escherichia coli was centrifuged and separated into supernatant and cell pellet. The cell pellet was purified with plasmid extraction kit (GeneAll). Sequence analysis of the purified plasmid was carried out in Cosmos TEX. The thus constructed transferrin expression plasmid is referred to as pcDNA3.1 (+) / preTf (SEQ ID NO: 5) in this specification (Fig. 1).

Transferrin present in the gene in order to use the restriction enzymes BamH I site when plasmid was produced by the process of the BamH I site subjected to regioselective mutation (site-direct mutagenesis) removing the BamH I restriction site. To remove the BamH I restriction site present in the transferrin gene, the nucleic acid was replaced with cytosine (C) in thymine (T), but the amino acid sequence was maintained as aspartic acid. (5'-CTATGGGTCAAAAGAGGACCCACAGACTTTCTATT-3 ', SEQ ID NO: 12) and an antisense primer (5'-AATAGAAAGTCTGTGGGTCCTCTTTTGGCCCATAG-3', SEQ ID NO: 13) to replace the BamH I restriction enzyme site, GGATCC with GGACCC, . The prepared pcDNA3.1 (+) / preTf plasmid was used as a template, and a site-directed mutagenesis kit (iNtRON) was used according to the manufacturer's instructions. After completion of the polymerase chain reaction, the gene was confirmed by electrophoresis on 1% agarose gel. The identified E. coli colonies were inoculated into LB liquid medium containing ampicillin antibiotics and cultured for 16 hours in a shaking incubator at 37 ° C. The cultured Escherichia coli was centrifuged, and the supernatant and cell pellet were separated. The cell pellet was purified by plasmid extraction kit (GeneAll), and the plasmid DNA was purified from the BamH I restriction site It was confirmed that the nucleic acid was substituted. The thus constructed plasmid is referred to as pcDNA3.1 (+) / preTf (B) (SEQ ID NO: 6) in this specification (FIG.

Transferrin is a secreted protein that contains a signal peptide that helps the protein synthesized at the N-terminus to be delivered to the cell membrane, and the amino acid sequence is MRLAVGALLVCAVLGLCLA (SEQ ID NO: 14). To insert the human growth hormone gene into the 5 'end of the transferrin gene, a signal peptide present in the transferrin was removed and a plasmid in which the restriction enzyme Bam HI was located at the 5' end was prepared. Xho I and Stop codon (TAA) were located at the 3 'end. The polymerase chain reaction was performed using pcDNA3.1 (+) / preTf (B) as a template and DNA polymerase. The primers used for the reaction were custom-made in Cosmos Tech. 10 pmol of a sense primer (5'-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3 ', SEQ ID NO: 15) and an antisense primer (5'-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3', SEQ ID NO: 11) was used. Distilled water was added to the DNA polymerase and buffer solution together with the template The total volume was 50 μl. The reaction was carried out at 98 ° C for 30 seconds using a PCR instrument. The circulation program was repeated 30 times at 98 ° C for 10 seconds, at 53 ° C for 30 seconds, at 72 ° C for 1 minute, and finally at 72 ° C for 10 minutes. The amplified transferrin gene was electrophoresed on a 1% agarose gel to confirm its size, and the agarose gel was cut and the DNA was purified using a DNA purification kit (iNtRoN). The purified transferrin gene and pcDNA3.1 (+) plasmid were treated with Bam HI (Enzynomics) and Xho I (Enzynomics) restriction enzymes, respectively. 10 units of restriction enzyme and 2 μl of buffer solution (10 mM Tris-HCL pH 7.9, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, 100 μg / ml BSA) were added and reacted at 37 ° C for 2 to 3 hours. After the reaction, electrophoresis was carried out on 1% agarose gel, and the size was confirmed. The agarose gel was cut and the DNA was purified using a DNA purification kit (iNtRoN). The purified transferrin gene and pcDNA3.1 (+) vector were electrophoresed on 1% agarose gel and DNA was quantified. The transferrin gene and pcDNA3.1 (+) plasmid were added with T4 DNA joining enzyme (Takara) And reacted at 16 DEG C for 16 hours. After the reaction, E. coli DH10B was transformed, applied to an agar plate containing ampicillin, and cultured at 37 DEG C for 16 hours to select E. coli colonies. Successful insertion of the transferrin gene into the pcDNA3.1 (+) plasmid was performed by polymerase chain reaction. Escherichia coli colonies were diluted in water and used as a template. Using the sense primer (5'-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3 ', SEQ ID NO: 15) and antisense primer (5'-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3', SEQ ID NO: 11) The reaction was carried out at 98 ° C for 30 seconds. The circulation program was repeated 30 times at 98 ° C for 10 seconds, at 53 ° C for 30 seconds, at 72 ° C for 1 minute, and finally at 72 ° C for 10 minutes. After completion of the polymerase chain reaction, the cells were electrophoresed on 1% agarose gel to confirm the insertion of the gene, and E. coli colonies with gene insertion were inoculated into LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl) And cultured in a 37 ° C shaking incubator for 16 hours. The cultured Escherichia coli was centrifuged and separated into supernatant and cell pellet. The cell pellet was purified using a plasmid purification kit (GeneAll). Sequence analysis of the purified plasmid confirmed the successful insertion of the signal peptide-deleted transferrin gene into pcDNA3.1 (+). The resulting plasmid was named pcDNA3.1 (+) / Tf (B) (SEQ ID NO: 7) in this specification (Fig. 3).

<1-2> GHC3 - P00 - Th  Production of expression plasmids

GH1 (untagged) -Human growth hormone 1 (GH1), transcript variant 1, NM_000515.3 (OriGene, SC300088) was purchased and human growth hormone contained in pCMV6-XL5 vector was used as a template. (5'-ATAGCTAGCTCCACCATGGCTACAGGCTCCCGGACCTACAGGCTCCCGGAC-3 ', SEQ ID NO: 16) and an antisense primer (5'-TATGGATCCGAAGCCACAGCTGCCCTCCA-3', SEQ ID NO: 17) were amplified by polymerase chain reaction using a DNA polymerase The gene was amplified. The amplified gene includes a signal peptide that helps the protein synthesized at the N-terminus to be delivered to the cell membrane, and the amino acid sequence is MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 18). The gene fragment was manipulated to have the NheI restriction site and the Kozak sequence at the 5 'end of the amplified gene and the BamHI restriction enzyme at the 3' end. Twenty units of Nhe I and Bam HI restriction enzymes were added to each of the amplified human growth hormone gene and pcDNA3.1 (+) / Tf (B) plasmid, and reacted at 37 ° C for 3 hours. Then, electrophoresis was performed on 1% agarose gel After DNA was isolated, DNA was purified using a DNA clean up kit (COSMO). The purified DNA was reacted with T4 DNA joining enzyme (Takara) at 16 ° C for 16 hours. After the reaction, E. coli DH10B was transformed and applied to an agar plate containing ampicillin and cultured at 37 ° C for 16 hours. E. coli colonies were selected on the plate, inoculated into LB liquid medium, and cultured with shaking at 37 DEG C for 16 hours. The cultured Escherichia coli was centrifuged and separated into supernatant and cell pellet. The cell pellet was purified using Exprep ^™ PlasmidSV, mini (GeneAll). The purified plasmids were identified by sequencing in Cosmogyne Tex. This plasmid was named pcDNA3.1 (+) / GHW-P00-Th (SEQ ID NO: 8) in the present specification.

