WO2021249443A1 - 一种甘精胰岛素衍生物及其制备方法和应用 - Google Patents

一种甘精胰岛素衍生物及其制备方法和应用 Download PDF

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WO2021249443A1
WO2021249443A1 PCT/CN2021/099243 CN2021099243W WO2021249443A1 WO 2021249443 A1 WO2021249443 A1 WO 2021249443A1 CN 2021099243 W CN2021099243 W CN 2021099243W WO 2021249443 A1 WO2021249443 A1 WO 2021249443A1
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insulin glargine
seq
fusion protein
protein
boc
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French (fr)
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陈卫
刘慧玲
骆莉
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宁波鲲鹏生物科技有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C07K2319/00—Fusion polypeptide
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of biotechnology, and more specifically to an insulin glargine derivative and its application.
  • Diabetes is a major disease threatening human health worldwide. In China, as the people’s lifestyle changes and the aging process accelerates, the prevalence of diabetes is showing a rapid upward trend.
  • the acute and chronic complications of diabetes, especially chronic complications accumulate multiple organs, causing disability and high fatality rate, which seriously affect the physical and mental health of patients, and bring a heavy burden to individuals, families and society.
  • Insulin Glargine achieves the purpose of long-term maintenance by changing the amino acids of recombinant human insulin Glargine and slightly adjusting the formula.
  • Insulin glargine replaces the asparagine at position 21 of the A chain of human insulin with neutral charge glycine to make the hexamer more stable.
  • Adding 2 arginines at the C-terminus of the B chain increases the isoelectric point from 5.4 to 6.7, making insulin glargine a transparent solution in a weak acid environment, and its solubility is greatly reduced under physiological conditions and precipitation occurs.
  • a small amount of zinc is added to the formula to form crystals under the skin during subcutaneous injection, thereby delaying the absorption time, and thus has a long-term effect of lowering blood sugar.
  • the preparation of insulin glargine generally uses genetic engineering technology to prepare precursors.
  • Sanofi US 5,663,291
  • E. coli E. coli for recombinant fermentation preparation, followed by Pichia pastoris fermentation expression (Biocon, IN2008CHE000310).
  • the classic preparation process of converting insulin glargine precursor to insulin glargine all adopts the transpeptidation process.
  • Sanofi (US 2009/0192073 A1) first prepares insulin glargine precursor B-Arg(B31)Arg(B32)-A -Gly(A21), insulin glargine and insulin glargine analogues B-Arg(B31)-A-Gly(A21) are obtained by trypsin digestion, insulin glargine analogues are converted to glargine after transpeptidation and deprotection insulin.
  • the process for preparing insulin glargine by this method is complicated, and the yield is low, resulting in extremely high production costs.
  • the purpose of the present invention is to provide an insulin glargine derivative and its application.
  • an insulin glargine fusion protein having the structure shown in formula I:
  • A is none or leader peptide
  • FP is the green fluorescent protein folding unit
  • TEV is a restriction site, preferably a TEV restriction site
  • R is arginine or lysine for digestion
  • G is the precursor of insulin glargine or its active fragment
  • the green fluorescent protein folding unit includes 2-6, preferably 2-3 ⁇ -sheet units selected from the following group:
  • the green fluorescent protein folding unit is u2-u3, u4-u5, u1-u2-u3, u3-u4-u5 or u4-u5-u6.
  • G is a Boc-modified insulin glargine precursor and has the structure shown in formula II:
  • GB is Boc-modified insulin glargine B chain, the amino acid sequence is shown in SEQ ID NO: 5, positions 1-32,
  • X is no or connecting peptide, preferably, the amino acid sequence of the connecting peptide is R, or as shown in SEQ ID NO: 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLA LEGSLQKR);
  • GA is the A chain of insulin glargine, and the amino acid sequence is shown in positions 33-53 of SEQ ID NO: 5.
  • the R is used for trypsin digestion.
  • G is Boc-modified insulin glargine whose sequence is shown in SEQ ID NO: 5.
  • X is none.
  • sequence of the insulin glargine fusion protein is as described in SEQ ID NO:1.
  • the 7th position of the insulin glargine B chain and the 7th position of the A chain (A7-B7), the 19th position of the B chain and the 20th position of the A chain (A20-B19) form an interchain Disulfide bond.
  • an intrachain disulfide bond is formed between the 6th position of the insulin glargine A chain and the 11th position (A6-A11) of the A chain.
  • a double-stranded insulin glargine fusion protein which has the structure shown in Formula III:
  • A is no or leader peptide, preferably a leader peptide whose sequence is shown in SEQ ID NO: 2,
  • FP is the green fluorescent protein folding unit
  • TEV is a restriction site, preferably a TEV restriction site (sequence is ENLYFQG, SEQ ID NO: 4);
  • R is arginine or lysine for digestion
  • D is Boc-modified double-stranded insulin glargine, and the main chain has the structure of the following formula IV;
  • GA is the A chain of insulin glargine, and the amino acid sequence is shown in SEQ ID NO: 5, 33-53,
  • X is the connecting peptide
  • GB is a Boc-modified insulin glargine B chain at position 29, and the amino acid sequence is shown in positions 1-32 of SEQ ID NO: 5;
  • the green fluorescent protein folding unit includes 2-6, preferably 2-3 ⁇ -sheet units selected from the following group:
  • the green fluorescent protein folding unit is u2-u3, u4-u5, u1-u2-u3, u3-u4-u5 or u4-u5-u6.
  • the C-terminus of the insulin glargine B chain is connected to the N-terminus of the insulin glargine A chain through a connecting peptide.
  • the X is a non-or connecting peptide, preferably, the amino acid sequence of the connecting peptide is R, or as shown in SEQ ID NO: 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR);
  • a Boc-modified insulin glargine precursor which has the structure shown in Formula II:
  • GB is a Boc-modified insulin glargine B chain at position 29, and the amino acid sequence is shown in SEQ ID NO: 5 at positions 1-32,
  • X is no or connecting peptide, preferably, the amino acid sequence of the connecting peptide is R, or as shown in SEQ ID NO: 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR);
  • GA is the A chain of insulin glargine, and the amino acid sequence is shown in positions 33-53 of SEQ ID NO: 5.
  • the protected lysine is N ⁇ -(tert-butoxycarbonyl)-lysine.
  • a Boc-modified insulin glargine which has the structure shown in formula IV:
  • GA is the A chain of insulin glargine, and the amino acid sequence is shown in SEQ ID NO: 5, 33-53,
  • GB is the B chain of insulin glargine, the amino acid sequence is shown in SEQ ID NO: 5, positions 1-32, and the 29th lysine of the B chain is N ⁇ -(tert-butoxycarbonyl)-lysine .
  • the purification treatment includes the steps:
  • step (ii) the temperature of the enzyme digestion is 15-25°C, preferably 18-22°C.
  • step (ii) succinic acid and L-lysine are added as an auxiliary agent for enzyme cleavage during the enzyme cleavage process.
  • the digestion system contains succinic acid, and the concentration of succinic acid is 10-50 mmol/L, preferably 15-30 mmol/L, More preferably, it is 30 mmol/L.
  • the enzyme digestion system contains L-lysine, and the concentration of L-lysine is 10-50 mmol/L, preferably 15- 30mmol/L, more preferably 30mmol/L.
  • the purity of the prepared insulin glargine is higher than 99%.
  • the prepared insulin glargine has insulin activity.
  • Boc-insulin glargine is insulin glargine with lysine protected at B29 (position 29 of the insulin B chain).
