WO2013042821A1

WO2013042821A1 - Selection of a permissive site for protein redesign, and method for producing a modified protein using same

Info

Publication number: WO2013042821A1
Application number: PCT/KR2011/007683
Authority: WO
Inventors: 이승구; 이상준; 김은미; 김수진; 손정훈; 나유진; 박순호
Original assignee: 한국생명공학연구원
Priority date: 2011-09-22
Filing date: 2011-10-14
Publication date: 2013-03-28
Also published as: KR20130032217A; KR101752775B1

Abstract

The present invention relates to a protein engineering technology for improving the effect of a target protein. More particularly, the present invention relates to a method for selecting a permissive site enabling genetic modification, chemical modification, or foreign protein fusion in a target protein library having a fusion protein in which a folding reporter protein is inserted to various inner positions of the target protein, and further relates to a method for producing a modified target protein by performing mutation, chemical modification, or foreign protein fusion at the selected permissive site, and further relates to a target protein produced by the method, and to a novel modified target protein.

Description

Acceptance site screening for protein redesign and method for producing modified protein using same

The present invention relates to protein engineering techniques for improving the efficacy of target proteins. More specifically, a method of selecting a allowable position where genetic modification, chemical modification, or heterologous protein fusion is possible in a target protein library in which the folding reporter protein has a fusion protein inserted at various internal positions of the target protein, and the allowance selected by the method A method of making a modified target protein by performing mutations, chemical modifications or heterologous protein fusions in position, relates to a target protein produced by the method and a novel modified target protein.

Human growth hormone (hGH), insulin, EGFR (Epidermal Growth Factor), EPO (Erythropoietin), hematopoietic stimulant, neutrophil promoter (G-CSF), platelet growth factor (Thrombopoietin), interferon, coagulation factor (coagulation) factors), follicle stimulating hormone, etc. are currently being applied variously as protein therapeutics.

Among them, hGH, also called somatotropin, is a hormone secreted by the somatotrophic cells of the anterior pituitary gland and is a monomer containing two disulfides (Cys53-Cys164 and Cys183-Cys190). Protein (191 amino acids, 22 kDa). Human growth hormone plays an important role in regulating the growth of human cells by acting directly on tissues such as bones and muscles or by indirect methods of stimulating the secretion of insulin-like growth factor-1. (Endocr Rev. (1995) Feb; 16 (1): 3-34).

Recombinant human growth hormone produced by genetic engineering technology is also known to exhibit the same biological efficacy as natural hormone (Nature Biotechnol. (1992) 10 (7): 812). Therefore, it is used to treat clinical symptoms caused by deficiency of human growth hormone, such as pediatric dwarf, which is caused by low growth hormone levels, and recently, it has been extended to be used as a muscle enhancer or anti-aging for cosmetic purposes. Pediatric Drugs (2002) 4 (1): 3747).

On the other hand, recombinant human growth hormones exhibit immunoactivity due to thermodynamic instability or aggregation due to thermodynamic instability during manufacturing, distribution and use, and are rapidly hydrolyzed by various enzymes in the bloodstream in the human body. The half life is very short, less than 10 minutes. In particular, enzymes such as plasmin and thrombin present in plasma are likely to be hydrolyzed at 134-150 residues. When used as an oral protein therapeutic, activity may be lost by proteolytic enzymes such as saliva enzyme, trypsin, pepsin, and the like. Since the instability problem of human growth hormone has a great influence on the bioavailability of the agent, protein engineering stabilization studies to improve the sustainability of the drug by improving the thermodynamic stability and resistance to proteolysis of human growth hormone are required.

In the case of the study of improving the activity or stability using protein engineering, the method of analyzing the tertiary structure of the protein and selecting or modifying the exposed site and modifying it chemically is used (Protein Sci. (2002) 11: 1452-). 1461). However, this method assumes a complete understanding of the structure-function correlation of proteins, and in reality it is unlikely to match the prediction. Therefore, there is a need for a method capable of improving more efficient protein activity and safety.

We use a rational analysis and experimental approach to the tertiary structure of human growth hormone to allow for artificial mutations and chemical modifications without interfering with the inherent folding and function of proteins. We have developed a new human growth hormone that is resistant to proteolytic enzymes (plasmin, thrombin, pepsin, etc.) in the body by introducing genetic modifications and chemical modifications. The present invention was completed by confirming that chemical modifications could be identified and at the same time, the new human growth hormone thus modified was resistant to proteolytic enzymes (plasmin, thrombin, pepsin, etc.) in the body.

One object of the present invention is to prepare a target protein library having a fusion protein in which a folding reporter protein is inserted at various internal positions of the target protein; (b) selecting from the target protein library a colony having a higher activity of the reporter protein than the control group in which the folding reporter protein is fused at the N-terminus or C-terminus of the target protein; And (c) analyzing a fusion protein sequence of the selected colonies to identify a position at which the folding reporter protein is inserted, thereby selecting a permissive site that can be modified among target proteins. .

Another object of the present invention is to provide a method for preparing a modified target protein, comprising performing a fusion of a mutation, chemical modification or heterologous protein at an allowable position selected by the method.

Another object of the present invention is to provide a modified protein produced by the above method.

Another object of the present invention is to provide a novel modified human growth hormone.

Acceptable site search techniques identified by random insertion of transposons containing nucleic acids encoding the folding reporter proteins of the present invention are very useful methods for improving the stability and activity of human growth hormone as well as most other protein therapeutics. Can be. In particular, such a method may be very useful in the case of improving the protein for use as a smart drug applicable only to a specific target such as cancer cells. It can also be used for the attachment of surfactants such as cholic acid to increase the solubility of the protein. In addition, through the glycosylation for stabilization at the position searched in this way it was possible to significantly increase the stability of the desired enzyme, thereby providing a method of resolving the instability of the enzyme in the bloodstream, the biggest weakness of the therapeutic protein do. Such stabilizing peptides, that is, stabilized hGH redesign proteins with sugar chains, are expected to be very useful in the medical industry. In addition, the acceptable positions of the present invention may be used as the position of a fusion protein that binds separate proteins (ligand, albumin, etc.).

1 is a view briefly illustrating the experimental development and the final result derivation process of the present invention.

Figure 2 is a schematic diagram showing the configuration of the hGH library for the detection of hGH tolerance site, a human growth hormone. (a) shows a transposon including a Tn5 transposase recognition site (Mosaic End, ME), a kanamycin resistance gene, a complementary Tn5 transposase recognition site (Mosaic End, ME), and an ultraviolet fluorescent gene It is a figure which shows (transposon). (b) is a figure which shows the structure of the library which inserted the transposon of (a).

