Proteins, culture media containing them, and their use

Abstract

The present invention provides a protein, a culture medium containing the protein, and its use. The protein is a variant of the amino acid sequence shown in Sequence ID No. 1. When the protein is added to the culture medium, the medium can effectively stimulate the potential of cells or microorganisms, accelerate the growth rate of those cells or microorganisms, and allow the cells or microorganisms to maintain their viability in sudden unfavorable conditions.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, specifically relates to a protein, and further relates to a medium containing the protein and its use for culturing cells or microorganisms.

Background Art

[0002] In cell culture and microbial fermentation, the medium is a basic requirement for cell growth as a cell nutrient. Currently, there are various methods to optimize the medium in order to improve the efficiency of cell culture. However, there is still a need for a medium that can stimulate the potential of cells or microorganisms, increase the growth rate of cells or microorganisms, maintain the survival rate even under sudden adverse conditions, and maintain the survival rate under sudden adverse conditions.

Summary of the Invention

[0003] Therefore, an object of the present invention is to provide a protein for culturing cells or microorganisms based on the prior art. The protein of the present invention acts as an additive to the cell medium and can significantly promote the survival and growth of cells or microorganisms.

[0004] The object of the present invention is achieved by the following technical solutions.

[0005] In a first aspect of the present invention, there is provided a protein which is a variant of the amino acid sequence shown in SEQ ID NO: 1, and the variant (a) adds S to the N-terminus, (b) the 4th position is H, G, Q, K or E, (c) the 5th position is D, H, G, S, A, T or does not exist, (d) the 6th position is V, I, P, N, A or E, (e) the 7th position is Q, H, P or does not exist, (f) the 8th position is N, H or D, (g) the 9th position is M, (h) the 10th position is D. (i) The 11th place is S or G. (j) The 12th place is S or E. (k) The 13th place is K. (l) M is in 14th place. (m) The 15th place is G or D. (n) The 16th place is S, T, or A. (o) G is in 18th place. (p)23rd place is C. (q) T is in 25th place. (r)R is ranked 28th. (s) D is in 32nd place. (t) The 38th position is Q, K, R, N, or S. (u) The 39th place is Y. (v) The 46th position is N or T. (w)49th place is V. (x) The 53rd place is K. (y) The 54th place is N. (z) The 56th place is K. (aa) The 59th place is either K or R. (bb)63rd place is F and / or (cc) The C-terminus contains at least one mutation of either the pentapeptide GLVPR or is absent.

[0006] This invention is based on the inventors' unexpected observation that proteins stimulate the survival and growth of cells or microorganisms.

[0007] In a specific embodiment, the protein is (1) Compared to the amino acid sequence shown in Sequence ID No. 1, the following mutations are present: T6→V6 and / or N11→S11. (2) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: T6→I6, N11→S11, R13→K13, I14→M14, E32→D32, P38→Q38, and Q59→K59. (3) Compared to the amino acid sequence shown in Sequence ID No. 1, the following mutations are present: H4→G4 and / or T6→P6 (4) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence contains one or more mutations including H4→Q4, E7→Q7, N11→S11, E32→D32, P38→Q38, R53→K53, and 59Q→59K. (5) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: N11→S11, R13→K13, E32→D32, P38→Q38, and Q59→K59. (6) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, E7→Q7, N11→S11, A12→S12, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (7) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, P16→S16, E32→D32, P38→K38, R53→K53, and Q59→K59. (8) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E15→G15, P38→Q38, I49→V49, R53→K53, and Q59→K59. (9) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E15→G15, E32→D32, P38→R38, I49→V49, R53→K53, and Q59→K59. (10) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (11) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, E7→Q7, E10→D10, N11→S11, E32→D32, P38→N38, R53→K53, and Q59→K59. (12) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: H4→K4, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, R56→K56, and Q59→K59. (13) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, T6→I6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (14) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, T6→N6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (15) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→E4, T6→A6, E7→Q7, V9→M9, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (16) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: H4→Q4, E7→H7, N11→S11, R13→K13, E25→T25, E32→D32, P38→Q38, R53→K53, and Q59→K59. (17) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, T6→P6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, and R53→K53, Q59→K59. (18) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, E7→Q7, V9→M9, E10→D10, N11→S11, R13→K13, Y23→C23, E32→D32, P38→N38, R53→K53, and Q59→R59. (19) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: H4→K4, N5→D5, T6→I6, E7→P7, P8→N8, N11→G11, A12→E12, R13→K13, P16→T16, K28→R28, E32→D32, P38→K38, K46→N46, R53→K53, R56→K56 and Q59→K59. (20) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, E7→Q7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (21) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, N5→del, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (22) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, N5→H5, E7→Q7, N11→S11, R13→K13, P16→A16, E32→D32, P38→N38, R53→K53, and Q59→K59. (23) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations of H4→K4, E7→Q7, E10→D10, N11→S11, R13→K13, D18→G18, E32→D32, P38→N38, H39→Y39, R53→K53 and Q59→K59. (24) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, N5→G5, T6→E6, E7→P7, P8→H8, N11→G11, R13→K13, P16→T16, E32→D32, P38→K38, K46→T46, R53→K53, R56→K56, and Q59→K59. (25) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→E4, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (26) containing one or more mutations of H4→K4, T6→N6, E7→P7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (27) containing one or more mutations of H4→K4, N5→S5, T6→P6, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (28) containing one or more mutations of T6→V6, N11→S11, R13→K13, E32→D32, P38→Q38, S54→N54, Q59→K59 and L63→F63 as compared with the amino acid sequence shown in SEQ ID NO: 1 (29) containing one or more mutations of H4→K4, N5→S5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (30) containing one or more mutations of H4→K4, N5→A5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (31) containing the mutation of T6→A6 and / or N11→S11 as compared with the amino acid sequence shown in SEQ ID NO: 1 (32) containing one or more mutations of H4→Q4, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (33) containing one or more mutations of H4→K4, N5→G5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53 and Q59→K59 as compared with the amino acid sequence shown in SEQ ID NO: 1 (34) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, E7→H7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (35) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more mutations are present, including H4→K4, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, R56→K56 and Q59→K59. (36) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, T6→P6, E7→H7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (37) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→del, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (38) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more mutations are present, including N11→S11, E15→D15, E32→D32, P38→Q38 and Q59→K59. (39) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, E15→D15, E32→D32, P38→K38, R53→K53, and Q59→K59. (40) Compared to the amino acid sequence shown in SEQ ID NO: 1, one or more mutations are present, including H4→Q4, E7→H7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (41) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: H4→K4, N5→D5, E7→P7, P8→D8, N11→G11, A12→E12, R13→K13, E32→D32, P38→Q38, K46→N46, R53→K53, S54→N54, R56→K56 and Q59→K59. (42) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more of the following mutations are present: H4→Q4, N5→G5, T6→E6, E7→P7, P8→H8, N11→G11, R13→K13, P16→T16, E32→D32, P38→K38, K46→T46, R53→K53, R56→K56, and Q59→K59. (43) Compared to the amino acid sequence shown in SEQ ID NO: 1, one or more mutations are present, including H4→K4, T6→P6, E7→H7, V9→M9, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53 and Q59→K59. (44) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more mutations are present, including H4→Q4, T6→I6, E7→P7, N11→S11, R13→K13, E32→D32, P38→Q38, R53→K53, R56→K56, and Q59→K59. (45) Compared to the amino acid sequence shown in SEQ ID NO: 1, the sequence includes one or more mutations: H4→K4, N5→T5, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53, and Q59→K59. (46) Compared to the amino acid sequence shown in SEQ ID NO: 1, one or more mutations are present, including H4→K4, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53 and Q59→K59. (47) Compared to the amino acid sequence shown in SEQ ID NO: 1, one or more mutations including R53→K53 and / or Q59→K59, (48) Compared to the amino acid sequence shown in Sequence ID No. 1, an S is added to the N-terminus and includes the mutations R53→K53 and / or Q59→K59. (49) Compared to the amino acid sequence shown in Sequence ID No. 1, an S is added to the N-terminus, a pentapeptide GLVPR is added to the C-terminus, and the R53→K53 and / or Q59→K59 mutations are included.

