A derivative of insulin aspart and a method for preparing and using the same

Abstract

A derivative of insulin aspart and a method for preparing the same, the derivative comprising a fusion protein of a green fluorescent protein folding unit and an insulin aspart precursor or an active fragment thereof. The fusion protein is expressed in a significantly increased amount, the insulin aspart precursor protein in the fusion protein is folded correctly and has biological activity. Furthermore, the green fluorescent protein folding unit in the fusion protein can be digested by a protease into small fragments, and has a large difference in molecular weight compared to the target protein, and is easy to separate. A method for preparing insulin aspart and an intermediate using the fusion protein is also provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly to a derivative of insulin aspart and application thereof. BACKGROUND

[0002] Diabetes mellitus is a major disease that threatens human health worldwide. In China, with the change of people's lifestyle and the acceleration of the aging process, the prevalence of diabetes is showing a rapid upward trend. The acute and chronic complications of diabetes, especially the chronic complications involving multiple organs, have high rates of disability and death, seriously affecting the physical and mental health of patients, and imposing a heavy burden on individuals, families and society.

[0003] Insulin aspart, as a rapid-acting insulin analogue, belongs to the third generation of insulin, which is bridged by two chains of half-amino acid disulfide bond. It is a genetically engineered DNA recombinant technology that replaces (mutates) proline (Pro) at position 28 of human insulin B chain with aspartic acid (Asp) with negative charge, which prevents self-aggregation of insulin monomers or dimers (B28 of one insulin molecule and B23 of another insulin molecule) by using the repulsive effect of charge, reducing intermolecular aggregation. It can well simulate the secretion pattern of human insulin, and its pharmacokinetic characteristics are about half of those of conventional human insulin, with an onset time of 10-20 minutes, a peak time of 40 minutes, and a duration of action shortened to 3-5 hours, which significantly reduces the incidence of nocturnal hypoglycemia.

[0004] The preparation of insulin aspart generally uses genetic engineering technology to prepare precursors. The original research company Novo Nordisk uses Saccharomyces cerevisiae as an expression host to produce insulin aspart precursors by recombinant DNA technology, and then prepares insulin aspart through a series of complex processes such as transpeptidation. This method has great difficulty in host strain modification and other technical aspects, and the process is complex. Because the expression ability of Saccharomyces cerevisiae is weak, the yield of insulin aspart is not high, and the highest yield of the engineering bacteria is 21.5 mg / L, which to some extent increases the cost of drug production.

[0005] Therefore, there is an urgent need in the art to develop a simple, environmentally friendly, high-yield and high-yield method for preparing and purifying insulin aspart. SUMMARY

[0006] The purpose of the present application is to provide a derivative of insulin aspart and application thereof.

[0007] In the first aspect of the present application, a derivative of insulin aspart is provided, which has the structure shown in formula I:

[0008] A-FP-TEV-R-G (I)

[0009] In the formula,

[0010] "-" represents a peptide bond;

[0011] A is nothing or a leader peptide,

[0012] FP is a green fluorescent protein folding unit,

[0013] TEV is an enzyme cleavage site, preferably a TEV enzyme cleavage site;

[0014] R is arginine or lysine for enzyme cleavage;

[0015] G is a Boc-modified proinsulin or an active fragment thereof;

[0016] The green fluorescent protein folding unit comprises 2-6, preferably 2-3, β-sheet units selected from the group consisting of:

[0017] Beta-sheet unit Amino acid sequence 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).

[0018] In another preferred embodiment, the green fluorescent protein folding unit is u8-u9, u9-u10-u11, or u10-u11.

[0019] In another preferred embodiment, the G is a Boc-modified proinsulin precursor having the structure shown in Formula II:

[0020] GB-X-GA (II)

[0021] In the formula,

[0022] GB is a Boc-modified proinsulin B chain, and the amino acid sequence is shown in SEQ ID NO: 5, 1-30,

[0023] X is a connecting peptide, preferably the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9 (RRGSKR, RRAAKR, RRYPGDVKR, or RREAEDLQVGQVELGGG PGAGSLQPLALEGSLQKR);

[0024] GA is a proinsulin A chain, and the amino acid sequence is shown in SEQ ID NO: 5, 32-52.

[0025] In another preferred embodiment, R is used for trypsin and carboxypeptidase enzyme cleavage.

[0026] In another preferred embodiment, G is a Boc-modified proinsulin with the sequence shown in SEQ ID NO: 5.

[0027] In another preferred embodiment, there is an intrachain disulfide bond between GB-X-GA.

[0028] In another preferred embodiment, the sequence of the aspart insulin fusion protein is as set forth in SEQ ID NO: 1, 22, 23.

[0029] In another preferred embodiment, there is an interchain disulfide bond between the 7th amino acid of the B chain and the 7th amino acid of the A chain (A7-B7), and the 19th amino acid of the B chain and the 20th amino acid of the A chain (A20-B19).

[0030] In another preferred embodiment, there is an intrachain disulfide bond between the 6th amino acid of the A chain and the 11th amino acid of the A chain (A6-A11).

[0031] In a second aspect of the present application, a double-chain aspart insulin fusion protein is provided, having a structure as set forth in Formula III:

[0032] A-FP-TEV-R-D (III)

[0033] In the formula,

[0034] "║" represents a disulfide bond;

[0035] A is a leader peptide, preferably a leader peptide having a sequence as set forth in SEQ ID NO: 2,

[0036] FP is a green fluorescent protein folding unit,

[0037] TEV is an enzyme cleavage site, preferably a TEV enzyme cleavage site (sequence ENLYFQG, SEQ ID NO: 4);

[0038] R is arginine or lysine for enzyme cleavage;

[0039] D is a Boc-modified double-chain aspart insulin, the main chain having a structure as set forth in Formula IV below;

[0040]

[0041] In the formula,

[0042] "║" represents a disulfide bond;

[0043] GA is an aspart insulin A chain, having an amino acid sequence as set forth in positions 32-52 of SEQ ID NO: 5,

[0044] X is a connecting peptide;

[0045] GB is an aspart insulin B chain having a Boc modification at the 29th amino acid, having an amino acid sequence as set forth in positions 1-30 of SEQ ID NO: 5;

[0046] The green fluorescent protein folding unit comprises 2-6, preferably 2-3, β-folding units selected from the following group:

[0047] Beta-sheet unit Amino acid sequence 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).