Figure 3 is a diagram showing the results of the image through the microscopic scanning of the library and the control inserting the folding reporter.

4 is a diagram showing the configuration and results of the hGH library in which the transposon is inserted.

5 is a diagram showing the results of position selection for the detection of the allowable region from the hGH library in which the transposon is inserted. (a) is a table showing the position of the fluorescent reporter insertion confirmed by sequencing the hits obtained from the yeast library. (b) is a diagram showing the amino acid sequence selected for each library during the allowable position search process.

Figure 6 is a diagram confirming the expression results in yeast of wild type (a) and variant hGH (b and c).

7 is a diagram confirming the activity level of wild-type and mutant hGH expressed in yeast through Nb2 cell proliferation experiment.

8a to 8c is a diagram confirming the stability of the mutant proteins through fragmentation confirmation by proteolytic enzymes (pepsin, plasmin, thrombin) in the human body.

As one aspect for achieving the above object, the present invention comprises the steps of (a) preparing a target protein library having a fusion protein in which a folding reporter protein is inserted at various internal positions of the target protein; (b) selecting from the target protein library a colony having a higher activity of the reporter protein than the control group in which the folding reporter protein is fused at the N-terminus or C-terminus of the target protein; And (c) analyzing the fusion protein sequence of the selected colony to identify a position at which the folding reporter protein is inserted, thereby selecting a permissive site that can be modified among target proteins.

As used herein, the term "permissive site" refers to a site that can be redesigned to enhance safety, delivery to a target, for example, without significantly affecting the original activity of the protein. Preferably, it may be a position where mutation, chemical modification or fusion of a heterologous protein can be performed without impairing the function of the target protein. Preferably, the chemical modification may be at a position to introduce a chemical modification including glycosylation, PEGylation, or the attachment of a surfactant. Such introduction can use without limitation methods known in the art.

When the surfactant is attached at the selected allowable position, the surfactant may be, but is not limited to, for example, cholic acid, and such a surfactant may increase the solubility of the protein to increase the permeability of the cell membrane to increase the protein therapeutics. It is possible to facilitate the injection of the living body. Attachment of such surfactants can be carried out using known methods without limitation. When the heterologous protein is fused to a selected acceptable position, the heterologous protein may be, but is not limited to, a ligand, a receptor, or the like for moving the protein therapeutic agent to a specific target such as cancer cells. Alternatively, heterologous proteins, such as immunoglobulin Fc regions or albumin, may enhance the half-life or stability of the protein therapeutic agent, but are not limited thereto. Fusion of such heterologous proteins can be performed using known methods without limitation.

The overall schematic diagram of the permissible position selection method of the present invention is as shown in FIG. The inventors constructed an in-frame fusion protein library into which a transposon containing a folding reporter protein was randomly inserted, and then selected candidate positions using protein engineering techniques, I went through the screening process.

In step (a), the nucleic acid encoding the folding reporter protein may be inserted into various internal positions of the nucleic acid encoding the target protein by transposon.

In the present invention, the term "transposon" refers to a kind of protein expression cassette used for random insertion into a protein or for insertion into a designed position. Preferably, the transposon may include a nucleic acid encoding a folding reporter protein and a transposase recognition site at both ends. In addition, a marker gene for transposon insertion clone selection may be included, and the marker gene may be an antibiotic resistance gene. A brief schematic diagram of such a transposon is shown in Fig. 2 (a). The translocation enzyme recognition sites are each complementary to each other. Preferably, the transposon may be a transposon consisting of the kanamycin resistance gene described in SEQ ID NO: 5, wherein the translocation enzyme recognition sites described in SEQ ID NOs: 2 and 3 are present at both ends.

The nucleic acid encoding the folding reporter protein, the antibiotic resistance gene for transposon insertion clone selection and the transposase recognition site at both ends may be in the form of an expression cassette operably linked.

The term "operably linked" in the present invention means that when one nucleic acid fragment is combined with another nucleic acid fragment, typically their respective function or expression is affected by the other nucleic acid fragment, but many possible Binding refers to binding in a state in which each fragment has no detectable effect on performing its function.

The transposon may be treated with a transposae to prepare a target protein library into which a transposon is randomly inserted.

Preferably, the target protein library of step (a) comprises (i) introducing a transposon comprising a nucleic acid encoding a folding reporter protein, a transposon insertion clone selection marker gene, and a translocation enzyme recognition site at both ends into a first host cell; ; (ii) selecting a colony expressing a selection marker gene from the first host cells to prepare a transposon insertion first library; (iii) removing the selection marker gene from the transposon from the first library of step (ii); And (iv) introducing the transposon from which the selection marker has been removed into a second host cell to prepare a second library.

The first host cell may be a prokaryotic cell, for example, but may not be limited to E. coli. The second host cell may be a eukaryotic cell, for example, but may not be limited to yeast.

As used herein, the term "folding reporter protein" refers to a protein capable of confirming whether or not the folding and solubility of a target protein is correct. The binding of the reporter protein to the target protein may reflect the success of the folding of the target protein. In other words, if the target protein is not properly folded, the activity of the reporter protein is weak. Therefore, even when the folding reporter protein is inserted, the reporter protein may exhibit activity if there is no effect on the folding of the target protein, but when the portion of the folding reporter protein is inserted is the position that affects the folding, the activity of the reporter protein is not perfect, You can see if the location is important. The folding reporter protein may include, without limitation, a protein capable of confirming proper folding, but is preferably a green fluorescent protein (GFP), a modified green fluorescent protein (mGFP), or an enhanced green fluorescent protein. enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), Yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP) or enhanced indigo fluorescent protein (ECFP), but is not limited thereto. According to one embodiment of the present invention using green fluorescent protein (GFP) as a folding reporter protein was confirmed whether the folding is correct.

Preferably step (b) may be performed by selecting from the target protein library colonies having higher activity of the reporter protein than the control group in which the folding reporter protein is fused to the N-terminus or C-terminus of the target protein.

In addition, (c) analyzing the fusion protein sequence of the selected colony may be performed to determine the position where the folding reporter protein is inserted.