[0008] In a specific embodiment, the protein is a sequence having two or more, three or more, four or more, or five or more and ten or fewer, eleven or fewer, twelve or fewer, thirteen or fewer, fourteen or fewer, or fifteen or fewer amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in any one of Sequence IDs 1 to 50.

[0009] Preferably, the protein contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 50, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

[0010] Preferably, the protein has the amino acid sequence shown in any one of SEQ ID NOs: 2 to 50.

[0011] A second aspect of the present invention provides an isolated nucleic acid molecule containing a nucleotide sequence encoding the protein.

[0012] A third aspect of the present invention provides an expression vector containing the isolated nucleic acid molecule.

[0013] A fourth aspect of the present invention provides a culture medium supplement containing at least one of the proteins or a combination thereof.

[0014] A fifth aspect of the present invention provides a culture medium containing at least one of the proteins or a combination thereof, or the supplement.

[0015] In a preferred embodiment, the working concentration of the protein is 0.02 to 1000 μg / mL, preferably 10 to 800 μg / mL, more preferably 20 to 600 μg / mL, and even more preferably 40 to 400 μg / mL or any concentration value between 0.02 and 1000 μg / mL. Specifically, the working concentrations of the protein are 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400, 600, 800, and 1000 μg / mL.

[0016] Preferably, the culture medium further comprises a basal culture medium.

[0017] Preferably, the basal culture medium is selected from ordinary culture media.

[0018] A sixth aspect of the present invention provides a method for culturing cells or microorganisms, the method being: A step of providing cells or microorganisms for culture, The steps of bringing the cells or microorganisms into contact with at least one or a combination thereof of the proteins, the culture medium supplement, or the culture medium, The procedure includes the step of collecting the culture.

[0019] Preferably, the cells are stem cells, cell lines, or primary cells, and more preferably, the cell lines are genetically modified cell lines.

[0020] The aforementioned genetically modified cell lines include, for example, Kit225, 293T, and CHO, and the aforementioned primary cells include, but are not limited to, PMO and PBMC.

[0021] Preferably, the microorganism is a genetically modified bacterium such as yeast or Escherichia coli.

[0022] A seventh aspect of the present invention provides the use of the protein, the culture medium supplement, or the culture medium for culturing cells or microorganisms.

[0023] Preferably, the cells are stem cells, cell lines, or primary cells, and more preferably, the cell lines are genetically modified cell lines.

[0024] The aforementioned genetically modified cell lines include, for example, Kit225, 293T, and CHO, and the aforementioned primary cells include, but are not limited to, PMO and PBMC.

