CN113354745A - Composition and method for large-scale production of fibroblast growth factor - Google Patents

Composition and method for large-scale production of fibroblast growth factor Download PDF

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CN113354745A
CN113354745A CN202110779452.4A CN202110779452A CN113354745A CN 113354745 A CN113354745 A CN 113354745A CN 202110779452 A CN202110779452 A CN 202110779452A CN 113354745 A CN113354745 A CN 113354745A
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growth factor
fibroblast growth
seq
human fibroblast
gly
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CN113354745B (en
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李校堃
苏志坚
林丽
李世军
冯治国
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Wenzhou Medical University
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Wenzhou Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Abstract

The invention relates to a composition and a method for producing fibroblast growth factor in a large scale. The fibroblast growth factor is fused with the green fluorescent protein and the small ubiquitin-like modifier to realize the soluble large-scale expression of the fibroblast growth factor, and then the small ubiquitin-like modifier protease expressed under the action of the cold shock promoter is promoted to carry out rapid hydrolysis on the fusion protein in cells by utilizing the synergistic effect of the tri (2-carbonyl ethyl) phosphate and the zinc sulfate, so that the production steps are simplified, and the fibroblast growth factor with biological activity is produced on a milligram scale to a kilogram scale.

Description

Composition and method for large-scale production of fibroblast growth factor
Technical Field
The invention relates to a composition for fermentation culture and purification of fibroblast growth factors, and a fusion protein containing the fibroblast growth factors, in particular to a method for large-scale culture and obtaining of the fibroblast growth factors.
Background
The Fibroblast Growth Factor (FGFs) superfamily consists of 22 members, mainly classified into secretory and intracellular types. The secretory fibroblast growth factor (eFGF) comprises 1-10 fibroblast growth factors and 16-23 fibroblast growth factors, and the Intracellular Fibroblast Growth Factor (iFGF) comprises 11-14 fibroblast growth factors. The eFGF must activate downstream signaling pathways by binding and activating Fibroblast Growth Factor Receptors (FGFRs) of cell surface tyrosine kinases (RTKs) family members, while the iFGF primarily interacts with other proteins to regulate cellular sodium channel switches. In humans, the fibroblast growth factor superfamily is expressed in almost all tissues and performs vital biological functions for embryonic development, organ formation and regeneration, tissue repair, and metabolism of the body.
The fibroblast growth factor superfamily of both human and mouse includes 22 members, whereas fibroblast growth factor-15 and fibroblast growth factor-19 are homologous genes in vertebrates, fibroblast growth factor-15 has not been found in the human genome, whereas fibroblast growth factor-19 is not found in large and small mice.
The genetic engineering technology is used to obtain a large amount of active fibroblast growth factors, which is a necessary condition for producing drugs related to the fibroblast growth factors. Compared with eukaryotic expression systems such as yeast, insect cells and mammalian cells, prokaryotic systems (especially Escherichia coli) are most convenient and economical to express recombinant proteins, and the time, production cost, site requirements and the like are required. However, the fibroblast is difficult to produce in large scale due to the problems of low expression level in a prokaryotic system, formation of inclusion bodies or toxicity to hosts and the like. In addition, although the expression level can be increased by means of molecular chaperone fusion, redundant amino acids occur, which results in increased difficulty of separation and purification processes and dramatically increased production cost. These factors all have serious effects on the pharmaceutical properties of fibroblast growth factor.
Disclosure of Invention
One aspect of the present invention provides a composition comprising tris (2-carbonylethyl) phosphate and zinc sulfate.
In one embodiment, the mass ratio of the tris (2-carbonylethyl) phosphonium hydrochloride to the zinc sulfate is 1 (1 to 50).
The second invention provides the use of the composition according to the first invention in the production of fibroblast growth factor, in particular in the large-scale production of fibroblast growth factor.
The invention also provides a fusion protein containing fibroblast growth factor, which comprises a visual reporter protein from the N end to the C end, a Small Ubiquitin-like Modifier (SUMO) and a fibroblast growth factor.
In a specific embodiment, the fibroblast growth factor is human fibroblast growth factor-1, human fibroblast growth factor-2, human fibroblast growth factor-3, human fibroblast growth factor-4, human fibroblast growth factor-5, human fibroblast growth factor-6, human fibroblast growth factor-7, human fibroblast growth factor-8, human fibroblast growth factor-9, human fibroblast growth factor-10, human fibroblast growth factor-11, human fibroblast growth factor-12, human fibroblast growth factor-13, human fibroblast growth factor-14, human fibroblast growth factor-16, human fibroblast growth factor-14, human fibroblast growth factor-6, human fibroblast growth factor-7, human fibroblast growth factor-8, human fibroblast growth factor-9, human fibroblast growth factor-10, human fibroblast growth factor-11, human fibroblast growth factor-12, human fibroblast growth factor-13, human fibroblast growth factor-14, human fibroblast growth factor-16, or human fibroblast growth factor-6, One of human fibroblast growth factor-17, human fibroblast growth factor-18, human fibroblast growth factor-19, human fibroblast growth factor-20, human fibroblast growth factor-21, human fibroblast growth factor-22 and human fibroblast growth factor-23.
In a specific embodiment, the amino acid sequence of the human fibroblast growth factor-1 is shown as SEQ ID No.1, the amino acid sequence of the human fibroblast growth factor-2 is shown as SEQ ID No.2, the amino acid sequence of the human fibroblast growth factor-3 is shown as SEQ ID No.3, the amino acid sequence of the human fibroblast growth factor-4 is shown as SEQ ID No.4, the amino acid sequence of the human fibroblast growth factor-5 is shown as SEQ ID No.5, the amino acid sequence of the human fibroblast growth factor-6 is shown as SEQ ID No.6, the amino acid sequence of the human fibroblast growth factor-7 is shown as SEQ ID No.7, and the amino acid sequence of the human fibroblast growth factor-8 is shown as SEQ ID No.8, the amino acid sequence of the human fibroblast growth factor-9 is shown as SEQ ID No.9, the amino acid sequence of the human fibroblast growth factor-10 is shown as SEQ ID No.10, the amino acid sequence of the human fibroblast growth factor-11 is shown as SEQ ID No.11, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.12, the amino acid sequence of the human fibroblast growth factor-13 is shown as SEQ ID No.13, the amino acid sequence of the human fibroblast growth factor-14 is shown as SEQ ID No.14, the amino acid sequence of the human fibroblast growth factor-16 is shown as SEQ ID No.15, the amino acid sequence of the human fibroblast growth factor-17 is shown as SEQ ID No.16, the amino acid sequence of the human fibroblast growth factor-18 is shown as SEQ ID No.17, the amino acid sequence of the human fibroblast growth factor-19 is shown as SEQ ID No.18, the amino acid sequence of the human fibroblast growth factor-20 is shown as SEQ ID No.19, the amino acid sequence of the human fibroblast growth factor-21 is shown as SEQ ID No.20, the amino acid sequence of the human fibroblast growth factor-22 is shown as SEQ ID No.21, and the amino acid sequence of the human fibroblast growth factor-23 is shown as SEQ ID No. 22.
In a specific embodiment, the visual reporter protein is a fluorescent protein.
In a specific embodiment, the visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein and cyan fluorescent protein; preferably, the amino acid sequence of the green fluorescent protein is shown as SEQ ID No. 23.
In a specific embodiment, the amino acid sequence of the small ubiquitin-like modifier is shown in SEQ ID No. 24.
In a specific embodiment, the visual reporter protein and the small ubiquitin-like modifier are linked by a polypeptide linker.
In one embodiment, the amino acid sequence of the polypeptide linker is shown in SEQ ID No. 25.
The fourth aspect of the present invention provides an expression cassette for expressing the fusion protein according to any one of the third aspect of the present invention, comprising a first promoter from 5 'end to 3' end, a nucleic acid encoding the fusion protein, and a first terminator. Wherein, the downstream of the first promoter and the upstream of the nucleic acid for encoding the fusion protein also comprise RBS, and the nucleotide sequence of the RBS is shown as SEQ ID No. 53. An enzyme cleavage site may also be included downstream of the first promoter and upstream of the nucleic acid encoding the fusion protein. Wherein the enzyme cleavage site is located downstream of the RBS.
In a specific embodiment, the first promoter is the T7 promoter and the first terminator is the T7 terminator.
In a specific embodiment, the nucleotide sequence encoding the human fibroblast growth factor-1 is shown as SEQ ID No.26, the nucleotide sequence encoding the human fibroblast growth factor-2 is shown as SEQ ID No.27, the nucleotide sequence encoding the human fibroblast growth factor-3 is shown as SEQ ID No.28, the nucleotide sequence encoding the human fibroblast growth factor-4 is shown as SEQ ID No.29, the nucleotide sequence encoding the human fibroblast growth factor-5 is shown as SEQ ID No.30, the nucleotide sequence encoding the human fibroblast growth factor-6 is shown as SEQ ID No.31, the nucleotide sequence encoding the human fibroblast growth factor-7 is shown as SEQ ID No.32, and the nucleotide sequence encoding the human fibroblast growth factor-8 is shown as SEQ ID No.33, the nucleotide sequence for coding the human fibroblast growth factor-9 is shown as SEQ ID No.34, the nucleotide sequence for coding the human fibroblast growth factor-10 is shown as SEQ ID No.35, the nucleotide sequence for coding the human fibroblast growth factor-11 is shown as SEQ ID No.36, the nucleotide sequence for coding the human fibroblast growth factor-12 is shown as SEQ ID No.37, the nucleotide sequence for coding the human fibroblast growth factor-13 is shown as SEQ ID No.38, the nucleotide sequence for coding the human fibroblast growth factor-14 is shown as SEQ ID No.39, the nucleotide sequence for coding the human fibroblast growth factor-16 is shown as SEQ ID No.40, and the nucleotide sequence for coding the human fibroblast growth factor-17 is shown as SEQ ID No.41, the nucleotide sequence for coding the human fibroblast growth factor-18 is shown as SEQ ID No.42, the nucleotide sequence for coding the human fibroblast growth factor-19 is shown as SEQ ID No.43, the nucleotide sequence for coding the human fibroblast growth factor-20 is shown as SEQ ID No.44, the nucleotide sequence for coding the human fibroblast growth factor-21 is shown as SEQ ID No.45, the nucleotide sequence for coding the human fibroblast growth factor-22 is shown as SEQ ID No.46, and the nucleotide sequence for coding the human fibroblast growth factor-23 is shown as SEQ ID No. 47.
In a specific embodiment, the nucleotide sequence encoding the visual reporter protein is shown in SEQ ID No. 48.
In a specific embodiment, the nucleotide sequence encoding the small ubiquitin-like modifier is shown in SEQ ID No. 49.
In one embodiment, the nucleotide sequence encoding the polypeptide linker is as set forth in SEQ ID No. 50.
The fifth aspect of the present invention provides a set of expression cassettes for expressing the fusion protein according to any one of the third aspect of the present invention or for expressing the fusion protein according to any one of the third aspect of the present invention and purifying the fibroblast growth factor, comprising the expression cassette according to the fourth aspect of the present invention and an expression cassette for expressing a small ubiquitin-like modifier protease (SUMO protease, e.g., Ulp 1).
In a specific embodiment, the expression cassette for expressing a small ubiquitin-like modifier protease comprises a second promoter from the 5 'end to the 3' end, a 3 '-UTR, a nucleic acid encoding a small ubiquitin-like modifier protease, and a 5' -UTR.
In a specific embodiment, the second promoter is a CspA promoter, the 3 '-UTR is a CspA 3' -UTR, and the 5 '-UTR is a CspA 5' -UTR.
In a specific embodiment, the amino acid sequence of the small ubiquitin-like modifier protease is shown in SEQ ID No. 51.
In one embodiment, the nucleotide sequence encoding the small ubiquitin-like modifier protease is shown in SEQ ID No. 52.
The sixth aspect of the present invention provides a method for obtaining the fusion protein according to any one of the third aspect of the present invention or the fibroblast growth factor therein, comprising the steps of:
1-1) ligating an expression cassette according to the fourth of the present invention or a nucleic acid encoding said fusion protein to a first expression vector to obtain a first recombinant expression vector; wherein the first expression vector is an isopropyl-beta-D-thiogalactoside (IPTG) inducible expression vector;
2-1) connecting the expression cassette for expressing the small ubiquitin-like modifier protease or the nucleic acid for encoding the small ubiquitin-like modifier protease in the expression cassette of the fifth invention to the first recombinant expression vector to obtain a second recombinant expression vector;
3-1) transforming the second recombinant expression vector into a first host to obtain a first recombinant organism;
4-1) culturing the first recombinant organism in a first culture solution until the fusion protein and the small ubiquitin-like modifier protease are expressed to obtain a first culture;
5-1) purifying said first culture to obtain said fibroblast growth factor;
or
1-2) same as 1-1);
2-2) connecting the expression cassette for expressing the small ubiquitin-like modifier protease or the nucleic acid for encoding the small ubiquitin-like modifier protease in the expression cassette of the fifth invention to the second expression vector to obtain a third recombinant expression vector;
3-2) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism; transforming the third recombinant expression vector into a third host to obtain a third recombinant organism;
4-2) culturing the second recombinant organism in a second culture fluid until the fusion protein is expressed to obtain a second culture; culturing the third recombinant organism in a third culture solution until the small ubiquitin-like modifier protease is expressed to obtain a third culture; mixing the second culture with the third culture to obtain a fourth culture;
5-2) purifying said fourth culture, thereby obtaining said fibroblast growth factor;
or
1-3) same as 1-1);
2-3) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism as in 3-2);
3-3) culturing the second recombinant organism in a second culture solution until the fusion protein is expressed to obtain a second culture;
4-3) purifying the second culture, thereby obtaining the fusion protein.
In a specific embodiment, the first culture liquid, the second culture liquid and the third culture liquid are independently LB culture liquid or LB culture liquid containing glucose.
In one embodiment, the first expression vector may be one of the pET series of expression vectors.
In a specific embodiment, the first expression vector is one of pET21a, pET20b, and pET28 a;
the second expression vector is pCold, which can be purchased from Dalibao bioengineering GmbH (Cathaki No: 3360);
the first host, the second host, and the third host are independently Escherichia coli (Escherichia coli); preferably, the strain of E.coli is BL21(DE 3).
In a specific embodiment, in step 4-2) or step 3-3), the second recombinant organism is cultured at a temperature of 28 to 37 ℃ for a time of 4 to 8 hours;
in step 4-1), culturing the first recombinant organism at a temperature of 28 to 37 ℃ for 4 to 8 hours under induction of isopropyl- β -D-thiogalactoside (IPTG), and then continuing culturing the first recombinant organism at a temperature of 10 to 15 ℃ for 15 to 60 minutes;
in step 4-2), the third recombinant organism is cultured at a temperature of 10 to 15 ℃ for 15 to 60 minutes.