[0048] In another preferred embodiment, the green fluorescent protein folding unit is u8-u9, u9-u10-u11, or u10-u11.

[0049] In another preferred embodiment, the green fluorescent protein folding unit has an amino acid sequence as shown in SEQ ID NO: 3, 24, or 25.

[0050] In another preferred embodiment, the C-terminus of the B chain of aspart insulin is connected to the N-terminus of the A chain of aspart insulin via a connecting peptide.

[0051] In another preferred embodiment, X is a connecting peptide, preferably, the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9 (RRGSKR, RRAAKR, RRYPGDVKR, or RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR).

[0052] In a third aspect of the present application, a Boc-modified aspart insulin precursor is provided, having a structure as shown in formula II:

[0053] GB-X-GA (II)

[0054] In the formula,

[0055] GB is a Boc-modified B chain of aspart insulin at position 29, having an amino acid sequence as shown in SEQ ID NO: 5 at positions 1-30,

[0056] X is a connecting peptide, preferably, the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9 (RRGSKR, RRAAKR, RRYPGDVKR, or RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR).

[0057] GA is an A chain of aspart insulin, having an amino acid sequence as shown in SEQ ID NO: 5 at positions 32-52.

[0058] In another preferred embodiment, the protected lysine is Nε-(tert-butyloxycarbonyl)-lysine.

[0059] In a fourth aspect of the present application, a Boc-modified aspart insulin is provided, having a structure as shown in formula IV:

[0060]

[0061] wherein,

[0062] "║" represents a disulfide bond;

[0063] GA is an A chain of aspart insulin, the amino acid sequence of which is shown in SEQ ID NO: 5, 32-52,

[0064] GB is a B chain of aspart insulin, the amino acid sequence of which is shown in SEQ ID NO: 5, 1-30, and the lysine at position 29 of the B chain is Nε-(tert-butyloxycarbonyl)-lysine.

[0065] In a fifth aspect of the present application, a method for preparing aspart insulin is provided, the method comprising steps of:

[0066] (i) preparing a fermentation broth of aspart insulin fusion protein (first protein) by fermentation with a recombinant bacterium;

[0067] (ii) enzymatically cleaving the aspart insulin fusion protein (first protein) to obtain a mixture I containing Boc-modified aspart insulin (second protein);

[0068] (iii) deprotecting the Boc-modified aspart insulin (second protein) to obtain a mixture II containing deprotected aspart insulin (third protein);

[0069] (iv) purifying the mixture II to obtain aspart insulin.

[0070] In another preferred embodiment, between step (ii) and step (iii), a step of:

[0071] (I) subjecting the mixture I to a first anion exchange chromatography to obtain a dry powder of Boc-modified aspart insulin (second protein) is further included.

[0072] In another preferred embodiment, the purifying treatment of step (iv) comprises steps of:

[0073] (II) subjecting the mixture II to a second anion exchange chromatography with glycine as the mobile phase to obtain an eluate II containing deprotected aspart insulin (third protein);

[0074] (III) subjecting the eluate II to a reverse phase chromatography to obtain aspart insulin.

[0075] In another preferred embodiment, the prepared aspart insulin has a purity higher than 99%.

[0076] In another preferred embodiment, the prepared aspart insulin has aspart insulin activity.

[0077] In another preferred embodiment, the Boc-insulin aspart is an insulin aspart with a protected lysine at B29 (position 29 of the B chain of insulin).

[0078] In another preferred embodiment, the protected lysine is a lysine with a protecting group.

[0079] In another preferred embodiment, the protected lysine is Nε-(tert-butoxycarbonyl)-lysine.

[0080] In another preferred embodiment, in step (i), the fermentation production of the insulin aspart fusion protein is performed using a recombinant bacterium.

[0081] In another preferred embodiment, the recombinant bacterium comprises or integrates an expression cassette for expressing the insulin aspart fusion protein.

[0082] In another preferred embodiment, in step (i), the insulin aspart fusion protein inclusion bodies are isolated from the fermentation broth of the recombinant bacterium.

[0083] In another preferred embodiment, in step (i), the step of denaturation and renaturation of the inclusion bodies is further included, thereby obtaining the protein-folded insulin aspart fusion protein (first protein).

[0084] In another preferred embodiment, the insulin aspart fusion protein comprises an intrachain disulfide bond between the A chain and the B chain of the insulin aspart in the protein-folded insulin aspart fusion protein.

[0085] In another preferred embodiment, the insulin aspart fusion protein is as described in the first aspect of the present application.

[0086] In another preferred embodiment, in step (ii), the enzymatic cleavage is performed using trypsin and carboxypeptidase B.

[0087] In another preferred embodiment, in step (ii), the mass ratio of carboxypeptidase B to insulin aspart fusion protein is 1:(20000-30000).

[0088] In another preferred embodiment, in step (ii), the trypsin is recombinant porcine trypsin.

[0089] In another preferred embodiment, in step (ii), the mass ratio of trypsin to insulin aspart fusion protein is 1:1000-10000, preferably 1:1000-3000.

[0090] In another preferred embodiment, in step (ii), the temperature for the enzymatic cleavage is 32-42°C, preferably 36-38°C.

