WO2020093789A1 - 用于改善活性蛋白或多肽性能的人工重组蛋白及其应用 - Google Patents

用于改善活性蛋白或多肽性能的人工重组蛋白及其应用 Download PDF

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WO2020093789A1
WO2020093789A1 PCT/CN2019/106092 CN2019106092W WO2020093789A1 WO 2020093789 A1 WO2020093789 A1 WO 2020093789A1 CN 2019106092 W CN2019106092 W CN 2019106092W WO 2020093789 A1 WO2020093789 A1 WO 2020093789A1
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polypeptide
protein
unit
fusion protein
active
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French (fr)
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黄岩山
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浙江道尔生物科技有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • 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
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    • 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
    • C07K14/495—Transforming growth factor [TGF]
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • 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
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03001—Arginase (3.5.3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to the field of biotechnology. More specifically, the present invention relates to an artificial recombinant protein for improving the performance of biologically active proteins or polypeptides and its application.
  • the carrier used for cross-linking is generally PEG or fatty acid, etc., and human serum albumin, immunoglobulin Fc fragment, transferrin, etc. are generally used for recombinant fusion, and most of them have corresponding successful marketed drugs.
  • new recombinant fusion carrier proteins have continuously emerged (WR Strohl, Biodrugs, 2015, 29 (4): 215-39), such as URP (Chinese patent ZL200780015899.2), XTEN (Chinese patent application CN201080011467.6; Volker Schellenberger Etc., Nature Biotechnology 27 (12): 1186, 2009), elastin-like ELP (MacEwan SR, etc., J Control Release.
  • the Chinese patent with the patent number ZL200780015899.2 discloses an unstructured recombinant polymer (URP), which basically cannot non-specifically bind to serum proteins, and is characterized by: (a) containing at least 100 contiguous amino acids; (b) Glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) contained in URP ) The sum of the residues accounts for more than about 80% of all amino acids in the URP; (c) at least 50% of the amino acids of the URP sequence do not form a secondary structure as determined by the Chou-Fasman algorithm; (d) the URP T epitope score is less than -4.
  • URP unstructured recombinant polymer
  • the Chinese patent application with the application number CN201080011467.6 discloses an isolated extended recombinant polypeptide (XTEN) containing more than about 400 to about 3000 amino acid residues, wherein the XTEN is characterized by: (a) glycine (G), The sum of alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residues accounts for more than about 80% of the total amino acid sequence of XTEN; (b ) The XTEN sequence is basically non-repetitive; (c) When analyzed by the TEPITOPE algorithm, the XTEN sequence lacks a predicted T cell epitope, wherein the TEPITOPE algorithm prediction of the epitope within the XTEN sequence is based on a score of -9 or higher ; (D) determined by the GOR algorithm, the XTEN sequence has more than 90% random coil formation; and (e) determined by the Chou-Fasman algorithm, the XTEN sequence has an
  • the Chinese patent with the patent number ZL200880019017 discloses a biologically active protein comprising at least two domains, wherein: (a) the first domain of the at least two domains contains and / or mediates the biological activity Amino acid sequence of; and (b) the second domain of the at least two domains comprises an amino acid sequence consisting of at least 10 amino acid residues forming a random coil conformation, wherein the second domain is composed of alanine , Serine and proline residues, wherein the random coil conformation mediates the increased stability of the biologically active protein in vivo and / or in vitro.
  • Elastin-like ELP is composed of (VPGXG) n, where X can be any amino acid except Proline (Pro).
  • the number of n is not fixed, ELP has a characteristic that the state will undergo a sharp transition at a specific temperature (span 2-3 ° C): below this temperature, the ELP is in a soluble state; above this temperature, ELP will occur rapidly Aggregate into microscopic particles visible to the naked eye; lower the temperature again, and the ELP will re-dissolve; this temperature is called the reverse conversion temperature, referred to as the phase transition temperature (Tt).
  • Tt phase transition temperature
  • ELP is an elastin, which is biodegradable and non-immunogenic, so it is suitable for use as a fusion protein that prolongs the half-life of drugs.
  • the Chinese patent with the patent number ZL200980103870.9 discloses a recombinant gelatin-like unit (GLK) for prolonging the half-life of protein in vivo, characterized in that the gelatin-like unit is a polypeptide with the following structure: (Gly-XY) n ;
  • Gly is a glycine residue;
  • X and Y are any amino acid residues of 20 natural amino acids except Cys, and Hyp; n is 20-300; and, the gelatin-like unit has the following characteristics: (a) The following hydrophilic amino acids Asn, Asp, Gln, Glu, Lys, Pro, Ser, Hyp, and Arg in the gelatin-like unit have a total percentage amino acid content of 40% to 2/3;
  • the gelatin In the sample unit the ratio of the sum of Pro and Hyp to n is ⁇ 0.6;
  • the ratio of the sum of Gly to n is ⁇ 1.15; and, according to the ProtParam formula, the
  • the XTEN sequence can be designed to have a net negative charge to minimize non-specific interactions between the XTEN-containing composition and various surfaces such as blood vessels, healthy tissues, or various receptors "(application number CN201080011467.6);
  • PAS focuses on imitating polyethylene glycol (PEG) and uses three uncharged amino acids: proline, alanine, and serine.
  • the XTEN sequence emphasizes the feature of "substantially non-repetitive”: "Repetitive amino acid sequences have a tendency to aggregate to form higher-order structures, examples of which are natural repetitive sequences such as collagen and leucine zipper, or contact, resulting Crystal or quasi-crystal structure. In contrast, the low tendency of non-repetitive sequence aggregation allows the design of long sequences of XTEN with relatively low frequency charged amino acids, which may aggregate if the sequence repeats. " The interpretation of "substantially non-repetitive" by XTEN technology is “referring to a lack or limited degree of internal homology in a peptide or polypeptide sequence.
  • the polypeptide has a subsequence score of 10 or lower, or there is no pattern of motifs constituting the polypeptide sequence in the order from N-terminus to C-terminus ".
  • XTEN, PAS, and URP are rich in S and T, the degree of glycosylation is particularly high when expressed in eukaryotic systems.
  • the active protein or polypeptide may significantly reduce its biological activity after fusion or cross-linking with these carrier proteins, as reported by Gething NC and others, the glucagon-XTEN fusion protein only exhibits the unmodified glucagon polypeptide 15 % Biological activity (Gething NC, etc., PLoS One, 2010, 5 (4): e10175), however, the improvement of physical and chemical properties such as stability and solubility after fusion or crosslinking can make up for this deficiency.
  • the purpose of the present invention is to provide an artificial recombinant protein that improves the performance of active protein or polypeptide and its application.
  • a polypeptide unit (U) which has the following characteristics: (1) is composed of proline (P), alanine (A) and glutamic acid (E); (2 ) There is more than 50% of the ⁇ -helix secondary structure; and (3) ⁇ 15 amino acids in length.
  • the polypeptide unit has an alpha-helix secondary level of more than 60%, preferably more than 70%, more preferably more than 80%, and even more preferably more than 90% structure.
  • the ratio of the ⁇ -helix secondary structure is calculated according to the Chou-Fasman algorithm; and / or ⁇ 20 amino acids in length.
  • the length of the polypeptide unit is 15-100 aa, preferably 16-60 aa; specifically, such as 18 aa, 19 aa, 20 aa, 22 aa, 25 aa, 30 aa, 40 aa, 50 aa, 60 aa, 70 aa, 80aa or 90aa.
  • the polypeptide unit includes a polypeptide unit selected from the group consisting of: SEQ ID NO: 19-47 amino acid sequence polypeptide unit.
  • a polypeptide composite unit (PU) is provided, the core structure of which is selected from:
  • U 1 , U 2 ,..., U n each represent one of the described polypeptide units, and n is a positive integer>2; and, the amino acid sequences of two or more of the polypeptide units are the same or different.
  • n is a positive integer from 3 to 100, for example, a positive integer from 4 to 90, and a positive integer from 5 to 80; more specifically, n is, for example, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 40, 50, 60, 70.
  • the number of amino acid residues in the core structure accounts for more than 70% of the total number of amino acid residues in the polypeptide complex unit, preferably more than 80%, more preferably 85%, further more preferably 90 % Or more, 95% or more, 99% or more or 100%.
  • the polypeptide complex unit is selected from: (a) a protein with an amino acid sequence shown in any one of SEQ ID NO: 48-105 or a repeated splicing protein of the polypeptide unit corresponding to SEQ ID NO: 48-76; Or any polypeptide defined in (b) (a) undergoes substitution, deletion or addition of one or more (eg 1-20; preferably 1-10; more preferably 1-5) amino acid residues And a polypeptide derived from (a) that has the function of (a) polypeptide; or (c) the amino acid sequence of the amino acid sequence and the polypeptide defined by (a) is more than 80% (preferably more than 85%; more preferably More than 90%; more preferably more than 95%, such as 98%, 99% or more) percent sequence similarity and having (a) polypeptide function polypeptide.
  • the polypeptide unit or the polypeptide composite unit itself has no biological function, and its properties are similar to PEG, and only functions as a "carrier".
  • a biologically active fusion protein comprising: the polypeptide unit or the polypeptide complex unit and the active protein or polypeptide.
  • the fusion protein includes a structure selected from any one or a combination of the following group:
  • PU 1 , PU 2 ,..., PU m are selected from the polypeptide complex units; D 1 , D 2 ,..., D m are the active polypeptides, and m is a positive integer>2;
  • (D 1 -PU 1 ) includes (PU 1 -D 1 ), (D 2 -PU 2 ) includes (PU 2 -D 2 ), and (PU m -D m ) includes (D m -PU m ).
  • D 1 , D 2 ,..., D m each represent one or more (including 2) interconnected active proteins or polypeptides, and the multiple active proteins or polypeptides have the same function or different.
  • m is a positive integer from 3 to 50, for example, a positive integer from 4 to 40, and a positive integer from 5 to 35; more specifically, m is, for example, 6, 7, 8, 9, 10, 15 , 20, 25, 30.
  • the length of the fusion protein is ⁇ 50 amino acids, preferably ⁇ 80 amino acids, more preferably ⁇ 100 amino acids; for example, 50-5000aa, preferably 80-4000aa, more A good place is 100 to 2000 aa; more specifically, for example, 150 aa, 200 aa, 300 aa, 50 aa, 60 aa, 700 aa, 800 aa, 1000 aa.
  • the ratio of the amino acid sequence occupied by the polypeptide unit or the polypeptide compound unit is more than 10%, preferably more than 20%.
  • the active protein or polypeptide includes (but is not limited to): GLP-2 analogue, AR VEGF , hGH, Arginase 1, G-CSF, Exendin-4, GLP-1 analogue, GDF15, glucacon, IL-2, IL-15, FGF19, EPO, IL-6, M-CSF, FGF21.
  • the fusion protein includes (but is not limited to) a fusion protein selected from the group consisting of: (a) a protein with an amino acid sequence shown in any one of SEQ ID NO: 106-131; or (b) (a) any defined polypeptide is formed by substitution, deletion or addition of one or more (eg 1-20; preferably 1-10; more preferably 1-5) amino acid residues, And the protein derived from (a) having (a) polypeptide activity; or (c) the amino acid sequence of the amino acid sequence of the polypeptide defined in (a) is more than 80% (preferably more than 85%; more preferably more than 90%) ; More preferably more than 95%, such as 98%, 99% or more) percent sequence similarity and having (a) polypeptide activity protein.
  • a fusion protein selected from the group consisting of: (a) a protein with an amino acid sequence shown in any one of SEQ ID NO: 106-131; or (b) (a) any defined polypeptide is formed by substitution, deletion or addition of one
  • the half-life or stability of the fusion protein in the body is statistically higher than that of the unfused active polypeptide (for example, more than 50% higher, preferably more than 100% higher, more preferably higher 200% or more, more specifically as high as 500%, 1000%, 2000%, 5000%, 10000% or more).
  • nucleic acid molecule that encodes the aforementioned polypeptide unit, polypeptide complex unit or fusion protein.
  • a recombinant expression vector which contains the nucleic acid molecule.
  • a genetically engineered cell in another aspect of the present invention, the cell contains the recombinant expression vector; or the cell genome is integrated with the nucleic acid molecule.
  • a conjugate comprising: (a) the polypeptide unit or the polypeptide complex unit; and, (b) an active protein or polypeptide; wherein, (b) and (a) Connected by coupling or adsorption.
  • the use of the polypeptide unit or the polypeptide composite unit is provided for improving the stability of an active polypeptide, preferably including thermal stability, enzyme resistance stability and serum stability; Extend the half-life of the active polypeptide, that is, extend the action time of the active polypeptide; and / or increase the solubility of the active polypeptide.
  • composition comprising: the fusion protein or the conjugate; and a pharmaceutically or food acceptable carrier.
  • FIG. 1 SEC-HPLC analysis results of PU-hArg1 fusion protein.
  • P1 is PUMix17-hArg1-PUMix49
  • P2 is PUMix35-hArg1-PUMix76
  • P3 is PUMix109-hArg1-PUMix163
  • P4 is PU98x5-hArg1-PU106x5
  • P5 is PU12x5-hArg1-PU12x5
  • P6 is PU98x10-hArg1
  • P7 is PU12x10-hArg1
  • P8 are mURP-hArg1-mURP
  • P9 is mXTEN-hArg1-mXTEN
  • P10 is mPAS-hArg1-mPAS.
  • Thyroglobulin (669kDa).
  • M is the protein molecular weight MARKER: 200, 116, 97.2, 66.4, 44.3KD.
  • Lane 1 PU27x28-GH-PU27x5 at 4 ° C for 20 hours;
  • Lane 2 PU27x28-GH-PU27x5 at 37 ° C for 20 hours;
  • Lane 3 PU27x28-GH-PU27x5 at 60 ° C for 20 hours;
  • Lane 4 PU98x28- GH-PU98x4 placed at 4 ° C for 20 hours;
  • lane 5 PU98x28-GH-PU98x4 placed at 37 ° C for 20 hours;
  • lane 6 PU98x28-GH-PU98x4 placed at 60 ° C for 20 hours;
  • Lane 7 PU130x28-GH-PU130x5 at 4 Place at 20 ° C for 20 hours;
  • Lane 8 PU130x28-GH-PU130x5 at 37 ° C for 20 hours;
  • Lane 9 PU130x28-GH-PU130x5 at
  • Figure 7 In vitro cytological activity of PU-GH fusion protein.
  • Figure 8 A graph of the effect of PU-GDF15 fusion protein on weight loss in DIO mice.
  • Figure 9 Effect of PU and GDF15 fusion protein on appetite suppression in DIO mice.
  • Figure 10 Graph of the results of in vitro cell activity tests of GLP-2G and PU fusion proteins.
  • Figure 12 PU-GH fusion protein incubated on day 7 in rat serum. 1-2 are PU27x28-GH-PU27x5 samples on day 0 and day 7, 3-4 are PU98x28-GH-PU98x4 samples on day 0 and day 7, 5-6 are on day 0 and day 7 respectively PU130x28-GH-PU130x5 samples.
  • FIG. 13 Stability results of PU-GH fusion protein and hGH in pancreatin.
  • A. Lanes 1-4 are the results of hGH incubation in pancreatin of 0, 0.02%, 0.1%, 0.5% for 40 min; M is the low molecular weight MARKER: 97.2KD, 66.4KD, 44KD, 29KD, 21KD and 14KD.
  • B. Lanes 1 and 2: PU27x28-GH-PU27x5; lanes 3 and 4: PU98x28-GH-PU98x4; lanes 5 and 6: PU130x28-GH-PU130x5.
  • M is high molecular weight MARKER: 220KD, 135KD, 90KD, 66KD, 45KD and 35KD.
  • a polypeptide unit (U) or a polypeptide complex unit (PU) that is very effective for extending the half-life of a protein / polypeptide in vivo or improving the physical and chemical properties of the protein / polypeptide in Proline (P), alanine (A) and glutamic acid (E).
  • the present invention first provides an artificially designed polypeptide unit (U), and has the following characteristics: (1) is composed of proline (P), alanine (A) and glutamic acid (E); (2) according to The Chou-Fasman formula calculates that at least 50% or more is an ⁇ -helix secondary structure; and (3) The length is ⁇ 15, preferably ⁇ 20 amino acids.
  • At least 60% of the polypeptide unit (U) is an ⁇ -helix secondary structure; preferably, at least 70% of the polypeptide unit (U) is ⁇ -helix secondary structure; preferably, at least 80% of said polypeptide unit (U) is an ⁇ -helix secondary structure; more preferably, at least 90% of said polypeptide unit (U) is an ⁇ -helix secondary structure.
  • the secondary structure of proteins or peptides can be detected using far ultraviolet circular dichroism (CD) spectroscopy.
  • CD far ultraviolet circular dichroism
  • the ⁇ -helix, ⁇ -sheet and random coil structures each form the characteristic peak and width of the CD spectrum.
  • the secondary structure of PU is predicted by the Chou-Fasman algorithm (Chou, P.Y. et al., Biochemistry, 1974, 13, 222-45).
  • polypeptide units (U) composed of P, A, and E contain more than 50% of the ⁇ -helix structure (such as SEQ ID NO: 17 and SEQ ID NO: 18), and SEQ ID NO: 17 and SEQ ID NO: 18 Because the content of proline is too high, in the preparation process of the present invention, it was found that the polypeptide composite unit (PU) composed of SEQ ID NO: 17 and SEQ ID NO: 18 is extremely difficult to obtain Expression products. Proline has a typical function of destroying secondary structure.
  • proline (P) appears in a highly repeating form, then the ⁇ -helix structure will be extremely low, and when alanine (A) or glutamic acid (E ) Highly repeated, the content of ⁇ -helical structure is significantly increased.
  • polypeptide units (U) are shown in SEQ ID NO: 19-47. It should be understood that other sequences of P, A, and E arranged in a sequence different from SEQ ID NO: 19 to 47 can also be included in the present invention, as long as it satisfies at least 50% of the ⁇ -helix secondary structure And the characteristic that the length is ⁇ 15, preferably ⁇ 20 amino acids.
  • polypeptide composite unit including the polypeptide unit (U), which includes 2 or more (including 2) of the polypeptide unit (U).
  • each polypeptide unit (U) may be arranged in the same sequence or different sequences.