[0025] Preferably, the microorganism is a genetically modified bacterium such as yeast or Escherichia coli. [Brief explanation of the drawing]

[0026] To more clearly explain the technical solution of the present invention, the technical solution will be described in detail below with reference to the drawings. [Figure 1] The electrophoresis diagrams for protein purification using sample codes B01-B14 are shown. [Figure 2] The electrophoresis diagrams for protein purification using sample codes B15-B28 are shown. [Figure 3] The electrophoresis diagrams for protein purification using sample codes B29-B42 are shown. [Figure 4] The electrophoresis diagrams for protein purification using sample codes B43-B47 are shown. [Figure 5] The electrophoresis diagrams for protein purification using sample codes B81-B83 are shown. [Figure 6] This shows the effect of the protein with sample code number B83 on the KIT225 cell growth curve under nutrient starvation conditions. [Figure 7] This shows the effect of the protein with sample code number B83 on the viability of KIT225 cells under nutrient starvation conditions. [Figure 8] This shows the effect of the protein with sample code number B83 on the growth curve of 293T cells under nutrient starvation conditions. [Figure 9] This shows the effect of the protein with sample code number B83 on 293T cell viability under nutrient starvation conditions. [Figure 10]This shows the effect of the protein with sample code number B83 on the CHO cell growth curve under nutrient starvation conditions. [Figure 11] This shows the effect of the protein with sample code number B83 on CHO cell viability under nutrient starvation conditions. [Figure 12] This shows the effect of the protein with sample code number B83 on the growth curve of PBMC cells under nutrient starvation conditions. [Figure 13] This shows the effect of the protein with sample code number B83 on PBMC cell viability under nutrient starvation conditions. [Figure 14] This shows the effect of the protein with sample code number B83 on the PMO cell growth curve under nutrient starvation conditions. [Figure 15] This shows the effect of the protein with sample code number B83 on PMO cell viability under nutrient starvation conditions. [Modes for carrying out the invention]

[0027] The technical solutions of the present invention will be described below with specific test examples, but the scope of protection of the present invention is not limited thereto.

[0028] The examples described herein merely enumerate ways in which the conceptual framework of the invention can be realized, and the scope of protection of the present invention should not be considered to be limited to the specific forms described herein. Rather, the scope of protection of the present invention extends to equivalent technical means that a person skilled in the art could conceive based on the conceptual framework of the present invention. Embodiments of the present invention are described below, but the present invention is not limited to the specific embodiments and fields of application described herein, and the specific embodiments described below are illustrative and teaching examples, not limiting. A person skilled in the art can make many more forms without departing from the suggestions herein and the scope protected by the claims of the present invention, all of which fall within the scope of protection of the present invention.

[0029] The tests described below in this invention are based on multiple creative tests and summarize the researchers' conclusive tests, which are based on the technical solutions that this invention seeks to protect. In the quantitative tests in the following examples, three repeated experiments are set up, and the data are the mean or mean ± standard deviation of the three repeated experiments.

[0030] definition The amino acids that form the protein of the present invention may be natural or unnatural amino acids.

[0031] The term "natural amino acids" refers to L-type amino acids expressed in natural proteins, namely alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0032] The term "non-natural amino acids" refers to the D-form of natural amino acids, isoforms of certain natural amino acids (such as arginine, lysine, phenylalanine, and serine), and the L-form of leucine and valine. It further includes synthetic amino acids, such as alpha-aminobutyric acid (Abu), agmatine (Agm), alpha-aminoisobutyric acid (Aib), N-formyl-Trp (F-trp), sarcosine, statin, ornithine, and desaminotyrosine. Desaminotyrosine is incorporated into the N-terminus of these peptides, while agmatine and statin are incorporated into the C-terminus of these peptides.

[0033] In all discussions herein, standard single-letter codes for amino acids and standard substitution notation are used. In formulas, 1) Amino acid residues are represented using the generally accepted IUPAC nomenclature and in single-letter code form. DNA nucleic acid sequences also use the generally accepted IUPAC nomenclature.

[0034] 2) Mutant identification uses the "amino acid substituted at the original amino acid position" to represent the mutated amino acid in the mutant. For example, T6→V6 indicates that the amino acid at position 6 has been substituted from threonine (Thr) to valine (Val), and T6→del indicates that the amino acid at position 6 has been deleted. The position number corresponds to the amino acid sequence number in SEQ ID NO: 1.

[0035] cell As used herein, the term “cell” refers to a single cell, but also to a population (i.e., multiple cells). A population may be a pure population containing one cell type, or it may contain multiple cell types. In the present invention, the number of cell types contained in a population is not limited. Furthermore, as used herein, “cell culture” refers to an in vitro culture of any cells. This term includes any other cell population maintained in vivo, including serial cell lines (i.e., having a persistent phenotype), primary cell cultures, finite cell lines (i.e., non-transformed cells), oocytes, and embryos.

[0036] In some embodiments, the cells are non-genetically modified cell lines, and in other embodiments, the cells are genetically modified cell lines. In some embodiments, the cells contain genetically modified molecules, and the present invention is not limited to combinations of cell types suitable for teratoma production or detection, identification and / or quantification of apoptosis in a sample, including mixed cells cultured with all cell types used that are not genetically modified cell lines, one or more cell types in the mixture are genetically modified cell lines and the rest are not, and all cell types in the mixture are genetically modified cell lines.

[0037] As used herein, the term “primary cells” refers to cells obtained directly from the tissues (e.g., blood) or organs of animals, including humans, without culture. While not mandatory, primary cells are typically passaged up to 10 times in vitro before dying and / or ceasing to grow. In contrast, “cultured cells” are cells that have already been maintained and / or grown more than 10 times in vitro. Compared to primary cells of the same origin, cultured cells refer to cells that can be passaged multiple times in vitro before ceasing to grow and / or dying. Cultured cells include “cell lines” and “primary cultured cells.”

