WO2020073825A1 - 一种改善生物活性蛋白性质的载体蛋白 - Google Patents

一种改善生物活性蛋白性质的载体蛋白 Download PDF

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WO2020073825A1
WO2020073825A1 PCT/CN2019/108430 CN2019108430W WO2020073825A1 WO 2020073825 A1 WO2020073825 A1 WO 2020073825A1 CN 2019108430 W CN2019108430 W CN 2019108430W WO 2020073825 A1 WO2020073825 A1 WO 2020073825A1
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gelatin
protein
sequence
amino acid
proteins
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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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    • 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)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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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    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
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    • C07K14/475—Growth factors; Growth regulators
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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
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    • 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
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • the invention relates to the field of biotechnology, in particular to a carrier protein that can improve the properties of active proteins.
  • 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.
  • the Chinese patent with the patent number ZL200780015899.2 discloses an unstructured recombinant polymer (URP), which is basically unable to 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 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, where 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 al
  • 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, 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.
  • Chinese Patent No. 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
  • 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 ⁇ 0.6;
  • the ratio of the sum of Gly to n is ⁇ 1.15; and, according to the ProtParam formula, the GRAVY value representing
  • the above-mentioned new carrier proteins differ from the traditional albumin and immunoglobulin IgG Fc fragments in that most of the sequences have fewer amino acid types, and generally consist of only a few specific amino acids.
  • VPGXG constituent unit of the elastin-like ELP there is no strict restriction on the amino acid charge and hydrophilicity at the X position.
  • the design of URP and XTEN emphasizes the use of hydrophilic amino acids and the addition of negatively charged aspartic acid and / or glutamic acid to further extend the half-life.
  • 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 "(CN201080011467.6); instead, PAS focuses on imitating polyethylene glycol (PEG) and uses three uncharged amino acids: proline, alanine and serine.
  • PEG polyethylene glycol
  • the XTEN sequence emphasizes the feature of "substantially non-repetitive”: "Repetitive amino acid sequences have a tendency to aggregate to form higher-level structures, examples of which are natural repetitive sequences such as collagen and leucine zipper, or contact, resulting in Crystal or quasi-crystal structure.
  • 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 the N-terminus to the C-terminus ".
  • the active protein or polypeptide may significantly weaken its biological activity after being fused or cross-linked with these carrier proteins, as reported by Gething NC, etc.
  • the glucagon-XTEN fusion protein only shows the unmodified glucagon polypeptide 15 % Biological activity (Gething NC, etc., Gcg-XTEN: an improved glucagon capable of of preventing hypoglycemia without increasing baseline blood, PLoS One, 2010, 5 (4): e10175), however, the stability and The improvement of solubility and other physical and chemical properties make up for the shortcomings in this regard.
  • This article provides a gelatin-like unit with the following repeating structure:
  • G is glycine, and X and Y are each independently selected from proline, alanine and glutamic acid; n is an integer of 5-20, preferably n is an integer of 6-20 or 9-15.
  • Exemplary gelatin-like units can be selected from any of the odd-numbered sequences in SEQ ID NO: 17-89; preferably, the gelatin-like units are selected from SEQ ID NO: 17, SEQ ID NO: 19. SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31.
  • a gelatin-like protein comprising at least two gelatin-like units described herein; the at least two gelatin-like units may be the same or different; preferably, in the gelatin-like protein, alanine
  • the acid content is greater than or equal to 10%, preferably greater than or equal to 12%, more preferably greater than or equal to 15%, more preferably greater than or equal to 18%, more preferably greater than or equal to 20%; preferably the content of alanine ⁇ 45%, such as ⁇ 40% or less Equal to 35%;
  • the GRAVY value representing hydrophilicity is greater than -1.1, preferably greater than or equal to -1.0, more preferably greater than or equal to -0.9, more preferably greater than or equal to -0.8, preferably the value is ⁇ 0 , Such as ⁇ -0.1 or ⁇ -0.2.
  • gelatin proteins have 100-2000 amino acids.
  • Exemplary gelatin-like proteins can be selected from the sequences shown by any odd numbers in SEQ ID NO: 91-185, or can contain two or more (eg 2-20) SEQ ID NO: 91-185 Any odd numbered sequence.
  • Exemplary gelatin-like proteins containing two or more sequences represented by any odd numbers in SEQ ID NO: 91-185 are preferably tandem repeats of two or more identical sequences, including but Not limited to SEQ ID NO: 231 amino acid residue sequence 1-231, SEQ ID NO: 239 amino acid residue sequence 1-573, SEQ ID NO: 263 amino acid residue sequence 1-915, SEQ ID NO : 265 amino acid residue sequence 1-864, SEQ ID NO: 267 amino acid residue sequence 1-864, SEQ ID NO: 269 amino acid residue sequence 1-864, SEQ ID NO: 271 sequence 1- 864 amino acid residue sequence, SEQ ID NO: 273 amino acid residue sequence 1-864, SEQ ID NO: 275 amino acid residue sequence 1-915, SEQ ID NO: 279 amino acid residue 1-216 Sequence, SEQ ID NO: 281 amino acid residue sequence 1-216, SEQ ID NO: 283 amino acid residue sequence 1-231, SEQ ID NO: 293 amino acid residue sequence 1-687, SEQ ID NO : 295 amino
  • the gelatin-like proteins include those having a percent identity of more than 80%, preferably a percent identity of more than 85%, more preferably a percent identity of more than 90%, more preferably a percent identity of more than 95% with any of the amino acid sequences described in this paragraph Amino acid sequence.
  • fusion protein containing the gelatin-like protein and biologically active protein disclosed herein.
  • exemplary fusion proteins can be selected from the fusion proteins shown in any of the odd-numbered sequences in SEQ ID NO: 211-239, 247-259, and 263-309.
  • This article also provides a polynucleotide sequence selected from:
  • nucleic acid construct comprising the polynucleotide sequence described herein; preferably, the nucleic acid construct is a cloning vector or an expression vector.
  • gelatin-like unit described herein or its coding sequence or the complementary sequence of the coding sequence in the preparation of gelatin-like protein or fusion protein containing the gelatin-like protein;
  • the method uses a chemical synthesis method or a recombinant technology to prepare the carrier protein; wherein, the structure of the carrier protein is GXY III Meta-repetitive structure, where G is glycine, X and Y are independently selected from proline, alanine and glutamic acid;
  • the recombination technology includes constructing an expression vector expressing the carrier protein, transforming the host cell with the expression vector, and cultivating the host cell to express and produce the carrier protein;
  • the chemical synthesis method includes, according to the structure of the carrier protein, amino acid residues selected from glycine, proline, alanine and glutamic acid are sequentially connected to the peptide chain to form a ternary repeat with GXY Structure of the carrier protein.
  • glycine, proline, alanine and glutamic acid in preparing carrier proteins that can improve the biological properties or functions of biologically active proteins.
  • FIG. 1 GS100R9-hArg1 fusion protein at Sepax SRT On the apparent molecular weight.
  • M1 thyroglobulin (Thyroglobulin, 669kDa); M2, ferritin (Ferritin, 440KD); M3, aldolase (Aldolase, 158KD); M4, conalbumin (Conalbumin, 75KD); M5 , Ovalbumin (Ovalbumin, 44KD).
  • Figure 2 Apparent molecular weight of GS-hArg1 fusion protein on Sepax SRT-1000 SEC.
  • Figure 3 Graph of GS-hArg1 fusion protein pharmacokinetic results.
  • Figure 4 Graph of glycosylation detection results of protein samples. A shows the result before sugar staining, B shows the result after sugar staining. Lanes 1 and 2 are positive control proteins; lane 3: GS100R9-hArg1-GS100R9; lane 4: GS100R35-hArg1-GS100R35; lane 5: GS100R52-hArg1-GS100R52; lane 6: GS100R74-hArg1-GS100R74; lane 7: GS100R77- hArg1-GS100R77; lane 8: GS100R98-hArg1-GS100R98; lane 9: GS100R112-hArg1-GS100R112; lanes 10-11 are two independent batches of rGLK116 4- hArg1, respectively.
  • Figure 5 SDS-PAGE electrophoresis of GS and GH fusion proteins after different temperature treatments.
  • Lanes 1 and 8 GS800R9-GH-GS100R9; lanes 2 and 9: GS800R35-GH-GS100R35; lanes 3 and 10: GS800R127-GH-GS100R127; lanes 4 and 11: GS800L91-GH-GS100L91; lanes 5 and 12: GS800L102 -GH-GS100L102; lanes 6 and 13: GS800L146-GH-GS100L146; lanes 7 and 14: GS800S203-GH-GS100S203.
  • Lanes 1-7 are samples left at room temperature for 30 minutes, and lanes 8-14 are samples treated at 85 ° C for 30 minutes.
  • M is the protein molecular weight MARKER: 200, 116, 97.2, 66.4, 44.3KD.
  • Figure 6 Diagram of the analysis of the fusion of GS and hGH fusion protein samples.
  • Figure 7 Graph of in vitro cell activity results of GS and hGH fusion proteins.
  • Figure 8 SDS-PAGE electrophoresis of GS and GDF15 fusion protein.
  • Lanes 1-4 are: GS600R9-GDF15, GS600L23-GDF15, GS600L136-GDF15, GS600S14-GDF15; lanes 5-8 are: GS400R9-GDF15, GS400L23-GDF15, GS400L136-GDF15, GS400S14-GDF15; lanes 9-12 They are: GS200R9-GDF15, GS200L23-GDF15, GS200L136-GDF15, GS200S14-GDF15.
  • Figure 9 A graph showing the effect of GS and GDF15 fusion protein on weight loss in DIO mice.
  • Figure 10 Graph of the effect of GS and GDF15 fusion protein on appetite suppression in DIO mice.
  • Figure 11 Result of in vitro cell activity test of GS and GLP2G fusion protein.
  • Figure 12 Graph of the cell activity results of GS and AR VEGF fusion protein in vitro.
  • Figure 13 GS-GH and rGLK116 4 -hArg1 fusion protein incubated on day 7 in rat serum.
  • Figure 14 Stability results of GS-GH fusion protein and hGH in pancreatin.
  • 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.
  • Lanes 1 and 2 GS800R9-GH-GS100R9; lanes 3 and 4: GS800R35-GH-GS100R35; lanes 5 and 6: GS800R127-GH-GS100R127; lanes 7 and 8: GS800L91-GH-GS100L91; lanes 9 and 10 : GS800L102-GH-GS100L102; lanes 11 and 12: GS800L146-GH-GS100L146; lanes 13 and 14: GS800S203-GH-GS100S203.
  • M is a high molecular weight MARKER: 220KD, 135KD, 90KD, 66KD, 45KD and 35KD.
  • biologically active protein / polypeptide refers to proteins, antibodies, polypeptides, and fragments and variants thereof, having one or more pharmacological and / or biological activities or functions (such as the pharmacokinetics described herein) Academic and physical and chemical properties), or targeted guidance, multimerization and other functions. They can be naturally occurring or artificially constructed.
  • Bioactive protein / polypeptide may include enzymes, enzyme inhibitors, antigens, antibodies, hormones, coagulation factors, interferons, cytokines, growth factors, differentiation factors, bone tissue growth-related factors, bone factor-related absorption Factors, chemokines, cell movement factors, mobility factors, resting factors, bactericidal factors, antifungal factors, plasma adhesion molecules, interstitial adhesion molecules and extracellular matrix, receptor ligands and fragments thereof.
  • the biologically active protein / polypeptide involved in the present invention is a protein / polypeptide that exhibits “therapeutic activity”, and this protein / polypeptide possesses one or more known organisms and / or therapies active. 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 (eg, 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 the following biological activities: such as increasing angiogenesis, inhibiting angiogenesis, regulating hematopoietic function, promoting nerve development, improving immune response, suppressing immune response, etc.
  • therapeutic activity or “activity” may refer to activity in humans, non-human mammals, or other species of organisms that achieves an effect consistent with the desired therapeutic result. 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,
  • 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 proteins / polypeptides newly discovered after the present invention having biological activity are also applicable to the present invention.
  • gelation refers to the fact that certain solutions gradually become viscous when cooled, and eventually lose their fluidity to become an elastic jelly. This phenomenon is called gelation.
  • Gelatin obtained by hydrolysis of natural collagen has certain specific properties. The properties of gelatin in aqueous solution are affected by temperature, pH, manufacturing process and concentration. Among them, reversible gelation to temperature is one of the most important properties of gelatin (GELATIN HANDBOOK, GMIA, 2012).
  • PEG and / or “PEGylated” refers to the covalent attachment of polyethylene glycol (PEG) polymer chains to the biologically active protein of interest.
  • Covalently linking PEG to a biologically active protein can mask the protein from the host's immune system and increase the hydrodynamic radius of the biologically active protein of interest, thereby extending the circulation time of the protein drug by reducing renal clearance.
  • 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 can be obtained from NCBI.
  • sequence similarity refers to the degree of similarity between several DNA, RNA, or protein sequences, which 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 of the same amino acid residues (percent identity, identity%) 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 invention discloses a gelatin-like unit (U).
  • the types of amino acids constituting the gelatin-like unit are composed of glycine (G), proline (P), alanine (A) and glutamic acid (E), and have GXY ternary monomer repeat structure, where G represents glycine (G), X and Y are each independently selected from proline (P), alanine (A) or glutamic acid (E).
  • the gelatin-like units of the invention may have the following repeating structure:
  • G is glycine
  • X and Y are each independently selected from proline, alanine or glutamic acid
  • n is an integer of 5-20.
  • the G-X-Y ternary monomer repeating structure is selected from: GPP, GEE, GAA, GEA, GAE, GAP, GPA, GPE and GEP. Therefore, in certain embodiments, the gelatin-like unit (U) disclosed in the present invention may be composed of two or more GXY ternary monomer repeating structures selected from the group consisting of: GPP, GEE, GAA, GEA, GAE, GAP, GPA, GPE and GEP.
  • the gelatin-like unit (U) of the present disclosure consists of at least 6 G-X-Y ternary monomers (ie, n ⁇ 6), such as 6 ⁇ n ⁇ 20 or 6 ⁇ n ⁇ 15. In certain embodiments, the gelatin-like unit (U) of the present disclosure consists of at least 9 G-X-Y ternary monomers, such as 9 ⁇ n ⁇ 20 or 9 ⁇ n ⁇ 15.
  • the gelatin-like units disclosed in the present invention are selected from the gelatin-like units shown in any odd-numbered sequence in SEQ ID NO: 17-89. In certain preferred embodiments, the gelatin-like units disclosed in the present invention are selected from SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31.
  • GS Gelatin-like protein
  • gelatin-like protein comprising at least two gelatin-like units (U) described herein.
  • the gelatin-like proteins herein can be used as carrier proteins for carrying biologically active proteins, especially active protein drugs.
  • the core structure of the gelatin-like protein described herein is: U 1 -U 2 or U 1 -U 2 -... U a , wherein U 1 , U 2 , ..., U a each represent the text
  • a is an integer ⁇ 3.
  • the various types of gelatin units in the gelatin-like proteins of the present invention may be the same or different. In certain embodiments, 3 ⁇ a ⁇ 150; in certain embodiments, 3 ⁇ a ⁇ 100; in certain embodiments, 3 ⁇ a ⁇ 50.
  • gelatin-like proteins described herein are selected so that the total number of amino acid residues of the gelatin-like proteins described herein is within the range described in any of the embodiments below.
  • the gelatin-like proteins described herein may contain other biological properties that do not affect the gelatin-like proteins (including but not limited to gelation, viscosity, product uniformity, serum stability, and enzyme resistance as described below Stability and immunogenicity, etc.).
  • the N-terminus, C-terminus of the gelatin protein and / or appropriate regions within the protein may contain those used to promote expression, secretion to the outside of the host cell and / or purification when the gelatin protein is produced by recombinant technology
  • Amino acid sequences including but not limited to suitable linker sequences, signal peptides, leader peptides, terminal extensions, etc.
  • the amino acid sequence is a protein tag, which may be FLAG, HA, Poly-His, GST, MBP, c-Myc, and the like. These tags can be used to purify proteins.
  • the total number of amino acid residues of the core structure accounts for more than 70% of the total number of amino acid residues of the gelatin protein, preferably more than 80%, more preferably more than 85%, further more preferably more than 90%, 95 % Or more or 99% or more.
  • the gelatin-like proteins of the invention consist of the gelatin-like units described in any of the embodiments herein.
  • the content of Ala in the gelatin-like protein herein is greater than or equal to 10%; more preferably, the content of Ala is greater than or equal to 12%; more preferably, the content of Ala is greater than or equal to 15%; more preferably, the content of Ala is greater than or equal to 18%; more preferably, the content of amino acid Ala is greater than or equal to 20%.
  • the content of amino acid Ala is not more than 45%, for example, not more than 40%, not more than 35%, or not more than 30%, etc.
  • the content of Ala in the GS of the present invention is within the range constituted by any two values listed above as endpoints, such as in the range of 10-45%, such as 12-45%, 15 -45%, 18-45%, 20-45% or 10-40%, 10-30%, 10-20% or 15-45%, etc.
  • the amino acid Ala is 1.800, Glu is -3.500, Pro is -1.600, and Gly is- 0.400. That is, Ala is a hydrophobic amino acid, and Glu, Pro, and Gly are hydrophilic amino acids.
  • the gelatin-like protein (GS) described herein represents a hydrophilic GRAVY value greater than -1.1; preferably, the GRAVY value is greater than or equal to -1.0; more preferably, the GRAVY The value is greater than or equal to -0.9; more preferably, the GRAVY value is greater than or equal to -0.8.
  • the GRAVY value is at most 0, such as at most -0.1 or at most -0.2.
  • the gelatin-like protein (GS) described herein represents a hydrophilic GRAVY value within the range of any two of the above values as endpoints, such as greater than -1.1 to ⁇ 0 , Such as greater than -1.1 to -0.1, -1.0 to 0, -0.9 to 0, -0.8 to 0 or -0.8 to -0.1, etc.
  • the gelatin-like proteins (GS) described herein represent hydrophilic GRAVY values between -1.0 and 0.0.
  • the gelatin-like proteins described herein generally have the following characteristics: (1) containing the gelatin-like units described herein; (2) the content of Ala is greater than or equal to 10%, preferably greater than or equal to 12%, and more preferably greater than or equal to 15%, More preferably 18% or more, more preferably 20% or more, preferably Ala content ⁇ 45%, such as ⁇ 40% or 35% or less; and (3) GRAVY value representing hydrophilicity is greater than -1.1, preferably greater than or equal to- 1.0, more preferably greater than or equal to -0.9, more preferably greater than or equal to -0.8, preferably the value is ⁇ 0, such as ⁇ -0.1 or ⁇ -0.2.