  • the polypeptide composite unit has 100-2000 amino acids.
  • Exemplary preferred modes include: having at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, and at least 400 amino acids. , At least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, at least 1000 amino acids or at least 1200 amino acids.
  • more than 80% of the polypeptide complex unit (PU) is composed of proline (P), alanine (A) and glutamic acid (E); more preferably, more than 85% Composed of proline (P), alanine (A) and glutamic acid (E); more preferably, more than 90% of proline (P), alanine (A) and glutamic acid (E ) Composition; more preferably, more than 95% is composed of proline (P), alanine (A) and glutamic acid (E); most preferably, 100% is composed of proline (P), alanine (A) and glutamic acid (E).
  • the polypeptide composite unit (PU) is formed by repeatedly splicing polypeptide units (U) of the same sequence, or by splicing different polypeptide units (U).
  • XTEN is a polypeptide consisting of 6 amino acids (A, E, G, P, S, T), of which 8% A, 12% E, 18% G, 17% P, 28% S and 17% T ( Volker Schellenberger et al., A combination of polypeptides extends the vivo in half-life of peptides and proteins in tunablemanner, Nature Biotechnology., 27 (12): 1186, 2009), rich in S and T.
  • PAS is composed of proline (P), alanine (A) and serine (S) and is also rich in S.
  • P proline
  • A alanine
  • S serine
  • the carrier protein composition rich in S and T is difficult to solve the problem of glycosylation.
  • glycosylation there are generally two main types of glycosylation: O-linked oligosaccharide glycosylation, the attachment site is on serine or threonine residues; N-linked oligosaccharide glycosylation, the attachment site is on Asn -Asparaginic acid residues of the X-Ser / Thr sequence, where X can be any amino acid except proline.
  • yeast the glycosylation system of yeast is different from humans.
  • the structure of the N-terminus of some proteins or peptides is closely related to activity.
  • the exposure of the N-terminus of Exendin-4 or GLP-1 is critical to its activity.
  • prokaryotic systems such as E. coli
  • fusion expression tags in front of the N-end of Exendin-4, such as the CBD tag (Volker Schellenberger, etc., Arecombinant polypeptide extends the the in vivo vivo half-life of peptides and proteins) in Tunablemanner, NatureBiotechnology., 27 (12 ): 1186, 2009), and then use protease to cut after the expression is completed; or add other amino acids before the GLP-1 sequence, such as two consecutive alanines to improve the cutting efficiency (M. Amiram et al., A depot-forming glucagon-like peptide-1 fusion proteins reduce blood glucose for five days with injection in J.
  • the polypeptide unit (U) or the polypeptide composite unit (PU) of the present invention is mainly composed of P, A and E. No matter whether it is prepared in a prokaryotic or eukaryotic expression system, the problem of glycosylation will not occur.
  • the polypeptide unit (U) or polypeptide composite unit (PU) provided by the present invention has non-uniformity caused by the deamidation of Asn (N) and Gln (Q) and is caused by a large number of amino acids. The possibility of degradation caused by the increase of potential protease sites is extremely small.
  • the polypeptide unit (U) or polypeptide complex unit (PU) has superior serum stability and enzyme resistance stability compared to the carrier protein of the prior art.
  • the present invention also provides a fusion protein with therapeutic activity, which comprises one or several same or different active protein drugs (D) and the polypeptide unit (U) or the polypeptide composite unit (PU) connected (such as tandem)
  • D active protein drugs
  • U polypeptide unit
  • PU polypeptide composite unit
  • the D 1 , D 2 , D 3 , D 4 ... are active proteins or polypeptides having therapeutic activity, and D 1 , D 2 , D 3 , D 4 ... may be the same or different; PU 1 , PU 2 , PU 3 , PU 4 ... Each represent one of the polypeptide composite units, and PU 1 , PU 2 , PU 3 , PU 4 ... May be the same or different.
  • the polypeptide unit (U) or polypeptide composite unit (PU) of the present invention is suitable for fusion with a variety of active proteins or polypeptides to improve the stability, half-life and other properties of the active proteins or polypeptides.
  • the active protein or polypeptide (D) may be selected from agonists, receptors, ligands, antagonists, enzymes or hormones.
  • the active protein or polypeptide may be a protein or polypeptide that has or will be applied to treat various diseases, including but not limited to: metabolic related diseases, cardiovascular diseases, coagulation / bleeding diseases, growth Disorders or disorders, tumors, vascular disorder diseases, inflammation, autoimmune disorders, etc. These diseases include metabolic-related diseases, cardiovascular diseases, coagulation / bleeding diseases, growth disorders or disorders, tumors, vascular disorder diseases, inflammation, autoimmune disorders, etc.
  • Type 1 diabetes type 2 diabetes, gestational diabetes, hypercholesterolemia, obesity, hyperglycemia, ultra hyperinsulinemia, reduced insulin production, insulin resistance, metabolic disorders, polycystic ovary syndrome , Dyslipidemia, eating disorders, hypertension, pulmonary hypertension, retinal neurodegeneration, metabolic disorders, glucagonoma, ulcerative colitis, renal failure, congestive heart failure, nephrotic syndrome, nephropathy, diarrhea, surgery Backward dumping syndrome, irritable bowel syndrome, critically ill polyneuropathy, systemic inflammatory response syndrome, blood Lipid disorders, stroke, coronary heart disease, hemophilia, GH deficiency in adults and children, Turner syndrome, chronic renal failure, intrauterine growth retardation, idiopathic short stature, AIDS consumption, obesity, multiple sclerosis, aging, Fibromyalgia, Crohn's disease, ulcerative colitis, muscular dystrophy, low bone density, etc.
  • the D 1 , D 2 , D 3 , D 4 may be selected from but not limited to the active proteins or polypeptides listed in Table 1 or analogs thereof.
  • the physical and chemical properties of the active protein (D) are significantly improved, manifested by increased water solubility, increased resistance to enzymes and thermal stability, hydrodynamic The increase in the academic radius, etc., these ideal properties make the active protein drug (D) half-life in vivo significantly extended.
  • the biological activity of the active protein drug (D) decreases, however, the occurrence of the half-life of the fusion protein is extremely significant The increase, this decrease in activity is still acceptable.
  • Antibody-drug conjugate is a therapeutic drug obtained by preparing antibodies and toxic compounds or radionuclides through lysine, cysteine, unnatural amino acids and engineered labels.
  • a prominent shortcoming of ADC drugs is that due to the cross-linking of highly hydrophobic toxic compounds or radionuclides, the entire ADC molecule is likely to aggregate or even produce insoluble precipitates, especially when the drug / antibody ratio (DAR) is high .
  • DAR drug / antibody ratio
  • chemically synthesized high hydrophilic polyethylene glycol (PEG) or biodegradable short-chain molecules can be used as linkers, such as PHF (or ). These measures can effectively improve the hydrophilicity of ADC molecules and increase their stability.
  • the protein having therapeutic activity when the protein having therapeutic activity has a very small molecular weight or is a polypeptide that is not suitable for recombinant expression, chemical cross-linking may be preferably used.
  • the protein with therapeutic activity can be prepared by chemical crosslinking of several different active proteins or polypeptides (D) and the said polypeptide unit (U) or polypeptide composite unit (PU). Chemical crosslinking can be performed on most amino acid residues, however, the nucleophilic primary amine group on lysine and the active sulfhydryl group on cysteine are the most commonly used crosslinking sites. In addition, tyrosine and selenocysteine will also be used for chemical crosslinking.
  • polypeptide unit (U) or polypeptide composite unit (PU) of the present invention is suitable for chemical coupling with a wide variety of active proteins or polypeptides, and the active proteins or polypeptides (D) are selected from agonists, receptors, and ligands , Antagonists, enzymes or hormones, including but not limited to the active proteins, polypeptides or analogs listed in Table 1.
  • the active protein or polypeptide may be a protein or polypeptide that has or will be applied to treat various diseases, including but not limited to: metabolic related diseases, cardiovascular diseases, coagulation / bleeding diseases, growth Disorders or disorders, tumors, vascular disorders, inflammation, autoimmune disorders, etc .; or specifically include type 1 diabetes, type 2 diabetes, gestational diabetes, hypercholesterolemia, obesity, hyperglycemia, ultra-hyperinsulinemia, Reduced insulin production, insulin resistance, metabolic disorders, polycystic ovary syndrome, dyslipidemia, eating disorders, hypertension, pulmonary hypertension, retinal neurodegeneration, metabolic disorders, glucagonoma, ulcerative colitis, kidney Failure, congestive heart failure, nephrotic syndrome, nephropathy, diarrhea, postoperative dumping syndrome, irritable bowel syndrome, critically ill polyneuropathy, systemic inflammatory response syndrome, dyslipidemia, stroke, coronary heart disease, hemophilia , GH deficiency in adults and children, Turner syndrome, chronic renal failure, intra
  • the present invention also provides isolated polynucleotides, encoding the polypeptide unit (U) or polypeptide complex unit (PU), or encoding the fusion protein.
  • the polynucleotide may be in the form of DNA or RNA.
  • the polynucleotide encoding the protein or polypeptide may include a polynucleotide encoding the protein or polypeptide, or may further include additional coding and / or non-coding sequences.
  • Polynucleotides encoding proteins or polypeptides can be obtained by PCR amplification, recombination, or synthetic methods.
  • the present invention also provides a recombinant expression vector comprising the polynucleotide. And providing a host cell, the cell containing the expression vector or genome integrated with the foreign polynucleotide, so that the protein or polypeptide unit (U), polypeptide complex unit (PU) or Fusion protein.
  • the present invention also provides a method for preparing the polypeptide unit (U) or polypeptide composite unit (PU) or fusion protein, including the following steps: 1) cultivating the host cell to express the polypeptide unit (U), Polypeptide composite unit (PU) or fusion protein; 2) Collect the culture containing the polypeptide unit (U), polypeptide composite unit (PU) or fusion protein; 3) Separate the polypeptide unit from the culture obtained in step 2 (U), Polypeptide Compound Unit (PU) or fusion protein.
  • the present invention also provides a composition containing the polypeptide unit (U) or polypeptide complex unit (PU), fusion protein or the host cell culture, and a pharmaceutically or food acceptable carrier.
  • “Pharmaceutically or food-acceptable” ingredients are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergies), ie substances with reasonable benefit / risk ratios; eg Pharmaceutical carriers or excipients commonly used in the art.
  • the polypeptide unit (U), polypeptide complex unit (PU), fusion protein, or the host cell culture is usually an "effective amount", and an "effective amount” refers to a function or activity that can produce function in humans and / or animals And the amount that can be accepted by humans and / or animals.
  • Protein viscosity Protein drugs are generally stored at high concentrations and are administered by injection during administration. Therefore, the lower the viscosity of the protein drug, the higher the protein concentration during storage and administration. Especially for eye-administered drugs, the decrease in protein drug viscosity can reduce the volume of administration, improve patient compliance, and has extremely high clinical significance.
  • the fusion protein containing the polypeptide unit (U) or polypeptide composite unit (PU) of the present invention has extremely low protein viscosity and extremely high solubility.
  • Immunogenicity As a carrier protein, in vivo immunogenicity is the most important factor. Factors that produce immunogenicity include amino acid sequence, production of aggregates, presence of impurities, the patient's immune status and genetic background, dosage, and route of administration.
  • the polypeptide composite unit (PU) provided by the present invention is composed of multiple polypeptide units (U) repeatedly spliced. If a single polypeptide unit (U) is immunogenic, such as having a T cell epitope, it is likely to cause strong body fluids immune response. In addition to potential cell epitopes, protein aggregation also increases the substantial cross-linking of B cell receptors, resulting in rapid B cell activation and enhanced antigen uptake, processing, and presentation.
  • the polypeptide unit (U) uses the prediction tool TEPITOPE (Sturniolo T et al., Nat. Biotechnol. 17: 555-561.) Based on the QAM method to calculate DRB1 * 01: 01, DRB1 * 01: 02 , DRB1 * 03: 01, DRB1 * 03: 02, DRB1 * 04: 01, DRB1 * 04: 02, DRB1 * 07: 01 and DRB1 * 15: 01 allele scores, the results are all less than or equal to the score -8, and the lizard-derived Exendin-4 (SEQ ID NO: 11) has a higher score, which means a higher risk of immunogenicity.
  • TEPITOPE Sturniolo T et al., Nat. Biotechnol. 17: 555-561.
  • polypeptide unit (U) or polypeptide composite unit (PU) when the polypeptide unit (U) or polypeptide composite unit (PU) is fused with human growth hormone with a high aggregation tendency, there is no tendency to aggregate at high temperature, indicating that it has a good The in vitro stability properties also indicate a low potential risk of immunogenicity.
  • no anti-drug antibodies against the portion of the polypeptide unit (U) or polypeptide complex unit (PU) were detected.
  • the polypeptide unit (U) or polypeptide composite unit (PU) of the present invention can enhance the pharmacokinetic properties of biologically active proteins, and the fusion of the polypeptide unit (U) or polypeptide composite unit (PU) )
  • the active protein or polypeptide half-life can be extended by more than 2 times, wherein the pharmacokinetic properties are determined by measuring the terminal half-life of the biologically active protein administered to the subject and administering the equivalent amount of the polypeptide unit fused (U) Or the biological activity of the polypeptide complex unit (PU) is determined by comparison.
  • the hydrodynamic radius of the active protein fused with the polypeptide unit (U) or the polypeptide composite unit (PU) is significantly increased, thereby reducing the renal clearance rate of the active protein.
  • the hydrodynamic radius of the resulting fusion protein can be detected by ultracentrifugation, size exclusion chromatography or light scattering. Increased hydrodynamic radius will cause a decrease in tissue permeability, which can be used to minimize the side effects of pharmaceutically active proteins. It has been documented that due to enhanced permeability and retention effect (EPR), hydrophilic polymers tend to accumulate selectively in tumor tissues. The potential causes of EPR effects are the leakage properties of tumor blood vessels (McDonald, D.M.
  • hydrophilic polymers can enhance the selectivity of pharmacologically active proteins used in tumor tissues.
  • fusion polypeptide units (U) or polypeptide composite units (PU) can increase the therapeutic index of a given pharmaceutically active protein.
  • the biologically active protein fused with the polypeptide unit (U) or the polypeptide composite unit (PU) has significantly improved solubility and stability, such as thermal stability, enzyme resistance stability and serum stability.
  • the hGH fusion protein fused with the polypeptide unit (U) or the polypeptide composite unit (PU) has a higher thermal stability at high temperature than unfused hGH, and hGH is easy to prepare Aggregation occurred during the process, but after fusion of the polypeptide unit (U) or the polypeptide composite unit (PU), no significant aggregation was observed on SEC-HPLC.
  • the serum stability was determined by measuring the integrity of the biologically active protein after exposure to 37 ° C and rat serum for at least 7 days.
  • the pancrease-added hGH fusion protein exhibits higher enzyme resistance stability than unfused hGH.
  • Bioly active protein / polypeptide refers to proteins, antibodies, polypeptides and fragments and variants thereof, having one or more pharmacological and / or biological activities, or targeted guidance, multimerization and other functions. They can be naturally occurring or artificially constructed.
  • Bioactive protein / polypeptide includes enzymes, enzyme inhibitors, antigens, antibodies, hormones, coagulation factors, interferons, cytokines, growth factors, differentiation factors, bone tissue growth related factors, and bone factor absorption related factors , Chemotactic factors, cell motility factors, migration factors, cytostatic factors, bactericidal factors, antifungal factors, plasma adhesion molecules, interstitial adhesion Molecules and extracellular matrix, receptor ligands and their fragments, etc.
  • the biologically active protein / polypeptide involved in the present invention is a protein / polypeptide that exhibits "therapeutic activity", such protein / polypeptide possessing one or more known biological and / or therapeutic activities . These activities are associated with one or more of the therapeutic proteins described herein or other known therapeutic proteins.
  • therapeutic protein (interchangeable with “therapeutic protein” or “active protein drug” herein) refers to a protein useful for treating, preventing, or ameliorating a disease, symptom, or dysfunction .
  • a “therapeutic protein” may be a protein that specifically binds to a specific cell type (such as lymphocytes or cancer cells) and is localized on the surface of the cell (or subsequently endocytosed into the cell).
  • “therapeutic protein” refers to a biologically active protein, especially a biologically active protein useful for treating, preventing, or ameliorating a disease.
  • Non-limiting therapeutic proteins include proteins with biological activities such as increasing angiogenesis, inhibiting angiogenesis, regulating hematopoietic function, promoting nerve development, improving immune response, and suppressing immune response.
  • therapeutic activity or “activity” may refer to activity in humans, non-human mammals, or other species of organisms that provides an effect consistent with the desired therapeutic outcome. Therapeutic activity can be measured in vivo or in vitro.
  • the "therapeutic protein” may include but is not limited to: VEGF receptor or its fragments, TNF receptor, HER-2 / neural membrane receptor, human ErbB3 receptor secreting morphological isomers, transforming growth factor bIII Type extracellular domain, transforming growth factor b type II extracellular domain, IL-1 receptor, IL-4 receptor, urokinase, ⁇ -glucocerebrosidase, arginine deiminase, Arginase, herstatin, epidermal growth factor, FGF-1, FGF-19, FGF-21, fibroblast growth factor-2, common fibroblast growth factor, nerve growth factor, platelet-derived growth factor, VEGF-1, IL-1 , IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-11, IL-12, IL-15, IL-18, IL-21, IL-24, IL-1RA, RANKL, RANK, OPG, LEPTIN, inter
  • Therapeutic proteins can also be antibodies and fragments thereof, especially antigen-binding fragments, including single chain antibody scFv and the like. These proteins and the nucleic acid sequences encoding these proteins are well known and can be found in public databases such as Chemical Abstracts Services Databases (eg CAS Registry), GenBank and GenSeq. For those skilled in the art, according to the spirit of the present invention, it is easy to understand that most of the biologically active proteins that have been found in the prior art are suitable for the present invention. Of course, it should also be understood that the proteins / polypeptides newly discovered after the present invention having biological activity are also applicable to the present invention.
  • sequence homology is used to describe the distance between species. If the two sequences have a common evolutionary ancestor, then they are homologous.