As such, steps (a) to (c) may mean "experimental step of selecting acceptable positions". The "experimental method of finding acceptable positions" can be used to construct an in-frame fusion protein library by random insertion and search by high-speed fluorescence analysis. That is, a transposon containing a fluorescent protein gene, which is a reporter protein, is prepared, and high-speed search of a library randomly inserted into a target protein is performed. In this case, the fluorescent protein gene functions as a kind of folding reporter. Therefore, when the target protein is normally folded in the target protein-fluorescence fusion protein, the fluorescence inherent in the fluorescence protein is strongly displayed, and thus, by selecting these and analyzing the gene sequence to confirm the insertion position, the target tolerance can be determined experimentally. The experimental method of the present invention enables accurate and rapid hit screening by the folding reporter, and can be easily applied to improvement studies of other proteins as well as protein therapeutics such as human growth hormone. Modifications such as performing various genetic variations and chemical modifications to the excavated allowable positions do not affect the intrinsic function of the protein, and improve the usability of the target protein as a protein therapeutic agent, such as resistance to proteolytic enzymes. You can.

Preferably, the method may further include designing an amino acid located within 5 ms (± 2 ms) around the folding reporter protein insertion position identified in step (c) as an acceptable position. This range is supported by the mutations in Example 5.

Preferably, the method may further include the step of (d) selecting an amino acid located within 5 Å (± 2 절단) of the folding reporter protein insertion sites identified in step (c) with respect to the cleavage site of the protease. It may include a site that is exposed to the outside. This step (d) can be performed not only by experimental methods through sequencing, but also by structure-based rational selection by tertiary structure substitution of proteins.

Preferably, the method may further include the step (e) of excluding the position at which the folding reporter protein insertion positions identified in step (c) remove the binding activity of the target protein or the intrinsic functional activity of the target protein. The binding activity of the target protein may include various binding activities such as receptor binding, ligand binding, antigen-antibody binding and the like. This step (e) can be confirmed experimentally on the basis of sequencing or cell system, as well as by the structure-based rational selection by tertiary structure substitution of the protein, more preferably the modification of the target protein during protein modification. The site of eliminating binding activity or intrinsic functional activity of the target protein can be identified by substituting the known tertiary structure of the target protein. Heterologous conserved sequences associated with the activity of the target protein may be excluded prior to assignment to the tertiary structure. Such heterologous preservation sequences may be selected using a known sequence database (GenBank, etc.), and then the sequence of the target protein may be selected using a known sequence analysis program (blast, etc.).

Steps (d) and (e) may be performed simultaneously, in reverse order or sequentially.

Preferably, (d) selecting an amino acid located within 5 이내에 (± 2 Å) of the folding reporter protein insertion sites identified in step (c) around the cleavage site of the protease; And (e) excluding the position at which the folding reporter protein insertion positions identified in step (c) remove the binding activity of the target protein or the position of eliminating the intrinsic functional activity of the target protein when the protein is modified. In this case it may mean "structure based rational selection step".

"Structural based rational selection" refers to sites where disallowing protein redesign through sequence and structural analysis, that is, directly or indirectly associated with the original activity of the target protein, resulting in mutation, glycosylation, or PEGylation. Alternatively, the step of classifying a site that is difficult to redesign the protein and a site that is easy to redesign the protein, such as chemical modification (incorporation of chemical modification) and protein fusion, including the attachment of a surfactant, may be performed according to the following steps. First, the protein amino acid sequences of various target proteins are compared to analyze the conserved common residues, and the structure of the target protein is analyzed. Then, the protein redesigning site is expected to influence the direct receptor binding site and the receptor binding site. Excluded as classified as unacceptable site. In addition, a site close to the site cleaved by the protease in the body and exposed to the outside to facilitate protein redesign can be selected as a candidate site for protein redesign. In addition, the protein redesign is excluded except the sites where protein redesign is not allowed among the positions of reporter proteins selected by the experimental method based on the protein redesign candidate candidate positions and the sites where the protein redesign is not allowed. Can select the possible positions.

The "structure-based rational selection" of the present invention can quickly and easily select only those positions capable of various genetic variations and chemical modifications among the positions of the folding reporter protein selected using the transposon.

The site for removing the binding activity of the protein target protein or the intrinsic functional activity of the target protein can be performed by substituting a known tertiary structure of the target protein.

The removal position may mean a position at which the function of the original protein does not operate when the protein redesign is applied, and may include, but is not limited to, a receptor binding position and a position expected to affect receptor binding. Does not. It may comprise an N-terminus or a C-terminus.

The tertiary structure of the protein therapeutic agent for structural analysis of the protein therapeutic agent may be provided from a database including all the structures of known human proteins such as PDB (Protein Data Bank), but are not limited thereto. The three-dimensional structure of a human protein known in the art may be provided through various databases.

In addition, enzyme diagrams, gene ontology functional assignments, 1D sequence annotations, and schematic diagrams of protein-protein interactions PDBsum (http://www.ebi.ac.uk/pdsum) containing protein structure information and SCOP (Structural Claasification of Protein) classified into groups according to the three-dimensional structure of each human protein 3D structure information of the protein may be provided using various databases such as, but is not limited thereto. Thereafter, the three-dimensional structure of the protein therapeutic agent, which is provided from a known tertiary structure database, is analyzed using a 3D molecular visualization program such as PyMOL and Discovery Studio Visualizer. Identify areas where design is not acceptable and areas where protein redesign is easy. Locations where protein redesign is not permitted include receptor binding sites and / or positions that are expected to affect receptor binding. In addition, in order to efficiently select only the insertion position of the folding reporter protein within the target protein, which is easily redesigned, using a transposon, the target of the protein modification during the modification of the protein is not allowed Protein redesign based on the protein redesign candidate candidate sites classified through the structural analysis and sites where protein redesign is not allowed, including the step of excluding the binding activity of the protein or the location of eliminating the native functional activity of the target protein. Only the position inserted in the easy design area is easily selected.

As used herein, the term "target protein" may refer to a protein for which to find an acceptable position for preparing a modified protein using the method of the present invention. Examples include, but are not limited to, human growth hormone, insulin, epidermal growth factor (EGFR), erythropoietin (EPO), hematopoietic stimulant, neutrophil promoter (G-CSF), platelet growth factor (Thrombopoietin), interferon, coagulation factor ( coagulation factor) or follicle stimulating hormone, or a protein therapeutic agent, but is preferably a human growth hormone. According to one embodiment of the present invention, acceptable positions were selected from human growth hormone.

