CN113773399B

CN113773399B - Insulin glargine derivative and application thereof

Info

Publication number: CN113773399B
Application number: CN202010526320.6A
Authority: CN
Inventors: 陈卫; 刘慧玲; 骆莉
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: 2023-05-12
Anticipated expiration: 2040-06-09
Also published as: CN113773399A; WO2021249443A1; CN115698089A

Abstract

The invention provides a insulin glargine derivative and a preparation method thereof. In particular, the present invention provides fusion proteins comprising a green fluorescent protein folding unit and insulin glargine or an active fragment thereof. The fusion protein of the invention has obviously improved expression quantity, correct folding of insulin glargine protein in the fusion protein and biological activity. In addition, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by protease, and compared with the target protein, the fusion protein has large molecular weight difference and is easy to separate. The invention also provides a method for preparing insulin glargine by using the fusion protein and a preparation intermediate.

Description

Insulin glargine derivative and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a insulin glargine derivative and application thereof.

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 glargine achieves the aim of long acting and maintaining time by changing amino acid of recombinant human insulin glargine and slightly adjusting the formula. Insulin glargine is prepared by substituting charge neutral glycine for asparagine at 21 position of human insulin A chain, so that hexamer is more stable. 2 arginines are added at the C end of the B chain, so that the isoelectric point is improved from 5.4 to 6.7, insulin glargine is transparent solution in weak acid environment, and the solubility of the insulin glargine is greatly reduced in physiological environment to generate precipitation. A small amount of zinc is added into the formula, so that crystals can be formed under the skin during subcutaneous injection, and the absorption time is delayed, thereby playing a role in reducing blood sugar for a long time.

The preparation of insulin glargine is generally carried out by preparing a precursor by genetic engineering technology, preparing Sainophenanthrene (US 5663291) by recombinant fermentation by using escherichia coli, and then carrying out fermentation expression by using pichia pastoris (bioconon Baiaokang, IN2008CHE 000310). The classical preparation processes for converting insulin glargine precursors into insulin glargine all use a transpeptidation process, such as the preparation of the insulin glargine precursor B-Arg (B31) Arg (B32) -a-Gly (a 21) by starting with celecoxib (US 2009/0192073 A1), the insulin glargine and insulin glargine analogues B-Arg (B31) -a-Gly (a 21) obtained by trypsin cleavage, and the insulin glargine analogues are converted into insulin glargine after transpeptidation and deprotection. The process for preparing insulin glargine by the method is complex, the yield is low, and the production cost is extremely high.

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

Disclosure of Invention

The invention aims to provide a glargine derivative and application thereof.

In a first aspect of the present invention, there is provided a recombinant insulin glargine fusion protein having the structure shown in formula I:

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

in the method, in the process of the invention,

"-" 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 glargine 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 u2-u3, u4-u5, u1-u2-u3, u3-u4-u5 or u4-u5-u6.

In another preferred embodiment, G is a Boc-modified insulin glargine precursor having the structure of formula II:

GB-X-GA (II)

in the method, in the process of the invention,

GB is Boc modified insulin glargine B chain, the amino acid sequence is shown in the 1 st-32 th positions of SEQ ID NO. 5,

X is a free or linked peptide, preferably the amino acid sequence of the linked peptide is R, or as shown in SEQ ID NO. 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLALE GSLQKR);

GA is insulin glargine A chain, and the amino acid sequence is shown in 33-53 positions of SEQ ID NO. 5.

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

In another preferred embodiment, G is Boc modified insulin glargine 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, X is absent.

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

In another preferred embodiment, the insulin glargine forms an inter-chain disulfide bond between position 7 of the B chain and position 7 of the A chain (A7-B7), and between position 19 of the B chain and position 20 of the A chain (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 invention, there is provided a double-stranded insulin glargine fusion protein having the structure shown in formula III:

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

in the method, in the process of the invention,

"-" 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 glargine, and the main chain has a structure shown in the following formula IV;

in the method, in the process of the invention,

"|" represents a disulfide bond;

GA is insulin glargine A chain, the amino acid sequence is shown in 33-53 positions of SEQ ID NO. 5,

x is none or a connecting peptide;

GB is a B chain of insulin glargine modified by Boc at 29 th site, and the amino acid sequence is shown in 1-32 th site of SEQ ID NO. 5;

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

In another preferred embodiment, X is a free or linked peptide, preferably the amino acid sequence of the linked peptide is R, or as shown in SEQ ID NO. 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR);

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

GB-X-GA(II)

in the method, in the process of the invention,

GB is the B chain of insulin glargine modified by Boc at 29 th site, the amino acid sequence is shown in 1-32 th site of SEQ ID NO. 5,

x is a free or linked peptide, preferably the amino acid sequence of the linked peptide is R, or as shown in SEQ ID NO. 6-9 (GSKR, AAKR, YPGDVKR or EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR);

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

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

in the method, in the process of the invention,

"|" represents a disulfide bond;

GB is insulin glargine B chain, the amino acid sequence is shown in the 1 st-32 rd position 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 invention, there is provided a method of preparing insulin glargine, the method comprising the steps of:

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

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

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

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

In another preferred embodiment, the purification process comprises the steps of:

(I) Performing first cationic chromatography on the mixed solution II to obtain an eluent I containing recombinant insulin glargine (third protein);

(II) subjecting said eluate I to a second cationic analysis, thereby obtaining an eluate II comprising recombinant insulin glargine (a third protein);

(III) carrying out reverse phase chromatography on the eluent II, thereby obtaining insulin glargine.