In a specific embodiment, in step 4-2) or step 3-3), the second recombinant organism is cultured at a temperature of 32 to 37 ℃ for a time of 4 to 6 hours;
in step 4-1), the first recombinant organism is cultured at a temperature of 32 to 37 ℃ for 4 to 6 hours under induction with IPTG, and then the first recombinant organism is cultured for a further 15 to 30 minutes at a temperature of 10 to 15 ℃;
in step 4-2), the third recombinant organism is cultured at a temperature of 10 to 15 ℃ for 15 to 30 minutes.
In a particular embodiment, in step 4-1), the composition according to one of the invention is added to said first culture broth when the culture temperature is lowered to said temperature of between 10 and 15 ℃.
In one embodiment, the final concentration of the tris (2-carbonylethyl) phosphonium hydrochloride in the first culture liquid is from 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the first broth is 1 to 5 mmol/l.
In a specific embodiment, in step 4-2), a composition according to one of the present invention is added to the second culture broth.
In one embodiment, the final concentration of the tris (2-carbonylethyl) phosphonium hydrochloride in the second culture liquid is from 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the second broth is 1 to 5 mmol/l.
In addition, in one embodiment, the activities of purified fibroblast growth factor-1, fibroblast growth factor-2, fibroblast growth factor-3, fibroblast growth factor-4, fibroblast growth factor-5, fibroblast growth factor-6, fibroblast growth factor-7, fibroblast growth factor-10, fibroblast growth factor-11, fibroblast growth factor-12, fibroblast growth factor-13, fibroblast growth factor-14, fibroblast growth factor-16, fibroblast growth factor-18, fibroblast growth factor-19, fibroblast growth factor-20, fibroblast growth factor-21, fibroblast growth factor-22, and fibroblast growth factor-23 are performed The corresponding analysis shows that they all show the respective due activity.
The invention has the beneficial effects that:
aiming at the problems encountered by prokaryotic expression in the prior art and aiming at further improving the quality control precision in the expression process of fibroblast growth factors, the invention provides a composition and designs a recombinant escherichia coli expression plasmid which is matched with the composition and contains two gene expression cassettes with different induction expression modes, wherein one expression cassette is used for expressing fusion protein containing the fibroblast growth factors, the fusion of the fibroblast growth factors with fluorescent protein and SUMO is favorable for the mass expression of soluble fusion protein, and the expression of the protein can be visually monitored by utilizing green fluorescence generated by green fluorescent protein, for example; the other is used for expressing small ubiquitin-like modifier proteases. After the induction of the fusion protein is finished, the compound combination is added in the low-temperature culture process, so that the hydrolysis effect of the small ubiquitin-like modifier protease on the fusion protein can be realized while the small ubiquitin-like modifier protease is induced and expressed, and the aim of large-scale production of the soluble fibroblast growth factor which has the corresponding biological function and is completely consistent with the amino acid sequence of the natural fibroblast growth factor is fulfilled.
The invention has the advantages that: 1) by visual green fluorescence, the soluble expression and expression quantity of the recombinant fusion protein can be evaluated or monitored in real time, and in order to achieve the purpose, the adopted conventional method at least needs more than 6 hours; 2) unlike other protease recognition sites, SUMO proteases recognize the three-dimensional structure of SUMO and, therefore, there is no case of indiscriminate cleavage (hydrolysis); in addition, when the SUMO protease is used for hydrolysis, any redundant amino acid is not brought into the target protein sequence, so that the fibroblast growth factor consistent with the natural protein sequence can be obtained; 3) the specific mode of the fusion protein of the invention is utilized to realize the soluble expression of fibroblast growth factor which is difficult to express (difficult-to-express) in an escherichia coli host, and the fusion protein is rapidly hydrolyzed while the intracellular SUMO protease expression is realized by the synergistic interaction between the compositions of the invention for the first time, thereby greatly simplifying the production and purification processes of the fibroblast growth factor. The present invention can shorten the purification time by at least 8 to 48 hours relative to the time required by the conventional method; 4) the process of the invention can realize the production of the fibroblast growth factor from milligram level to kilogram level by only enlarging the production scale, such as fermentation scale, thallus crushing and scale of a centrifugal apparatus, gel chromatography scale and the like, without complex operation process and special apparatus.
Drawings
FIG. 1 is a schematic drawing of a map of a pET-GSFGF9-CspA-Ulp1 recombinant plasmid
FIG. 2 fluorescent observation of pET-GSFGF17-CspA-Ulp1/BL21 after induced expression
A. Observation results of the fermentation liquid under natural light, wherein the fermentation liquid in the left test tube (after pET-GSFGF17-CspA-Ulp1/BL21 IPTG induced expression) shows green fluorescence, and the fermentation liquid in the right test tube (before pET-GSFGF17-CspA-Ulp1/BL21 IPTG induced expression) does not show green fluorescence; and B, observing the fermentation liquor subjected to induced expression of pET-GSFGF17-CspA-Ulp1/BL21 IPTG under ultraviolet light.
FIG. 3 analysis of expression of fusion protein GFP-SUMO-FGFn
A. Lane 1: IPTG induced pET-GSFGF8-CspA-Ulp1/BL21 total protein; lane 2: IPTG induced pET-GSFGF9-CspA-Ulp1/BL21 total protein; lane 3: IPTG induced pET-GFP-SUMO/BL21 total protein; lane M: protein molecular weight markers; lane 4: pET-GSFGF8-CspA-Ulp1/BL21 total protein before IPTG induction; lane 5: IPTG induced pET-GSFGF3-CspA-Ulp1/BL21 total protein; lane 6: IPTG induced pET-GSFGF16-CspA-Ulp1/BL21 total protein; lane 7: IPTG induced pET-GSFGF17-CspA-Ulp1/BL21 total protein; lane 8: IPTG induced pET-GSFGF19-CspA-Ulp1/BL21 total protein; lane 9: IPTG induced pET-GSFGF20-CspA-Ulp1/BL21 total protein;
B. lane 1: IPTG induced pET-GSFGF4-CspA-Ulp1/BL21 total protein; lane 2: IPTG induced pET-GSFGF6-CspA-Ulp1/BL21 total protein; lane 3: IPTG induced pET-GSFGF7-CspA-Ulp1/BL21 total protein; lane 4: IPTG induced pET-GFP-SUMO/BL21 total protein; lane M: protein molecular weight markers; lane 5: pET-GSFGF4-CspA-Ulp1/BL21 total protein before IPTG induction; lane 6: IPTG induced pET-GSFGF10-CspA-Ulp1/BL21 total protein; lane 7: IPTG induced pET-GSFGF18-CspA-Ulp1/BL21 total protein; lane 8: IPTG induced pET-GSFGF21-CspA-Ulp1/BL21 total protein; lane 9: IPTG induced pET-GSFGF22-CspA-Ulp1/BL21 total protein;
C. lane 1: IPTG induced pET-GSFGF11-CspA-Ulp1/BL21 total protein; lane 2: IPTG induced pET-GSFGF23-CspA-Ulp1/BL21 total protein; lane 3: IPTG induced pET-GSFGF5-CspA-Ulp1/BL21 total protein; lane 4: IPTG induced pET-GFP-SUMO/BL21 total protein; lane M: protein molecular weight markers; lane 5: pET-GSFGF11-CspA-Ulp1/BL21 total protein before IPTG induction; lane 6: IPTG induced pET-GSFGF12-CspA-Ulp1/BL21 total protein; lane 7: IPTG induced pET-GSFGF13-CspA-Ulp1/BL21 total protein; lane 8: and (3) IPTG-induced pET-GSFGF14-CspA-Ulp1/BL21 total protein.
FIG. 4 solubility analysis of fusion protein GFP-SUMO-FGFn after IPTG inducible expression
A: lane 1: pET-GSFGF8-CspA-Ulp1/BL21 total protein; lane 2: the protein in the supernatant of pET-GSFGF8-CspA-Ulp1/BL 21; lane 3: the protein in the pET-GSFGF8-CspA-Ulp1/BL21 precipitate; lane M: protein molecular weight markers; lane 4: pET-GSFGF5-CspA-Ulp1/BL21 total protein; lane 5: the protein in the supernatant of pET-GSFGF5-CspA-Ulp1/BL 21; lane 6: the protein in the pET-GSFGF5-CspA-Ulp1/BL21 precipitate; lane 7: pET-GSFGF23-CspA-Ulp1/BL21 total protein; lane 8: the protein in the supernatant of pET-GSFGF23-CspA-Ulp1/BL 21; lane 9: the protein in the pET-GSFGF23-CspA-Ulp1/BL21 precipitate;
b: lane 1: pET-GSFGF4-CspA-Ulp1/BL21 total protein; lane 2: the protein in the supernatant of pET-GSFGF4-CspA-Ulp1/BL 21; lane 3: the protein in the pET-GSFGF4-CspA-Ulp1/BL21 precipitate; lane M: protein molecular weight markers; lane 4: pET-GSFGF16-CspA-Ulp1/BL21 total protein; lane 5: the protein in the supernatant of pET-GSFGF16-CspA-Ulp1/BL 21; lane 6: the protein in the pET-GSFGF16-CspA-Ulp1/BL21 precipitate; lane 7: pET-GSFGF14-CspA-Ulp1/BL21 total protein samples; lane 8: the protein in the supernatant of pET-GSFGF14-CspA-Ulp1/BL 21; lane 9: the protein in the pET-GSFGF14-CspA-Ulp1/BL21 precipitate;
c: lane 1: pET-GSFGF11-CspA-Ulp1/BL21 total protein; lane 2: the protein in the supernatant of pET-GSFGF11-CspA-Ulp1/BL 21; lane 3: the protein in the pET-GSFGF11-CspA-Ulp1/BL21 precipitate; lane 4: pET-GSFGF12-CspA-Ulp1/BL21 total protein; lane 5: the protein in the supernatant of pET-GSFGF12-CspA-Ulp1/BL 21; lane 6: the protein in the pET-GSFGF12-CspA-Ulp1/BL21 precipitate; lane M: protein molecular weight markers;
d: lane 1: pET-GSFGF19-CspA-Ulp1/BL21 total protein; lane 2: the protein in the supernatant of pET-GSFGF19-CspA-Ulp1/BL 21; lane 3: the protein in the pET-GSFGF19-CspA-Ulp1/BL21 precipitate; lane M: protein molecular weight markers; lane 4: pET-GSFGF17-CspA-Ulp1/BL21 total protein; lane 5: the protein in the supernatant of pET-GSFGF17-CspA-Ulp1/BL 21; lane 6: pET-GSFGF17-CspA-Ulp1/BL 21.
FIG. 5 functional analysis of tri (2-carbonylethyl) phosphate, zinc sulfate and combination on small ubiquitin-like modifier proteolytic fusion proteins
Black arrows indicate the fusion protein GFP-SUMO-FGF 12; the gray arrows indicate GFP-SUMO after hydrolysis of the fusion protein GFP-SUMO-FGF 12; the hollow arrow indicates the fibroblast growth factor-12 after hydrolysis of the fusion protein GFP-SUMO-FGF 12.
A: shake flask 1 and shake flask I, influence of the addition of zinc sulfate at a final concentration of 5 mmol/l at the initiation of the Low temperature Induction at 15 ℃ on the hydrolyzed fusion protein of Small ubiquitin-like modifier protease
Lane 1: low-temperature induction is carried out on pET-GSFGF12-CspA-Ulp1/BL21 for 30 minutes; lane 2: low-temperature induction is carried out on pET-GSFGF12-CspA-Ulp1/BL21 for 60 minutes; lane 3: low-temperature induction is carried out on pET-GSFGF12-CspA-Ulp1/BL21 for 120 minutes; lane 4: pET-GFP-SUMO/BL21 was induced at low temperature for 120 min;
b: influence of the addition of tris (2-carbonylethyl) phosphate to the hydrolyzed fusion protein of small ubiquitin-like modifier protease at a final concentration of 1 mmol/l at the initiation of 2, 15 ℃ low temperature induction in shake flask
Lane 1: low-temperature induction for 0 minute; lane 2: low-temperature induction for 15 minutes; lane 3: low-temperature induction for 30 minutes; lane 4: low-temperature induction for 60 minutes; lane 5: low-temperature induction for 90 minutes; lane 6: inducing at low temperature for 120 minutes;
c: effect of the addition of a combination of tris (2-carbonylethyl) phosphate and zinc sulfate at a final concentration of 1 mmol/l on the hydrolysis of fusion proteins by Small ubiquitin-like modifier proteases at the initiation of Low temperature Induction at 3, 15 ℃ in Shake flasks
Lane 1: low-temperature induction for 0 minute; lane 2: low-temperature induction for 15 minutes; lane 3: low-temperature induction for 30 minutes; lane 4: low-temperature induction for 60 minutes; lane 5: low-temperature induction for 90 minutes; lane 6: inducing at low temperature for 120 minutes;
d: effect of the addition of a combination of tris (2-carbonylethyl) phosphate and zinc sulfate at a final concentration of 1 mmol/l and 3 mmol/l on the hydrolysis of fusion proteins by Small ubiquitin-like modifier proteases at the initiation of Low temperature Induction at 4, 15 ℃ in Shake flasks
Lane 1: low-temperature induction for 0 minute; lane 2: low-temperature induction for 15 minutes; lane 3: low-temperature induction for 30 minutes; lane 4: low-temperature induction for 60 minutes; lane 5: low-temperature induction for 90 minutes; lane 6: inducing at low temperature for 120 minutes;
e: effect of the combination of Tri (2-carbonylethyl) phosphate and Zinc sulfate added to a final concentration of 0.1 mmol/L at the initiation of the Low temperature Induction at 5, 15 ℃ in Shake flask on Small ubiquitin-like modifier proteolytic fusion proteins
Lane 1: low-temperature induction for 0 minute; lane 2: low-temperature induction for 15 minutes; lane 3: low-temperature induction for 30 minutes; lane 4: low-temperature induction for 60 minutes; lane 5: low-temperature induction for 90 minutes; lane 6: low temperature induction for 120 min.
FIG. 6 combination of tris (2-carbonylethyl) phosphate and zinc sulfate to promote proteolysis of small ubiquitin-like modifier proteases by other fusion proteins GFP-SUMO-FGFn
And adding tris (2-carbonyl ethyl) phosphate and 5 mmol/L zinc sulfate at a final concentration of 0.1 mmol/L in the low-temperature induction process at 15 ℃, and observing the hydrolysis capacity of the small ubiquitin-like modifier protease on GFP-SUMO-FGFn fusion protein.
Lane M: protein molecular weight markers; lane 1: low-temperature induction is carried out on pET-GSFGF9-CspA-Ulp1/BL21 for 0 minute; lane 2: low-temperature induction is carried out on pET-GSFGF9-CspA-Ulp1/BL21 for 30 minutes; lane 3: low-temperature induction is carried out on pET-GSFGF8-CspA-Ulp1/BL21 for 30 minutes; lane 4: low-temperature induction is carried out on pET-GSFGF16-CspA-Ulp1/BL21 for 30 minutes; lane 5: low-temperature induction is carried out on pET-GSFGF17-CspA-Ulp1/BL21 for 30 minutes; lane 6: pET-GSFGF19-CspA-Ulp1/BL21 was induced at low temperature for 30 min.