[0091] In another preferred embodiment, in step (ii), the time for the enzyme digestion is 10-30 h, preferably 14-20 h.

[0092] In another preferred embodiment, in step (ii), the pH of the digestion system of the insulin B16-A14 fusion protein is 7.0-9.5, preferably 8.0-9.5.

[0093] In another preferred embodiment, in step (iii), TFA (trifluoroacetic acid) is added to the reaction system to perform the deprotection treatment.

[0094] In another preferred embodiment, in step (iii), the ratio of the Boc-modified insulin B16-A14 fusion protein (the second protein) to TFA is 1 g: (4-10 mL).

[0095] In another preferred embodiment, in step (iii), the temperature of the deprotection reaction is 4-37°C, preferably 18-25°C.

[0096] In another preferred embodiment, in step (iii), the time for the deprotection reaction is 0.5-8 h, preferably 0.5-3 h.

[0097] In another preferred embodiment, the Boc-insulin B16-A14 fusion protein is Nε-(tert-butyloxycarbonyl)-lysine insulin B16-A14 fusion protein.

[0098] In another preferred embodiment, in step (I), the loading amount of the Boc-modified insulin B16-A14 fusion protein (the second protein) is ≤45 mg / ml.

[0099] In another preferred embodiment, in step (I), a linear gradient elution is performed using 100-500 mmol / L sodium chloride.

[0100] In another preferred embodiment, in step (II), an isocratic gradient elution is performed using 100-500 mmol / L sodium chloride.

[0101] In another preferred embodiment, in step (II), the loading amount of the insulin B16-A14 fusion protein in the eluent I is ≤15 mg / ml, preferably the loading amount is ≤10 mg / ml.

[0102] In another preferred embodiment, in step (III), an isocratic elution is performed using 150-250 mmol / L, preferably 180-220 mmol / L sodium acetate in acetonitrile as the mobile phase.

[0103] In another preferred embodiment, in step (III), the loading amount of the insulin B16-A14 fusion protein in the eluent II is ≤6 mg / ml, preferably the loading amount is ≤5 mg / ml.

[0104] In another preferred embodiment, after step (III), the method further comprises the steps of crystallizing and lyophilizing the aspart insulin produced, thereby producing a lyophilized product.

[0105] In a sixth aspect of the present application, there is provided an aspart insulin formulation prepared using the method of the fifth aspect of the present application.

[0106] In another preferred embodiment, the aspart insulin formulation comprises aspart insulin having a purity of greater than 99%.

[0107] In another preferred embodiment, the aspart insulin formulation comprises aspart insulin having a biological activity.

[0108] In a seventh aspect of the present application, there is provided an isolated polynucleotide encoding the aspart insulin fusion protein of the first aspect of the present application, the aspart insulin backbone fusion protein of the second aspect of the present application, the Boc-modified aspart insulin precursor of the third aspect of the present application, or the Boc-modified aspart insulin backbone of the fourth aspect of the present application.

[0109] In an eighth aspect of the present application, there is provided a vector comprising the polynucleotide of the seventh aspect of the present application.

[0110] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentivirus vector, adenovirus vector, retrovirus vector, transposon, or a combination thereof.

[0111] In a ninth aspect of the present application, there is provided a host cell comprising the vector of the eighth aspect of the present application, or having integrated into its chromosome exogenously the polynucleotide of the seventh aspect of the present application, or expressing the aspart insulin fusion protein of the first aspect of the present application, the aspart insulin backbone fusion protein of the second aspect of the present application, the Boc-modified aspart insulin precursor of the third aspect of the present application, or the Boc-modified aspart insulin backbone of the fourth aspect of the present application.

[0112] In another preferred embodiment, the host cell is E. coli, B. subtilis, yeast cell, insect cell, mammalian cell, or a combination thereof.

[0113] In a tenth aspect of the present application, there is provided a formulation or pharmaceutical composition comprising the aspart insulin fusion protein of the first aspect of the present application, the aspart insulin backbone fusion protein of the second aspect of the present application, the Boc-modified aspart insulin precursor of the third aspect of the present application, or the Boc-modified aspart insulin backbone of the fourth aspect of the present application, and a pharmaceutically acceptable carrier.

[0114] It should be understood that, in the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0115] Figure 1 A map of plasmid pBAD-FP-TEV-R-G is shown.

[0116] Figure 2 A map of plasmid pEvol-pylRs-pylT is shown.

[0117] Figure 3 An SDS-PAGE electropherogram of the aspart insulin fusion protein after inclusion body denaturation is shown.

[0118] Figure 4 An SDS-PAGE electropherogram of Boc-aspart insulin after the first chromatography is shown.

[0119] Figure 5 An HPLC detection map of aspart insulin after deprotection is shown.

[0120] Figure 6 An HPLC detection map of aspart insulin after the second chromatography is shown.

[0121] Figure 7 An HPLC detection map of aspart insulin after the third chromatography is shown.

[0122] Figure 8 A crystal map of aspart insulin crystallization is shown.

[0123] Figure 9 A mass spectrum of Boc-aspart insulin is shown. DETAILED DESCRIPTION

[0124] The present inventors have made extensive and in-depth research and found an aspart insulin derivative and a preparation method thereof. Specifically, the present application provides a fusion protein comprising a green fluorescent protein folding unit and an aspart insulin precursor or an active fragment thereof. The fusion protein of the present application has significantly improved expression, the aspart insulin protein in the fusion protein is correctly folded, and has biological activity. Moreover, the green fluorescent protein folding unit in the fusion protein of the present application can be digested into small fragments by a protease, and has a large difference in molecular weight compared with the target protein, and is easy to separate. The present application also provides a method for preparing aspart insulin and an intermediate in the preparation using the fusion protein.