[0038] As used herein, the term “cell line” refers to in vitro cultured cells, including primary cell lines, finite cell lines, serial cell lines, and transformed cell lines. This term does not require that the number of cultured cells be infinite. Cell lines may be produced spontaneously or by transformation.

[0039] As used herein, the terms “culture medium” and “cell culture medium” refer to a medium suitable for in vitro culture of cells (i.e., a cell culture). This term is not limited to any specific medium. For example, this definition includes by-products in addition to maintenance media. In fact, this term includes any medium suitable for cell growth.

[0040] As used herein, the term “in vitro” refers to an artificial environment and the processes and reactions within it. In vitro environments are exemplified by, but are not limited to, test tubes and cell cultures.

[0041] As used herein, the term "in vivo" refers to a natural environment (animal or cell) and the processes and reactions within it.

[0042] As used herein, the terms “proliferation” and “growth” can be used interchangeably to refer to an increase in cell number. In contrast, “maintenance” refers to the continuous survival of a cell or cell population, but does not necessarily have to be survival with an increased cell number.

[0043] As used herein, the terms “stem cells,” “non-functional cells,” and “undifferentiated cells” refer to cells that possess the unique ability to self-replicate and produce specific types of cells that make up the tissues and organs of the body. Embryonic stem cells do not possess tissue-specific structure and tissue-specific function (i.e., cardiomyocytes, nerve cells, etc.). Stem cells may be derived from embryo (i.e., embryonic stem cells), fetal, and adult tissues. The terms “specific” and “differentiated” refer to cells that possess tissue-specific structure and / or function (i.e., cardiomyocytes, nerve cells, etc.). When used in reference to cells, the term “differentiation” refers to the process by which non-functional cells acquire a specific function (i.e., heart, liver, or muscle cells).

[0044] Examples In the following examples, experimental methods where specific conditions are not explicitly stated will be carried out according to standard methods and conditions, or in accordance with the product description.

[0045] Plasmid pET30b and BL21 competent cells were purchased from Novagen.

[0046] Restriction endonucleases such as NdeI and HindIII, DNA markers, plasmid extraction kits, and DNA gel recovery and purification kits were all purchased from Takara.

[0047] The Superdex 200 molecular sieves and CM ion exchange chromatography columns were purchased from Nanomicro.

[0048] All of the chemical reagents are analytical reagents as defined in the Chinese Pharmacopoeia.

[0049] The plasmid extraction procedure steps were followed according to the instructions included with the plasmid mini-extraction kit.

[0050] The DNA gel retrieval procedure was followed according to the instructions included with the DNA gel retrieval kit.

[0051] The DNA fragment ligation procedure followed the instructions for the T4 ligase.

[0052] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the remainder is water.

[0053] E. coli culture medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the remainder is water.

[0054] Saccharomyces cerevisiae medium: 20 g / L yeast powder, 40 g / L peptone, 40 g / L glucose, and the remainder is water.

[0055] The human T lymphocyte KIT225 cell line was purchased from ATCC.

[0056] The human kidney epithelial cell line 293T was purchased from ATCC.

[0057] The hamster ovary cell CHO cell line was purchased from ATCC.

[0058] The SD rats were purchased from Suzhou Aermaite Biotechnology Co., Ltd.

[0059] The culture medium 1640 was purchased from Gibco, product number 22400121.

[0060] The FBS (fetal bovine serum) was purchased from Gibco, product number 10270-106.

[0061] The HBSS medium was purchased from Biyuntian, product number C0219.

[0062] Example 1: Protein expression and purification Using a whole-gene synthesis method, the coding DNA sequences (not shown) of the target proteins (shown in SEQ ID NOs. 1-50) were obtained, double-enzyme cleavage with NdeI and HindIII, and then ligated to pET30b plasmid (Novagen) to obtain recombinant plasmids expressing the target proteins. After transforming BL21 competent cells with the recombinant plasmids, they were plated and positive clones were screened. Twenty clones were selected and each was inoculated into LB medium and cultured. 600 When the ratio reached 0.6, 0.1 mM IPTG was added to induce expression, and it was confirmed that the molecular weight met the requirements. A series of recombinant strains were constructed using the above method, and the protein sequences expressed by the strains are shown in Table 1.

[0063] Table 1 Protein Sequences [Table 1-1]

[0064] [Table 1-2]

[0065] Using the sample preparation process for code number B83 as an example, the bacterial strain was inoculated into a shaking flask medium for high-volume expression, and the cells were collected after IPTG induction. The cells were added to a buffer at a ratio of 1:10, and then disrupted by sonication to obtain a bacterial lysate. The bacterial lysate was purified through the following steps: (1) Separation and purification in the first step was performed using a Nanomicro CM ion exchange chromatography column, and (2) a target product (55 mg) with a purity of more than 95% was obtained using molecular sieve chromatography with a Nanomicro Superdex 200, and its molecular weight was 8,400 daltons (shown in Figure 5).

[0066] The sample preparation process for code numbers B1-B47, B81, and B82 was the same as for B83, and the electrophoretic diagrams of the obtained target products are shown in Figures 1-5.

[0067] Example 2: The protein of the present invention significantly improves the growth rate of E. coli. Prepare an E. coli culture medium and dispense it into test tubes numbered 1 to 51. Each test tube contains a parallel sample, arranged in a triple configuration, and includes a control group. Sterilize all test tubes and prepare them for use.

