CN113773391B

CN113773391B - Preparation method of insulin aspart

Info

Publication number: CN113773391B
Application number: CN202010519559.0A
Authority: CN
Inventors: 唐亚连; 陈卫; 李克朗
Original assignee: Ningbo Kunpeng Biotech Co Ltd
Current assignee: Ningbo Kunpeng Biotech Co Ltd
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2023-10-20
Anticipated expiration: 2040-06-09
Also published as: CN113773391A

Abstract

The application provides an insulin aspart derivative and a preparation method thereof. Specifically, the method of the application expresses the insulin aspart fusion protein containing the green fluorescent protein folding unit in high density in escherichia coli, and carries out enzyme digestion and purification on the fusion protein to prepare the insulin aspart. The method of the application is not needed to be carried out under a solid-phase organic system, reduces the process steps, has little environmental pollution and lower cost, and is suitable for popularization.

Description

Preparation method of insulin aspart

Technical Field

The application relates to the technical field of biology, in particular to a preparation method of insulin aspart.

Background

Diabetes is a major disease that threatens human health worldwide. In China, along with the change of the life style of people and the acceleration of the aging process, the prevalence of diabetes mellitus is rapidly rising. The acute and chronic complications of diabetes, especially the chronic complications accumulate a plurality of organs, have high disability and mortality, seriously affect the physical and mental health of patients, and bring heavy burden to individuals, families and society.

Insulin aspart is a quick-acting insulin analogue, belongs to the third generation of insulin, is bridged by two chains of A, B by means of two semi-amino acid disulfide bonds, replaces (mutates) proline (Pro) at the 28 th position of a human insulin B chain with aspartic acid (Asp) with negative charges by utilizing a genetic engineering DNA recombination technology, and prevents self-polymerization of insulin monomers or dimers (formed by B28 of one insulin molecule and B23 of the other insulin molecule) by utilizing the repulsive effect of charges, so that intermolecular polymerization is reduced. The human insulin secretion mode can be well simulated, the pharmacokinetic characteristic of the human insulin secretion mode is about half of that of the conventional human insulin, the acting time is 10-20 minutes, the peak time is 40 minutes, and the action duration is shortened to 3-5 hours, so that a patient can obtain good blood sugar control and is not easy to be overlapped with insulin action before meals or at night, and the occurrence rate of hypoglycemia at night is obviously reduced.

The preparation of the insulin aspart generally adopts a genetic engineering technology to prepare a precursor, the original research company Noand Norde uses Saccharomyces cerevisiae as an expression host, the recombinant DNA technology is utilized to produce the insulin aspart precursor, and then the insulin aspart is prepared through a series of complex processes such as transpeptidation and the like. The method has great technical difficulty and complex process on the reverse side of host bacterium transformation and the like. The saccharomyces cerevisiae has weak self expression capability, so that the yield of insulin aspart is low, and the maximum engineering bacteria shake cultivation is 21.5mg/L, so that the production cost of the medicine is increased to a certain extent.

Therefore, there is an urgent need in the art to develop a method for preparing and purifying insulin aspart with simple process, environmental friendliness, high yield and high yield.

Disclosure of Invention

The application aims to provide a preparation method of insulin aspart.

In a first aspect of the present application, there is provided a recombinant insulin aspart fusion protein having a structure represented by formula I:

A-FP-TEV-R-G (I)

in the method, in the process of the application,

"-" represents a peptide bond;

a is a non-or leader peptide,

FP is a green fluorescent protein folding unit,

TEV is a cleavage site, preferably a TEV cleavage site;

r is arginine or lysine for enzyme digestion;

g is insulin aspart or an active fragment thereof;

wherein said green fluorescent protein folding units comprise 2-6, preferably 2-3 beta-folding units selected from the group consisting of:

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)。

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

In another preferred embodiment, G is a Boc-modified insulin aspart precursor having a structure according to formula II:

GB-X-GA (II)

in the method, in the process of the application,

GB is the B chain of the insulin aspart modified by Boc, the amino acid sequence is shown in the 1 st-30 th positions of SEQ ID NO. 5,

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);

GA is an insulin aspart A chain, and the amino acid sequence is shown in 32-52 positions of SEQ ID NO. 5.