We constructed a transposon comprising translocation enzyme recognition site (Mosaic End, Me) of transposon 5 (Tn5) and GFP as a folding reporter, kanamycin resistance gene for primary selection for acceptable site search (FIG. 2), To make a random insertion library, a primary library was prepared by reacting transposon, human growth hormone (hGH) DNA, and Tn5 translocation enzyme, a secondary library from which kanamycin resistance gene was removed, and a folding reporter was included in hGH. The transposons were selected randomly inserted colonies. Next, hGH-GFP was isolated from the colonies, and then introduced into yeast to obtain a yeast library. Using the hGH-GFP prepared by serially connecting GFP outside the ORF of hGH in the yeast as a positive control, colonies having higher fluorescence values were selected and sequenced to confirm that the reporter was introduced at position 8 ( 5). This is indicated in the tertiary structure of the known hGH, and contains four amino acid conserved sequences, 40 amino acids that are the binding surface to the hGH receptor, 17 N-terminals, and 10 C-terminals, with four acceptable positions (Q49, E129, R134, Q141) were selected (FIG. 5). This acceptable position is close to the site hydrolyzed by pepsin (No. 44), 134 is the position hydrolyzed by thrombin in the blood, 141 is the position hydrolyzed by plasmin, and 129 is not hydrolyzed It is located in a loop that is not directly related but has a high degree of freedom. These results suggest that protein engineering tolerances discovered through GFPuv insertion are related to the high degree of freedom and probable attack by proteolysis.

As another aspect, the present invention provides a method for preparing a modified target protein, comprising rinsing a mutation, chemical modification or fusion of a heterologous protein at an acceptable position selected by the method.

Chemical formulas include glycosylation, PEGylation or attachment of surfactants as described above. Surfactants include cholic acid and the like, which can facilitate their introduction into target cells.

Preferably, the modified target protein may be a target protein having improved safety compared to a wild type target protein. In addition, the modified target protein may be a target protein having improved introduction into the target cell compared to the wild type target protein. This enhances the solubility of the protein to increase the permeability of the cell membrane to facilitate the injection into the living body, or the ligand or receptor that can specifically bind to a receptor or ligand that is specifically expressed on the target cell surface is fused with a heterologous protein to the target cell. Facilitates the delivery of

Modified target proteins can be prepared by introducing new amino acids, artificial sugar chains, artificial polymers (eg PEGylation), surfactants, new fusion proteins, and the like into accepted positions discovered by experimental and theoretical methods. For example, when artificial sugar chains are introduced into the allowable position searched from hGH through the method of the present invention, hGH having high biological stability against pepsin, thrombin, plasmin, etc., can be produced compared to wild-type proteins. As used herein, the term "biological stability" means having resistance to degradation by enzymes in or in vitro that can cause denaturation to natural or unnatural proteins.

The method for producing a modified target protein in the present invention can be carried out by "search for and validate the location through cell-based activity assay". For example, by introducing a peptide sequence required for sugar chain binding at an allowable position and purifying the hGH protein produced by fermentation in a yeast strain, and applying various stresses such as temperature-pH-proteinase, a cell line having an hGH receptor ( For example using NB2 cell line). As a result, it is possible to produce a new hGH that is significantly improved in stability while maintaining efficacy compared to the existing wild type hGH.

Preferably, the protein therapeutic agent may be a human growth hormone, wherein the wild type of human growth hormone is an amino acid set forth in SEQ ID NO: 1, the allowable position is 49th glutamine (Q), 50th threonine (T), 51st Serine (S), 129th Glutamic Acid (E), 130th Aspartic Acid (D), 131th Glycine (G), 133th Proline (P), 134th Arginine (R), 135 Threonine (T), 136th glycine (G), 141th glutamine (Q), 142th threonine (T), 143th tyrosine (Y), 144th serine (S) , 155th alanine (A), 156th leucine (L), 157th leucine (L) and 158th lysine (K) amino acid, and at least one amino acid selected from the group consisting of One of the 49th glutamine (Q), the 129th glutamic acid (E), the 134th alginine (R), or the 141st glutamine (Q) amino acid One can.

The inventors performed the mutation for glycosylation at the allowable position in Example 5 to confirm that the mutation can be introduced into the protein without affecting the function (Table 1).

In another embodiment, the present invention may be a target protein produced by the above method.

The method for preparing a modified target protein by attaching a surfactant to the selected allowable position may include, but is not limited to, a surfactant, for example, cholic acid, and the surfactant may be used to determine the solubility of the protein. Increasing the permeability of the cell membrane can be facilitated to facilitate the introduction of the protein therapeutic agent in vivo. Attachment of such surfactants can be carried out using known methods without limitation. In addition, in the method for producing a modified target protein by fusing a heterologous protein in a selected acceptable position, the heterologous protein may be a ligand, a receptor, or the like for moving the protein therapeutic agent to a specific target such as cancer cells, but is not limited thereto. Does not. Alternatively, heterologous proteins, such as immunoglobulin Fc regions or albumin, may enhance the half-life or stability of the protein therapeutic agent, but are not limited thereto. Fusion of such heterologous proteins can be performed using known methods without limitation.

Preferably human growth hormone.

In another aspect, the present invention provides the human growth hormone of SEQ ID NO: 1, wherein the allowable position is 49th glutamine (Q), 50th threonine (T), 51st serine (S), 129 Glutamic acid (E), 130th aspartic acid (D), 131th glycine (G), 133th proline (P), 134th arginine (R), 135th threonine (T), 136th glycine (G), 141th glutamine (Q), 142th threonine (T), 143th tyrosine (Y), 144th serine (S), 155th alanine (A), Modified human growth hormone that fuses mutations, chemical modifications or heterologous proteins to one or more amino acids selected from the group consisting of 156 leucine (L), 157 leucine (L) and 158 lysine (K) amino acids To provide.

Mutation of the amino acid may be substituted with asparagine (N) or cysteine (C).

Preferably, the modified human growth hormone is in the human growth hormone set forth in SEQ ID NO: 1, the 49th glutamine (Q), the 129th glutamic acid (E), the 134th arginine (R), and 141 are acceptable positions. And one or more amino acids selected from the group consisting of the first glutamine (Q) amino acid, which is mutated to asparagine (N). The modified human growth hormone may be a modified human growth hormone that has mutated one, two, three, or four acceptable positions with asparagine, which may increase cell growth than the wild type.

Preferably, the modified human growth hormone is in the human growth hormone set forth in SEQ ID NO: 1, the 49th glutamine (Q), the 129th glutamic acid (E), the 134th arginine (R), and 141 are acceptable positions. And one or more amino acids selected from the group consisting of the first glutamine (Q) amino acid, each with cysteine (C), followed by pegylated modified human growth hormone. The pegylated modified human growth hormone may be a pegylated modified human growth hormone after substitution of one, two, three or four acceptable positions with cysteine, which may increase the half-life than the wild type. Can be.