[0125] TERMS

[0126] To enable a clearer understanding of the present disclosure, certain terms are defined first. As used in this application, unless specifically stated otherwise, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

[0127] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0128] Aspart insulin

[0129] Insulin products are the first largest drug varieties in the diabetes market, accounting for about 53% market share, among which the third generation of recombinant insulin is mainly used. Aspart insulin belongs to the third generation of recombinant insulin, which is a rapid-acting insulin (or called mealtime insulin), and has an onset of action 10-15 minutes after subcutaneous injection, reaches a peak in 1-2 hours, and has a duration of action of 4-6 hours.

[0130] Construction of aspart insulin expression plasmid

[0131] The FP-TEV-R-G target gene is synthesized, which has recognition sites of restriction endonuclease Nco I and Xho I at both ends, and contains the coding gene A of aspart insulin. The sequence is codon-optimized, which can realize high-level expression of functional protein in Escherichia coli. After expression, the expression vector "pBAD / His A (Kana R )" and the plasmid containing the "FP-TEV-R-G" target gene are digested with restriction endonuclease Nco I and Xho I, the digestion products are separated by agarose electrophoresis, then extracted using an agarose gel DNA recovery kit, and finally the two DNA fragments are ligated using T4 DNA ligase. The ligation product is chemically transformed into Escherichia coli Top10 cells, and the transformed cells are cultured on LB agar medium (10 g / L yeast extract peptone, 5 g / L yeast extract powder, 10 g / L NaCl, 1.5% agar) containing 50 μg / mL kanamycin overnight. Three live colonies are picked and cultured in 5 mL of liquid LB medium (10 g / L yeast extract peptone, 5 g / L yeast extract powder, 10 g / L NaCl) containing 50 μg / mL kanamycin overnight, and plasmid extraction is performed using a plasmid miniprep kit. Then, the extracted plasmid is sequenced to confirm correct insertion. The final plasmid is named "pBAD-FP-TEV-R-G".

[0132] Fusion protein

[0133] The present application constructs two fusion proteins, i.e. the insulin aspart fusion protein comprising single-chain insulin aspart precursor according to the first aspect of the present application and the double-chain insulin aspart fusion protein comprising double-chain insulin aspart according to the second aspect of the present application, by using the green fluorescent protein folding unit. In fact, the protection scope of the two fusion proteins of the present application can partially overlap, for example, the double-chain insulin aspart contained in the fusion protein, the C-terminus of the B chain of which can also be connected with the N-terminus of the A chain by a peptide, can also be identified as single-chain containing intrachain disulfide bond.

[0134] The green fluorescent protein folding unit FP contained in the fusion protein of the present application comprises 2-6, preferably 2-3, β-folding units selected from the following group:

[0135] Amino acid sequence 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).

[0136] In another preferred embodiment, the green fluorescent protein folding unit FP can be selected from the following group: 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, or u10-I-u11.

[0137] In another preferred embodiment, the green fluorescent protein folding unit is u8-u9, u9-u10-u11, or u10-u11.

[0138] As used herein, the term "fusion protein" also includes variants having the above-mentioned activities. These variants include (but are not limited to) deletion, insertion and / or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, and addition or deletion of one or several (usually within 3, more preferably within 2, most preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical amino acids usually does not change the function of the protein. For another example, addition or deletion of 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 monomeric and polymeric forms of the polypeptides of the present application. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0139] The present application also encompasses active fragments, derivatives, and analogs of the fusion proteins described above. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially maintain the function or activity of the fusion proteins of the present application. Polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or several conservative or non-conservative amino acid residue substitutions, preferably conservative amino acid residue substitutions, or (ii) polypeptides having a substituent group at one or more amino acid residues, or (iii) polypeptides formed by fusing the polypeptide to another compound, such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (fusion proteins formed by fusing leader sequences, secretion sequences, or tag sequences such as 6His). These fragments, derivatives, and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0140] One preferred class of active derivatives are polypeptides having up to three, preferably up to two, and more preferably up to one amino acid replaced by a similar or related amino acid compared to the amino acid sequence of the present application. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0141] Table A

[0142] Initial residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; 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

[0143] Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0144] The present application also provides analogs of the fusion proteins of the present application. These analogs can differ from the polypeptides of the present application in terms of amino acid sequence, in terms of modifications that do not affect the sequence, or both. Analogues also include those having residues other than naturally occurring L-amino acids, for example D-amino acids, and those having non-naturally occurring or synthetic amino acids, for example β, γ-amino acids. It is understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

[0145] In addition, the fusion proteins of the present application can be modified. Modifications, which generally do not alter the primary structure, include chemical derivatization of the polypeptide in vivo or in vitro, for example acetylation or carboxylation. Modifications also include glycosylation, for example those polypeptides that have been glycosylated during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that glycosylate or deglycosylate, for example mammalian glycosylation enzymes. Modifications also include sequences having phosphorylated amino acid residues, for example phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

[0146] The term "polynucleotide encoding a fusion protein of the present application" can be a polynucleotide including a polynucleotide encoding a fusion protein of the present application, or a polynucleotide further including additional coding and / or non-coding sequences.

[0147] The present application also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of the polypeptides or fusion proteins having the same amino acid sequence as the present application. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which can be one or more nucleotides different from the polynucleotide, but which do not change the functional properties of the fusion protein encoded by the polynucleotide.

[0148] The present application also relates to polynucleotides which hybridize to the above sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides which hybridize to the polynucleotides of the present application under stringent conditions (or stringency conditions). In the present application, "stringent conditions" means: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more.

[0149] The fusion proteins and polynucleotides of the present application are preferably provided in an isolated form, more preferably, purified to homogeneity.

[0150] The full-length sequence of the polynucleotides of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art 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 the fragments amplified in each amplification are spliced together in the correct order.