  • the protected lysine is a lysine with a protecting group.
  • the protected lysine is N ⁇ -(tert-butoxycarbonyl)-lysine.
  • step (i) recombinant bacteria are used to fermentatively produce insulin glargine fusion protein.
  • the recombinant bacteria contains or integrates an expression cassette for expressing insulin glargine fusion protein.
  • step (i) the insulin glargine fusion protein inclusion body is isolated from the fermentation broth of the recombinant bacteria.
  • step (i) it further includes the step of denaturing and renaturing the inclusion body, so as to obtain the insulin glargine fusion protein (the first protein) with correct protein folding.
  • the insulin glargine fusion protein with correct protein folding contains intrachain disulfide bonds between the insulin glargine A chain and the B chain.
  • the insulin glargine fusion protein is as described in the first aspect of the present invention.
  • the trypsin in step (ii), is recombinant porcine trypsin.
  • the mass ratio of the trypsin to the precursor of insulin glargine is 1:1000-40000, preferably 1:3000-10000.
  • step (ii) the digestion auxiliary agent contained in the digestion system can effectively increase the digestion yield.
  • step (ii) the time of the enzyme digestion is 10-25h, preferably 14-20h.
  • step (ii) the pH of the insulin glargine precursor solution is 7.5-9.0.
  • step (iii) hydrochloric acid is used for deprotection treatment.
  • step (iii) the temperature of the deprotection reaction is 25-40°C, preferably 36-38°C.
  • step (iii) the deprotection reaction time is 2-6h, preferably 4-5h.
  • the Boc-insulin glargine is N ⁇ -(tert-butoxycarbonyl)-lysine insulin glargine.
  • step (I) a weak cation exchange filler is used to perform cation chromatography.
  • step (I) a 40-60 mmol/L acetic acid counterion column is used.
  • step (I) the loading amount of insulin glargine (third protein) is ⁇ 12 mg/ml.
  • step (I) ammonium acetate containing isopropanol is used for linear gradient elution.
  • the step (I) also includes a desalination step.
  • the target protein is precipitated by the isoelectric point method.
  • the standing temperature is 2-8°C.
  • the standing time is 1 to 5 hours.
  • a microfiltration membrane with a pore size of 0.1-0.4 ⁇ m is selected for filtration.
  • microfiltration membrane after the microfiltration membrane is filtered, it is replaced with an ammonium acetate solution.
  • step (II) a sodium chloride solution containing isopropanol is used as the mobile phase.
  • step (II) the concentration of the sodium chloride solution is 0.1-0.5 mol/L.
  • step (II) a mobile phase is used for linear elution.
  • the loading amount of insulin glargine in the eluent I is less than or equal to 5 mg/ml, preferably, the loading amount is less than or equal to 4 mg/ml.
  • step (III) an acetonitrile solution containing sodium citrate is used as the mobile phase.
  • step (III) the concentration of sodium citrate in the mobile phase is 80-120 mmol/L, preferably 90-110 mmol/L.
  • the pH of the mobile phase is 4.0-4.5, preferably 4.1-4.2.
  • step (III) the mobile phase is used for gradient elution.
  • the loading amount of insulin glargine in the eluent II is less than or equal to 6 mg/ml, preferably, the loading amount is less than or equal to 5 mg/ml.
  • step (III) the step of precipitating and freeze-drying the prepared insulin glargine is further included, thereby preparing a freeze-dried product.
  • an insulin glargine preparation is provided, which is prepared using the method described in the fifth aspect of the present invention.
  • the purity of the insulin glargine contained in the insulin glargine preparation is higher than 99%.
  • the insulin glargine contained in the insulin glargine preparation has biological activity.
  • the seventh aspect of the present invention provides an isolated polynucleotide encoding the insulin glargine fusion protein of the first aspect of the present invention and the insulin glargine backbone of the second aspect of the present invention A fusion protein, the Boc-modified insulin glargine precursor of the third aspect of the present invention, or the Boc-modified insulin glargine backbone of the fourth aspect of the present invention.
  • the eighth aspect of the present invention provides a vector, which includes the polynucleotide according to the seventh aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the ninth aspect of the present invention provides a host cell containing the vector of the eighth aspect of the present invention, or the polynucleotide of the seventh aspect of the present invention integrated into the chromosome, Or express the insulin glargine fusion protein described in the first aspect of the present invention, the insulin glargine backbone fusion protein described in the second aspect of the present invention, the Boc modified insulin glargine precursor described in the third aspect of the present invention, or The Boc modified insulin glargine backbone described in the fourth aspect of the present invention.
  • the host cell is Escherichia coli, Bacillus subtilis, yeast cells, insect cells, mammalian cells or a combination thereof.
  • the tenth aspect of the present invention provides a preparation or a pharmaceutical composition comprising the insulin glargine fusion protein according to the first aspect of the present invention and the insulin glargine backbone fusion protein according to the second aspect of the present invention ,
  • the Boc modified insulin glargine precursor of the third aspect of the present invention, or the Boc modified insulin glargine backbone of the fourth aspect of the present invention and a pharmaceutically acceptable carrier.
  • Figure 1 shows the map of plasmid pBAD-FP-TEV-R-G.
  • Figure 2 shows the map of plasmid pEvol-pylRs-pylT.
  • Figure 3 shows the SDS-PAGE electrophoresis of insulin glargine after the first chromatography.
  • Figure 4 shows the HPLC detection profile of insulin glargine after the second chromatography.
  • Figure 5 shows the HPLC detection profile of insulin glargine after the third chromatography.
  • the present invention provides a fusion protein comprising a green fluorescent protein folding unit and an insulin glargine precursor or an active fragment thereof.
  • the expression level of the fusion protein of the present invention is significantly improved, and the insulin glargine protein in the fusion protein is folded correctly and has biological activity.
  • the green fluorescent protein folding unit in the fusion protein of the present invention can be digested into small fragments by protease, which has a large molecular weight difference compared with the target protein and is easy to separate.
  • the invention also provides a method for preparing insulin glargine and preparing intermediates by using the fusion protein.
  • Insulin products are the largest drug product in the diabetes market, occupying about 53% of the market share, of which third-generation recombinant insulin is the main product.
  • Insulin glargine belongs to the third generation of recombinant insulin. It is a long-acting insulin without obvious peaks and the risks of hypoglycemia and sudden death caused by it. Due to its safety and long-acting characteristics, insulin glargine Lantus has continued to become The product with the largest market share in the insulin market accounts for more than 30% of the overall insulin market.
  • Insulin glargine achieves the purpose of maintaining a long time by changing the amino acids of recombinant human insulin and slightly adjusting the formula.
  • Insulin glargine replaces the asparagine at position 21 of the A chain of human insulin glargine with neutral charge glycine to make the hexamer more stable.
  • Two arginines are added to the C-terminal of the B chain to increase the isoelectric point from 5.4 to 6.7.
  • Insulin glargine is a transparent solution in a weak acid environment, and its solubility is greatly reduced under physiological conditions and precipitation occurs.
  • a small amount of zinc is added to the formula to form crystals under the skin during subcutaneous injection, thereby delaying the absorption time, and thus has a long-term effect of lowering blood sugar.
  • the present invention constructs two fusion proteins, namely the single-chain insulin glargine fusion protein described in the first aspect of the present invention and the double-stranded insulin glargine fusion protein described in the third aspect of the present invention.
  • Double-chain insulin glargine fusion protein of chain insulin glargine may partially overlap.