Positive mutation was performed using pcDNA3.1 (+) / GHW-P00-Th as a template to substitute 85 amino acid serine of human growth hormone with cysteine. The gene was amplified through chain polymerization by inserting the sense primer (5'-GCTGCTCATCCAGTGTTGGCTGGAGCCCG-3 ', SEQ ID NO: 19) and the antisense primer (5'-CGGGCTCCAGCCAACACTGGATGAGCAGC-3', SEQ ID NO: 20) . The amplified DNA was reacted with Dpn I (Enzynomics) at 37 ° C for 1 hour to remove the DNA used as a template. After the reaction, 5 μl was transformed into DH10B, applied to a plate containing ampicillin, and cultured at 37 ° C for 16 hours. E. coli colonies were selected and cultured again in LB liquid medium for 16 hours. After centrifugation, the cells were separated into supernatant and cell pellet, and the plasmid DNA was purified. The purified DNA was identified by sequencing.

Additional positional selective mutagenesis was performed to replace the 144 amino acid serine with cysteine, using the DNA in which the 85th serine was replaced with cysteine as a template. The gene was amplified using a sense primer (5'-TCAAGCAGACCTACTGCAAGTTCGACACACCC-3 ', SEQ ID NO: 21) and an antisense primer (5'-GTTTGTGTCGAACTTGCAGTAGGTCTGCTTGA-3', SEQ ID NO: 22). The amplified DNA was reacted with Dpn I (Enzynomics) at 37 ° C for 1 hour to remove the DNA used as a template. After the reaction, 5 μl was transformed into DH10B, applied to a plate containing ampicillin, and cultured at 37 ° C for 16 hours. E. coli colonies were selected and cultured again in LB liquid medium for 16 hours. After centrifugation, the cells were separated into supernatant and cell pellet, and the plasmid DNA was purified. The purified DNA was identified by sequencing. The thus constructed plasmid was named pcDNA3.1 (+) / GHC3-P00-Th (SEQ ID NO: 9) in this specification (FIG.

< Example  2> through transient transduction GHC3 - P00 - Th  Expression of protein

The proteins used in the present invention were expressed by expressing proteins outside the cells using Expi293F ^TM cells (Gibco), a modified human embryonic kidney cancer cell line, for suspension culture. Inoculate Expi293F ^TM Expression system kit (Gibco) after the Expi293F ^TM cells contained in the extract, 30 ml suspension culture a serum-free medium (Serum-free media for animal cell culture, Gioco) is filled with a sterile 125 ml flask (Corning) for And incubated at 37 DEG C in a 5% carbon dioxide incubator at a stirring speed of 125 rpm.

Maintain Expi293F ^TM cell lines in cell density of ^{3 ~ 5 x 10 6 cells /} ml and serum-free medium to a cell density of 2 x 10 ⁶ cells / ml for transfection was subcultured to stabilize the cells every 3-4 days And cultured with shaking for 24 hours. The cell density was again adjusted to 2 x 10 ⁶ cells / ml just before transfection and cell viability was determined to be 96% or more.

Prepared via bulk purified plasmid pcDNA3.1 (+) / GHC3-P00 -Th DNA to prepare the 22.5 ug prepared and diluted with Opti-MEM (Gibco) and ExpiFectamin ^TM 45 uL of 293 Reagent was also diluted with Opti-MEM® medium and left at room temperature for 5 minutes. Plasmid and ExpiFectamin ^TM 293Reagent mixture were mixed well, allowed to stand at room temperature for 20 minutes, and placed evenly in the prepared cells. To increase protein expression between 16 and 18 hours after the transformation, the additives Enhancer 1, 2 (Gibco) were added and cultured for another 78 hours in the shaking culture conditions. The cell culture was centrifuged at 2,000 xg for 10 minutes at 4 ° C to separate the supernatant and cell pellet. Protease inhibitor (Roche) and 80% sucrose were added to the separated supernatant to 5%, and stored at -70 ° C.

To confirm protein expression, the supernatant was mixed with 5 × loading dye (Biosesang), reacted at 95 ° C for 20 minutes, and loaded on a 10% SDS PAGE gel and electrophoresed at 160 V for 1 hour. Gel is separated, washed with purified distilled water and reacted with protein staining solution (Thermo) for 1 hour to develop color. The transferrin was about 75 kDa and the molecular weight of GHC3-P00-Th fused with the human growth hormone mutant protein was 97 kDa, indicating that the protein was normally expressed (FIG. 5A).

To further confirm the protein expression by Western blotting, the sample was loaded on a 10% SDS PAGE gel, electrophoresed, and the gel was separated and loaded on a PVDF Immobilon-P-Transfer Membrane (Milipore) . 5% Skim milk was mixed with a phosphate buffer solution (phosphate buffer solution containing 0.05% Tween 20), and Membrane was added thereto, followed by reaction at room temperature for 1 hour. After completion of the reaction, the cells were washed three times with phosphate buffered saline, and human growth hormone antibody (Santa Cruz) was diluted in 2,000-fold phosphate buffered saline and reacted at room temperature for 2 hours. After completion of the reaction, the cells were washed three times with phosphate-buffered saline, diluted with phosphate buffered saline (10,000 ×), and reacted at room temperature for 1 hour with a secondary antibody (Santa Crux) conjugated with Horseradish perixidase. After completion of the reaction, the protein is washed three times with phosphate-buffered saline and developed using SuperSignal®WestPicoChemiluminescentSubstrate (Thermo). GH and GHC3-P00-Th were confirmed by human growth hormone antibody (Fig. 5B).

< Example  3> protein purification

<3-1> GHC3 - P00 - Th  Protein Q- Sepharose  Chromatography

The GHC3-P00-Th culture was dialyzed in 20 mM potassium phosphate buffer, pH 7.5 solution (dialysis time: at least 16 hours at 4 ° C). AKTA prime plus FPLC was loaded with Hitrap Q HP, 5 mL column, and then flowed into 20 mM K ₂ HPO ₄ , pH 7.5 buffer at a flow rate of 3 mL / min. The column was equilibrated and the dialyzed culture fluid was injected. Then, a target protein adsorbed to the resin was eluted using a NaCl concentration gradient (Fig. 6).

<3-2> Iron Fe ^{3 +} )this Combined  Form manufacturing

The holo-form production of the GHC3-P00-Th of the present invention is described in Zhang et al. BMC Biotechnology 2012, 12: 92]. The purified GHC3-P00-Th protein was subjected to SDS-PAGE to determine the approximate concentration, and then treated with iron ammonium citrate, which was twice the amount of protein, and reacted at 4 ° C for 16 hours or more. (Fig. 7).

<3-3> GHC3 - P00 - Th  protein you exclusion  Chromatography

Size exclusion chromatography was performed to remove the remaining free iron ions. The column was equilibrated with 20 mM Tris, 75 mL NaCl, pH 8.0 buffer at a flow rate of 1 ml / min in a 120 mL column of Hi-load 16/60 sepharcryl, and 5 mL of the iron-bound protein was loaded. Free iron ions were removed (Fig. 8). Purity was determined by reverse phase-HPLC (RP-HPLC) for the target peak during iron ion removal. The Waters alliance HPLC instrument was equipped with an RP-HPLC column of ZORBAX 300SB-C18 (4.6 X 50 mm) and equilibrated with 0.1% TFA / Water. The concentration of 0.1% TFA / acetonitrile was gradually increased to 50 minutes, and the concentration of GHC3-P00-Th protein in equilibrated column was 40 mL. (Fig. 9). In addition, a region containing a peak estimated as an impurity was also found, and a region where the 28th peak was excluded was excluded and a protein was obtained (Fig. 9).