FIG. 7 hybridization analysis of fusion protein GFP-SUMO-FGFn before and after hydrolysis
Solid arrows indicate the fusion protein GFP-SUMO-FGFn; the open arrow indicates fibroblast growth factor-n.
A: lane M: protein molecular weight markers; lane 1: before induction by pET-GSFGF9-CspA-Ulp1/BL21 IPTG; lane 2: low-temperature induction is carried out on pET-GSFGF9-CspA-Ulp1/BL21 for 30 minutes; lane 3: pET-GSFGF9-CspA-Ulp1/BL21 low temperature induction is 0 min;
b: lane M: protein molecular weight markers; lane 1: low-temperature induction is carried out on pET-GSFGF18-CspA-Ulp1/BL21 for 30 minutes; lane 2: low-temperature induction is carried out on pET-GSFGF18-CspA-Ulp1/BL21 for 5 minutes; lane 3: low-temperature induction of pET-GSFGF18-CspA-Ulp1/BL21 was initiated at 0 min.
FIG. 8 analysis of the purification Process of fibroblast growth factor-17 (FGF-17)
A: CM ion chromatography gel separation of FGF-17, lane 1: buffer 1 containing 0.1M sodium chloride; lane 2: buffer 2 containing 0.3M sodium chloride: lanes 3 and 4: buffer 3 containing 0.6M sodium chloride; lane 4: buffer 6 containing 1.0 mol/l sodium chloride; lane M: protein molecular weight markers;
b: heparin affinity chromatography gel purified FGF-17, in lanes 1 and 2: buffer 4 containing 1.2 mol/l sodium chloride.
FIG. 9 Activity assay of fibroblast growth factor to promote proliferation of leydig cells
Detailed Description
The above-described aspects of the invention are explained in more detail below by means of preferred embodiments, but they are not intended to limit the invention.
The reagents, enzymes and the like in the examples of the present invention are commercially available unless otherwise specified.
Escherichia coli pET-21a expression vector (cat # 69740) and Escherichia coli BL21(DE3) (cat # 69450) were purchased from Sigma-Aldrich.
pCold (cat. No. 3360) was purchased from Takara bioengineering (Dalian) Inc.
Heparin affinity chromatography gel (cat # 17-0998-03) and ion chromatography gel (CM Sepharose Fast Flow, cat # 17-0719-01; Q Sepharose Fast Flow, cat # 17-0510-01) used for purification of fusion proteins were purchased from GE, USA.
The amino acid sequence of the human fibroblast growth factor-1 is shown as SEQ ID No.1, the amino acid sequence of the human fibroblast growth factor-2 is shown as SEQ ID No.2, the amino acid sequence of the human fibroblast growth factor-3 is shown as SEQ ID No.3, the amino acid sequence of the human fibroblast growth factor-4 is shown as SEQ ID No.4, the amino acid sequence of the human fibroblast growth factor-5 is shown as SEQ ID No.5, the amino acid sequence of the human fibroblast growth factor-6 is shown as SEQ ID No.6, the amino acid sequence of the human fibroblast growth factor-7 is shown as SEQ ID No.7, the amino acid sequence of the human fibroblast growth factor-8 is shown as SEQ ID No.8, the amino acid sequence of the human fibroblast growth factor-9 is shown as SEQ ID No.9, the amino acid sequence of the human fibroblast growth factor-10 is shown as SEQ ID No.10, the amino acid sequence of the human fibroblast growth factor-11 is shown as SEQ ID No.11, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.12, the amino acid sequence of the human fibroblast growth factor-13 is shown as SEQ ID No.13, the amino acid sequence of the human fibroblast growth factor-14 is shown as SEQ ID No.14, the amino acid sequence of the human fibroblast growth factor-16 is shown as SEQ ID No.15, the amino acid sequence of the human fibroblast growth factor-17 is shown as SEQ ID No.16, the amino acid sequence of the human fibroblast growth factor-18 is shown as SEQ ID No.17, the amino acid sequence of the human fibroblast growth factor-19 is shown as SEQ ID No.18, the amino acid sequence of the human fibroblast growth factor-20 is shown as SEQ ID No.19, the amino acid sequence of the human fibroblast growth factor-21 is shown as SEQ ID No.20, the amino acid sequence of the human fibroblast growth factor-22 is shown as SEQ ID No.21, and the amino acid sequence of the human fibroblast growth factor-23 is shown as SEQ ID No. 22.
The amino acid sequence of the green fluorescent protein is shown as SEQ ID No. 23; the amino acid sequence of the small ubiquitin-like modifier is shown in SEQ ID No. 24; the amino acid sequence of the polypeptide joint between the green fluorescent protein and the small ubiquitin-like modifier is shown in SEQ ID No. 25.
The nucleotide sequence for coding the human fibroblast growth factor-1 is shown as SEQ ID No.26, the nucleotide sequence for coding the human fibroblast growth factor-2 is shown as SEQ ID No.27, the nucleotide sequence for coding the human fibroblast growth factor-3 is shown as SEQ ID No.28, the nucleotide sequence for coding the human fibroblast growth factor-4 is shown as SEQ ID No.29, the nucleotide sequence for coding the human fibroblast growth factor-5 is shown as SEQ ID No.30, the nucleotide sequence for coding the human fibroblast growth factor-6 is shown as SEQ ID No.31, the nucleotide sequence for coding the human fibroblast growth factor-7 is shown as SEQ ID No.32, the nucleotide sequence for coding the human fibroblast growth factor-8 is shown as SEQ ID No.33, the nucleotide sequence for coding the human fibroblast growth factor-9 is shown as SEQ ID No.34, the nucleotide sequence for coding the human fibroblast growth factor-10 is shown as SEQ ID No.35, the nucleotide sequence for coding the human fibroblast growth factor-11 is shown as SEQ ID No.36, the nucleotide sequence for coding the human fibroblast growth factor-12 is shown as SEQ ID No.37, the nucleotide sequence for coding the human fibroblast growth factor-13 is shown as SEQ ID No.38, the nucleotide sequence for coding the human fibroblast growth factor-14 is shown as SEQ ID No.39, the nucleotide sequence for coding the human fibroblast growth factor-16 is shown as SEQ ID No.40, the nucleotide sequence for coding the human fibroblast growth factor-17 is shown as SEQ ID No.41, the nucleotide sequence for coding the human fibroblast growth factor-18 is shown as SEQ ID No.42, the nucleotide sequence for coding the human fibroblast growth factor-19 is shown as SEQ ID No.43, the nucleotide sequence for coding the human fibroblast growth factor-20 is shown as SEQ ID No.44, the nucleotide sequence for coding the human fibroblast growth factor-21 is shown as SEQ ID No.45, the nucleotide sequence for coding the human fibroblast growth factor-22 is shown as SEQ ID No.46, and the nucleotide sequence for coding the human fibroblast growth factor-23 is shown as SEQ ID No. 47.
The nucleotide sequence of the coded green fluorescent protein is shown as SEQ ID No. 48; the nucleotide sequence of the coded small ubiquitin-like modifier is shown in SEQ ID No. 49; the nucleotide sequence of the polypeptide joint between the coded green fluorescent protein and the small ubiquitin-like modifier is shown in SEQ ID No. 50.
The amino acid sequence of the small ubiquitin-like modifier protease is shown in SEQ ID No. 51; the nucleotide sequence of the gene for coding the small ubiquitin-like modifier protease is shown as SEQ ID No. 52. The CspA promoter, CspA 3 '-UTR and CspA 5' -UTR are located on the pCold vector.
Example 1 construction of prokaryotic expression vector of visualized fibroblast growth factor fusion protein and acquisition of recombinant Strain
Designing a fusion protein GFP-SUMO-FGFn (wherein N is 1 to 23, and N is not 15, and is consistent with N in human fibroblast growth factor-N), sequentially from N end to C end, a green fluorescent protein, a polypeptide linker, a small ubiquitin-like modifier and a human fibroblast growth factor-N (wherein N is 1 to 23, and N is not 15), wherein the amino acid sequences of all parts are as described above; the nucleic acid for coding the fusion protein comprises green fluorescent protein, a polypeptide linker, a small ubiquitin-like modifier and a human fibroblast growth factor-n (wherein n is 1 to 23, and n is not 15) from 5 'end to 3' end in sequence, and the nucleotide sequences of all parts are as described above.
The nucleic acid for coding the fusion protein GFP-SUMO-FGFn is synthesized by Nanjing Kingsrie Biotechnology GmbH, and then the synthesized nucleic acid fragments are respectively cloned into an escherichia coli expression plasmid vector pET-21a by using a genetic engineering method to obtain recombinant plasmids and transform the escherichia coli. The correctness of the nucleic acid sequence is detected by nucleic acid sequencing. The recombinant plasmid containing the nucleic acid encoding the fusion protein GFP-SUMO-FGFn was designated pET-GFP-SUMO-hFGFn (n is 1 to 23, respectively, and n is not 15).
The recombinant plasmid pET-GFP-SUMO-hFGFn is transformed into an Escherichia coli host BL21(DE3), and positive recombinant bacteria are screened out by ampicillin resistance and are respectively named as pET-GFP-SUMO-hFGFn/BL 21.
Example 2 construction of prokaryotic expression vector of Small ubiquitin-like modifier protease Ulp1 and acquisition of recombinant Strain
The amino acid sequence of the small ubiquitin-like modifier protease Ulp1 and the nucleotide sequence of the nucleic acid encoding it. The nucleic acid for coding the small ubiquitin-like modifier protease Ulp1 is synthesized by Nanjing Kinsrui Biotechnology GmbH, and then the synthesized nucleic acid fragment is cloned into an escherichia coli expression plasmid vector pCold by using a genetic engineering method to obtain a recombinant plasmid, and escherichia coli is transformed. The correctness of the nucleic acid sequence was checked by nucleic acid sequencing, and the correct recombinant plasmid was named pCold-Ulp 1.
The pCold-Ulp1 plasmid is subjected to single enzyme digestion by Pfo I to obtain a DNA fragment containing a CspA promoter, a 3 '-UTR, a nucleic acid for coding a small ubiquitin-like modifier protease and a small ubiquitin-like modifier protease expression cassette of a 5' -UTR, and then the DNA fragment of the expression cassette is subjected to enzyme digestion site end filling by T4 polymerase to form a blunt end. pET-GFP-SUMO-hFGFn is singly cut by using a blunt-end restriction endonuclease EcoRI to obtain a carrier framework, and the carrier framework is connected with a small ubiquitin-like modifier protease expression cassette and is transformed into escherichia coli. The correctness of the nucleic acid sequence was checked by nucleic acid sequencing, and the correct recombinant plasmid was named pET-GSFGFn-CspA-Ulp1(n ═ 1,2,3,4,5,6,7,8,9,10,11,12,13,14,16,17,18,19,20,21,22, 23). The plasmid map shown in FIG. 1 is shown by taking pET-GSFGF9-CspA-Ulp1 as an example.
The recombinant pET-GSFGFn-CspA-Ulp1 was transformed into E.coli host BL21(DE3), and ampicillin resistance was used to select a positive recombinant bacterium, which was named pET-GSFGFn-CspA-Ulp1/BL 21.
Example 3 construction of prokaryotic expression vector of fusion protein GFP-SUMO and acquisition of recombinant Strain
Designing the fusion protein GFP-SUMO to be sequentially a green fluorescent protein, a polypeptide joint and a small ubiquitin-like modifier from the N end to the C end, wherein the amino acid sequences of all parts are as described above; the nucleic acid for coding the fusion protein is provided with green fluorescent protein, polypeptide linker and small ubiquitin-like modifier in sequence from 5 'end to 3' end, and the nucleotide sequence of each part is as described above.
The nucleic acid for coding the fusion protein GFP-SUMO is synthesized by Nanjing Kingsrey Biotechnology, Inc., and then the synthesized nucleic acid fragment is cloned into an escherichia coli expression plasmid vector pET-21a by using a genetic engineering method to obtain a recombinant plasmid, and escherichia coli is transformed. The correctness of the nucleic acid sequence is detected by nucleic acid sequencing. The recombinant plasmid containing the nucleic acid encoding GFP-SUMO was designated pET-GFP-SUMO.
The recombinant plasmid pET-GFP-SUMO is transformed into an escherichia coli host BL21(DE3), ampicillin resistance is used for screening out positive recombinant bacteria, the positive recombinant bacteria are respectively named as pET-GFP-SUMO/BL21, and the recombinant strain is used as a control strain of pET-GSFGFn-CspA-Ulp1/BL21 expression protein.
Example 4 inducible expression of recombinant strains
pET-GSFGFn-CspA-Ulp1/BL21 was inoculated in an inoculum size of 0.1% (v/v) into 50 ml of fresh liquid LB medium (peptone 10 g/l, yeast extract 5 g/l, sodium chloride 10 g/l, ampicillin 100 mg/l, pH 7.0), and cultured with shaking at 37 ℃ for 14 hours at 220 rpm. Then, the cells were inoculated into 1000 ml of fresh liquid LB medium at an inoculum size of 5% (v/v), incubated at 220rpm and 37 ℃ to OD with shaking600When the concentration is 1.0 (about 2-3 hours), Isopropyl-beta-D-thiogalactoside (IPTG) is added at the final concentration of 1 millimole/liter, shaking culture is carried out at 37 ℃ and 160 revolutions/minute, and after 1 hour, the fermentation liquor gradually shows green fluorescence, as shown in FIG. 2, fluorescence observation after IPTG induced expression of pET-GSFGF17-CspA-Ulp1/BL21 is carried out, wherein, panel A is the observation result of the fermentation liquor under natural light, the fermentation liquor in the left test tube shows obvious green fluorescence (after IPTG induced expression of pET-GSFGF17-CspA-Ulp1/BL21 for 1 hour), and the fermentation liquor in the right test tube does not show green fluorescence (pET-GSFGF17-CspA-Ulp1/BL21 IPTG induced expression before IPTG induced expression); and the graph B shows that the green fluorescence is more obvious when the pET-GSFGF17-CspA-Ulp1/BL21 fermentation liquor is observed under the ultraviolet light. The same results were obtained with other recombinant strains of fibroblast growth factor. After 4 hours of IPTG induction, the cells were centrifuged at 12000rpm, and the precipitates showing green fluorescence, that is, the recombinant cells after expression induction were collected and resuspended in a phosphate buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM sodium dihydrogen phosphate, 2 mM potassium dihydrogen phosphate, 0.2% NP-40, pH 7.0) to obtain a bacterial suspension. The bacterial suspension was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection. For specific procedures of SDS-PAGE, reference is made to Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989。
pET-GFP-SUMO/BL21 subjected to the same induction operation was used as a control; meanwhile, pET-GSFGF8-CspA-Ulp1/BL21 (FIG. 3A) before IPTG induction, pET-GSFGF4-CspA-Ulp1/BL21 (FIG. 3B) before IPTG induction and pET-GSFGF11-CspA-Ulp1/BL21 (FIG. 3C) before IPTG induction were used as additional controls, i.e., 1 ml of the inoculum was taken out of the fermentation broth for SDS-PAGE of the subsequent total protein before IPTG addition.