[0151] Once the relevant sequences are obtained, recombination can be used to obtain the relevant sequences in large quantities. This is usually done by cloning the sequences into vectors, transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods.

[0152] In addition, artificial synthesis can be used to synthesize the relevant sequences, particularly when the length of the fragment is short. Usually, a long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together.

[0153] At present, it is possible to obtain the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of the present application by chemical synthesis alone. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

[0154] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotide of the present application. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-cDNA rapid amplification of the end) is preferably used, primers for PCR can be appropriately selected based on the sequence information of the present application disclosed herein, and can be synthesized by a conventional method. The amplified DNA / RNA fragment can be separated and purified by a conventional method such as gel electrophoresis.

[0155] Expression vector

[0156] The present application also relates to a vector comprising the polynucleotide of the present application, and a host cell genetically engineered with the vector of the present application or the fusion protein coding sequence of the present application, and a method of producing the polypeptide of the present application by recombinant technology.

[0157] The polynucleotide sequence of the present application can be used to express or produce a recombinant fusion protein by conventional recombinant DNA technology. In general, the following steps are taken:

[0158] (1). Transform or transduce a suitable host cell with the polynucleotide of the present application encoding the fusion protein of the present application (or a variant), or with a recombinant expression vector containing the polynucleotide;

[0159] (2). Culture the host cell in a suitable medium;

[0160] (3). Isolate and purify the protein from the culture medium or cells.

[0161] In the present application, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vector well known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the expression vector is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.

[0162] Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding the fusion proteins of the present application and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequences are operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E. coli, the PL promoter of bacteriophage lambda, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for initiation of translation and a transcription terminator.

[0163] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance for eukaryotic and prokaryotic cell culture, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0164] Vectors containing the appropriate DNA sequences as described above, and appropriate promoters or control sequences, can be employed to transform appropriate host cells to enable them to express the proteins.

[0165] The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples of useful host cells are prokaryotic cells, such as bacterial cells of E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast, plant cells (e.g., ginseng cells), and mammalian cells.

[0166] The polynucleotides of the present application, when expressed in higher eukaryotic cells, can be enhanced by the insertion of enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, which act to increase the transcription of a gene. Examples of enhancers include the SV40 enhancer, which is in the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and enhancers associated with adenovirus.

[0167] The selection of appropriate vectors, promoters, enhancers, and host cells is well within the level of skill in the art.

[0168] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. Another method is the use of MgCl2. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0169] The transformants obtained can be cultured in conventional media using conventional procedures to express the polypeptides encoded by the genes of the present application. The medium used to culture the host cells is selected to provide an appropriate cell density, and the culture is conducted under conditions suitable for growth of the host cells. When the host cells have grown to an appropriate density, the selected promoter is induced by the appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0170] The recombinant polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation procedures, treatment with protein precipitants (salting-out procedures), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0171] The main advantages of the present application include:

[0172] (1) The method of the present application does not require dilution, ultrafiltration, or other methods to remove excess inorganic salts in the supernatant of the fermentation broth. The inclusion bodies obtained have high purity and less pigment, which reduces the separation materials and the purification cost in subsequent purification. In the cation chromatography separation of the present method, the yield of the insulin is more than 80% in one step.

[0173] (2) Because of the protection of Boc lysine at position B 29 , trypsin will not recognize the lysine at position B 29 , and des(B 30 ) byproducts will not be produced, which can improve the yield of enzymatic digestion and reduce the impurities of the insulin analog, providing convenience for subsequent purification and separation.

[0174] (3) In the process of enzymatic digestion, the enzyme addition ratio is optimized and the enzyme digestion temperature is controlled, thereby improving the yield of enzymatic digestion.

[0175] (4) In the deprotection step, Boc-aspartic insulin is converted into aspartic insulin without the need of being carried out in an organic system, reducing the process steps, less environmental pollution, and lower cost.

[0176] (5) The present application adopts two steps of ion exchange chromatography and one step of reverse phase chromatography for separation and purification, replacing the conventional four steps of chromatography, reducing the production cycle, and reducing the use of organic solvents, saving cost.

[0177] (6) The fusion protein of the present application contains a high proportion of aspartic insulin (increased fusion ratio), and the FP or A-FP in the fusion protein contains arginine and lysine, which can be digested into small fragments by protease, and the molecular weight difference compared with the target protein is large, and it is easy to separate.

[0178] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0179] Example 1 Construction and expression of aspartic insulin expression strain

[0180] The construction of aspartic insulin expression plasmid is referred to the description of the examples in Chinese patent application No. 201910210102.9. The DNA fragment containing the fusion protein FP-TEV-R-G is cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD / His A (purchased from NTCC company, kanamycin resistance) to obtain the plasmid pBAD-FP-TEV-R-G. The plasmid map is shown as Figure 1 .

[0181] The DNA sequence of pylRs is further cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (purchased from NTCC company, chloramphenicol resistance), and at the same time, the DNA sequence of lysyl-tRNA synthetase tRNA (pylTcua) is inserted downstream of the proK promoter by PCR method. The plasmid is named pEvol-pylRs-pylT. The plasmid map is shown as Figure 2 .

[0182] The constructed plasmid pBAD-FP-TEV-R-G and pEvol-pylRs-pylT were co-transformed into E. coli TOP10 strain, and a recombinant E. coli strain expressing insulin aspart fusion protein FP-TEV-R-G was screened. The sequence of FP is u8-u9 (SEQ ID NO: 3), and the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1.