<3-4> GHC3 - P00 - Th  Protein quantification

Purified GHC3-P00-Th protein was quantitated by SDS-PAGE. 0.1, 0.2, 0.4, and 0.6 μg were injected into the wells for standard quantification curves using human growth hormone protein quantified by ELISA. The purified samples were diluted and treated with 5 mL and 10 mL, Protein was quantified by comparison with quantitative curve.

<3-5> GHC3 - P00 - Th  Check protein purity

Reverse phase-HPLC and size exclusion chromatography-HPLC of purified GHC3-P00-Th proteins confirmed impurity and purity measurements. ZORBAX GF-250 (4.6 X 250 mm) was used for the SEC-HPLC column and ZORBAX 300SB-C18 (4.6 X 50 mm) was used for the RP-HPLC column. Waters HPLC instrument was equipped with SEC-HPLC column, equilibrated with 20 mM tris + 75 mM NaCl, pH 8.0, and 50 mL of GHC3-P00-Th protein was injected for 20 min. As a result of measurement, the target peak eluted at 4.3 minutes, and when the basic peak of the buffer was removed, the purity was measured to be 99.9%. In addition, a reverse phase-HPLC column was mounted on a Waters HPLC instrument and equilibrated with 0.1% TFA / Water. Then, 50 mL of GHC3-P00-Th protein was injected and the concentration of 0.1% TFA / acetonitrile was gradually increased. , The target peak eluted at 27 minutes and when the basic peak of the buffer was removed, the purity was measured to be 96.8% (FIG. 10).

< Example  4> Protein Free  Cysteine Residue  Check whether

In the case of GHC3-P00-Th protein, the 85th serine and 144th serine in the amino acid sequence of native GH were substituted with cysteine and induced to form disulfide bonds with each other. In the case of wild type GH, the 53rd amino acid is combined with 165, 182 and 189 cysteines, resulting in a total of two disulfide bonds and no free cysteine (free cysteine). Therefore, the presence of free cysteine of the GHC3-P00-Th protein substituted with cysteine at positions 85 and 144 was confirmed to indirectly confirm the disulfide bond of cysteines 85 and 144.

A commercially available product (Cayman Chemical) was purchased and confirmed for free cysteine or free thiol. GHC3-P00-Th, and BSA 500 nM were reacted with a thiol fluorometric detector and fluorescence was measured. BSA is a 66 kDa protein with a single free cysteine residue. Fluorescence measurements were performed at 385 nm and the fluorescence intensity at 515 nm was measured.

The results of reaction of each protein with a fluorescent detector are shown in Fig. Free cysteine residues were found to be lower than those of BSA containing one free cysteine residue. It means that there is little free cysteine in the GHC3-P00-Th protein.

< Example  5> Measurement of biological activity of human growth hormone mutant protein

In order to compare the intracellular activity of the purified human growth hormone mutant protein, the following experiment was conducted.

Specifically, intracellular activity was comparatively analyzed using Nb2-11 cells (European Collection of Cell Cultures (ECACC) # 97041101), which is a rat node lymphoma cell line that is a human growth hormone-dependent mitotic dividing cell .

Nb2-11 cells were cultured in Fischer's medium supplemented with 10% fetal bovine serum (FBS), 0.075% NaCO ₃ , 50 μM 2-mercaptoethanol, 2 mM glutamine and 10% -deficient horse serum) at 37 ° C under 5% carbon dioxide, and replaced with fresh medium at 2-day-3-day intervals. Cells are not adhesion-dependent.

For analysis, Nb2-11 cells were adjusted to about 2.5 x 10 ⁵ cells / ml using 1% FBS culture in 10% FBS, and then cultured for 24 hours. After 24 hours of incubation, the Nb2-11 cell line was harvested by centrifugation, washed with the same medium except for 10% FBS, and 100 μl was added to each well of the 96-well plate to give about 2 x 10 ⁴ cells per well. Human growth hormone (hGH, GenScript) used as a control and human growth hormone mutant protein (GHC3-P00-Th) as a test substance were respectively diluted and added to each well containing Nb2-11 cells by concentration. After 72 hours And cultured in a 5% carbon dioxide incubator at 37 ° C. The cell proliferation activity of the test substance was the same as that of human growth hormone (hGH). 10 μl of alamarBlue (ThermoFisher SCIENTIFIC) reagent was added to the cell culture and the plate was incubated in the incubator for 4 hours. Plates were gently shaken to evenly react cells in each well, and the absorbance at 570 nm was measured to determine the number of increased cell lines and analyzed. The concentration of the sample that allowed 50% of the cells to survive with the absorbance value obtained by the alamarBlue assay was determined as EC ₅₀ (50% effective concentration).

The results of the intracellular activity analysis of human growth hormone (hGH) and human growth hormone mutant protein (GHC3-P00-Th) of the present invention are shown in Fig. As shown in FIG. 12, GHC3-P00-Th showed lower cell proliferation activity than hGH. Based on the above results, the value of EC ₅₀ was calculated. For each calculated EC ₅₀ value, it was confirmed that hGH was 0.07 ng / ml and GHC3-P00-Th was 0.47 ng / ml. In the case of GHC3-P00-Th, it was confirmed that the biological efficacy of the hGH-domain was preserved even with increasing molecular weight.

< Example  6> Measurement of degradation resistance to proteolytic enzymes in blood

In order to measure the degree of degradation resistance of the GHC3-P00-Th protein of the present invention to the proteolytic enzyme in blood, the amount of GH remaining in the serum was measured at a predetermined time after the protein was reacted with human serum (Serum).

Specifically, human serum (SIGMA) was inactivated at 56 ° C for about 30 minutes, and a 2.8 μg / ml protein sample was reacted with serum at a ratio of 24: 1 (v / v) at 37 ° C. The reaction time was 0, 3, 6, 9, 12, and 15 hours. The samples were treated with complete protease inhibitor cocktail (Roche) at each time point and stored at -70 ° C to terminate the reaction. GH ELISA (RnDSystems) was performed to determine the amount of residual protein. The procedure was performed with reference to the manufacturer's manual.

The concentration conversion according to the absorbance was calculated based on the calibration curve of the protein concentration-absorbance for the standard solution. The standard curve was created using an Excel program. The average value of the absorbance corresponding to each GH and GHC3-P00-Th protein standard solution and the difference value of the absorbance of the well treated only with the buffer were used. The amount of residual GH protein (% GH) was expressed based on 100% of the protein concentration (pm / ml) of GH when the reaction time was 0 hour.

GH, and GHC3-P00-Th proteins of the present invention are shown in FIG. In the case of natural type GH, the amount of residual GHC3-P00-Th protein was found to be 30% at 15 hours, as the reaction time with serum increased. have. It was confirmed that disulfide bond formation between cysteine 85 and 144 helps increase the resistance of protein to protease.