[0068] Each group was given a 10 μM sterile recombinant protein sample (code numbers B1-B47, B81, B82, and B83), and an equal volume of sterile physiological saline was added to the control group.

[0069] All groups were inoculated with a 1:20 ratio of E. coli suspension. After inoculation, the cells were placed in a constant temperature shaking incubator and cultured under conditions of 37°C and 225 rpm. Samples were taken and detected after 2 hours. 600 The value was recorded.

[0070] The results are shown in Table 2, and all of the proteins of the present invention showed the 2-hour OD of E. coli. 600 The values ​​can be improved, particularly for proteins represented by code numbers B12, B14, B15, B16, B18, B23, B24, B26, B27, B30, B31, B34, B35, B37, B44, B46, B81, B82, and B83, of which B81, B82, and B83 show particularly significant effects, especially B83.

[0071] Example 3: The protein of the present invention significantly improves the growth rate of Saccharomyces cerevisiae. Prepare a culture medium for yeast growth, dispense it into test tubes numbered 1 to 51, each containing a parallel sample in a triple-tube arrangement, and include a control group. Sterilize all test tubes and prepare them for use.

[0072] Each group was given a 10 μM sterile recombinant protein sample (code numbers B1-B47, B81, B82, and B83), and an equal volume of sterile physiological saline was added to the control group.

[0073] All groups were inoculated with yeast solution in a 1:20 ratio. After inoculation, they were placed in a constant temperature shaking incubator and cultured under conditions of 37°C and 225 rpm. Samples were taken and detected after 2 hours, and OD was measured. 600 The value was recorded.

[0074] The results are shown in Table 2, and as can be seen from Table 2, these proteins are similar to the OD of Saccharomyces cerevisiae 2h. 600 The values ​​can be improved, particularly for proteins represented by code numbers B12, B14, B15, B16, B18, B23, B24, B26, B27, B30, B31, B34, B35, B37, B44, B46, B81, B82, and B83, of which B81, B82, and B83 show particularly significant effects, especially B83.

[0075] Table 2. OD of Escherichia coli and Saccharomyces cerevisiae 600 value [Table 2-1]

[0076] [Table 2-2]

[0077] Example 4: The protein of the present invention significantly improves the survival rate of human T lymphocyte KIT225 cell line under nutrient starvation conditions. 1. Resuscitation of cell lines: 1) Prepare a medium 1640:FBS=90:10, and put 3 mL of the medium into a 15 mL centrifuge tube. 2) Remove the cells from liquid nitrogen and dissolve them in a 37°C water bath for 1 minute. 3) Add a small amount of medium to the cell cryopreservation tube, transfer the cells to a 15 mL centrifuge tube, 4) Centrifuge at 1000 × g for 3 minutes, remove the supernatant, add 4 mL of medium, and slowly pipette to mix uniformly. 5) Pipette the cells uniformly into a culture dish and transfer to an incubator.

[0078] 2. Subculturing the cell line: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) transfer it to a 15 mL centrifuge tube, centrifuge at 1000 × g for 3 min, remove the supernatant, add 4 mL of culture medium, mix thoroughly by slow pipetting, and subculturing at a ratio of 1:4.

[0079] 3. Cell line plating: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) Centrifuge at 1000 × g for 3 min, 3) Remove the supernatant, add HBSS medium and mix uniformly by gently pipetting, and 4) Plate into a 24-well plate according to the experimental grouping requirements below.

[0080] The cells were divided into four groups: B83 protein was added to each group until the final concentrations reached 0 μg / mL, 4 μg / mL, 40 μg / mL, and 400 μg / mL. Each group consisted of three wells, with 380,000 cells / well and a volume of 1 mL / well. Cells were cultured for 6 hours, 24 hours, and 30 hours, then sampled, stained with trypan blue, and cell viability was detected.

[0081] Cell viability = (Total number of cells - Number of blue cells) / Total number of cells * 100% The results are shown in Figures 6 and 7. In nutrient-starved HBSS, the number of viable KIT225 cells decreased significantly over time. However, after adding B83, the number of viable cells increased significantly at the same time point, and the viability improved significantly. In particular, a working concentration of 400 μg / mL significantly improved cell viability.

[0082] Example 5: The protein of the present invention significantly improves the survival rate of human kidney epithelial cell line 293T under nutrient starvation conditions. 1. Resuscitation of cell lines: 1) Prepare a medium 1640:FBS=90:10, and put 3 mL of the medium into a 15 mL centrifuge tube. 2) Remove the cells from liquid nitrogen and dissolve them in a 37°C water bath for 1 minute. 3) Add a small amount of medium to the cell cryopreservation tube, transfer the cells to a 15 mL centrifuge tube, 4) Centrifuge at 1000 × g for 3 minutes, remove the supernatant, add 4 mL of medium, and slowly pipette to mix uniformly. 5) Pipette the cells uniformly into a culture dish and transfer to an incubator.

[0083] 2. Subculturing the cell line: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) transfer it to a 15 mL centrifuge tube, centrifuge at 1000 × g for 3 min, remove the supernatant, add 4 mL of culture medium, mix thoroughly by slow pipetting, and subculturing at a ratio of 1:4.

[0084] 3. Cell line plating: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) Centrifuge at 1000 × g for 3 min, 3) Remove the supernatant, add HBSS medium and mix uniformly by gently pipetting, and 4) Plate into a 24-well plate according to the experimental grouping requirements below.

[0085] The cells were divided into four groups: B83 protein was added to each group until the final concentrations reached 0 μg / mL, 4 μg / mL, 40 μg / mL, and 400 μg / mL. Each group consisted of three wells, with 380,000 cells / well and a volume of 1 mL / well. The cells were cultured for 6 hours and 24 hours, then sampled, stained with trypan blue, and cell viability was detected.