[0183]

[0184] Using the same method, the following expression plasmids were constructed:

[0185] pBAD-FP(u9-u10-u11)-TEV-R-G, amino acid sequence of the fusion protein

[0186]

[0187] pBAD-FP(u10-u11)-TEV-R-G, amino acid sequence of the fusion protein

[0188]

[0189] pBAD-FP(gⅢ)-R-G, amino acid sequence of the fusion protein

[0190]

[0191] (SEQ ID NO: 10). Among them, FP(gⅢ) represents gⅢ signal peptide derived from green fluorescent protein.

[0192] Using conventional methods in the art, the corresponding expression strains were constructed, and the expression amount of insulin aspart fusion protein was detected by electrophoresis.

[0193] Fusion protein structure Expression amount of insulin aspart fusion protein

[0194] (g / L fermentation broth) FP(u8-u9)-TEV-R-G 6.5 FP(u9-u10-u11)-TEV-R-G 6.8 FP(u10-u11)-TEV-R-G 6.3 FP(gIII)-R-G 5.3

[0195] Seed culture medium was prepared, inoculated, and two-stage culture was performed to obtain secondary seed liquid. After 20 h of culture, OD600 reached about 180, fermentation was completed, about 3 L of fermentation broth was obtained, and about 130 g / L of wet bacteria were obtained by centrifugation. After centrifugation of the fermentation broth, break buffer was added, and the bacteria were broken twice using a high-pressure homogenizer. After centrifugation, Tween 80 and EDTA-2Na were added for washing, followed by water washing once. The inclusion bodies were obtained by centrifugation to collect the precipitate. About 43 g of wet weight inclusion bodies per liter of fermentation broth were obtained.

[0196] Example 2: Solubilization and renaturation of inclusion bodies

[0197] The obtained inclusion body was added with 8 mol / L urea solution, and the pH was adjusted to 9.0-10.0 by sodium hydroxide, and then stirred at room temperature for 1-3 h, the protein concentration was controlled to be 10-20 mg / mL, and β-mercaptoethanol was added to a final concentration of 15-20 mmol / L, and then stirred for 0.5-1.0 h.

[0198] The inclusion body solution was added dropwise into the renaturation buffer, and the renaturation was diluted by 5-10 times, the pH of the renaturation solution was maintained at 9.0-10.0, and the stirring time of the renaturation was 10-20 h.

[0199] After 20 h of renaturation, the content of aspart insulin fusion with correct folding was detected by HPLC, and the renaturation rate was more than 75%. Figure 3 The SDS-PAGE electrophoretogram of aspart insulin fusion protein after the renaturation of the inclusion body is shown.

[0200] Example 3 Enzymatic digestion of the fusion protein

[0201] To the renaturation solution, dilute hydrochloric acid was added to adjust the pH to 8.0-9.5, and then recombinant trypsin was added at a mass ratio of 1:3000 based on the total protein of the renaturation solution, and carboxypeptidase B was added at a mass ratio of 1:15000 based on the total protein of the renaturation solution, the enzymatic digestion temperature was 36-38°C, the enzymatic digestion time was 14-20 h, and then Boc-aspart insulin was obtained after the enzymatic digestion.

[0202] After 16 h of enzymatic digestion, the content of Boc-aspart insulin in the enzymatic digestion solution was detected by HPLC, and when the concentration of Boc-aspart insulin detected in two consecutive hours differed by less than 3%, the enzymatic digestion was completed. Finally, the concentration of Boc-aspart insulin in the enzymatic digestion solution was 0.4-0.6 g / L, and the enzymatic digestion rate was more than 80%.

[0203] Example 4 First chromatography

[0204] According to the difference in the isoelectric point of the protein, Boc-aspart insulin was coarsely extracted by using anion exchange filler. The chromatography column was equilibrated with 5-20 mmol / L sodium carbonate buffer at pH 8.0-9.0 for 3-5 column volumes, Boc-aspart insulin was fully combined with the anion filler, and the loading capacity was less than 45 g / L. After the loading was completed, 100-500 mmol / L sodium chloride was linearly eluted for 15 column volumes, and the eluted protein solution was collected. The detection results of SDS protein gel electrophoresis are shown in Figure 4 . The yield of Boc-aspart insulin was more than 90%, and the purity was more than 70%.

[0205] Example 5 Deprotection

[0206] The Boc-aspartic insulin dry powder was obtained after drying the Boc-aspartic insulin crude extract by anion exchange chromatography. Mass spectrum analysis was performed on the obtained Boc-aspartic insulin dry powder, and the mass spectrum chart is shown in Figure 9 The results show that the measured molecular weight of Boc-aspartic insulin is 5921.644 Da, and the calculated value is 5925.6 Da, indicating that the target protein is obtained.

[0207] To 1 g of Boc-aspartic insulin dry powder, 4-10 mL of TFA was added, and the reaction was stirred at room temperature for 0.5-3.0 h. After the reaction was completed, NaOH was added to adjust the pH of the protein solution to above 2.5 to terminate the deprotection reaction. Finally, aspartic insulin was obtained, and the deprotection yield was above 90% and the purity was above 75% determined by HPLC. The HPLC detection results are shown in Figure 5 .

[0208] Example 6 Second chromatography

[0209] According to the difference in the charge of the substance, anion exchange chromatography technology was used to purify aspartic insulin to remove part of the impurities. The ion column was equilibrated with 20 mmol / L glycine, pH 9.0 buffer solution for 3-5 column volumes. The aspartic insulin protein solution was combined with the cationic filler, and the loading capacity of aspartic insulin was controlled to be not more than 10 mg / mL. Finally, it was eluted with a solution containing 0.27 mol / L sodium chloride, and the aspartic insulin sample was collected. The purity of aspartic insulin in the collected solution was 97.83%, and the yield was 87.96%. The HPLC detection chart is shown in Figure 6 .