  • the C-terminus of the B chain can also be connected to the N-terminus of the A chain. It can be considered as a single chain containing intrachain disulfide bonds.
  • the green fluorescent protein folding unit FP contained in the fusion protein of the present invention contains 2-6, preferably 2-3 ⁇ -sheet units selected from the following group:
  • the green fluorescent protein folding unit FP can be selected from: u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3- u4-u5, u5-u6-u7, u8-u9-u10, u9-u10-u11, u3-u5-u7, u3-u4-u6, u4-u7-u10, u6-u8-u10, u1-u2- u3-u4, u2-u3-u4-u5, u3-u4-u3-u4, u3-u5-u7-u9, u5-u6-u7-u8, u1-u3-u7-u9, u2-u2-u7- u8, u7-u2- u5-u11, u3-u4-u7-u10, u1-I-u
  • the green fluorescent protein folding unit is u3-u4-u5.
  • the term "fusion protein” also includes variant forms having the above-mentioned activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminus and/or N-terminus One or several (usually within 3, preferably within 2, more preferably within 1) amino acid is missing. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention can be (i) one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fusion A polypeptide formed from this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence, or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of the present invention, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn
  • the present invention also provides analogs of the fusion protein of the present invention.
  • the difference between these analogs and the polypeptide of the present invention may be the difference in amino acid sequence, the difference in modification form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or to optimize their solubility.
  • polynucleotide encoding the fusion protein of the present invention may include the polynucleotide encoding the fusion protein of the present invention, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the fusion protein encoded by it.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%.
  • the fusion protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA end rapid amplification method
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the fusion protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as ginseng cells).
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell.
  • a suitable method such as temperature conversion or chemical induction
  • the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the method of the present invention does not need to use dilution, ultrafiltration, and other methods to remove the excess inorganic salts in the supernatant of the fermentation broth, and the obtained inclusion bodies have higher purity and less pigments, which reduces the separation of substances for subsequent purification. Reduce purification costs.
  • the cation chromatography in the method separates insulin glargine with a one-step yield of more than 80%.
  • B 29 Boc lysine Due to the protection of B 29 Boc lysine, B 29 lysine will not be recognized during trypsin digestion, and des (B 30 ) by-products will not be produced, which can increase the digestion yield and reduce glycerol Impurities of insulin analogues provide convenience for subsequent purification and separation.
  • Boc-insulin glargine is converted into insulin glargine, which does not need to be carried out in an organic system, reducing process steps, reducing environmental pollution and lowering costs.
  • the target protein is precipitated by the isoelectric point method, which can purify to a certain extent and remove part of the impurity protein.
  • the use of a filter column with a membrane pore size of 0.1-0.2 ⁇ m instead of an ultrafiltration membrane with a small pore size greatly shortens the filtration time.
  • the present invention adopts two-step ion exchange chromatography and one-step reversed phase chromatography for separation and purification, instead of conventional four-step chromatography, which reduces the production cycle, reduces the use of organic solvents, and saves costs.
  • the fusion protein of the present invention contains high proportion of insulin glargine (the fusion ratio is increased), the FP or A-FP in the fusion protein contains arginine and lysine, which can be digested into small fragments by protease, and the purpose Compared with the protein, the molecular weight has a big difference and it is easy to separate.
  • the construction method refers to the prior art in the field, and specifically can refer to the description of the example in Chinese Patent Application No. 201910210102.9.
  • the DNA fragment of the fusion protein FP-TEV-RG was cloned into the expression vector plasmid pBAD/His A (purchased from NTCC, kanamycin resistant) at the downstream NcoI-XhoI site of the araBAD promoter to obtain the plasmid pBAD-FP -TEV-RG.
  • the plasmid map is shown in Figure 1.
  • the constructed insulin glargine expression vector was transformed into an E. coli strain, and a recombinant strain expressing the insulin glargine precursor (namely the insulin glargine fusion protein) was obtained by screening.
  • the amino acid sequence of the precursor of insulin glargine is shown in SEQ ID NO:1, and the 73rd position (the 29th position of insulin glargine) of the precursor sequence is a Boc-protected lysine.
  • the structure of the aforementioned recombinant insulin glargine precursor is as follows:
  • A is the leader peptide sequence, the sequence is MVSKGEELFTGV (SEQ ID NO: 2)
  • FP is the green fluorescent protein folding unit, the sequence is
  • TEV is the restriction site of TEV, and the sequence is ENLYFQG (SEQ ID NO: 4);
  • R is arginine or lysine used for trypsin digestion
  • G insulin glargine modified by Boc at position 29, the sequence is
  • the seed culture medium is prepared, inoculated, and the secondary seed liquid is obtained after two-stage culture. After culturing for 20 hours, the OD600 reaches about 180. After the fermentation is completed, about 3L of fermentation broth is obtained, and about 150g/L of wet bacteria is obtained by centrifugation. After the fermentation broth is centrifuged, the crushing buffer is added, and the bacteria are broken twice with a high-pressure homogenizer. After centrifugation, Tween 80 and EDTA-2Na are added for washing, and then washed with water once, and the precipitate is collected by centrifugation to obtain inclusion bodies. Each liter of fermentation broth can finally obtain about 43g wet weight inclusion bodies.
  • Example 2 Dissolution and renaturation of inclusion bodies
  • the initial protein mixture contains a large amount of bacterial protein residues, and secondly contains the by-products produced during the digestion process and the hydrolyzed protein produced by deprotection. According to the difference of protein isoelectric point, cation exchange packing (CM) was selected for crude extraction of insulin glargine.
  • CM cation exchange packing
  • the purity of insulin glargine in the initial mixed solution of this step is about 40%, and the insulin glargine analogue lacking B 32 arginine is very similar in structure to insulin glargine, so it is difficult to remove. According to the difference in charge of the substance, high Resolution polymer cation chromatography technology purifies insulin glargine to remove some impurities.
  • the C8 reversed-phase chromatography column technology is used to finely purify the insulin glargine, which mainly removes the hydrolysate of the insulin glargine.
  • the insulin glargine solution obtained by the secondary chromatography is diluted more than 4 times with pure water and combined with the C8 reverse phase filler. Control the loading capacity of insulin glargine not to be higher than 5mg/mL, and perform gradient elution with acetonitrile solution containing 100mmol/L sodium citrate and pH 4.2, collect the elution peak of insulin glargine, and finally obtain the insulin glargine yield The rate is 77.3%, and the purity is 99.18%.
  • the HPLC detection result is shown in Figure 5.
  • Example 1 The method similar to Example 1 was used to construct and express the fusion protein expression strain, the difference is only that the amino acid sequence of the fusion protein used for expression is shown in SEQ ID NO: 10.
  • the above-mentioned fusion protein contains the B chain and A chain of insulin glargine, as well as the gIII signal peptide.
  • the expression level of the fusion protein of the present invention is significantly increased, and the insulin glargine protein in the fusion protein is folded correctly and has biological activity.