In another preferred embodiment, R is used for trypsin and carboxypeptidase cleavage.

In another preferred embodiment, G is Boc modified insulin aspart having the sequence shown in SEQ ID NO. 5.

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

In another preferred embodiment, the sequence of the recombinant insulin aspart fusion protein is shown in SEQ ID NO. 1, 22 and 23.

In another preferred embodiment, an inter-chain disulfide bond is formed between the B-chain position 7 and the A-chain position 7 (A7-B7), and between the B-chain position 19 and the A-chain position 20 (A20-B19).

In another preferred embodiment, an intra-chain disulfide bond is formed between position 6 of the A chain and position 11 of the A chain (A6-A11).

In a second aspect of the application, there is provided a double-stranded insulin aspart fusion protein having the structure shown in formula III:

A-FP-TEV-R-D (III)

in the method, in the process of the application,

"-" represents a peptide bond;

a is a null or leader peptide, preferably a leader peptide having the sequence shown in SEQ ID NO. 2,

FP is a green fluorescent protein folding unit,

TEV is a cleavage site, preferably a TEV cleavage site (sequence ENLYFQG, SEQ ID NO: 4);

r is arginine or lysine for enzyme digestion;

d is Boc modified double-chain insulin aspart, and the main chain has a structure shown in the following formula IV;

in the method, in the process of the application,

represents a disulfide bond;

GA is an insulin aspart A chain, the amino acid sequence is shown in 32-52 positions of SEQ ID NO. 5,

x is none or a connecting peptide;

GB is a B chain of the insulin aspart modified by Boc at 29 th site, and the amino acid sequence is shown in 1-30 th site of SEQ ID NO. 5;

In another preferred embodiment, the amino acid sequence of the green fluorescent protein folding unit is shown in SEQ ID NO. 3, 24 and 25.

In another preferred embodiment, the C-terminus of the insulin aspart B chain is linked to the N-terminus of the insulin aspart A chain by a linker peptide.

In another preferred embodiment, X is a linker 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).

In a third aspect of the application, there is provided a Boc-modified insulin aspart precursor having the structure shown in formula II:

GB-X-GA (II)

in the method, in the process of the application,

GB is the B chain of the insulin aspart modified by Boc at 29 th site, the amino acid sequence is shown in 1-30 th site of SEQ ID NO. 5,

In another preferred embodiment, the protecting lysine is N [ epsilon ] - (t-butoxycarbonyl) -lysine.

In a fourth aspect of the application, there is provided a Boc-modified insulin aspart having the structure shown in formula IV:

in the method, in the process of the application,

represents a disulfide bond;

GB is insulin aspart B chain, the amino acid sequence is shown in the 1 st-30 th positions of SEQ ID NO. 5, and lysine at the 29 th position of the B chain is N epsilon- (tert-butoxycarbonyl) -lysine.

In a fifth aspect of the present application, there is provided a method of preparing insulin aspart, the method comprising the steps of:

(i) Fermenting by utilizing recombinant bacteria to prepare recombinant insulin aspart fusion protein (first protein) fermentation liquor;

(ii) Performing enzyme digestion on the recombinant insulin aspart fusion protein (first protein) to obtain a mixed solution I containing Boc modified insulin aspart (second protein);

(iii) Deprotecting the Boc-modified insulin aspart (second protein) to obtain a mixture II comprising deprotected insulin aspart (third protein);

(iv) Purifying the mixed solution II to obtain the insulin aspart.

In another preferred embodiment, between step (ii) and step (iii), the method further comprises the steps of:

(I) And carrying out anion exchange chromatography on the mixed solution I for the first time, thereby obtaining the dry powder of the insulin aspart (second protein) modified by Boc.