< Example  7> hGH  In vivo half-life measurement of mutant proteins

When the GHC3-P00-Th fusion protein of the present invention was administered to a living body, the amount of Growth Hormone present in the serum was measured after administration of the protein in mice to determine the biological half-life of the protein. The above-described "in vivo half-life time" is expressed as a "half-life of protein in plasma &quot;, i.e., a time period during which growth hormone protein circulates in plasma and remains at about 50% at initial concentration. The pharmacokinetic calculation was performed using the R program.

<7-1> Growth Hormone  Animal administration test of mutant protein

Animal experiments were conducted to determine the duration of the GHC3-P00-Th fusion protein of the present invention in the body. The entire process of purchasing, breeding, administering and collecting blood samples was conducted by the Korean Institute of Science and Technology.

Male Sprague-Dawley (SPF) male mice (6 weeks old) were purchased from Orient BioRotor. After a minimum of 7 days of acclimation, individuals were weighed at the end of the quarantine and refinement period, and healthy animals were selected for weight gain and general symptoms based on the weight at that time. (Within 200g ± 20% per animal) were randomly assigned to each test group.

On the day of administration of the test substance, 50 μg of growth hormone mutant or its transferrin fusion protein per kg of body weight (1 mL / kg) was weighed (5 mL / kg) , 26 G needle). The day of administration was defined as day 1 of the test.

Blood samples were collected at each point (0, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h) using the jugular vein. mL. The blood was collected and centrifuged at 3.000 rpm for 10 minutes in a heparin tube. The samples were stored in a deep freezer set at -70 ° C or lower.

<7-2> Growth Hormone  Measurement of in vivo plasma half-life of mutant proteins

The amount of active growth hormone in the plasma was quantified by ELISA after thawing the sample from ice. The quantification of plasma growth hormone by ELISA was performed by purchasing the R & D Human Growth Hormone ELISA Kit (R & D, Cat. DGH00) and the procedure was carried out according to the manual of the reagent. Calibration curve of absorbance according to protein concentration in standard solution was prepared and regression analysis was performed to determine the content of proteins of Growth Hormone mutation in the test solution.

Non-compartment pharmacokinetic analysis from the mean concentration-time profile of each test compound was performed by applying a non-compartment analysis input to the R program. Pharmacokinetic parameters were evaluated for terminal half-life (t _1/2).

The half-life of proteins in plasma of mutant proteins of Growth Hormone is shown in Table 1 below. As shown in Table 1, the GHC3-P00-Th fusion protein showed a higher blood half-life than the native human growth hormone. This is a result of slower filtration rate in the kidney, as it has a relatively larger protein radius than Growth Hormone. In addition, fusion of Growth Hormone mutant protein with increased resistance to blood proteolytic enzymes to transferrin resulted in a decrease in renal filtration rate and improvement in blood stability, thus maximizing blood half-life in the living body (FIG. 14).

Protein half-life in plasma The parameter (H) Growth Hormone GHC3-P00-Th t _1/2 1.62 12.16

< Example  8 > GHC3 - P00 - Th In vivo activity test

Five-week-old male rats (hypophysectomized Sprague Dawley rats, SLC, Japan) in which the pituitary gland was removed were used as experimental animals. The experimental group was divided into four groups and each group was subjected to weight gain test using 5 mice per group. Mice were separated by a randomized method so that the weight of the animals determined to be healthy during the 15 day purification period was measured and weighted according to the weighted average weight distribution of each group as uniform as possible. Human gonadotropin (hGH), 30 ㎍ per rat, human growth hormone mutant protein (GHC3-P00-Th), 30 ㎍ per rat, and vehicle control were administered once / day in male rats from which pituitary- (GHC3-P00-Th) was administered at a dose of 200 μg per rat. The dose and method as shown in Table 2 below were subcutaneously injected and the body weight of the rats after injection was measured daily.

The results of the weight gain analysis of the rat pituitary gland of GHC3-P00-Th of the present invention are shown in FIG. As shown in FIG. 15, in the case of a rat in which a pituitary gland was removed from which a solvent control was administered, weight gain hardly occurred, but human growth hormone (hGH) and human growth hormone mutation protein (GHC3-P00-Th) (GHC3-P00-Th) increased by 3.53% more than human growth hormone (hGH) at 10 days after the administration for 10 days. Respectively. When the human growth hormone mutant protein (GHC3-P00-Th) was administered once, body weight was continuously increased until the third day after administration, and up to day 7, the activity was superior to that of human growth hormone (hGH) . When human growth hormone (GHC3-P00-Th) was administered in a single dose, the efficacy was remarkably high, and even when administered once a day, it was found to have an efficacy equal to or higher than that of human growth hormone (hGH). In addition, in rats that received a single dose of human growth hormone mutation protein (GHC3-P00-Th) in comparison with rats injected daily with human growth hormone (hGH) at a weight gain rate, (Fig. 15).

In addition, as shown in Fig. 17, in the case of GHC3-P00-Th in which free iron ions were removed, it was also confirmed that the body weight was rapidly increased over the first 3 days in case of single administration (Fig. 17).

Therefore, the GHC3-P00-Th of the present invention is expected to be able to solve the limitation of human growth hormone which has an excellent continuous activity as compared with hGH and has to be injected every day.

number Test substance N Route Dose (ug / head) Amount of solution (mL / head) Method of administration One Vehicle 5 s.c. 1 time / day, repeated administration for 10 days 2 hGH (GenScript) 5 s.c. 30 7 1 time / day, repeated administration for 10 days 3 GHC3-P00-Th 5 s.c. 30 7 1 time / day, repeated administration for 10 days 4 GHC3-P00-Th 5 s.c. 200 7 Single administration

< Example  9> IGF -1 protein in vivo

When the transferrin fusion protein of Growth hormone of the present invention was administered to a living body, the amount of IGF-1 protein present in the serum was measured. The entire process of purchasing, breeding, administering and collecting blood samples, analyzing IGF-1 concentration, etc., was performed by Korean scientific research institute.

Blood samples were taken from the jugular vein using a syringe once per day before and 10 days after dosing. Blood was injected into a vacutainer tube containing a clot activator, allowed to stand at room temperature for about 15 minutes, and then coagulated. The serum was centrifuged at 3,000 rpm for 10 minutes, and IGF-1 was quantified using an ELISA kit.

As shown in FIG. 16, the test substance GHC3-P00-Th 30 ug / rat (Daily) and hGH 30 ug / rat (Daily) were significantly higher than vehicle (p <0.001 or p <0.01). GHC3-P00-Th 200 ug / rat (Single) was significantly higher up to day 5 than vehicle (p <0.001 or p <0.05). In addition, the GHC3-P00-Th 30 ug / rat (Daily) was significantly higher than the hGH 30 ug / rat (Daily) until the 10th day (p <0.001). GHC3-P00-Th 200 ug / rat (Single) was significantly higher up to day 3 than hGH 30 ug / rat (Daily) (p <0.001) (Fig.