  • sequence homology the sequence to be studied is generally added to a set of multiple sequences from different species to determine the homology relationship between the sequence and other sequences. Commonly used analysis tools are CLUSTAL.
  • sequence identity refers to the percentage of identical residues in the sequences involved in the comparison.
  • sequence identity of two or more entry sequences can be calculated using calculation software well known in the art, and these software are available from NCBI, for example.
  • sequence similarity refers to the degree of similarity between several DNA, RNA, or protein sequences, and is understood as the percentage of identical residues in the sequences involved in the comparison (identity percentage, identity%) or similar physical chemistry Percentage of residues in nature (% similarity,% similarity).
  • sequence similarity of two different protein sequences can be understood as the percentage (identity percentage) of the same amino acid residues present in the two sequences or the similar physical and chemical properties present in the two protein sequences The percentage of amino acid residues (% similarity, similarity%).
  • the polypeptide unit (U) is mainly composed of or only composed of P, A, and E amino acids. Within each unit ( ⁇ 20 amino acids), the chance of consecutive occurrence of the same amino acid is extremely high unless This amino acid arrangement affects its recombinant expression.
  • the secondary structure of protein or polypeptide chain refers to the regular repeating conformation in protein or polypeptide chain, mainly including ⁇ -helix, ⁇ -sheet, ⁇ -turn and irregular coil.
  • the following term explanations are consistent with the definitions in classical molecular biology.
  • Alpha-helix A common secondary structure.
  • the main chain of the peptide chain is coiled into a spiral around the imaginary central axis. It is generally a right-handed spiral structure.
  • the helix is maintained by hydrogen bonds within the chain.
  • the carbonyl oxygen of each amino acid residue (nth) forms a hydrogen bond with the amide nitrogen of the fourth residue (n + 4) in the C-terminal direction of the polypeptide chain.
  • the pitch is 0.54 nm, each turn contains 3.6 amino acid residues, and each residue rises 0.15 nm along the long axis of the helix.
  • ⁇ -sheet The secondary structure common in proteins is composed of stretched polypeptide chains. The conformation of the folded sheet is maintained by the hydrogen bond formed between the carbonyl oxygen of a peptide bond and another amide hydrogen located in the same peptide chain or adjacent peptide chain. The hydrogen bonds are substantially perpendicular to the long axis of the peptide chain. These peptide chains can be arranged in parallel; or they can be arranged in antiparallel.
  • ⁇ -turn a non-repetitive polypeptide region that connects the ⁇ -helix and ⁇ -sheet in the protein molecule to change the direction of the peptide chain. It generally contains 2 to 16 amino acid residues. Corners containing more than 5 amino acid residues are often called loops. Common corners contain 4 amino acid residues and there are two types. The characteristic of corner I is that: the first amino acid residue forms a hydrogen bond with the fourth residue; the third residue of corner II is often glycine. The second residue in these two corners is mostly proline.
  • Random coil This structure is the secondary structure conformation of the part of the peptide chain that has no regularity except for the above-mentioned more regular conformations in the polypeptide chain.
  • Secondary structure prediction At present, the secondary structure prediction methods of proteins or peptides are: Chou-Fasman algorithm, PHD algorithm, multi-sequence alignment prediction, neural network-based sequence prediction, prediction method based on existing knowledge (knowledge based method) ) And hybrid methods (hybrid system), these methods are well known to those skilled in the art.
  • the present invention uses the result of the Chou-Fasman algorithm (http://www.biogem.org/tool/chou-fasman/index.php).
  • Hydrophilic amino acids examples are arginine, lysine, threonine, asparagine, glutamine, proline and glutamic acid.
  • Hydrophobic amino acids examples are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine and valine, alanine.
  • PEG polyethylene glycol
  • PEGylated refers to the covalent attachment of polyethylene glycol (PEG) polymer chains to the biologically active protein or polypeptide of interest. It is generally believed that covalently linking PEG to a biologically active protein or polypeptide can mask the protein or polypeptide from the host's immune system and increase the hydrodynamic radius of the biologically active protein or polypeptide of interest, thereby extending by reducing renal clearance Its internal circulation time.
  • the polypeptide unit (U) consists of three amino acids P, A and E.
  • the preferred exemplary sequences and their corresponding TEPITOPE scores are shown in Table 2.
  • the ⁇ -helix content is calculated using the Chou-Fasman algorithm, and the ⁇ -helix content of exemplary polypeptide units (U) is shown in Table 3.
  • AAEAAPAAPAAPEPAEEAAP 90 41 APEAAPAAAEPAPAPAAPEP 90 42 APAEAAEAAPEAPEPAAPAA 95 43 AEAAAPAEAAPAPAPEAPAP 90 44 AAEAAEAAPAEPEPPAPPAP 65 45 AEAAEAPAPAAPPAAPAPEP 90 46 APEAPAEEAEPAAAPPAEAP 90 47 APAPPAEAEAEAPPAAPEPA 85
  • Example 2 Obtaining high expression low molecular weight polypeptide composite unit ( PU )
  • the polypeptide composite unit ( PU ) is formed by splicing the polypeptide unit (U), and can be realized by one of the following steps:
  • the polypeptide unit (U) is spliced under the action of T4 DNA ligase by complementary sticky ends, and then carried out Agarose gel electrophoresis to recover DNA fragments of appropriate size.
  • the polypeptide units (U) involved in the splicing may have the same sequence or different.
  • a 6 ⁇ His affinity purification tag is added to the N-terminus or C-terminus of the polypeptide complex unit ( PU ).
  • the corresponding nucleotide (DNA) fragment of the polypeptide unit (U) is synthesized: for example, the BglI and SfiI cleavage sites are introduced at both ends of the DNA fragment corresponding to AEPAAPAPAEPAAPAPEAPA (SEQ ID NO: 33), and the base sequence is E. coli codon optimization, the sequence was inserted into pUC57 through the blunt end of EcoRV, and the plasmid was named pUC57-U 1 .
  • the fragment obtained by digesting pUC57-U 1 with Bgl I was ligated with a vector digested with SfiI and dephosphorylated to obtain a dimer.
  • PU polypeptide composite unit
  • U polypeptide units
  • PUMix17 including U35, U35, U34, U46, etc.
  • PUMix357 including U79, U27, U12, etc.
  • PU polypeptide complex unit sequences are shown in Table 4. It is worth noting that the peptide composite unit (PU) has low dyeing efficiency under conventional Coomassie brilliant blue dyeing conditions and requires copper staining (Chris Lee et al., Analytical Biochemistry 166: 308-312, 1987). The specific steps are as follows: 1. Prepare 0.3M CuCl 2 aqueous solution; 2. After removing the electrophoresis gel, rinse with double distilled water for 2-3min; 3. Infiltrate the gel into the 0.3M CuCl 2 solution and stain for 2-5min; 4. After removing the glue, take a picture with an imager.
  • the spliced PU fragment in Example 2 was fused with the coding sequence of hArg1 (Chinese name: human arginase; the sequence is shown in SEQ ID NO: 7) (as shown in Table 5), and C-terminal was His-6
  • the tags are connected and constructed into the pET41a vector.
  • the plasmid was transformed into E. coli competent BL21 (DE3) Gold, and the single clone was picked to LB kanamycin resistance liquid medium, cultured at 37 °C, 250RPM, and grown to OD 0.4-0.6 (about 3h) 200 ⁇ L of pre-induction culture served as a negative control.
  • IPTG was added to the remaining culture to a final concentration of 1 mM, and 200 ⁇ L was taken after induction at 37 ° C for 2.5 h.
  • the samples before and after induction were centrifuged at 5000 rpm and 4 min, discard the supernatant, add 2% SDS 40 ⁇ L to resuspend, add 10 ⁇ L 5 * Loading Buffer and mix, heat at 100 ° C for 8-10 min.
  • mPAS mPAS
  • mXTEN mXTEN
  • mURP three kinds of carrier proteins in the prior art.
  • Protein purification methods vary according to different expression systems. Existing technologies already have a lot of knowledge to provide guidance for protein purification, such as GE Healthcare's classic purification guidebook "Antibody Purification Handbook", or “METHODS” published by Elsevier Press INZYMOLOGY, Guide to Protein Purification, 2nd Edition, etc., affinity chromatography, size exclusion chromatography, ion exchange chromatography and hydrophobic chromatography, etc., are well-known technologies for those skilled in the art.
  • the following purification process is an exemplary illustration of the purification method in a specific case where the expression host is E. coli and fermentation conditions. When the fermentation conditions are different, the purification conditions should also be adjusted accordingly, which will not be repeated here.
  • Purification steps of PU-hArg1 fusion protein include ammonium sulfate precipitation, metal ion heating precipitation, cation exchange, anion exchange, and hydrophobic chromatography.
  • PU polypeptide composite unit
  • purification with a larger pore size filler is required to obtain a higher load.
  • the purification of different structures is slightly different. For example, as the percentage of glutamic acid in the fused peptide composite unit (PU) sequence increases, the weaker the binding of the cation exchange column, the lower the pH to bind, and the lower the salt. The concentration can be washed off.
  • the stronger the binding of the anion exchange column only lower pH is required for binding, and higher salt concentration is required for elution.
  • the binding elution properties of the ion exchange column will also be similar to the phenomenon described above when the percentage of glutamic acid increases.
  • Loading buffer 1M (NH 4 ) 2 SO 4 , 20 mM NaAc-HAc, pH 6.0, elution buffer: 20 mM NaAc-HAc, pH 6.0.
  • the eluted sample was desalted on a G25 (Sephadex G-25, Coarse) chromatography column with 10 mM Tris-HCl, pH 8.0, and then loaded onto 20 mL SuperQ (SuperQ-650M, TOSOH) equilibrated with 20 mM Tris-HCl, pH 8.0. ) Chromatography column to collect the effluent, the target protein is in the effluent. 2M NaCl 20mM Tris-HCl, pH 8.0 elution, discard the eluent.
  • the detection method is as follows: detection wavelength: 214 nm; chromatography column: column temperature 25 ° C., Sepax SRT-1000SEC 5 ⁇ m (300 ⁇ 7.8 mm), mobile phase: 50 mM PB, 150 mM NaCl, pH 7.2; running time: 20 minutes.
  • the detection method is as follows: detector: 173 degree light scattering detector; detection temperature: 25 ° C; exemplary particle size is shown in FIG. 2, and the particle size in the aqueous solution of PU98x5-hArg1-PU106x5 reaches 17 nm.
  • the live sample to be tested was diluted to 1 ⁇ M, 45 ⁇ l of the diluted sample was mixed with 5 ⁇ l of 500 mM CoCl 2 and activated at 60 ° C. for 10 min.
  • Add 50 ⁇ l of the activated sample add 450 ⁇ l of 500mM L-arginine (pH7.4), mix well, hydrolyze at 37 °C for 15min, sample 20 ⁇ L, add it to 2mL of urea nitrogen reagent mixture (Nanjing Jiancheng Bioengineering Research Institute), immediately Place the boiling water in an accurate water bath for 15 minutes, cool with ice water for 5 minutes, and then measure the OD value at 520 nm. Calculate the urea nitrogen content according to the standard curve.
  • SD rats were randomly divided into groups of 10, each immunized with different PU-hArg1 and unfused hArg1 protein (R & D Systems, Cat: 5868-AR), 3mg / kg, subcutaneous injection, once weekly injection, continuous Four weeks after injection, a group of PBS was injected simultaneously as a negative control. Two weeks after the last immunization, blood was taken after sacrifice and serum was isolated. Serum PU antibody production was detected by ELISA. Specifically, ELISA plates were coated with different PU-hArg1 fusion proteins, SEQ ID NO: 113-115 control proteins, and unfused hArg1 protein of the corresponding immunized rats.
  • mice 6 rats were randomly divided into groups, 10 rats in each group were injected with different PU-hArg1 protein and SEQ ID NO: 113-115 control protein, 2mg / kg, subcutaneous injection, before and 3h, 8h, 12h Blood was collected at 24h, 36h, 48h, 72h, 96h, 120h, 144h and 168h, and serum was isolated.
  • IPTG was added to the remaining culture to a final concentration of 1 mM, and 200 ⁇ L was taken after induction at 37 ° C for 2.5 h.
  • the samples before and after induction were centrifuged at 5000 rpm for 4 minutes, discard the supernatant, add 2% SDS 40 ⁇ L to resuspend, add 10 ⁇ L of 5 * Loading Buffer and mix, heat at 100 ° C for 8-10 min.
  • the expression strain was screened by SDS-PAGE electrophoresis.
  • the precipitate was dissolved with a 20 mM PB (pH 7.0) solution, and then precipitated with ammonium sulfate at a conductivity of 180 mS / cm. Take the precipitate to dissolve with 20mM NaAc (pH5.0) solution, and dilute with water to a conductivity below 4mS / cm. Purify with Super Q-650M (TOSOH) chromatography column (Buffer A: 20mM NaAc pH5; Buffer B: 0.5M NaCl 20mM NaAc pH5), and elute at 20% B, 70% B, 100% B at a time.
  • TOSOH Super Q-650M
  • K 1 and K 2 are constants, and M r is the relative molecular mass.
  • the detection method is as follows: detection wavelength: 214 nm; chromatography column: column temperature 25 ° C., Sepax SRT-1000SEC 5 ⁇ m (300 ⁇ 7.8 mm), mobile phase: 50 mM PB, 150 mM NaCl, pH 7.2; running time: 20 minutes.
  • the results of the thermal stability measurement are shown in Fig. 6, which shows that each fusion protein has good thermal stability. Due to high temperature treatment, hGH aggregates and precipitates, and no liquid phase analysis is performed.
  • Example 13 In vitro cell activity detection of PU-GH samples
  • Ba / f3-GHR cells were starved with IL-3-free RPMI 1640 medium (containing 5% FBS and 1 mg / mL G418) for 4-6 hours, then transferred to centrifuge tubes and centrifuged at 1000 RPM for 5 minutes. After resuspending with the above medium, count, adjust to 2 x 10 5 / mL, plate 96-well plates, 100 ⁇ L per well, that is, 20,000 cells / well. Each protein to be tested was diluted to the appropriate concentration with the above medium, and 10 ⁇ L was added to each well. After 48 hours of stimulation, the protein was detected by MTT method. As in Table 8 and Figure 7.
  • the cytological activity of the fusion protein is somewhat reduced, but within an acceptable range.
  • SD rats were randomly divided into groups of 10, each subcutaneously injected with different PU-GH fusion protein or hGH recombinant protein (Sino Biological, Cat: 16122-H07E), 2mg / kg, before and 3h, 8h, Blood was collected at 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, 168h, and serum was isolated. Sandwich ELISA method was used to detect the pharmacokinetics of PU-GH protein in rats.
  • hGH antibody (Sino Biological, Cat: 16122-R101) was added to ELISA enzyme plate at 100ng / well, coated at 4 ° C overnight, washed 3 times with PBST and blocked with 5% milk powder for 2 hours, washed 3 times with PBST again, each time Dilute the spotted serum to the specified multiple and add to the ELISA plate.
  • the half-life of the fusion protein has been significantly extended, compared with the pre-fusion hGH, the extension is higher than 50 times, even higher than 100 times.
  • the spliced PU fragment in Example 2 was fused and expressed with GDF 15 (growth and differentiation factor, SEQ ID NO: 15) (as shown in Table 10), the N-terminal was connected to His6, and the nucleotide fragment was subcloned into plasmid pPIC9 (Life Technologies), to construct an expression vector, in the methylotrophic yeast Pichia pastor GS115 (His -) as expression hosts, transformed by electroporation of the linearized expression plasmids were transformed into GS115 and. Incubate at 30 ° C for 3 days until single colonies appear.
  • GDF 15 growth and differentiation factor, SEQ ID NO: 15
  • SEQ ID NO: 15 growth and differentiation factor
  • the centrifuged supernatant of the fermentation broth was first precipitated with 40% ammonium sulfate, reconstituted with deionized water, and then loaded onto 50ml of Chelating Sepharose equilibrated with an equilibrium buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5) Fast Flow chromatography column (GE Healthcare), after re-equilibration, elution with 10%, 50%, 100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0).
  • an equilibrium buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5
  • Fast Flow chromatography column GE Healthcare
  • mice 7-week-old male C57BL / 6J male mice were given high-fat feed (60% kcal from fat) and continued to feed for 16 weeks (total 23 weeks), when the body weight was about 55g.
  • Feeding conditions 12h light / 12h darkness, free feeding, single cage feeding, mice were grouped (8 / group) according to body weight and body weight growth curve the day before dosing, and subcutaneously administered the next day.
  • the control group was injected with equal volume of normal saline (PBS); the fusion protein was administered once every 4 days for 28 consecutive days.
  • the body weight and food intake of mice were measured every day. They were sacrificed on the 5th day after the last dose. Calculate the average weight change of each group of animals before administration and at the time of sacrifice.
  • the fusion protein can significantly reduce the weight of obese animals, indicating that the fusion protein retains the biological activity of GDF15 itself.
  • the spliced PU fragment in Example 2 was fused with the glucagon-like peptide 2 analog GLP-2G (SEQ ID NO: 1), the C-terminus was connected to the His-6 tag, and the nucleotide fragment was subcloned into the plasmid pPIC9 (Life Technologies), to construct an expression vector, in the methylotrophic yeast Pichia pastor GS115 (His -) as expression hosts, transformed by electroporation of the linearized expression plasmids were transformed into GS115 and. Incubate at 30 ° C for 3 days until single colonies appear.
  • the centrifuged supernatant of the fermentation broth was first precipitated with 40% ammonium sulfate, reconstituted with deionized water, and then loaded onto 50ml of Chelating Sepharose equilibrated with an equilibrium buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5) Fast Flow chromatography column (GE Healthcare), after re-equilibration, elution with 10%, 50%, 100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0).
  • an equilibrium buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5
  • Fast Flow chromatography column GE Healthcare
  • Example 18 Activity determination of PU and GLP-2G fusion protein
  • GLP-2G fusion protein in vitro cytological activity detection using luciferase reporter gene detection method.
  • the GLP-2R (GLP-2 receptor) gene was cloned into mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-2R, and the full-length gene of luciferase (luciferase) was cloned into pCRE -Replace the EGFP gene on the EGFP plasmid to obtain the pCRE-Luc recombinant plasmid.