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Abstract

一种甘精胰岛素衍生物及其制备方法，包含绿色荧光蛋白折叠单元和甘精胰岛素前体或其活性片段的融合蛋白，以及利用该融合蛋白制备甘精胰岛素及制备中间体的方法。所述融合蛋白表达量显著提高，融合蛋白中的甘精胰岛素前体蛋白折叠正确，具有生物活性。并且，所述融合蛋白中的绿色荧光蛋白折叠单元可以被蛋白酶消化成小片段，和目的蛋白相比分子量差别大，容易分离。

Description

一种甘精胰岛素衍生物及其制备方法和应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种甘精胰岛素衍生物及其应用。
背景技术
糖尿病是全球范围内威胁人类健康的一大疾病。在中国，随着人民生活方式的改变和老龄化进程的加快，糖尿病的患病率呈快速上升趋势。糖尿病的急慢性并发症，尤其是慢性并发症累计多个器官，致残、致死率高，严重影响患者的身心健康，并给个人、家庭和社会带来沉重的负担。
甘精胰岛素通过改变重组人甘精胰岛素的氨基酸和略微调整配方以达到作用维持时间长的目的。甘精胰岛素是以电荷中性的甘氨酸取代人胰岛素A链21位置天冬酰胺，使六聚体更加稳定。在B链C端增加2个精氨酸，使等电点从5.4提高到6.7，使甘精胰岛素在弱酸环境下为透明溶液，在生理环境下溶解度大大降低出现沉淀。在配方中加入少量的锌，在皮下注射时使其能在皮下形成结晶，从而延缓吸收时间，从而起到长效降血糖的作用。
甘精胰岛素的制备一般采用基因工程技术制备前体，原研公司赛诺菲(US 5663291)采用大肠杆菌进行重组发酵制备，其次有采用毕赤酵母发酵表达(Biocon百奥康，IN2008CHE000310)。甘精胰岛素前体转变为甘精胰岛素的经典制备工艺均采用转肽工艺，比如赛诺菲(US 2009/0192073 A1)先制备甘精胰岛素前体B-Arg(B31)Arg(B32)-A-Gly(A21)，通过胰蛋白酶切获得甘精胰岛素与甘精胰岛素类似物B-Arg(B31)-A-Gly(A21)，甘精胰岛素类似物在转肽与脱保护后转换为甘精胰岛素。这种方法制备获得甘精胰岛素的工序复杂，收率较低，导致生产成本极高。
因此，本领域迫切需要开发工艺简单、环境友好、收率高的甘精胰岛素的制备及纯化方法。
发明内容
本发明的目的在于提供一种甘精胰岛素衍生物及其应用。
在本发明的第一方面，提供了一种甘精胰岛素融合蛋白，具有式I所示的结构：
A-FP-TEV-R-G    (I)
式中，
“-”代表肽键；
A为无或前导肽，
FP为绿色荧光蛋白折叠单元，
TEV为酶切位点，较佳地为TEV酶酶切位点；
R为用于酶切的精氨酸或赖氨酸；
G为甘精胰岛素前体或其活性片段；
其中，所述的绿色荧光蛋白折叠单元包含2-6个，较佳地2-3个选自下组的β-折叠单元：
β-折叠单元 氨基酸序列
u1 VPILVELDGDVNG(SEQ ID NO:11)
u2 HKFSVRGEGEGDAT(SEQ ID NO:12)
u3 KLTLKFICTT(SEQ ID NO:13)
u4 YVQERTISFKD(SEQ ID NO:14)
u5 TYKTRAEVKFEGD(SEQ ID NO:15)
u6 TLVNRIELKGIDF(SEQ ID NO:16)
u7 HNVYITADKQ(SEQ ID NO:17)
u8 GIKANFKIRHNVED(SEQ ID NO:18)
u9 VQLADHYQQNTPIG(SEQ ID NO:19)
u10 HYLSTQSVLSKD(SEQ ID NO:20)
u11 HMVLLEFVTAAGI(SEQ ID NO:21)。
在另一优选例中，其特征在于，所述的绿色荧光蛋白折叠单元为u2-u3、u4-u5、u1-u2-u3、u3-u4-u5或u4-u5-u6。
在另一优选例中，其特征在于，所述的G为Boc修饰的甘精胰岛素前体，具有式II所示的结构:
GB-X-GA   (II)
式中，
GB为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，
X为无或连接肽，较佳地，连接肽的氨基酸序列为R，或如SEQ ID NO:6-9所示(GSKR，AAKR，YPGDVKR或EAEDLQVGQVELGGGPGAGSLQPLA LEGSLQKR)；
GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示。
在另一优选例中，所述的R用于胰蛋白酶酶切。
在另一优选例中，G为序列如SEQ ID NO:5所示的Boc修饰的甘精胰岛素。
在另一优选例中，GB-X-GA之间存在链内二硫键。
在另一优选例中，X为无。
在另一优选例中，所述的甘精胰岛素融合蛋白的序列如SEQ ID NO:1所述。
在另一优选例中，所述甘精胰岛素B链第7位与A链第7位(A7-B7)、B链第19位与A链第20位(A20-B19)之间形成链间二硫键。
在另一优选例中，所述甘精胰岛素A链第6位与A链第11位(A6-A11)之间形成链内二硫键。
在本发明的第二方面，提供了双链甘精胰岛素融合蛋白，具有式III所示的结构：
A-FP-TEV-R-D    (III)
式中，
“-”代表肽键；
A为无或前导肽，较佳地为序列如SEQ ID NO:2所示的前导肽，
FP为绿色荧光蛋白折叠单元，
TEV为酶切位点，较佳地为TEV酶酶切位点(序列为ENLYFQG，SEQ ID NO:4)；
R为用于酶切的精氨酸或赖氨酸；
D为Boc修饰的双链甘精胰岛素，所述主链具有下式IV的结构；
Figure PCTCN2021099243-appb-000001
式中，
“║”代表二硫键；
GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示，
X为连接肽；
GB为第29位为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示；
其中，所述的绿色荧光蛋白折叠单元包含2-6个，较佳地2-3个选自下组的β-折叠单元：
β-折叠单元 氨基酸序列
u1 VPILVELDGDVNG(SEQ ID NO:11)
u2 HKFSVRGEGEGDAT(SEQ ID NO:12)
u3 KLTLKFICTT(SEQ ID NO:13)
u4 YVQERTISFKD(SEQ ID NO:14)
u5 TYKTRAEVKFEGD(SEQ ID NO:15)
u6 TLVNRIELKGIDF(SEQ ID NO:16)
u7 HNVYITADKQ(SEQ ID NO:17)
u8 GIKANFKIRHNVED(SEQ ID NO:18)
u9 VQLADHYQQNTPIG(SEQ ID NO:19)
u10 HYLSTQSVLSKD(SEQ ID NO:20)
u11 HMVLLEFVTAAGI(SEQ ID NO:21)。
在另一优选例中，所述的绿色荧光蛋白折叠单元为u2-u3、u4-u5、u1-u2-u3、u3-u4-u5或u4-u5-u6。
在另一优选例中，所述甘精胰岛素B链的C端通过连接肽与甘精胰岛素A链的N端相连。
在另一优选例中，所述的X为无或连接肽，较佳地，连接肽的氨基酸序列为R，或如SEQ ID NO:6-9所示(GSKR，AAKR，YPGDVKR或EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR)；
在本发明的第三方面，提供了一种Boc修饰的甘精胰岛素前体，具有式II所示的结构：
GB-X-GA    (II)
式中，
GB为第29位为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，
X为无或连接肽，较佳地，连接肽的氨基酸序列为R，或如SEQ ID NO:6-9所示(GSKR，AAKR，YPGDVKR或EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR)；
GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示。
在另一优选例中，所述的保护赖氨酸为Nε-(叔丁氧羰基)-赖氨酸。
在本发明的第四方面，提供了一种Boc修饰的甘精胰岛素，具有式IV所示的结构：
Figure PCTCN2021099243-appb-000002
式中，
“║”代表二硫键；
GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示，
GB为甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，且所述B链的第29位赖氨酸为Nε-(叔丁氧羰基)-赖氨酸。
在本发明的第五方面，提供了一种制备甘精胰岛素的方法，所述方法包括步骤：
(i)利用重组菌进行发酵，制备甘精胰岛素融合蛋白(第一蛋白)发酵液；
(ii)对所述甘精胰岛素融合蛋白(第一蛋白)进行酶切，从而获得含Boc修饰的甘精胰岛素(第二蛋白)的混合液I；
(iii)对所述的Boc修饰的甘精胰岛素(第二蛋白)进行脱保护处理，从而获得含脱保护的甘精胰岛素(第三蛋白)的混合液II；
(iv)对混合液II进行纯化处理，从而获得甘精胰岛素。
在另一优选例中，所述的纯化处理包括步骤：
(I)对所述的混合液II进行第一次阳离子层析，从而获得含甘精胰岛素(第三蛋白)的洗脱液I；
(II)对所述的洗脱液I进行第二次阳离子层析，从而获得含甘精胰岛素(第三蛋白)的洗脱液II；
(III)对所述的洗脱液II进行反相层析，从而获得甘精胰岛素。
在另一优选例中，在步骤(ii)中，所述酶切的温度为15-25℃，较佳地为18-22℃。
在另一优选例中，在步骤(ii)中，在所述的酶切过程中，加入丁二酸和L-赖氨酸作为酶切辅助剂。
在另一优选例中，在步骤(ii)中，所述酶切体系中包含丁二酸，并且所述 丁二酸的浓度为10-50mmol/L，较佳地为15-30mmol/L，更佳地为30mmol/L。
在另一优选例中，在步骤(ii)中，所述酶切体系中包含L-赖氨酸，并且所述L-赖氨酸的浓度为10-50mmol/L，较佳地为15-30mmol/L，更佳地为30mmol/L。
在另一优选例中，制得的甘精胰岛素纯度高于99％。
在另一优选例中，制得的甘精胰岛素具有胰岛素活性。
在另一优选例中，Boc-甘精胰岛素是在B29(胰岛素B链29位)为保护赖氨酸的甘精胰岛素。
在另一优选例中，所述的保护赖氨酸为带有保护基团的赖氨酸。
在另一优选例中，所述的保护赖氨酸为Nε-(叔丁氧羰基)-赖氨酸。
在另一优选例中，在步骤(i)中，利用重组菌进行甘精胰岛素融合蛋白的发酵生产。
在另一优选例中，所述的重组菌中包含或整合有表达甘精胰岛素融合蛋白的表达盒。
在另一优选例中，在步骤(i)中，从所述重组菌的发酵液中分离获得甘精胰岛素融合蛋白包涵体。
在另一优选例中，在步骤(i)中，还包括对所述包涵体进行变性和复性，从而获得蛋白折叠正确的甘精胰岛素融合蛋白(第一蛋白)的步骤。
在另一优选例中，所述的蛋白折叠正确的甘精胰岛素融合蛋白中的甘精胰岛素A链和B链间包含链内二硫键。
在另一优选例中，所述的甘精胰岛素融合蛋白如本发明第一方面所述。
在另一优选例中，在步骤(ii)中，所述的胰蛋白酶为重组的猪胰蛋白酶。
在另一优选例中，在步骤(ii)中，所述胰蛋白酶与甘精胰岛素前体的质量比为1:1000-40000，较佳地为1:3000-10000。
在另一优选例中，在步骤(ii)中，所述酶切体系中包含的酶切辅助剂可有效提高酶切收率。
在另一优选例中，在步骤(ii)中，所述酶切的时间为10-25h，较佳地为14-20h。
在另一优选例中，在步骤(ii)中，所述的甘精胰岛素前体溶液的pH为7.5-9.0。
在另一优选例中，在步骤(iii)中，利用盐酸进行脱保护处理。
在另一优选例中，在步骤(iii)中，所述脱保护反应的温度为25-40℃，较佳 地为36-38℃。
在另一优选例中，在步骤(iii)中，所述脱保护反应的时间为2-6h，较佳地为4-5h。
在另一优选例中，所述的Boc-甘精胰岛素是Nε-(叔丁氧羰基)-赖氨酸甘精胰岛素。
在另一优选例中，在步骤(I)中，利用弱阳离子交换填料进行阳离子层析。
在另一优选例中，在步骤(I)中，利用40-60mmol/L的乙酸平衡离子柱。
在另一优选例中，在步骤(I)中，甘精胰岛素(第三蛋白)的上样量≤12mg/ml。
在另一优选例中，在步骤(I)中，利用含异丙醇的乙酸铵进行线性梯度洗脱。
在另一优选例中，所述的步骤(I)还包括脱盐处理的步骤。
在另一优选例中，在脱盐处理中，利用等电点法沉淀目的蛋白。
在另一优选例中，在脱盐处理中，在搅拌条件下，加入1-3mmol/L乙酸锌溶液，随后滴加氢氧化钠调节pH至6.0～7.0，搅拌后静置。
在另一优选例中，所述静置的温度为2～8℃。
在另一优选例中，所述静置的时间为1～5h。
在另一优选例中，在所述静置后，选用孔径为0.1～0.4μm微滤膜进行过滤。
在另一优选例中，在微滤膜过滤后，利用乙酸铵溶液进行置换。
在另一优选例中，在步骤(II)中，以含异丙醇的氯化钠溶液作为流动相。
在另一优选例中，在步骤(II)中，所述氯化钠溶液的浓度为0.1-0.5mol/L。
在另一优选例中，在步骤(II)中，利用流动相进行线性洗脱。
在另一优选例中，在步骤(II)中，所述的洗脱液I中甘精胰岛素的上样量≤5mg/ml，较佳地，所述的上样量≤4mg/ml。
在另一优选例中，在步骤(III)中，以含柠檬酸钠的乙腈溶液作为流动相。
在另一优选例中，在步骤(III)中，所述流动相中柠檬酸钠的浓度为80-120mmol/L，较佳地为90-110mmol/L。在另一优选例中，在步骤(III)中，所述的流动相的pH为4.0-4.5，较佳地为4.1-4.2。
在另一优选例中，在步骤(III)中，利用流动相进行梯度洗脱。
在另一优选例中，在步骤(III)中，所述的洗脱液II中甘精胰岛素的上样量≤6mg/ml，较佳地，所述的上样量≤5mg/ml。
在另一优选例中，在步骤(III)之后，还包括对制得的甘精胰岛素进行沉淀和冻干的步骤，从而制得冻干产品。
在本发明的第六方面，提供了一种甘精胰岛素制剂，所述的甘精胰岛素制剂使用本发明第五方面所述的方法制备的。
在另一优选例中，所述的甘精胰岛素制剂中包含的甘精胰岛素纯度高于99％。
在另一优选例中，所述的甘精胰岛素制剂中包含的甘精胰岛素具有生物活性。
本发明的第七方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的甘精胰岛素融合蛋白、本发明第二方面所述的甘精胰岛素主链融合蛋白、本发明第三方面所述的Boc修饰的甘精胰岛素前体、或本发明第四方面所述的Boc修饰的甘精胰岛素主链。
本发明的第八方面，提供了一种载体，所述载体包括本发明第七方面所述的多核苷酸。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、或其组合。
本发明的第九方面，提供了一种宿主细胞，所述的宿主细胞含有本发明第八方面所述的载体、或染色体中整合有外源的本发明第七方面所述的多核苷酸、或表达本发明第一方面所述的甘精胰岛素融合蛋白、本发明第二方面所述的甘精胰岛素主链融合蛋白、本发明第三方面所述的Boc修饰的甘精胰岛素前体、或本发明第四方面所述的Boc修饰的甘精胰岛素主链。
在另一优选例中，所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、酵母细胞、昆虫细胞、哺乳动物细胞或其组合。
本发明的第十方面，提供了一种制剂或药物组合物，所述制剂包含本发明第一方面所述的甘精胰岛素融合蛋白、本发明第二方面所述的甘精胰岛素主链融合蛋白、本发明第三方面所述的Boc修饰的甘精胰岛素前体、或本发明第四方面所述的Boc修饰的甘精胰岛素主链，以及药学上可接受的载体。