<110> EQUIS & ZAROO <120> Pharmaceutical compositions comprising mutant proteins of human          growth hormone or transferrin fusion proteins thereof <130> 2015P-10-034 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> human growth hormone <400> 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg   1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu              20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro          35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg      50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu  65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val                  85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp             100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu         115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser     130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe                 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe             180 185 190 <210> 2 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Transferrin protein <400> 2 Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala   1 5 10 15 Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser              20 25 30 Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys          35 40 45 Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala      50 55 60 Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val  65 70 75 80 Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr                  85 90 95 Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu             100 105 110 Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp         115 120 125 Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys     130 135 140 Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro 145 150 155 160 Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly                 165 170 175 Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe             180 185 190 Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His Ser         195 200 205 Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu     210 215 220 Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp 225 230 235 240 Cys His Leu Ala Gln Val Ser Ser His Thr Val Val Ala Arg Ser Met                 245 250 255 Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu             260 265 270 His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro         275 280 285 His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys     290 295 300 Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val 305 310 315 320 Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr                 325 330 335 Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg             340 345 350 Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys         355 360 365 Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly     370 375 380 Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly 385 390 395 400 Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp                 405 410 415 Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Val             420 425 430 Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys         435 440 445 Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met     450 455 460 Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe 465 470 475 480 Ser Glu Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys                 485 490 495 Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu             500 505 510 Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly         515 520 525 Asp Val Ala Phe Val Lys His Gln Thr Val Gln Asn Thr Gly Gly     530 535 540 Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu 545 550 555 560 Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn                 565 570 575 Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp             580 585 590 Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe         595 600 605 Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser     610 615 620 Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys 625 630 635 640 Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val                 645 650 655 Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu             660 665 670 Ala Cys Thr Phe Arg Arg Pro         675 <210> 3 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> GH variation protein <400> 3 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg   1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu              20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro          35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg      50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu  65 70 75 80 Leu Leu Ile Gln Cys Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val                  85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp             100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu         115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Cys     130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe                 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe             180 185 190 <210> 4 <211> 872 <212> PRT <213> Artificial Sequence <220> <223> GHC3-P00-Th <400> 4 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg   1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu              20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro          35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg      50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu  65 70 75 80 Leu Leu Ile Gln Cys Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val                  85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp             100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu         115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Cys     130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe                 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly             180 185 190 Ser Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu         195 200 205 Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro     210 215 220 Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp 225 230 235 240 Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp                 245 250 255 Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro             260 265 270 Val Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr         275 280 285 Tyr Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln     290 295 300 Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly 305 310 315 320 Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg                 325 330 335 Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala             340 345 350 Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro         355 360 365 Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala     370 375 380 Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His 385 390 395 400 Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr                 405 410 415 Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys             420 425 430 Asp Cys His Leu Ala Gln Val Ser Ser His Thr Val Val Ala Arg Ser         435 440 445 Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln     450 455 460 Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser 465 470 475 480 Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu                 485 490 495 Lys Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr             500 505 510 Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro         515 520 525 Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu     530 535 540 Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu 545 550 555 560 Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn                 565 570 575 Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala             580 585 590 Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser         595 600 605 Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val     610 615 620 Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys 625 630 635 640 Lys Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro                 645 650 655 Met Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe             660 665 670 Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys         675 680 685 Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys     690 695 700 Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys 705 710 715 720 Gly Asp Val Ala Phe Val Lys His Gln Thr Val Gln Asn Thr Gly                 725 730 735 Gly Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr             740 745 750 Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala         755 760 765 Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys     770 775 780 Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu 785 790 795 800 Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg                 805 810 815 Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala             820 825 830 Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr         835 840 845 Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu     850 855 860 Glu Ala Cys Thr Phe Arg Arg Pro 865 870 <210> 5 <211> 2097 <212> DNA <213> Artificial Sequence <220> <223> preTf <400> 5 atgaggctcg ccgtgggagc cctgctggtc tgcgccgtcc tggggctgtg tctggctgtc 60 cctgataaaa ctgtgagatg gtgtgcagtg tcggagcatg aggccactaa gtgccagagt 120 ttccgcgacc atatgaaaag cgtcattcca tccgatggtc ccagtgttgc ttgtgtgaag 180 aaagcctcct accttgattg catcagggcc attgcggcaa acgaagcgga tgctgtgaca 240 ctggatgcag gtttggtgta tgatgcttac ctggctccca ataacctgaa gcctgtggtg 300 gcagagttct atgggtcaaa agaggatcca cagactttct attatgctgt tgctgtggtg 360 aagaaggata gtggcttcca gatgaaccag cttcgaggca agaagtcctg ccacacgggt 420 ctaggcaggt ccgctgggtg gaacatcccc ataggcttac tttactgtga cttacctgag 480 ccacgtaaac ctcttgagaa agcagtggcc aatttcttct cgggcagctg tgccccttgt 540 gcggatggga cggacttccc ccagctgtgt caactgtgtc cagggtgtgg ctgctccacc 600 cttaaccaat acttcggcta ctcaggagcc ttcaagtgtc tgaaggatgg tgctggggat 660 gtggcctttg tcaagcactc gactatattt gagaacttgg caaacaaggc tgacagggac 720 cagtatgagc tgctttgcct ggacaacacc cggaagccgg tagatgaata caaggactgc 780 cacttggccc aggtcccttc tcataccgtc gtggcccgaa gtatgggcgg caaggaggac 840 ttgatctggg agcttctcaa ccaggcccag gaacattttg gcaaagacaa atcaaaagaa 900 ttccaactat tcagctctcc tcatgggaag gacctgctgt ttaaggactc tgcccacggg 960 tttttaaaag tcccccccag gatggatgcc aagatgtacc tgggctatga gtatgtcact 1020 gccatccgga atctacggga aggcacatgc ccagaagccc caacagatga atgcaagcct 1080 gtgaagtggt gtgcgctgag ccaccacgag aggctcaagt gtgatgagtg gagtgttaac 1140 agtgtaggga aaatagagtg tgtatcagca gagaccaccg aagactgcat cgccaagatc 1200 atgaatggag aagctgatgc catgagcttg gatggagggt ttgtctacat agcgggcaag 1260 tgtggtctgg tgcctgtctt ggcagaaaac tacaataaga gcgataattg tgaggataca 1320 ccagaggcag ggtattttgc tgtagcagtg gtgaagaaat cagcttctga cctcacctgg 1380 gacaatctga aaggcaagaa gtcctgccat acggcagttg gcagaaccgc tggctggaac 1440 atccccatgg gcctgctcta caataagatc aaccactgca gatttgatga atttttcagt 1500 gaaggttgtg cccctgggtc taagaaagac tccagtctct gtaagctgtg tatgggctca 1560 ggcctaaacc tgtgtgaacc caacaacaaa gagggatact acggctacac aggcgctttc 1620 aggtgtctgg ttgagaaggg agatgtggcc tttgtgaaac accagactgt cccacagaac 1680 actgggggaa aaaaccctga tccatgggct aagaatctga atgaaaaaga ctatgagttg 1740 ctgtgccttg atggtaccag gaaacctgtg gaggagtatg cgaactgcca cctggccaga 1800 gccccgaatc acgctgtggt cacacggaaa gataaggaag cttgcgtcca caagatatta 1860 cgtcaacagc agcacctatt tggaagcaac gtaactgact gctcgggcaa cttttgtttg 1920 ttccggtcgg aaaccaagga ccttctgttc agagatgaca cagtatgttt ggccaaactt 1980 catgacagaa acacatatga aaaatactta ggagaagaat atgtcaaggc tgttggtaac 2040 ctgagaaaat gctccacctc atcactcctg gaagcctgca ctttccgtag accttaa 2097 <210> 6 <211> 2097 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > preTf (B) <400> 6 atgaggctcg ccgtgggagc cctgctggtc tgcgccgtcc tggggctgtg tctggctgtc 60 cctgataaaa ctgtgagatg gtgtgcagtg tcggagcatg aggccactaa gtgccagagt 120 ttccgcgacc atatgaaaag cgtcattcca tccgatggtc ccagtgttgc ttgtgtgaag 180 aaagcctcct accttgattg catcagggcc attgcggcaa acgaagcgga tgctgtgaca 240 ctggatgcag gtttggtgta tgatgcttac ctggctccca ataacctgaa gcctgtggtg 300 gcagagttct atgggtcaaa agaggaccca cagactttct attatgctgt tgctgtggtg 360 aagaaggata gtggcttcca gatgaaccag cttcgaggca agaagtcctg ccacacgggt 420 ctaggcaggt ccgctgggtg gaacatcccc ataggcttac tttactgtga cttacctgag 480 ccacgtaaac ctcttgagaa agcagtggcc aatttcttct cgggcagctg tgccccttgt 540 gcggatggga cggacttccc ccagctgtgt caactgtgtc cagggtgtgg ctgctccacc 600 cttaaccaat acttcggcta ctcaggagcc ttcaagtgtc tgaaggatgg tgctggggat 660 gtggcctttg tcaagcactc gactatattt gagaacttgg caaacaaggc tgacagggac 720 cagtatgagc tgctttgcct ggacaacacc cggaagccgg tagatgaata caaggactgc 780 cacttggccc aggtcccttc tcataccgtc gtggcccgaa gtatgggcgg caaggaggac 840 ttgatctggg agcttctcaa ccaggcccag gaacattttg gcaaagacaa atcaaaagaa 900 ttccaactat tcagctctcc tcatgggaag gacctgctgt ttaaggactc tgcccacggg 960 tttttaaaag tcccccccag gatggatgcc aagatgtacc tgggctatga gtatgtcact 1020 gccatccgga atctacggga aggcacatgc ccagaagccc caacagatga atgcaagcct 1080 gtgaagtggt gtgcgctgag ccaccacgag aggctcaagt gtgatgagtg gagtgttaac 1140 agtgtaggga aaatagagtg tgtatcagca gagaccaccg aagactgcat cgccaagatc 1200 atgaatggag aagctgatgc catgagcttg gatggagggt ttgtctacat agcgggcaag 1260 tgtggtctgg tgcctgtctt ggcagaaaac tacaataaga gcgataattg tgaggataca 1320 ccagaggcag ggtattttgc tgtagcagtg gtgaagaaat cagcttctga cctcacctgg 1380 gacaatctga aaggcaagaa gtcctgccat acggcagttg gcagaaccgc tggctggaac 1440 atccccatgg gcctgctcta caataagatc aaccactgca gatttgatga atttttcagt 1500 gaaggttgtg cccctgggtc taagaaagac tccagtctct gtaagctgtg tatgggctca 1560 ggcctaaacc tgtgtgaacc caacaacaaa gagggatact acggctacac aggcgctttc 1620 aggtgtctgg ttgagaaggg agatgtggcc tttgtgaaac accagactgt cccacagaac 1680 actgggggaa aaaaccctga tccatgggct aagaatctga atgaaaaaga ctatgagttg 1740 ctgtgccttg atggtaccag gaaacctgtg gaggagtatg cgaactgcca cctggccaga 1800 gccccgaatc acgctgtggt cacacggaaa gataaggaag cttgcgtcca caagatatta 1860 cgtcaacagc agcacctatt tggaagcaac gtaactgact gctcgggcaa cttttgtttg 1920 ttccggtcgg aaaccaagga ccttctgttc agagatgaca cagtatgttt ggccaaactt 1980 catgacagaa acacatatga aaaatactta ggagaagaat atgtcaaggc tgttggtaac 2040 ctgagaaaat gctccacctc atcactcctg gaagcctgca ctttccgtag accttaa 2097 <210> 7 <211> 2040 <212> DNA <213> Artificial Sequence <220> <223> Tf (B) <400> 7 gtccctgata aaactgtgag atggtgtgca gtgtcggagc atgaggccac taagtgccag 60 agtttccgcg accatatgaa aagcgtcatt ccatccgatg gtcccagtgt tgcttgtgtg 120 aagaaagcct cctaccttga ttgcatcagg gccattgcgg caaacgaagc ggatgctgtg 180 acactggatg caggtttggt gtatgatgct tacctggctc ccaataacct gaagcctgtg 240 gtggcagagt tctatgggtc aaaagaggac ccacagactt tctattatgc tgttgctgtg 300 gtgaagaagg atagtggctt ccagatgaac cagcttcgag gcaagaagtc ctgccacacg 360 ggtctaggca ggtccgctgg gtggaacatc cccataggct tactttactg tgacttacct 420 gagccacgta aacctcttga gaaagcagtg gccaatttct tctcgggcag ctgtgcccct 480 tgtgcggatg ggacggactt cccccagctg tgtcaactgt gtccagggtg tggctgctcc 540 acccttaacc aatacttcgg ctactcagga gccttcaagt gtctgaagga tggtgctggg 600 gatgtggcct ttgtcaagca ctcgactata tttgagaact tggcaaacaa ggctgacagg 660 gaccagtatg agctgctttg cctggacaac acccggaagc cggtagatga atacaaggac 720 tgccacttgg cccaggtccc ttctcatacc gtcgtggccc gaagtatggg cggcaaggag 780 gacttgatct gggagcttct caaccaggcc caggaacatt ttggcaaaga caaatcaaaa 840 gaattccaac tattcagctc tcctcatggg aaggacctgc tgtttaagga ctctgcccac 900 gggtttttaa aagtcccccc caggatggat gccaagatgt acctgggcta tgagtatgtc 960 actgccatcc ggaatctacg ggaaggcaca tgcccagaag ccccaacaga tgaatgcaag 1020 cctgtgaagt ggtgtgcgct gagccaccac gagaggctca agtgtgatga gtggagtgtt 1080 aacagtgtag ggaaaataga gtgtgtatca gcagagacca ccgaagactg catcgccaag 1140 atcatgaatg gagaagctga tgccatgagc ttggatggag ggtttgtcta catagcgggc 1200 aagtgtggtc tggtgcctgt cttggcagaa aactacaata agagcgataa ttgtgaggat 1260 acaccagagg cagggtattt tgctgtagca gtggtgaaga aatcagcttc tgacctcacc 1320 tgggacaatc tgaaaggcaa gaagtcctgc catacggcag ttggcagaac cgctggctgg 1380 aacatcccca tgggcctgct ctacaataag atcaaccact gcagatttga tgaatttttc 1440 gtk tcaggcctaa acctgtgtga acccaacaac aaagagggat actacggcta cacaggcgct 1560 ttcaggtgtc tggttgagaa gggagatgtg gcctttgtga aacaccagac tgtcccacag 1620 aacactgggg gaaaaaaccc tgatccatgg gctaagaatc tgaatgaaaa agactatgag 1680 ttgctgtgcc ttgatggtac caggaaacct gtggaggagt atgcgaactg ccacctggcc 1740 agagccccga atcacgctgt ggtcacacgg aaagataagg aagcttgcgt ccacaagata 1800 ttacgtcaac agcagcacct atttggaagc aacgtaactg actgctcggg caacttttgt 1860 ttgttccggt cggaaaccaa ggaccttctg ttcagagatg acacagtatg tttggccaaa 1920 cttcatgaca gaaacacata tgaaaaatac ttaggagaag aatatgtcaa ggctgttggt 1980 aacctgagaa