  • CHO cells were transfected with pCDNA3.1-GLP-2R and pCRE-Luc plasmids at a molar ratio of 1:10, and stable transfected expression strains were selected to obtain recombinant GLP-2R / Luc-CHO stable transfected cell strains.
  • the purified recombinant protein or GLP-2 (Hangzhou Sinopeptide Biochemical Co., Ltd., Catalog No. GLUC-002A) was diluted to a series with DMEM / F12 medium containing 0.1% FBS Specified concentration, added to cell culture wells, 100 ⁇ l / well, tested after 6h stimulation.
  • DMEM / F12 medium containing 0.1% FBS Specified concentration
  • FBS Specified concentration 0.1% FBS Specified concentration
  • Example 19 Pharmacokinetic detection test of PU and GLP-2G fusion protein
  • SD rats were randomly divided into groups of 10, each group was injected with different fusion protein, 2mg / kg, subcutaneously, before and after injection 3h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h At 168h, blood was collected and serum was separated. The pharmacokinetics of the fusion protein in rats were detected by sandwich ELISA.
  • GLP-2 antibody (Abcam, catalog number ab14183) was added to ELISA enzyme plate at 100ng / well, coated at 4 ° C overnight, washed 3 times with PBST and blocked with 5% milk powder for 2 hours, washed again with PBST 3 times, The serum was diluted to the specified multiple and added to the ELISA enzyme plate.
  • PBST was washed three times.
  • Biotin-labeled GLP-2 polyclonal antibody (Abcam, catalog number ab48292) was added and incubated at 37 ° C for 2h.
  • PBST After washing for 5 times, the HRP-labeled streptavidin was diluted 50,000 times and added to the ELISA plate. After incubation at 37 ° C for 1 hour, the TMB method was used to detect the OD450 value.
  • the spliced PU in Example 2 was fused with an VEGF-bound anchor repeat protein (Ankyrin repeat proteins (AR VEGF ), SEQ ID NO: 3) (Table 14), and the C-terminal was connected to a 6His tag to construct a pET41a vector in.
  • the plasmid was transformed into E. coli competent BL21 (DE3) gold, and the monoclonal was picked to LB Kana resistant liquid medium, cultured at 37 ° C and 250 RPM, and grown to OD 0.4-0.6 (about 3h), 200 ⁇ L was taken before induction The culture serves as a negative control.
  • IPTG was added to the remaining culture to a final concentration of 1 mM, and 200 ⁇ L was taken after induction at 37 ° C for 2.5 h.
  • Pre-induction and post-induction samples were centrifuged at 5000 rpm for 4 minutes, discard the supernatant, add 2% SDS 40 ⁇ L to resuspend, add 10 ⁇ L of 5 * loading Buffer to mix, and heat at 100 ° C for 8-10 min.
  • the expression strain was screened by SDS-PAGE electrophoresis.
  • the eluent was precipitated with 45% saturated ammonium sulfate, centrifuged at 8000 rpm for 20 min to collect the precipitate, and reconstituted with deionized water.
  • the reconstituted sample was desalted on a G25 (Sephadex G-25, Coarse) chromatography column with 10 mM Tris-HCl, pH 8.0.
  • Amino acid sequence (SEQ ID NO :) Code 130 PU73x49-AR VEGF 131 PU98x43-AR VEGF
  • Example 21 Affinity determination of PU and AR VEGF fusion protein
  • BLI Bio-layer inteferometry, ForteBio
  • Biotin Biotin Thermo, Prod # 21338, Sulfo-NHS
  • VEGF vascular endothelial growth factor
  • PBS containing 0.1% Tween-20
  • the gradient is diluted
  • the fusion protein and control antibody were added to the predetermined position of the 96-well black plate (Greiner, 655209) according to the program settings.
  • the fusion protein is combined and then dissociated in PBST solution to obtain the experimental curve; according to the analysis software of Octet-QK results, the experimental results are fitted with the local full curve to calculate kon, kdis, Kd.
  • Table 15 summarizes the Kd of the fusion protein and the control drug Bevacizumab (Medchemexpress, Cat. No.:HY-P9906). From the table, it can be seen that the average affinity of AR VEGF to VEGF before and after fusion with PU is not significantly different from that of Bevacizumab In the same order of magnitude.
  • PU-AR VEGF activity was measured by VEGF receptor competition inhibition method. Prepare two 96-well plates, ELISA plate and cell plate. The two boards are processed as follows:
  • VEGF Receptor 2 (KDR) (Abcam, ab155628) to the ELISA plate, 50 ⁇ L per well, and place at 37 ° C for 2h.
  • KDR VEGF Receptor 2
  • the cells were blocked with 1% BSA / TBS and placed at 37 ° C for 1h; 1% BSA / TBS was added to the cell plate, 200 ⁇ L per well, and placed at 37 ° C for 2h.
  • the PU-AR VEGF and the control substance Bevacizumab were diluted with PBS into a 100 ⁇ g / mL mother liquor, and then the mother liquor was diluted 3 times, a total of 11 concentrations were diluted, and 80 uL of the diluted sample was mixed with an equal volume of 1 ⁇ g / mL VEGF, 37 Place at °C for 1h.
  • the KDR-coated ELISA plate was washed twice, after patting dry, the mixed solution was transferred to the ELISA plate in turn, placed at 37 ° C for 1 h, and then washed 6 times.
  • Protein samples IC50 (nM) PU73x49-AR VEGF 0.55 PU98x43-AR VEGF 0.51 AR VEGF 0.65 Bevacizumab 0.56
  • Fusion protein samples (PU27x28-GH-PU27x5, PU98x28-GH-PU98x4, PU130x28-GH-PU130x5) were prepared with 40mM PB, pH7.4 to 2.0-3.0mg / ml, and sterilized and filtered (0.22 ⁇ m, Millipore). Dilute rat serum 10 times, mix well, and distribute into sterile centrifuge tubes; place in a 37 ° C incubator, take samples on day 0 and day 7 for Western analysis, and use HRP-labeled Anti-6X His Antibody (Abcam, ab1187) was used as detection antibody. The results are shown in Figure 12, which shows that the fusion protein is very stable in serum.

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Abstract

提供了一种对于延长蛋白或多肽的生物活性、半衰期或改善蛋白或多肽体内外性质的多肽单元(U)或多肽复合单元(PU)，所述的多肽单元(U)主要由脯氨酸(P)、丙氨酸(A)和谷氨酸(E)组成，所述PU含有多个所述U。还提供了具有生物活性的融合蛋白，其包括所述的多肽单元(U)或多肽复合单元(PU)以及与之连接的活性蛋白或多肽。

Description

用于改善活性蛋白或多肽性能的人工重组蛋白及其应用 技术领域
本发明涉及生物技术领域，更具体地，本发明涉及一种用于改善生物活性蛋白或多肽性能的人工重组蛋白及其应用。
背景技术
众所周知，分子量小于70kDa的蛋白质或多肽容易通过肾过滤而被体内清除(Jevsevar S等，5:113–28，2010)。因此，一般采用融合或交联分子量较大的载体蛋白、聚乙二醇(PEG)、脂肪酸等来增加其表观分子量和流体动力学半径，从而降低其肾小球滤过率(Kontermann RE，BioDrugs，23:93-109；2009；Kang JS等，Expert Opin Emerg Drugs.，14:363–80，2009)，并最终达到延长蛋白或多肽体内半衰期的目的。
用作交联使用的载体一般是PEG或脂肪酸等，而人血清白蛋白、免疫球蛋白Fc片段、转铁蛋白等则普遍用于重组融合，并且大都有对应的成功上市药物。近年来，新型的重组融合载体蛋白不断涌现(WR Strohl，Biodrugs，2015，29(4):215-39)，如URP(中国专利ZL200780015899.2)、XTEN(中国专利申请CN201080011467.6；Volker Schellenberger等，Nature Biotechnology 27(12):1186，2009)、类弹性蛋白ELP(MacEwan SR等，J Control Release.2014；190:314-30.)、PAS(专利号ZL200880019017，M Schlapschy等，Protein Engineering Design&Selection Peds.2013，26(8):489-501)和GLK(中国专利号200980103870.9)等。其中XTEN和ELP融合制备的蛋白药物已经用于临床试验中(Yuen KC等，J Clin Endocrinol Metab.,98(6):2595-603.2013；Christiansen M等，Weekly Subcutaneous Doses of Glymera(PB1023)a Novel GLP-1Analogue Reduce Glucose Exposure Dose Dependently，http://phasebio.com/)。这些人工设计的非天然蛋白质，在跟某些活性蛋白或多肽通过重组表达或者交联后形成的融合蛋白或产物，相比于单独的活性蛋白和多肽，显著地提高了血清稳定性并延长了其体内半衰期，最终改善了治疗效果。
专利号为ZL200780015899.2的中国专利公开了一种非结构化重组聚合物(URP)，其基本不能非特异性结合血清蛋白，其特征在于：(a)含有至少100个毗连氨基酸；(b)所述URP中所含的甘氨酸(G)、天冬氨酸(D)、丙氨酸(A)、丝氨酸(S)、苏氨酸(T)、谷氨酸(E)和脯氨酸(P)残基之和占所述URP中全部氨基酸的约80％以上；(c)经Chou-Fasman算法测定，所述URP序列的至少50％氨基酸不形成二级结构；(d)所述URP的T表位评分小于-4。
申请号为CN201080011467.6的中国专利申请公开了一种分离的延伸重组多肽(XTEN)，包含超过大约400至大约3000个氨基酸残基，其中该XTEN的特征在于：(a)甘氨酸(G)、丙氨酸(A)、丝氨酸(S)、苏氨酸(T)、谷氨酸(E)和脯氨酸(P)残基的总和占XTEN的总氨基酸序列的超过大约80％；(b)该XTEN序列基本是非重复的；(c)当通过TEPITOPE算法分析时，该XTEN序列缺乏预测的T细胞表位，其中对XTEN序列内表位的 TEPITOPE算法预测是基于-9或更高的得分；(d)通过GOR算法确定，该XTEN序列具有超过90％的无规卷曲形成；和(e)通过Chou-Fasman算法确定，该XTEN序列具有少于2％的α螺旋和少于2％的β-折叠。此公开中并未强调氨基酸的种类数，然而在文献报道中，XTEN的氨基酸含量包括：8％A，12％E，18％G，17％P，28％S和17％T(Volker Schellenberger等，Nature Biotechnology.，27(12):1186，2009)，这是因为在构建无结构化的序列时发现2-5个氨基酸的组成会代来强免疫原性和低水溶性，而且对于半衰期的延长效果有限。
专利号为ZL200880019017的中国专利公开了包含至少两个结构域的生物学活性蛋白，其中：(a)所述至少两个结构域的第一结构域包含具有和/或介导所述生物学活性的氨基酸序列；以及(b)所述至少两个结构域的第二结构域包含形成无规卷曲构象的由至少10个氨基酸残基组成的氨基酸序列，其中所述第二结构域由丙氨酸、丝氨酸和脯氨酸残基组成，其中所述无规卷曲构象介导所述生物学活性蛋白体内和/或体外稳定性的增加。
类弹性蛋白ELP是由(VPGXG)n组成，其中X可以是除脯氨酸(Pro)之外的任意氨基酸。n的数目不固定，ELP有在特定温度(跨度为2-3℃)下状态会发生急剧转变的特性：低于这一温度，ELP呈可溶状态；高于这一温度，ELP会发生快速聚集成微米级肉眼可见的颗粒物；再次降低温度，ELP又会重新溶解；这一温度称之为反向转换温度，简称相变温度(Tt)。ELP属于弹性蛋白，具有生物降解性和非免疫原性，因此适用于作为延长药物半衰期的融合蛋白使用。
专利号为ZL200980103870.9的中国专利公开了一种用于延长蛋白体内半衰期的重组明胶样单元(GLK)，其特征在于，所述的明胶样单元是结构如下的多肽：(Gly-X-Y)n；式中，Gly为甘氨酸残基；X和Y分别为20种天然氨基酸除了Cys之外的任意氨基酸残基，以及Hyp；n为20-300；并且，所述的明胶样单元具有以下特征：(a)所述明胶样单元中以下亲水性氨基酸Asn，Asp，Gln，Glu，Lys，Pro，Ser，Hyp，Arg的氨基酸百分比含量总和为40％至2/3；(b)所述明胶样单元中，Pro和Hyp的数量之和与n的比值≥0.6；(c)Gly的数量之和与n的比值≤1.15；且，根据ProtParam公式计算，代表亲水性的GRAVY值小于-1.5；附加条件是，所述的明胶样单元不是天然的明胶蛋白。