应理解，在本发明范围内中，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，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了质粒pBAD-FP-TEV-R-G图谱。
图2显示了质粒pEvol-pylRs-pylT图谱。
图3显示了第一次层析后甘精胰岛素的SDS-PAGE电泳图。
图4显示了第二次层析后甘精胰岛素的HPLC检测图谱。
图5显示了第三次层析后甘精胰岛素的HPLC检测图谱。
具体实施方式
本发明人经过广泛而深入地研究，发现了一种甘精胰岛素衍生物及其制备方法。具体地，本发明提供了包含绿色荧光蛋白折叠单元和甘精胰岛素前体或其活性片段的融合蛋白。本发明的融合蛋白表达量显著提高，融合蛋白中的甘精胰岛素蛋白折叠正确，具有生物活性。并且，本发明融合蛋白中的绿色荧光蛋白折叠单元可以被蛋白酶消化成小片段，和目的蛋白相比分子量差别大，容易分离。本发明还提供了利用该融合蛋白制备甘精胰岛素及制备中间体的方法。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
甘精胰岛素
胰岛素类产品是糖尿病市场第一大用药品种，占据53％左右市场份额，其中又以三代重组胰岛素为主。甘精胰岛素属于第三代重组胰岛素，是一种长效胰岛素，没有明显峰值及其引发的低血糖、猝死等风险，由于其安全性、长效性的特点，甘精胰岛素Lantus持续数年成为胰岛素市场中市场份额最大的产品，占整体胰岛素市场的30％以上。
甘精胰岛素通过改变重组人胰岛素的氨基酸和略微调整配方以达到作用维持时间长的目的。甘精胰岛素是以电荷中性的甘氨酸取代人甘精胰岛素A链21位置天冬酰胺，使六聚体更加稳定。在B链C端增加2个精氨酸，使等电点从5.4提高到6.7，甘精胰岛素在弱酸环境下为透明溶液，在生理环境下溶解度大大降低出现沉淀。在配方中加入少量的锌，在皮下注射时使其能在皮下形成结晶，从而延缓吸收时间，从而起到长效降血糖的作用。
融合蛋白
利用绿色荧光蛋白折叠单元，本发明构建了两种融合蛋白，即本发明第一方面所述的包含单链甘精胰岛素前体的甘精胰岛素融合蛋白和本发明第三方面所述的包含双链甘精胰岛素的双链甘精胰岛素融合蛋白。事实上，本发明的两种融合蛋白的保护范围可能部分重叠，例如融合蛋白中包含的双链形式的甘精胰岛素，其B链的C端也可以连接肽与A链的N端相连，也可以被认定为包含链内二硫键的单链。
本发明的融合蛋白中包含的绿色荧光蛋白折叠单元FP包含2-6个，较佳地2-3个选自下组的β-折叠单元：
β-折叠单元 氨基酸序列
u1 VPILVELDGDVNG(SEQ ID NO:11)
u2 HKFSVRGEGEGDAT(SEQ ID NO:12)
u3 KLTLKFICTT(SEQ ID NO:13)
u4 YVQERTISFKD(SEQ ID NO:14)
u5 TYKTRAEVKFEGD(SEQ ID NO:15)
u6 TLVNRIELKGIDF(SEQ ID NO:16)
u7 HNVYITADKQ(SEQ ID NO:17)
u8 GIKANFKIRHNVED(SEQ ID NO:18)
u9 VQLADHYQQNTPIG(SEQ ID NO:19)
u10 HYLSTQSVLSKD(SEQ ID NO:20)
u11 HMVLLEFVTAAGI(SEQ ID NO:21)。
在另一优选例中，所述的绿色荧光蛋白折叠单元FP可以选自：u8、u9、u2-u3、u4-u5、u8-u9、u1-u2-u3、u2-u3-u4、u3-u4-u5、u5-u6-u7、u8-u9-u10、u9-u10-u11、u3-u5-u7、u3-u4-u6、u4-u7-u10、u6-u8-u10、u1-u2-u3-u4、u2-u3-u4-u5、u3-u4-u3-u4、u3-u5-u7-u9、u5-u6-u7-u8、u1-u3-u7-u9、u2-u2-u7-u8、u7-u2- u5-u11、u3-u4-u7-u10、u1-I-u2、u1-I-u5、u2-I-u4、u3-I-u8、u5-I-u6、或u10-I-u11。
在另一优选例中，所述的绿色荧光蛋白折叠单元为u3-u4-u5。
如本文所用，术语“融合蛋白”还包括具有上述活性的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与本发明的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与本发明的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
此外，还可以对本发明融合蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
术语“编码本发明融合蛋白的多核苷酸”可以是包括编码本发明融合蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或融合蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实 质上改变其编码的融合蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的融合蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发 明融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA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。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。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、反转录病毒的LTRs和其他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。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如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。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
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；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、植物细胞(如人参细胞)。
本发明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时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。可举的例子包括在复制起始点晚 期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点包括：
(1)本发明的方法不需要对发酵液上清中过量的无机盐采用稀释、超滤换液等方法去除，获得的包涵体纯度较高，并且色素比较少，为后续纯化减少分离物质，降低纯化成本。且本方法中的阳离子层析分离甘精胰岛素，一步收率达80％以上。
(2)由于B 29位Boc赖氨酸的保护，胰蛋白酶酶切时不会识别B 29位赖氨酸，不产生des(B 30)的副产物，能够提升酶切收率，并且减少甘精胰岛素类似物杂质，为后续纯化分离提供方便。
(3)在酶切过程中，通过优化胰蛋白酶比例、控制酶切温度及添加酶切辅助剂，从而提高酶切收率。
(4)脱保护步骤中Boc-甘精胰岛素转变为甘精胰岛素，无需在有机体系下 进行，减少工艺步骤，环境污染小，成本更低。