  • the gelatin-like proteins (GS) described herein have at least 100 amino acids, preferably at least 200 amino acids, more preferably at least 300 amino acids, more preferably at least 400 amino acids, more preferably at least 500 amino acids, and more Preferably it has at least 600 amino acids, more preferably at least 700 amino acids, more preferably at least 800 amino acids, more preferably at least 900 amino acids, more preferably at least 1000 amino acids, more preferably at least 1200 amino acids.
  • the gelatin-like proteins described herein have 100-2000 amino acids, such as 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000 or 1200-2000 amino acids.
  • the gelatin-like proteins described herein are formed by repeating splicing of gelatin-like units (U) of the same sequence. In other preferred embodiments, the gelatin-like proteins described herein are spliced from different gelatin-like units (U). In certain embodiments, there may be linker sequences between various types of gelatin units, such as linker sequences formed from amino acid sequences containing glycine (G) and / or proline (P).
  • G glycine
  • P proline
  • the exemplary gelatin-like protein of the present invention is selected from the sequence shown by any odd number in SEQ ID NO: 91-185.
  • any two or more e.g., 2-20, or 2-10) selected from the sequence shown by the odd numbers in SEQ ID NO: 91-185 2-8) Splicing to form the gelatin-like protein of the present invention. Therefore, in these embodiments, the gelatin-like protein of the present invention contains 2-20 sequences selected from any odd-numbered sequence selected from SEQ ID NO: 91-185.
  • two or more of the sequences used for splicing to form the gelatin-like protein of the present invention are the same sequence.
  • the splicing sequences may be connected between each other, or may be connected by a linker sequence well known in the art (such as a linker sequence formed by an amino acid sequence containing glycine (G) and / or proline (P)).
  • a linker sequence well known in the art (such as a linker sequence formed by an amino acid sequence containing glycine (G) and / or proline (P)).
  • the exemplary gelatin-like proteins of the present invention are selected from the group consisting of the fusion proteins shown in any odd-numbered sequence of SEQ ID NO: 211-239, 247-259, and 263-309 Gelatin-like proteins, including but not limited to SEQ ID NO: 231 amino acid residue sequence 1-231 (GS200R9), SEQ ID NO: 239 amino acid residue sequence 1-573 (GS500R9), SEQ ID NO: 263 1-915 amino acid residue sequence (GS800R9), SEQ ID NO: 265, 1-864 amino acid residue sequence (GS800R35), SEQ ID NO: 267, 1-864 amino acid residue sequence (GS800R127), SEQ ID NO: 269 amino acid residues 1-864 sequence (GS800L91), SEQ ID NO: 271 amino acid residues 1-864 sequence (GS800L102), SEQ ID NO: 273 amino acid residues 1-864 sequence (GS800L146) ), SEQ ID NO: 275 amino acid residues 1-915 sequence (GS800S203),
  • the amino acid sequence of the gelatin-like protein described herein has a percent identity of more than 80%, preferably a percent identity of more than 85%, more preferably a percent identity of more than 90% with any of the amino acid sequences mentioned in this paragraph Amino acid sequences with a percentage of identity, more preferably 95% or greater.
  • Natural gelatin is made from the hydrolysis of collagen in animal fur, bones and other connective tissues by strong acids or bases. The properties of gelatin are greatly affected by temperature, pH and concentration. Natural gelatin is easily soluble in hot water (> 40 ° C) and tends to form a gel at low temperatures.
  • the gelatin-like protein (GS) provided by the present invention not only has no obvious gelation phenomenon, but also has a very low viscosity, and is a more ideal protein drug carrier.
  • the freezing strength of the gelatin-like protein of the present invention determined by the method of national standard "Food Additive Gelatin" GB6783-94 is ⁇ 10g, preferably ⁇ 5g, and more preferably ⁇ 3g.
  • the gel strength of the gelatin-like protein of the present invention may be between 1-10g, or between 1-5g or 1-3g.
  • the viscosity of the gelatin-like protein of the present invention determined by the ND-2 Brinell viscosity according to the national standard "Food Additive Gelatin" GB6783-94 is ⁇ 3 mPa ⁇ s, preferably ⁇ 2 mPa ⁇ s, and more preferably ⁇ 1mPa ⁇ s.
  • the viscosity of the gelatin-like protein of the present invention is in the range of 0.01-3 mPa ⁇ s, preferably in the range of 0.05-1 mPa ⁇ s.
  • 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
  • glycosylation O-linked oligosaccharide glycosylation, the attachment site is on serine or threonine residues; N-linked oligosaccharide glycosylation, the attachment site is on Asn -At the position of the asparagine residue of the X-Ser / Thr sequence, here, X may be any amino acid except proline.
  • the glycosylation system of yeast is different from humans. High glycosylation, especially O-glycosylation, is prone to strong immunogenicity, and the problem of batch heterogeneity in the production process is difficult to solve.
  • sequences containing a large amount of S or T such as XTEN, PAS, GLK, or URP are expressed in expression systems other than the prokaryotic
  • severe glycosylation and heterogeneity are extremely serious problems, and the only solutions that can be taken are
  • These sequences are obtained in the prokaryotic expression system, and then cross-linked with the active protein or polypeptide obtained by the eukaryotic expression system by chemical cross-linking (it is impossible or difficult to express in the prokaryotic expression system).
  • chemical cross-linking it is well known that the products caused by chemical crosslinking are not uniform, and the process is cumbersome, etc., which are currently unsolvable problems.
  • 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
  • they often carry additional methionine at the N-terminus making it difficult to obtain active products directly. Therefore, it is generally necessary to add fusion expression tags in front of the N-end of Exendin-4, such as CBD tags (Volker Schellenberger, etc., Arecombinant polypeptide extends the the in vivo vivo half-life of peptides and proteins) in Tunablemanner, NatureBiotechnology.
  • the gelatin-like protein (GS) of the present invention is composed of glycine (G), proline (P), alanine (A) and glutamic acid (E), whether prepared in prokaryotic or eukaryotic expression systems There will be problems with glycosylation.
  • GLK's non-uniformity caused by the deamidation of Asn (N) and Gln (Q) and the non-uniformity of products caused by the degradation caused by the increase of potential protease sites due to the large number of amino acids are provided in the present invention.
  • the possibility of the existence of gelatin-like proteins (GS) is extremely small. As shown in the examples, the gelatin-like protein of the present invention has superior serum stability and enzyme resistance stability compared to GLK.
  • the present disclosure further provides fusion proteins containing bioactive proteins and gelatin-like proteins described herein.
  • the biologically active protein may be a protein known in the art to have one or more pharmacological and / or biological activities, or targeted guidance, multimerization and other functions. They can be naturally occurring or artificially constructed.
  • Biologically active proteins can include enzymes, enzyme inhibitors, antigens, antibodies, hormones, blood coagulation factors, interferons, cytokines, growth factors, differentiation factors, bone tissue growth-related factors, bone factor-related absorption-related factors, chemotaxis Factors, cell movement factors, mobility factors, resting factors, bactericidal (fine) factors, antifungal factors, plasma adhesion molecules, interstitial adhesion molecules and extracellular matrix, receptor ligands and fragments thereof.
  • the fusion protein herein contains the active protein drug (D) and the gelatin-like protein (GS) described herein.
  • Active protein drugs suitable for use herein include but are not limited to agonists, receptors, ligands, antagonists, enzymes and hormones. More specifically, the active protein drug suitable for use herein may be an active protein drug used in the treatment and / or prevention of various diseases and / or the improvement of symptoms, which are well known in the art. Such diseases include but are not limited to: Metabolic-related diseases, cardiovascular diseases, coagulation / bleeding diseases, growth disorders or disorders, tumors, vascular disorder diseases, inflammation, autoimmune disorders, etc.
  • the diseases include type 1 diabetes, type 2 diabetes, gestational diabetes, hypercholesterolemia, obesity, hyperglycemia, ultrahyperinsulinemia, reduced insulin production, insulin resistance, metabolic disorders, multiple Cystic ovary syndrome, dyslipidemia, eating disorders, hypertension (such as pulmonary hypertension), retinal neurodegeneration, metabolic disorders, glucagonoma, ulcerative colitis, renal failure, congestive heart failure, nephrotic syndrome , Kidney disease, 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 synthesis Signs, chronic kidney failure, intrauterine growth retardation, idiopathic short stature, AIDS depletion, obesity, multiple sclerosis, aging, fibromyalgia, Crohn's disease, ulcerative colitis, muscular dystrophy, low bone Density etc.
  • the active such as pulmonary hyper
  • Bioactive proteins (especially active protein drugs) and gelatin-like proteins can be fused in series in a manner well known in the art.
  • the bioactive protein is fused at the N-terminus or C-terminus of the gelatin-like protein, or the gelatin-like protein is fused at both ends of the bioactive protein, or the bioactive protein is fused at both ends of the gelatin-like protein.
  • the fusion protein may contain two or more biologically active proteins, and the biologically active proteins may be the same or different.
  • the fusion protein may also contain two or more kinds of gelatin-like proteins, which may be the same or different.
  • the tandem connection between the biologically active proteins and the gelatin-like proteins may be diverse. Exemplary tandem fusion includes, but is not limited to the following structures:
  • D 1 , D 2 , D 3 and D 4 are active protein drugs, and D 1 , D 2 , D 3 and D 4 may be the same or different;
  • GS 1 , GS 2 , GS 3 and GS 4 are gelatin-like Proteins (GS), GS 1 , GS 2 , GS 3 and GS 4 may be the same or different.
  • the biologically active proteins are usually connected by GS, and the GS are usually not directly connected. , But through biologically active proteins.
  • the active protein drugs listed in Table 1 below or analogs thereof are preferably used herein.
  • GLP-2 analogue 1 Glucagon 2 AR VEGF 3 IL-2 4 hGH 5 IL-15 6 Arginase1 7 FGF19 8 G-CSF 9 EPO 10 Exendin-4 11 IL-6 12 GLP-1 analogue 13 M-CSF 14 GDF15 15 FGF-21 16
  • the biologically active proteins especially the active protein drug (D)
  • the biologically active proteins have significantly improved physical and chemical properties, which are manifested as an increase in water solubility, resistance to enzymes and heat stability, Increased hydrodynamic radius, etc., these ideal properties make the half-life of biologically active proteins significantly longer.
  • the half-life of the fusion protein is more than 10 times longer than when it is not fused.
  • the amino terminus and / or carboxy terminus of the fusion protein herein may also contain one or more polypeptide fragments as protein tags.
  • Any suitable label can be used for this article.
  • the tags can be FLAG, HA, Poly-His, GST, MBP, c-Myc, and these tags can be used for protein purification.
  • a suitable linker sequence may be provided between the bioactive protein and the GS, between the two bioactive proteins, or even between the two GS, such as a linker sequence containing G (glycine) and / or S (serine). Any linker sequence known in the art can be used in the fusion protein herein.
  • the gelatin-like protein in the fusion protein herein may be selected from the sequence shown in any odd-numbered sequence of SEQ ID NO: 91-185, and amino acid residues 1-231 of SEQ ID NO: 231 Sequence (GS200R9), SEQ ID NO: 239 amino acid residue sequence 1-753 (GS500R9), SEQ ID NO: 263 amino acid residue sequence 1-915 (GS800R9), SEQ ID NO: 265 sequence 1-864 Amino acid residue sequence (GS800R35), SEQ ID NO: 267 first amino acid residue sequence 1-864 (GS800R127), SEQ ID NO: 269 first amino acid residue sequence 1-864 (GS800L91), SEQ ID NO: 271 Amino acid residue sequence at position 1-864 (GS800L102), SEQ ID NO: 273 Amino acid residue sequence at position 1-864 (GS800L146), SEQ ID NO: 275 amino acid residue sequence at position 1-915 (GS800S203), SEQ ID NO: 279 amino acid residue sequence 1-2
  • Base sequence (GS800S14), SEQ ID NO: 305 amino acid residues 34-948 (GS800S203) and SEQ ID NO: 309 amino acid residues 1-687 (GS900R9), or any An amino acid sequence has an amino acid sequence of 80% or more identity, preferably 85% or more identity, more preferably 90% or more identity, more preferably 95% or more identity.
  • Exemplary fusion proteins of the present invention are selected from the fusion proteins shown in any odd-numbered sequences in SEQ ID NO: 211-239, 247-259 and 263-309, or any one of these fusion proteins
  • a fusion protein having 80% or more identity, preferably 85% or more identity, more preferably 90% or more identity, more preferably 95% or more identity.
  • the gelatin-like protein (GS) of the present invention can enhance the pharmacokinetic properties of the bioactive protein or polypeptide after being fused with the bioactive protein or polypeptide, and the half-life of the bioactive protein or polypeptide fused with the gelatin-like protein (GS) can be at least extended More than 2 times, where the pharmacokinetic properties are determined by measuring the terminal half-life of the biologically active protein administered to the subject compared to the administration of a comparable dose of the biologically active protein fused with gelatin-like protein (GS). It can play a role in prolonging the half-life in the body. The essence is that the hydration radius of gelatin-like protein (GS) is extremely large.
  • the size of gelatin-like protein (GS) can reach the nanometer level due to the full extension .
  • the apparent molecular weight of GS100R9-hArg1 with a molecular weight of about 140KD is between 669KD and 440KD; GS100R9-hArg1-GS100R9 with a molecular weight of about 170KD
  • the apparent molecular weight of GS100R35-hArg1-GS100R35 is already greater than 669KD, while GS200R9-hArg1-GS200R9 with a molecular weight of approximately 220KD is much larger than 669KD.
  • Human arginase 1 (hArg1) is a natural trimer structure with a monomer molecular weight of about 35KD and a trimer molecular weight of about 105KD. Although the molecular weight has far exceeded the glomerular filtration pore size, humans The half-life of arginase 1 in the body is surprisingly short, only a few minutes (PNCheng, TLLam, WMLam, SMTsui, AWCheng, WHLo, et al., Pegylated recombinant human arginase (rhArg-PEG5,000mw ) inhibits the vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion, Cancer Res. 67 (2007) 309–317).
  • PEGylation modification is generally used to extend its half-life in vivo. After administration of PEG-hArg1 in mice, the half-life can be extended to 63 hours.
  • the fusion of gelatin-like proteins (GS) with similar amino acid lengths (about 100 amino acids) and different sequences significantly increases the half-life of hArg1.
  • the C-terminal (S-shaped tail) of hArg1 is involved in the formation of trimers, and surprisingly, in the present invention, the fusion of gelatin-like protein (GS) at the C-terminus of hArg1 does not affect the formation of trimers .
  • the fusion of gelatin-like protein (GS) at one end of hArg1 or fusion of gelatin-like protein (GS) at both ends can be observed to have obvious improvement of pharmacokinetic properties, and the same length of gelatin-like protein (GS) In this case, when the gelatin-like protein (GS) is fused at both ends, the half-life is longer.
  • human growth hormone hGH
  • growth differentiation factor is used 15 (GDF15) to verify the function of gelatin-like protein (GS) to improve the pharmacokinetic properties
  • GDF15 growth differentiation factor 15
  • an artificially designed anchor protein Designed ankyrin repeat proteins
  • GLP2G is used to verify the function of gelatin-like protein (GS) to improve pharmacokinetic properties.
  • bioactive proteins fused with gelatin-like proteins have significantly improved solubility and stability, such as thermal stability, enzyme resistance stability, and serum stability.
  • the thermal stability of GH protein fused with gelatin-like protein (GS) at 85 ° C is significantly higher than that of unfused GH.
  • GS gelatin-like protein
  • no significant aggregation phenomenon was observed on SEC-HPLC.
  • the potential human circulation stability was determined by measuring the integrity of the biologically active protein exposed to 37 ° C for 7 days. As shown in the results of FIG. 13WB, the GS fusion protein is highly stable in serum, while the control rGLK116 4 -Arg1 protein has been dispersed in a degraded state.
  • Antibody-drug conjugate is a therapeutic drug obtained by preparing antibodies and toxic compounds or radionuclides through lysine, cysteine, unnatural amino acids and engineered tags.
  • 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
  • the chemically synthesized high hydrophilic polyethylene glycol (PEG) or biodegradable short-chain molecules can be used as the linker (Linker), such as PHF (or called ). These methods can effectively improve the hydrophilicity of ADC molecules and increase their stability.
  • therapeutically active proteins can be prepared by chemical crosslinking of several different active protein drugs (D) and gelatin-like proteins (GS). Chemical cross-linking can be performed on most amino acid residues. For example, the nucleophilic primary amine group on lysine and the active sulfhydryl group on cysteine are the most commonly used cross-linking sites. In addition, tyrosine and selenocysteine will also be used for chemical crosslinking.
  • This document includes coding sequences and complementary sequences for various gelatin-like units, gelatin-like proteins, and fusion proteins provided herein.
  • Exemplary coding sequence of gelatin-like units is shown in any even-numbered sequence in SEQ ID NO: 18-90; exemplary coding sequence of gelatin-like protein is shown in any even-numbered sequence in SEQ ID NO: 92-186 The sequence is shown, or the coding sequence of the gelatin-like protein contained in the fusion protein coding sequence shown in any of the even-numbered sequences of SEQ ID NO: 212-240, 248-260 and 264-310; exemplary fusion The protein coding sequence is shown in any even-numbered sequence of SEQ ID NO: 212-240, 248-260 and 264-310.
  • the polynucleotide sequence can be prepared by conventional methods in the art. For example, a small-sized gelatin-like protein unit (U) fragment can be obtained first by gene synthesis, and then a larger-molecular-weight gelatin-like protein (GS) obtained by repeating the splicing of the gelatin-like protein unit (U) can be obtained by gene splicing. ).
  • GS larger-molecular-weight gelatin-like protein
  • a nucleic acid construct is an artificially constructed nucleic acid segment that can be introduced into target cells or tissues.
  • the nucleic acid construct contains the coding sequence described herein or its complement, and one or more regulatory sequences operatively linked to these sequences.
  • the control sequence may be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the amino acid sequence to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be extracellular encoding homologous or heterologous to the host cell Or the gene of the intracellular polypeptide is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is connected to the 3 'end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequences described herein, especially gelatin-like proteins or fusion proteins can be cloned into many types of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and mucous grain.
  • the vector may be an expression vector or a cloning vector.
  • suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
  • promoters are: the lac or trp promoter of E. coli; the phage lambda PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Bi The methanol oxidase promoter of S. cerevisiae and some other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Marker genes can be used to provide phenotypic traits for selection of transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or for the large intestine Bacillus tetracycline or ampicillin resistance.