[0086] Cell viability = (Total number of cells - Number of blue cells) / Total number of cells * 100% The results are shown in Figures 8 and 9. In nutrient-starved HBSS, the number of viable 293T cells decreased significantly over time. However, after adding B83, the number of viable cells increased significantly at the same time point, and the viability improved significantly. In particular, a working concentration of 400 μg / mL significantly improved cell viability.

[0087] Example 6: The protein of the present invention significantly improves the survival rate of hamster ovary cell CHO cell line under nutrient starvation conditions. 1. Resuscitation of cell lines: 1) Prepare a medium 1640:FBS=90:10, and put 3 mL of the medium into a 15 mL centrifuge tube. 2) Remove the cells from liquid nitrogen and dissolve them in a 37°C water bath for 1 minute. 3) Add a small amount of medium to the cell cryopreservation tube, transfer the cells to a 15 mL centrifuge tube, 4) Centrifuge at 1000 × g for 3 minutes, remove the supernatant, add 4 mL of medium, and slowly pipette to mix uniformly. 5) Pipette the cells uniformly into a culture dish and transfer to an incubator.

[0088] 2. Subculturing the cell line: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) transfer it to a 15 mL centrifuge tube, centrifuge at 1000 × g for 3 min, remove the supernatant, add 4 mL of culture medium, mix thoroughly by slow pipetting, and subculturing at a ratio of 1:4.

[0089] 3. Cell line plating: 1) Collect the old culture medium from the culture dish into a 15 mL centrifuge tube, 2) Centrifuge at 1000 × g for 3 min, 3) Remove the supernatant, add HBSS medium and mix uniformly by gently pipetting, and 4) Plate into a 24-well plate according to the experimental grouping requirements below.

[0090] The cells were divided into four groups: B83 protein was added to each group until the final concentrations reached 0 μg / mL, 4 μg / mL, 40 μg / mL, and 400 μg / mL. Each group consisted of three wells, with 380,000 cells / well and a volume of 1 mL / well. The cells were cultured for 6 hours and 24 hours, then sampled, stained with trypan blue, and cell viability was detected.

[0091] Cell viability = (Total number of cells - Number of blue cells) / Total number of cells * 100% The results are shown in Figures 10 and 11. In nutrient-starved HBSS, the number of viable CHO cells decreased significantly over time. However, after adding B83, the number of viable cells increased significantly at the same time point, and the viability improved significantly. In particular, working concentrations between 40 and 400 μg / mL significantly improved cell viability.

[0092] Example 7: The protein of the present invention significantly improves the viability of primary rat peritoneal macrophage (PMO) cells under nutrient starvation conditions. 1. Extraction and Culture of Rat Peritoneal Macrophages: Three days prior to the experiment, 5 mL of 3% sodium thioglycolate or starch soup is injected intraperitoneally into each rat, and then the following steps are initiated. 1) The rats are killed by cervical dislocation, immersed in 75% alcohol for 1-2 minutes, transferred to a clean bench, placed on a dissection table, the limbs are secured with needles, and the skin is torn apart with forceps in both hands and pulled to the sides to expose the peritoneum, without damaging the peritoneal wall. 2) After further scrubbing the peritoneal wall with 70% alcohol, 20 mL of serum-free medium is injected into the peritoneal cavity with a syringe, and at the same time, the peritoneal wall is massaged with fingers from both sides to allow the fluid to flow sufficiently into the peritoneal cavity. 3) The abdominal wall is gently lifted with a needle, the animal's body is slightly tilted to one side, and the fluid in the peritoneal cavity is collected below the needle and aspirated into the needle tube. 4) Carefully withdraw the needle, inject the liquid into a centrifuge tube, centrifuge at 1200 r / min at 4°C for 10 minutes, discard the supernatant, repeat twice, and resuspend with complete culture medium. 5) Count using the trypan blue exclusion method, dilute the cells to the desired concentration, and then inoculate. 6) When using as feeder cells, the concentration should be 2 × 10⁻⁶ 5 Prepare the solution at 100-200 μL per well and inoculate it into a 96-well plate. When used for other experiments, use 2 × 10⁶ 6 The volume may be adjusted to 1 mL / mL, and added to a 24-well flat-bottom culture plate, with 1 mL / well. After incubation for 12 hours in a 5% CO2 incubator, the fluid is changed, and the mixture is washed 1-2 times with RPMI1640 medium. Non-adherent cells (erythrocytes) are discarded, and the adherent cells are monolayer PMO cells.

[0093] 2. Cell Plating: 1) Collect the cells in the culture dish into a 15 mL centrifuge tube, 2) Centrifuge at 1000 × g for 3 min, 3) Remove the supernatant, add HBSS medium and mix uniformly by gently pipetting, and 4) Plate the cells into a 24-well plate according to the experimental grouping requirements below.

[0094] The cells were divided into four groups: B83 protein was added to each group until the final concentrations reached 0 μg / mL, 4 μg / mL, 40 μg / mL, and 400 μg / mL. Each group consisted of three wells, with 380,000 cells / well and a volume of 1 mL / well. The cells were cultured for 6 hours and 24 hours, then sampled, stained with trypan blue, and cell viability was detected.