Preferably, the modified human growth hormone is in the human growth hormone set forth in SEQ ID NO: 1, the 49th glutamine (Q), the 50th threonine (T), the 51st serine (S), 129th glutamic acid (E), 130th aspartic acid (D), 131th glycine (G), 133th proline (P), 134th arginine (R), 135th threonine (T) 136th glycine (G), 141th glutamine (Q), 142th threonine (T), 143th tyrosine (Y), 144th serine (S), 155th alanine (A) At least one amino acid selected from the group consisting of 156 leucine (L), 157 leucine (L) and 158 lysine (K) amino acids, preferably 49th glutamine (Q), 129th A sugar chain is attached to each of one or more amino acids selected from the group consisting of glutamic acid (E), 134th arginine (R), and 141th glutamine (Q) amino acids. It provides a modified human growth hormone. The modified human growth hormone to which the sugar chains are added may be a modified human growth hormone that is glycosylated by adding sugar chains to one, two, three or four acceptable positions, which is proteolytic degradation in vivo than the wild type. It may be stable to enzymes.

Such mutations, glycosylation, or PEGylation can be carried out by known methods. According to one embodiment of the invention, each of the 49th glutamine (Q), 129th glutamic acid (E), 134th arginine (R) and 141th glutamine (Q) amino acids of human growth hormone is asparagine. It was confirmed that the modified human growth hormone mutated to (N) increased the cell growth of Nb2 compared to the wild type (FIG. 7), wherein the human growth hormone glycated at the allowable position was pepsin, an in vivo proteolytic enzyme, It was confirmed that the stability to plasmin, thrombin (Fig. 8).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

실시예 1: 허용위치 탐색을 위한 트랜스포존 제작Example 1 Preparation of Transposon for Permissible Location Search

The process of constructing the transposon and the transposon for the detection of the allowable position is briefly shown in FIG. The transposon includes transposase recognition sites (Mosaic End, ME), translocation enzymes of Tn5 shown in SEQ ID NOs: 2 and 3, and ultraviolet fluorescence (GFPuv) gene of SEQ ID NO: 5 as folding reporters at both ends. In addition, the transposon contains the kanamycin resistance gene of SEQ ID NO: 4 as a cloning marker for primary selection.

The present inventors prepared the transposon for the allowable position search by the following method. Using clones contained in the EZ-Tn5 In-Frame Linker Insertion Kit (Epicentre, USA) as a template, the kanamycin resistance gene and the Tn5 translocation enzyme recognition site of the C-terminal region were PCR (Polymerase) using primers of SEQ ID NOs: 8 and 9. obtained by performing a chain reaction. PCR conditions are as follows. First, the previous stage of denaturation was performed once at 98 degrees for 2 minutes, then the denaturation was repeated 30 times at 98 degrees for 30 seconds, the coupling reaction at 55 degrees for 15 seconds, and the renal response at 74 degrees for 23 seconds. The reaction was reacted for 2 minutes at 74 degrees. The transposase recognition sequence and the fluorescent gene portion of the N-terminal region of the transposon sequence were PCR using primers of SEQ ID NOs: 6 and 7, and then purified by DNA using a Qiagen gel purification kit. Its PCR conditions are as follows. First, the denaturation reaction was performed once at 98 ° C for 2 minutes, then the denaturation reaction was repeated 30 times at 98 ° C for 30 seconds, the coupling reaction at 55 ° C for 15 seconds, and the renal response at 74 ° C for 37 seconds. Reacted for 2 minutes at 74 degrees. The GFPuv gene amplified by PCR and the kanamycin resistance gene were finally linked by overlap PCR. In this case, a forward primer (SEQ ID NO: 6) and a reverse primer (SEQ ID NO: 9) were used. The overlap PCR conditions are as follows. First, the denaturation reaction was performed once at 98 degrees for 2 minutes, then the denaturation reaction was repeated at 98 degrees for 15 seconds, the coupling reaction at 55 degrees for 15 seconds, and the renal reaction was repeated 30 times for 1 minute at 74 degrees. Reacted for 2 minutes at 74 degrees. KOD polymerase (TOYOBO) was used for all PCR reactions. The transposon DNA obtained by the overlap PCR was performed using pBluescriptII SK (+) vector.PvuIIThe vector obtained by cleavage with the restriction enzyme was subcloned.

Primer sequences used in this example are as follows.

SEQ ID NO: 5'-ctgtctcttgtacacatctgagtaaaggagaagaacttttca-3 '

SEQ ID NO: 5'-ctgtctcttgtacacatctgcgtcagc-3 '

SEQ ID NO: 5'-catggcatggatgagctctacaaagctagcatcatgaacaat-3 '

SEQ ID NO: 5'-ctgtctcttgtacacatctggctagctgaagcttgcatgc-3 '

Finally, the transposon required for the Tn5-transposition reaction was obtained by cutting the transposon cloned into the pBluescriptII vector with PvuII restriction enzyme and gel purification.

실시예 2: hGH 내부에 트랜스포존이 삽입된 라이브러리 구축Example 2: Constructing a Library with a Transposon Inserted in hGH

First, by inserting the hGH having the amino acid sequence of SEQ ID NO: 1 in place of the PvuII restriction enzyme pBluscript-SK2 (+) vector to prepare a pBlue-hGH clones. In addition, the high purity transposon prepared in Example 1 was used to make a random insertion library.

As the transposon transferase, Tn5 transferase (Epicentre, USA, Cat No. TNP92110) capable of gene transfer and insertion in in vitro DNA was used. 200 ng of hGH DNA, 1 unit of Tn5 transferase and 1x reaction buffer were added to perform transposition at 37 ° C. for 2 hours, and 1% SDS (sodium dodecyl sulfate) was added, followed by heat at 70 ° C. for 10 minutes. The reaction was stopped by adding. The reaction DNA product was purified using a DNA purification kit (Qiagen, Germany), concentrated to 5 μl of ethanol and introduced into host E. coli (DH5a) by electroporation. After recovering the strain for 1 hour at 37 ° C., it was applied to LB solid medium to which 10 μg / ml of kanamycin (transpozone selection marker) and 100 μg / ml of empicillin were added, and 16 hours at 37 ° C. Incubated for The size of the library obtained was 1.08 × 10 ⁵ levels. Colonies generated after the culture were recovered in storage buffer (1X TY buffer, 15% glycerol, 2% glucose). The recovered microbial library was purified by plasmid pBlue-hGH-GFP-Km using Qiagen plasmid prep kit.