In another preferred embodiment, the purification treatment of step (iv) comprises the steps of:

(II) subjecting said mixture II to a second anion exchange chromatography with glycine as mobile phase, thereby obtaining an eluate II comprising deprotected insulin aspart (third protein);

(III) performing reverse phase chromatography on the eluent II, thereby obtaining insulin aspart.

In another preferred embodiment, insulin aspart is produced having a purity of greater than 99%.

In another preferred embodiment, insulin aspart is produced having insulin aspart activity.

In another preferred embodiment, the recombinant Boc-insulin aspart is insulin aspart with a lysine at position B29 (insulin B chain 29).

In another preferred embodiment, the protecting lysine is a lysine with a protecting group.

In another preferred embodiment, in step (i), the recombinant production of the recombinant insulin aspart fusion protein is performed using recombinant bacteria.

In another preferred embodiment, the recombinant bacterium comprises or incorporates an expression cassette for expressing a recombinant insulin aspart fusion protein.

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

In another preferred embodiment, in step (i), the method further comprises the step of denaturing and renaturating the inclusion bodies to obtain a recombinant insulin aspart fusion protein (first protein) having a correctly folded protein.

In another preferred embodiment, the protein fold is correct and the insulin aspart fusion protein comprises an intra-chain disulfide bond between the A and B chains.

In another preferred embodiment, the recombinant insulin aspart fusion protein is as described in the first aspect of the application.

In another preferred embodiment, in step (ii), cleavage is performed using trypsin and carboxypeptidase B.

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

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

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

In another preferred embodiment, in step (ii), the temperature of the cleavage is 32-42 ℃, preferably 36-38 ℃.

In another preferred embodiment, in step (ii), the time for the cleavage is from 10 to 30 hours, preferably from 14 to 20 hours.

In another preferred embodiment, in step (ii), the pH of the recombinant insulin aspart fusion protein cleavage system is from 7.0 to 9.5, preferably from 8.0 to 9.5.

In another preferred embodiment, in step (iii), TFA (trifluoroacetic acid) is added to the reaction system and the deprotection treatment is performed.

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

In another preferred embodiment, in step (iii), the temperature of the deprotection reaction is 4-37 ℃, preferably 18-25 ℃.

In another preferred embodiment, in step (iii), the time of the deprotection reaction is from 0.5 to 8 hours, preferably from 0.5 to 3 hours.

In another preferred embodiment, the Boc-insulin aspart is N [ epsilon ] -t-butoxycarbonyl) -lysine insulin aspart.

In another preferred embodiment, in the step (I), the loading amount of the insulin aspart (second protein) containing the Boc modification is less than or equal to 45mg/ml.

In another preferred embodiment, in step (I), a linear gradient elution is carried out with 100-500mmol/L sodium chloride.

In another preferred embodiment, in step (II), an isocratic gradient elution is carried out with 100-500mmol/L sodium chloride.

In another preferred embodiment, in step (II), the loading of insulin aspart in the eluent I is less than or equal to 15mg/ml, preferably less than or equal to 10mg/ml.

In a further preferred embodiment, in step (III), the isocratic elution is carried out with a solution of 150 to 250mmol/L, preferably 180 to 220mmol/L, sodium acetate in acetonitrile as mobile phase.

In another preferred embodiment, in step (III), the loading of insulin aspart in the eluent II is less than or equal to 6mg/ml, preferably less than or equal to 5mg/ml.

In another preferred embodiment, after step (III), the method further comprises the steps of crystallizing and freeze-drying the prepared insulin aspart, thereby preparing a freeze-dried product.

In a sixth aspect of the application there is provided an insulin aspart formulation prepared using the method of the fifth aspect of the application.

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

In another preferred embodiment, the insulin aspart contained in the insulin aspart formulation has biological activity.