The results of SDS-PAGE are shown in FIG. 3. As can be seen from FIG. 3, the fusion protein GFP-SUMO-FGFn can be expressed in large amounts. The density grayscale scanning of the SDS-PAGE result by using Image J software shows that the expression level of the fusion protein GFP-SUMO-FGFn accounts for more than 40 percent of the total protein.
EXAMPLE 5 inducible expression of recombinant strains
The temperature of IPTG induction was 28 ℃ and the induction time was 8 hours, as in example 4. The results are comparable to example 4.
Example 6 solubility analysis of the Induction of recombinant strains
IPTG-induced expression of the fusion protein GFP-SUMO-FGFn was carried out in the same manner as in example 4. After induction expression, the cells were centrifuged at 12000rpm, and the precipitate showing green fluorescence, i.e., the recombinant cells after induction expression, was collected and weighed.
And (3) collecting the recombinant thallus, and performing the following steps of: the phosphate buffer was 1: phosphate buffer (137 mmol/l sodium chloride, 2.7 mmol/l potassium chloride, 10 mmol/l sodium dihydrogen phosphate, 2 mmol/l potassium dihydrogen phosphate, 0.2% NP-40, pH 7.0) was added at a ratio of 10 (mass/volume) to resuspend the recombinant cells, thereby obtaining a bacterial suspension. And (3) placing the bacterial suspension into a high-pressure homogenizer, and continuously homogenizing for 2 times at 4 ℃ under the pressure conditions of 800 bar (bar) and 1200bar (bar) successively to obtain a lysate. The lysate was placed in a sterile centrifuge tube and centrifuged at 12000rpm for 1 minute, and the lysate, supernatant and pellet were subjected to SDS-PAGE, respectively.
The results of SDS-PAGE of the fraction pET-GSFGFn-CspA-Ulp1/BL21 total protein, protein in supernatant and protein in pellet are shown in FIG. 4. As can be seen from FIG. 4, the soluble expression amounts of the fusion proteins GFP-SUMO-FGF4, GFP-SUMO-FGF5, GFP-SUMO-FGF8, GFP-SUMO-FGF11, GFP-SUMO-FGF12, GFP-SUMO-FGF14, GFP-SUMO-FGF16, GFP-SUMO-FGF17, GFP-SUMO-FGF19 and GFP-SUMO-FGF23 account for 80% or more of the total amount of the fusion proteins; moreover, the fusion proteins are not hydrolyzed, which shows that the normal expression of the small ubiquitin-like modifier protease Ulp1 is not caused in the process of IPTG induction at 37 ℃, so that the phenomenon that the fusion protein GFP-SUMO-FGFn is hydrolyzed does not occur.
The results of pET-GSFGFn-CspA-Ulp1/BL21 which are not shown after the same induction expression operation are equivalent, namely the expression amount of the soluble fusion protein GFP-SUMO-FGFn accounts for more than 80 percent of the total amount of the fusion protein GFP-SUMO-FGFn; moreover, the fusion proteins are not hydrolyzed.
The results are combined to show that the soluble fusion protein GFP-SUMO-FGFn containing the fibroblast growth factor-n can be efficiently expressed by utilizing a fusion expression mode, and the yield can completely meet the requirement of scale production of the later fibroblast growth factor.
Example 7 hydrolysis of fusion proteins containing fibroblast growth factor
The recombinant strain pET-GSFGFn-CspA-Ulp1/BL21 was fermented at 37 ℃ as described in example 4 and rapidly reduced to 15 ℃ after 4 hours induction with IPTG, a portion of the fermentation broth was taken and distributed into 5 flasks of 1000 ml volume, numbered flask 1 to flask 5, and 200 ml of the fermentation broth was added to each flask.
pET-GFP-SUMO/BL21 subjected to the same induction procedure was used as a control, only 1 control flask, numbered flask I.
In flask 1 and flask I, aqueous zinc sulphate solution was added to give a final concentration of 5 mmol/l zinc sulphate in the broth.
In flask 2I, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to give a final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth of 1 mmol/l.
In the flask 3, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to give a final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth of 1 mmol/l; an aqueous solution of zinc sulfate was added to give a final concentration of 1 mmol/l zinc sulfate in the fermentation broth.
In the flask 4, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to give a final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth of 1 mmol/l; an aqueous solution of zinc sulfate was added to give a final concentration of 3 mmol/l zinc sulfate in the fermentation broth.
In flask 5, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to give a final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth of 0.1 mmol/l; an aqueous solution of zinc sulfate was added to give a final concentration of 5 mmol/l zinc sulfate in the fermentation broth.
After the addition of the above components, the cultivation of the fermentation broth in flask 1 to 5 and I was continued for 2 hours at 15 ℃. Wherein, during the fermentation, samples are taken at 0 min, 30 min, 60 min, 90 min and 120 min respectively for subsequent SDS-PAGE.
And respectively centrifuging samples taken at different time points of different shake flasks at high speed of 12000 r/min, and respectively collecting recombinant thalli, wherein the ratio of the recombinant thalli: the phosphate buffer was 1: and (3) adding phosphate buffer solution in the proportion (mass/volume) of 10 respectively to resuspend the recombinant bacteria to obtain recombinant bacteria suspension. The suspension is placed in a high-pressure homogenizer and is continuously homogenized for 2 times at 4 ℃ under the pressure of 800 bar (bar) and 1200bar (bar) in sequence, thus obtaining the lysate. The lysate was placed in a sterile centrifuge tube and centrifuged at 12000rpm for 1 minute, and the supernatant was collected and analyzed by SDS-PAGE. FIG. 5 shows the results of pET-GSFGF12-CspA-Ulp1/BL 21. The electrophoresis results according to fig. 5 show that the addition of aqueous zinc sulfate alone, even when induced at low temperature for 2 hours, did not promote the hydrolysis of fusion protein GFP-SUMO-FGF12 by SUMO protease at all (fig. 5A); the addition of tris (2-carbonylethyl) phosphate solution alone at a final concentration of 1 mmol/l promoted SUMO protease hydrolysis of fusion protein GFP-SUMO-FGF12 to obtain fibroblast growth factor-12, but the hydrolysis efficiency was low, and about 30% to 40% of the fusion protein was not hydrolyzed after 120 minutes of treatment (fig. 5B); when tris (2-carbonylethyl) phosphate and zinc sulfate are added simultaneously, the amount of fusion protein hydrolyzed by SUMO protease is obviously increased, and more than 90% of fusion protein GFP-SUMO-FGFn can be hydrolyzed within 0.5 to 1 hour basically, which shows that the combination has synergistic effect and can remarkably improve the hydrolysis capacity of SUMO protease on the fusion protein (fig. 5C-E); among them, the results of adding a combination of tris (2-carbonylethyl) phosphate and zinc sulfate at a final concentration of 0.1 mmol/l and 5 mmol/l during low-temperature induced expression showed that the fusion protein was completely hydrolyzed by SUMO protease expressed at low-temperature induction within 15 minutes (fig. 5E).
In addition, the results of testing for the other fusion proteins GFP-SUMO-FGFn showed agreement with GFP-SUMO-FGF 12. Wherein FIG. 6 shows SDS-PAGE of the supernatant of fractions pET-GSFGFn-CspA-Ulp1/BL21 cultured at 15 ℃ for 30 minutes using a combination of tris (2-carbonylethyl) phosphate and zinc sulfate at a final concentration of 0.1 mmole/liter in combination, showing that the fusion protein GFP-SUMO-FGFn is completely hydrolyzed by the small ubiquitin-like modifier protease within 30 minutes of low temperature induction at 15 ℃.
To more clearly understand the progress of stepwise hydrolysis of the fusion protein GFP-SUMO-FGFn by SUMO protease, Western immunoblot analysis was performed on supernatants of pET-GSFGF9-CspA-Ulp1/BL21 and pET-GSFGF18-CspA-Ulp1/BL21 by using anti-human fibroblast growth factor-9 (Sammer Feishel technologies, Inc., China, Cat.: PA5-111618) and anti-human fibroblast growth factor-18 antibody (Sammer Feishel technologies, Inc., Cat.: PA5-78261) for 5 minutes and 30 minutes, respectively. The results are shown in FIG. 7, and the Western blot demonstrates that the fusion protein GFP-SUMO-FGFn can be completely hydrolyzed within 30 minutes.
EXAMPLE 8 hydrolysis of fibroblast growth factor-containing fusion proteins
The recombinant strain pET-GSFGFn-CspA-Ulp1/BL21 was fermented at 32 ℃ by the method of example 4 and rapidly lowered to 10 ℃ after 6 hours of IPTG induction, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to give a final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth of 0.1 mmol/l, an aqueous solution of zinc sulfate was added to give a final concentration of zinc sulfate in the fermentation broth of 5 mmol/l, and the fermentation broth was cultured for a further 2 hours. Wherein, during the fermentation, samples are taken at 0 min, 30 min, 60 min, 90 min and 120 min respectively for subsequent SDS-PAGE. The western blot results also showed that the fusion protein GFP-SUMO-FGFn was able to be completely hydrolyzed by SUMO protease within 30 minutes.
Example 9 Large Scale fermentation and isolation purification of fibroblast cytokine-n
The pET-GSFGF17-CspA-Ulp1/BL21 was taken out from a-80 ℃ refrigerator and the strain was frozen in a 1: 100 (v/v) was inoculated into 3.7L of fresh LB medium (peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, ampicillin 100 mg/L, pH 7.0), and cultured at 37 ℃ for 4 hours with shaking at 220rpm (rev/min). Inoculating the bacterial liquid into 200L (L) LB culture medium containing 5% (mass/volume) glucose at an inoculum size of 10% (volume/volume), and culturing at 37 deg.C for 4-6 hr until the thallus density reaches OD600About.0.6, the whole was transferred to 800L (liter) of LB medium containing 5% (mass/volume) glucose and cultured for 4 hours, and then inoculated to 8000L of LB medium containing 5% (mass/volume) glucose and cultured (total volume about 9000L). When the glucose content in the culture medium is reduced to 0.1% (mass/volume), feeding materials, wherein the feeding medium is an LB culture medium containing 20% (mass/volume) glucose, and setting fermentation parameters: pH is 7.0-7.2; dissolved oxygen>30 percent; the stirring speed was 250 to 650rpm (revolutions per minute). When the wet weight of the bacteria reached 35. + -.1 g/L (g/L), the inducer was added to IPTG to a final concentration of 1 mmol/L and the induction was carried out for 4 hours. Then, the temperature of the fermentation broth was rapidly decreased to 15 ℃, and at the same time, an aqueous solution of tris (2-carbonylethyl) phosphonium hydrochloride was added to the fermentation broth so that the final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the fermentation broth became 0.1 mmol/l, and an aqueous solution of zinc sulfate was added so that the final concentration of zinc sulfate in the fermentation broth became 5 mmol/l, with the parameters set as: pH 7.0; dissolved oxygen>30 percent; stirring speed 400rpm (revolution per minute), culturing for 1 hour, finishing the whole fermentation and induction expression process, finally obtaining the total volume of the fermentation liquor about 10000L, centrifugally collecting thalli and weighing to obtain 513 KG (KG) of wet weight recombinant thalli, and calculating to achieve 51.3g/L of wet weight recombinant thalli.
Because the fibroblast growth factors all have heparin binding domains, the fibroblast growth factors hydrolyzed from the fusion protein can be purified by a method of direct adsorption of heparin affinity chromatography gel.
All the recombinant cells collected were disrupted by a high-pressure homogenizer in the same manner as in example 6 to obtain a lysate. And (3) placing the lysate in a sterile centrifuge tube, centrifuging at 12000rpm for 1 minute, collecting supernatant, loading the supernatant on a CM ion exchange gel chromatographic column, and performing gradient elution on the fibroblast growth factor-17 by using buffer solutions with different sodium chloride concentrations. The buffer used was in this order buffer 1(50 mmol/l Tris-HCl, 0.1 mol/l sodium chloride, pH 8.0); buffer 2(50 mmol/l Tris-HCl, 0.3 mol/l sodium chloride, pH 8.0); buffer 3(50 mmol/l Tris-HCl, 0.6 mol/l sodium chloride, pH 8.0) and buffer 6(50 mmol/l Tris-HCl, 1.0 mol/l sodium chloride, pH 8.0). SDS-PAGE detection and results are shown in FIG. 8A, and analysis shows that fibroblast growth factor-17 is mainly present in the eluate of buffer 3. The fibroblast growth factor-17-containing eluate was loaded onto a heparin affinity chromatography gel column at a flow rate of 8 to 10 ml/min, and then eluted sequentially using buffer 3(50 mmol/l Tris-HCl, 0.6 mol/l sodium chloride, pH 8.0), buffer 4(50 mmol/l Tris-HCl, 1.2 mol/l sodium chloride, pH 8.0), and buffer 5(50 mmol/l Tris-HCl, 2.0 mol/l sodium chloride, pH 8.0). SDS-PAGE is shown in figure 8B, and the detection result shows that the fibroblast growth factor-17 is positioned in the eluent of the buffer 4, and the density gray scale scanning (ImageJ software) result of the electrophoretic band shows that the purity of the fibroblast growth factor-17 reaches more than 95%.
And quantifying the purified fibroblast growth factor-17 by using a bicinchoninic acid (BCA) protein quantification method, and converting the wet weight of the recombinant thallus based on pET-GSFGF17-CspA-Ulp1/BL21 to obtain the yield of the fibroblast growth factor-17, wherein the yield of the finally purified fibroblast growth factor-17 is 5.048 mg/g wet weight.
Similar fermentation results were obtained using the same fermentation procedure for other pET-GSFGFn-CspA-Ulp1/BL21 fermentations and purified fibroblast growth factor-n. The yields of purified fibroblast growth factors were: fibroblast growth factor-16.066 mg/g wet weight, fibroblast growth factor-28.62 mg/g wet weight, fibroblast growth factor-39.329 mg/g wet weight, fibroblast growth factor-47.14 mg/g wet weight, fibroblast growth factor-55.224 mg/g wet weight, fibroblast growth factor-61.34 mg/g wet weight, fibroblast growth factor-72.019 mg/g wet weight, fibroblast growth factor-82.46 mg/g wet weight, fibroblast growth factor-94.984 mg/g wet weight, fibroblast growth factor-106.673 mg/g wet weight, fibroblast growth factor-111.243 mg/g wet weight, fibroblast growth factor-121.758 mg/g wet weight, fibroblast growth factor-133.153 mg/g wet weight, fibroblast growth factor-141.585 mg/g wet weight, fibroblast growth factor-161.892 mg/g wet weight, fibroblast growth factor-182.298 mg/g wet weight, fibroblast growth factor-191.957 mg/g wet weight, fibroblast growth factor-201.466 mg/g wet weight, fibroblast growth factor-213.428 mg/g wet weight, fibroblast growth factor-221.206 mg/g wet weight, fibroblast growth factor-232.694 mg/g wet weight.