(5)脱盐处理步骤通过等电点法沉淀目的蛋白，在一定程度上有提纯作用，去除部分杂蛋白。另外，使用0.1-0.2μm膜孔径的过滤柱代替小孔径的超滤膜，大大缩短了过滤时间。
(6)本发明共采用两步离子交换层析与一步反相层析进行分离纯化，替代常规的四步层析，减少了生产周期，并减少有机溶剂的使用，节约成本。
(7)本发明的融合蛋白中包含甘精胰岛素的比重高(融合比增加)，融合蛋白中的FP或A-FP包含精氨酸与赖氨酸，可以被蛋白酶消化成小片段，和目的蛋白相比分子量差别大，容易分离。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1 甘精胰岛素表达菌株的构建及表达
构建甘精胰岛素表达质粒，构建方法参照本领域的现有技术，具体地可以参照中国专利申请号201910210102.9中实施例的记载。将融合蛋白FP-TEV-R-G的DNA片段，克隆至表达载体质粒pBAD/His A(购自NTCC公司，卡那霉素抗性)的araBAD启动子下游NcoI-XhoI位点，得到质粒pBAD-FP-TEV-R-G。质粒图谱如图1所示。
再将pylRs的DNA序列，克隆至表达载体质粒pEvol-pBpF(购自NTCC公司，氯霉素抗性)的araBAD启动子下游SpeI-SalI位点，同时在proK启动子下游，以PCR方法插入赖氨酰-tRNA合成酶的tRNA(pylTcua)的DNA序列。该质粒命名为pEvol-pylRs-pylT。质粒图谱如图2所示。
将构建的甘精胰岛素表达载体转化大肠杆菌菌株，筛选获得表达甘精胰岛素前体(即甘精胰岛素融合蛋白)的重组菌株。其中甘精胰岛素前体的氨基酸序列如SEQ ID NO:1所示，前体序列第73位(甘精胰岛素第29位)为Boc保护的赖氨酸。
Figure PCTCN2021099243-appb-000003
Figure PCTCN2021099243-appb-000004
前述的重组的甘精胰岛素前体的结构如下：
A-FP-TEV-R-G
式中，
“-”代表肽键；
A为前导肽序列，序列为MVSKGEELFTGV(SEQ ID NO:2)
FP为绿色荧光蛋白折叠单元，序列为
Figure PCTCN2021099243-appb-000005
TEV为TEV酶酶切位点，序列为ENLYFQG(SEQ ID NO:4)；
R为用于胰蛋白酶酶切的精氨酸或赖氨酸；
G为第29位被Boc修饰的甘精胰岛素，序列为
Figure PCTCN2021099243-appb-000006
配制种子培养基，接种，经过两级培养制得二级种子液，培养20h，OD600达到180左右，发酵结束，得到约3L发酵液，离心得到约150g/L的湿菌体。发酵液离心后，加入破碎缓冲液，利用高压均质机破菌两次，离心后加入吐温80和EDTA-2Na等洗涤，接着水洗一次，离心收集沉淀得到包涵体。每升发酵液最终可获得约43g湿重包涵体。
实施例2 包涵体的溶解和复性
向所得的包涵体中加入8mol/L脲溶解液，氢氧化钠调节pH至8.0-9.0，室温搅拌1-3h，控制蛋白浓度为10-20mg/mL，补加β-巯基乙醇至终浓度为10-20mmol/L，继续搅拌0.5～1.0h。
将包涵体溶解液滴加至含有0.2～1.0mmol/L L-胱氨酸、5～20mmol/L碳酸钠、5～20mmol/L甘氨酸、0.3～1.0mmol/L EDTA-2Na的复性缓冲液，5-10倍稀释复性，维持复性液pH 9.0-10.5，温度2-8℃，搅拌复性时间10～20h。
实施例3 融合蛋白酶切
选用10KD超滤膜浓缩复性液8～10倍。向复性液中加入稀盐酸调节pH至7.5-9.0。以Bradford法测定复性浓缩液的蛋白浓度，计算总蛋白量。搅拌下加入重组胰蛋白酶，重组胰蛋白酶与复性液总蛋白质量比为1:3000～1:10000，加入30mmol/L丁二酸或30mmol/L L-赖氨酸，酶切温度为15-25℃，酶切时间为14～20h。
酶切10h后，HPLC检测酶切液中Boc-甘精胰岛素含量，当连续两小时检测 的Boc-甘精胰岛素浓度相差小于3％，酶切完成。最终，酶切液中Boc-甘精胰岛素浓度为0.9～1.3mg/mL，酶切率为30％～40％。
实施例4 脱保护
酶切液中加入酶切液体积的7％～10％稀盐酸，25-40℃下反应4-5h脱除Boc保护基，加入氢氧化钠调节pH至3.0～3.5终止脱保护反应。脱保护收率约75％～80％，甘精胰岛素纯度约20％。
实施例5 第一次层析
初始蛋白混合液中含有大量的菌体蛋白残留，其次含有酶切过程中产生的酶切副产物与脱保护产生的水解蛋白。根据蛋白质等电点的差异，选用阳离子交换填料(CM)对甘精胰岛素进行粗提取。以50mmol/L乙酸，pH3.0-3.5的缓冲液平衡离子柱3-5个柱体积，使甘精胰岛素与阳离子填料结合，控制甘精胰岛素上样载量低于12mg/ml，上样结束后，以含有异丙醇的1mol/L乙酸铵线性梯度洗脱层析柱20个柱体积，收集洗脱的目的蛋白峰，收集洗脱峰，SDS蛋白凝胶电泳检测结果如图3。层析Ⅰ的甘精胰岛素收率为80～85％，纯度为30％左右，该步骤能去除大多数菌体蛋白与部分酶切副产物。
层析Ⅰ洗脱的收集液加入2mmol/L乙酸锌溶液，搅拌2～5min。在搅拌条件下滴加氢氧化钠调节pH至6.0～7.0，继续搅拌2～10min后静置，温度2～8℃，静置时间1～5h。选用孔径为0.1～0.4μm微滤膜浓缩样品至10倍以上，再以6倍体积的pH6.0～7.0乙酸铵溶液进行置换。
结果显示，甘精胰岛素收率高于95％，纯度提高40％左右。
实施例6 第二次层析
该步骤的初始混合溶液中甘精胰岛素纯度在40％左右，其中缺B 32精氨酸的甘精胰岛素类似物与甘精胰岛素结构极为相似，因此难去除，根据物质带电荷的差异，采用高分辨率聚合物阳离子层析技术对甘精胰岛素进行纯化，以除去部分杂质。
将甘精胰岛素蛋白溶液澄清后，加入醋酸调节pH至2.5-4.5。以75mmol/L甘氨酸，30％异丙醇，pH3.4的缓冲液平衡离子柱3-5个柱体积，甘精胰岛素蛋白溶液与阳离子填料结合，控制甘精胰岛素的上样载量不超过4mg/mL，以含有异丙醇的0.3mol/L氯化钠线性洗脱，收集甘精胰岛素样品。最终获得纯度高于97％的甘精胰岛素，收率达75.6％，其中缺B 32精氨酸的甘精胰岛素含量控制在0.5％以内，其HPLC检测结果见图4。
实施例7 第三次层析
根据物质疏水性的差异，采用C8反相层析柱技术对甘精胰岛素进行精细纯化，主要去除甘精胰岛素的水解产物。将二次层析获得的甘精胰岛素溶液以纯水稀释4倍以上，与C8反相填料结合。控制甘精胰岛素上样载量不高于5mg/mL，以含有100mmol/L柠檬酸钠，pH4.2的乙腈溶液进行梯度洗脱，收集甘精胰岛素的洗脱峰，最终获得甘精胰岛素收率达77.3％，纯度99.18％，其HPLC检测结果见图5。
实施例8 沉淀及冻干
向三次层析洗脱收集液中加入注射用水稀释至乙腈含量不超过15％(v/v)，加入乙酸锌至浓度为2mmol/L，利用氢氧化钠调节pH至6.8-7.1，在4-8℃静置沉淀。收集沉淀，以100倍以上注射用水洗涤沉淀并收集洗涤后的沉淀样品，经干燥获得甘精胰岛素。
对比例
采用实施例1类似的方法进行融合蛋白表达菌株的构建及表达，其区别仅在于用于表达的融合蛋白的氨基酸序列如SEQ ID NO:10所示。
Figure PCTCN2021099243-appb-000007
上述融合蛋白中，包含甘精胰岛素B链和A链，同时还包含gⅢ信号肽。
结果显示，培养20h，OD 600达到140左右，发酵结束，得到约3L发酵液，离心得到约105g/L的湿菌体。发酵液离心后，加入破碎缓冲液，利用高压均质机破菌两次，并离心收集沉淀得到包涵体。每升发酵液最终可获得约30g湿重包涵体。
上述结果表明，与常规结构融合蛋白的表达相比，本发明的融合蛋白表达量显著提高，并且融合蛋白中的甘精胰岛素蛋白折叠正确，具有生物活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种甘精胰岛素融合蛋白，其特征在于，具有式I所示的结构：