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

In an eighth aspect thereof the present application provides a vector comprising a polynucleotide according to the seventh aspect of the present application.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

In a ninth aspect of the application there is provided a host cell comprising a vector according to the eighth aspect of the application, or a polynucleotide according to the seventh aspect of the application integrated into the chromosome, or expressing a recombinant insulin aspart fusion protein according to the first aspect of the application, a insulin aspart backbone fusion protein according to the second aspect of the application, a Boc modified insulin aspart precursor according to the third aspect of the application, or a Boc modified insulin aspart backbone according to the fourth aspect of the application.

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

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

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the map of plasmid pBAD-FP-TEV-R-G.

FIG. 2 shows a map of plasmid pEvol-pylRs-pylT.

FIG. 3 shows SDS-PAGE electrophoresis of insulin aspart fusion proteins after inclusion body renaturation.

FIG. 4 shows SDS-PAGE of Boc-insulin aspart after the first chromatography.

Figure 5 shows the HPLC detection profile of insulin aspart after deprotection.

FIG. 6 shows the HPLC detection pattern of insulin aspart after the second chromatography.

FIG. 7 shows the HPLC detection pattern of insulin aspart after the third chromatography.

FIG. 8 shows a crystal diagram of insulin aspart crystals.

Detailed Description

The present inventors have studied extensively and intensively to find an insulin aspart derivative and a method for producing the same. Specifically, the method of the application expresses the insulin aspart fusion protein containing the green fluorescent protein folding unit in high density in escherichia coli, and carries out enzyme digestion and purification on the fusion protein to prepare the insulin aspart. . The method of the application is not needed to be carried out under an organic system, reduces the process steps, has little environmental pollution and lower cost, and is suitable for popularization.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

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

Insulin aspart

Insulin products are the first large drug varieties in the diabetes market, occupy about 53% of market share, and are mainly three-generation recombinant insulin. Insulin aspart belongs to the third generation of recombinant insulin, is quick-acting insulin (or called meal insulin), takes effect 10-15 minutes after subcutaneous injection, reaches peak value 1-2 hours, and has action duration of 4-6 hours.

Construction of insulin aspart expression plasmid

The target FP-TEV-R-G gene is synthesized, and the gene has recognition sites of restriction endonucleases NcoI and XhoI at two ends and contains encoding gene A of insulin aspart. The sequence is codon optimized, and can realize high-level expression of functional protein in colibacillus. After expression, the expression vector "pBAD/His A" was digested with restriction enzymes NcoI and XhoI (Kana ^R ) And a plasmid containing the target gene of FP-TEV-R-G, separating the enzyme digestion product by agarose electrophoresis, extracting by using an agarose gel DNA recovery kit, and finally connecting two DNA fragments by using T4DNA ligase. The ligation product is subjected toThe transformed cells were chemically transformed into E.coli Top10 cells, and the transformed cells were cultured on LB agar medium (10 g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl,1.5% agar) containing 50. Mu.g/mL kanamycin overnight. 3 viable colonies were picked, cultured overnight in 5mL of liquid LB medium (10 g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl) containing 50. Mu.g/mL kanamycin, and plasmid extraction was performed using a plasmid miniextraction kit. The extracted plasmid was then sequenced to confirm correct insertion. The resulting plasmid was designated "pBAD-FP-TEV-R-G".

Fusion proteins

By using the green fluorescent protein folding unit, two fusion proteins are constructed, namely the recombinant insulin aspart fusion protein containing the single-chain insulin aspart precursor according to the first aspect of the application and the double-chain insulin aspart fusion protein containing the double-chain insulin aspart according to the second aspect of the application. In fact, the protective scope of the two fusion proteins of the application may overlap, for example, the double-stranded form of insulin aspart contained in the fusion protein, the C-terminus of which may also be linked to the N-terminus of the A-chain by a linker peptide, and may also be considered as a single chain comprising an intrachain disulfide bond.