[0210] Example 7 Third chromatography

[0211] According to the difference in the hydrophobicity of the substance, reverse phase chromatography column technology was used to purify aspartic insulin. The aspartic insulin solution obtained by twice chromatography was diluted by more than 4 times with pure water, and combined with C8 reverse phase filler. The loading capacity of aspartic insulin was controlled to be not higher than 5 mg / mL, and eluted with 10 CV of a solution containing 200 mmol / L sodium acetate and 26% acetonitrile. The results show that the yield of aspartic insulin is 93.8%, and the purity is 99.70%. The detection results are shown in Figure 7 .

[0212] Example 8 Crystallization and freeze-drying

[0213] To the elution collected solution of the third chromatography, distilled water was added to dilute the acetonitrile solution to a content of not more than 15% (v / v). The solution was sequentially added with 0.7 mol / L glycine, 0.8 mol / L sodium chloride, and 0.5% saturated phenol, and then zinc acetate was added according to a molar ratio of 1:3. The pH was adjusted to 5.6-6.0 with acetic acid, and the solution was statically placed at 4-8°C for crystallization for more than 16 h. Under a microscope, rod-shaped crystals were observed to form (seeFigure 8 ). The crystals were collected and lyophilized to obtain the raw material of aspart insulin.

[0214] All documents referred to in this disclosure are incorporated by reference herein as if each were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications will occur to those skilled in the art upon reading the above teaching. It is intended to include all such alterations and modifications within the scope of the application. Therefore, the above disclosure is intended to be illustrative, but not restrictive. Changes can be made by one of ordinary skill in the art which are within the scope of the application. In addition, where a description of a technological interaction of structure and / or function of a specific compound is given, one of ordinary skill in the art will recognize that the same technological interaction of structure and / or function can occur for other compounds. SEQUENCE LISTING <110> Ningbo Kaimeng Biotechnology Co., Ltd. <120> Aspart insulin derivative and preparation method and application thereof <130> P2022-2979 <150> CN202010526819.7 <151> 2020-06-09 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Aspart insulin fusion protein <400> 1 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Val Gln 1 5 10 15 Leu Ala Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Glu Asn Leu Tyr 20 25 30 Phe Gin Gly Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val 35 40 45 Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp 50 55 60 Lys Thr Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu 65 70 75 80 Tyr Gin Leu Glu Asn Tyr Cys Asn 85 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Leader Peptide <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 1 5 10 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Green Fluorescent Protein Folding Unit <400> 3 Gly lie Lys Ala Asn Phe Lys lie Arg His Asn Val Glu Asp Val Gin 1 5 10 15 Leu Ala Asp His Tyr Gin Gin Asn Thr Pro lie Gly 20 25 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> TEV Enzyme Cleavage Site <400> 4 Glu Asn Leu Tyr Phe Gin Gly 1 5 <210> 5 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> Insulin B29K <400> 5 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr Arg Gly 20 25 30 Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu 35 40 45 Asn Tyr Cys Asn 50 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 6 Arg Arg Gly Ser Lys Arg 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 7 Arg Arg Ala Ala Lys Arg 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 8 Arg Arg Tyr Pro Gly Asp Val Lys Arg 1 5 <210> 9 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 9 Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly 1 5 10 15 Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu 20 25 30 Gln Lys Arg 35 <210> 10 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 10 Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser 1 5 10 15 His Ser Thr Met Glu Leu Glu Ile Cys Ser Trp Tyr His Met Gly Ile 20 25 30 Arg Ser Phe Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser 35 40 45 Ala Val Asp Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val 50 55 60 Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp 65 70 75 80 Lys Thr Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu 85 90 95 Tyr Gin Leu Glu Asn Tyr Cys Asn 100 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Beta-sheet unit <400> 11 Val Pro lie Leu Val Glu Leu Asp Gly Asp Val Asn Gly 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Beta-sheet unit <400> 12 His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly Asp Ala Thr 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 13 Lys Leu Thr Leu Lys Phe lie Cys Thr Thr 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 14 Tyr Val Gin Glu Arg Thr lie Ser Phe Lys Asp 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 15 Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 1 5 10 <210> 16 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 16 Thr Leu Val Asn Arg lie Glu Leu Lys Gly lie Asp Phe 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 17 His Asn Val Tyr Ile Thr Ala Asp Lys Gln 1 5 10 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 18 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 19 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> beta-sheet unit <400> 20 His Tyr Leu Ser Thr Gln Ser Val Leu Ser Lys Asp 1 5 10 <210> 21 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Beta-sheet unit <400> 21 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 1 5 10 <210> 22 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 22 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly His Tyr 1 5 10 15 Leu Ser Thr Gln Ser Val Leu Ser Lys Asp His Met Val Leu Leu Glu 20 25 30 Phe Val Thr Ala Ala Gly Ile Glu Asn Leu Tyr Phe Gln Gly Arg Phe 35 40 45 Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu 50 55 60 Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr Arg Gly Ile 65 70 75 80 Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn 85 90 95 Tyr Cys Asn <210> 23 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 23 His Tyr Leu Ser Thr Gin Ser Val Leu Ser Lys Asp His Met Val Leu 1 5 10 15 Leu Gin Phe Val Thr Ala Ala Gly He Gin Asn Leu Tyr Phe Gin Gin 20 25 30 Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu 35 40 45 Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr Arg 50 55 60 Gly He Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu 65 70 75 80 Glu Asn Tyr Cys Asn 85 <210> 24 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Green Fluorescent Protein Foldon <400> 24 Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro He Gly His Tyr 1 5 10 15 Leu Ser Thr Gin Ser Val Leu Ser Lys Asp His Met Val Leu Leu Glu 20 25 30 Phe Val Thr Ala Ala Gly lie 35 <210> 25 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Green fluorescent protein folding unit <400> 25 His Tyr Leu Ser Thr Gin Ser Val Leu Ser Lys Asp His Met Val Leu 1 5 10 15 Leu Glu Phe Val Thr Ala Ala Gly lie 20 25