Example 10 analysis of the proliferation-promoting Activity of fibroblast growth factor-8, fibroblast growth factor-9 and fibroblast growth factor-17 on Leydig cells
In humans, 95% of androgens are synthesized by leydig cells, and a decrease in the number of leydig cells leads to a significant decrease in the production of androgens in humans.
Human primary leydig cells (cat # 4510, science cell Research Laboratories, Inc., usa) were inoculated into Modified Eagle's Medium (Dulbecco's Modified Eagle Medium, DMEM/F-12, available from sequoise corporation, cat # 11320033), cultured at 37 ℃ to logarithmic growth phase, digested with trypsin solution, transferred to 96-well cell culture plates (8000 cells/well, 200 microliters volume/well), and then added with purified fibroblast growth factor-8 (FGF-8), fibroblast growth factor-9 (FGF-9), and fibroblast growth factor-17 (FGF-17) at a final concentration of 100 ng/ml, respectively, with the addition of an equal volume of PBS buffer as a negative control. After 24 hours of incubation, 100. mu.l of the medium was discarded, and 100. mu.l of fresh modified eagle's medium containing 5-ethyl-2' -deoxyuridine (EdU, a tracer compound, which requires replication of deoxyribonucleic acid (DNA) for cell proliferation, at a final concentration of 10. mu.M, during which EdU is inserted into the newly formed nucleotide chain and emits green fluorescence under illumination with a wavelength of 450 and 540 nm was added, and incubation was continued for 24 hours, as observed by fluorescence microscopy, as shown in FIG. 9. The results in FIG. 9 show that FGF-8, FGF-9, and FGF-17 significantly promote the synthesis of deoxyribonucleic acid (DNA) from mesenchymal cells, relative to the negative control group.
Sequence listing
<110> Wenzhou university of medical science
Su Zhijian
<120> a composition and a method for mass production of fibroblast growth factor
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Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr
1 5 10 15
Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val
20 25 30
Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser
35 40 45
Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
50 55 60
Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro
65 70 75 80
Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn
85 90 95
Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu
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Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln
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Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
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<211> 155
<212> PRT
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Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
20 25 30
Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
35 40 45
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
50 55 60
Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn
65 70 75 80
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys
85 90 95
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys
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Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
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Met Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly
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Arg Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys
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Leu Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg
35 40 45
Val Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr
50 55 60
Ala Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg
65 70 75 80
Tyr Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr
85 90 95
Ser Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn
100 105 110
Thr Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala
115 120 125
Arg Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly
130 135 140
Lys Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser
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Ser Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val
165 170 175
Arg Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln
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Pro Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser
195 200 205
His Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala Ser Ala His
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Met Gly Arg Gly Gly Ala Ala Ala Pro Thr Ala Pro Asn Gly Thr Leu
1 5 10 15
Glu Ala Glu Leu Glu Arg Arg Trp Glu Ser Leu Val Ala Leu Ser Leu
20 25 30
Ala Arg Leu Pro Val Ala Ala Gln Pro Lys Glu Ala Ala Val Gln Ser
35 40 45
Gly Ala Gly Asp Tyr Leu Leu Gly Ile Lys Arg Leu Arg Arg Leu Tyr
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Cys Asn Val Gly Ile Gly Phe His Leu Gln Ala Leu Pro Asp Gly Arg
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Ile Gly Gly Ala His Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu Ser
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Pro Val Glu Arg Gly Val Val Ser Ile Phe Gly Val Ala Ser Arg Phe
100 105 110
Phe Val Ala Met Ser Ser Lys Gly Lys Leu Tyr Gly Ser Pro Phe Phe
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Thr Asp Glu Cys Thr Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr Asn
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Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala Leu Ser Lys
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Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val
165 170 175
Thr His Phe Leu Pro Arg Leu
180
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Met Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro
1 5 10 15
Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln
20 25 30
Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala
35 40 45
Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln
50 55 60
Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly
65 70 75 80
Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser
85 90 95
His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln
100 105 110
Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met
115 120 125
Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys
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Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser
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Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu
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Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro
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Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln
195 200 205
Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro
210 215 220
Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr
225 230 235 240
Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly
245 250
<210> 6
<211> 169
<212> PRT
<213> Intelligent (Homo sapiens)
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Met Gly Thr Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly
1 5 10 15
Thr Leu Leu Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly
20 25 30
Val Asn Trp Glu Ser Gly Tyr Leu Val Gly Ile Lys Arg Gln Arg Arg
35 40 45
Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu Gln Val Leu Pro Asp
50 55 60
Gly Arg Ile Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu
65 70 75 80
Ile Ser Thr Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser
85 90 95
Ala Leu Phe Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro
100 105 110
Ser Phe Gln Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn
115 120 125
Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu
130 135 140
Ser Lys Tyr Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met
145 150 155 160
Thr Val Thr His Phe Leu Pro Arg Ile
165
<210> 7
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<212> PRT
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Met Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile Arg Val Arg Arg Leu
1 5 10 15
Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val
20 25 30
Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile Arg
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Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly Val Glu Ser Glu Phe
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Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys
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Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu Glu Asn His Tyr Asn
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Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val
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Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly Lys Lys Thr Lys Lys
115 120 125
Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala Ile Thr
130 135 140
<210> 8
<211> 194
<212> PRT
<213> Intelligent (Homo sapiens)
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Met Gln Val Thr Val Gln Ser Ser Pro Asn Phe Thr Gln His Val Arg
1 5 10 15
Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg Arg Leu Ile Arg Thr
20 25 30
Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Leu Ala
35 40 45
Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly Asp Pro Phe Ala Lys
50 55 60
Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Val Arg Gly
65 70 75 80
Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys Lys Gly Lys Leu Ile
85 90 95
Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val Phe Thr Glu Ile Val
100 105 110
Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala Lys Tyr Glu Gly Trp
115 120 125
Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg Lys Gly Ser Lys Thr
130 135 140
Arg Gln His Gln Arg Glu Val His Phe Met Lys Arg Leu Pro Arg Gly
145 150 155 160
His His Thr Thr Glu Gln Ser Leu Arg Phe Glu Phe Leu Asn Tyr Pro
165 170 175
Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg Thr Trp Ala Pro Glu
180 185 190
Pro Arg
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Met Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala Val
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Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu Leu
20 25 30
Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly Pro
35 40 45
Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg Gln
50 55 60
Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr
65 70 75 80
Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu Phe
85 90 95
Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser Gly
100 105 110
Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu Lys
115 120 125
Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr
130 135 140
Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg Arg
145 150 155 160
Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr Arg
165 170 175
Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp
180 185 190
Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser
195 200 205
<210> 10
<211> 170
<212> PRT
<213> Intelligent (Homo sapiens)
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Met Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser
1 5 10 15
Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr
20 25 30
Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr
35 40 45
Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys
50 55 60
Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile
65 70 75 80
Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met
85 90 95
Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys
100 105 110
Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser
115 120 125
Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly
130 135 140
Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser
145 150 155 160
Ala His Phe Leu Pro Met Val Val His Ser
165 170
<210> 11
<211> 225
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 11
Met Ala Ala Leu Ala Ser Ser Leu Ile Arg Gln Lys Arg Glu Val Arg
1 5 10 15
Glu Pro Gly Gly Ser Arg Pro Val Ser Ala Gln Arg Arg Val Cys Pro
20 25 30
Arg Gly Thr Lys Ser Leu Cys Gln Lys Gln Leu Leu Ile Leu Leu Ser
35 40 45
Lys Val Arg Leu Cys Gly Gly Arg Pro Ala Arg Pro Asp Arg Gly Pro
50 55 60
Glu Pro Gln Leu Lys Gly Ile Val Thr Lys Leu Phe Cys Arg Gln Gly
65 70 75 80
Phe Tyr Leu Gln Ala Asn Pro Asp Gly Ser Ile Gln Gly Thr Pro Glu
85 90 95
Asp Thr Ser Ser Phe Thr His Phe Asn Leu Ile Pro Val Gly Leu Arg
100 105 110
Val Val Thr Ile Gln Ser Ala Lys Leu Gly His Tyr Met Ala Met Asn
115 120 125
Ala Glu Gly Leu Leu Tyr Ser Ser Pro His Phe Thr Ala Glu Cys Arg
130 135 140
Phe Lys Glu Cys Val Phe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Ala
145 150 155 160
Leu Tyr Arg Gln Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp
165 170 175
Lys Glu Gly Gln Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Ala
180 185 190
Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu
195 200 205
Pro Ser Leu His Ser Val Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala
210 215 220
Pro
225
<210> 12
<211> 243
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 12
Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala
1 5 10 15
Arg Glu Ser Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser
20 25 30
Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val
35 40 45
Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg
50 55 60
Arg Pro Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln
65 70 75 80
Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr
85 90 95
Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly
100 105 110
Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala
115 120 125
Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu
130 135 140
Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser
145 150 155 160
Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly
165 170 175
Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Lys Lys Thr
180 185 190
Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys Met Tyr
195 200 205
Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Arg
210 215 220
Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln
225 230 235 240
Asp Ser Thr
<210> 13
<211> 245
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 13
Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala
1 5 10 15
Arg Glu Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser
20 25 30
Lys Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg
35 40 45
Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro Glu Pro
50 55 60
Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His
65 70 75 80
Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp Gly Thr Lys Asp Glu Asp
85 90 95
Ser Thr Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val
100 105 110
Ala Ile Gln Gly Val Gln Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu
115 120 125
Gly Tyr Leu Tyr Thr Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys
130 135 140
Glu Ser Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr
145 150 155 160
Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu
165 170 175
Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala
180 185 190
His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser
195 200 205
Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr
210 215 220
Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser
225 230 235 240
His Asn Glu Ser Thr
245
<210> 14
<211> 247
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 14
Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala
1 5 10 15
Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser
20 25 30
Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe
35 40 45
Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln
50 55 60
Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly
65 70 75 80
Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp
85 90 95
Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg
100 105 110
Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn
115 120 125
Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys
130 135 140
Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met
145 150 155 160
Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu Asn
165 170 175
Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val Lys Lys Thr Lys Pro
180 185 190
Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu
195 200 205
Pro Ser Leu His Asp Val Gly Glu Thr Val Pro Lys Pro Gly Val Thr
210 215 220
Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met Asn Gly Gly Lys Pro
225 230 235 240
Val Asn Lys Ser Lys Thr Thr
245
<210> 15
<211> 207
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 15
Met Ala Glu Val Gly Gly Val Phe Ala Ser Leu Asp Trp Asp Leu His
1 5 10 15
Gly Phe Ser Ser Ser Leu Gly Asn Val Pro Leu Ala Asp Ser Pro Gly
20 25 30
Phe Leu Asn Glu Arg Leu Gly Gln Ile Glu Gly Lys Leu Gln Arg Gly
35 40 45
Ser Pro Thr Asp Phe Ala His Leu Lys Gly Ile Leu Arg Arg Arg Gln
50 55 60
Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr
65 70 75 80
Val His Gly Thr Arg His Asp His Ser Arg Phe Gly Ile Leu Glu Phe
85 90 95
Ile Ser Leu Ala Val Gly Leu Ile Ser Ile Arg Gly Val Asp Ser Gly
100 105 110
Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Tyr Gly Ser Lys Lys
115 120 125
Leu Thr Arg Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr
130 135 140
Asn Thr Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp Ser Glu Arg Gln
145 150 155 160
Tyr Tyr Val Ala Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg
165 170 175
Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp
180 185 190
Pro Ser Lys Leu Pro Ser Met Ser Arg Asp Leu Phe His Tyr Arg
195 200 205
<210> 16
<211> 195
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 16
Met Thr Gln Gly Glu Asn His Pro Ser Pro Asn Phe Asn Gln Tyr Val
1 5 10 15
Arg Asp Gln Gly Ala Met Thr Asp Gln Leu Ser Arg Arg Gln Ile Arg
20 25 30
Glu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Thr
35 40 45
Gly Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly Asn Lys Phe Ala Lys
50 55 60
Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Ile Lys Gly
65 70 75 80
Ala Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys Arg Gly Lys Leu Ile
85 90 95
Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val Phe Thr Glu Ile Val
100 105 110
Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala Arg His Glu Gly Trp
115 120 125
Phe Met Ala Phe Thr Arg Gln Gly Arg Pro Arg Gln Ala Ser Arg Ser
130 135 140
Arg Gln Asn Gln Arg Glu Ala His Phe Ile Lys Arg Leu Tyr Gln Gly
145 150 155 160
Gln Leu Pro Phe Pro Asn His Ala Glu Lys Gln Lys Gln Phe Glu Phe
165 170 175
Val Gly Ser Ala Pro Thr Arg Arg Thr Lys Arg Thr Arg Arg Pro Gln
180 185 190
Pro Leu Thr
195
<210> 17
<211> 174
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 17
Met Ala Glu Glu Asn Val Asp Phe Arg Ile His Val Glu Asn Gln Thr
1 5 10 15
Arg Ala Arg Asp Asp Val Ser Arg Lys Gln Leu Arg Leu Tyr Gln Leu
20 25 30
Tyr Ser Arg Thr Ser Gly Lys His Ile Gln Val Leu Gly Arg Arg Ile
35 40 45
Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln Leu Leu Val Glu
50 55 60
Thr Asp Thr Phe Gly Ser Gln Val Arg Ile Lys Gly Lys Glu Thr Glu
65 70 75 80
Phe Tyr Leu Cys Met Asn Arg Lys Gly Lys Leu Val Gly Lys Pro Asp
85 90 95
Gly Thr Ser Lys Glu Cys Val Phe Ile Glu Lys Val Leu Glu Asn Asn
100 105 110
Tyr Thr Ala Leu Met Ser Ala Lys Tyr Ser Gly Trp Tyr Val Gly Phe
115 120 125
Thr Lys Lys Gly Arg Pro Arg Lys Gly Pro Lys Thr Arg Glu Asn Gln
130 135 140
Gln Asp Val His Phe Met Lys Arg Tyr Pro Lys Gly Gln Pro Glu Leu
145 150 155 160
Gln Lys Pro Phe Lys Tyr Thr Thr Val Thr Lys Arg Ser Arg
165 170
<210> 18
<211> 195
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 18
Met Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly
1 5 10 15
Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His
20 25 30
Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp
35 40 45
Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val
50 55 60
Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu
65 70 75 80
Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu
85 90 95
Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val
100 105 110
Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys
115 120 125
Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
130 135 140
Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly
145 150 155 160
His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met
165 170 175
Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser
180 185 190
Phe Glu Lys
195
<210> 19
<211> 210
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 19
Met Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly Leu
1 5 10 15
Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala Gly Glu Arg
20 25 30
Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala Arg
35 40 45
Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu Arg
50 55 60
Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu Pro
65 70 75 80
Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly Ile
85 90 95
Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly Val
100 105 110
Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr Gly
115 120 125
Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu Glu
130 135 140
Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp Thr
145 150 155 160
Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Asp
165 170 175
Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg
180 185 190
Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu Met
195 200 205
Tyr Thr
210
<210> 20
<211> 182
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 20
Met His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln
1 5 10 15
Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala
20 25 30
His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln
35 40 45
Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile
50 55 60
Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp
65 70 75 80
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe
85 90 95
Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
100 105 110
His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp
115 120 125
Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
130 135 140
Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp
145 150 155 160
Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg
165 170 175
Ser Pro Ser Tyr Ala Ser
180
<210> 21
<211> 150
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 21
Met Gly Thr Pro Ser Ala Ser Arg Gly Pro Arg Ser Tyr Pro His Leu
1 5 10 15
Glu Gly Asp Val Arg Trp Arg Arg Leu Phe Ser Ser Thr His Phe Phe
20 25 30
Leu Arg Val Asp Pro Gly Gly Arg Val Gln Gly Thr Arg Trp Arg His
35 40 45
Gly Gln Asp Ser Ile Leu Glu Ile Arg Ser Val His Val Gly Val Val
50 55 60
Val Ile Lys Ala Val Ser Ser Gly Phe Tyr Val Ala Met Asn Arg Arg
65 70 75 80
Gly Arg Leu Tyr Gly Ser Arg Leu Tyr Thr Val Asp Cys Arg Phe Arg
85 90 95
Glu Arg Ile Glu Glu Asn Gly His Asn Thr Tyr Ala Ser Gln Arg Trp
100 105 110
Arg Arg Arg Gly Gln Pro Met Phe Leu Ala Leu Asp Arg Arg Gly Gly
115 120 125
Pro Arg Pro Gly Gly Arg Thr Arg Arg Tyr His Leu Ser Ala His Phe
130 135 140
Leu Pro Val Leu Val Ser
145 150
<210> 22
<211> 228
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 22
Met Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly Gly Leu
1 5 10 15
Ile His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His Leu Gln Ile
20 25 30
His Lys Asn Gly His Val Asp Gly Ala Pro His Gln Thr Ile Tyr Ser
35 40 45
Ala Leu Met Ile Arg Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly
50 55 60
Val Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe
65 70 75 80
Gly Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr
85 90 95
Leu Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu
100 105 110
Val Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro
115 120 125
Pro Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu Ile
130 135 140
His Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg Ser Ala Glu Asp
145 150 155 160
Asp Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg Met
165 170 175
Thr Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp
180 185 190
Asn Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg
195 200 205
Val Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro Phe
210 215 220
Ala Lys Phe Ile
225
<210> 23
<211> 253
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Met Lys Ser Ser His His His His His His Gly Ser Ser Val Ser Lys
1 5 10 15
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
20 25 30
Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly
35 40 45
Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly
50 55 60
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly
65 70 75 80
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
85 90 95
Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser
100 105 110
Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
115 120 125
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys
130 135 140
Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser
145 150 155 160
His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala
165 170 175
Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala
180 185 190
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
195 200 205
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro
210 215 220
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
225 230 235 240
Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Ile
245 250
<210> 24
<211> 98
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro
1 5 10 15
Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser
20 25 30
Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu
35 40 45
Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg
50 55 60
Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp
65 70 75 80
Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile
85 90 95
Gly Gly
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Gly Gly Ser Gly Gly Gly Ser
1 5
<210> 26
<211> 426
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 26
atgttcaacc tgccgccggg caactacaag aaaccgaagc tgctgtattg cagcaacggt 60
ggccactttc tgcgtatcct gccggatggt accgtggatg gtacccgtga ccgtagcgat 120
cagcacatcc agctgcaact gagcgcggag agcgtgggcg aagtttacat taaaagcacc 180
gagaccggcc agtatctggc gatggacacc gatggtctgc tgtacggcag ccaaaccccg 240
aacgaggaat gcctgttcct ggagcgtctg gaggaaaacc actacaacac ctatatcagc 300
aagaaacacg cggaaaagaa ctggtttgtg ggtctgaaga aaaacggcag ctgcaagcgt 360
ggtccgcgta cccactatgg tcaaaaagcg attctgttcc tgccgctgcc ggttagcagc 420
gactaa 426
<210> 27
<211> 468
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 27
atggcggcgg gtagcatcac caccctgccg gcgctgccgg aagatggtgg cagcggtgcg 60
tttccgccgg gtcacttcaa ggacccgaaa cgtctgtact gcaagaacgg tggcttcttt 120
ctgcgtattc acccggacgg tcgtgtggat ggcgttcgtg aaaagagcga cccgcacatc 180
aaactgcagc tgcaagcgga ggaacgtggt gtggttagca ttaaaggcgt gtgcgcgaac 240
cgttatctgg cgatgaaaga ggacggccgt ctgctggcga gcaaatgcgt taccgatgaa 300
tgcttctttt tcgagcgtct ggaaagcaac aactacaaca cctatcgtag ccgtaagtac 360
accagctggt acgttgcgct gaaacgtacc ggccagtaca agctgggcag caaaaccggt 420
ccgggccaaa aggcgatcct gtttctgccg atgagcgcga aaagctaa 468
<210> 28
<211> 675
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 28
atgccggcgg cgggtccggg tgcgcgtctg cgtcgtgatg cgggtggccg tggtggcgtg 60
tatgagcacc tgggtggtgc gccgcgtcgt cgtaagctgt actgcgcgac caaatatcac 120
ctgcagctgc acccgagcgg tcgtgtgaac ggcagcctgg aaaacagcgc gtacagcatc 180
ctggagatta ccgcggtgga agttggtatc gttgcgattc gtggtctgtt cagcggccgt 240
tatctggcga tgaacaagcg tggccgtctg tacgcgagcg agcactatag cgcggagtgc 300
gaatttgtgg aacgtatcca cgaactgggt tacaacacct atgcgagccg tctgtaccgt 360
accgttagca gcaccccggg tgcgcgtcgt cagccgagcg cggagcgtct gtggtatgtg 420
agcgttaacg gtaaaggccg tccgcgtcgt ggtttcaaga cccgtcgtac ccagaagagc 480
agcctgtttc tgccgcgtgt gctggaccac cgtgatcacg aaatggttcg tcagctgcaa 540
agcggtctgc cgcgtccgcc gggcaagggc gtgcaaccgc gtcgtcgtcg tcagaaacaa 600
agcccggata acctggaacc gagccatgtt caggcgagcc gtctgggtag ccaactggaa 660
gcgagcgcgc actaa 675
<210> 29
<211> 552
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 29
atgggtcgtg gtggcgcggc ggcgccgacc gcgccgaacg gtaccctgga ggcggaactg 60
gagcgtcgtt gggaaagcct ggtggcgctg agcctggcgc gtctgccggt tgcggcgcag 120
ccgaaagagg cggcggtgca aagcggtgcg ggtgactacc tgctgggtat caaacgtctg 180
cgtcgtctgt attgcaacgt tggtattggt tttcacctgc aggcgctgcc ggatggtcgt 240
atcggtggcg cgcatgcgga cacccgtgat agcctgctgg aactgagccc ggtggagcgt 300
ggtgtggtta gcatttttgg cgtggcgagc cgtttctttg ttgcgatgag cagcaagggt 360
aaactgtacg gcagcccgtt ctttaccgac gaatgcacct tcaaggagat tctgctgccg 420
aacaactaca acgcgtatga aagctacaaa tatccgggca tgtttatcgc gctgagcaag 480
aacggtaaaa ccaagaaagg caaccgtgtg agcccgacca tgaaggttac ccacttcctg 540
ccgcgtctgt aa 552
<210> 30
<211> 759
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 30
atggcgtggg cgcatggtga gaagcgtctg gcgccgaagg gtcagccggg tccggcggcg 60
accgaccgta acccgatcgg tagcagcagc cgtcaaagca gcagcagcgc gatgagcagc 120
agcagcgcga gcagcagccc ggcggcgagc ctgggcagcc agggtagcgg tctggaacag 180
agcagctttc agtggagccc gagcggtcgt cgtaccggta gcctgtactg ccgtgttggt 240
atcggctttc acctgcagat ttatccggat ggcaaagtta acggtagcca cgaggcgaac 300
atgctgagcg tgctggaaat tttcgctgtg agccaaggta tcgttggcat tcgtggtgtg 360
ttcagcaaca agtttctggc gatgagcaag aaaggcaagc tgcacgcgag cgcgaaattt 420
accgacgatt gcaagttccg tgagcgtttt caggaaaaca gctacaacac ctatgcgagc 480
gcgatccacc gtaccgagaa aaccggtcgt gaatggtacg tggcgctgaa caagcgtggc 540
aaggcgaaac gtggttgcag cccgcgtgtg aaaccgcaac acattagcac ccacttcctg 600
ccgcgtttta agcagagcga gcaaccggaa ctgagcttca ccgtgaccgt tccggagaag 660
aaaaacccgc cgagcccgat caagagcaaa attccgctga gcgcgccgcg taaaaacacc 720
aacagcgtga agtatcgtct gaaattccgt tttggttaa 759
<210> 31
<211> 510
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 31
atgggtaccc gtgcgaacaa caccctgctg gatagccgtg gttggggtac cctgctgagc 60
cgtagccgtg cgggtctggc gggtgaaatt gcgggcgtta actgggaaag cggttacctg 120
gtgggcatca agcgtcagcg tcgtctgtat tgcaacgttg gtattggctt ccacctgcaa 180
gtgctgccgg atggtcgtat cagcggcacc cacgaggaaa acccgtacag cctgctggag 240
atcagcaccg ttgaacgtgg tgtggttagc ctgttcggcg tgcgtagcgc gctgtttgtt 300
gcgatgaaca gcaagggtcg tctgtatgcg accccgagct tccaggaaga gtgcaaattt 360
cgtgagaccc tgctgccgaa caactacaac gcgtatgaaa gcgacctgta ccaaggcacc 420
tatattgcgc tgagcaaata cggtcgtgtg aagcgtggca gcaaagttag cccgatcatg 480
accgtgaccc actttctgcc gcgtatttaa 510
<210> 32
<211> 426
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 32
atgagctacg actatatgga gggtggcgat atccgtgtgc gtcgtctgtt ctgccgtacc 60
cagtggtacc tgcgtattga caagcgtggc aaggttaaag gcacccaaga gatgaagaac 120
aactacaaca tcatggaaat ccgtaccgtg gcggttggta tcgtggcgat taagggcgtt 180
gagagcgaat tctacctggc gatgaacaag gaaggtaaac tgtatgcgaa gaaagagtgc 240
aacgaagatt gcaactttaa ggagctgatc ctggaaaacc actacaacac ctatgcgagc 300
gcgaaatgga cccacaacgg tggcgagatg ttcgtggcgc tgaaccagaa gggtatcccg 360
gttcgtggca agaaaaccaa gaaagaacaa aaaaccgcgc actttctgcc gatggcgatt 420
acctaa 426
<210> 33
<211> 585
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 33
atgcaggtga ccgttcaaag cagcccgaac ttcacccagc acgtgcgtga gcaaagcctg 60
gttaccgacc agctgagccg tcgtctgatc cgtacctacc aactgtatag ccgtaccagc 120