  • GFP green fluorescent protein
  • the expression vectors containing the polynucleotide sequences described herein and appropriate transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on.
  • host cells comprising the polynucleotide sequences described herein, their nucleic acid constructs, and / or expressing amino acid sequences containing the gelatin-like units described herein (especially the gelatin-like proteins described herein).
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells of Salmonella typhimurium fungal cells such as yeast, filamentous fungi, plant cells
  • insect cells of Drosophila S2 or Sf9 CHO, COS, 293 cells, or Bowes black Animal cells such as tumor cells.
  • compositions containing the fusion proteins described herein may also contain various suitable pharmaceutically acceptable carriers or excipients well known in the art.
  • the dosage and concentration of the pharmaceutically acceptable carrier or excipient are non-toxic to the recipient, including but not limited to: buffering agents such as acetate, Tris, phosphate, citrate and other organic acids ; Antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethylene chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl Alcohols; hydrocarbyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); protein , Such as serum albumin, gelatin, or immunoglobulin; hydrophilic polyureasulin; hydro
  • a suitable pharmaceutically acceptable carrier or excipient can be selected according to the dosage form of the pharmaceutical composition.
  • the pharmaceutical composition can be prepared into different dosage forms according to different uses of the composition.
  • the pharmaceutical composition of the present invention can be prepared into commonly used dosage forms such as tablets, injections, and lyophilizates.
  • the pharmaceutical compositions generally contain a therapeutically or prophylactically effective amount of the fusion protein described herein.
  • a therapeutically effective amount generally refers to a dose sufficient to demonstrate its benefit to the subject to be administered. The actual amount administered, as well as the rate and time course of administration will depend on the individual's condition and severity. The prescription of treatment (for example, the decision on the dosage, etc.) is ultimately the responsibility of the general practitioner and other doctors and depends on them to make decisions, usually considering the disease to be treated, the individual patient's condition, the delivery site, the method of administration, and the Know other factors.
  • a prophylactically effective amount refers to an amount that is effective to achieve the desired preventive effect at the necessary dose and time. Usually, but not necessarily, since the prophylactic dose is applied to the subject before the onset of the disease or early in the disease, the prophylactically effective amount will be lower than the therapeutically effective amount.
  • the amino acid sequence of the present invention may be a product of chemical synthesis, or a recombinant polypeptide produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host eg, bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells
  • the active protein or polypeptide portion of the invention may be glycosylated or may be non-glycosylated.
  • a method for preparing a protein therapeutic drug including the following steps:
  • the cell culture method can be determined according to different cell types, and is a conventional culture method in the art.
  • the present invention also provides a method for treating or preventing a disease, which method comprises administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the fusion protein described herein or a pharmaceutical composition thereof.
  • the disease to be treated is related to the biological activity or function of the biologically active protein in the fusion protein.
  • growth hormone can promote bone, visceral and systemic growth, promote protein synthesis, affect fat and mineral metabolism, and can be used for growth disorders caused by insufficient secretion of endogenous pituitary growth hormone, short stature dwarfism, short stature Children with disease can also be used to treat burns, fractures, trauma, hemorrhagic ulcers, muscular dystrophy, osteoporosis and other diseases. Therefore, when the active protein drug in the fusion protein is GH, the fusion protein can be used to treat growth disorders caused by insufficient secretion of endogenous pituitary growth hormone, children with short stature, dwarfism, and stunt disease, and can also be used to treat burns.
  • IL-2 plays an important role in the body's immune response and antiviral infection, and it is used clinically as an immune enhancer, mainly for kidney cancer, melanoma, and non-Hodgkin's lymphoma. Therefore, when treating patients with kidney cancer, melanoma, or non-Hodgkin's lymphoma, the fusion protein herein containing IL-2 as an active protein drug may be administered.
  • GDF15 can be used to treat diseases related to obesity and underweight. Therefore, the subject fusion protein containing GDF15 can be administered to a subject in need thereof.
  • the fusion protein described herein can be used to treat or prevent: metabolic related diseases, cardiovascular diseases, coagulation / bleeding diseases, growth disorders or conditions, tumors, blood vessels Obstacle diseases, inflammation, autoimmune disorders, etc.
  • the diseases include type 1 diabetes, type 2 diabetes, gestational diabetes, hypercholesterolemia, obesity, hyperglycemia, ultrahyperinsulinemia, reduced insulin production, insulin resistance, metabolic disorders, multiple Cystic ovary syndrome, dyslipidemia, eating disorders, hypertension (such as pulmonary hypertension), retinal neurodegeneration, metabolic disorders, glucagonoma, ulcerative colitis, renal failure, congestive heart failure, nephrotic syndrome , Kidney disease, 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 synthesis Signs, chronic kidney failure, intrauterine growth retardation, idiopathic short stature, AIDS depletion, obesity, multiple sclerosis, aging, fibromyalgia, Crohn's disease, ulcerative colitis, muscular dystrophy and low bone Density etc.
  • This document includes methods for treating or
  • Also provided herein is a method for enhancing the pharmacokinetic properties of a biologically active protein, especially an active protein drug, the method comprising fusing the gelatin-like protein described herein at the C-terminus and / or N-terminus of the biologically active protein A step of.
  • pharmacokinetic properties include but are not limited to in vivo half-life.
  • a method for improving the physicochemical properties of a biologically active protein (especially an active protein drug) the method comprising fusing the C-terminus and / or N-terminus of the biologically active protein as described herein Gelatin-like steps.
  • the biologically active protein may be any one or more of the biologically active proteins described above.
  • Methods of fusion or chemical cross-linking are known in the art.
  • the fusion protein can be prepared using the method for preparing a fusion protein described above, thereby enhancing the pharmacokinetic properties and / or improving the physical and chemical properties of the biologically active protein.
  • the article also provides the use of the gelatin-like units or gelatin-like proteins described herein in enhancing the pharmacokinetic properties of biologically active proteins (especially active protein drugs) and / or improving their physicochemical properties;
  • gelatin-like units or gelatin-like proteins described herein for enhancing the pharmacokinetic properties of biologically active proteins (especially active protein drugs) and / or improving their physicochemical properties, and fusion proteins for treatment or prevention .
  • a protein composed of glycine, proline, alanine and glutamic acid can be used as a carrier protein to extend the half-life of biologically active proteins or polypeptides and improve their properties in vitro and in vivo. Therefore, the use of glycine, proline, alanine, and glutamic acid in the preparation of carrier proteins that can improve the biological properties or functions of biologically active proteins (eg, pharmacokinetic and physicochemical properties) is also included in the scope of this article within.
  • a method of preparing a carrier protein that can improve the biological properties or function of a biologically active protein The method may be a chemical synthesis method or a biological recombination method.
  • the structure of the carrier protein is a ternary repeating structure of G-X-Y, where G is glycine, and X and Y are each independently selected from proline, alanine and glutamic acid. More preferably, the carrier protein is a gelatin-like protein as described in any of the embodiments herein.
  • the chemical synthesis method may be various chemical synthesis methods well known in the art, including sequentially linking amino acid residues selected from glycine, proline, alanine and glutamic acid to the peptide according to the structure of the carrier protein On the chain, the carrier protein having a GXY ternary repeat structure is formed.
  • Chemical synthesis methods usually include solid-phase synthesis and liquid-phase synthesis, of which solid-phase synthesis is more commonly used. Solid-phase synthesis methods include but are not limited to Fmoc and tBoc.
  • resin is used as an insoluble solid phase carrier, and the amino acids are usually connected to the peptide chain one by one from the C-terminus (carboxyl-terminus) to the N-terminus (amino-terminus).
  • the protected amino acid must use a deprotection solvent to remove the protecting group of the amino group; 2) Activation: the carboxyl group of the amino acid to be connected is activated by the activator; and 3) Coupling: the activated carboxyl group is exposed to the previous amino acid The amino group reacts to form a peptide bond. The cycle is repeated until the peptide chain reaches the desired length. Finally, the connection between the peptide chain and the solid phase carrier is cut with a cutting solution, and the desired amino acid sequence can be obtained.
  • the above-mentioned chemical synthesis can be carried out on an automated peptide synthesizer controlled by a program. Such instruments include but are not limited to Tribute dual-channel peptide synthesizer launched by Protein Technologies, UV Biosystem's UV Online Monitor system, and Aapptec's Focus XC Three-channel synthesizer, etc.
  • the biological recombination method includes preparing a polynucleotide sequence encoding the amino acid sequence according to the amino acid sequence of the carrier protein, using the polynucleotide sequence to construct an expression vector, using the expression vector to transform or transfect a host cell, and culturing the host cell to express it
  • the carrier protein is produced. This can be achieved using technical means well known in the art.
  • Protein purification methods vary according to different expression systems. Existing technology already has 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 IN, ENZYMOLOGY, Guide to Protein Purification, 2nd Edition, etc. Affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic chromatography and other purification tools are well known in the art by the principles, methods of use, and combined use. The purification processes involved in the following examples are exemplary to show that the expression host is methanol yeast GS115 and the purification method under the specific fermentation conditions.
  • the purification conditions should be adjusted accordingly. Since it is a well-known technology, it will not be repeated here. However, as a general standard, the final purity of the target protein should exceed 95% (SDS-PAGE purity and HPLC identification purity).
  • the gelatin-like unit (U) is mainly composed of the following G-X-Y ternary monomer structures: GPP, GEE, GAA, GEA, GAE, GAP, GPA, GPE, GEP. Different G-X-Y ternary monomer structures are randomly combined to form a gelatin-like protein unit (U). Exemplary combinations are shown in Table 2.
  • GS low molecular weight recombinant gelatin-like protein
  • U xyz-1 a nucleotide sequence containing U x , U y, and U z (referred to as U xyz-1 for short) was added with the ⁇ -factor signal peptide sequence of yeast GS115 (with Xho I site) at the 5 ′ end of the synthesis.
  • the recognition site of endonuclease DraIII, and the 3 'end has Van91I and EcoRI recognition sites, and is connected to the cloning vector pMD18-T (TaKaRa Company) to construct the plasmid pMD-U xyz-1 .
  • the plasmid pMD-U xyz-1 was first double-digested with Van 91I / Dra III. Electrophoresis was performed on 1% agarose gel, and the U xyz-1 fragment was recovered by excising the gel. At the same time, the pMD-U xyz-1 plasmid was digested with Van91I. The digested plasmid was recovered by tapping as above and dissolved in 30 ⁇ L of TE solution. It is then treated with Alkaline Phosphatase (BAP).
  • BAP Alkaline Phosphatase
  • the dephosphorylated pMD-U xyz-1 and Van 91I / Dra III double digested and recovered U xyz-1 fragments were ligated with T4 DNA ligase in a 1:10 molar ratio.
  • the ligation product transforms E.coli DH5 ⁇ competent cells.
  • the target genes U xyz-3 and U xyz-4 containing 3 or 4 U xyz-1 fragments can be constructed.
  • U abc-1 a nucleotide sequence containing U a , U b and U c (abbreviated as U abc-1 ) and U xyz-1 are spliced into U abcxyz-1 by the above method, and U abcxyz-1 can be spliced into The dimerized U abcxyz-2 may be spliced with other sequences of gelatin-like protein units (U), and so on.
  • the gelatin-like protein unit (U) was spliced by complementary sticky ends under the action of T4 DNA ligase, and then subjected to agarose gel electrophoresis to recover DNA fragments of appropriate size.
  • the gelatin-like protein units (U) involved in the splicing may have the same sequence or different.
  • 6His affinity purification tag is added to the N-terminus or C-terminus of gelatin-like protein (GS).
  • GS Gelatin class (GS) at the N-terminal 6His tag fusion, nucleotide fragment was subcloned into plasmid pPIC9 (Life Technologies, Inc.), to construct an expression vector, in the methylotrophic yeast Pichia pastor GS115 (His -) is an expression host strain by electroporation Transformation
  • the linearized expression plasmid was transformed into GS115. Incubate at 30 ° C for 3 days until single colonies appear. Inoculate the transformed recombinant yeast single colony into 10ml BMGY liquid medium and incubate at 30 ° C and 250rpm for 24 hours.
  • gelatin-like protein has lower dyeing efficiency under conventional Coomassie brilliant blue dyeing conditions, and adopts negative staining methods, such as copper staining (Chris Lee et al., Analytical Biochemistry 166: 308-312, 1987). high.
  • 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. Immerse the gel in 0.3M CuCl 2 solution and dye for 2-5min; 4. After removing the glue, take a picture with an imager.
  • the supernatant of the culture solution was centrifuged to precipitate at 8000 rpm, and then precipitated with 40% ammonium sulfate. After the precipitate was reconstituted with deionized water, the sample was loaded and equilibrated with an equilibration buffer (0.5 M NaCl, 20 mM imidazole, 20 mM Tris-HCl, pH 7.5). After the 50ml Chelating Sepharose Fast Flow column (GE Healthcare), after re-equilibration, linear elution with 0-100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0). After the 50% eluent was mixed, a 35% saturated ammonium sulfate precipitate was added, and the precipitate was collected by centrifugation at 8000 rpm for 20 minutes and reconstituted with deionized water.
  • an equilibration buffer 0.5 M NaCl, 20 mM
  • Table 3 exemplarily lists low molecular weight gelatin-like proteins (GS) composed of gelatin-like units (U) and their corresponding sequences.
  • Viscosity and reversible gelation of temperature in aqueous solutions are the most important properties of natural gelatin.
  • a natural gelatin aqueous solution with a concentration greater than 0.5% When cooled to about 35-40 ° C, it first increases the viscosity and then forms a gel.
  • the rigidity or strength of the gel depends on the concentration of gelatin, the inherent strength of gelatin, pH, temperature and the presence of additives (GELATIN HANDBOOK, GMIA, 2012).
  • Animal-derived gelatin is made from collagen by acid or alkali hydrolysis.
  • the molecular weight distribution of 20KD-25KD is generally low Bloom value, 25-50KD is medium Bloom value, and 50KD-100KD is high Bloom value.
  • the preferred high-expression low molecular weight gelatin-like protein (GS) in Example 2 was prepared as a dimer or by sticky end splicing, so that its molecular weight reached 40KD or more, and then with natural animal-derived Compare with gelatin.
  • GLK containing 4-16 amino acid compositions was also prepared at the same time.
  • This example refers to the national standard "Food Additive Gelatin” GB6783-94 method to determine the freezing strength and Brinell viscosity of the sample.
  • the purified sample of GS protein in Table 4 was prepared into a 6.67% (W / W) aqueous solution, and then the freezing strength was measured using a freezing force tester, and the viscosity was measured using the ND-2 Brinell viscosity tester.
  • Animal gelatin (48722-100G-F, Sigma) was used as a control. Each measurement was repeated three times, and the results are shown in Table 4.
  • Human arginase 1 (hArg1) is a natural trimer structure with a monomer molecular weight of about 35KD and a trimer molecular weight of about 105KD. Although the molecular weight has far exceeded the glomerular filtration pore size, humans The half-life of arginase 1 in the body is surprisingly short, only a few minutes (PNCheng, TLLam, WMLam, SMTsui, AWCheng, WHLo, et al., Pegylated recombinant human human arginase (rhArg-peg5,000mw ) inhibits the vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion, Cancer Res. 67 (2007) 309–317). Currently, PEGylation modification is generally used to extend its half-life in vivo.
  • the spliced GS fragment and the control fragment (the 6His purification tag was introduced at the N-terminal) in Example 2 were fused with human arginase 1 (SEQ ID NO: 7) (as shown in Table 5), and the nucleotide fragment was cloned into plasmid pPIC9 (Life Technologies, Inc.), 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.
  • Fusion protein code Amino acid sequence, nucleotide sequence (SEQ ID NO :) GS100R9-hArg1 211, 212 GS100R35-hArg1 213, 214 GS100R52-hArg1 215, 216 GS100R9-hArg1-GS100R9 217, 218 GS100R35-hArg1-GS100R35 219, 220 GS100R52-hArg1-GS100R52 221, 222 GS100R74-hArg1-GS100R74 223, 224 GS100R77-hArg1-GS100R77 225, 226 GS100R98-hArg1-GS100R98 227, 228 GS100R112-hArg1-GS100R112 229, 230 GS200R9-hArg1 231, 232 GS200R9-hArg1-GS200R9 233, 234 GS400R9-hArg1-GS400R9 235, 236 GS400R77-hArg
  • Protein purification methods vary according to different expression systems. Existing technology already has 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, molecular 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 example to show that the expression host is methanol yeast GS115 and the purification method under specific conditions of fermentation conditions. When the fermentation conditions are different, the purification conditions should also be adjusted accordingly, which will not be repeated here.
  • the 2L fermentation supernatant was concentrated by ultrafiltration with a 30kDa filter membrane to 250mL, the concentrated liquid was added to 0.5M ammonium sulfate and centrifuged to take the supernatant, and the supernatant was added to 1.3M ammonium sulfate to centrifuge to discard the supernatant, and the precipitate was 1M ammonium sulfate 20mM PB pH6.
  • the sample is loaded onto a 50ml Chelating Sepharose Fast Flow chromatographic column equilibrated with equilibration buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5) (GE Healthcare), after re-equilibration, elute with 10%, 50%, 100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0).
  • equilibration buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5
  • the spliced GS or GLK fragment in Example 2 was fused with G-CSF (SEQ ID NO: 9), and the N terminal was connected to 6His (as shown in Table 6).
  • Nucleotide fragment was subcloned into plasmid pPIC9 (Life Technologies, Inc.), 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 methanol induction process is shown in Example 2. The supernatant of the culture solution was mixed with 5 times of loading buffer, and heated at 100 °C for 8-10min.
  • 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 after being equilibrated with an equilibrium buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5).
  • an equilibrium buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5.
  • Fast Flow column GE Healthcare
  • linear elution with 0-100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0).
  • the 50% eluent was added with 35% saturated ammonium sulfate to precipitate, and the precipitate was collected by centrifugation at 8000 rpm for 20 minutes and reconstituted with deionized water.
  • Fusion protein code Amino acid sequence, nucleotide sequence (SEQ ID NO :) GS100R9-GCSF 247, 248 GS100R35-GCSF 249, 250 GS100R52-GCSF 251,252 GS100R74-GCSF 253, 254 GS100R77-GCSF 255, 256 GS100R98-GCSF 257, 258 GS100R112-GCSF 259, 260 rGLK116 4 -GCSF 261, 262
  • GS-hArg1 fusion protein sample (GS-hArg1) and hArg1 (R & D Systems, Cat: 5868-AR) to be tested were 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.