Claims

1. A fusion protein of insulin aspart, characterized in that, A-FP-TEV-R-G (I) wherein, "-" represents a peptide bond; A is a leader peptide, FP is a green fluorescent protein folding unit, TEV is an enzyme cleavage site; R is arginine or lysine for enzyme cleavage; G is a Boc-modified insulin precursor, having a structure as shown in formula II: GB-X-GA (II) wherein, 2. The fusion protein of claim 1, wherein, GB is a Boc-modified insulin B chain at the 29th position, having an amino acid sequence as shown in SEQ ID NO: 5 from 1st to 30th position, 3. The fusion protein of claim 1, wherein, X is a connecting peptide, and the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9; GA is an insulin A chain, having an amino acid sequence as shown in SEQ ID NO: 5 from 32nd to 52nd position. There is an intrachain disulfide bond between GB-X-GA. TEV is a TEV enzyme cleavage site. A-FP-TEV-R-D (III) wherein, 4. The fusion protein of claim 3, wherein, "-" represents a peptide bond; 5. The fusion protein of claim 1, wherein, A is a leader peptide, 6. A double-chain insulin aspart fusion protein, characterized in that, FP is a green fluorescent protein folding unit, TEV is an enzyme cleavage site; R is arginine or lysine for enzyme cleavage; D is a Boc-modified double-chain insulin, having a structure as shown in formula IV: wherein, "║" represents a disulfide bond; GA is an insulin A chain, having an amino acid sequence as shown in SEQ ID NO: 5 from 32nd to 52nd position, X is a connecting peptide, and the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9; GB is a Boc-modified insulin B chain at the 29th position, having an amino acid sequence as shown in SEQ ID NO: 5 from 1st to 30th position; wherein, the green fluorescent protein folding unit is a β-folding unit selected from the group consisting of u8-u9, u9-u10-u11, u10-u11, u2-u3, u4-u5, u1-u2-u3, u3-u4-u5, and u4-u5-u6; TEV is a TEV enzyme cleavage site. The method comprises the steps of: (i) fermenting with a recombinant bacterium, isolating a recombinant insulin precursor fusion protein inclusion body from the fermentation broth of the recombinant bacterium, and preparing the fusion protein of claim 1; (ii) cleaving the fusion protein to obtain a Boc-modified insulin precursor, and the structure of the Boc-modified insulin precursor is as shown in formula II: GB-X-GA (II) 7. The double-chain insulin fusion protein of claim 6, wherein, wherein, 8. A method of preparing a Boc-modified insulin proline characterized in that, GB is a Boc-modified insulin B chain at the 29th position, having an amino acid sequence as shown in SEQ ID NO: 5 from 1st to 30th position, X is a connecting peptide, and the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9; ​ ​ ​ ​ X is a connecting peptide, and the amino acid sequence of X is R, RR, RRR, or as shown in SEQ ID NO: 6-9; GA is an A chain of aspart insulin, and the amino acid sequence is shown in SEQ ID NO: 5, 32-52.

9. A process for the preparation of Boc modified double chain aspart insulin, characterized by, The method comprises the steps of: (i) fermentation by using a recombinant bacterium, separation of aspart insulin fusion protein inclusion bodies from the fermentation broth of the recombinant bacterium, and preparation of the fusion protein of claim 1; (ii) enzyme digestion of the fusion protein to obtain Boc-modified double-chain aspart insulin, The structure of the Boc-modified double-chain aspart insulin is shown in formula IV: In the formula, "║" represents a disulfide bond; GA is an A chain of aspart insulin, and the amino acid sequence is shown in SEQ ID NO: 5, 32-52. GB is a B chain of aspart insulin, and the amino acid sequence is shown in SEQ ID NO: 5, 1-30, and the lysine at the 29th position of the B chain is Nε-(tert-butoxycarbonyl)-lysine.

10. An isolated polynucleotide, comprising: The polynucleotide encodes the aspart insulin fusion protein of claim 1, or the double-chain aspart insulin fusion protein of claim 6.

11. A vector, characterized in that, The vector comprises the polynucleotide of claim 10.

12. A host cell, characterized in that, The host cell contains the vector of claim 11, or the exogenous polynucleotide of claim 10 integrated in the chromosome, or expresses the aspart insulin fusion protein of claim 1, or the double-chain aspart insulin fusion protein of claim 6.

13. A method of preparing insulin aspart, characterized by, The method comprises the steps of: (i) fermentation by using a recombinant bacterium, preparation of a fermentation broth containing the aspart insulin fusion protein of claim 1; (ii) enzyme digestion of the aspart insulin fusion protein to obtain a mixture I containing Boc-modified aspart insulin; (iii) deprotection treatment of the Boc-modified aspart insulin to obtain a mixture II containing deprotected aspart insulin; (iv) purification treatment of the mixture II to obtain aspart insulin.

14. The method of claim 13, wherein, Between step (ii) and step (iii), a step is further included: (I) first anion exchange chromatography of the mixture I to obtain a dry powder containing Boc-modified aspart insulin.

15. The method of claim 13, wherein, The purification treatment of step (iv) comprises the steps of: (II) second anion exchange chromatography of the mixture II using glycine as the mobile phase to obtain eluate II containing deprotected aspart insulin; (III) reverse phase chromatography of the eluate II to obtain aspart insulin.

16. The method of claim 13, wherein, In step (i), aspart insulin fusion protein inclusion bodies are separated from the fermentation broth of the recombinant bacterium, and then the inclusion bodies are denatured and renatured to obtain aspart insulin fusion protein with correct protein folding.

17. The method of claim 13, wherein, In step (ii), trypsin and carboxypeptidase B are used for enzyme digestion.

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