ggcaagcacg tgcaggttct ggcgaacaaa cgtatcaacg cgatggcgga ggacggtgat 180
ccgttcgcga agctgattgt ggaaaccgac acctttggca gccgtgtgcg tgttcgtggt 240
gcggaaaccg gcctgtacat ctgcatgaac aagaaaggta aactgattgc gaaaagcaac 300
ggcaagggca aagattgcgt gttcaccgag attgttctgg aaaacaacta taccgcgctg 360
caaaacgcga agtacgaggg ctggtatatg gcgttcaccc gtaaaggtcg tccgcgtaag 420
ggcagcaaaa cccgtcagca ccaacgtgaa gttcacttta tgaaacgtct gccgcgtggt 480
caccacacca ccgagcagag cctgcgtttc gaatttctga actacccgcc gtttacccgt 540
agcctgcgtg gcagccaacg tacctgggcg ccggagccgc gttaa 585
<210> 34
<211> 624
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 34
atgccgctgg gtgaagtggg caactacttc ggcgtgcagg acgcggttcc gtttggtaac 60
gtgccggttc tgccggtgga cagcccggtt ctgctgagcg atcacctggg ccaaagcgag 120
gcgggtggcc tgccgcgtgg tccggcggtt accgacctgg atcacctgaa gggcatcctg 180
cgtcgtcgtc agctgtattg ccgtaccggt ttccacctgg aaatctttcc gaacggtacc 240
attcaaggca cccgtaaaga ccacagccgt ttcggcatcc tggagtttat cagcattgcg 300
gtgggtctgg ttagcattcg tggtgtggat agcggcctgt acctgggtat gaacgagaag 360
ggcgaactgt atggtagcga gaaactgacc caggaatgcg ttttccgtga gcaatttgag 420
gaaaactggt acaacaccta tagcagcaac ctgtacaagc acgtggacac cggtcgtcgt 480
tactatgttg cgctgaacaa agatggcacc ccgcgtgaag gtacccgtac caagcgtcac 540
cagaaattca cccactttct gccgcgtccg gtggacccgg ataaggttcc ggagctgtat 600
aaagatattc tgagccaaag ctaa 624
<210> 35
<211> 513
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 35
atgctgggtc aggacatggt tagcccggag gcgaccaaca gcagcagcag cagctttagc 60
agcccgagca gcgcgggtcg tcatgtgcgt agctacaacc acctgcaagg cgatgttcgt 120
tggcgtaaac tgttcagctt taccaagtat tttctgaaga tcgagaaaaa cggcaaagtt 180
agcggcacca agaaagaaaa ctgcccgtac agcatcctgg agattaccag cgtggaaatc 240
ggtgtggttg cggttaaagc gattaacagc aactactatc tggcgatgaa caagaaaggt 300
aaactgtatg gcagcaagga gttcaacaac gactgcaagc tgaaagaacg tattgaggaa 360
aacggttaca acacctatgc gagctttaac tggcagcaca acggccgtca aatgtatgtg 420
gcgctgaacg gtaaaggtgc gccgcgtcgt ggtcagaaaa cccgtcgtaa gaacaccagc 480
gcgcacttcc tgccgatggt ggttcacagc taa 513
<210> 36
<211> 678
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 36
atggcggcgc tggcgagcag cctgatccgt cagaagcgtg aggtgcgcga gccgggtggc 60
agccgtccgg ttagcgcgca acgtcgtgtt tgcccgcgtg gcaccaagag cctgtgccag 120
aaacaactgc tgattctgct gagcaaagtg cgtctgtgcg gtggccgtcc ggcgcgtccg 180
gaccgtggtc cggagccgca gctgaagggt atcgttacca aactgttctg ccgtcagggc 240
ttttacctgc aagcgaaccc ggacggtagc attcaaggca ccccggaaga taccagcagc 300
ttcacccact ttaacctgat cccggttggt ctgcgtgtgg ttaccattca gagcgcgaag 360
ctgggccact acatggcgat gaacgcggag ggtctgctgt atagcagccc gcacttcacc 420
gcggaatgcc gtttcaaaga gtgcgtgttt gaaaactact atgttctgta cgcgagcgcg 480
ctgtatcgtc agcgtcgtag cggtcgtgcg tggtacctgg gtctggataa agagggtcaa 540
gtgatgaaag gtaaccgtgt taagaaaacc aaggcggcgg cgcactttct gccgaaactg 600
ctggaagtgg cgatgtatca agaaccgagc ctgcacagcg ttccggaagc gagcccgagc 660
agcccgccgg cgccgtaa 678
<210> 37
<211> 732
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 37
atggcggcgg cgatcgcgag cagcctgatt cgtcagaagc gtcaagcgcg tgagagcaac 60
agcgaccgtg tgagcgcgag caagcgtcgt agcagcccga gcaaagatgg ccgtagcctg 120
tgcgaacgtc acgtgctggg tgttttcagc aaagtgcgtt tttgcagcgg ccgtaaacgt 180
ccggttcgtc gtcgtccgga gccgcagctg aaaggtatcg ttacccgtct gttcagccag 240
caaggctact ttctgcaaat gcacccggac ggtaccattg atggcaccaa ggacgaaaac 300
agcgattata ccctgttcaa cctgatcccg gtgggcctgc gtgtggttgc gattcagggt 360
gtgaaagcga gcctgtacgt tgcgatgaac ggcgagggct acctgtatag cagcgacgtt 420
tttaccccgg aatgcaagtt caaagagagc gtgtttgaaa actactatgt tatctacagc 480
agcaccctgt atcgtcagca agagagcggt cgtgcgtggt tcctgggtct gaacaaggaa 540
ggccaaatta tgaaaggtaa ccgtgtgaag aaaaccaagc cgagcagcca ctttgtgccg 600
aaaccgatcg aggtttgcat gtatcgtgaa ccgagcctgc acgagattgg tgaaaagcag 660
ggccgtagcc gtaaaagcag cggcaccccg accatgaacg gtggcaaggt ggttaaccaa 720
gatagcacct aa 732
<210> 38
<211> 738
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 38
atggcggcgg cgatcgcgag cagcctgatt cgtcaaaagc gtcaggcgcg tgagcgtgaa 60
aagagcaacg cgtgcaaatg cgtgagcagc ccgagcaagg gcaaaaccag ctgcgacaag 120
aacaaactga acgtgttcag ccgtgttaaa ctgtttggta gcaagaagcg tcgtcgtcgt 180
cgtccggagc cgcaactgaa gggcatcgtt accaaactgt acagccgtca gggttatcac 240
ctgcaactgc aggcggacgg taccattgat ggcaccaagg acgaagatag cacctacacc 300
ctgttcaacc tgatcccggt gggcctgcgt gtggttgcga ttcaaggtgt tcagaccaaa 360
ctgtatctgg cgatgaacag cgagggctac ctgtatacca gcgagctgtt taccccggaa 420
tgcaagttca aagagagcgt gtttgaaaac tactatgtta cctacagcag catgatctat 480
cgtcagcaac agagcggtcg tggctggtac ctgggtctga acaaagaggg tgaaattatg 540
aaaggtaacc acgtgaagaa aaacaaaccg gcggcgcact tcctgccgaa gccgctgaaa 600
gttgcgatgt ataaagagcc gagcctgcac gatctgaccg aatttagccg tagcggtagc 660
ggcaccccga ccaagagccg tagcgtgagc ggtgttctga acggtggcaa aagcatgagc 720
cacaacgaaa gcacctaa 738
<210> 39
<211> 744
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 39
atggcggcgg cgattgcgag cggtctgatt cgtcagaagc gtcaagcgcg tgaacaacac 60
tgggaccgtc cgagcgcgag ccgtcgtcgt agcagcccga gcaagaaccg tggtctgtgc 120
aacggcaacc tggtggacat cttcagcaaa gttcgtattt ttggtctgaa gaaacgtcgt 180
ctgcgtcgtc aggacccgca gctgaaaggc attgtgaccc gtctgtactg ccgtcagggt 240
tactatctgc aaatgcaccc ggacggtgcg ctggatggca ccaaggacga tagcaccaac 300
agcaccctgt tcaacctgat cccggtgggc ctgcgtgtgg ttgcgattca gggtgttaaa 360
accggcctgt atatcgcgat gaacggcgag ggctacctgt atccgagcga gctgtttacc 420
ccggaatgca agttcaaaga gagcgtgttc gaaaactact acgttatcta cagcagcatg 480
ctgtatcgtc agcaagagag cggtcgtgcg tggttcctgg gtctgaacaa ggaaggccaa 540
gcgatgaaag gtaaccgtgt gaagaaaacc aagccggcgg cgcactttct gccgaaaccg 600
ctggaagtgg cgatgtaccg tgaaccgagc ctgcacgatg tgggcgaaac cgttccgaag 660
ccgggtgtga ccccgagcaa aagcaccagc gcgagcgcga tcatgaacgg tggcaagccg 720
gttaacaaga gcaaaaccac ctaa 744
<210> 40
<211> 624
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 40
atggcggaag tgggtggtgt gttcgcgagc ctggactggg atctgcacgg ctttagcagc 60
agcctgggta acgtgccgct ggcggacagc ccgggcttcc tgaacgagcg tctgggtcag 120
atcgaaggca agctgcaacg tggcagcccg accgattttg cgcacctgaa aggtatcctg 180
cgtcgtcgtc agctgtactg ccgtaccggt ttccacctgg agatttttcc gaacggtacc 240
gtgcatggta cccgtcatga ccacagccgt ttcggcatcc tggaatttat tagcctggcg 300
gtgggtctga tcagcattcg tggtgttgat agcggcctgt acctgggtat gaacgagcgt 360
ggcgaactgt atggtagcaa gaaactgacc cgtgagtgcg ttttccgtga acaatttgag 420
gaaaactggt acaacaccta tgcgagcacc ctgtacaagc acagcgacag cgagcgtcag 480
tactatgtgg cgctgaacaa agatggcagc ccgcgtgaag gttatcgtac caagcgtcac 540
caaaaattca cccactttct gccgcgtccg gttgacccga gcaagctgcc gagcatgagc 600
cgtgacctgt tccactatcg ttaa 624
<210> 41
<211> 588
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 41
atgacccaag gcgagaacca cccgagcccg aacttcaacc agtacgtgcg tgaccaaggt 60
gcgatgaccg atcagctgag ccgtcgtcaa attcgtgaat accagctgta tagccgtacc 120
agcggcaagc acgtgcaggt taccggtcgt cgtattagcg cgaccgcgga ggacggcaac 180
aagttcgcga aactgatcgt ggaaaccgat acctttggca gccgtgttcg tatcaagggt 240
gcggagagcg aaaaatacat ttgcatgaac aagcgtggta aactgatcgg caaaccgagc 300
ggcaagagca aagactgcgt gttcaccgag attgttctgg aaaacaacta taccgcgttt 360
caaaacgcgc gtcacgaggg ctggttcatg gcgtttaccc gtcagggtcg tccgcgtcag 420
gcgagccgta gccgtcagaa ccaacgtgaa gcgcacttca tcaagcgtct gtatcagggc 480
caactgccgt ttccgaacca cgcggagaag cagaaacaat tcgaatttgt tggtagcgcg 540
ccgacccgtc gtaccaaacg tacccgtcgt ccgcagccgc tgacctaa 588
<210> 42
<211> 525
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 42
atggcggagg aaaacgtgga cttccgtatc cacgttgaga accagacccg tgcgcgtgac 60
gatgtgagcc gtaagcagct gcgtctgtac caactgtata gccgtaccag cggcaaacac 120
atccaagttc tgggtcgtcg tattagcgcg cgtggtgagg acggcgataa gtacgcgcag 180
ctgctggttg agaccgacac cttcggcagc caagttcgta tcaagggtaa agagaccgaa 240
ttttatctgt gcatgaaccg taagggtaaa ctggtgggca agccggatgg taccagcaaa 300
gaatgcgtgt tcattgagaa ggttctggaa aacaactaca ccgcgctgat gagcgcgaaa 360
tacagcggct ggtacgttgg ttttaccaag aaaggccgtc cgcgtaaggg tccgaaaacc 420
cgtgagaacc agcaagatgt tcacttcatg aagcgttacc cgaaaggcca gccggaactg 480
caaaagccgt ttaaatatac caccgttacc aaacgtagcc gttaa 525
<210> 43
<211> 588
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 43
atgcgtccgc tggcgtttag cgatgcgggt ccgcatgtgc actatggttg gggcgatccg 60
atccgtctgc gtcacctgta taccagcggt ccgcatggtc tgagcagctg ctttctgcgt 120
attcgtgcgg acggtgtggt tgattgcgcg cgtggtcaga gcgcgcacag cctgctggaa 180
atcaaggcgg tggcgctgcg taccgttgcg attaaaggtg tgcacagcgt tcgttacctg 240
tgcatgggtg cggacggcaa gatgcagggc ctgctgcaat atagcgagga agactgcgcg 300
ttcgaggaag agatccgtcc ggatggctac aacgtgtatc gtagcgagaa acaccgtctg 360
ccggttagcc tgagcagcgc gaagcagcgt caactgtaca aaaaccgtgg tttcctgccg 420
ctgagccact ttctgccgat gctgccgatg gttccggaag agccggaaga cctgcgtggc 480
cacctggaga gcgatatgtt cagcagcccg ctggaaaccg acagcatgga cccgtttggt 540
ctggtgaccg gcctggaagc ggttcgtagc ccgagctttg agaagtaa 588
<210> 44
<211> 633
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 44
atgccgctgg cggaagtggg tggtttcctg ggtggtctgg aaggtctggg ccagcaagtt 60
ggcagccact ttctgctgcc gccggcgggt gaacgtccgc cgctgctggg cgagcgtcgt 120
agcgcggcgg aacgtagcgc gcgtggtggc ccgggtgcgg cgcagctggc gcacctgcac 180
ggtatcctgc gtcgtcgtca gctgtactgc cgtaccggtt tccacctgca aattctgccg 240
gatggtagcg tgcagggtac ccgtcaagat cacagcctgt tcggcatcct ggaatttatt 300
agcgtggcgg ttggtctggt tagcatccgt ggtgttgaca gcggcctgta cctgggtatg 360
aacgataagg gcgagctgta tggtagcgaa aaactgacca gcgagtgcat cttccgtgaa 420
cagtttgagg aaaactggta caacacctat agcagcaaca tttacaagca cggtgacacc 480
ggccgtcgtt attttgttgc gctgaacaaa gatggtaccc cgcgtgatgg tgcgcgtagc 540
aagcgtcacc aaaaattcac ccactttctg ccgcgtccgg tggacccgga gcgtgttccg 600
gaactgtaca aggatctgct gatgtatacc taa 633
<210> 45
<211> 549
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 45
atgcatccga ttccggacag cagcccgctg ctgcagtttg gtggccaagt gcgtcaacgt 60
tacctgtata ccgacgatgc gcagcaaacc gaggcgcacc tggagattcg tgaagacggt 120
accgttggtg gcgcggcgga tcagagcccg gaaagcctgc tgcaactgaa ggcgctgaaa 180
ccgggtgtga tccagattct gggcgttaag accagccgtt ttctgtgcca acgtccggat 240
ggtgcgctgt atggcagcct gcacttcgat ccggaggcgt gcagctttcg tgagctgctg 300
ctggaagacg gctacaacgt gtatcaaagc gaagcgcatg gtctgccgct gcacctgccg 360
ggtaacaaga gcccgcaccg tgatccggcg ccgcgtggtc cggcgcgttt cctgccgctg 420
ccgggtctgc cgccggcgct gccggagccg ccgggtatcc tggcgccgca gccgccggac 480
gtgggcagca gcgatccgct gagcatggtt ggtccgagcc aaggccgtag cccgagctat 540
gcgagctaa 549
<210> 46
<211> 453
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 46
atgggtaccc cgagcgcgag ccgtggtccg cgtagctacc cgcacctgga gggtgacgtt 60
cgttggcgtc gtctgttcag cagcacccac ttctttctgc gtgttgaccc gggtggccgt 120
gtgcagggta cccgttggcg tcacggccaa gatagcatcc tggaaattcg tagcgttcac 180
gtgggtgtgg ttgtgatcaa ggcggttagc agcggcttct atgtggcgat gaaccgtcgt 240
ggtcgtctgt acggcagccg tctgtatacc gtggactgcc gttttcgtga gcgtattgag 300
gaaaacggtc acaacaccta cgcgagccag cgttggcgtc gtcgtggtca accgatgttc 360
ctggcgctgg atcgtcgtgg tggcccgcgt ccgggtggcc gtacccgtcg ttatcacctg 420
agcgcgcact ttctgccggt tctggttagc taa 453
<210> 47
<211> 687
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 47
atgtatccga acgcgagccc gctgctgggt agcagctggg gtggcctgat ccacctgtac 60
accgcgaccg cgcgtaacag ctatcacctg cagattcaca aaaacggtca tgtggatggt 120
gcgccgcacc aaaccatcta tagcgcgctg atgattcgta gcgaggatgc gggttttgtg 180
gttatcaccg gcgttatgag ccgtcgttac ctgtgcatgg acttccgtgg taacattttt 240
ggcagccact atttcgatcc ggagaactgc cgtttccagc accaaaccct ggaaaacggc 300
tacgacgtgt atcacagccc gcagtaccac tttctggtta gcctgggtcg tgcgaagcgt 360
gcgttcctgc cgggtatgaa cccgccgccg tatagccaat ttctgagccg tcgtaacgag 420
atcccgctga ttcacttcaa caccccgatt ccgcgtcgtc acacccgtag cgcggaggac 480
gatagcgaac gtgatccgct gaacgtgctg aaaccgcgtg cgcgtatgac cccggcgccg 540
gcgagctgca gccaggagct gccgagcgcg gaagacaaca gcccgatggc gagcgatccg 600
ctgggtgtgg ttcgtggtgg ccgtgttaac acccatgcgg gtggcaccgg tccggaaggt 660
tgccgtccgt tcgcgaaatt catctaa 687
<210> 48
<211> 762
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
atgaagagca gccatcacca ccaccaccat ggcagcagcg tgagcaaagg cgaggaactg 60
ttcaccggcg tggttccgat cctggtggaa ctggacggcg atgttaacgg tcacaagttt 120
agcgttcgtg gtgagggcga aggtgatgcg accaacggca agctgaccct gaaattcatt 180
tgcaccaccg gtaaactgcc ggtgccgtgg ccgaccctgg ttaccaccct gacctacggt 240
gtgcagtgct ttagccgtta tccggaccac atgaaacaac acgatttctt taagagcgcg 300
atgccggaag gttacgttca ggagcgtacc atcagcttca aagacgatgg cacctataag 360
acccgtgcgg aagtgaaatt tgaaggtgat accctggtta accgtatcga actgaaaggc 420
attgacttca aagaggacgg caacatcctg ggtcacaagc tggaatacaa ctttaacagc 480
cacaacgtgt atattaccgc ggacaagcag aaaaacggta tcaaggcgaa ctttaaaatt 540
cgtcacaacg tggaggacgg cagcgttcaa ctggcggatc actaccagca aaacaccccg 600
attggtgatg gtccggttct gctgccggat aaccactatc tgagcaccca gagcaagctg 660
agcaaagacc cgaacgaaaa gcgtgatcac atggtgctgc tggagttcgt taccgcggcg 720
ggcatcaccc tgggtatgga tgaactgtac aaaggtattg ag 762
<210> 49
<211> 294
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
atgagcgaca gcgaggtgaa ccaagaagcg aagccggaag tgaaaccgga agttaagccg 60
gagacccaca tcaacctgaa agttagcgac ggcagcagcg agatcttctt taaaattaag 120
aaaaccaccc cgctgcgtcg tctgatggaa gcgttcgcga aacgtcaggg caaggagatg 180
gacagcctgc gttttctgta tgatggcatc cgtattcagg cggaccaaac cccggaagac 240
ctggatatgg aggacaacga tatcattgaa gcgcaccgtg agcaaattgg tggc 294
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ggcggcggct ccggcggcgg ctcc 24
<210> 51
<211> 232
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Met Lys Ser Ser His His His His His His Gly Ser Ser Leu Val Pro
1 5 10 15
Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln Lys Ala Leu Ala Ser
20 25 30
Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn Ile Glu Ile Thr Val
35 40 45
Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp Leu Asn Asp Thr Ile
50 55 60
Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser Thr Pro Asn Thr Val
65 70 75 80
Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser Glu Arg Gly Tyr Gln
85 90 95
Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr Gln Ile Asp Lys Leu
100 105 110
Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln Ser His Trp Ala Leu
115 120 125
Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly Tyr Val Asp Ser Leu
130 135 140
Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile Leu Thr Asp Leu Gln
145 150 155 160
Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile Gly Glu Asp Phe Asp
165 170 175
Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn Gly Tyr Asp Cys Gly
180 185 190
Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser Ala Asp Ala Pro Leu
195 200 205
Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg Arg Phe Ile Ala His
210 215 220
Leu Ile Leu Thr Asp Ala Leu Lys
225 230
<210> 52
<211> 699
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
atgaaaagca gccaccacca ccaccaccac ggtagcagcc tggtgccgga gctgaacgaa 60
aaagacgatg accaggttca aaaggcgctg gcgagccgtg agaacaccca gctgatgaac 120
cgtgataaca tcgaaattac cgtgcgtgac ttcaagaccc tggcgccgcg tcgttggctg 180
aacgatacca tcatcgagtt ctttatgaag tacatcgaaa agagcacccc gaacaccgtg 240
gcgtttaaca gcttctttta caccaacctg agcgagcgtg gttatcaggg cgttcgtcgt 300
tggatgaagc gtaagaaaac ccaaatcgat aaactggaca agatcttcac cccgattaac 360
ctgaaccaga gccactgggc gctgggcatc attgatctga agaaaaagac catcggttac 420
gtggacagcc tgagcaacgg cccgaacgcg atgagcttcg cgattctgac cgatctgcaa 480
aaatatgtta tggaggaaag caagcacacc atcggtgaag attttgacct gattcacctg 540
gattgcccgc agcaaccgaa cggttacgac tgcggcatct atgtttgcat gaacaccctg 600
tatggcagcg cggatgcgcc gctggatttc gactataaag acgcgattcg tatgcgtcgt 660
tttatcgcgc acctgattct gaccgacgcg ctgaagtaa 699
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tttgtttaac tttaagaagg aga 23

Claims (11)