  • SD rats were randomly divided into groups of 10, each immunized with the protein in Table 7, 3 mg / kg, subcutaneously, once a week, continuously for 4 weeks, and one group was injected with PBS as a negative control. Blood was taken before administration, and two weeks after the last immunization, rats were sacrificed, blood was taken, and serum was isolated. ELISA test was used to detect the production of GS antibody in serum.
  • GS-GCSF fusion protein coated ELISA plate 100ng / well
  • the sera of immunized animals were diluted 100-fold and 500-fold, and incubated with them at 37 ° C for 2h
  • HRP-labeled goat anti-rat secondary antibody EarthOX, E030140-01) detection read OD450 value.
  • Table 8 The results are shown in Table 8 below.
  • GS-hArg1 fusion protein was positive when coated with GS-hArg1 fusion protein, but negative when coated with hArg1 unrelated protein (GS-GCSF fusion protein), indicating that hArg1 produced a stronger Immunogenic but not GS carrier protein.
  • rGLK116 4 -hArg1 is always positive, indicating that rGLK116 4 has strong immunogenicity in rats.
  • the OD450 value is more than twice that of the sample before administration, which is positive;
  • the OD450 value is less than twice that of the sample before administration, which is negative.
  • SD rats were randomly divided into groups of 7, each group was subcutaneously injected with the fusion protein in Table 7, 2mg / kg, GS-hArg1 fusion protein administration group before injection and 3h, 8h, 12h, 24h, 36h, 48h after injection , 72h, 96h, 120h, 144h, 168h blood was collected and serum was isolated.
  • the hArg1 protein (R & D Systems, Cat: 5868-AR) administration group was bled before injection and 3h and 8h after injection.
  • Sandwich ELISA method was used to detect the pharmacokinetics of the fusion protein in rats.
  • 100ng / well of hArg1 rabbit polyclonal antibody (self-made) was coated overnight and washed 3 times with PBST. After blocking with 5% skimmed milk powder, wash with PBST 3 times, dilute the serum at each time point to the specified multiple, add 100 ⁇ l / well to the ELISA enzyme plate, incubate at 37 °C for 2h, wash with PBST 3 times, add biotin Labeled hArg1 rabbit polyclonal antibody (self-made), incubate at 37 ° C for 2 hours, wash 3 times with PBST, and finally add 50,000 times HRP-labeled streptavidin to the ELISA plate, then incubate at 37 ° C for 1 hour with conventional TMB Method to detect and read the OD450 value.
  • the GRAVY value of the gelatin-like protein (GS) shown in Table 3 is between -1.0 and 0, while GLK116 4 is -1.815, which is quite different.
  • GEE151 in GEE151-hArg1 (SEQ ID NO: 245) is composed of glycine G and glutamic acid E, and has a very low GRAVY value (-2.467).
  • GLK RD GRAVY value -0.785
  • GLK RD -hArg1 does not have the structure of Gly-XY, nor does it have the same effect as the GS protein.
  • Protein samples were detected by the periodate Schiff base (PAS) reagent method: first, the samples were loaded on 10% SDS-PAGE, after electrophoresis, using Thermo Scientific Staining Kit (Cat. No. 24562, Lot PE201610B) Glycosyl staining: completely immerse the acrylamide gel after electrophoresis in 100ml of 50% methanol for 30 minutes to fix the gel; then use 100ml of 3% acetic acid and gently shake for 10 minutes to clean the gel; transfer the gel to 25ml of oxidizing solution, After shaking gently for 15 minutes, the gel was washed by shaking gently with 100 ml of 3% acetic acid for 5 minutes. Repeat this step twice. Transfer the gel to 25 ml of glycoprotein staining reagent (Thermo Scientific, Cat. No. 24562, Lot PE201610B) and shake gently for 15 minutes.
  • PAS periodate Schiff base
  • Human growth hormone (hGH, SEQ ID NO: 5) has a significant tendency to aggregate, and when recombinantly expressed alone, a large number of irreversible aggregates are often produced.
  • the spliced GS fragment in Example 2 was fused and expressed with the hGH gene (as shown in Table 9), and the N terminal was connected to 6His.
  • 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.
  • Example 2 The methanol induction process is shown in Example 2. After centrifuging the supernatant of the fermentation broth, add 5x sample buffer to mix well, and heat at 100 °C for 8-10min. The expression strain was screened by SDS-PAGE electrophoresis. As a classic theoretical guide, specific steps can be found in Life Technologies' product manual "Pichia Expression Kit, For Expression of Recombinant Proteins in Pichia pastoris, Catalog no. K1710-01".
  • Ammonium sulfate was added to the supernatant of the fermentation broth to a conductivity of 180 mS / cm, 8000 rpm, 15 min, and centrifugation at 10 ° C to collect protein precipitates.
  • the precipitate was dissolved with 20 mM PB pH 7 solution, and then precipitated with ammonium sulfate at a conductivity of 180 mS / cm. Take the precipitate to dissolve with 20mM NaAc pH5 solution and dilute with water to a conductivity below 4mS / cm.
  • Example 13 Thermal stability of GS-GH fusion protein
  • the purified GS-GH fusion protein prepared in Example 12 was quantified by C18RP-HPLC, the concentration was adjusted to about 1.0 mg / ml, and treated at room temperature and 85 ° C for 30 minutes respectively, centrifuged to remove the precipitate, and the supernatant was taken for SDS-PAGE. The results are shown in Figure 5.
  • Example 14 SEC-HPLC analysis of aggregation of GS and hGH fusion protein samples
  • the detection method is as follows: detection wavelength: 280 nm; chromatography column: column temperature 25 ° C., Sepax SRT-1000 SEC 5 ⁇ m (300 ⁇ 7.8 mm), mobile phase: 50 mM PB, 150 mM NaCl, pH 7.2; running time: 20 minutes.
  • detection wavelength 280 nm
  • chromatography column column temperature 25 ° C., Sepax SRT-1000 SEC 5 ⁇ m (300 ⁇ 7.8 mm)
  • mobile phase 50 mM PB, 150 mM NaCl, pH 7.2
  • running time 20 minutes.
  • Example 15 In vitro cell activity detection of GS-GH samples
  • Ba / f3-GHR cells were starved with RPMI 1640 medium without IL-3 (containing 5% FBS and 1 mg / ml G418) for 4-6 hours, then transferred to a centrifuge tube and centrifuged at 1000 RPM for 5 min. After the above medium were resuspended count was adjusted to 2x 10 5 / ml, 96 well plates plated, 100 L per well, i.e., 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 stimulation for 48 hours, the protein was detected by MTT method. The results are shown in Table 10 and Figure 7 below.
  • Fusion protein code EC 50 (nM) GS800R9-GH-GS100R9 7.2 GS800R35-GH-GS100R35 8.2 GS800R127-GH-GS100R127 7.6 GS800L91-GH-GS100L91 6.1
  • SD rats were randomly divided into groups of 10, each subcutaneously injected with different GS-GH fusion protein or hGH recombinant protein (Sino Biological, Cat: 16122-H07E), 2mg / kg, before and 3h, 8h after injection. 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 GS-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 again with PBST 3 times, each time Dilute the spotted serum to the specified multiple and add it to the ELISA plate.
  • the spliced GS fragment in Example 2 was fused and expressed with GDF 15 (SEQ ID NO: 15) (as shown in Table 12), and the N-terminal was connected to 6His.
  • 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. The methanol induction process is shown in Example 2.
  • Fusion protein code Amino acid sequence, nucleotide sequence (SEQ ID NO :) GS200R9-GDF15 277, 278 GS200L23-GDF15 279, 280 GS200L136-GDF15 281, 282 GS200S14-GDF15 283, 284 GS400R9-GDF15 285, 286 GS400L23-GDF15 287, 288 GS400L136-GDF15 289, 290 GS400S14-GDF15 291, 292 GS600R9-GDF15 293, 294 GS600L23-GDF15 295, 296 GS600L136-GDF15 297, 298 GS600S14-GDF15 299, 300
  • 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 after being equilibrated with an equilibrium buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5).
  • an equilibrium buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5.
  • Fast Flow column GE Healthcare
  • linear elution with 0-100% elution buffer (0.15M NaCl, 0.5M imidazole, 20mM Tris-HCl, pH 8.0).
  • Add 35-50% saturated AS precipitate to the eluent, centrifuge at 8000 rpm for 20 min to collect the precipitate, and reconstitute with deionized water.
  • 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, and the body weight and food intake of mice were measured every day. They were sacrificed on the 5th day after the last dose. Blood was taken from the orbit and the plasma specimens were stored at -80 ° C. Calculate the average body weight change and feeding change of each group of animals before administration and at the time of sacrifice. The results are shown in Figures 9 and 10.
  • the spliced GS fragment in Example 2 was fused with the glucagon-like peptide 2 analog GLP-2G (SEQ ID NO: 1) (as shown in Table 13), the C-terminus was connected to the 6His tag, 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.
  • Fusion protein code Amino acid sequence, nucleotide sequence (SEQ ID NO :) GLP2G-GS800R9 301,302 GLP2G-GS800S14 303, 304 GLP2G-GS800S203 305, 306
  • 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 after being equilibrated with an equilibrium buffer (0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5).
  • an equilibrium buffer 0.5M NaCl, 20mM imidazole, 20mM Tris-HCl, pH 7.5
  • Fast Flow chromatography column GE Healthcare
  • After mixing the eluent add 30-50% saturated ammonium sulfate precipitate, centrifuge at 8000 rpm for 20 min to collect the precipitate, and reconstitute with deionized water.
  • Example 20 Activity determination of GS and GLP-2G fusion protein
  • GLP-2G fusion protein in vitro cytological activity detection using luciferase reporter gene detection method.
  • the GLP-2R gene was cloned into mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-2R, and the full-length luciferase gene was cloned into pCRE-EGFP (preserved in this experiment) Replace the EGFP gene on the plasmid to obtain the pCRE-Luc recombinant plasmid.
  • CHO cells were transfected with pCDNA3.1-GLP-2R and pCRE-Luc plasmid 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.
  • DMEM / F12 medium containing 10% FBS Dilute the cells with DMEM / F12 medium containing 10% FBS to 3x 10 5 / ml. Spread 100 ⁇ L per well in a 96-well plate, that is, 30,000 cells per well. After attaching, change to DMEM / F12 culture containing 0.1% FBS Culture overnight.
  • 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. According to lucifersae reporter kit (Ray Biotech, Cat: 68-LuciR-S200) manual for detection. The results are shown in Table 14 and Figure 11.
  • Example 21 Pharmacokinetic test of GS and GLP2G fusion protein
  • SD rats were randomly divided into groups of 10, each group was injected with different fusion protein, 2mg / kg, subcutaneous injection, before and after injection 3h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h At 168h, blood was collected and serum was isolated.
  • Sandwich ELISA method was used to detect the pharmacokinetics of the fusion protein in rats.
  • GLP-2 antibody (Abcam, catalog number ab14183) was added to the 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, Serum was diluted to the specified multiple and added to the ELISA enzyme plate.
  • the spliced GS fragment in Example 2 is fused with VEGF-bound anchor repeat protein (Ankyrin repeat proteins, SEQ ID NO: 3) (as shown in Table 16), the C-terminal is connected to the 6His tag, and the nucleotide fragment 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.
  • VEGF-bound anchor repeat protein Alignin repeat proteins, SEQ ID NO: 3
  • Fusion protein code Amino acid sequence, nucleotide sequence (SEQ ID NO :) GS600R9-AR VEGF 307, 308 GS900R9-AR VEGF 309, 310
  • Example 23 Determination of affinity of GS and AR VEGF fusion protein
  • BLI Bio-layer inteferometry, ForteBio
  • biotin Biotin Thermo, Prod # 21338, Sulfo-NHS
  • VEGF Molar ratio 2 1, label, and remove the biotin that did not participate in labeling by dialysis; then, according to the instructions for use of Octet-QK, Select a high-sensitivity experimental program, load biotin-labeled VEGF on the avidin probe SA (forteBIO, Part # 18-5019); the buffer used in the experiment is PBS (containing 0.1% Tween-20), and the gradient is diluted
  • the fusion protein and the control antibody were added to the predetermined position of the 96-well black plate (Greiner, 655209) according to the program settings. According to the program settings, the fusion protein is combined and then dissociated in the PBST solution to obtain the experimental curve; using the Octet-QK result analysis software, the experimental result is locally fitted to the curve to
  • Table 17 summarizes the Kd of the fusion protein and the control drug Bevacizumab. From the table, it can be seen that the average affinity of AR VEGF to VEGF before and after fusion with GS is not significantly different from that of Bevacizumab (Medchemexpress, Cat.No.:HY-P9906) In the same order of magnitude.
  • Example 24 In vitro activity study of GS and AR VEGF fusion protein
  • AR VEGF activity was measured by VEGF receptor competition inhibition method.
  • 5 ⁇ g / mL VEGF receptor 2 / KDR (Abcam, ab155628) was added to the ELISA plate, 50 ⁇ L per well, and placed at 37 ° C for 2h. Sealed with 1% BSA / TBS and placed at 37 ° C for 2h.
  • AR VEGF and the control substance Bevacizumab were respectively diluted with PBST as a 3-fold gradient. 80 ⁇ L of the diluted sample was mixed with an equal volume of 1 ⁇ g / mL VEGF, and placed at 37 ° C for 1 h.
  • the KDR-coated ELISA plate was washed twice, after patting dry, the gradient diluted mixture samples were sequentially transferred to the ELISA plate, placed at 37 ° C for 1 h, and then washed 5 times.
  • 1000 diluted HRP-labeled goat anti-mouse secondary antibody (Pierce, 31432, QA1969921), 50 ⁇ L per well, let stand at 37 ° C for 1 h, then wash the plate 6 times.
  • Table 18 IC50 of GS and AR VEGF fusion protein
  • the GS-GH fusion protein sample was prepared into 40-3.0 mg / ml with 40 mM PB, pH 7.4, sterilized and filtered (0.22 ⁇ m, Millipore), diluted 10 times with rat serum, mixed, and divided into sterile centrifuges In the tube; place in a 37 ° C incubator, take samples on day 0 and day 7 for Western-blot analysis, and use HRP-labeled Anti-6X His Antibody (Abcam, ab1187) was used as detection antibody. The results are shown in Figure 13.