aatgctccac ctcatcactc ctggaagcct gcactttccg tagaccttaa 2040                                                                         2040 <210> 8 <211> 2697 <212> DNA <213> Artificial Sequence <220> <223> GHW-P00-Th <400> 8 atggctacag gctcccggac gtccctgctc ctggcttttg gcctgctctg cctgccctgg 60 cttcaagagg gcagtgcctt cccaaccatt cccttatcca ggctttttga caacgctatg 120 ctccgcgccc atcgtctgca ccagctggcc tttgacacct accaggagtt tgaagaagcc 180 tatatcccaa aggaacagaa gtattcattc ctgcagaacc cccagacctc cctctgtttc 240 tcagagtcta ttccgacacc ctccaacagg gaggaaacac aacagaaatc caacctagag 300 ctgctccgca tctccctgct gctcatccag tcgtggctgg agcccgtgca gttcctcagg 360 agtgtcttcg ccaacycct ggtgtacggc gcctctgaca gcaacgtcta tgacctccta 420 aaggacctag aggaaggcat ccaaacgctg atggggaggc tggaagatgg cagcccccgg 480 actgggcaga tcttcaagca gacctacagc aagttcgaca caaactcaca caacgatgac 540 gcactactca agaactacgg gctgctctac tgcttcagga aggacatgga caaggtcgag 600 acattcctgc gcatcgtgca gtgccgctct gtggagggca gctgtggctt cggatccgtc 660 cctgataaaa ctgtgagatg gtgtgcagtg tcggagcatg aggccactaa gtgccagagt 720 ttccgcgacc atatgaaaag cgtcattcca tccgatggtc ccagtgttgc ttgtgtgaag 780 aaagcctcct accttgattg catcagggcc attgcggcaa acgaagcgga tgctgtgaca 840 ctggatgcag gtttggtgta tgatgcttac ctggctccca ataacctgaa gcctgtggtg 900 gcagagttct atgggtcaaa agaggaccca cagactttct attatgctgt tgctgtggtg 960 aagaaggata gtggcttcca gatgaaccag cttcgaggca agaagtcctg ccacacgggt 1020 ctaggcaggt ccgctgggtg gaacatcccc ataggcttac tttactgtga cttacctgag 1080 ccacgtaaac ctcttgagaa agcagtggcc aatttcttct cgggcagctg tgccccttgt 1140 gcggatggga cggacttccc ccagctgtgt caactgtgtc cagggtgtgg ctgctccacc 1200 cttaaccaat acttcggcta ctcaggagcc ttcaagtgtc tgaaggatgg tgctggggat 1260 gtggcctttg tcaagcactc gactatattt gagaacttgg caaacaaggc tgacagggac 1320 cagtatgagc tgctttgcct ggacaacacc cggaagccgg tagatgaata caaggactgc 1380 cacttggccc aggtcccttc tcataccgtc gtggcccgaa gtatgggcgg caaggaggac 1440 ttgatctggg agcttctcaa ccaggcccag gaacattttg gcaaagacaa atcaaaagaa 1500 ttccaactat tcagctctcc tcatgggaag gacctgctgt ttaaggactc tgcccacggg 1560 tttttaaaag tcccccccag gatggatgcc aagatgtacc tgggctatga gtatgtcact 1620 gccatccgga atctacggga aggcacatgc ccagaagccc caacagatga atgcaagcct 1680 gtgagtggt gtgcgctgag ccaccacgag aggctcaagt gtgatgagtg gagtgttaac 1740 agtgtaggga aaatagagtg tgtatcagca gagaccaccg aagactgcat cgccaagatc 1800 atgaatggag aagctgatgc catgagcttg gatggagggt ttgtctacat agcgggcaag 1860 tgtggtctgg tgcctgtctt ggcagaaaac tacaataaga gcgataattg tgaggataca 1920 ccagaggcag ggtattttgc tgtagcagtg gtgaagaaat cagcttctga cctcacctgg 1980 gacaatctga aaggcaagaa gtcctgccat acggcagttg gcagaaccgc tggctggaac 2040 atccccatgg gcctgctcta caataagatc aaccactgca gatttgatga atttttcagt 2100 gaaggttgtg cccctgggtc taagaaagac tccagtctct gtaagctgtg tatgggctca 2160 ggcctaaacc tgtgtgaacc caacaacaaa gagggatact acggctacac aggcgctttc 2220 aggtgtctgg ttgagaaggg agatgtggcc tttgtgaaac accagactgt cccacagaac 2280 actgggggaa aaaaccctga tccatgggct aagaatctga atgaaaaaga ctatgagttg 2340 ctgtgccttg atggtaccag gaaacctgtg gaggagtatg cgaactgcca cctggccaga 2400 gccccgaatc acgctgtggt cacacggaaa gataaggaag cttgcgtcca caagatatta 2460 cgtcaacagc agcacctatt tggaagcaac gtaactgact gctcgggcaa cttttgtttg 2520 ttccggtcgg aaaccaagga ccttctgttc agagatgaca cagtatgttt ggccaaactt 2580 catgacagaa acacatatga aaaatactta ggagaagaat atgtcaaggc tgttggtaac 2640 ctgagaaaat gctccacctc atcactcctg gaagcctgca ctttccgtag accttaa 2697 <210> 9 <211> 2697 <212> DNA <213> Artificial Sequence <220> <223> GHC3-P00-Th <400> 9 atggctacag gctcccggac gtccctgctc ctggcttttg gcctgctctg cctgccctgg 60 cttcaagagg gcagtgcctt cccaaccatt cccttatcca ggctttttga caacgctatg 120 ctccgcgccc atcgtctgca ccagctggcc tttgacacct accaggagtt tgaagaagcc 180 tatatcccaa aggaacagaa gtattcattc ctgcagaacc cccagacctc cctctgtttc 240 tcagagtcta ttccgacacc ctccaacagg gaggaaacac aacagaaatc caacctagag 300 ctgctccgca tctccctgct gctcatccag tgttggctgg agcccgtgca gttcctcagg 360 agtgtcttcg ccaacycct ggtgtacggc gcctctgaca gcaacgtcta tgacctccta 420 aaggacctag aggaaggcat ccaaacgctg atggggaggc tggaagatgg cagcccccgg 480 actgggcaga tcttcaagca gacctactgc aagttcgaca caaactcaca caacgatgac 540 gcactactca agaactacgg gctgctctac tgcttcagga aggacatgga caaggtcgag 600 acattcctgc gcatcgtgca gtgccgctct gtggagggca gctgtggctt cggatccgtc 660 cctgataaaa ctgtgagatg gtgtgcagtg tcggagcatg aggccactaa gtgccagagt 720 ttccgcgacc atatgaaaag cgtcattcca tccgatggtc ccagtgttgc ttgtgtgaag 780 aaagcctcct accttgattg catcagggcc attgcggcaa acgaagcgga tgctgtgaca 840 ctggatgcag gtttggtgta tgatgcttac ctggctccca ataacctgaa gcctgtggtg 900 gcagagttct atgggtcaaa agaggaccca cagactttct attatgctgt tgctgtggtg 960 aagaaggata gtggcttcca gatgaaccag cttcgaggca agaagtcctg ccacacgggt 1020 ctaggcaggt ccgctgggtg gaacatcccc ataggcttac tttactgtga cttacctgag 1080 ccacgtaaac ctcttgagaa agcagtggcc aatttcttct cgggcagctg tgccccttgt 1140 gcggatggga cggacttccc ccagctgtgt caactgtgtc cagggtgtgg ctgctccacc 1200 cttaaccaat acttcggcta ctcaggagcc ttcaagtgtc tgaaggatgg tgctggggat 1260 gtggcctttg tcaagcactc gactatattt gagaacttgg caaacaaggc tgacagggac 1320 cagtatgagc tgctttgcct ggacaacacc cggaagccgg tagatgaata caaggactgc 1380 cacttggccc aggtcccttc tcataccgtc gtggcccgaa gtatgggcgg caaggaggac 1440 ttgatctggg agcttctcaa ccaggcccag gaacattttg gcaaagacaa atcaaaagaa 1500 ttccaactat tcagctctcc tcatgggaag gacctgctgt ttaaggactc tgcccacggg 1560 tttttaaaag tcccccccag gatggatgcc aagatgtacc tgggctatga gtatgtcact 1620 gccatccgga atctacggga aggcacatgc ccagaagccc caacagatga atgcaagcct 1680 gtgagtggt gtgcgctgag ccaccacgag aggctcaagt gtgatgagtg gagtgttaac 1740 agtgtaggga aaatagagtg tgtatcagca gagaccaccg aagactgcat cgccaagatc 1800 atgaatggag aagctgatgc catgagcttg gatggagggt ttgtctacat agcgggcaag 1860 tgtggtctgg tgcctgtctt ggcagaaaac tacaataaga gcgataattg tgaggataca 1920 ccagaggcag ggtattttgc tgtagcagtg gtgaagaaat cagcttctga cctcacctgg 1980 gacaatctga aaggcaagaa gtcctgccat acggcagttg gcagaaccgc tggctggaac 2040 atccccatgg gcctgctcta caataagatc aaccactgca gatttgatga atttttcagt 2100 gaaggttgtg cccctgggtc taagaaagac tccagtctct gtaagctgtg tatgggctca 2160 ggcctaaacc tgtgtgaacc caacaacaaa gagggatact acggctacac aggcgctttc 2220 aggtgtctgg ttgagaaggg agatgtggcc tttgtgaaac accagactgt cccacagaac 2280 actgggggaa aaaaccctga tccatgggct aagaatctga atgaaaaaga ctatgagttg 2340 ctgtgccttg atggtaccag gaaacctgtg gaggagtatg cgaactgcca cctggccaga 2400 gccccgaatc acgctgtggt cacacggaaa gataaggaag cttgcgtcca caagatatta 2460 cgtcaacagc agcacctatt tggaagcaac gtaactgact gctcgggcaa cttttgtttg 2520 ttccggtcgg aaaccaagga ccttctgttc agagatgaca cagtatgttt ggccaaactt 2580 catgacagaa acacatatga aaaatactta ggagaagaat atgtcaaggc tgttggtaac 2640 ctgagaaaat gctccacctc atcactcctg gaagcctgca ctttccgtag accttaa 2697 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Tf-F primer <400> 10 catgctagct ccaccatgag gctcgccgtg ggagcc 36 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Tf-R primer <400> 11 agactcgagt taaggtctac ggaaagtgca g 31 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Tf (B) -F primer <400> 12 ctatgggtca aaagaggacc cacagacttt ctatt 35 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Tf (B) -R primer <400> 13 aatagaaagt ctgtgggtcc tcttttgacc catag 35 <210> 14 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Tranferrin signal peptide <400> 14 Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu   1 5 10 15 Cys Leu Ala             <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> preTf (B) primer <400> 15 ctcggatccg tccctgataa aactgtgaga tg 32 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GHC3-P00-Th F primer <400> 16 atagctagct ccaccatggc tacaggctcc cggac 35 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> GHC3-P00-ThR primer <400> 17 tatggatccg aagccacagc tgccctcca 29 <210> 18 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> hGH signal peptide <400> 18 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Ala Phe Gly Leu Leu   1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala              20 25 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> GHW-P00-T F primer <400> 19 gctgctcatc cagtgttggc tggagcccg 29 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> GHW-P00-T R primer <400> 20 cgggctccag ccaacactgg atgagcagc 29 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GHC3-P00-T F primer <400> 21 tcaagcagac ctactgcaag ttcgacacac cc 32 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> GHC3-P00-T R primer <400> 22 gtttgtgtcg aacttgcagt aggtctgctt ga 32