    A-FP-TEV-R-G  (I)
    式中，
    “-”代表肽键；
    A为无或前导肽，
    FP为绿色荧光蛋白折叠单元，
    TEV为酶切位点，较佳地为TEV酶酶切位点；
    R为用于酶切的精氨酸或赖氨酸；
    G为甘精胰岛素前体或其活性片段；
    其中，所述的绿色荧光蛋白折叠单元包含2-6个选自下组的β-折叠单元：
    β-折叠单元 氨基酸序列 u1 VPILVELDGDVNG(SEQ ID NO:11) u2 HKFSVRGEGEGDAT(SEQ ID NO:12) u3 KLTLKFICTT(SEQ ID NO:13) u4 YVQERTISFKD(SEQ ID NO:14) u5 TYKTRAEVKFEGD(SEQ ID NO:15) u6 TLVNRIELKGIDF(SEQ ID NO:16) u7 HNVYITADKQ(SEQ ID NO:17) u8 GIKANFKIRHNVED(SEQ ID NO:18) u9 VQLADHYQQNTPIG(SEQ ID NO:19) u10 HYLSTQSVLSKD(SEQ ID NO:20) u11 HMVLLEFVTAAGI(SEQ ID NO:21)。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述的绿色荧光蛋白折叠单元为u2-u3、u4-u5、u1-u2-u3、u3-u4-u5或u4-u5-u6。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述的G为Boc修饰的甘精胰岛素前体，具有式II所示的结构：
    GB-X-GA  (II)
    式中，
    GB为第29位为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，
    X为无或连接肽；
    GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示。
  4. 如权利要求1所述的融合蛋白，其特征在于，所述的甘精胰岛素融合蛋白的序列如SEQ ID NO:1所述。
  5. 一种双链甘精胰岛素融合蛋白，其特征在于，具有式III所示的结构：
    A-FP-TEV-R-D  (III)
    式中，
    “-”代表肽键；
    A为无或前导肽，
    FP为绿色荧光蛋白折叠单元，
    TEV为酶切位点，较佳地为TEV酶酶切位点；
    R为用于酶切的精氨酸或赖氨酸；
    D为Boc修饰的双链甘精胰岛素，具有下式IV的结构；
    Figure PCTCN2021099243-appb-100001
    式中，
    Figure PCTCN2021099243-appb-100002
    代表二硫键；
    GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示，
    X为连接肽；
    GB为第29位为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示；
    其中，所述的绿色荧光蛋白折叠单元包含2-6个选自下组的β-折叠单元：
    β-折叠单元 氨基酸序列 u1 VPILVELDGDVNG(SEQ ID NO:11) u2 HKFSVRGEGEGDAT(SEQ ID NO:12) u3 KLTLKFICTT(SEQ ID NO:13) u4 YVQERTISFKD(SEQ ID NO:14) u5 TYKTRAEVKFEGD(SEQ ID NO:15) u6 TLVNRIELKGIDF(SEQ ID NO:16) u7 HNVYITADKQ(SEQ ID NO:17) u8 GIKANFKIRHNVED(SEQ ID NO:18)
    u9 VQLADHYQQNTPIG(SEQ ID NO:19) u10 HYLSTQSVLSKD(SEQ ID NO:20) u11 HMVLLEFVTAAGI(SEQ ID NO:21)。
  6. 一种Boc修饰的甘精胰岛素前体，其特征在于，具有式II所示的结构：
    GB-X-GA  (II)
    式中，
    GB为第29位为Boc修饰的甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，
    X为无或连接肽，所述连接肽的氨基酸序列为R，或如SEQ ID NO:6-9所示；
    GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示。
  7. 一种Boc修饰的双链甘精胰岛素，其特征在于，具有式IV所示的结构：
    Figure PCTCN2021099243-appb-100003
    式中，
    Figure PCTCN2021099243-appb-100004
    代表二硫键；
    GA为甘精胰岛素A链，氨基酸序列如SEQ ID NO:5的第33-53位所示，
    GB为甘精胰岛素B链，氨基酸序列如SEQ ID NO:5的第1-32位所示，且所述B链的第29位赖氨酸为Nε-(叔丁氧羰基)-赖氨酸。
  8. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1所述的甘精胰岛素融合蛋白、权利要求5所述的双链甘精胰岛素融合蛋白、权利要求6所述的Boc修饰的甘精胰岛素前体、或权利要求7所述的Boc修饰的双链甘精胰岛素。
  9. 一种载体，其特征在于，所述载体包括权利要求8所述的多核苷酸。
  10. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求9的载体、或染色体中整合有外源的权利要求8的多核苷酸、或表达权利要求1所述的甘精胰岛素融合蛋白、权利要求5所述的双链甘精胰岛素融合蛋白、权利要求6所述的Boc修饰的甘精胰岛素前体、或权利要求7所述的Boc修饰的双链甘精胰岛素。
  11. 一种制备甘精胰岛素的方法，其特征在于，所述方法包括步骤：
    (i)利用重组菌进行发酵，制备包含权利要求1所述的甘精胰岛素融合蛋白的发酵液；
    (ii)对所述甘精胰岛素融合蛋白(第一蛋白)进行酶切，从而获得含Boc修饰的甘精胰岛素(第二蛋白)的混合液I；
    (iii)对所述的Boc修饰的甘精胰岛素(第二蛋白)进行脱保护处理，从而获得含脱保护的甘精胰岛素(第三蛋白)的混合液II；
    (iv)对混合液II进行纯化处理，从而获得甘精胰岛素。
  12. 如权利要求11所述的方法，其特征在于，所述的纯化处理包括步骤：
    (I)对所述的混合液II进行第一次阳离子层析，从而获得含甘精胰岛素(第三蛋白)的洗脱液I；
    (II)对所述的洗脱液I进行第二次阳离子层析，从而获得含甘精胰岛素(第三蛋白)的洗脱液II；
    (III)对所述的洗脱液II进行反相层析，从而获得甘精胰岛素。
  13. 如权利要求12所述的方法，其特征在于，在步骤(III)之后，还包括对制得的甘精胰岛素进行沉淀和冻干的步骤，从而制得冻干产品。
  14. 如权利要求11所述的方法，其特征在于，在步骤(i)中，从所述重组菌的发酵液中分离获得甘精胰岛素融合蛋白包涵体，随后对所述包涵体进行变性和复性，从而获得蛋白折叠正确的甘精胰岛素融合蛋白(第一蛋白)。
  15. 如权利要求11所述的方法，其特征在于，在步骤(ii)中，在所述的酶切过程中，加入丁二酸和L-赖氨酸作为酶切辅助剂。
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