The green fluorescent protein folding units FP comprised in the fusion proteins of the application comprise 2-6, preferably 2-3 β -sheet units selected from the group consisting of:

	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)。

In another preferred embodiment, the green fluorescent protein folding unit FP may 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, U8-U5U 3-U4-U3-U4, U3-U5-U7-U9, U5-U6-U7-U8, U1-U3-U7-U9, 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.

The term "fusion protein" also includes variants having the above-described activities. These variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (typically 1-2, more preferably 1) amino acids, and additions or deletions of one or several (typically within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus generally does not alter the structure or function of the protein. Furthermore, the term also includes polypeptides of the application in monomeric and multimeric form. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The application also includes active fragments, derivatives and analogues of the fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the function or activity of the fusion proteins of the application. The polypeptide fragment, derivative or analogue of the present application may be (i) a polypeptide having one or several conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound such as a compound which extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (fusion protein formed by fusion with a tag sequence such as a leader sequence, a secretory sequence or 6 His). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides in which up to 3, preferably up to 2, more preferably up to 1 amino acid is replaced by an amino acid of similar or similar nature, as compared to the amino acid sequence of the application. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

The application also provides analogs of the fusion proteins of the application. These analogs may differ from the polypeptides of the application by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

In addition, the fusion proteins of the application may also be modified. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.

The term "polynucleotide encoding a fusion protein of the application" may include polynucleotides encoding a fusion protein of the application, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The 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 application. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the fusion protein it encodes.

The application also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The application relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the application. In the present application, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

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

The full-length polynucleotide sequence of the present application can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present application, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the application (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the application. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present application disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Expression vector

The application also relates to vectors comprising the polynucleotides of the application, host cells genetically engineered with the vectors of the application or the fusion protein coding sequences of the application, and methods for producing the polypeptides of the application by recombinant techniques.

The polynucleotide sequences of the present application can be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a fusion protein of the application, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present application, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

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

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, plant cells (e.g., ginseng cells).

When the polynucleotide of the present application is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene. Examples include the SV40 enhancer 100 to 270 base pairs on the late side of the origin of replication, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

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, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present application. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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.

The main advantages of the application include:

(1) The method does not need to remove excessive inorganic salt in the supernatant of the fermentation broth by adopting methods such as dilution, ultrafiltration liquid exchange and the like, and the obtained inclusion body has higher purity and less pigment, reduces separation substances for subsequent purification and reduces purification cost. In addition, the positive ion chromatography in the method separates the insulin aspart, and the yield of one step is more than 80 percent.

(2) Due to B ₂₉ Protection of Boc lysine at position, and trypsin cleavage does not recognize B ₂₉ Lysine in position, does not produce des (B ₃₀ ) The by-product of the method can improve the digestion yield, reduce the impurities of insulin aspart analogues and provide convenience for subsequent purification and separation.

(3) In the enzyme digestion process, the enzyme digestion yield is improved by optimizing the enzyme addition proportion and controlling the enzyme digestion temperature.

(4) In the deprotection step, boc-insulin aspart is converted into insulin aspart, and the method is not required to be carried out under an organic system, so that the process steps are reduced, the environmental pollution is small, and the cost is lower.

(5) The application adopts two-step ion exchange chromatography and one-step reversed phase chromatography to separate and purify, replaces the conventional four-step chromatography, reduces the production period, reduces the use of organic solvents and saves the cost.

(6) The fusion protein of the application contains insulin aspart with high specific gravity (the fusion ratio is increased), and the green fluorescent protein in the fusion protein contains arginine and lysine, can be digested into small fragments by protease, has large molecular weight difference compared with the target protein, and is easy to separate.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

EXAMPLE 1 construction and expression of insulin aspart expression Strain

Construction of an insulin aspart expression plasmid is described in the examples of patent application No. 201910210102.9. The DNA fragment containing the fusion protein FP-TEV-R-G was cloned into the expression vector plasmid pBAD/His A (purchased from NTCC company, kanamycin resistance) at the NcoI-XhoI site downstream of the araBAD promoter, resulting in plasmid pBAD-FP-TEV-R-G. The plasmid map is shown in FIG. 1.