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Abstract

本文提供一种用于改善生物活性蛋白的性质的载体蛋白，该载体蛋白的结构为G-X-Y三元重复结构，其中G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸和谷氨酸。本文还提供含有该载体蛋白和生物活性蛋白的融合蛋白。本文的融合蛋白具有改善的生物学性质，例如改善的药代动力学和理化性质。

Description

一种改善生物活性蛋白性质的载体蛋白 技术领域
本发明涉及生物技术领域，具体涉及一种可以改善活性蛋白性质的载体蛋白。
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,Kunstelj M,Porekar VG，PEGylation of therapeutic proteins，Biotechnol J.，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，Strategies to extend plasma half-lives of recombinant antibodies，BioDrugs，23:93–109；2009；Kang JS等，Emerging PEGylated drugs.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，Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters，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，A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner.Nature Biotechnology 27(12):1186，2009)、类弹性蛋白ELP(MacEwan SR,Chilkoti A.,Applications of elastin-like polypeptides in drug delivery.J Control Release.2014；190:314-30.)、PAS(专利号ZL200880019017，M Schlapschy等，PASylation:a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.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，A long-acting human growth hormone with delayed clearance(VRS-317):results of a double-blind,placebo-controlled,single ascending dose study in growth hormone-deficient adults,J Clin Endocrinol Metab.,98(6):2595-603.2013；Christiansen M等，Weekly Subcutaneous Doses of Glymera(PB1023)a Novel GLP-1 Analogue 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。
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-1.1；附加条件是，所述的明胶样单元不是天然的明胶蛋白。
上述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，一般只由少数几种特定氨基酸组成。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”(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”。
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，Gcg-XTEN:an improved glucagon capable of preventing hypoglycemia without increasing baseline blood glucose,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弥补了这方面的缺陷。
发明内容
本文提供一种类明胶单元，其具有以下重复结构：
(G-X-Y) n
其中，G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸和谷氨酸；n为5-20的整数，优选n为6-20或9-15的整数。示例性的类明胶单元可选自SEQ ID NO:17-89中任一奇数编号的序列所示的类明胶单元；优选地，所述类明胶单元选自SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29和SEQ ID NO:31。
本文还提供一种类明胶蛋白，该类明胶蛋白含有至少两个本文所述的类明胶单元；所述至少两个类明胶单元可相同或可不同；优选地，所述类明胶蛋白中，丙氨酸的含量大于 等于10％，优选大于等于12％，更优选大于等于15％，更优选大于等于18％，更优选大于等于20％；优选丙氨酸含量≤45％，如≤40％或小于等于35％；优选地，所述类明胶单元中，代表亲水性的GRAVY值大于-1.1，优选大于等于-1.0，更优选大于等于-0.9，更优选大于等于-0.8，优选该值≤0，如≤-0.1或≤-0.2。优选地，该类明胶蛋白具有100-2000个氨基酸。示例性的类明胶蛋白可选自SEQ ID NO:91-185中任一奇数编号所示的序列，或可含有两条或两条以上(如2-20条)SEQ ID NO:91-185中任一奇数编号所示的序列。示例性的含有两条或两条以上SEQ ID NO:91-185中任一奇数编号所示的序列的类明胶蛋白优选是两条或两条以上相同的所述序列的串联重复序列，包括但不限于SEQ ID NO:231第1-231位氨基酸残基序列、SEQ ID NO:239第1-573位氨基酸残基序列、SEQ ID NO:263第1-915位氨基酸残基序列、SEQ ID NO:265第1-864位氨基酸残基序列、SEQ ID NO:267第1-864位氨基酸残基序列、SEQ ID NO:269第1-864位氨基酸残基序列、SEQ ID NO:271第1-864位氨基酸残基序列、SEQ ID NO:273第1-864位氨基酸残基序列、SEQ ID NO:275第1-915位氨基酸残基序列、SEQ ID NO:279第1-216位氨基酸残基序列、SEQ ID NO:281第1-216位氨基酸残基序列、SEQ ID NO:283第1-231位氨基酸残基序列、SEQ ID NO:293第1-687位氨基酸残基序列、SEQ ID NO:295第1-648位氨基酸残基序列、SEQ ID NO:297第1-648位氨基酸残基序列、SEQ ID NO:299第1-687位氨基酸残基序列、SEQ ID NO:303第34-948位氨基酸残基序列、SEQ ID NO:305第34-948位氨基酸残基序列和SEQ ID NO:309第1-1029位氨基酸残基序列。优选地，类明胶蛋白包括与本段所述任一氨基酸序列具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的氨基酸序列。
本文还提供一种融合蛋白，该融合蛋白含有本文公开的类明胶蛋白和生物活性蛋白。示例性的融合蛋白可选自：SEQ ID NO:211-239、247-259和263-309中任一奇数编号的序列所示的融合蛋白。
本文还提供一种多核苷酸序列，选自：
(1)编码本文所述的类明胶单元、类明胶蛋白或融合蛋白的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列。
本文还提供一种核酸构建体，其包含本文所述的多核苷酸序列；优选地，所述核酸构建体为克隆载体或表达载体。
本文还提供一种宿主细胞，所述宿主细胞：
(1)含有本文所述的多核苷酸序列和/或核酸构建体；和/或
(2)表达本文所述的类明胶单元、类明胶蛋白和/或融合蛋白。
本文还提供选自以下的应用：
(1)本文所述的类明胶单元或其编码序列或该编码序列的互补序列在制备类明胶蛋白或含该类明胶蛋白的融合蛋白中的应用；
(2)本文所述的类明胶蛋白或其编码序列或该编码序列的互补序列在制备含有该类明胶蛋白的融合蛋白中的应用，或在提高生物活性蛋白的药代动力学和/或增强生物活性蛋白的理化性质中的应用；
(3)本文所述的融合蛋白、其编码序列或含该编码序列或其互补序列的核酸构建体在制备药物中的应用。
还提供一种制备能改善生物活性蛋白的生物学性质或生物学功能的载体蛋白的方法，该方法采用化学合成方法或重组技术制备所述载体蛋白；其中，所述载体蛋白的结构为G-X-Y三元重复结构，其中G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸和谷氨酸；
其中，所述重组技术包括构建表达该载体蛋白的表达载体，使用该表达载体转化宿主细胞，以及培养宿主细胞以使其表达产生所述载体蛋白；
其中，所述化学合成方法包括，按照所述载体蛋白的结构将选自甘氨酸、脯氨酸、丙氨酸和谷氨酸中的氨基酸残基依次连接到肽链上，形成具有G-X-Y三元重复结构的所述载体蛋白。
还提供的是甘氨酸、脯氨酸、丙氨酸和谷氨酸在制备能改善生物活性蛋白的生物学性质或功能的载体蛋白中的应用。
下文将对本文公开的各方面进行详细的描述。
附图说明
图1：GS100R9-hArg1融合蛋白在Sepax SRT
Figure PCTCN2019108430-appb-000001
上的表观分子量。1、GS100R9-hArg1；M1、甲状腺球蛋白(Thyroglobulin,669kDa)；M2、铁蛋白(Ferritin,440KD)；M3、醛缩酶(Aldolase，158KD)；M4、伴清蛋白(Conalbumin，75KD)；M5、卵清蛋白(Ovalbumin,44KD)。
图2：GS-hArg1融合蛋白在Sepax SRT-1000 SEC上的表观分子量。1、GS200R9-hArg1-GS200R9；2、GS100R9-hArg1-GS100R9；3、GS100R35-hArg1-GS100R35；M1、甲状腺球蛋白(Thyroglobulin,669kDa)；M2、铁蛋白(Ferritin,440KD)+醛缩酶(Aldolase,158KD)+伴清蛋白(Conalbumin,75KD)+卵清蛋白(Ovalbumin,44KD)。
图3：GS-hArg1融合蛋白药代结果图。
图4：蛋白样品糖基化检测结果图。A显示糖染前的结果，B显示糖染后的结果。泳道1、2为阳性对照蛋白；泳道3：GS100R9-hArg1-GS100R9；泳道4：GS100R35-hArg1-GS100R35；泳道5：GS100R52-hArg1-GS100R52；泳道6： GS100R74-hArg1-GS100R74；泳道7：GS100R77-hArg1-GS100R77；泳道8：GS100R98-hArg1-GS100R98；泳道9：GS100R112-hArg1-GS100R112；泳道10-11分别为两个独立批次的rGLK116 4-hArg1。
图5：经过不同温度处理之后的GS与GH融合蛋白的SDS-PAGE电泳图。泳道1和8：GS800R9-GH-GS100R9；泳道2和9：GS800R35-GH-GS100R35；泳道3和10：GS800R127-GH-GS100R127；泳道4和11：GS800L91-GH-GS100L91；泳道5和12：GS800L102-GH-GS100L102；泳道6和13：GS800L146-GH-GS100L146；泳道7和14：GS800S203-GH-GS100S203。其中泳道1-7为室温放置30分钟样品，泳道8-14为85℃下处理30分钟的样品。M为蛋白分子量MARKER：200、116、97.2、66.4、44.3KD。
图6：GS与hGH融合蛋白样品的聚体分析情况图。
图7：GS与hGH融合蛋白的体外细胞活性结果图。
图8：GS与GDF15融合蛋白的SDS-PAGE电泳图。泳道1-4分别为：GS600R9-GDF15、GS600L23-GDF15、GS600L136-GDF15、GS600S14-GDF15；泳道5-8分别为：GS400R9-GDF15、GS400L23-GDF15、GS400L136-GDF15、GS400S14-GDF15；泳道9-12分别为：GS200R9-GDF15、GS200L23-GDF15、GS200L136-GDF15、GS200S14-GDF15。
图9：GS与GDF15融合蛋白对DIO小鼠的体重减轻效果图。
图10：GS与GDF15融合蛋白对DIO小鼠的食欲抑制效果图。
图11：GS与GLP2G融合蛋白的体外细胞活性检测结果图。
图12：GS与AR VEGF融合蛋白的体外细胞活性结果图。
图13：在大鼠血清中温育第7天的GS-GH及rGLK116 4-hArg1融合蛋白。1、GS800R9-GH-GS100R9；2、GS800R35-GH-GS100R35；3、GS800R127-GH-GS100R127；4、GS800L91-GH-GS100L91；5、GS800L102-GH-GS100L102；6、GS800L146-GH-GS100L146；7、rGLK116 4-hArg1；8、GS800S203-GH-GS100S203。
图14：GS-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：GS800R9-GH-GS100R9；泳道3和4：GS800R35-GH-GS100R35；泳道5和6：GS800R127-GH-GS100R127；泳道7和8：GS800L91-GH-GS100L91；泳道9和10：GS800L102-GH-GS100L102；泳道11和12：GS800L146-GH-GS100L146；泳道13和14：GS800S203-GH-GS100S203。M为高分子量MARKER:220KD、135KD、90KD、66KD、45KD及35KD。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
术语
本文中，“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/或生物学活性或功能(如本文所述的药代动力学和理化性质)，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”可包括酶、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、细胞运动因子、移动因子、静止因子、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，本发明的“生物学活性”或“生物活性”表现为“治疗活性”。因此，在这些实施方案中，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以下生物活性的蛋白：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。
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， TGF-β，TGF-β-1，TGF-β-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 1 domain，plasminogen kringle 5 domain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，“凝胶化”指某些溶液在冷却时会逐渐变得粘稠，最后失去流动性而成为具有弹性的胶冻，这种现象叫做凝胶化现象。通过天然胶原水解获得的明胶具有某些特定的性质，水溶液中明胶性质受温度、pH、制作工艺及浓度等的影响。其中，对温度产生可逆转的凝胶化是明胶最重要的一种性质(GELATIN HANDBOOK，GMIA，2012)。
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上。将PEG共价连接至生物活性蛋白能够掩蔽所述蛋白免于宿主的免疫系统攻击，并增加目的生物活性蛋白的水动力学半径，从而通过降低肾清除率来延长该蛋白药物的循环时间。
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％)。
除上述术语外，对于本文中使用到的其它术语，除非另有说明，否则其含义为本领域的通用含义。
类明胶单元(U)
本发明公开提供一种类明胶单元(U)，构成该类明胶单元的氨基酸种类由甘氨酸(G)、脯氨酸(P)、丙氨酸(A)和谷氨酸(E)组成，并具有G-X-Y三元单体重复结构，其中G代表甘氨酸(G)，X和Y各自独立选自脯氨酸(P)、丙氨酸(A)或谷氨酸(E)。
在某些实施方案中，本发明的类明胶单元可具有以下重复结构：
(G-X-Y)n
其中，G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸或谷氨酸；n为5-20的整数。
在某些实施方案中，所述G-X-Y三元单体重复结构选自：GPP、GEE、GAA、GEA、GAE、GAP、GPA、GPE和GEP。因此，在某些实施方案中，本发明公开的类明胶单元(U)可由选自以下的两种或两种以上的G-X-Y三元单体重复结构组成：GPP、GEE、GAA、GEA、GAE、GAP、GPA、GPE和GEP。
在某些实施方案中，本公开的类明胶单元(U)至少由6个G-X-Y三元单体组成(即n≥6)，如6≤n≤20或6≤n≤15。在某些实施方案中，本公开的类明胶单元(U)至少由9个G-X-Y三元单体组成，如9≤n≤20或9≤n≤15。
通常，基于表达量的优化，相同的G-X-Y三元单体并不连续出现，以避免潜在的同源重组事件发生。
在某些实施方案中，本发明公开的类明胶单元选自SEQ ID NO:17-89中任一奇数编号的序列所示的类明胶单元。在某些优选的实施方案中，本发明公开的类明胶单元选自SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29和SEQ ID NO:31。
类明胶蛋白(GS)
进一步地，本文提供一种包括至少两个本文所述类明胶单元(U)的类明胶蛋白(GS)。本文的类明胶蛋白可用作载体蛋白，用于承载生物活性蛋白，尤其是活性蛋白药物。
在某些实施方案中，本文所述的类明胶蛋白的核心结构为：U 1-U 2或U 1-U 2-…U a，其中，U 1、U 2、…、U a各自代表本文任一实施方案所述的任意一种类明胶单元，a为≥3的整数。本发明类类明胶蛋白中的各类明胶单元可相同或不同。在某些实施方案中，3≤a≤150；在某些实施方案中，3≤a≤100；在某些实施方案中，3≤a≤50。应理解，a的选 择应使得本文所述的类明胶蛋白的氨基酸残基总数在下文任意实施方案所述的范围内。除核心结构外，本文所述类明胶蛋白可含有其它不影响到该类明胶蛋白的生物学性质(包括但不限于下文所述的凝胶化、粘度、产物均一性、血清稳定性、耐酶稳定性和免疫原性等)的氨基酸序列。例如，该类明胶蛋白N-末端、C-末端和/或蛋白内的合适区域内可含有采用重组技术生产该类明胶蛋白时为促进其表达、分泌到宿主细胞外和/或纯化而使用的氨基酸序列，包括但不限于适合的接头序列、信号肽、前导肽、末端延伸等。在某些实施方案中，所述氨基酸序列为蛋白标签，可以是FLAG、HA、Poly-His、GST、MBP和c-Myc等。这些标签可用于对蛋白进行纯化。在某些实施方案中，所述核心结构的氨基酸残基总数占该类明胶蛋白氨基酸残基总数的70％以上，较佳80％以上，更佳85％以上，进一步更佳90％以上、95％以上或99％以上。在某些实施方案中，本发明的类明胶蛋白由本文任一实施方案所述的类明胶单元组成。
优选地，本文的类明胶蛋白中Ala的含量大于等于10％；更优选地，Ala的含量大于等于12％；更优选地，Ala的含量大于等于15％；更优选地，Ala的含量大于等于18％；更优选地，氨基酸Ala的含量大于等于20％。优选地，氨基酸Ala的含量不超过45％，例如不超过40％、不超过35％或不超过30％等。因此，在某些实施方案中，本发明GS中Ala的含量在上述列出的任意两个数值作为端点构成的范围之内，如在10-45％的范围内，如12-45％、15-45％、18-45％、20-45％或10-40％、10-30％、10-20％或15-45％等。
根据代表亲水性的GRAVY值(Kyte J.,Doolittle R.F.，J.Mol.Biol.157:105-132，1982)划分，氨基酸Ala为1.800，Glu为-3.500，Pro为-1.600，Gly为-0.400。即Ala为疏水性氨基酸，Glu、Pro和Gly为亲水性氨基酸。在某些实施方案中，根据ProtParam公式计算，本文所述的类明胶蛋白(GS)代表亲水性的GRAVY值大于-1.1；优选地，该GRAVY值大于等于-1.0；更优选地，该GRAVY值大于等于-0.9；更优选地，该GRAVY值大于等于-0.8。优选地，该GRAVY值最大为0，如最大为-0.1或最大为-0.2。因此，在某些实施方案中，本文所述的类明胶蛋白(GS)代表亲水性的GRAVY值在上述任意两个数值作为端点构成的范围之内，如大于-1.1到≤0的范围内，如大于-1.1到-0.1、-1.0到0、-0.9到0、-0.8到0或-0.8到-0.1等。在某些实施方案中，本文所述的类明胶蛋白(GS)代表亲水性的GRAVY值在-1.0到0.0之间。
因此，本文所述的类明胶蛋白通常具有以下特征：(1)含有本文所述的类明胶单元；(2)Ala的含量大于等于10％，优选大于等于12％，更优选大于等于15％，更优选大于等于18％，更优选大于等于20％，优选Ala含量≤45％，如≤40％或小于等于35％；和(3)代表亲水性的GRAVY值大于-1.1，优选大于等于-1.0，更优选大于等于-0.9，更优选大于等于-0.8，优选该值≤0，如≤-0.1或≤-0.2。
通常，本文所述的类明胶蛋白(GS)具有至少100个氨基酸，优选具有至少200个氨基酸，更优选具有至少300个氨基酸，更优选具有至少400个氨基酸，更优选具有至少500个氨基酸，更优选具有至少600个氨基酸，更优选具有至少700个氨基酸，更优选具有至少800个氨基酸，更优选具有至少900个氨基酸，更优选具有至少1000个氨基酸，更优选具有至少1200个氨基酸。在某些实施方案中，本文所述的类明胶蛋白具有100-2000个氨基酸，例如200-2000、300-2000、400-2000、500-2000、600-2000、700-2000、800-2000、900-2000、1000-2000或1200-2000个氨基酸。
在某些优选的实施方案中，本文所述的类明胶蛋白由相同序列的类明胶单元(U)通过重复拼接而成。在其它优选的实施方案中，本文所述的类明胶蛋白由不同的类明胶单元(U)拼接而成。在某些实施方案中，各类明胶单元之间可存在接头序列，如由含有甘氨酸(G)和/或脯氨酸(P)的氨基酸序列形成的接头序列。