Subsequently, in order to remove the kanamycin resistance gene contained in the transposon, a secondary library was prepared by cleaving NheI restriction enzyme sites at both ends of the kanamycin resistance gene and restoring it into a circular clone through self-ligation. Secondary library size was 1.8 × 10 ⁴ . The plasmid pBlue-hGH-GFP was purified and recovered in the same manner as before. The pBlue-hGH-GFP plasmid from which the kanamycin resistance gene has been removed has a form in which a gene encoding ultraviolet fluorescence (GFPuv) is inserted into various positions of the hGH open reading frame (ORF).

As a result of analyzing the gene sequence by separating 15 colonies from the initial library, it was confirmed that random insertion was successfully performed as intended in the present invention because the base sequences and insertion positions were different in all 15. On the other hand, in 15 colony sequencing, the probability that the reading frame fits hGH (N) -GFP-hGH (C) correctly matches exactly one-third of general expectations.

실시예 3: 형광 리포터 발현을 위한 재조합 효모발현 라이브러리의 제조Example 3: Preparation of Recombinant Yeast Expression Library for Fluorescent Reporter Expression

The pBlue-hGH-GFP plasmid prepared in Example 2 was treated with HindIII / SpeI restriction enzymes present at both ends of the hGH to recover the hGH-GFP gene, and the recovered hGH-GFP gene was transferred to the Yeast / E.coli shuttle vector. Introduced to YGaC9. This cloning induced a homologous recombination by introducing a linear hGH-GFP gene (1 kb) into the yeast ( Saccharomyces cerevisiae YT2805) containing the shuttle vector through heat shock transformation. . The LB was smeared on a solid medium containing empicillin and kanamycin antibiotics, and then cultured at 37 ° C. for 16 hours to finally obtain a yeast library having a diversity of 2 × 10 ³ levels. Since the shuttle vector has a yeast-specific secretory signal sequence, hGH-GFP is secreted out of the cells through vesicles (ER) and Golgi, which are secretory cell organelles, and can be easily observed through cellular fluorescence. have.

The obtained yeast library was performed by analyzing fluorescence of 2 × 10 ⁴ colonies in agar plate medium using AZ-M100 fluorescence microscope (Nikon, Japan) (FIG. 3). Thus, the screening is at a level of rescanning the library about 10 times.

When compared with hGH-GFP prepared by tandem fusion of GFP outside the ORF of hGH as a positive control, fluorescence levels similar to the control were observed in about 1% of the total colonies. Of these, 78 clones with high fluorescence values were selected and subjected to gene sequencing. As a result, eight groups were identified (Fig. 5), and each GFP insertion site was assigned to a well-known hGH structure (PDF entry: 3hhr). Most of the results confirmed that insertion occurred at residues exposed to the tertiary structure surface. Therefore, the experimental search of the allowable position was performed through rational analysis of the selected site through the GFP folding reporter experiment.

실시예 4: 구조 분석 및 합리적 선발 과정Example 4 Structural Analysis and Rational Selection Process

Growth hormone (GH) has a 4-helix bundle structure in which four α-helixes are linked by a linker. The way GH transmits a signal to a cell of interest is to bind two receptors (GHR1 and GHR2) on the cell surface to form an active dimer. Thus, hGH has a binding surface with two receptors, which are called site1 and site2.

Among the GHs derived from various mammals, one type of hGH (Human growth hormone) is known, and seven types of GH (porcine, bovine, ovine_ovisaries, horse_Equus caballus, rat_Nannospalax ehrenbergi, monkey_Callithrix jacchas and avian_Gallus gallus) are selected. Sequence comparison showed that 96 of the 191 amino acid residues were well conserved in all hGH. Most of these conserved sequences were conserved in site1 (8/26), site2 (9/13), and additional important positions, and it was determined that these sites play an important role in the function of hGH. Therefore, amino acid residues located at the binding side with hGH receptors that are well-preserved and possibly affect the activity among heterologous species are inadequate as acceptable sites. In addition, the N-terminus and C-terminus of hGH have been reported to be severely degraded when added to water-soluble polymers (PEG) in the existing literature and invention, which is not suitable for the purpose of preparing high activity and high stability hGH of the present invention. Judging

Indeed, the positions of the 78 clones obtained in the search of Example 3 using the GFP folding reporter could be classified into eight characteristic positions if they were marked on the known hGH structure and classified according to characteristics. 40 of these absolute majority were located on the binding side with the hGH receptors (38 Site1 and 2 Site2) and 17 and 10 were observed at the N- and C-terminus, respectively. Eventually, among the total 78 positions, the positions where protein engineering or chemical modification is possible without impairing the folding and efficacy of the hGH raised in the present invention are narrowed down to 4 positions (referred to as amino acid residues of 49, 129, 134 and 141). It could be lost (Fig. 5 (b)).

As a result of substituting the four residue positions into the hGH protein structure, position 49 is close to the site hydrolyzed by pepsin (number 44), number 134 is the position hydrolyzed by blood thrombin, and number 141 is the plasmin. 129 was located in a loop with high degree of freedom, although not directly related to hydrolysis. These results suggest that protein engineering tolerances discovered through GFPuv insertion are related to the high degree of freedom and probable attack by proteolysis.

실시예 5: 허용위치 재설계를 통한 단백질공학 및 당쇄화, 페길화 발현, 정제Example 5 Protein Engineering and Glycosylation, Pegylation Expression, Purification through Redesigned Tolerance

Various protein engineering modifications were added to the above amino acid positions obtained through experimental and structural methods, and active expression and purification were performed in yeast.

First, in order to induce protein sugar chain addition to the four acceptable positions selected in Example 4, the sequence around each site was intended to be changed. In other words, using the web-based N-glycosylation prediction program (http://www.cbs.dtu.dk/services/NetNGlyc/) to design the amino acid sequence with the highest probability of sugar chain addition reaction, and to fit the amino acid sequence Altered protein engineering was performed. Asnazine (N), asparagine, was introduced into glutamine Q49, residue 49, glutamic acid E129, residue 129, and arginine R134, residue 134, and glutamine Q141, residue 141, as shown in FIG. The produced mutant hGH gene was introduced into the yeast-E. Coli shuttle vector YGaC9 and expressed in the yeast strain YT2805 to induce glycosylation. Expression of the engineered protein in S. cerevisiae was confirmed by SDS-PAGE on 15% acrylamide gels.

This glycosylation introduction is shown in Table 1 below. Amino acid residue 155 is an acceptable position obtained through structural analysis.

Table 1

Sequence (position)	Mutation sequence
Q49-T50-S51	N49-T50-T51
P133-R134-T135-G136	G133-N134-T135-T136
E129-D130-G131	N129-S130-T131
Q141-T142-Y143-S144	G141-N142-V143-T144
A155-L156-L157-K158	N155-L156-T157-N158

As a result, it was confirmed that sugar chain addition was successful in 49-NTS and 141NVTK which performed N-glycosylation, and in the case of 129NSTS and 134NTT mutant protein, glycosylation was not performed unlike expectations, and the same as wild type. Recombinant protein expression was observed only at 22 kDa size (FIG. 6).