上述的几种新型载体蛋白与传统的白蛋白和免疫球蛋白IgG FC片段的不同之处在于，其大部分序列中氨基酸种类较少。在类弹性蛋白ELP的VPGXG组成单元里，对于X位的氨基酸电荷及亲水性并无严格限定。而URP和XTEN的设计强调采用亲水性氨基酸，并加入了带负电荷的天冬氨酸和/或谷氨酸进一步延长半衰期，这是“由于人或动物的大多数组织和表面具有净负电荷，XTEN序列可以设计为具有净负电荷以使含有XTEN的组合物与各种表面如血管、健康组织或各种受体之间的非特异性相互作用最小化”(申请号CN201080011467.6)；相反，PAS则着重于模仿聚乙二醇(PEG)，采用不带电的三种氨基酸：脯氨酸、丙氨酸和丝氨酸组成。另一方面，XTEN序列强调“基本上非重复”这一特征：“重复氨基酸序列具有聚集形成更高级结构的倾向，其例子为天 然重复序列如胶原蛋白和亮氨酸拉链，或者形成接触，导致晶体或拟晶体结构。相反，非重复序列聚集的低倾向使得能够设计具有相对较低频率的带电荷氨基酸的长序列XTEN，如果序列重复它可能聚集”。XTEN技术对于“基本上非重复”的解释为“指在肽或多肽序列中缺乏或有限程度的内部同源性。例如，在该序列的四个连续氨基酸中极少或者没有一个是相同的氨基酸类型，或者该多肽具有10或更低的亚序列得分，或者在从N端到C端的顺序中没有构成该多肽序列的基序的模式”。但是，XTEN和PAS、URP由于富含S和T，在真核系统中表达时糖基化程度会特别高。
尽管活性蛋白或多肽与这些载体蛋白融合或交联后可能会显著减弱其生物学活性，例如Gething NC等报道，胰高血糖素-XTEN融合蛋白仅表现出未修饰的胰高血糖素多肽的15％生物活性(Gething NC等，PLoS One，2010，5(4):e10175)，然而融合或交联后带来的稳定性和溶解性等理化性质的提高却可弥补这方面的缺陷。
综上，本领域还需要进一步开发其它的能够改善多肽稳定性、延长多肽半衰期的物质或方法，以期进一步提高活性多肽的体内活性。
发明内容
本发明的目的在于提供一种改善活性蛋白或多肽性能的人工重组蛋白及其应用。
在本发明的第一方面，提供一种多肽单元(U)，其具有以下特征：(1)由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；(2)存在50％以上的α-螺旋二级结构；和(3)长度≥15个氨基酸。
在一个优选例中，根据Chou-Fasman公式计算，所述的多肽单元存在60％以上，较佳地70％以上，更佳地80％以上，进一步更佳地90％以上的α-螺旋二级结构。
在另一优选例中，所述的α-螺旋二级结构的比例按照Chou-Fasman算法计算；和/或长度≥20个氨基酸。
在另一优选例中，所述的多肽单元的长度为15～100aa，较佳地16～60aa；具体的，如18aa，19aa，20aa，22aa，25aa，30aa，40aa，50aa，60aa，70aa，80aa或90aa。
在另一优选例中，所述多肽单元包括选自下组的多肽单元：SEQ ID NO:19～47所示氨基酸序列的多肽单元。
在本发明的另一方面，提供一种多肽复合单元(PU)，其核心结构选自：
U 1-U 2或U 1-U 2-…U n；
U 1，U 2，…，U n各自代表一个所述的多肽单元，n为＞2的正整数；且，两个或多个所述多肽单元的氨基酸序列相同或不同。
在另一优选例中，n为3～100的正整数，例如为4～90的正整数，5～80的正整数；更具体地，n例如为6，7，8，9，10，15，20，25，30，40，50，60，70。
在另一优选例中，所述核心结构的氨基酸残基数占所述多肽复合单元总氨基酸残基数的70％以上，较佳地80％以上，更佳地85％，进一步更佳地90％以上，95％以上， 99％以上或100％。
在另一优选例中，所述多肽复合单元选自：(a)SEQ ID NO:48～105任一所示的氨基酸序列的蛋白或SEQ ID NO:48～76对应多肽单元的重复拼接蛋白；或(b)(a)限定的任一多肽经过一个或多个(如1-20个；较佳地1-10个；更佳地1-5个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；或(c)氨基酸序列与(a)限定的多肽的氨基酸序列有80％以上(较佳地85％以上；更佳地90％以上；进一步更佳地95％以上，如98％，99％以上)序列相似性百分比且具有(a)多肽功能的多肽。所述多肽单元或多肽复合单元本身并无生物学功能，其性质与PEG类似，仅仅起到“载体”的功能。
在本发明的另一方面，提供一种具有生物活性的融合蛋白，其包括：所述的多肽单元或所述的多肽复合单元以及活性蛋白或多肽。
在另一优选例中，所述的融合蛋白包括选自下组任一或组合的结构：
(D 1-PU 1)；
(D 1-PU 1)-(D 2-PU 2)；或
(D 1-PU 1)-(D 2-PU 2)-…(D m-PU m)；
其中，PU 1，PU 2，…，PU m选自所述的多肽复合单元；D 1，D 2，…，D m为所述活性多肽，m为＞2的正整数；
且，(D 1-PU 1)包括(PU 1-D 1)，(D 2-PU 2)包括(PU 2-D 2)，(PU m-D m)包括(D m-PU m)。
在另一优选例中，D 1，D 2，…，D m各自代表一条或多条(包括2条)相互连接的活性蛋白或多肽，所述的多条活性蛋白或多肽之间功能相同或不同。
在另一优选例中，m为3～50的正整数，例如为4～40的正整数，5～35的正整数；更具体地，m例如为6，7，8，9，10，15，20，25，30。
在另一优选例中，所述的融合蛋白的长度≥50个氨基酸，较佳地≥80个氨基酸，更佳地≥100个氨基酸；如为50～5000aa，较佳地如80～4000aa，更佳地如100～2000aa；更具体地，例如150aa，200aa，300aa，50aa，60aa，700aa，800aa，1000aa。
在另一优选例中，所述的融合蛋白中的氨基酸序列中，所述的多肽单元或所述的多肽复合单元所占氨基酸序列占比为10％以上，较佳地20％以上。
在另一优选例中，所述活性蛋白或多肽包括(但不限于)：GLP-2类似物，AR VEGF，hGH，Arginase 1，G-CSF，Exendin-4，GLP-1类似物，GDF15，glucacon，IL-2，IL-15，FGF19，EPO，IL-6，M-CSF，FGF21。
在另一优选例中，所述的融合蛋白包括(但不限于)选自下组的融合蛋白：(a)SEQ ID NO:106～131任一所示的氨基酸序列的蛋白；或(b)(a)限定的任一多肽经过一个或多个(如1-20个；较佳地1-10个；更佳地1-5个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽活性的由(a)衍生的蛋白；或(c)氨基酸序列与(a)限定的多肽的氨基酸序列有80％以上(较佳地85％以上；更佳地90％以上；进一步更佳地95％以上， 如98％，99％以上)序列相似性百分比且具有(a)多肽活性的蛋白。
在另一优选例中，所述的融合蛋白在体内的半衰期或稳定性在统计学上高于未融合的活性多肽(例如，高50％以上，较佳地高100％以上，更佳地高200％以上，更具体地如高500％，1000％，2000％，5000％，10000％或更高)。
在本发明的另一方面，提供一种核酸分子，所述的核酸分子编码前面所述的多肽单元，多肽复合单元或融合蛋白。
在本发明的另一方面，提供一种重组表达载体，它含有所述的核酸分子。
在本发明的另一方面，提供一种遗传工程化的细胞，所述的细胞含有所述的重组表达载体；或所述的细胞基因组中整合有所述的核酸分子。
在本发明的另一方面，提供一种偶联物，其包括：(a)所述的多肽单元或所述的多肽复合单元；以及，(b)活性蛋白或多肽；其中，(b)与(a)以偶联或吸附的方式连接。
在本发明的另一方面，提供所述的多肽单元或所述的多肽复合单元的用途，用于提高活性多肽的稳定性，较佳地包括热稳定性、耐酶稳定性和血清稳定性；延长活性多肽的半衰期，即延长活性多肽的作用时间；和/或提高活性多肽的溶解度。
在本发明的另一方面，提供一种组合物，其包括：所述的融合蛋白或所述的偶联物；和药学上或食品学上可接受的载体。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1.PU-hArg1融合蛋白的SEC-HPLC分析结果图。其中，P1为PUMix17-hArg1-PUMix49、P2为PUMix35-hArg1-PUMix76、P3为PUMix109-hArg1-PUMix163、P4为PU98x5-hArg1-PU106x5、P5为PU12x5-hArg1-PU12x5、P6为PU98x10-hArg1、P7为PU12x10-hArg1、P8为mURP-hArg1-mURP、P9为mXTEN-hArg1-mXTEN、P10为mPAS-hArg1-mPAS。其中箭头所指之处为甲状腺球蛋白(Thyroglobulin，669kDa)。
图2.PU98x5-hArg1-PU106x5的DLS分析结果图。
图3.不同PU-hArg1融合蛋白在SD大鼠体内的免疫原性结果(血清1：500稀释)。
图4.PU-hArg1融合蛋白药代结果。
图5.PU-GH融合蛋白的热稳定性(铜染)。M为蛋白分子量MARKER：200、116、97.2、66.4、44.3KD。泳道1：PU27x28-GH-PU27x5在4℃放置20小时；泳道2：PU27x28-GH-PU27x5在37℃放置20小时；泳道3：PU27x28-GH-PU27x5在60℃放置20小时；泳道4：PU98x28-GH-PU98x4在4℃放置20小时；泳道5：PU98x28-GH-PU98x4在37℃放置20小时；泳道6：PU98x28-GH-PU98x4在60℃放置20小时；泳道7：PU130x28-GH-PU130x5在4℃放置20小时；泳道8：PU130x28-GH-PU130x5在37℃放置20小时；泳道9：PU130x28-GH-PU130x5在60℃放置20小时；泳道10：PU98x28-GH-PU98x4在80℃放置5小时；泳道11：PU130x28-GH-PU130x5在80℃放 置5小时。
图6.实施例11中的PU-GH融合蛋白的热稳定性。
图7.PU-GH融合蛋白的体外细胞学活性。
图8.PU-GDF15融合蛋白对DIO小鼠的体重减轻效果图。
图9.PU与GDF15融合蛋白对DIO小鼠的食欲抑制效果图。
图10.GLP-2G与PU融合蛋白的体外细胞活性检测结果图。
图11.PU与AR VEGF融合蛋白的体外细胞活性结果图。
图12.在大鼠血清中温育第7天的PU-GH融合蛋白。1-2分别为0天和第7天的PU27x28-GH-PU27x5样品、3-4分别为0天和第7天的PU98x28-GH-PU98x4样品、5-6分别为0天和第7天的PU130x28-GH-PU130x5样品。
图13.PU-GH融合蛋白及hGH在胰酶中的稳定性结果图。A.泳道1-4分别为hGH在0、0.02％、0.1％、0.5％的胰酶中孵育40min的结果；M为低分子量MARKER:97.2KD、66.4KD、44KD、29KD、21KD及14KD。B.泳道1和2：PU27x28-GH-PU27x5；泳道3和4：PU98x28-GH-PU98x4；泳道5和6：PU130x28-GH-PU130x5。M为高分子量MARKER:220KD、135KD、90KD、66KD、45KD及35KD。
具体实施方式
本发明人经过深入的研究，提供对于延长蛋白/多肽体内半衰期或改善蛋白/多肽体内外理化性质非常有效的多肽单元(U)或多肽复合单元(PU)，所述的多肽单元(U)由脯氨酸(P)、丙氨酸(A)和谷氨酸(E)组成。
一、单或重复的多肽单元(U)
本发明首先提供一种人工设计的多肽单元(U)，并具有以下特征：(1)由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；(2)根据Chou-Fasman公式计算，至少50％以上为α-螺旋二级结构；和(3)长度≥15个，较佳地≥20个氨基酸。
作为本发明的优选方式，根据Chou-Fasman公式计算，所述的多肽单元(U)至少60％以上为α-螺旋二级结构；优选地，所述的多肽单元(U)至少70％以上为α-螺旋二级结构；优选地，所述的多肽单元(U)至少80％以上为α-螺旋二级结构；更优选地，所述的多肽单元(U)至少90％以上为α-螺旋二级结构。
可以利用远紫外圆二色谱(CD)光谱来检测蛋白或多肽的二级结构。α-螺旋、β-折叠和无规卷曲结构各自形成CD谱的特征峰和宽度。作为本发明的优选实施方式，通过Chou-Fasman算法(Chou，P.Y.等，Biochemistry，1974,13,222-45)预测PU的二级结构。
根据Chou-Fasman公式计算，并非所有由P、A和E组成的多肽单元(U)都含有超过50％的α-螺旋结构(如SEQ ID NO:17和SEQ ID NO:18)，而且SEQ ID NO:17和SEQ ID NO:18由于脯氨酸含量太高，在本发明的制备过程中发现由SEQ ID NO:17和SEQ ID  NO:18组合而成的多肽复合单元(PU)极难获得表达产物。脯氨酸具有典型的破坏二级结构的功能，例如脯氨酸(P)以高度重复的形式出现，则α-螺旋结构会极低，而当丙氨酸(A)或谷氨酸(E)高度重复出现，则α-螺旋结构含量显著提高。
一些示例性的多肽单元(U)如SEQ ID NO:19～47所示。应理解，序列不同于SEQ ID NO:19～47的其它由P、A、E排布而成的序列也可被涵盖在本发明中，只要其满足至少50％以上的α-螺旋二级结构以及长度≥15个，较佳地≥20个氨基酸的特征。
进一步地，本发明提供一种包括所述多肽单元(U)的多肽复合单元(PU)，其中包含2个或多个(包括2个)所述多肽单元(U)。当所述多肽单元(U)为多个时，各个多肽单元(U)之间可以是相同的序列排布或不同的序列排布。
作为本发明的优选方式，所述的多肽复合单元(PU)具有100-2000个氨基酸，示例性的优选方式包括：具有至少100个氨基酸，至少200个氨基酸，至少300个氨基酸，至少400个氨基酸，至少500个氨基酸，至少600个氨基酸，至少700个氨基酸，至少800个氨基酸，至少900个氨基酸，至少1000个氨基酸或至少1200个氨基酸。
作为本发明的优选方式，所述的多肽复合单元(PU)的80％以上由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；更优选地，85％以上由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；更优选地，90％以上由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；更优选地，95％以上由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成；最优选地，100％由脯氨酸(P)、丙氨酸(A)及谷氨酸(E)组成。在一个优选的实施方案中，所述多肽复合单元(PU)由相同序列的多肽单元(U)通过重复拼接而成，或由不同的多肽单元(U)拼接而成。
XTEN为一种由6种氨基酸(A，E，G，P，S，T)组成的多肽，其中8％A，12％E，18％G，17％P，28％S和17％T(Volker Schellenberger等，A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner，Nature Biotechnology.，27(12):1186，2009)，富含S和T。PAS由脯氨酸(P)、丙氨酸(A)和丝氨酸(S)组成，也富含S。然而，在本发明的研究过程中发现，S和T的加入会导致在真核表达系统中带来严重的糖基化现象。对于某些不适合于原核表达系统内重组表达、又不适合化学合成的大分子蛋白而言，富含S和T的载体蛋白组成很难解决糖基化的问题。众所周知，糖基化一般有两种主要类型：O-连接的寡糖糖基化，连接位点在丝氨酸或苏氨酸残基上；N-连接的寡糖糖基化，连接位点在Asn-X-Ser/Thr序列的天冬酰胺酸残基位点上，在此，X可以是除了脯氨酸以外的任意氨基酸。尤其是在酵母中，酵母的糖基化系统与人类的不同，高度糖基化尤其是O-糖基化容易产生强免疫原性，而且生产过程批次不均一性问题很难控制。理论上，XTEN、PAS、GLK或者URP等包含大量S或T的序列在原核以外的表达系统中表达时，糖基化严重且产物不均一是极其严峻的问题。
另外，有些蛋白或多肽N端的结构与活性密切相关，比如Exendin-4或GLP-1的N端暴露对于其活性至关重要。然而外源蛋白在原核系统(如大肠杆菌E.coli)表达时往往N端带有额外的甲硫氨酸，很难直接获得活性产物。因此一般需要在Exendin-4的N端前面加入融合表达标 签，如CBD标签(Volker Schellenberger等，A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner，Nature Biotechnology.，27(12):1186，2009)，表达完成后再用蛋白酶进行切割；或者在GLP-1序列前加上其他氨基酸，如两个连续的丙氨酸以提高切割效率(M.Amiram等，A depot-forming glucagon-like peptide-1fusion protein reduces blood glucose for five days with a single injection，J Control Release.，172(1):144-51，2013)，只有这样才能获得具有天然生物学活性的GLP-1融合蛋白。而直接用酵母或者哺乳动物细胞表达GLP-1融合蛋白则不需要额外的蛋白酶切就可以直接获得具有天然N端序列的GLP-1活性融合蛋白。
本发明的所述的多肽单元(U)或多肽复合单元(PU)则主要由P、A和E组成，无论在原核还是真核表达系统中制备，都不会出现糖基化的问题。
另外，本发明提供的所述多肽单元(U)或多肽复合单元(PU)相比现有技术，Asn(N)及Gln(Q)脱酰胺化造成的不均一现象以及由于氨基酸种类较多导致的潜在蛋白酶位点增多而引起的降解等问题的可能性都极小。在本发明的一个实施例中，所述的多肽单元(U)或多肽复合单元(PU)相比现有技术的载体蛋白具有更优越的血清稳定性和耐酶稳定性。
二、具有治疗活性的融合蛋白
融合蛋白
本发明还提供了具有治疗活性的融合蛋白，其包括一个或若干个相同或不同的活性蛋白药物(D)和所述的多肽单元(U)或多肽复合单元(PU)相连接(如串联)的结构，示例性的此类融合包括，但不限于如下结构：
D-PU
PU-D
D 1- PU-D 2
PU 1-D-PU 2
PU 1-D 1-PU 2-D 2-PU 3-D 3
PU 1-D 1-D 2-PU 2-D 3
PU 1-D 1-PU 2-D 2-D 3
PU 1-D 1-PU 2-D 2-D 3-PU 3
D 1-PU 1-D 2-PU 2-D 3
PU 1-D 1-PU 2-D 2-PU 3-D 3-PU 4-D 4
D 1-PU 1-D 2-PU 2-D 3-PU 3-D 4-PU 4
本发明中，所述D 1、D 2、D 3、D 4…为具有治疗活性的活性蛋白或多肽，且D 1、D 2、D 3、D 4…之间可以相同或不相同；PU 1、PU 2、PU 3、PU 4…各自代表一个所述的多肽复合单元，PU 1、PU 2、PU 3、PU 4…之间可以相同或不相同。