在某些优选的实施方案中，示例性的本发明类明胶蛋白选自SEQ ID NO:91-185中任一奇数编号所示的序列。在某些优选的实施方案中，可将选自SEQ ID NO:91-185中奇数编号所示的序列中的任意两条或任意多条(如2-20条，或2-10条，或2-8条)拼接形成本发明的类明胶蛋白。因此，在这些实施方案中，本发明的类明胶蛋白含有2-20条选自SEQ ID NO:91-185中任一奇数编号所示的序列。优选地，用于拼接形成本发明类明胶蛋白的两条或多条所述序列为相同的序列。同样地，各拼接序列之间可之间连接，或者可通过本领域周知的接头序列(如由含有甘氨酸(G)和/或脯氨酸(P)的氨基酸序列形成的接头序列)相连。在某些优选的实施方案中，示例性的本发明类明胶蛋白选自SEQ ID NO:211-239、247-259和263-309中任一奇数编号的序列所示的融合蛋白中所包含的类明胶蛋白，包括但不限于SEQ ID NO:231第1-231位氨基酸残基序列(GS200R9)、SEQ ID NO:239第1-573位氨基酸残基序列(GS500R9)、SEQ ID NO:263第1-915位氨基酸残基序列(GS800R9)、SEQ ID NO:265第1-864位氨基酸残基序列(GS800R35)、SEQ ID NO:267第1-864位氨基酸残基序列(GS800R127)、SEQ ID NO:269第1-864位氨基酸残基序列(GS800L91)、SEQ ID NO:271第1-864位氨基酸残基序列(GS800L102)、SEQ ID NO:273第1-864位氨基酸残基序列(GS800L146)、SEQ ID NO:275第1-915位氨基酸残基序列(GS800S203)、SEQ ID NO:279第1-216位氨基酸残基序列(GS200L23)、SEQ ID NO:281第1-216位氨基酸残基序列(GS200L136)、SEQ ID NO:283第1-231位氨基酸残基序列(GS200S14)、SEQ ID NO:293第1-687位氨基酸残基序列(GS600R9)、SEQ ID NO:295第1-648位氨基酸残基序列(GS600L23)、SEQ ID NO:297第1-648位氨基酸残基序列(GS600L136)、SEQ ID NO:299第1-687位氨基酸残基序列(GS600S14)、SEQ ID NO:303第34-948位氨基酸残基序列(GS800S14)、SEQ ID NO:305第34-948位氨基酸残基序列(GS800S203)和SEQ  ID NO:309第1-1029位氨基酸残基序列(GS900R9)。在某些实施方案中，本文所述的类明胶蛋白的氨基酸序列为与此段提及的任一氨基酸序列具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的氨基酸序列。
绝大部分治疗用生物活性蛋白，因其具有高级构象，因此一般都需要低温储存。因此作为生物活性蛋白的载体蛋白而言，在低温及常温下均需要有极好的溶解度和较低的粘度。如果在低温下形成了凝胶状，则溶解度下降，粘度显著增加，对于生物药物制备以及患者的给药都会增加额外障碍。天然的明胶是由动物毛皮、骨骼等结缔组织中的胶原部分通过强酸或强碱水解而成。明胶的性质受温度、pH及浓度的影响很大。天然明胶易溶于热水(>40℃)并在低温下趋于形成胶状。另外，当水溶液中的明胶浓度高于0.5％时，在35-40℃的温度下粘度首先会提高，进而形成胶状(Gelatin handbook,Gelatin Manufacturers Institute of America,2012)。ZL200980103870.9及Werten MW等(Werten MW等,Secreted production of a custom-designed,highly hydrophilic gelatin in Pichia pastoris.Protein Eng.14(6):447-54.2001)分别报道了人工设计的重组表达制备的类明胶蛋白。然而本文的实施例显示，取自ZL200980103870.9的GLK序列其理化性质对于温度的变化非常敏感，即这些类明胶样的序列虽然经过重组表达，但是很大程度上仍然保留了天然明胶的凝胶化性质。此外，在保持G-X-Y三元单体结构的基础上，类明胶蛋白的氨基酸种类越多，其凝胶化的性质越接近天然明胶，即越容易受温度的影响而凝胶化。如本文实施例所示，氨基酸种类超过4个的蛋白都带有凝胶化的性质。而当X和Y分别独立地选自脯氨酸(P)、丙氨酸(A)或谷氨酸(E)时，则可大幅地消除凝胶化的性质，粘度显著降低。
另外，除静脉输注以外，皮下注射、肌肉注射、玻璃体注射等注射给药途径，对于给药容量都有限制。例如眼科玻璃体给药，注射部位超过100微升就会导致眼压升高，需要通过玻璃体引流等来降低眼压；皮下注射的最大给药容量一般不超过2毫升，如果超出则需要进行多部位注射。更大的给药体积往往意味着给药途径的负担加重，增加患者的痛苦。为了减少给药体积，很多时候需要将药品制剂制备成较高浓度。因此，在常温下粘度高对于蛋白药物是一个严重的缺陷；作为载体蛋白，除了免疫原性低，无其他生理学活性外，很重要的一个条件是溶解度高，粘度低。
但是，现有的载体蛋白，如取自ZL200980103870.9的GLK与活性蛋白融合而成的重组融合蛋白，在25℃下或者在浓度高于10mg/ml时，凝胶化现象已经开始显现，而且粘度较大，并不适合临床应用，即并不适合作为蛋白药物载体。
与现有载体蛋白不同的是，本发明提供的类明胶蛋白(GS)不仅无明显凝胶化的现象，而且粘度极低，是一种更为理想的蛋白药物载体。在某些实施方案中，采用冻力测试 仪，按照国家标准《食品添加剂明胶》GB6783－94方法测定得到的本发明类明胶蛋白的凝冻强度≤10g，优选≤5g，更优选≤3g。例如，本发明类明胶蛋白的凝冻强度可在1-10g之间，或在1-5g或1-3g之间。在某些实施方案中，采用ND-2勃氏粘度，按照国家标准《食品添加剂明胶》GB6783－94方法测定得到的本发明类明胶蛋白的粘度≤3mPa·s，优选≤2mPa·s，更优选≤1mPa·s。例如，在某些实施方案中，本发明类明胶蛋白的粘度在0.01-3mPa·s的范围内，优选在0.05-1mPa·s的范围内。
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。对于某些不适合原核表达系统重组表达、又不适合化学合成的大分子蛋白而言，富含S和T的载体蛋白很难解决糖基化的问题。例如，在本发明的一个实施例中，富含S和/或T的序列在毕赤氏酵母(Pichia pastoris)中表达时，糖基化程度非常高。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。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解决。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，糖基化严重且不均一都是个极其严峻的问题，能够采取的解决办法只有先在原核表达系统中获得这些序列，再通过化学交联的方式与真核表达系统获得的活性蛋白或多肽(无法或很难在原核表达系统中表达)交联在一起。然而众所周知，化学交联带来的产物不均一，过程繁琐等都是目前无法解决的问题。
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TEV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-1 fusion 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。
本发明的类明胶蛋白(GS)由甘氨酸(G)、脯氨酸(P)、丙氨酸(A)和谷氨酸(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，GLK由于Asn(N)及Gln(Q)的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造成的产物不均一等问题在本发明提供的类明胶蛋白(GS)中存在的可能性都极小。如实施例所示，本发明的类明胶蛋白相比GLK具有更优越的血清稳定性和耐酶稳定性。
最后，作为一种载体蛋白，体内免疫原性是最重要的因素。在本发明的免疫原性检测实施例中，在大鼠体内多次给药后，针对rGLK116 4(取自中国专利号200980103870.9)的抗体滴度实际上比较高，而本发明提供的类明胶蛋白(GS)则几乎无免疫原性迹象。此结果与该专利实施例4中的结果不一致，推测可能的原因是该专利中采用的是rGLK116 4包被(说明书213段)，rGLK116 4是极为亲水的序列，作为包被蛋白是极不合适的。因为酶标板与蛋白之间是通过疏水作用而结合，而rGLK116 4本身亲水性极高，因而包被的量极少，造成了假阴性的结果。
融合蛋白
本公开进一步提供融合蛋白，其含有生物活性蛋白和本文所述的类明胶蛋白。如前文所述，生物活性蛋白可以是本领域周知的具有一种或者多种药理学和/或生物学活性，或靶向引导，多聚化等功能的蛋白。它们可以是天然就存在的，也可以是人工构建的。生物活性蛋白可包括酶、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、细胞运动因子、移动因子、静止因子、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，本文的融合蛋白含有活性蛋白药物(D)和本文所述的类明胶蛋白(GS)。
适用于本文的活性蛋白药物包括但不限于激动剂、受体、配体、拮抗剂、酶和激素。更具体而言，适用于本文的活性蛋白药物可以是本领域周知的在各种疾病的治疗和/或预防和/或症状的改善中使用到的活性蛋白药物，这类疾病包括但不限于：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。更具体而言，所述疾病包括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(如肺性高血压)、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。在某些实施方案中，本文所述的活性蛋白药物为前文所述的治疗性蛋白。
生物活性蛋白(尤其是活性蛋白药物)与类明胶蛋白可以本领域周知的方式串联融合。例如，生物活性蛋白融合在类明胶蛋白的N端或C端，或类明胶蛋白融合在生物活性蛋白的两端，又或者生物活性蛋白融合在类明胶蛋白的两端。融合蛋白中可含有两种或两种以上的生物活性蛋白，该生物活性蛋白可相同也可不同。同样地，融合蛋白中也可含有两种或两种以上的类明胶蛋白，该类明胶蛋白可相同也可不同。当含有两种或两种以上生物活性蛋白和/或两种或两种以上的类明胶蛋白时，生物活性蛋白与类明胶蛋白之间的串联方式可以是多样的。示例性的串联融合包括，但不限于如下结构：
D-GS；
GS-D；
D 1-GS-D 2；
GS 1-D-GS 2；
GS 1-D 1-GS 2-D 2-GS 3-D 3；
GS 1-D 1-D 2-GS 2-D 3；
GS 1-D 1-GS 2-D 2-D 3；
GS 1-D 1-GS 2-D 2-D 3-GS 3；
D 1-GS 1-D 2-GS 2-D 3；
GS 1-D 1-GS 2-D 2-GS 3-D 3-GS 4-D 4；
D 1-GS 1-D 2-GS 2-D 3-GS 3-D 4-GS 4；
其中D 1、D 2、D 3、D 4为活性蛋白药物，D 1、D 2、D 3、D 4之间可以相同或不相同；GS 1、GS 2、GS 3、GS 4为类明胶蛋白(GS)，GS 1、GS 2、GS 3、GS 4之间可以相同或不相同。
通常，在融合蛋白中存在两种或两种以上生物活性蛋白和/或两种或两种以上GS时，所述生物活性蛋白之间通常通过GS相连，所述GS之间通常也不直接相连，而是通过生物活性蛋白相连。在某些实施方案中，本文优选使用下表1所列的活性蛋白药物或其类似物。
表1
活性蛋白 SEQ ID NO: 活性蛋白 SEQ ID NO:
GLP-2类似物 1 Glucagon 2
AR VEGF 3 IL-2 4
hGH 5 IL-15 6
Arginase 1 7 FGF19 8
G-CSF 9 EPO 10
Exendin-4 11 IL-6 12
GLP-1类似物 13 M-CSF 14
GDF15 15 FGF-21 16
在融合了本文所公开的类明胶蛋白(GS)后，生物活性蛋白，尤其是活性蛋白药物(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生物活性蛋白的体内半衰期显著延长。在本发明某些实施例中，融合蛋白的半衰期比未融合时延长了10倍以上。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸(例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等)添加至重组蛋白的N-末端、C-末端和/或该蛋白内的其它合适区域内。因此，本文融合蛋白的氨基端和/或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本文。例如，所述的标签可以是FLAG、HA、Poly-His、GST、MBP、c-Myc，这些标签可用于对蛋白进行纯化。
此外，在生物活性蛋白与GS之间、两个生物活性蛋白之间甚至两个GS之间可设有合适的接头序列，如含有G(甘氨酸)和/或S(丝氨酸)的接头序列。任何本领域周知的接头序列都可用于本文的融合蛋白。
优选的实施方案中，本文融合蛋白中的类明胶蛋白可选自SEQ ID NO:91-185中任一奇数编号的序列所示的序列，以及SEQ ID NO:231第1-231位氨基酸残基序列(GS200R9)、SEQ ID NO:239第1-573位氨基酸残基序列(GS500R9)、SEQ ID NO:263第1-915位氨基酸残基序列(GS800R9)、SEQ ID NO:265第1-864位氨基酸残基序列(GS800R35)、SEQ ID NO:267第1-864位氨基酸残基序列(GS800R127)、SEQ ID NO:269第1-864位氨基酸残基序列(GS800L91)、SEQ ID NO:271第1-864位氨基酸残基序列(GS800L102)、SEQ ID NO:273第1-864位氨基酸残基序列(GS800L146)、SEQ ID NO:275第1-915位氨基酸残基序列(GS800S203)、SEQ ID NO:279第1-216位氨基酸残基序列(GS200L23)、SEQ ID  NO:281第1-216位氨基酸残基序列(GS200L136)、SEQ ID NO:283第1-231位氨基酸残基序列(GS200S14)、SEQ ID NO:287第1-432位氨基酸残基序列(GS400L23)、SEQ ID NO:289第1-432位氨基酸残基序列(GS400L136)、SEQ ID NO:291第1-459位氨基酸残基序列(GS400S14)、SEQ ID NO:293第1-687位氨基酸残基序列(GS600R9)、SEQ ID NO:295第1-648位氨基酸残基序列(GS600L23)、SEQ ID NO:297第1-648位氨基酸残基序列(GS600L136)、SEQ ID NO:299第1-648位氨基酸残基序列(GS600S14)、SEQ ID NO:303第34-948位氨基酸残基序列(GS800S14)、SEQ ID NO:305第34-948位氨基酸残基序列(GS800S203)和SEQ ID NO:309第1-687位氨基酸残基序列(GS900R9)，或为与本段所述任一氨基酸序列具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的氨基酸序列。
本发明示例性的融合蛋白选自SEQ ID NO:211-239、247-259和263-309中任一奇数编号的序列所示的融合蛋白，或为与这些融合蛋白中的任意一种融合蛋白具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的融合蛋白。
本发明类明胶蛋白(GS)在与生物活性蛋白或多肽融合后，能够增强生物活性蛋白或多肽的药代动力学性质，融合该类明胶蛋白(GS)的生物活性蛋白或多肽半衰期可以至少延长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类明胶蛋白(GS)的生物活性蛋白相比较来确定。能够起到延长体内半衰期的作用，其本质是由于类明胶蛋白(GS)的水合半径极大，与相同分子量的常规球形蛋白相比，类明胶蛋白(GS)由于充分伸展，尺寸能到达纳米级别。在本发明的一个实施例中(结果如图1和2所示)，分子量约为140KD的GS100R9-hArg1的表观分子量介于669KD与440KD之间；分子量约为170KD的GS100R9-hArg1-GS100R9及GS100R35-hArg1-GS100R35的表观分子量已经大于669KD，而分子量约为220KD的GS200R9-hArg1-GS200R9更是远远大于669KD。
人精氨酸酶1(hArg1)是一种天然的三聚体结构，单体分子量约为35KD，三聚体分子量为105KD左右，虽然该分子量已经远超过肾小球的滤过孔径，然而人精氨酸酶1在体内的半衰期却出奇地短，只有数分钟(P.N.Cheng,T.L.Lam,W.M.Lam,S.M.Tsui,A.W.Cheng,W.H.Lo,et al.,Pegylated recombinant human arginase(rhArg-PEG5,000mw)inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion,Cancer Res.67(2007)309–317)。目前一般采用PEG化修饰来延长其体内半衰期。在小鼠体内施用PEG-hArg1后，半衰期能延长至63小时。在本发明的一个实施例中，融合了相似氨基酸长度(100个氨基酸左右)且序列不相同的类明胶蛋白(GS)后，hArg1 的半衰期显著延长。hArg1的C末端(S-shaped tail)参与三聚体的形成,而让人惊奇的是，在本发明中，在hArg1的C末端融合类明胶蛋白(GS)却毫不影响其形成三聚体。而且只在hArg1的一端融合类明胶蛋白(GS)或者在两端皆融合类明胶蛋白(GS)都可观察到明显的药代动力学性质的改善，而且在类明胶蛋白(GS)长度相同的情况下，两端皆融合类明胶蛋白(GS)时半衰期更长。
在本发明的另一个实施例中，采用了人生长激素(hGH)来验证类明胶蛋白(GS)改善药代动力学性质的功能；在本发明的另一个实施例中，采用了生长分化因子15(GDF15)来验证类明胶蛋白(GS)改善药代动力学性质的功能；在本发明的另一个实施例中，采用了能结合VEGF的人工设计锚定蛋白(Designed ankyrin repeat proteins)来验证类明胶蛋白(GS)改善药代动力学性质的功能；在本发明的另一个实施例中，采用了GLP2G来验证类明胶蛋白(GS)改善药代动力学性质的功能。在这些实施例中，尽管采用的类明胶蛋白(GS)序列不同，但是这些类明胶蛋白(GS)在延长体内半衰期的作用都是类似的，而且效果都与长度成正比。与同样长度的GLK相比，类明胶蛋白(GS)延长半衰期的效果更显著。
此外，融合了类明胶蛋白(GS)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，融合了类明胶蛋白(GS)的GH蛋白在85℃下的热稳定性明显高于未融合的GH。而且GH容易在制备过程中产生聚集，但是融合了类明胶蛋白(GS)后，在SEC-HPLC上并无观察到显著的聚集现象。另外，通过测定生物活性蛋白暴露于37℃下7天保持的完整性来判断其潜在的人体循环稳定性。如图13WB结果所示，GS融合蛋白在血清中高度稳定，而对照的rGLK116 4-Arg1蛋白已经呈现被降解的弥散状。
具有治疗活性的化学偶联物
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 PCTCN2019108430-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。在本公开中，具有治疗活性的蛋白可由若干不同的活性蛋白药物 (D)和类明胶蛋白(GS)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，例如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。
多核苷酸序列、核酸构建体和宿主细胞
本文包括编码本文提供的各种类明胶单元、类明胶蛋白以及融合蛋白的编码序列及其互补序列。示例性的类明胶单元的编码序列如SEQ ID NO:18-90中任一偶数编码的序列所示；示例性的类明胶蛋白的编码序列如SEQ ID NO:92-186中任一偶数编码的序列所示，或者如SEQ ID NO:212-240、248-260和264-310中任一偶数编号的序列所示的融合蛋白编码序列中所包含的类明胶蛋白的编码序列；示例性的融合蛋白的编码序列如SEQ ID NO:212-240、248-260和264-310中任一偶数编号的序列所示。可采用本领域常规的方法制备得到所述多核苷酸序列。例如，可先通过基因合成法获得小片段的类明胶蛋白单元(U)片段，再通过基因拼接法获得更大分子量的由该类明胶蛋白单元(U)重复拼接而成的类明胶蛋白(GS)。
本文还包括核酸构建体。核酸构建体是可以被引入靶细胞或组织中的人工构建的核酸区段。该核酸构建体含有本文所述的编码序列或其互补序列，以及与这些序列操作性连接的一个或多个调控序列。调控序列可以是合适的启动子序列。启动子序列通常与待表达氨基酸序列的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端相连，在选择的宿主细胞中有功能的任何终止子都可用于本文。
在某些实施方案中，所述核酸构建体是载体。具体而言，可将本文所述的编码序列，尤其是类明胶蛋白或融合蛋白克隆入许多类型的载体，这些类型的载体包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。载体可以是表达载体，或者是克隆载体。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、毕赤酵母的甲醇氧化酶启动子和其它一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。标记基因可用于提供用于选择转化的宿主细胞的表型性状，包括但不限于真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。当本文所述的多核苷酸 在高等真核细胞中表达时，如果在载体中插入增强子序列，则将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域一般技术人员清楚如何选择适当的载体、启动子、增强子和宿主细胞。可采用本领域技术人员熟知的方法构建含本文所述的多核苷酸序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本文还包括包含本文所述多核苷酸序列、其核酸构建体，和/或表达含有本文所述类明胶单元(尤其是本文所述类明胶蛋白)的氨基酸序列的宿主细胞。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；丝状真菌细胞、或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、丝状真菌、植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。
可采用常规的方法将本文的载体导入宿主细胞中，这些方法包括显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等。
药物组合物
本文提供含有本文所述融合蛋白的药物组合物。药物组合物中还可含有本领域周知的各种合适的药学上可接受的载体或赋形剂。药学上可接受的载体或赋形剂在所采用的剂量和浓度对接受者是无毒的，包括但不限于：缓冲剂，如乙酸盐、Tris、磷酸盐、柠檬酸盐和其它有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如氯化十八烷基二甲基苄基铵；氯己双铵；苯扎氯铵、苄索氯铵；酚、丁醇或苄醇；对羟基苯甲酸烃基酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；蛋白质，如血清清蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；张力调节剂，诸如海藻糖和氯化钠；糖类，诸如蔗糖、甘露醇、海藻糖或山梨醇；表面活性剂，如聚山梨醇酯；成盐抗衡离子，如钠；金属复合物(如Zn-蛋白质复合物)；和/或非离子表面活性剂，如
Figure PCTCN2019108430-appb-000003
Figure PCTCN2019108430-appb-000004