The DNA sequence of the pylRs was cloned into the expression vector plasmid pEvol-pBpF (from NTCC, chloramphenicol resistant) downstream of the araBAD promoter at the SpeI-SalI site, and the DNA sequence of the tRNA of lysyl-tRNA synthetase (pylTcua) was inserted downstream of the proK promoter by PCR. This plasmid was designated pEvol-pylRs-pylT. The plasmid map is shown in FIG. 2.

The constructed plasmids pBAD-FP-TEV-R-G and pEvol-pylRs-pylT are transformed into an escherichia coli TOP10 strain together, and the recombinant escherichia coli strain expressing the insulin aspart fusion protein FP-TEV-R-G is obtained by screening. Wherein the FP sequence is u8-u9 (SEQ ID NO: 3), and the amino acid sequence of the fusion protein is shown as SEQ ID NO:1.

GIKANFKIRHNVEDVQLADHYQQNTPIGENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTDK(Boc)TRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:1)

Using the same method, the following expression plasmids were constructed:

amino acid sequence of pBAD-FP (u 9-u10-u 11) -TEV-R-G, fusion protein

VQLADHYQQNTPIGHYLSTQSVLSKDHMVLLEFVTAAGIENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTDK(Boc)TRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:22)。

Amino acid sequence of pBAD-FP (u 10-u 11) -TEV-R-G, fusion protein

HYLSTQSVLSKDHMVLLEFVTAAGIENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTDK(Boc)TRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:23)。

Amino acid sequence of pBAD-FP (gIII) -R-G, fusion protein

MKKLLFAIPLVVPFYSHSTMELEICSWYHMGIRSFLEQKLISEEDLNSAVDRFVNQHLCGSHLVEALYLVCGERGFFYTDK(Boc) TRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 10). Wherein FP (gIII) represents a gIII signal peptide derived from a green fluorescent protein.

And constructing a corresponding expression strain by using a conventional method in the field, and carrying out electrophoresis detection on the expression quantity of the insulin aspart fusion protein.

Preparing a seed liquid culture medium, inoculating, performing two-stage culture to obtain a second-stage seed liquid, culturing for 20h, wherein the OD600 reaches about 180, fermenting to obtain about 3L of fermentation liquid, and centrifuging to obtain about 130g/L of wet thalli. After the fermentation liquor is centrifugated, adding a crushing buffer solution, using a high-pressure homogenizer to perform bacteria breaking twice, adding tween 80 with a certain concentration, EDTA-2Na and the like for washing after centrifugation, washing once, and centrifugally collecting sediment to obtain the inclusion body. Approximately 43g of wet weight inclusion bodies per liter of fermentation broth are finally obtained.

EXAMPLE 2 solubilization and renaturation of inclusion bodies

Adding 8mol/L urea solution into the inclusion body, regulating the pH to 9.0-10.0 by sodium hydroxide, stirring for 1-3h at room temperature, controlling the protein concentration to 10-20 mg/mL, adding beta-mercaptoethanol to a final concentration of 15-20mmol/L, and continuously stirring for 0.5-1.0 h.

Adding inclusion body solution into renaturation buffer solution in a liquid drop way, diluting and renaturating by 5-10 times, maintaining the pH value of the renaturation solution to be 9.0-10.0, and stirring and renaturating for 10-20 h.

After 20h of renaturation, HPLC is used for detecting the fusion content of the insulin aspart with correct renaturation folding, and the renaturation rate is more than 75%.

EXAMPLE 3 cleavage of fusion proteins

Adding dilute hydrochloric acid into the renaturation solution to adjust the pH value to 8.0-9.5, adding recombinant trypsin according to a ratio of 1:3000, adding carboxypeptidase B according to a ratio of 1:15000, and carrying out enzyme digestion at 36-38 ℃ for 14-20h to obtain the Boc-insulin aspart after enzyme digestion.