本发明的多肽单元(U)或多肽复合单元(PU)适用于与多种多样的活性蛋白或多肽进行融合，提高活性蛋白或多肽的稳定性、半衰期等性能。所述活性蛋白或多肽(D)可以选自激动剂、受体、配体、拮抗剂、酶或激素等。所述的活性蛋白或多肽可以是已经或将被应用于治疗多种疾病的具有药效的蛋白或多肽，所述疾病包括但不限于：代谢相关疾病、心血管疾病、凝血/出血疾病、生长障碍或病症、肿瘤、血管障碍疾病、炎症、自身免疫病症等所述疾病包括代谢相关疾病、心血管疾病、凝血/出血疾病、生长障碍或病症、肿瘤、血管障碍疾病、炎症、自身免疫病症等；或具体地包括1型糖尿病，2型糖尿病、妊娠糖尿病、高胆固醇血症、肥胖症、高血糖症、超高胰岛素血症、胰岛素产生减少、胰岛素抗性、代谢紊乱、多囊卵巢综合征、血脂异常、进食障碍、高血压、肺性高血压、视网膜神经变性、代谢紊乱、胰高血糖素瘤、溃疡性结肠炎、肾衰竭、充血性心力衰竭、肾病综合征、肾病、腹泻、术后倾倒综合征、肠易激综合征、危重病性多神经病、全身炎症反应综合征、血脂异常、中风、冠心病、血友病、成人和儿童的GH缺乏、Turner综合征、慢性肾衰竭、宫内发育迟缓、特发性身材矮小、AIDS消耗、肥胖症、多发性硬化、衰老、纤维肌痛、克罗恩病、溃疡性结肠炎、肌营养不良症、低骨密度等。
例如，所述D 1、D 2、D 3、D 4…可选自但不限于表1中所列的活性蛋白或多肽或其类似物。
表1
Figure PCTCN2019106092-appb-000001
在融合了所述的多肽单元(U)或多肽复合单元(PU)后，活性蛋白(D)的理化性质显著改善，表现为水溶性的提高、耐酶及耐热稳定性的提高，水动力学半径的增大等，这些理想的性质使得活性蛋白药物(D)的体内半衰期显著延长。在本发明的一个实施例中，融合了所述的多肽单元(U)或多肽复合单元(PU)后，活性蛋白药物(D)的生物学活性下降，然而由于该融合蛋白的半衰期发生极为显著的提高，这种活性的下降仍然是可以接受的。
三、化学偶联物
抗体-药物偶联物(ADC)是抗体与毒性化合物或放射性核素通过赖氨酸、半胱氨酸、非天然氨基酸及工程构建的标签等制备获得的治疗药物。作为ADC药物的一个突出缺点是，由于高疏水性毒性化合物或放射性核素的交联导致整个ADC分子容易聚集甚至产生不可溶的沉淀，尤其是当药物/抗体比率(DAR)较高的情况下。为了解决这个问题，可以通过将化学合成的高亲水聚乙二醇(PEG)或者可生物降解的短链分子作为连接链(Linker)，如PHF(或称
Figure PCTCN2019106092-appb-000002
)。这些手段能有效地改善ADC分子的亲水性并增加其稳定性。
同样地，当具有治疗活性的蛋白分子量极小或者为不适合重组表达的多肽时，优选地可采用化学交联的方式。在本发明中，具有治疗活性的蛋白可由若干不同的活性蛋白或多肽(D)和所述的多肽单元(U)或多肽复合单元(PU)通过化学交联的方式制备而成。化学交联可以在大部分氨基酸残基上进行，然而赖氨酸上亲核型的伯胺基团和半胱氨酸上的活性巯基是最常用的交联位点。此外，酪氨酸和硒代半胱氨酸也会被用于化学交联。
本发明的多肽单元(U)或多肽复合单元(PU)适用于与多种多样的活性蛋白或多肽进行化学偶联，所述活性蛋白或多肽(D)选自激动剂、受体、配体、拮抗剂、酶或激素等，包括但不限于表1中所列的活性蛋白、多肽或其类似物。所述的活性蛋白或多肽可以是已经或将被应用于治疗多种疾病的具有药效的蛋白或多肽，所述疾病包括但不限于：代谢相关疾病、心血管疾病、凝血/出血疾病、生长障碍或病症、肿瘤、血管障碍疾病、炎症、自身免疫病症等；或具体地包括1型糖尿病，2型糖尿病、妊娠糖尿病、高胆固醇血症、肥胖症、高血糖症、超高胰岛素血症、胰岛素产生减少、胰岛素抗性、代谢紊乱、多囊卵巢综合征、血脂异常、进食障碍、高血压、肺性高血压、视网膜神经变性、代谢紊乱、胰高血糖素瘤、溃疡性结肠炎、肾衰竭、充血性心力衰竭、肾病综合征、肾病、腹泻、术后倾倒综合征、肠易激综合征、危重病性多神经病、全身炎症反应综合征、血脂异常、中风、冠心病、血友病、成人和儿童的GH缺乏、Turner综合征、慢性肾衰竭、宫内发育迟缓、特发性身材矮小、AIDS消耗、肥胖症、多发性硬化、衰老、纤维肌痛、克罗恩病、溃疡性结肠炎、肌营养不良症、低骨密度等。
四、多肽单元(U)、多肽复合单元(PU)或融合蛋白的制备及组合物
本发明还提供了分离的多核苷酸，编码所述的多肽单元(U)或多肽复合单元(PU)，或编码所述的融合蛋白。所述的多核苷酸可以是DNA形式或RNA形式。编码蛋白或多肽的多核苷酸可以是包括编码此蛋白或多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。编码蛋白或多肽的多核苷酸可以用PCR扩增法、重组法或人工合成的方法获得。
本发明还提供了重组表达载体，包含所述多核苷酸。以及提供了宿主细胞，所述细胞含有所述表达载体或基因组中整合有外源的所述的多核苷酸，从而可表达所述的蛋白或多 肽单元(U)、多肽复合单元(PU)或融合蛋白。
本发明还提供了所述的多肽单元(U)或多肽复合单元(PU)或融合蛋白的制备方法，包括如下步骤：1)培养所述宿主细胞，使之表达所述多肽单元(U)、多肽复合单元(PU)或融合蛋白；2)收集含有所述多肽单元(U)、多肽复合单元(PU)或融合蛋白的培养物；3)从步骤2所得培养物中分离出所述多肽单元(U)、多肽复合单元(PU)或融合蛋白。
本发明还提供了一种组合物，含有所述多肽单元(U)或多肽复合单元(PU)、融合蛋白或所述宿主细胞培养物，以及药学上或食品学上可接受的载体。“药学上或食品学上可接受的”的成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质；如本领域常用的药物载体或赋形剂。所述多肽单元(U)、多肽复合单元(PU)、融合蛋白或所述宿主细胞培养物通常是“有效量”的，“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
五、融合蛋白的性能
蛋白粘度：蛋白药物一般都高浓度下储存，给药时通过注射给药。因此蛋白药物的粘度越低，储存及给药时的蛋白浓度可以越高。尤其是对于眼部给药的药物而言，蛋白药物粘度下降可以减少给药体积，提高患者的依从性，具有极高的临床意义。包含本发明的多肽单元(U)或多肽复合单元(PU)的融合蛋白，具有极低的蛋白粘度和极高的溶解度。
免疫原性：作为一种载体蛋白，体内免疫原性是最重要的因素。免疫原性产生的因素包括氨基酸序列、聚体的产生、杂质的存在、患者的免疫状态及遗传背景、剂量和给药途径等。本发明提供的多肽复合单元(PU)由多个多肽单元(U)重复拼接组成，如果单个多肽单元(U)具有免疫原性，例如具有T细胞表位，则极有可能会引起强烈的体液免疫反应。除了潜在的细胞表位，蛋白聚集也会增加B细胞受体的大幅交联，导致B细胞快速活化，并增强抗原摄取、加工和递呈。在本发明的一个实施例中，多肽单元(U)用基于QAM方法的预测工具TEPITOPE(Sturniolo T等，Nat.Biotechnol.17:555-561.)计算DRB1*01:01，DRB1*01:02，DRB1*03:01，DRB1*03:02，DRB1*04:01，DRB1*04:02，DRB1*07:01和DRB1*15:01等位基因的得分，结果其得分均低于或等于-8，而蜥蜴来源的Exendin-4(SEQ ID NO:11)则具有更高的得分，意味着更高的免疫原性风险。而在另一个实施例中，所述的多肽单元(U)或多肽复合单元(PU)与具有高度聚集倾向的人生长激素融合后，在高温下完全没有产生聚集的倾向，说明其具有良好的体外稳定性性质，同时也预示着免疫原性的潜在风险较低。在本发明的另一个实施例中，融合蛋白在大鼠体内多次给药后并未检测到针对所述的多肽单元(U)或多肽复合单元(PU)部分的抗药抗体。
药代动力学性质：本发明所述的多肽单元(U)或多肽复合单元(PU)能够增强生物活性蛋白的药代动力学性质，融合所述的多肽单元(U)或多肽复合单元(PU)的活性蛋白或多肽半衰期可以延长2倍以上，其中药物代谢动力学性质通过测定施用于受试者的生物 活性蛋白的终末半衰期与施用了相当剂量的融合了所述的多肽单元(U)或多肽复合单元(PU)的生物活性蛋白相比较来确定。
融合所述的多肽单元(U)或多肽复合单元(PU)的活性蛋白流体力学半径显著增大，从而降低该活性蛋白的肾脏清除率。可利用超速离心、大小排阻色谱或光散射检测所得融合蛋白的流体力学半径。流体力学半径增加会引起组织渗透性减少，可利用这点将药学活性蛋白的副作用降至最小。已有记载，由于增强的渗透性和滞留效应(EPR)，亲水性聚合物趋于选择性蓄积于肿瘤组织中。EPR效应的潜在原因是肿瘤血管的渗漏属性(McDonald，D.M.等，Cancer Res，2002,62,5381-5)，以及肿瘤组织中缺少淋巴流出。因此，通过加入亲水性聚合物可增强用于肿瘤组织的药学活性蛋白的选择性。同样，融合多肽单元(U)或多肽复合单元(PU)可提高给定药学活性蛋白的治疗指数。
理化性质：融合了所述的多肽单元(U)或多肽复合单元(PU)的生物活性蛋白具有显著提升的溶解度和稳定性，比如热稳定性、耐酶稳定性和血清稳定性。在本发明的一个实施例中，融合了所述的多肽单元(U)或多肽复合单元(PU)的hGH融合蛋白在高温下的热稳定性明显高于未融合的hGH，而且hGH容易在制备过程中产生聚集，但是融合了所述的多肽单元(U)或多肽复合单元(PU)后，在SEC-HPLC上并无观察到显著的聚集现象。另外，在其中一个实施例中，通过测定生物活性蛋白暴露于37℃及大鼠血清中至少7天后保持的完整性，来判断其血清稳定性。在另一个实施例中，加入了胰酶的hGH融合蛋白比未融合的hGH显示出更高的耐酶稳定性。
六、术语解释
“生物活性蛋白/多肽”指的是蛋白质、抗体、多肽及其片段和变异体，具有一种或者多种药理学和/或生物学活性，或靶向引导，多聚化等功能。它们可以是天然就存在的，也可以是人工构建的。“生物活性蛋白/多肽”包括酶，酶抑制剂，抗原，抗体，激素，凝血因子，干扰素，细胞因子，生长因子，分化因子，骨组织生长有关的因子，与骨质因子吸收相关的因子，趋化因子(chemotactic factors)，细胞运动因子(cell motility factors)，移动因子(migration factors)，静止因子(cytostatic factors)，杀(细)菌因子，抗真菌因子，血浆黏附分子，间质黏附分子和细胞外基质，受体配基及其片段等。
在某些实施方案中，本发明所涉及的生物学活性蛋白/多肽为表现出“治疗活性”的蛋白/多肽，这种蛋白/多肽拥有一种或者多种已知的生物和/或治疗活性。这些活性与一种或多种本文描述的或者其他已知的治疗蛋白相关。作为一个非限制性例子，“治疗蛋白”(在本文中可与“治疗性蛋白”或“活性蛋白药物”互换)是指一种对于治疗、预防或者改善疾病、症状或者机能紊乱有用的蛋白。作为一个非限制性例子，“治疗蛋白”可以是一种特异性地结合到特定细胞类型(例如淋巴细胞或者癌细胞)并且定位于该细胞表面(或随后内吞至胞内)的蛋白。在另外的非限制性例子中，“治疗蛋白”是指有生物活性的蛋白，特别是一种对于治疗、预防或改善疾病有用的生物活性蛋白。非限制性的治疗蛋白包括拥有如增加血管新生、抑制 血管新生、调节造血功能、促进神经发育、提高免疫反应、抑制免疫反应等生物活性的蛋白。
正如上述提及中的一样，“治疗活性”或者“活性”可以指在人类、非人哺乳动物或其他种属生物体中得到与理想的治疗结果一致的效果的活性。治疗活性可以在体内或者体外测量。
本发明所述的“治疗蛋白”可包括但并不限于：VEGF受体或其片段，TNF受体，HER-2/神经膜受体，人ErbB3受体分泌形态异构体，转化生长因子bⅢ型受体胞外区，转化生长因子bⅡ型受体胞外区，IL-1受体，IL-4受体，尿激酶，β-葡糖脑苷脂酶，精氨酸脱亚胺酶，Arginase，herstatin，表皮生长因子，FGF-1，FGF-19，FGF-21，纤维原细胞生长因子-2，普通纤维细胞生长因子，神经生长因子，血小板来源生长因子，VEGF-1，IL-1，IL-2，IL-3，IL-4，IL-6，IL-8，IL-10，IL-11，IL-12，IL-15，IL-18，IL-21，IL-24，IL-1RA，RANKL，RANK，OPG，LEPTIN，干扰素α，干扰素β，干扰素γ，干扰素Ω，TGFβ，TGFβ-1，TGFβ-3，TNFα，心房钠尿肽，B型钠尿肽，促性腺激素，人黄体生成素，促卵泡激素，人生长激素，EPO，G-CSF，GM-CSF，TPO，M-CSF，SCF，VEGF，EPO模拟肽，TPO模拟肽，FLT3配体，Apo2配体，骨细胞抑止因子，BMP-2，BMP-7，GLP-1及其类似物，GLP-2及其类似物，Exendin-3，Exendin-4及其类似物，胰岛素及其类似物，GIP及其类似物，胰高血糖素及其类似物，内皮抑制素(endostatin)，plasminogen kringle 1domain，plasminogen kringle 5domain和血管抑素等。治疗性蛋白也还可以是抗体及其片段，尤其是抗原结合片段，包括单链抗体scFv等。这些蛋白以及编码这些蛋白的核酸序列都是大家熟知的，并且在如Chemical Abstracts Services Databases(例如CAS Registry)、GenBank和GenSeq这样的公共数据库中可以找到。对于本领域技术人员来说，根据本发明的精神，容易理解的是现有已经发现的绝大部分生物活性蛋白都适用于本发明。当然，同样应理解的是，在本发明以后新发现的具有生物学活性的蛋白/多肽也同样适用于本发明。
本文中，序列同源性(sequence homology)用来描述物种亲缘关系的远近。如果两条序列有一个共同的进化祖先，那么它们是同源的。对序列同源性进行分析时，一般是将待研究序列加入到一组来自不同物种的多序列中以确定该序列与其他序列的同源关系。常用的分析工具是CLUSTAL等。
本文中，序列同一性(sequence identity)指参与对比的序列中相同残基的百分比。可采用本领域周知的计算软件计算两条或多条目的序列的序列同一性，这些软件可获自如NCBI。
本文中，序列相似性(sequence similarity)指若干条DNA、RNA或蛋白序列之间的相似程度，理解为参与对比的序列中相同残基的百分比(同一性百分比，identity％)或具有相似物理化学性质的残基百分比(相似性百分比，similarity％)。例如，2条不同蛋白质序列的序列相似性可以理解为此2条序列中存在的相同氨基酸残基的百分比(同一性百分比，identity％)或者此2条蛋白质序列中存在的具有相似物理化学性质的氨基酸残基的百分比(相似性百分比，similarity％)。
重复序列：在本发明中，多肽单元(U)主要由或仅由P、A、E三种氨基酸组成，在 每个单元内部(≥20个氨基酸)，连续出现相同氨基酸的几率极高，除非这种氨基酸排列影响了其重组表达。
蛋白或多肽链的二级结构：指蛋白或多肽链中有规则重复的构象，主要有α-螺旋、β-折叠、β-转角及无规则卷曲。以下术语解释皆与经典分子生物学中的定义一致。
α-螺旋：常见的一种二级结构，肽链主链绕假想的中心轴盘绕成螺旋状，一般都是右手螺旋结构，螺旋是靠链内氢键维持的。每个氨基酸残基(第n个)的羰基氧与多肽链C端方向的第4个残基(第n+4个)的酰胺氮形成氢键。在典型的右手α-螺旋结构中，螺距为0.54nm，每一圈含有3.6个氨基酸残基，每个残基沿着螺旋的长轴上升0.15nm。
β-折叠：蛋白质中常见的二级结构，是由伸展的多肽链组成的。折叠片的构象是通过一个肽键的羰基氧和位于同一个肽链或相邻肽链的另一个酰胺氢之间形成的氢键维持的。氢键基本上垂直于肽链的螺旋长轴，这些肽链可以是平行排列；或者是反平行排列。
β-转角：连接蛋白质分子中的α-螺旋和β-折叠，使肽链走向改变的一种非重复多肽区，一般含有2～16个氨基酸残基。含有5个氨基酸残基以上的转角又常称之为环(loops)。常见的转角含有4个氨基酸残基，有两种类型。转角I的特点是：第1个氨基酸残基与第4个残基之间形成氢键；转角II的第3个残基往往是甘氨酸。这两种转角中的第2个残基大都是脯氨酸。
无规卷曲：此种结构为多肽链中除以上几种比较规则的构象外，其余没有确定规律性的那部分肽链的二级结构构象。
二级结构预测：目前，蛋白或多肽的二级结构预测方法有：Chou-Fasman算法、PHD算法、多序列列线预测、基于神经网络的序列预测、基于已有知识的预测方法(knowledge based method)及混合方法(hybrid system method)，这些方法为本领域技术人员所熟知的方法。本发明采用的是根据Chou-Fasman算法的结果(http://www.biogem.org/tool/chou-fasman/index.php)。
亲水性氨基酸：例子为精氨酸、赖氨酸、苏氨酸、天冬酰胺、谷胺酰胺、脯氨酸和谷氨酸。
疏水性氨基酸；例子为色氨酸、酪氨酸、苯丙氨酸、甲硫氨酸、亮氨酸、异亮氨酸和缬氨酸、丙氨酸。
本文中，“PEG”和/或“PEG化”指将聚乙二醇(PEG)聚合物链共价连接至目的生物活性蛋白或多肽上。一般认为将PEG共价连接至生物活性蛋白或多肽能够掩蔽所述蛋白或多肽免于宿主的免疫系统攻击，并增加目的生物活性蛋白或多肽的水动力学半径，从而通过降低肾清除率来延长其体内循环时间。
下述具体的实施方式，如无特别说明，均为本领域技术人员熟知的常规方法。如本发明的实施例使用免疫学、生物化学、微生物学、细胞生物学、遗传学和重组DNA常 规技术，例如可以参照如《分子克隆实验指南》第三版(Sambrook J，Russell DW，Molecular cloning：A laboratory manual.3rd edition，New York：Cold Spring Harkbor Laboratory Press，2001)或商业公司提供的操作说明书中的技术方案。
实施例1、 多肽单元(U)的获得
多肽单元(U)由P、A和E三种氨基酸组成，优选的示例性序列及其对应的TEPITOPE得分见如表2。
表2
Figure PCTCN2019106092-appb-000003
Figure PCTCN2019106092-appb-000004
利用Chou-Fasman算法计算α-螺旋含量，示例性的多肽单元(U)的α-螺旋含量如表3。
表3
SEQ ID NO: 序列 α-螺旋含量(％)
17 PPPPPPPPPPPPPPPAEAE 10.5
18 PPPAEPPPAPPPEPPPPPP 0
19 APPAEEPAEAPAEPPAAPEA 95
20 AEPAPPEEAAPAAPAAPEPE 80
21 APEEPAPEAPAAPAEAPPAP 90
22 AAPAEEAEAPEAPPAPAPEP 55
23 AEPAPPAAEPAPPAAEAAEP 75
24 APAAEAAPPAEAAEPEPEAP 90
25 APAPPEAEEPPEAAPAPAPA 75
26 AAEAEAAEAEPPAPPAPEPA 60
27 APPEAPEAPEAAPAAPEPAE 80
28 APPEAEPPEAPEAAEAAPAP 90
29 APAEAPAAAEEAPPAEPAPE 95
30 AAEAPAAPPAPEPEAEPEPA 80
31 AAEAPAPEPAAEPEPAPEAP 90
32 AEAEPAAPAPAEPAEPEAPA 95
33 AEPAAPAPAEPAAPAPEAPA 95
34 APAPEAPAAEPAAPAPAEPA 95
35 AAPPAPAPAAAEEAPAEAPA 75
36 AEPAAPAAPEPEPAAPAEAA 95
37 APPEPPAEAAPAAAEAPAEA 80
38 AAPAAAEPPAAEAEAPAPPA 80
39 APAAPPEEAAAEPPAAEAAP 80
40 AAEAAPAAPAAPEPAEEAAP 90
41 APEAAPAAAEPAPAPAAPEP 90