或聚乙二醇(PEG)。用于体内施用的药物配制剂一般是无菌的。这易于通过无菌滤膜过滤来实现。
可根据药物组合物的剂型选择合适的药学上可接受的载体或赋形剂。可根据药物组合物的不同用途将其制备成不同的剂型。例如，可将本发明的药物组合物制备成片剂、注射剂、冻干剂等常用剂型。
药物组合物中通常含有治疗或预防有效量的本文所述的融合蛋白。治疗有效量通常指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。预防有效量指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的，因此预防有效量将低于治疗有效量。
方法和用途
本发明的氨基酸序列可以是化学合成的产物，或是使用重组技术从原核或真核宿主(例如，细菌、酵母、丝状真菌、高等植物、昆虫和哺乳动物细胞)中产生的重组多肽。根据重组生产方案所用的宿主，本发明的活性蛋白或多肽部分可以是糖基化的，或可以是非糖基化的。
因此，在某些实施方案中，本文提供一种蛋白治疗药物的制备方法，包括如下步骤：
1)培养含有本文所述融合蛋白的编码序列或其表达载体的宿主细胞，使之表达所述融合蛋白；
2)收集含有所述融合蛋白的培养物；和
3)从所述培养物中分离出所述融合蛋白。
细胞培养方法可根据不同细胞类型加以确定，为本领域常规的培养方法。
在某些实施方案中，本文还提供一种疾病治疗或预防方法，该方法包括给予需要的对象治疗有效量或预防有效量的本文所述的融合蛋白或其药物组合物。所治疗的疾病与该融合蛋白中的生物活性蛋白的生物学活性或功能相关。例如，生长激素(GH)能促进骨骼、内脏和全身生长，促进蛋白质合成，影响脂肪和矿物质代谢，可用于内源性垂体生长激素分泌不足而引起的生长障碍、躯体矮小的侏儒症、矮小病患儿，还可用于治疗烧伤、骨折、创伤、出血性溃疡、肌肉萎缩症、骨质疏松等疾病。因此，当融合蛋白中的活性蛋白药物为GH时，该融合蛋白可用于治疗内源性垂体生长激素分泌不足而引起的生长障碍、躯体矮小的侏儒症、矮小病患儿，还可用于治疗烧伤、骨折、创伤、出血性溃疡、肌肉萎缩症、骨质疏松等疾病。又例如，IL-2对机体的免疫应答和抗病毒感染具有重要作用，临床上用作免疫增强剂，主要用于肾癌、黑色素瘤和非何杰金淋巴瘤。因此，当治疗肾癌、黑色素瘤或非何杰金淋巴瘤患者时，可给予含有IL-2作为活性蛋白药物的本文融合蛋白。又例如，GDF15可用于治疗过度肥胖和体重不足相关疾病。因此，可给予需要的对象含GDF15的本文融合蛋白。
因此，取决于所含有的生物活性蛋白的生物学活性或功能，本文所述的融合蛋白可用于治疗或预防：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。更具体而言，所述疾病包括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(如肺性高血压)、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、肌营养不良症和低骨密度等。本文包括治疗或预防上述任意一种疾病的方法，包括给予治疗有效量或预防有效量的并含有治疗或预防该疾病的活性蛋白药物的本文所述的融合蛋白。
本文还提供一种增强生物活性蛋白(尤其是活性蛋白药物)的药代动力学性质的方法，所述方法包括在该生物活性蛋白的C端和/或N端融合本文所述的类明胶蛋白的步骤。本文中，药代动力学性质包括但不限于体内半衰期。在某些实施方案中，本文还提供一种提高生物活性蛋白(尤其是活性蛋白药物)理化性质的方法，所述方法包括在该生物活性蛋白的C端和/或N端融合本文所述的类明胶蛋白的步骤。本文中，理化性质包括但不限于水溶性、血清稳定性、耐酶稳定性和热稳定性中的任意一种、任意两种、任意三种或全部四种。上述方法中，生物活性蛋白可以是前文所述的任意一种或多种生物活性蛋白。融合或化学交联的方法为本领域所知。例如，可采用前文所述的融合蛋白的制备方法制备得到所述融合蛋白，从而增强生物活性蛋白的药代动力学性质和/或提高其理化性质。
因此，本文也提供本文所述的类明胶单元或类明胶蛋白在增强生物活性蛋白(尤其是活性蛋白药物)的药代动力学性质和或提高其理化性质中的应用；本文所述的类明胶单元或类明胶蛋白在制备药代动力学增强和/或理化性质提高的生物活性蛋白(尤其是活性蛋白药物)中的应用；本文所述的多核苷酸序列、核酸构建物、宿主细胞和/或融合蛋白在制备药物中的应用。还提供的是用于增强生物活性蛋白(尤其是活性蛋白药物)的药代动力学性质和或提高其理化性质的本文所述的类明胶单元或类明胶蛋白，用于治疗或预防的融合蛋白。
本文首次发现，由甘氨酸、脯氨酸、丙氨酸和谷氨酸组成的蛋白可作为载体蛋白，用于延长生物活性蛋白或多肽半衰期并改善其体内外性质。因此，甘氨酸、脯氨酸、丙氨酸和谷氨酸在制备能改善生物活性蛋白的生物学性质或功能(例如药代动力学和理化性质)的载体蛋白中的应用也包括在本文的范围之内。在某些实施方案中，本文还提供一种制备 能改善生物活性蛋白的生物学性质或功能的载体蛋白的方法。该方法可以是化学合成方法，也可以是生物重组方法。该载体蛋白的结构为G-X-Y三元重复结构，其中G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸和谷氨酸。更优选地，该载体蛋白为本文任一实施方案所述的类明胶蛋白。
所述化学合成方法可以是本领域周知的各种化学合成方法，包括按照所述载体蛋白的结构将选自甘氨酸、脯氨酸、丙氨酸和谷氨酸中的氨基酸残基依次连接到肽链上，形成具有G-X-Y三元重复结构的所述载体蛋白。化学合成方法通常包括固相合成法和液相合成法，其中固相合成法较常用。固相合成方法包括但不限于Fmoc和tBoc两种常用方法。通常，使用树脂作为不溶性的固相载体，通常从C端(羧基端)向N端(氨基端)逐个将氨基酸连接在肽链上，每个氨基酸连接循环由以下三步反应构成：1)脱保护：被保护的氨基酸必须用一种脱保护溶剂去除氨基的保护基团；2)活化：待连接的氨基酸的羧基被活化剂所活化；和3)偶联：活化的羧基与前一个氨基酸裸露的氨基反应，形成肽键。反复循环直到肽链延伸至所需长度时即可完成。最后用切割液切割肽链和固相载体之间的连接，就可获得所需的氨基酸序列。可以在程序控制的自动化多肽合成仪上进行上述化学合成，这类仪器包括但不限于Protein Technologies公司推出的Tribute双通道多肽合成仪、C S Bio公司的UV Online Monitor系统、Aapptec公司推出的Focus XC三通道合成仪等。
生物重组方法包括按照载体蛋白的氨基酸序列制备编码该氨基酸序列的多核苷酸序列，使用该多核苷酸序列构建表达载体，使用该表达载体转化或转染宿主细胞，以及培养宿主细胞以使其表达产生所述载体蛋白。这可采用本领域熟知的技术手段实现。
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。本发明实施例使用的免疫学、生物化学、微生物学、细胞生物学、遗传学和重组DNA的常规技术可以参见如《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。
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纯化工具的原理及使用方法以及组合使用，已是本领域技术人员所熟知的技术。如下实施例中涉及的纯化过程为示例性的表明在表达宿主为甲醇酵母GS115及该特定发酵条件下的纯化方式。当发酵条件不同时，杂质的组分及含量也不相同，纯化条件亦应当相应稍作调整。由于是公知 技术，在此不再赘述。但是作为一个普遍的标准，目的蛋白的最终纯度应该超过95％(SDS-PAGE纯度及HPLC鉴定纯度)。
实施例1：类明胶蛋白单元(U)的获得
类明胶单元(U)主要由以下几种G-X-Y三元单体结构组成：GPP、GEE、GAA、GEA、GAE、GAP、GPA、GPE、GEP。不同的G-X-Y三元单体结构任意组合形成类明胶蛋白单元(U)。示例性的组合如表2所示。
表2
Figure PCTCN2019108430-appb-000005
Figure PCTCN2019108430-appb-000006
实施例2：高表达的低分子量重组类明胶蛋白(GS)的制备
低分子量重组类明胶蛋白(GS)的获取可通过如下步骤之其一实现：
(1)先设计一段由不同或相同类明胶蛋白单元(U)组成的类明胶蛋白(GS)蛋白序列；再将该类明胶蛋白(GS)的蛋白序列转为DNA序列，并通过基因合成法获得全长DNA。
(2)通过类明胶蛋白单元(U)的对应核苷酸序列拼接而成，如Marc W.T.Werten等(Marc W.T.Werten等，Secreted production of a custom-designed,highly hydrophilic gelatin in Pichia pastoris，Marc W.T.Werten等，Protein Engineering,Design and Selection,14：447–454，2001)所示，先通过基因合成法获得类明胶蛋白单元(U)的DNA序列，再通过基因拼接法获得更大分子量的由该类明胶蛋白单元(U)重复拼接而成的类明胶蛋白(GS)。示例性的GS序列如表3所示。
比如，一段含有U x、U y和U z的核苷酸序列(简称为U xyz-1)在合成时5'端加入酵母GS115的α-factor信号肽序列(带Xho I位点)，紧接着为内切酶DraIII的识别位点，3'端则带有Van91I及EcoRI识别位点，并连接在克隆载体pMD18-T(TaKaRa公司)上，构建成质粒pMD-U xyz-1。
为了获得U xyz-1的二聚体，首先将质粒pMD-U xyz-1用Van 91I/Dra III双酶切。1％琼脂 糖胶进行电泳，切胶回收U xyz-1片段。同时，pMD-U xyz-1质粒用Van91I单酶切。酶切的质粒如上割胶回收，溶于30μL的TE溶液。然后用碱性磷酸酶(Alkaline Phosphatase，BAP)处理。
去磷酸化处理后的pMD-U xyz-1与Van 91I/Dra III双酶切回收的U xyz-1片段用T4DNA连接酶按1：10摩尔比进行连接。连接产物转化E.coli DH5α感受态细胞。在转化平板上挑取单克隆至氨苄抗性的LB液体培养基培养，按常规方法提取质粒,用XhoI/EcoRI双酶切鉴定。酶切阳性克隆测序鉴定。此阳性克隆即为二聚体的pMD-U XYZ-2。将U XYZ-1或U XYZ-2片段连接于pMD-U xyz-2上，即可构建成含3个或4个U xyz-1片段的目的基因U xyz-3和U xyz-4。
同样地，一段含有U a、U b和U c的核苷酸序列(简称为U abc-1)与U xyz-1通过上述方法拼接而成U abcxyz-1，U abcxyz-1可再拼接成二聚体的U abcxyz-2或是与其他序列的类明胶蛋白单元(U)拼接，以此类推。
又或者如Martin Schlapschy等人报道(Martin Schlapschy等，Fusion of a recombinant antibody fragment with a homo-amino-acid polymer:effects on biophysical properties and prolonged plasma half-life，Protein Engineering,Design&Selection，20：273–284,2007)将类明胶蛋白单元(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，这些参与拼接的类明胶蛋白单元(U)可以是序列相同的，也可以是不同的。为了纯化方便，在类明胶蛋白(GS)的N末端或C末端加入6His亲和纯化标签。
类明胶蛋白(GS)在N端融合6His标签，核苷酸片段亚克隆至质粒pPIC9(Life Technologies公司)，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。培养液上清加5×加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。作为经典的理论指南，具体的细节步骤可参见Life Technologies公司的产品手册《Pichia Expression Kit，For Expression of Recombinant Proteins in Pichia pastoris，Catalog no.K1710-01》。值得注意的是，类明胶蛋白(GS)在常规的考马斯亮蓝染色条件下染色效率较低，采用负染方式，比如铜染(Chris Lee等，Analytical Biochemistry 166：308-312，1987)效率更高。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、将胶浸入0.3M CuCl 2溶液中，染色2-5min；4、取出胶后，用成像仪拍照。
培养液上清8000rpm离心去沉淀，先用40％硫酸铵沉淀，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。50％洗脱液混合后，加入35％饱和度的硫酸铵沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。
表3示例性地列出了由类明胶单元(U)组成的低分子量类明胶蛋白(GS)及其相应序列。
表3：由类明胶单元(U)组成的低分子量类明胶蛋白(GS)
Figure PCTCN2019108430-appb-000007
实施例3：凝胶化及粘度测定
粘度以及在水溶液中对温度产生可逆转的凝胶化是天然明胶最重要的性质。当浓度大于0.5％的天然明胶水溶液冷却至约35-40℃时，其首先增加粘度，然后形成凝胶。凝胶的刚性或强度取决于明胶浓度、明胶的固有强度、pH值、温度和添加剂的存在(GELATIN HANDBOOK，GMIA，2012)。动物来源的明胶由胶原经酸或碱水解制备而成，分子量分布在20KD-25KD的一般属于低Bloom值、25-50KD的属于中等Bloom值、而50KD-100KD之间的则属于高Bloom值。在检测凝冻强度时，将实施例2中优选的高表达的低分子量类明胶蛋白(GS)制备成二聚体或者通过粘端拼接的方式，使其分子量达到40KD以上，再与天然动物来源的明胶进行比较。作为对比，还同时制备了含4-16种氨基酸组成的GLK。
本实施例参照国家标准《食品添加剂明胶》GB6783－94方法测定样品的凝冻强度及勃氏粘度。先将表4中的GS蛋白纯化样品配制成6.67％(W/W)的水溶液，然后采用冻力测试仪测定凝冻强度，ND-2勃氏粘度测定仪测定粘度。以动物明胶(48722-100G-F，Sigma)为对照。每次测定重复三次，结果如表4所示。
表4结果表明，各种长度和不同序列的GS都没有明显的天然动物明胶性质，而rGLK116 4则与天然明胶性质接近。
表4
Figure PCTCN2019108430-appb-000008
Figure PCTCN2019108430-appb-000009
实施例4：hArg1融合蛋白的表达
人精氨酸酶1(hArg1)是一种天然的三聚体结构，单体分子量约为35KD，三聚体分子量为105KD左右，虽然该分子量已经远超过肾小球的滤过孔径，然而人精氨酸酶1在体内的半衰期却出奇的短，只有数分钟(P.N.Cheng,T.L.Lam,W.M.Lam,S.M.Tsui,A.W.Cheng,W.H.Lo,et al.,Pegylated recombinant human arginase(rhArg-peg5,000mw)inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion,Cancer Res.67(2007)309–317)。目前一般采用PEG化修饰来延长其体内半衰 期。
将实施例2中经拼接后的GS片段和对照片段(N端引入6His纯化标签)与人精氨酸酶1(SEQ ID NO:7)融合(如表5所示)，核苷酸片段亚克隆至质粒pPIC9(Life Technologies公司)，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。培养液上清加5倍加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。作为经典的理论指南，具体的细节步骤可参见Life Technologies公司的产品手册《PichiaExpression Kit，For Expression of Recombinant Proteins in Pichia pastoris，Catalog no.K1710-01》。
表5：示例性的hArg1融合蛋白
融合蛋白代号 氨基酸序列、核苷酸序列(SEQ ID NO:)
GS100R9-hArg1 211、212
GS100R35-hArg1 213、214
GS100R52-hArg1 215、216
GS100R9-hArg1-GS100R9 217、218
GS100R35-hArg1-GS100R35 219、220
GS100R52-hArg1-GS100R52 221、222
GS100R74-hArg1-GS100R74 223、224
GS100R77-hArg1-GS100R77 225、226
GS100R98-hArg1-GS100R98 227、228
GS100R112-hArg1-GS100R112 229、230
GS200R9-hArg1 231、232
GS200R9-hArg1-GS200R9 233、234
GS400R9-hArg1-GS400R9 235、236
GS400R77-hArg1-GS400R77 237、238
GS500R9-hArg1 239、240
GLK RD-hArg1 241、242
rGLK116 4-hArg1 243、244
GEE151-hArg1 245、246
实施例5：hArg1融合蛋白的分离纯化
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甲醇酵母GS115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。
将2L发酵上清用30kDa滤膜超滤浓缩至250mL，浓缩液加入0.5M硫酸铵离心取上清，上清中再加入1.3M硫酸铵离心弃上清，沉淀用1M硫酸铵20mM PB pH6.0复溶，然后上用1M硫酸铵20mM PB pH 6.0缓冲液平衡后的5mL Phenyl Bestarose High Performance层析柱(博格隆(上海)生物技术有限公司)，再平衡后用0-100％20mM PB pH6.0缓冲液洗脱15BV(5mL/min，15min)。洗脱液加入CoCl 2至体系中浓度为50mM，于60℃活化10min，离心去除沉淀。上清用20mM NaAc-HAc pH6.0 Buffer进行G25脱盐，脱盐后样品pH6.0上1ml SuperQ-650M层析柱(Tosoh BioScience)浓缩0-100％B 20CV洗脱，平衡缓冲液：20mM NaAc-HAc(pH6.0)，洗脱缓冲液：0.5M NaCl+20mM NaAc-HAc(pH6.0)。
当样品经SDS-PAGE鉴定纯度不高于95％时，样品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,pH8.0)洗脱。
实施例6：G-CSF的融合蛋白制备
将实施例2中经拼接后的GS或GLK片段与G-CSF(SEQ ID NO:9)融合，N端与6His相连(如表6所示)。核苷酸片段亚克隆至质粒pPIC9(Life Technologies公司)，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。甲醇诱导过程如实施例2所示。培养液上清加5倍加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。作为经典的理论指南，具体的细节步骤可参见Life Technologies公司的产品手册《PichiaExpression Kit，For Expression of Recombinant Proteins in Pichia pastoris，Catalog no.K1710-01》。
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0-100％洗脱缓冲液(0.15M NaCl,0.5M咪唑,20mM Tris-HCl,pH 8.0)线性洗脱。50％洗脱液加入35％饱和度的硫酸铵沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。
表6：示例性的G-CSF融合蛋白
融合蛋白代号 氨基酸序列、核苷酸序列(SEQ ID NO:)
GS100R9-GCSF 247、248
GS100R35-GCSF 249、250
GS100R52-GCSF 251、252
GS100R74-GCSF 253、254
GS100R77-GCSF 255、256
GS100R98-GCSF 257、258
GS100R112-GCSF 259、260
rGLK116 4-GCSF 261、262
实施例7：SEC-HPLC分析GS-Arg1表观分子量
将GS与hArg1融合蛋白1mg/ml样品和分子量标准(gel filtration standard proteins，Agilent)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。分别采用Sepax SRT
Figure PCTCN2019108430-appb-000010
5μm(300×7.8mm)及Sepax SRT
Figure PCTCN2019108430-appb-000011
5μm(300×7.8mm)层析柱进行检测，方法如下：检测波长：214nm；柱温25℃，流动相：150mM PB(pH7.0)+5％异丙醇；流速1.0ml/min，运行时间：20分钟。
结果如图1和2所示。从Sepax SRT
Figure PCTCN2019108430-appb-000012
的结果看，GS100R9-hArg1的表观分子量介于669KD与440KD之间。从Sepax SRT
Figure PCTCN2019108430-appb-000013
结果来看，GS100R9-hArg1-GS100R9及GS100R35-hArg1-GS100R35的表观分子量已经大于669KD，而GS200R9-hArg1-GS200R9更是远远大于669KD。
实施例8：GS-Arg1体外精氨酸水解活性检测
将待测活GS与hArg1融合蛋白样品(GS-hArg1)和hArg1(R&D Systems,Cat:5868-AR)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15min，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)x样品浓度/稀释倍数/分子量(mmol/mL)]。酶的比活是指每毫克的蛋白质中所含的精氨酸酶的催化活力，比活＝1/MW*Kcat*60*1000。
实验结果如下表7所示，由于融合GS序列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7：GS–Arg1融合蛋白的精氨酸水解活性
融合蛋白代号 Kcat(s -1) 比活(IU/mg)
GS100R9-hArg1 177.9 240.0
GS100R35-hArg1 177.5 243.8
GS100R52-hArg1 174.3 239.4
GS100R9-hArg1-GS100R9 167.4 185.2
GS100R35-hArg1-GS100R35 169.4 192.9
GS100R52-hArg1-GS100R52 167.5 190.7
GS100R74-hArg1-GS100R74 165.4 187.8
GS100R77-hArg1-GS100R77 164.2 187.0
GS100R98-hArg1-GS100R98 160.9 183.2
GS100R112-hArg1-GS100R112 169.3 192.8