1. A composition comprising tris (2-carbonylethyl) phosphonium hydrochloride and zinc sulfate.
2. The composition of claim 1, wherein the mass ratio of the tris (2-carbonylethyl) phosphorus hydrochloride to the zinc sulfate is 1 (1 to 50).
3. A fibroblast growth factor-containing fusion protein comprising, from N-terminus to C-terminus, a visual reporter protein, a small ubiquitin-like modifier and a fibroblast growth factor.
4. The fusion protein of claim 3, wherein the fibroblast growth factor is human fibroblast growth factor-1, human fibroblast growth factor-2, human fibroblast growth factor-3, human fibroblast growth factor-4, human fibroblast growth factor-5, human fibroblast growth factor-6, human fibroblast growth factor-7, human fibroblast growth factor-8, human fibroblast growth factor-9, human fibroblast growth factor-10, human fibroblast growth factor-11, human fibroblast growth factor-12, human fibroblast growth factor-13, human fibroblast growth factor-14, or a combination thereof, One of human fibroblast growth factor-16, human fibroblast growth factor-17, human fibroblast growth factor-18, human fibroblast growth factor-19, human fibroblast growth factor-20, human fibroblast growth factor-21, human fibroblast growth factor-22 and human fibroblast growth factor-23;
preferably, the amino acid sequence of the human fibroblast growth factor-1 is shown as SEQ ID No.1, the amino acid sequence of the human fibroblast growth factor-2 is shown as SEQ ID No.2, the amino acid sequence of the human fibroblast growth factor-3 is shown as SEQ ID No.3, the amino acid sequence of the human fibroblast growth factor-4 is shown as SEQ ID No.4, the amino acid sequence of the human fibroblast growth factor-5 is shown as SEQ ID No.5, the amino acid sequence of the human fibroblast growth factor-6 is shown as SEQ ID No.6, the amino acid sequence of the human fibroblast growth factor-7 is shown as SEQ ID No.7, the amino acid sequence of the human fibroblast growth factor-8 is shown as SEQ ID No.8, the amino acid sequence of the human fibroblast growth factor-9 is shown as SEQ ID No.9, the amino acid sequence of the human fibroblast growth factor-10 is shown as SEQ ID No.10, the amino acid sequence of the human fibroblast growth factor-11 is shown as SEQ ID No.11, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.12, the amino acid sequence of the human fibroblast growth factor-13 is shown as SEQ ID No.13, the amino acid sequence of the human fibroblast growth factor-14 is shown as SEQ ID No.14, the amino acid sequence of the human fibroblast growth factor-16 is shown as SEQ ID No.15, the amino acid sequence of the human fibroblast growth factor-17 is shown as SEQ ID No.16, the amino acid sequence of the human fibroblast growth factor-18 is shown as SEQ ID No.17, the amino acid sequence of the human fibroblast growth factor-19 is shown as SEQ ID No.18, the amino acid sequence of the human fibroblast growth factor-20 is shown as SEQ ID No.19, the amino acid sequence of the human fibroblast growth factor-21 is shown as SEQ ID No.20, the amino acid sequence of the human fibroblast growth factor-22 is shown as SEQ ID No.21, and the amino acid sequence of the human fibroblast growth factor-23 is shown as SEQ ID No. 22.
5. The fusion protein of claim 3, wherein the visual reporter protein is a fluorescent protein, preferably wherein the visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein and cyan fluorescent protein; preferably, the amino acid sequence of the green fluorescent protein is shown as SEQ ID No. 23; and/or
The amino acid sequence of the small ubiquitin-like modifier is shown in SEQ ID No. 24;
preferably, the visual reporter protein and the small ubiquitin-like modifier are connected by a polypeptide linker; preferably, the amino acid sequence of the polypeptide linker is shown as SEQ ID No. 25.
6. An expression cassette for expressing the fusion protein of any one of claims 3 to 5, comprising a first promoter from 5 'end to 3' end, a nucleic acid encoding the fusion protein, and a first terminator;
preferably, the first promoter is the T7 promoter and the first terminator is the T7 terminator;
preferably, the nucleotide sequence for coding the human fibroblast growth factor-1 is shown as SEQ ID No.26, the nucleotide sequence for coding the human fibroblast growth factor-2 is shown as SEQ ID No.27, the nucleotide sequence for coding the human fibroblast growth factor-3 is shown as SEQ ID No.28, the nucleotide sequence for coding the human fibroblast growth factor-4 is shown as SEQ ID No.29, the nucleotide sequence for coding the human fibroblast growth factor-5 is shown as SEQ ID No.30, the nucleotide sequence for coding the human fibroblast growth factor-6 is shown as SEQ ID No.31, the nucleotide sequence for coding the human fibroblast growth factor-7 is shown as SEQ ID No.32, the nucleotide sequence for coding the human fibroblast growth factor-8 is shown as SEQ ID No.33, the nucleotide sequence for coding the human fibroblast growth factor-9 is shown as SEQ ID No.34, the nucleotide sequence for coding the human fibroblast growth factor-10 is shown as SEQ ID No.35, the nucleotide sequence for coding the human fibroblast growth factor-11 is shown as SEQ ID No.36, the nucleotide sequence for coding the human fibroblast growth factor-12 is shown as SEQ ID No.37, the nucleotide sequence for coding the human fibroblast growth factor-13 is shown as SEQ ID No.38, the nucleotide sequence for coding the human fibroblast growth factor-14 is shown as SEQ ID No.39, the nucleotide sequence for coding the human fibroblast growth factor-16 is shown as SEQ ID No.40, and the nucleotide sequence for coding the human fibroblast growth factor-17 is shown as SEQ ID No.41, the nucleotide sequence for coding the human fibroblast growth factor-18 is shown as SEQ ID No.42, the nucleotide sequence for coding the human fibroblast growth factor-19 is shown as SEQ ID No.43, the nucleotide sequence for coding the human fibroblast growth factor-20 is shown as SEQ ID No.44, the nucleotide sequence for coding the human fibroblast growth factor-21 is shown as SEQ ID No.45, the nucleotide sequence for coding the human fibroblast growth factor-22 is shown as SEQ ID No.46, and the nucleotide sequence for coding the human fibroblast growth factor-23 is shown as SEQ ID No. 47;
preferably, the nucleotide sequence encoding the visual reporter protein is shown as SEQ ID No. 48;
preferably, the nucleotide sequence for coding the small ubiquitin-like modifier is shown as SEQ ID No. 49;
preferably, the nucleotide sequence encoding the polypeptide linker is shown in SEQ ID No. 50.
7. A set of expression cassettes for expressing the fusion protein according to any one of claims 3 to 5 or for expressing the fusion protein according to any one of claims 3 to 5 and purifying the fibroblast growth factor, comprising the expression cassette according to claim 6 and an expression cassette for expressing a small ubiquitin-like modifier protease;
preferably, the expression cassette for expressing a small ubiquitin-like modifier protease comprises a second promoter from 5 'to 3', a 3 '-UTR, a nucleic acid encoding a small ubiquitin-like modifier protease and a 5' -UTR;
preferably, the second promoter is a CspA promoter, the 3 '-UTR is a CspA 3' -UTR, and the 5 '-UTR is a CspA 5' -UTR;
preferably, the amino acid sequence of the small ubiquitin-like modifier protease is shown in SEQ ID No. 51;
preferably, the nucleotide sequence encoding the small ubiquitin-like modifier protease is shown in SEQ ID No. 52.
8. A method of obtaining the fusion protein of any one of claims 3 to 5 or the fibroblast growth factor therein, comprising the steps of:
1-1) ligating the expression cassette of claim 6 or a nucleic acid encoding said fusion protein to a first expression vector to obtain a first recombinant expression vector; wherein the first expression vector is an isopropyl-beta-D-thiogalactoside inducible expression vector;
2-1) connecting the expression cassette for expressing a small ubiquitin-like modifier protease or a nucleic acid encoding a small ubiquitin-like modifier protease in the expression cassette of claim 7 to the first recombinant expression vector to obtain a second recombinant expression vector;
3-1) transforming the second recombinant expression vector into a first host to obtain a first recombinant organism;
4-1) culturing the first recombinant organism in a first culture solution until the fusion protein and the small ubiquitin-like modifier protease are expressed to obtain a first culture;
5-1) purifying said first culture to obtain said fibroblast growth factor;
or
1-2) same as 1-1);
2-2) connecting the expression cassette for expressing a small ubiquitin-like modifier protease or a nucleic acid encoding a small ubiquitin-like modifier protease in the expression cassette of claim 7 to the second expression vector to obtain a third recombinant expression vector;
3-2) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism; transforming the third recombinant expression vector into a third host to obtain a third recombinant organism;
4-2) culturing the second recombinant organism in a second culture fluid until the fusion protein is expressed to obtain a second culture; culturing the third recombinant organism in a third culture solution until the small ubiquitin-like modifier protease is expressed to obtain a third culture; mixing the second culture with the third culture to obtain a fourth culture;
5-2) purifying said fourth culture, thereby obtaining said fibroblast growth factor;
or
1-3) same as 1-1);
2-3) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism as in 3-2);
3-3) culturing the second recombinant organism in a second culture solution until the fusion protein is expressed to obtain a second culture;
4-3) purifying the second culture, thereby obtaining the fusion protein;
preferably, the first culture liquid, the second culture liquid and the third culture liquid are independently LB culture liquid or LB culture liquid containing glucose.
9. The method of claim 8, wherein the first expression vector is one of the pET series of expression vectors;
preferably, the first expression vector is one of pET21a, pET20b, and pET28 a;
the second expression vector is pCold;
the first host, the second host, and the third host are independently Escherichia coli (Escherichia coli); preferably, the strain of E.coli is BL21(DE 3).
10. The method according to claim 8 or 9, wherein in step 4-2) or step 3-3), the second recombinant organism is cultured at a temperature of 28 to 37 ℃ for 4 to 8 hours;
in step 4-1), culturing the first recombinant organism at a temperature of 28 to 37 ℃ for 4 to 8 hours under induction of isopropyl- β -D-thiogalactoside, and then continuing culturing the first recombinant organism at a temperature of 10 to 15 ℃ for 15 to 60 minutes;
in step 4-2), the third recombinant organism is cultured at a temperature of 10 to 15 ℃ for 15 to 60 minutes.
11. The method according to claim 8, wherein the composition of claim 1 or 2 is added to the first culture solution while the culture temperature is decreased to the 10 to 15 ℃ in step 4-1);
preferably, the final concentration of the tris (2-carbonylethyl) phosphonium hydrochloride in the first culture liquid is from 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the first broth is 1 to 5 mmol/l;
in step 4-2), adding the composition of claim 1 or 2 to the second culture liquid;
preferably, the final concentration of the tris (2-carbonylethyl) phosphonium hydrochloride in the second culture liquid is from 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the second broth is 1 to 5 mmol/l.
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