42 APAEAAEAAPEAPEPAAPAA 95
43 AEAAAPAEAAPAPAPEAPAP 90
44 AAEAAEAAPAEPEPPAPPAP 65
45 AEAAEAPAPAAPPAAPAPEP 90
46 APEAPAEEAEPAAAPPAEAP 90
47 APAPPAEAEAEAPPAAPEPA 85
实施例2、高表达低分子量多肽复合单元( PU)的获得
所述的多肽复合单元( PU)由多肽单元(U)拼接而成，可通过如下步骤之其一实现：
a.先设计一段由不同或相同多肽单元(U)组成的所述多肽复合单元( PU)序列；再将该所述多肽复合单元( PU)的蛋白序列转为DNA序列，并通过基因合成法获得全长DNA。
b.或者，如Martin Schlapschy等人报道(Martin Schlapschy等，Protein Engineering，Design&Selection，20：273-284，2007)，将多肽单元(U)通过互补粘端的方式在T4DNA连接酶作用下拼接，再进行琼脂糖凝胶电泳，回收适当大小的DNA片段。同样地，这些参与拼接的多肽单元(U)可以是序列相同的，也可以是不同的。为了纯化方便，在所述多肽复合单元( PU)的N末端或C末端加入6×His亲和纯化标签。
根据方法b，合成多肽单元(U)的对应核苷酸(DNA)片段：例如在AEPAAPAPAEPAAPAPEAPA(SEQ ID NO:33)对应的DNA片段两端引入BglI与SfiI酶切位点，其碱基序列经大肠杆菌密码子优化，将该序列通过EcoRV平末端插入pUC57，质粒命名为pUC57-U 1。pUC57-U 1经Bgl I酶切获得的片段与经SfiI酶切及去磷酸化的载体连接以获得二聚体，菌落PCR并酶切验证挑选二聚体克隆。同样操作，直至拼接挑选到理想的长度为止。可以通过此方法拼接，形成由不同序列的多肽单元(U)构成的多肽复合单元(PU)，如将表3中20个氨基酸长度的多肽单元(U)混合拼接组成的PUMix17(包括了U35、U34、U46等)、PUMix357(包括了U79、U27、U12等)等的多肽复合单元(PU)；也可以将相同序列的多肽单元(U)通过不断重复拼接，获得如PU12x5(U12重复拼接5次)、PU23x10(U23重复拼接10次)等的所述多肽复合单元(PU)。示例性的多肽复合单元(PU)序列见表4所示。值得注意的是，多肽复合单元(PU)在常规的考马斯亮蓝染色条件下染色效率较低，需要用铜染的方式(Chris Lee等，Analytical Biochemistry 166：308-312，1987)。具体步骤如下：1.配制0.3M CuCl 2水溶液；2.将电泳胶拆下后，用双蒸水漂洗2-3min；3.将胶侵入0.3M CuCl 2溶液中，染色2-5min；4.取出胶后，用成像仪拍照。
表4、示例性的多肽复合单元(PU)序列
Figure PCTCN2019106092-appb-000005
实施例3、PU-hArg1融合蛋白的制备
将实施例2中经拼接后的PU片段与hArg1(中文名：人精氨酸酶；其序列见SEQ ID NO:7)的编码序列融合(如表5所示)，C端与His-6标签相连，构建到pET41a载体中。 将该质粒转化到大肠杆菌感受态BL21(DE3)Gold中，挑取单克隆至LB卡那霉素抗性液体培养基，37℃、250RPM培养，长至OD 0.4-0.6(大约3h)，取200μL诱导前培养物，作为阴性对照。之后在剩余培养物中加IPTG至终浓度为1mM，在37℃诱导2.5h后取200μL。将诱导前和诱导后样品5000rpm、4min离心后，弃上清，加2％SDS 40μL重悬，加10μL 5*Loading Buffer混匀，100℃加热8-10min。
表5、示例性的PU-hArg1融合蛋白序列
SEQ ID NO: 代号
106 PU12x10-hArg1
107 PU12x5-hArg1-PU12x5
108 PU98x10-hArg1
109 PU98x5-hArg1-PU106x5
110 PUMix17-hArg1-PUMix49
111 PUMix35-hArg1-PUMix76
112 PUMix109-hArg1-PUMix163
113 mPAS-hArg1-mPAS
114 mXTEN-hArg1-mXTEN
115 mURP-hArg1-mURP
其中，mPAS，mXTEN，mURP是现有技术中的3种载体蛋白。
实施例4、PU-hArg1类似蛋白的分离纯化
蛋白纯化的方法根据不同的表达系统有所差异，现有技术早已有大量的知识提供蛋白纯化的指导，如GE Healthcare公司的经典纯化指南手册《Antibody Purification Handbook》，或者Elsevier出版社出版的《METHODS IN ENZYMOLOGY，Guide to Protein Purification，2nd Edition》等，亲和层析、分子排阻层析、离子交换层析及疏水层析等，已是本领域技术人员所熟知的技术。如下纯化过程为示例性的表明在表达宿主为大肠杆菌及发酵条件特定情况下的纯化方式。当发酵条件不同时，纯化条件亦应当相应稍作调整，在此不再赘述。
PU-hArg1融合蛋白纯化步骤依次包括硫酸铵沉淀，金属离子加热沉淀，阳离子交换，阴离子交换，疏水层析。融合较长多肽复合单元(PU)序列后表观分子量过大需要使用更大孔径的填料纯化才可获得较高的载量。不同结构的纯化稍有差别，例如随着所融合的多肽复合单元(PU)序列中谷氨酸百分比的增大，阳离子交换柱的结合越弱相应需要更低的pH去结合，并且较低的盐浓度就可洗下，相反阴离子交换柱的结合越强，只需要较低的pH就可以结合，并且需要较高的盐浓度才可洗脱。随着融合的多肽复合单元(PU)长度的延长，离子交换柱的结合洗脱性质也会发生与前述谷氨酸百分比增大时类似的现象。当融合在蛋白一端的一定长度的多肽复合单元(PU)序列分散到蛋白的两端时， 阳离子交换柱的结合能力减弱，阴离子交换柱的结合能力增强。
将50g菌体与300ml 20mM PB，pH 7.0缓冲液混合后，用Ф15超声探头破碎2h，超3s停3s，破菌液8000rpm离心30min取上清，再用1μm滤膜过滤。破菌上清液调pH至5.0，上样到用0.2M NaCl，20mM NaAc-HAc，pH 5.0平衡后的25ml MMC(Bestarose Diamond MMC)层析柱上。先用2M NaCl，20mM NaAc-HAc，pH 5.0洗脱杂蛋白，再用洗脱液(2M NaCl，20mM Tris-HCl，pH8.0)洗脱目的蛋白。洗脱液调pH至6.0后加50mM CoCl 2于60℃活化10min，加入(NH 4) 2SO 4调电导至140ms/cm上样至5ml Phenyl(Phenyl Bestarose HP，博格隆(上海)生物技术有限公司)层析柱，50％洗脱液洗脱目的蛋白，100％洗脱杂质及聚体。上样缓冲液：1M(NH 4) 2SO 4，20mM NaAc-HAc，pH6.0，洗脱冲液：20mM NaAc-HAc，pH6.0。洗脱样品用10mM Tris-HCl，pH 8.0于G25(Sephadex G-25，Coarse)层析柱上脱盐，之后上样到用20mM Tris-HCl，pH 8.0平衡后的20mL SuperQ(SuperQ-650M，TOSOH)层析柱，收集流出液，目的蛋白在流出液中。2M NaCl 20mM Tris-HCl，pH8.0洗脱，弃洗脱液。
实施例5、SEC-HPLC分析PU-hArg1表观分子量
将1mg/mL样品和分子量标准混合溶液，使用SEC-HPLC-UV分析。以相对分子量(Mr)为横坐标，实际测得的洗脱体积(V e)为纵坐标，线性回归：V e＝K 1-K 2logM r。K 1与K 2为常数，M r为相对分子质量。检测方法如下：检测波长：214nm；色谱柱：柱温25℃，Sepax SRT-1000SEC 5μm(300×7.8mm)，流动相：50mM PB，150mM NaCl，pH7.2；运行时间：20分钟。
从结果来看，各个PU-hArg1的表观分子量大于669KDa，如图1。
实施例6、DLS分析PU-hArg1水化半径
将1mg/ml样品吸取1.5ml置于比色皿中。马尔文Zetasizer Nano ZS中重复测定三次。
检测方法如下：检测器：173度光散射检测器；检测温度：25℃；示例性的粒径如图2所示，PU98x5-hArg1-PU106x5的水溶液中粒径达到17nm。
实施例7、PU-hArg1体外水解精氨酸活性检测
将待测活样品稀释到1μM，将45μl稀释后的样品与5μl 500mM CoCl 2混合后60℃活化10min。50μl活化后的样品，加450μl 500mM L-精氨酸(pH7.4)，混匀后37℃水解15min，取样20μL，加至2mL尿素氮试剂混合液中(南京建成生物工程研究所)，立刻置沸水中准确水浴15分钟，冰水冷却5min，之后520nm处测定OD值。根据标准曲线计算尿素氮含量。Kcat(s -1)是指每摩尔酶每秒催化底物分解生成产物的摩尔数，Kcat(s -1)＝尿素氮浓度(mmol/mL)/[反应时间(s)×(样品浓度/稀释倍数/分子量)(mmol/mL)]。酶的比 活是指每毫克质量的蛋白质中所含的某种酶的催化活力，比活＝(1/MW)×Kcat×60×1000。实验结果如下表6所示，由于融合所述的多肽单元(U)或多肽复合单元(PU)后每个样品分子量不同，因此单位质量(mg)内的IU(比活)有差异，然而通过Kcat值可以明显看出PU-hArg1融合蛋白水解精氨酸的活性并不降低，反而相对于hArg1而言略微上升。
表6、PU-hArg1融合蛋白的精氨酸水解活性
样品名 比活(IU/mg) Kcat(s -1)
hArg1 252.6 147.9
PU12x10-hArg1 185.1 165.2
PU12x5-hArg1-PU12x5 184.1 164.3
PU98x10-hArg1 192.5 168.1
PU98x5-hArg1-PU106x5 193.9 169.3
PUMix17-hArg1-PUMix49 100.4 155.3
PUMix35-hArg1-PUMix76 100.1 157.9
PUMix109-hArg1-PUMix163 101.6 160.2
mPAS-hArg1-mPAS 120.0 152.5
mXTEN-hArg1-mXTEN 150.5 153.7
mURP-hArg1-mURP 120.4 155.1
实施例8、不同PU-hArg1类似蛋白的免疫原性试验
SD大鼠随机均分组，每组10只，分别用不同的PU-hArg1及未融合的hArg1蛋白(R&D Systems,Cat:5868-AR)免疫，3mg/kg，皮下注射，每周注射一次，连续注射4周，同时一组注射PBS作为阴性对照。最后一次免疫后两周，处死后取血，分离获得血清。用ELISA实验检测血清中PU抗体生成情况。具体地，用相应免疫大鼠的不同PU-hArg1融合蛋白、SEQ ID NO:113-115对照蛋白及未融合的hArg1蛋白包被ELISA板，PU-hArg1融合蛋白给药组血清的免疫动物血清分别稀释100倍、500倍及1000倍后加入过量hArg1蛋白拮抗结合hArg1的抗体，在37℃孵育2h，最后用HRP标记的羊抗大鼠二抗(EarthOX，E030140-01)检测，读取OD450值。给药后OD450为给药前2倍以上为阳性，反之为阴性。图3为血清稀释500倍后的检测结果。Arg1给药组抗药抗体较高，但是PU-hArg1融合蛋白组在拮抗掉针对Arg1的抗药抗体后，基本为阴性，而对照组超出阴性值2倍以上，即为弱阳性。
实施例9、不同PU-hArg1类似蛋白的药代检测试验
6只大鼠随机均分组，每组10只，分别注射不同的PU-hArg1蛋白和SEQ ID NO:113-115对照蛋白，2mg/kg，皮下注射，注射前和注射后3h，8h，12h，24h，36h，48h，72h，96h，120h，144h，168h取血，分离获得血清。
用夹心ELISA法检测融合蛋白在大鼠体内的药代情况。100ng/孔的hArg1兔多抗(杭州华安生物技术有限公司)包被过夜，PBST洗涤3次。5％脱脂奶粉封闭后，PBST洗涤3次，各时间点的血清稀释至指定的倍数，按100μl/孔加入到ELISA酶标板中，在37℃孵育2h后，PBST洗涤3次，加入生物素标记的hArg1兔多抗(杭州华安生物技术有限公司)，37℃孵育2小时，PBST洗涤3次，最后HRP标记的链霉亲和素稀释5万倍后加入到ELISA板中，37℃孵育1小时后PBST洗涤5次，用常规TMB法检测，读取OD450值。结果如图4所示，PU-hArg1融合蛋白的半衰期比对照蛋白有明显的改善。
实施例10、PU-GH融合蛋白的制备
将实施例2中经拼接后的所述的多肽单元(U)或多肽复合单元(PU)与hGH(人类生长素)片段(SEQ ID NO:5)连接(如表7所示)，N端与His6相连，构建到pET41a载体中。将该质粒转化到大肠杆菌感受态BL21(DE3)Gold，挑取单克隆至LB卡那抗性液体培养基，37℃、250RPM培养，长至OD 0.4-0.6(大约3h)，取200μL诱导前培养物，作为阴性对照。之后在剩余培养物中加IPTG至终浓度为1mM，在37℃诱导2.5h后取200μL。将诱导前和诱导后样品5000rpm、4min离心后，弃上清，加2％SDS 40μL重悬，加10μL5*Loading Buffer混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。
将30g菌体与300ml 20mM PB缓冲液(pH 7.0)混合后，用Ф15超声探头破碎2h，超3s停3s，破菌液8000rpm离心30min取上清，再用1μm滤膜过滤。向破菌上清中加入硫酸铵至电导180mS/cm，8000rpm、15min、10℃离心收集蛋白沉淀。沉淀用20mM PB(pH 7.0)溶液溶解，之后再用硫酸铵在180mS/cm的电导下沉淀。取沉淀用20mM NaAc(pH5.0)溶液溶解，并用水稀释至电导4mS/cm以下。用Super Q-650M(TOSOH)层析柱进行纯化(Buffer A：20mM NaAc pH5；Buffer B:0.5M NaCl 20mM NaAc pH5)，按20％B、70％B、100％B一次洗脱。取70％B洗脱样品，调pH至6.0，并用硫酸铵调电导至140mS/cm。用Phenyl HP(博格隆(上海)生物技术有限公司)层析柱进行纯化，直接用50mM PB，pH6洗脱。洗脱样品80℃水浴30min灭活蛋白酶。待样品温度恢复至室温后，调pH到4.0，并稀释溶液至电导4mS/cm以下。
最后用Diamond SP Mustang层析柱(博格隆(上海)生物技术有限公司)进行纯化(缓冲液A：20mM NaAc，pH4.0；缓冲液B1:20mM NaAc，pH5.0；缓冲液B2:20mM PB，pH7.0)，用B1和B2先后洗脱，收集B2洗脱样品。
当样品SDS-PAGE纯度低于95％时，进行如下步骤：洗脱液上样到用平衡缓冲液(0.5M NaCl，20mM咪唑，20mM Tris-HCl，pH 7.5)平衡后的50ml Chelating Sepharose Fast Flow层析柱(GE Healthcare)，再平衡后用10％、50％、100％洗脱缓冲液(0.15M NaCl，0.5M咪唑，20mM Tris-HCl，pH 8.0)洗脱。
表7、PU-GH融合蛋白
SEQ ID NO: 代号
116 PU27x28-GH
117 PU27x28-GH-PU27x5
118 PU98x28-GH
119 PU98x28-GH-PU98x4
120 PU130x28-GH
121 PU130x28-GH-PU130x5
实施例11、PU-GH融合蛋白的热稳定性
将经纯化的PU-GH融合蛋白样品调节成同样浓度(1mg/ml)，在无菌条件下过滤除菌，吸取相同体积到无菌的1.5ml离心管中，分别置于4℃、37℃及60℃中处理20小时或者在80℃放置5小时，SDS-PAGE电泳观察纯度。hGH由于在高温下出现聚集沉淀，不作电泳分析。结果如图5，可见所述融合蛋白具有良好的热稳定性。
实施例12、SEC-HPLC分析PU-GH样品的聚集
将实施例11中经加热处理的样品(PU27x28-GH-PU27x5在60℃放置20小时、PU98x28-GH-PU98x4在60℃放置20小时、PU130x28-GH-PU130x5在60℃放置20小时、PU98x28-GH-PU98x4在80℃放置5小时及PU130x28-GH-PU130x5在80℃放置5小时)用SEC-HPLC-UV分析。以相对分子量(Mr)为横坐标，实际测得的洗脱体积(V e)为纵坐标，线性回归：V e＝K 1-K 2logM r。K 1与K 2为常数，M r为相对分子质量。检测方法如下：检测波长：214nm；色谱柱：柱温25℃，Sepax SRT-1000SEC 5μm(300×7.8mm)，流动相：50mM PB，150mM NaCl，pH7.2；运行时间：20分钟。热稳定性测定结果如图6，可见各融合蛋白具有良好的热稳定性。hGH由于高温处理出现聚集沉淀，不进行液相分析。
实施例13、PU-GH样品的体外细胞活性检测
Ba/f3-GHR细胞用无IL-3的RPMI 1640培养基(含5％FBS和1mg/mL G418)饥饿处理4-6h后，转移至离心管中，1000RPM离心5min。用上述培养基重悬后计数，调整至2 x 10 5/mL，铺96孔板，每孔100μL，即2万个细胞/孔。各待检蛋白用上述培养基稀释至合适的浓度，每孔加入10μL，刺激48h后，用MTT法检测。如表8和图7。
表8.PU-GH融合蛋白的体外细胞学活性
代号 EC50(nM)
PU27x28-GH 3.12
PU27x28-GH-PU27x5 6.45
PU98x28-GH 2.33
PU98x28-GH-PU98x4 5.13
PU130x28-GH 2.11
PU130x28-GH-PU130x5 6.23
hGH 0.54
由上表可见，融合蛋白的细胞学活性发生一定的减弱，但是在可被接受的范围内。
实施例14、不同PU-GH类似蛋白的药代检测试验
SD大鼠随机均分组，每组10只，分别皮下注射不同的PU-GH融合蛋白或hGH重组蛋白(Sino Biological,Cat:16122-H07E)，2mg/kg，注射前和注射后3h、8h、12h、24h、36h、48h、72h、96h、120h、144h、168h取血，分离获得血清。用夹心ELISA方法检测PU-GH蛋白在大鼠体内的药代情况。hGH抗体(Sino Biological,Cat:16122-R101)按100ng/孔加至ELISA酶标板，4℃包被过夜，PBST洗涤3次并5％奶粉封闭2小时，再次PBST洗涤3次，将各时间点的血清稀释至指定的倍数并加入到ELISA酶标板中，在37℃孵育2h后，PBST洗涤3次，加入生物素标记的hGH多克隆抗体(Sino Biological,Cat:16122-T24，生物素标记自制)，37℃孵育2h，PBST洗涤5次，最后HRP标记的链霉亲和素稀释5万倍后加入到ELISA板中，37℃孵育1h后常规TMB法检测，读取OD450值。PU-GH融合蛋白的半衰期结果如表9。
表9
融合蛋白代号 半衰期(t 1/2，小时)
PU27x28-GH 10.1
PU27x28-GH-PU27x5 16.7
PU98x28-GH 10.4
PU98x28-GH-PU98x4 17.6
PU130x28-GH 11.5
PU130x28-GH-PU130x5 16.5
hGH 0.17
由上表可见，融合蛋白的半衰期发生了极为显著的延长，与融合前hGH相比延长幅度高于50倍，甚至高于100倍。