GS200R9-hArg1 158.6 176.3
GS200R9-hArg1-GS200R9 161.1 132.0
GS400R9-hArg1-GS400R9 163.9 88.4
GS400R77-hArg1-GS400R77 160.6 90.2
GS500R9-hArg1 160.5 90.2
GLK RD-hArg1 154.6 112.5
rGLK116 4-hArg1 165.5 132.1
GEE151-hArg1 170.0 202.3
hArg1 147.8 256.2
PBS / /
实施例9：GS与hArg1融合蛋白的免疫原性试验
SD大鼠随机均分组，每组10只，分别用表7中的蛋白免疫，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。给药前取血，最后一次免疫后两周，处死大鼠，取血，分离获得血清。用ELISA实验检测血清中GS抗体生成情况。具体地，GS-GCSF融合蛋白包被ELISA板(100ng/孔)，免疫动物血清稀释分别稀释100倍及500倍，与之在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。结果如下表8所示。GS-hArg1融合蛋白在用GS-hArg1融合蛋白包被时呈阳性，当用与hArg1无关蛋白包被(GS-GCSF融合蛋白)时则呈阴性，说明是hArg1在大鼠体内产生了较强的免疫原性而GS载体蛋白没有。而rGLK116 4-hArg1则始终呈阳性，说明rGLK116 4在大鼠体内产生了较强的免疫原性。
表8：GS与hArg1融合蛋白的免疫原性结果
Figure PCTCN2019108430-appb-000014
+：OD450值为给药前样品的2倍以上，为阳性；
－：OD450值为给药前样品的2倍以下，为阴性。
实施例10：不同GS-hArg1类似蛋白的药代检测试验
SD大鼠随机均分组，每组7只，分别皮下注射表7中融合蛋白，2mg/kg，GS-hArg1融合蛋白给药组在注射前和注射后3h,8h,12h,24h,36h,48h,72h,96h,120h,144h,168h取血，分离获得血清。hArg1蛋白(R&D Systems,Cat:5868-AR)给药组在注射前及注射后3h,8h取血。
用夹心ELISA方法检测融合蛋白在大鼠体内的药代情况。100ng/孔的hArg1兔多抗(自制)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(自制)，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小时后用常规TMB法检测，读取OD450值。结果如图3所示。表3中所示的类明胶蛋白(GS)的GRAVY值介于-1.0到0之间，而GLK116 4则为-1.815，有着较大差异。另外，GEE151-hArg1(SEQ ID NO:245)中的GEE151由甘氨酸G及谷氨酸E组成，具有极低的GRAVY值(-2.467)，然而其对于药代的效果并不理想，甚至还远不如rGLK116 4，而且制备极其困难。同样，GLK RD-hArg1(SEQ ID NO:241)中的GLK RD(GRAVY值为-0.785)由于不具备Gly-X-Y的结构，也没有起到与GS蛋白同样程度的效果。
实施例11：糖基化检测
蛋白样品通过高碘酸希夫碱(PAS)试剂法检测糖基含量：首先样品上样于10％SDS-PAGE，电泳结束后，采用Thermo Scientific糖蛋白染色试剂盒(货号24562，Lot PE201610B)进行糖基染色：将电泳后的丙烯酰胺凝胶完全浸入在100ml50％甲醇中30分钟以固定凝胶；再用100ml 3％乙酸，轻轻摇晃10分钟清洗凝胶；转移凝胶至25ml氧化溶液，轻轻摇晃15分钟后，通过用100ml 3％乙酸轻轻摇晃5分钟洗凝胶。重复此步骤两次。转移凝胶至25ml糖蛋白染色试剂(Thermo Scientific，货号24562，Lot PE201610B)，并轻轻摇晃15分钟。
转移凝胶至25ml还原性溶液，轻轻摇晃5分钟。用3％乙酸轻轻摇晃5分钟洗涤凝胶，然后用超纯水清洗。糖蛋白出现紫红色条带后，于3％乙酸中保存凝胶。
结果如图4所示。经过染色，只有阳性对照蛋白和rGLK116 4-hArg1被染色，而且rGLK116 4-hArg1条带呈弥散状，表明此样品含有各种不同修饰导致的分子量大小有异的目的蛋白，因此极不均一。
实施例12：GS与hGH融合蛋白的制备
人生长激素(hGH，SEQ ID NO:5)具有显著的聚集倾向，当单独进行重组表达时，往往会产生大量无法逆转的聚体。将实施例2中经拼接后的GS片段与hGH基因融合表达(如表9所示)，N端与6His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。甲醇诱导过程如实施例2所示。发酵液离心取上清后，加5x加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。作为经典的理论指南，具体的细节步骤可参见Life Technologies公司的产品手册《PichiaExpression Kit，For Expression of Recombinant Proteins in Pichia pastoris，Catalog no.K1710-01》。
表9：GS与hGH融合蛋白
Figure PCTCN2019108430-appb-000015
发酵液上清加入硫酸铵至电导180mS/cm，8000rpm，15min，10℃离心收集蛋白沉淀。沉淀用20mM PB pH7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溶液溶解，并用水稀释至电导4mS/cm以下。用Super Q-650M(TOSOH)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＝6，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)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，并稀释溶液至电导4mS/cm以下。
最后用Diamond SP Mustang(博格隆(上海)生物技术有限公司)进行纯化(缓冲液A：20mM NaAc，pH4；缓冲液B1:20mM NaAc，pH5；缓冲液B2:20mM PB，pH7)，用B1 和B2先后洗脱，收集B2洗脱样品。
当样品纯度低于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。
实施例13：GS-GH融合蛋白的热稳定性
用C18RP-HPLC定量实施例12制备得到的纯化后的GS-GH融合蛋白，调整浓度至约1.0mg/ml，分别置于室温及85℃下处理30分钟，离心去沉淀，取上清液进行SDS-PAGE。结果如图5所示。
实施例14：SEC-HPLC分析GS与hGH融合蛋白样品的聚集
将实施例12中经85℃加热处理的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：280nm；色谱柱：柱温25℃，Sepax SRT-1000 SEC 5μm(300×7.8mm)，流动相：50mM PB，150mM NaCl，pH7.2；运行时间：20分钟。当有明显聚体产生时，将会在SEC-HPLC图谱中出现高分子量的杂峰。结果如图6所示。hGH由于高温处理大量聚集沉淀，不进行液相分析。
实施例15：GS-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2x 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，结果如下表10和图7所示。
表10
融合蛋白代号 EC 50(nM)
GS800R9-GH-GS100R9 7.2
GS800R35-GH-GS100R35 8.2
GS800R127-GH-GS100R127 7.6
GS800L91-GH-GS100L91 6.1
GS800L102-GH-GS100L102 8.8
GS800L146-GH-GS100L146 7.5
GS800S203-GH-GS100S203 8.8
hGH 0.69
实施例16：不同GS-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GS-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GS-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，结果如下表11所示。
表11
融合蛋白代号 半衰期(t 1/2，小时) Cmax(μg/mL) AUC ∞(μg/mL*h)
GS800R9-GH-GS100R9 17.2 2.4 77.9
GS800R35-GH-GS100R35 16.8 2.4 76.5
GS800R127-GH-GS100R127 16.3 2.3 76.3
GS800L91-GH-GS100L91 16.5 2.5 78.5
GS800L102-GH-GS100L102 17.5 2.5 79.0
GS800L146-GH-GS100L146 16.4 2.3 75.8
GS800S203-GH-GS100S203 16.7 2.2 74.9
hGH 0.14 1.8 0.54
实施例17：GS与GDF 15融合蛋白的制备
将实施例2中经拼接后的GS片段与GDF 15(SEQ ID NO:15)融合表达(如表12所示)，N端与6His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。甲醇诱导过程如实施例2所示。发酵液离 心取上清后，加5x加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。作为经典的理论指南，具体的细节步骤可参见Life Technologies公司的产品手册《PichiaExpression Kit，For Expression of Recombinant Proteins in Pichia pastoris，Catalog no.K1710-01》。
表12
融合蛋白代号 氨基酸序列、核苷酸序列(SEQ ID NO:)
GS200R9-GDF15 277、278
GS200L23-GDF15 279、280
GS200L136-GDF15 281、282
GS200S14-GDF15 283、284
GS400R9-GDF15 285、286
GS400L23-GDF15 287、288
GS400L136-GDF15 289、290
GS400S14-GDF15 291、292
GS600R9-GDF15 293、294
GS600L23-GDF15 295、296
GS600L136-GDF15 297、298
GS600S14-GDF15 299、300
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35-50％饱和度的AS沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。
GS与GDF15融合蛋白的SDS-PAGE电泳图如图8所示。
实施例18：GS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。眼眶取血，将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化和进食变化。结果如图9和10所示。
实施例19：GS与GLP-2类似物融合蛋白的制备
将实施例2中经拼接后的GS片段与胰高血糖素样肽2类似物GLP-2G(SEQ ID NO:1)融合(如表13所示)，C端与6His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13
融合蛋白代号 氨基酸序列、核苷酸序列(SEQ ID NO:)
GLP2G-GS800R9 301、302
GLP2G-GS800S14 303、304
GLP2G-GS800S203 305、306
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。
实施例20：GS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(本实验保存)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，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3x 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14及图11所示。
表14
融合蛋白代号 EC 50(nM)
GLP2G-GS800R9 269.9
GLP2G-GS800S14 293.2
GLP2G-GS800S203 315.6
GLP-2 4.7
实施例21：GS与GLP2G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，结果如表15所示。
表15
融合蛋白代号 半衰期(t 1/2，小时) Cmax(μg/mL) AUC ∞(μg/mL*h)
GLP2G-GS800R9 41.5 7.1 550.2
GLP2G-GS800S14 42.8 7.0 545.7
GLP2G-GS800S203 45.2 7.3 589.4
实施例22：GS与AR VEGF融合蛋白的制备
将实施例2中经拼接后的GS片段与结合VEGF的锚定重复蛋白(Ankyrin repeat proteins，SEQ ID NO:3)融合(如表16所示)，C端与6His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。培养液上清加5倍加样缓冲液混匀，100℃加热8-10min。经SDS-PAGE电泳筛选表达菌株。发酵液先用80℃加热处理20分钟，离心沉淀杂蛋白后，上清用40％硫酸铵沉淀，用去离子水复溶并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35-50％饱和度的AS沉淀，8000rpm离心20min收集沉淀，用去离子水复溶。
表16
融合蛋白代号 氨基酸序列、核苷酸序列(SEQ ID NO:)
GS600R9-AR VEGF 307、308
GS900R9-AR VEGF 309、310
实施例23：GS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；采用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17概括了融合蛋白与对照药物Bevacizumab的Kd，从表中可以看出融合GS前后，AR VEGF对VEGF的平均亲和力无明显差异，与Bevacizumab(Medchemexpress，Cat.No.:HY-P9906)相比在同一个数量级。
表17：GS与AR VEGF融合蛋白的解离平衡常数(K d)
Figure PCTCN2019108430-appb-000016
实施例24：GS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AR VEGF的活性。ELISA板中加入5μg/mL的VEGF受体2/KDR(Abcam，ab155628)，每孔50μL，在37℃中放置2h。用1％BSA/TBS封闭，37℃放置2h。将AR VEGF和对照品Bevacizumab分别用PBST作3倍梯度稀释，将稀释好的样品80μL与等体积的1μg/mL的VEGF混合，37℃放置1h。将包被KDR的ELISA板洗板两次，拍干后，将梯度稀释后的混合物样品依次转移至该ELISA板中，37℃放置1h，然后洗板5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5次。再加入1：1000稀释的HRP标记的羊抗鼠二抗(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℃避光显色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18和图12所示。
表18：GS与AR VEGF融合蛋白的IC50
蛋白样品 IC50(nM)
GS600R9-AR VEGF 0.59
GS900R9-AR VEGF 0.59
AR VEGF 0.64
Bevacizumab 0.55
实施例25：血清稳定性
GS-GH融合蛋白样品用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37℃培养箱，取0天、及第7天样品进行Western-blot分析，采用HRP标记的Anti-6X His
Figure PCTCN2019108430-appb-000017
抗体 (Abcam,ab1187)作为检测抗体。结果如图13所示。
实施例26：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GS-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并煮沸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％SDS-PAGE，GS-GH融合蛋白取0％及0.5％胰酶处理组分别上样于8％SDS-PAGE。如图14所示，hGH在0.02％胰酶处理下已经几乎无完整蛋白，而GS-GH融合蛋白则几乎没有任何降解。
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. 一种类明胶单元，具有以下重复结构：
    (G-X-Y) n
    其中，G为甘氨酸，X和Y各自独立选自脯氨酸、丙氨酸和谷氨酸；n为5-20的整数，优选n为6-20或9-15的整数。
  2. 如权利要求1所述的类明胶单元，其特征在于，所述类明胶单元由选自以下的两种或两种以上的G-X-Y三元单体重复结构组成：GPP、GEE、GAA、GEA、GAE、GAP、GPA、GPE和GEP。
  3. 如权利要求1所述的类明胶单元，其特征在于，所述类明胶单元选自SEQ ID NO:17-89中任一奇数编号的序列所示的类明胶单元。
  4. 一种类明胶蛋白，其特征在于，所述类明胶蛋白的核心结构为：U 1-U 2或U 1-U 2-…U a，其中，U 1、U 2、…、U a各自代表权利要求1-3中任一项所述的任意一种类明胶单元，a为≥3的整数，且所述类明胶单元相同或不同。
  5. 如权利要求4所述的类明胶蛋白，其特征在于，所述核心结构的氨基酸残基总数占该类明胶蛋白氨基酸残基总数的70％以上，较佳80％以上，更佳85％以上，进一步更佳90％以上、95％以上或99％以上。
  6. 如权利要求4或5所述的类明胶蛋白，其特征在于，
    所述类明胶蛋白中，丙氨酸的含量大于等于10％；和/或
    所述类明胶单元中，代表亲水性的GRAVY值大于-1.1。
  7. 如权利要求6所述的类明胶蛋白，其特征在于，
    所述类明胶蛋白中，丙氨酸的含量在10-45％的范围内；和/或
    所述类明胶蛋白中，代表亲水性的GRAVY值小于等于0。
  8. 如权利要求4-7中任一项所述的类明胶蛋白，其特征在于，所述类明胶蛋白具有100-2000个氨基酸。
  9. 如权利要求4-8中任一项所述的类明胶蛋白，其特征在于，
    所述类明胶蛋白的凝冻强度≤10g；和/或
    所述类明胶蛋白的粘度≤3mPa·s。
  10. 如权利要求4所述的类明胶蛋白，其特征在于，所述类明胶蛋白的氨基酸序列选自：
    (1)SEQ ID NO:91-185中任一奇数编号所示的氨基酸序列；
    (2)含两条或两条以上(1)所述的氨基酸序列的氨基酸序列；和
    (3)与(1)或(2)所述的氨基酸序列具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的氨基酸序列。
  11. 一种融合蛋白，其特征在于，所述融合蛋白含有权利要求4-10中任一项所述的类明胶蛋白和生物活性蛋白。
  12. 如权利要求11所述的融合蛋白，其特征在于，所述生物活性蛋白选自：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、细胞运动因子、移动因子、静止因子、抗真菌因子、血浆黏附分子、间质黏附分子、细胞外基质、受体配基及其片段。
  13. 如权利要求11所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列选自与选自SEQ ID NO:211-239、247-259和263-309中任一奇数编号所示的氨基酸序列具有80％以上同一性百分比、优选85％以上同一性百分比、更优选90％以上同一性百分比、更优选95％以上同一性百分比的氨基酸序列。
  14. 一种多核苷酸序列，选自：
    (1)编码权利要求1-3中任一项所述的类明胶单元、权利要求4-10中任一项所述的类明胶蛋白或权利要求11-13中任一项所述的融合蛋白的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列。
  15. 一种核酸构建体，其包含权利要求14所述的多核苷酸序列；
    优选地，所述核酸构建体为克隆载体或表达载体。
  16. 一种宿主细胞，其特征在于，所述宿主细胞：
    (1)含有权利要求14所述的多核苷酸序列，和/或含有权利要求15所述的核酸构建体；和/或
    (2)表达权利要求1-3中任一项所述的类明胶单元、权利要求4-10中任一项所述的类明胶蛋白和/或权利要求11-13中任一项所述的融合蛋白。
  17. 选自以下的应用：
    (1)权利要求1-3中任一项所述的类明胶单元或其编码序列或该编码序列的互补序列在制备类明胶蛋白或含该类明胶蛋白的融合蛋白中的应用；
    (2)权利要求4-10中任一项所述的类明胶蛋白或其编码序列或该编码序列的互补序列在制备含有该类明胶蛋白的融合蛋白中的应用，或在提高生物活性蛋白的药代动力学和/或增强生物活性蛋白的理化性质中的应用；
    (3)权利要求11-13中任一项所述的融合蛋白、其编码序列或含该编码序列或其互补序列的核酸构建体在制备药物中的应用。
  18. 甘氨酸、脯氨酸、丙氨酸和谷氨酸在制备能改善生物活性蛋白的生物学性质或生物学功能的载体蛋白中的应用。
PCT/CN2019/108430 2018-10-12 2019-09-27 一种改善生物活性蛋白性质的载体蛋白 WO2020073825A1 (zh)

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CN113880954B (zh) * 2021-09-29 2024-05-14 江苏大学 一种重组人生长激素及其构建方法和应用
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CN101970678A (zh) * 2007-06-21 2011-02-09 慕尼黑科技大学 具有增加的体内和/或体外稳定性的生物学活性蛋白
CN103641896A (zh) * 2009-11-19 2014-03-19 浙江大学 明胶样单元的用途

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CN101970678A (zh) * 2007-06-21 2011-02-09 慕尼黑科技大学 具有增加的体内和/或体外稳定性的生物学活性蛋白
CN103641896A (zh) * 2009-11-19 2014-03-19 浙江大学 明胶样单元的用途

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