Claims (19)

A human growth hormone mutant protein consisting of an amino acid sequence in which the 85th and 144th serines are replaced with hydrophobic amino acids, respectively, in the amino acid sequence of human growth hormone protein.
2. The human growth hormone mutation protein according to claim 1, wherein the amino acid sequence of the human growth hormone protein is represented by SEQ ID NO:
The human growth hormone mutation protein according to claim 1, wherein the hydrophobic amino acid is an amino acid capable of sulfidation bonding.
The human growth hormone mutation protein according to claim 1, wherein the hydrophobic amino acid is cysteine.
A gene encoding the protein of claim 1.
An expression vector comprising a gene encoding the protein of claim 1.
A transformant wherein the expression vector of claim 6 is introduced into any one host cell selected from the group consisting of Escherichia coli, yeast, fungi, plant cells and animal cells other than human.
A fusion protein of human growth hormone mutant protein and transferrin, wherein transferrin is bound to the end of the human growth hormone mutant protein of claim 1.
9. The fusion protein of claim 8, wherein the fusion protein is represented by SEQ ID NO: 4.
A gene encoding the protein of claim 8.
An expression vector comprising a gene encoding the protein of claim 8.
12. The expression vector according to claim 11, wherein the gene encoding the protein is obtained by replacing the base sequence of the restriction enzyme recognition site in the gene with another base sequence encoding the same amino acid as the amino acid encoding the base sequence.
12. The expression vector according to claim 11, wherein the gene encoding the protein is a gene encoding a Bam HI restriction enzyme recognition site sequence (GGATCC) in the gene by substituting Cytosine for the Thymine.
12. The expression vector according to claim 11, wherein the gene encoding the protein further comprises a restriction enzyme recognition site at the end of the gene.
12. The expression vector according to claim 11, wherein the expression vector comprises any one of the sequences selected from the group consisting of SEQ ID NOS: 6, 7, 8 and 9.
The transformant according to claim 11, wherein the expression vector is introduced into any one host cell selected from the group consisting of Escherichia coli, yeast, fungi, plant cells and animal cells except humans.
A growth factor containing the human growth hormone mutation protein of claim 1, the fusion protein of claim 8, the vector of claim 6, the vector of claim 11, the transformant of claim 7 or the transformant of claim 16 as an active ingredient, Or growth retardation.
18. The method according to claim 17, wherein the growth failure or growth retardation is caused by pituitary gland hormone secretion disorder, chronic renal disease, Turner's syndrome, cachexia or AIDS wasting For the treatment of growth retardation or growth retardation.
A childhood-onset deficiency or an adult disease comprising the human growth hormone mutation protein of claim 1, the fusion protein of claim 8, the vector of claim 6, the vector of claim 11, the transformant of claim 7, or the transformant of claim 16, A pharmaceutical composition for the prevention or treatment of starvation deficiency.

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