Meanwhile, studies have been actively conducted to increase stability in blood and improve treatment efficiency by adding PEG (polyethylene glycol), a soluble polymer material, to protein therapeutics. In particular, studies of exposing cysteine Cys (C) residues on the surface of proteins and specifically adding PEG have been widely used in the development of persistent and stable protein therapeutics.

In the present invention, by introducing a Cys residue in the allowable positions (Q49, E129, R134, Q141) discovered in Example 4 and performing a PEGylation, the development of a new recombinant hGH having a sustainable effect without affecting the efficacy of hGH Tried.

The wild type and mutant proteins were produced through fed-batch cultivation using a 5L fermentor. Prior to the incubation, the cells were initially cultured in 50 ml YNB (yield substrate lacking 0.67% amino acid, 0.5% cassamino acid, 2% glucose) medium, and then cultured in 200 ml YEPD liquid medium and activated. Inoculation was incubated at 30 ° C. for 48 hours. HGH secreted in the medium was confirmed by SDS-PAGE analysis of the culture supernatant of the expected size of protein expression. hGH was solved by incubating poloxamer, which is a non-ionic detergent, with 0.5% of non-ionic detergent, due to the problem of aggregation during culturing in yeast.

The fermented protein is centrifuged (3,500rpm X 20min X 4 ℃) for purification, then passed through a 0.1 micron filtration membrane to completely remove the cells, and then the culture medium through ultrafiltration (molecular weight 30 kDa cut-off) Concentrated and exchanged with 20 mM Tris buffer (pH 8.0). First, in the case of wild type hGH, anion exchange chromatography was purified by a primary purification method. The method is as follows. The wild-type hGH protein fermentation supernatant in the above example was adsorbed at a rate of 2 ml / min on a previously equilibrated DEAE Sepharose fast flow column (1.5 × 6 cm; GE healthcare). Tris buffer was passed through the column again to remove all remaining free protein in the column. 100 ml of the adsorbed protein was eluted with a NaCl linear concentration gradient from 0 to 0.5 M using 20 mM Tris buffer (pH 8.0) and 20 mM Tris buffer (pH 8.0) containing 0.5 M NaCl. As a result of confirming the eluted fraction by SDS-PAGE, the band was confirmed as shown in Figure 6, it was confirmed that most of the eluted at 0.2M NaCl concentration.

As a final step of the purification, the fraction of the previous step was concentrated using a superdex 75 (1.6 × 61.5 cm) column using amicon (mwco: 10,000), and gel filtration chromatography was performed by reducing the amount. Chromatography using a method to separate the size of the protein was passed through 20mM Tris + 0.15M NaCl (pH8.0) buffer at a rate of 1 mL / min. As a result of SDS-PAGE, wild-type hGH recovered a fraction of 22 kDa with a purity of 95% or more, and a glycated mutated hGH recovered a fraction near 50 KDa (FIG. 6).

실시예 6: 세포기반 활성분석을 통한 허용위치 활성 검증Example 6: Validation of Acceptable Site Activity Through Cell-Based Activity Assay

The biological activity of the hGH protein was analyzed by measuring the proliferation of Nb2 cells by binding of hGH to prolactin receptors external to the Nb2 cells.

Nb2-11 rat lymphoma cells were purchased from the European Collection of Cell Culture (ECACC). Cells were cultured in RPMI1640 medium (GIB-11875, invitrogen) with the addition of 2 mM merethanethanol, anti-contaminant antibiotics (invitrogen, cat No. 15240-062) and 10% horse serum. The medium was passaged when the cells reached 80-90% of the surface of the culture flask, and exchanged every 2 to 3 days. After a 1X10 ⁴ cells cultured into a triple (triplate) in a 96-well plate, was diluted in the same medium so that the maximum of the hGH protein 1 nM for at least 10 ^-4 nM was added to the well and stimulate the cells for 48 hours . The final volume of each well was fixed at 100 μl. Yellow MTS (Promega, Cell-titer 96 well Aqueous non-radioactive cell proliferation assay kit, Cat No.G5421) is converted to a water-soluble purple substance by the activity of mitochondrial dehydrogenase in living cells. Cell viability was measured. 20 μl of MTS was added to Nb2 cells incubated in 96-well plates, and then reacted in a 37 ° C., 5% carbon dioxide incubator for 2 hours. The final reaction was carried out using an activity assay to measure absorbance at 490 nm.

Effect of proteolytic modification on the activity of various proteins in which mutation, glycosylation, and pegylation were introduced at the allowable positions (Q49, E129, R134, and Q141 residues) discovered in Example 4 above. Was investigated.

As a result, the effect of promoting the growth of NB2 cells was hardly affected or increased despite various modifications of the allowable position (FIG. 7). These results clearly show that the allowable positions of the present invention selected through experimental exploration, theoretical filtering, are not only inhibiting protein folding, but also very effective target sites that do not inhibit the inherent physiological efficacy at all. Therefore, in the present invention, the addition of sugar chains and PEG large molecular weight soluble polymers in the allowable position was to investigate whether the expected effects of stability, resistance to clearance, and the like generally expected in the blood protease.

To test the stability of pepsin, plasmin, and thrombin enzymes of mutant hGH, 4 μg of hGH protein was treated with 300 ng of enzyme plasmin and thrombin and 100 ng of pepsin. First, to test the stability against plasmin and thrombin, PBS was added so that the final reaction volume was 20 μl according to the buffer condition of pH 8. Enzyme reaction conditions were incubated at 37 ° C. for 4 hours, followed by 95 ° C. for 10 minutes to inactivate enzyme inactivation. Fragment formation of the protein was confirmed via 15% acrylamide SDS-PAGE gel and cell activity measurement methods. First, the Q49N glycosylation induced protein showed high stability against pepsin compared to the wild type, and the Q141N glycosylation induced protein showed stability against plasmin. For the R134N mutant protein, it showed stability against thrombin (FIG. 8).

As a result, the allowable position of the present invention showed little folding inhibition or deactivation of the protein drug, and was confirmed to be an excellent target position capable of producing a form showing better efficacy and stability through protein engineering or chemical modification.

On the other hand, the permissible position detection technology of the present invention is not limited to the phosphorus growth hormone (hGH) shown in the examples, and can be used as a source technology for developing a superior "super biosimilar" through the modification of various protein therapeutic agents. Being able to do that is obvious to an expert in protein engineering. Therefore, using the method of the present invention it is possible to develop a variety of protein drugs with excellent activity and stability.