[0095] Cell viability = (Total number of cells - Number of blue cells) / Total number of cells * 100% The results are shown in Figures 12 and 13. In nutrient-starved HBSS, the number of viable PMO cells decreased significantly over time. However, after adding B83, the number of viable cells increased significantly at the same time point, and the viability improved significantly. In particular, working concentrations between 40 and 400 μg / mL significantly improved cell viability.

[0096] Example 8: The protein of the present invention significantly improves the survival rate of primary PBMC cells in rat peripheral blood mononuclear cells under nutrient starvation conditions. 1. Isolation of rat peripheral blood mononuclear cells (PBMCs): (1) Transfer 10 mL of whole blood to a 50 mL centrifuge tube, dilute with 10 mL of PBS solution, and mix gently and uniformly. (2) Take two 15 mL centrifuge tubes and first add 5 mL of lymphocyte separatory solution. Next, gently add the diluted blood to the upper layer of lymphocyte separatory solution in the two centrifuge tubes, and leave gently so that the two solutions do not mix. Add 10 mL of diluted blood to each centrifuge tube. (3) Centrifuge at 2,000 rpm for 30 min, taking care to set the deceleration setting to no break or only 10-20% brake. (4) The cell layer containing PBMCs will be white. At this time, the cells in this layer can be aspirated with a pipette into another clean 15 mL centrifuge tube. (5) Add 10-15 mL of PBS, centrifuge at 1,500 rpm for 10 min, remove the supernatant, add culture medium and wash in the same manner. The resulting cells are PBMCs.

[0097] 2. Cell Plating: Resuspend the PBMC cells in 5-10 mL of 1640 medium, then perform counting culture or plating.

[0098] The cells were divided into four groups: B83 protein was added to each group until the final concentrations reached 0 μg / mL, 4 μg / mL, 40 μg / mL, and 400 μg / mL. Each group consisted of three wells, with 380,000 cells / well and a volume of 1 mL / well. The cells were cultured for 6 h, 24 h, 36 h, and 48 h, sampled, stained with trypan blue, and cell viability was detected.

[0099] Cell viability = (Total number of cells - Number of blue cells) / Total number of cells * 100% The results are shown in Figures 14 and 15. In HBSS under nutrient starvation, the number of viable PBMC cells decreased significantly over time. However, after adding B83, the number of viable cells increased significantly at the same time point, and the viability improved significantly. In particular, working concentrations of 40-400 μg / mL significantly improved cell viability.

[0100] Those skilled in the art will know that some further improvements and additions can be made without departing from the methods of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A protein which is a variant of the amino acid sequence shown in Sequence ID No. 1, The aforementioned mutant is (a) Add S to the N-terminus, (b) The fourth place is H, G, Q, K, or E. (c) The 5th place is D, H, G, S, A, T or does not exist. (d) The 6th place is V, I, P, N, A, or E. (e) The 7th place is either Q, H, or P, or does not exist. (f) The 8th place is N, H, or D. (g) M is in 9th place. (h) The 10th place is D. (i) The 11th place is S or G. (j) The 12th place is S or E. (k) K is in 13th place. (l) M is in 14th place. (m) 15th place is G or D. (n) The 16th place is S, T, or A. (o) G is in 18th place. (p) C is in 23rd place. (q) T is in 25th place. (r) The 28th place is R. (s) D is in 32nd place. (t) The 38th position is Q, K, R, N, or S. (u) Y is in 39th place. (v) The 46th position is N or T. (w) V is in 49th place. (x) The 53rd place is K. (y) The 54th place is N. (z) K is in 56th place. (aa) The 59th place is K or R. (bb) 63rd place is F, and / or (cc) A protein characterized by containing at least one mutation in the C-terminus, which is either the pentapeptide GLVPR or absent.