After 16h of digestion, the content of Boc-insulin aspart in the digestion liquid is detected by HPLC, and when the concentration difference of the Boc-insulin aspart detected in two continuous hours is less than 3%, the digestion is completed. Finally, the concentration of Boc-insulin aspart in the enzyme cutting liquid is 0.4-0.6g/L, and the enzyme cutting rate is more than 80%.

EXAMPLE 4 first chromatography

According to the difference of isoelectric points of proteins, an anion exchange filler is selected to carry out crude extraction on Boc-insulin aspart. Balancing 3-5 column volumes of the chromatographic column by using 5-20mmol/L sodium carbonate and buffer solution with pH of 8.0-9.0, fully combining Boc-insulin aspart with anion packing, loading the sample to be lower than 45g/L, linearly eluting 15 column volumes by using 100-500mmol/L sodium chloride after loading, collecting eluted protein solution, and detecting a result of SDS protein gel electrophoresis, wherein the result is shown in figure 4. The yield of Boc-insulin aspart is above 90% and the purity is above 70%.

EXAMPLE 5 deprotection

And drying the Boc-insulin aspart which is subjected to anion exchange chromatography crude extraction to obtain Boc-insulin aspart dry powder. Adding 4-10mL of TFA into 1g of Boc-insulin aspart dry powder, stirring at room temperature of 18-25 ℃ for reaction for 0.5-3.0h, adding NaOH to adjust the pH of protein solution to be more than 2.5 after the reaction is finished, and stopping deprotection reaction to finally obtain insulin aspart, wherein the deprotection yield is more than 90% and the purity is more than 75% by HPLC measurement, and the HPLC detection result is shown in figure 5.

EXAMPLE 6 second chromatography

According to the difference of charges of substances, the insulin aspart is purified by adopting an anion exchange chromatography technology to remove part of impurities. And (3) balancing the volume of the ion column for 3-5 columns by using 20mmol/L glycine and a buffer solution with the pH of 9.0, combining the insulin aspart protein solution with a cationic filler, controlling the loading amount of the insulin aspart to be not more than 10mg/mL, and finally isocratically eluting by using a sodium chloride solution with the concentration of 0.27mol/L, and collecting an insulin aspart sample. The purity of insulin aspart in the collected liquid was 97.83%, and the yield was 87.96%. The HPLC detection pattern is shown in FIG. 6.

EXAMPLE 7 third chromatography

According to the difference of the hydrophobicity of the substances, the reversed phase chromatographic column technology is adopted to carry out fine purification on the insulin aspart. Diluting insulin aspart solution obtained by secondary chromatography with pure water for more than 4 times, and combining with C8 reversed-phase filler. The loading of insulin aspart is controlled to be not higher than 5mg/mL, and 10CV is eluted at an isocratic containing 200mmol/L sodium acetate and 26% acetonitrile. The results show that: the yield of the insulin aspart is 93.8 percent and the purity is 99.70 percent. The detection results are shown in FIG. 7.

EXAMPLE 8 crystallization and lyophilization

Adding distilled water into the triple chromatography elution collecting solution to dilute acetonitrile solution until the content is not more than 15% (v/v), sequentially adding glycine with the final concentration of 0.7mol/L, sodium chloride with the final concentration of 0.8mol/L and saturated phenol with the concentration of 0.5%, and then, according to the mole ratio of 1:3 adding zinc acetate, adjusting pH to 5.6-6.0 with acetic acid, standing at 4-8deg.C for crystallization for more than 16 hr, and observing rod-like crystal formation under microscope (see figure 8). Collecting crystals, and lyophilizing to obtain crude drug of insulin aspart.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

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Claims

1. A method of preparing insulin aspart, the method comprising the steps of:

(i) Fermenting by utilizing recombinant bacteria to prepare recombinant insulin aspart fusion protein fermentation liquor;