In addition, the present invention can be a very useful invention even when the purpose of improving the protein for use as a smart drug (smart drug) applicable only to a specific target such as cancer cells. It can also be used for the attachment of surfactants such as cholic acid to increase the solubility of proteins.

Claims

(a) preparing a target protein library having a fusion protein in which the folding reporter protein is inserted at various internal positions of the target protein;

(b) selecting from the target protein library a colony having a higher activity of the reporter protein than the control group in which the folding reporter protein is fused at the N-terminus or C-terminus of the target protein; And

(c) analyzing the fusion protein sequence of the selected colonies to identify the position where the folding reporter protein is inserted, and selecting a permissive site that can be modified among the target proteins.
The method of claim 1, further comprising the step of designing an amino acid located within 5 ms (± 2 ms) about the folding reporter protein insertion position identified in step (c) as an acceptable position.
The method of claim 1, further comprising selecting an amino acid located within 5 μs (± 2 μs) around the cleavage site of the protease among the folding reporter protein insertion sites identified in step (c).
The method of claim 1, further comprising excluding from the folding reporter protein insertion positions identified in step (c) a position that removes the binding activity of the target protein or the intrinsic functional activity of the target protein when modifying the protein. .
5. The method of claim 4, wherein the site that removes the binding activity of the target protein or the intrinsic functional activity of the target protein when modifying the protein is identified by substitution into a known tertiary structure of the target protein.
The method of claim 1, wherein the nucleic acid encoding the folding reporter protein is inserted at various internal positions of the nucleic acid encoding the target protein by transposon.
7. The method of claim 6, wherein the transposon comprises a nucleic acid encoding a folding reporter protein and a transposase recognition site at both ends.
The method of claim 1, wherein step (a)

(i) introducing a transposon comprising a nucleic acid encoding a folding reporter protein, a transposon insertion clone selection marker gene, and a translocation enzyme recognition site at both ends into a first host cell;

(ii) selecting a colony expressing a selection marker gene from the first host cells to prepare a transposon insertion first library;

(iii) removing the selection marker gene from the transposon from the first library of step (ii); And

(iv) introducing the transposon from which the selection marker has been removed into a second host cell to prepare a second library.
The method of claim 8, wherein the first host cell is Escherichia coli and the second host cell is yeast.
The method of claim 8, wherein the nucleic acid encoding the folding reporter protein is a nucleic acid encoding a green fluorescent protein (GFP) as set forth in SEQ ID NO: 5, and the selection marker gene is a kanamycin resistance gene as set forth in SEQ ID NO: 4, and a translocation enzyme recognition. And the site is the sequence set forth in SEQ ID NOs: 2 and 3.
The method of claim 1, wherein said acceptable position is a position capable of performing mutation, chemical modification, or fusion of a heterologous protein without impairing the function of the protein therapeutic agent.
The method of claim 11, wherein the chemical modification comprises glycosylation, PEGylation or surfactant attachment.
The method of claim 1, wherein the folding reporter protein is a green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein ( RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein ( EYFP), indigo blue fluorescent protein (CFP) and enhanced indigo blue fluorescent protein (ECFP).
The method of claim 1, wherein the target protein is human growth hormone, insulin, EGFR (Epidermal Growth Factor), EPO (Erythropoietin), hematopoietic stimulant, neutrophil promoter (G-CSF), platelet growth factor (Thrombopoietin), interferon, coagulation And a protein therapeutic agent selected from the group consisting of coagulation factor and follicle stimulating hormone.
15. A method of making a modified target protein, comprising performing fusion of a mutation, chemical modification or heterologous protein at an acceptable position selected by the method of any one of claims 1-14.
The method of claim 15, wherein the modified target protein has improved safety compared to wild type.
The method of claim 15, wherein the modified target protein has improved introduction into the target cell compared to the wild type.
The method of claim 15, wherein the target protein is human growth hormone.
The wild type of human growth hormone according to claim 18 is an amino acid set forth in SEQ ID NO: 1, the allowable position is 49th glutamine (Q), 50th threonine (T), 51st serine (S), 129th glutamic acid (E), 130th aspartic acid (D), 131th glycine (G), 133th proline (P), 134th arginine (R), 135th threonine (T) 136th glycine (G), 141th glutamine (Q), 142th threonine (T), 143th tyrosine (Y), 144th serine (S), 155th alanine (A) And 156 leucine (L), 157 leucine (L) and 158 lysine (K) amino acids.
In the human growth hormone of SEQ ID NO: 1, the allowable position is the 49th glutamine (Q), the 50th threonine (T), the 51st serine (S), the 129th glutamic acid (E), and the 130th Aspartic Acid (D), 131th Glycine (G), 133th Proline (P), 134th Arginine (R), 135th Threonine (T), 136th Glycine (G), 141 Glutamine (Q), 142th threonine (T), 143th tyrosine (Y), 144th serine (S), 155th alanine (A), 156th leucine (L), A modified human growth hormone, wherein a mutation, chemical modification, or heterologous protein is fused to one or more amino acids selected from the group consisting of 157th leucine (L) and 158th lysine (K) amino acids.
The modified human growth hormone of claim 20, wherein the amino acid mutation is substituted with asparagine (N) or cysteine (C).
The at least one amino acid of claim 20, wherein the amino acid is at least one amino acid selected from the group consisting of the 49th glutamine (Q), 129th glutamic acid (E), 134th arginine (R) and 141th glutamine (Q) amino acids. Modified human growth hormone glycosylated by mutating each with asparagine (N).
The at least one amino acid of claim 20, wherein the amino acid is at least one amino acid selected from the group consisting of the 49th glutamine (Q), 129th glutamic acid (E), 134th arginine (R) and 141th glutamine (Q) amino acids. Modified human growth hormone pegylated after each substitution with cysteine (C).
21. The acceptable position of claim 20, wherein the allowable position is 49th glutamine (Q), 50th threonine (T), 51st serine (S), 129th glutamic acid (E), 130th aspartic acid (D) , 131th glycine (G), 133th proline (P), 134th arginine (R), 135th threonine (T), 136th glycine (G), 141th glutamine (Q) ), 142 th threonine (T), 143 th tyrosine (Y), 144 th serine (S), 155 th alanine (A), 156 th leucine (L), 157 th leucine A modified human growth hormone, wherein a sugar chain is added to each of at least one amino acid selected from the group consisting of (L) and 158th lysine (K) amino acids.