2. (1) Compared to the amino acid sequence shown in Sequence ID No. 1, the following mutations are present: T6 → V6 and / or N11 → S11 (2) Compared to the amino acid sequence shown in Sequence ID No. 1, it contains one or more mutations including T6→I6, N11→S11, R13→K13, I14→M14, E32→D32, P38→Q38 and Q59→K59. (3) Compared to the amino acid sequence shown in Sequence ID No. 1, the mutations include H4→G4 and / or T6→P6. (4) Compared to the amino acid sequence shown in Sequence ID No. 1, it contains one or more mutations including H4→Q4, E7→Q7, N11→S11, E32→D32, P38→Q38, R53→K53 and 59Q→59K. (5) Compared to the amino acid sequence shown in Sequence ID No. 1, it contains one or more mutations of N11→S11, R13→K13, E32→D32, P38→Q38 and Q59→K59. (6) Compared to the amino acid sequence shown in Sequence ID No. 1, it contains one or more mutations including H4→Q4, E7→Q7, N11→S11, A12→S12, R13→K13, E32→D32, P38→K38, R53→K53 and Q59→K59. (7) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, P16→S16, E32→D32, P38→K38, R53→K53, and Q59→K59. (8) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E15→G15, P38→Q38, I49→V49, R53→K53, and Q59→K59. (9) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E15→G15, E32→D32, P38→R38, I49→V49, R53→K53, and Q59→K59. (10) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (11) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, E7→Q7, E10→D10, N11→S11, E32→D32, P38→N38, R53→K53, and Q59→K59. (12) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, R56→K56, and Q59→K59. (13) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→I6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (14) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→N6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (15) Compared to the amino acid sequence shown in Sequence ID No. 1, it contains one or more mutations including H4→E4, T6→A6, E7→Q7, V9→M9, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53 and Q59→K59. (16) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E25→T25, E32→D32, P38→Q38, R53→K53, and Q59→K59. (17) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→P6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38 and R53→K53, Q59→K59. (18) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, E7→Q7, V9→M9, E10→D10, N11→S11, R13→K13, Y23→C23, E32→D32, P38→N38, R53→K53, and Q59→R59. (19) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→D5, T6→I6, E7→P7, P8→N8, N11→G11, A12→E12, R13→K13, P16→T16, K28→R28, E32→D32, P38→K38, K46→N46, R53→K53, R56→K56, and Q59→K59. (20) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, E7→Q7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (21) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→del, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (22) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→H5, E7→Q7, N11→S11, R13→K13, P16→A16, E32→D32, P38→N38, R53→K53, and Q59→K59. (23) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, E7→Q7, E10→D10, N11→S11, R13→K13, D18→G18, E32→D32, P38→N38, H39→Y39, R53→K53, and Q59→K59. (24) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, N5→G5, T6→E6, E7→P7, P8→H8, N11→G11, R13→K13, P16→T16, E32→D32, P38→K38, K46→T46, R53→K53, R56→K56, and Q59→K59. (25) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→E4, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (26) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→N6, E7→P7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (27) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→S5, T6→P6, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (28) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: T6→V6, N11→S11, R13→K13, E32→D32, P38→Q38, S54→N54, Q59→K59, and L63→F63. (29) Compared to the amino acid sequence shown in Sequence ID No. 1, it includes one or more mutations of H4→K4, N5→S5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53 and Q59→K59. (30) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→A5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53 and Q59→K59. (31) Compared to the amino acid sequence shown in Sequence ID No. 1, the following mutations are present: T6 → A6 and / or N11 → S11. (32) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (33) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→G5, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (34) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, E7→H7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (35) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→A6, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, R56→K56, and Q59→K59. (36) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→P6, E7→H7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (37) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→del, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (38) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more mutations are present, including N11→S11, E15→D15, E32→D32, P38→Q38 and Q59→K59. (39) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→H7, N11→S11, R13→K13, E15→D15, E32→D32, P38→K38, R53→K53, and Q59→K59. (40) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, E7→H7, N11→S11, R13→K13, E32→D32, P38→K38, R53→K53, and Q59→K59. (41) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→D5, E7→P7, P8→D8, N11→G11, A12→E12, R13→K13, E32→D32, P38→Q38, K46→N46, R53→K53, S54→N54, R56→K56 and Q59→K59. (42) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, N5→G5, T6→E6, E7→P7, P8→H8, N11→G11, R13→K13, P16→T16, E32→D32, P38→K38, K46→T46, R53→K53, R56→K56, and Q59→K59. (43) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, T6→P6, E7→H7, V9→M9, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (44) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→Q4, T6→I6, E7→P7, N11→S11, R13→K13, E32→D32, P38→Q38, R53→K53, R56→K56, and Q59→K59. (45) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, N5→T5, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→S38, R53→K53, and Q59→K59. (46) Compared to the amino acid sequence shown in Sequence ID No. 1, the sequence includes one or more mutations: H4→K4, E7→Q7, E10→D10, N11→S11, R13→K13, E32→D32, P38→N38, R53→K53, and Q59→K59. (47) Compared to the amino acid sequence shown in Sequence ID No. 1, one or more mutations including R53→K53 and / or Q59→K59, (48) Compared to the amino acid sequence shown in Sequence ID No. 1, an S is added to the N-terminus and includes the mutations R53→K53 and / or Q59→K59. (49) The protein according to claim 1, wherein, compared to the amino acid sequence shown in Sequence ID No. 1, S is added to the N-terminus, pentapeptide GLVPR is added to the C-terminus, and the mutations R53→K53 and / or Q59→K59 are included.

3. A sequence having two or more, three or more, four or more, or five or more and ten or fewer, eleven or fewer, twelve or fewer, thirteen or fewer, fourteen or fewer, or fifteen or fewer amino acid substitutions, deletions, or additions compared to any one of the amino acid sequences shown in SEQ ID NOs: 1 to 50, Preferably, the protein contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 50, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. Preferably, the protein is the amino acid sequence shown in any one of SEQ ID NOs: 2 to 50, according to claim 1 or 2.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein according to any one of claims 1 to 3.

5. An expression vector comprising an isolated nucleic acid molecule as described in claim 4.

6. A culture medium supplement comprising at least one or a combination thereof of the proteins described in any one of claims 1 to 3.

7. A culture medium comprising at least one protein or a combination thereof as described in any one of claims 1 to 3, or the supplement as described in claim 6.

8. The culture medium is a liquid culture medium, and / or In the culture medium, the working concentration of the protein is 0.02 to 1000 μg / mL, preferably 10 to 800 μg / mL, more preferably 20 to 600 μg / mL, and even more preferably 40 to 400 μg / mL. Preferably, the culture medium further comprises a basal culture medium, according to the culture medium supplement of claim 6 or the culture medium of claim 7.

9. A method for culturing cells or microorganisms, A step of providing cells or microorganisms for culture, The steps include contacting the cells or microorganisms with at least one or a combination thereof of proteins according to any one of claims 1 to 3, the culture medium supplement according to claim 6, or the culture medium according to claim 7, and collecting the culture, Preferably, the cells are stem cells, cell lines, or primary cells, and more preferably, the cell lines are genetically modified cell lines. A method for culturing cells or microorganisms, preferably characterized in that the microorganism is a genetically modified bacterium, and more preferably a yeast or Escherichia coli.

10. Use of the protein according to any one of claims 1 to 3, the culture medium supplement according to claim 6, or the culture medium according to claim 7 for culturing cells or microorganisms.

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