(ii) Performing enzyme digestion on the recombinant insulin aspart fusion protein, so as to obtain a mixed solution I containing Boc modified insulin aspart;

(iii) Deprotecting the Boc modified insulin aspart to obtain a mixed solution II containing deprotected insulin aspart;

(iv) Purifying the mixed solution II to obtain insulin aspart;

the recombinant insulin aspart fusion protein has a structure shown in a formula I:

A-FP-TEV-R-G(I)

in the method, in the process of the application,

"-" represents a peptide bond;

a is a non-or leader peptide,

FP is a green fluorescent protein folding unit,

TEV is the cleavage site;

r is arginine or lysine for enzyme digestion;

g is insulin aspart or an active fragment thereof;

wherein the green fluorescent protein folding unit is a beta-folding unit selected from the group consisting of: u8-u9, u9-u10-u11, u10-u11;

beta-sheet unit Amino acid sequence u8 GIKANFKIRHNVED(SEQ ID NO:18) u9 VQLADHYQQNTPIG(SEQ ID NO:19) u10 HYLSTQSVLSKD(SEQ ID NO:20) u11 HMVLLEFVTAAGI(SEQ ID NO:21)

And G is a Boc (t-butoxycarbonyl) -modified insulin aspart precursor, having a structure represented by formula II:

GB-X-GA(II)

in the method, in the process of the application,

x is a connecting peptide;

2. The method of claim 1, further comprising, between step (ii) and step (iii), the steps of:

(I) And carrying out anion exchange chromatography on the mixed solution I for the first time, thereby obtaining dry powder of the insulin aspart containing Boc modification.

3. The method of claim 1, wherein the purification treatment of step (iv) comprises the steps of:

(II) using glycine as a mobile phase, and carrying out anion exchange chromatography on the mixed solution II for the second time, thereby obtaining eluent II containing deprotected insulin aspart;

4. The method of claim 1, wherein in step (i), recombinant insulin aspart fusion protein inclusion bodies are isolated from the fermentation broth of the recombinant bacterium.

5. The method of claim 4, further comprising the step of denaturing and renaturing the inclusion bodies to obtain recombinant insulin aspart fusion proteins having correctly folded proteins in step (i).

6. The method of claim 1, wherein in step (ii) trypsin and carboxypeptidase B are used for cleavage.

7. The method of claim 1, wherein the recombinant insulin aspart fusion protein has the sequence set forth in SEQ ID NOs 1, 22, 23.

8. The method of claim 1, wherein in step (ii) trypsin and carboxypeptidase B are used for cleavage.

9. The method of claim 8, wherein in step (ii) the mass ratio of carboxypeptidase B to recombinant insulin aspart fusion protein is 1: (20000-30000).

10. The method of claim 9, wherein in step (ii), the trypsin is recombinant porcine trypsin.

11. The method according to claim 1, wherein in step (iii), TFA (trifluoroacetic acid) is added to the reaction system to perform the deprotection treatment.

12. The method of claim 11, wherein in step (iii), the ratio of Boc-modified insulin aspart to TFA is 1g: (4-10 mL).

13. The method of claim 1, wherein the TEV is a TEV cleavage site.

14. A method of preparing an insulin aspart formulation comprising the steps of:

(iv) Purifying the mixed solution II to obtain insulin aspart, crystallizing and freeze-drying the prepared insulin aspart to obtain a freeze-dried product, namely an insulin aspart preparation;

wherein, the recombinant insulin aspart fusion protein has a structure shown in a formula I:

A-FP-TEV-R-G(I)

in the method, in the process of the application,

"-" represents a peptide bond;

a is a non-or leader peptide,

FP is a green fluorescent protein folding unit,

TEV is the cleavage site;

r is arginine or lysine for enzyme digestion;

g is insulin aspart or an active fragment thereof;

GB-X-GA(II)

in the method, in the process of the application,

x is a connecting peptide;