实施例15、PU与GDF 15融合蛋白的制备
将实施例2中经拼接后的PU片段与GDF 15(生长分化因子，SEQ ID NO:15)融合表达(如表10所示)，N端与His6相连，核苷酸片段亚克隆至质粒pPIC9(Life Technologies)，构建成表达载体，以甲醇酵母Pichia pastor GS115(His -)为表达宿主菌，通过电转化将线性化的表达质粒转化到GS115中。30℃培养3天，至单菌落出现。将上述转化的重组酵母单菌落接种至10ml BMGY液体培养基中，30℃，250rpm培养24小时后，静置过夜，弃上清，加入10ml含1％甲醇的BMMY液体培养基，30℃，250rpm诱导表达。培养液离心取上清后加 5*加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。
表10
氨基酸序列(SEQ ID NO:) 融合蛋白代号
122 PUMix76-GDF15
123 PUMix257-GDF15
124 PU58x5-GDF15
125 PU98x10-GDF15
126 PU184x10-GDF15
发酵液离心上清先用40％硫酸铵沉淀，用去离子水复溶后，上样到用平衡缓冲液(0.5M NaCl,20mM咪唑,20mM Tris-HCl,pH 7.5)平衡后的50ml Chelating Sepharose Fast Flow层析柱(GE Healthcare)，再平衡后用10％，50％，100％洗脱缓冲液(0.15M NaCl,0.5M咪唑,20mM Tris-HCl,pH 8.0)洗脱。洗脱液混合后，加入30-50％饱和度的硫酸铵沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。复溶样品用10mM Tris-HCl，pH 8.0于G25(Sephadex G-25，Coarse)层析柱上脱盐。
实施例16、PU与GDF15融合蛋白在DIO小鼠中的药效研究
7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下给药处理。按照30nmol/千克体重的剂量给药，对照组注射等体积生理盐水(PBS)；融合蛋白4天一次，连续给药28天，每天测量小鼠体重及进食量。最后一次给药后的第5天处死。计算每组动物给药前及处死时的平均体重变化。
结果如图8和图9所示，融合蛋白可以使得肥胖的动物体重显著地减少，说明融合蛋白保留了GDF15本身的生物学活性。
实施例17、PU与GLP-2类似物融合蛋白的制备
将实施例2中经拼接后的PU片段与胰高血糖素样肽2类似物GLP-2G融合(SEQ ID NO:1)，C端与His-6标签相连，核苷酸片段亚克隆至质粒pPIC9(Life Technologies)，构建成表达载体，以甲醇酵母Pichia pastor GS115(His -)为表达宿主菌，通过电转化将线性化的表达质粒转化到GS115中。30℃培养3天，至单菌落出现。将上述转化的重组酵母单菌落接种至10ml BMGY液体培养基中，30℃，250rpm培养24小时后，静置过夜，弃上清，加入10ml含1％甲醇的BMMY液体培养基，30℃，250rpm诱导表达。培养液离心取上清后加5*加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。
表11
氨基酸序列(SEQ ID NO:) 融合蛋白代号
127 GLP2G-PU69x45
128 GLP2G-PU98x43
129 GLP2G-PU141x47
发酵液离心上清先用40％硫酸铵沉淀，用去离子水复溶后，上样到用平衡缓冲液(0.5M NaCl,20mM咪唑,20mM Tris-HCl,pH 7.5)平衡后的50ml Chelating Sepharose Fast Flow层析柱(GE Healthcare)，再平衡后用10％，50％，100％洗脱缓冲液(0.15M NaCl,0.5M咪唑,20mM Tris-HCl,pH 8.0)洗脱。洗脱液混合后，加入30-50％饱和度的硫酸铵沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。复溶样品用10mM Tris-HCl，pH 8.0于G25(Sephadex G-25，Coarse)层析柱上脱盐。
实施例18、PU与GLP-2G融合蛋白的活性测定
GLP-2G融合蛋白体外细胞学活性检测采用荧光素酶报告基因检测法。将GLP-2R(GLP-2受体)基因克隆至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GLP-2R，同时荧光素酶(luciferase)全长基因克隆至pCRE-EGFP质粒上，替换EGFP基因，得到pCRE-Luc重组质粒。pCDNA3.1-GLP-2R和pCRE-Luc质粒按摩尔比1:10的比例转染CHO细胞，筛选稳转表达株，获得重组GLP-2R/Luc-CHO稳转细胞株。
在10-cm细胞培养皿中用含10％FBS和300μg/ml G418的DMEM/F12培养基培养细胞，等汇合度至90％左右时，弃去培养上清，加入2ml胰酶消化2min后，吸掉上清，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基中和，转移至15ml离心管中，800rpm离心5min后，弃去上清，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基重悬，计数。用含10％FBS的DMEM/F12培养基稀释细胞至3*10 5/mL，96孔板中每孔铺100μl，即每孔3万细胞，贴壁后换成含0.1％FBS的DMEM/F12培养基培养过夜。
铺在96孔板的细胞弃去上清后，将纯化的重组蛋白或GLP-2(杭州中肽生化有限公司，货号GLUC-002A)用含0.1％FBS的DMEM/F12培养基稀释至一系列指定浓度，加入到细胞培养孔中，100μl/孔，刺激6h后检测。根据lucifersae reporter kit(Ray Biotech，Cat:68-LuciR-S200)说明书进行检测。结果如表12及图10所示。
表12
融合蛋白代号 EC50(nM)
GLP2G-PU69x45 278.9
GLP2G-PU98x43 311.1
GLP2G-PU141x47 298.6
GLP-2 5.2
由表12可见，融合蛋白的细胞学活性发生一定的减弱，但是在可被接受的范围内。
实施例19、PU与GLP-2G融合蛋白的药代检测试验
SD大鼠随机均分组，每组10只，分别注射不同的融合蛋白，2mg/kg，皮下注射，注射前和注射后3h、8h、12h、24h、36h、48h、72h、96h、120h、144h、168h取血，分离获得血清。用夹心ELISA法检测融合蛋白在大鼠体内的药代情况。GLP-2抗体(Abcam，货号ab14183)按100ng/孔加至ELISA酶标板，4℃包被过夜，PBST洗涤3次并5％奶粉封闭2小时，再次PBST洗涤3次，将各时间点的血清稀释至指定的倍数并加入到ELISA酶标板中，在37℃孵育2h后，PBST洗涤3次，加入生物素标记的GLP-2多抗(Abcam，货号ab48292)，37℃孵育2h，PBST洗涤5次，最后HRP标记的链霉亲和素稀释5万倍后加入到ELISA板中，37℃孵育1h后常规TMB法检测，读取OD450值。
表13
融合蛋白代号 半衰期(t 1/2，小时)
GLP2G-PU69x45 42.7
GLP2G-PU98x43 40.5
GLP2G-PU141x47 41.2
由表13可见，GLP-2在体内半衰期仅有数分钟，而融合蛋白的半衰期发生了极为显著的延长。
实施例20、PU与AR VEGF融合蛋白的制备
将实施例2中经拼接后的PU与结合VEGF的锚定重复蛋白(Ankyrin repeat proteins(AR VEGF)，SEQ ID NO:3)融合(表14)，C端与6His标签相连，构建到pET41a载体中。将该质粒转化到大肠杆菌感受态BL21(DE3)gold，挑取单克隆至LB卡那抗性液体培养基，37℃、250RPM培养，长至OD 0.4-0.6(大约3h)，取200μL诱导前培养物，作为阴性对照。之后在剩余培养物中加IPTG至终浓度为1mM，在37℃诱导2.5h后取200μL。将诱导前和诱导后样品5000rpm、4min离心后，弃上清，加2％SDS 40μL重悬，加10μL 5*loading Buffer混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。
将40g菌体与300ml 20mM PB，pH 7.0缓冲液混合后，用Ф15超声探头破碎2h，超3s停3s，破菌液8000rpm离心30min取上清，再用1μm滤膜过滤。破菌上清先在80℃加热处理20分钟，离心沉淀杂蛋白。用40％硫酸铵沉淀，用去离子水复溶后，上样到用平衡缓冲液(0.5M NaCl，20mM咪唑，20mM Tris-HCl，pH 7.5)平衡后的50ml Chelating Sepharose Fast Flow层析柱(GE Healthcare)，再平衡后用0-100％洗脱缓冲液(0.15M NaCl，0.5M咪唑，20mM Tris-HCl，pH 8.0)线性洗脱。洗脱液加入45％饱和度的硫酸铵沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。复溶样品用10mM Tris-HCl，pH 8.0于G25(Sephadex G-25，Coarse)层析柱上脱盐。
表14
氨基酸序列(SEQ ID NO:) 代号
130 PU73x49-AR VEGF
131 PU98x43-AR VEGF
实施例21、PU与AR VEGF融合蛋白的亲和力测定
采用BLI(Bio-layer inteferometry，ForteBio)对融合蛋白的结合亲和力进行测定。首先，将生物素Biotin(Thermo，Prod#21338，Sulfo-NHS)和VEGF按摩尔比2:1混合，进行标记，通过透析除去未参与标记的生物素；然后，根据Octet-QK的使用说明，选择高灵敏度实验程序，在亲和素探针SA(forteBIO，Part#18-5019)上加载生物素标记的VEGF；实验中所用的缓冲液是PBS(含0.1％Tween-20)，将梯度稀释的融合蛋白和对照抗体，根据程序的设置，加入96孔黑色板(Greiner，655209)的预定位置中。根据程序设置，结合融合蛋白，然后在PBST溶液中解离，获得实验曲线；根据Octet-QK的结果分析软件，实验结果用local full拟合曲线，计算kon、kdis、Kd。表15概括了融合蛋白与对照药物Bevacizumab(Medchemexpress，Cat.No.:HY-P9906)的Kd，从表中可以看出融合PU前后，AR VEGF对VEGF的平均亲和力无明显差异，与Bevacizumab相比在同一个数量级。
表15、PU-AR VEGF融合蛋白的解离平衡常数(Kd)
Figure PCTCN2019106092-appb-000006
实施例22、PU与AR VEGF融合蛋白的体外活性研究
用VEGF受体竞争抑制法测定PU-AR VEGF的活性。准备两块96孔板，分别为ELISA板和细胞板。两块板分别进行如下处理：
ELISA板中加入5μg/mL的VEGF Receptor 2(KDR)(Abcam，ab155628)，每孔50μL，在37℃中放置2h。用1％BSA/TBS封闭，37℃放置1h；细胞板中加入1％BSA/TBS，每孔200μL，在37℃中放置2h。
将PU-AR VEGF和对照品Bevacizumab分别用PBS稀释成100μg/mL的母液，再将母液进行3倍稀释，共稀释11个浓度，将稀释好的样品80uL与等体积的1μg/mLVEGF混合，37℃放置1h。将包被KDR的ELISA板洗板两次，拍干后，将混合液依次转移至ELISA板中，37℃放置1h，然后洗板6次。向ELISA板中各孔加入1∶1000稀释的鼠 抗人VEGF单克隆抗体(sigma，V4758-.5mg)，每孔50μL，37℃放置1h，然后洗板6次。再加入1∶1000稀释的羊抗鼠二抗(Pierce，31432，QA1969921)，每孔50μL，37℃放置1h，然后洗板6次。反应完后加入显色液，37℃显色15min，加入终止液终止显色反应，在酶标仪上读取OD450的数值，结果如表16和图11所示，可见，融合后的蛋白的生物学活性与融合前的蛋白相当。
表16、PU与AR VEGF融合蛋白的IC50
蛋白样品 IC50(nM)
PU73x49-AR VEGF 0.55
PU98x43-AR VEGF 0.51
AR VEGF 0.65
Bevacizumab 0.56
实施例23、血清稳定性
融合蛋白样品(PU27x28-GH-PU27x5、PU98x28-GH-PU98x4、PU130x28-GH-PU130x5)用40mM PB，pH7.4配制成2.0-3.0mg/ml，除菌过滤(0.22μm，Millipore)后，用大鼠血清稀释10倍，混匀，分装到无菌离心管中；置于37℃培养箱，取0天、及第7天样品进行Western分析，采用HRP标记的Anti-6X His
Figure PCTCN2019106092-appb-000007
抗体(Abcam，ab1187)作为检测抗体。结果如图12所示，可见融合蛋白在血清中稳定性非常好。
实施例24、耐酶稳定性
称取适量胰蛋白酶(生工生物工程上海(股份)有限公司，货号A620627-0250)，用经高温灭菌的20mM PB(含0.15M NaCl，pH7.5)缓冲液溶解为质量浓度10％的溶液。将PU-GH融合蛋白(5mg/ml)和hGH(Sino Biological,Cat:16122-H07E，配制成1mg/ml)，分别加入质量终浓度0、0.02％、0.1％、0.5％的胰酶溶液混匀，用20mM PB(含0.15M NaCl，pH7.5)补齐体积。然后放37℃孵育40min；取出后加入电泳缓冲液煮沸10min终止反应。hGH样品的0、0.02％、0.1％、0.5％胰酶处理组分别上样于12％进行SDS-PAGE，PU-GH融合蛋白取0％及0.5％胰酶处理组分别上样于8％SDS-PAGE。结果如图13所示，hGH在0.02％胰酶处理下已经几乎无完整蛋白，而PU-GH融合蛋白则几乎没有任何降解，表明融合蛋白的耐酶稳定性优异。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (18)

  1. 一种多肽单元，其具有以下特征：
    (1)由脯氨酸、丙氨酸及谷氨酸组成；
    (2)存在50％以上的α-螺旋二级结构；和
    (3)长度≥15个氨基酸。
  2. 如权利要求1的多肽单元，其特征在于，根据Chou-Fasman公式计算，所述的多肽单元存在60％以上，较佳地70％以上，更佳地80％以上，进一步更佳地90％以上的α-螺旋二级结构。
  3. 如权利要求1的多肽单元，其特征在于，所述的α-螺旋二级结构的比例按照Chou-Fasman算法计算；和/或长度≥20个氨基酸。
  4. 如权利要求1的多肽单元，其特征在于，其包括选自下组的多肽单元：SEQ ID NO:19～47所示氨基酸序列的多肽单元。
  5. 一种多肽复合单元，其核心结构选自：
    U 1-U 2或U 1-U 2-…U n；
    U 1，U 2，…，U n各自代表一个权利要求1的多肽单元，n为＞2的正整数；且，两个或多个所述多肽单元的氨基酸序列相同或不同。
  6. 如权利要求5所述的多肽复合单元，其特征在于，所述多肽复合单元选自：
    (a)SEQ ID NO:48～105任一所示的氨基酸序列的蛋白或SEQ ID NO:48～76对应多肽单元的重复拼接蛋白；或
    (b)(a)限定的任一多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽活性的由(a)衍生的多肽；或
    (c)氨基酸序列与(a)限定的多肽的氨基酸序列有80％以上相同性且具有(a)多肽活性的多肽。
  7. 一种具有生物活性的融合蛋白，其包括：权利要求1所述的多肽单元或权利要求5所述的多肽复合单元以及活性蛋白或多肽。
  8. 如权利要求7所述的融合蛋白，其特征在于，其包括选自下组任一或组合的结构：
    (D 1-PU 1)；
    (D 1-PU 1)-(D 2-PU 2)；或
    (D 1-PU 1)-(D 2-PU 2)-…(D m-PU m)；
    其中，PU 1，PU 2，…，PU m选自权利要求1所述的多肽单元或权利要求5所述的多肽复合单元；D 1，D 2，…，D m为所述活性多肽，m为＞2的正整数；
    且，(D 1-PU 1)包括(PU 1-D 1)，(D 2-PU 2)包括(PU 2-D 2)，(PU m-D m)包括(D m-PU m)。
  9. 如权利要求8所述的融合蛋白，其特征在于，D 1，D 2，…，D m各自代表一条或多条相互连接的活性蛋白或多肽，所述的多条活性蛋白或多肽之间功能相同或不同。
  10. 如权利要求7所述的融合蛋白，其特征在于，所述活性蛋白或多肽包括：GLP-2类似物，AR VEGF，hGH，Arginase 1，G-CSF，Exendin-4，GLP-1类似物，GDF15，glucagon，IL-2，IL-15，FGF19，EPO，IL-6，M-CSF，FGF21。
  11. 如权利要求7所述的融合蛋白，其特征在于，其包括选自下组的融合蛋白：
    (a)SEQ ID NO:106～131任一所示的氨基酸序列的蛋白；或
    (b)(a)限定的任一多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽活性的由(a)衍生的蛋白；或
    (c)氨基酸序列与(a)限定的多肽的氨基酸序列有80％以上相同性且具有(a)多肽活性的蛋白。
  12. 如权利要求7～11任一所述的融合蛋白，其特征在于，所述的融合蛋白在体内的半衰期或稳定性在统计学上高于未融合的活性多肽。
  13. 一种核酸分子，其特征在于，所述的核酸分子编码：
    权利要求1～4任一所述的多肽单元；或
    权利要求5～6任一所述的多肽复合单元；或
    权利要求7～12任一所述的融合蛋白。
  14. 一种重组表达载体，其特征在于，它含有权利要求13所述的核酸分子。
  15. 一种遗传工程化的细胞，其特征在于，
    所述的细胞含有权利要求14所述的重组表达载体；或
    所述的细胞基因组中整合有权利要求13所述的核酸分子。
  16. 一种偶联物，其特征在于，包括：(a)权利要求1所述的多肽单元或权利要求5 所述的多肽复合单元；以及，(b)活性蛋白或多肽；其中，(b)与(a)以偶联或吸附的方式连接。
  17. 权利要求1～4任一所述的多肽单元或权利要求5或6所述的多肽复合单元的用途，用于：
    提高活性多肽的稳定性，较佳地包括热稳定性、耐酶稳定性和血清稳定性；
    提高活性多肽的半衰期；
    延长活性多肽的作用时间；和/或
    提高活性多肽的溶解度。
  18. 一种组合物，其包括：
    权利要求7～12任一所述的融合蛋白或权利要求16所述的偶联物；和
    药学上或食品学上可接受的载体。
PCT/CN2019/106092 2018-11-07 2019-09-17 用于改善活性蛋白或多肽性能的人工重组蛋白及其应用 WO2020093789A1 (zh)

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