In another preferred embodiment, in step (ii), the temperature of the cleavage is 15-25 ℃, preferably 18-22 ℃.

In another preferred embodiment, in step (ii), succinic acid and L-lysine are added as cleavage aids during the cleavage process.

In another preferred embodiment, in step (ii), succinic acid is contained in the cleavage system and the concentration of succinic acid is 10-50mmol/L, preferably 15-30mmol/L, more preferably 30mmol/L.

In another preferred embodiment, in step (ii), L-lysine is contained in the cleavage system and the concentration of L-lysine is 10-50mmol/L, preferably 15-30mmol/L, more preferably 30mmol/L.

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

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

In another preferred embodiment, the recombinant Boc-insulin glargine is insulin glargine 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 recombinant insulin glargine 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 glargine fusion protein.

In another preferred embodiment, in step (i), recombinant insulin glargine 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 glargine fusion protein (first protein) having a correctly folded protein.

In another preferred embodiment, the protein fold is correct and the insulin glargine A and B chains in the recombinant insulin glargine fusion protein comprise intra-chain disulfide bonds.

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

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 glargine precursor is 1:1000-40000, preferably 1:3000-10000.

In another preferred embodiment, in step (ii), the cleavage auxiliary agent is included in the cleavage system to effectively increase the cleavage yield.

In another preferred embodiment, in step (ii), the time for the cleavage is from 10 to 25 hours, preferably from 14 to 20 hours.

In another preferred embodiment, in step (ii), the pH of the recombinant insulin glargine precursor solution is 7.5-9.0.

In another preferred embodiment, in step (iii), the deprotection treatment is performed with hydrochloric acid.

In another preferred embodiment, in step (iii), the temperature of the deprotection reaction is 25-40 ℃, preferably 36-38 ℃.

In another preferred embodiment, in step (iii), the time of the deprotection reaction is 2 to 6 hours, preferably 4 to 5 hours.

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

In another preferred embodiment, in step (I), cation chromatography is performed using a weak cation exchange filler.

In another preferred embodiment, in step (I), the ion column is equilibrated with 40-60mmol/L acetic acid.

In another preferred embodiment, in step (I), the loading of recombinant insulin glargine (third protein) is 12mg/ml or less.

In another preferred embodiment, in step (I), a linear gradient elution is performed with ammonium acetate containing isopropanol.

In another preferred embodiment, the step (I) further comprises a step of desalting treatment.

In another preferred example, the target protein is precipitated by isoelectric point method in desalting treatment.

In another preferred example, in the desalting treatment, 1-3mmol/L zinc acetate solution is added under stirring, then sodium hydroxide is added dropwise to adjust pH to 6.0-7.0, and the mixture is left to stand after stirring.

In another preferred embodiment, the temperature of the standing is 2 to 8 ℃.

In another preferred embodiment, the time for the standing is 1 to 5 hours.

In another preferred example, after the standing, a microfiltration membrane with a pore size of 0.1-0.4 μm is selected for filtration.

In another preferred embodiment, the displacement is performed with an ammonium acetate solution after microfiltration membrane filtration.

In another preferred embodiment, in step (II), a sodium chloride solution containing isopropyl alcohol is used as the mobile phase.

In another preferred embodiment, in step (II), the concentration of the sodium chloride solution is 0.1 to 0.5mol/L.

In another preferred embodiment, in step (II), the linear elution is performed with a mobile phase.

In another preferred embodiment, in step (II), the loading of insulin glargine in the eluent I is less than or equal to 5mg/ml, preferably the loading is less than or equal to 4mg/ml.

In another preferred embodiment, in step (III), an acetonitrile solution containing sodium citrate is used as the mobile phase.

In another preferred embodiment, in step (III), the sodium citrate concentration in the mobile phase is 80-120mmol/L, preferably 90-110mmol/L. In another preferred embodiment, in step (III), the pH of the mobile phase is 4.0-4.5, preferably 4.1-4.2.

In another preferred embodiment, in step (III), a gradient elution is performed with a mobile phase.

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

In another preferred embodiment, after step (III), the method further comprises the steps of precipitating and freeze-drying the produced insulin glargine, thereby producing a freeze-dried product.

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

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

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

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

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

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

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

It is understood that within the scope of the present invention, the above-described technical features of the present invention 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 of insulin glargine after the first chromatography.

Figure 4 shows the HPLC detection profile of insulin glargine after the second chromatography.

Figure 5 shows the HPLC detection profile of insulin glargine after the third chromatography.

Detailed Description

The present inventors have studied extensively and intensively to find a insulin glargine derivative and a method for producing the same. In particular, the present invention provides fusion proteins comprising a green fluorescent protein folding unit and insulin glargine or an active fragment thereof. The fusion protein of the invention has obviously improved expression quantity, correct folding of insulin glargine protein in the fusion protein and biological activity. In addition, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by protease, and compared with the target protein, the fusion protein has large molecular weight difference and is easy to separate. The invention also provides a method for preparing insulin glargine by using the fusion protein and a preparation intermediate.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly 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 glargine

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 glargine belongs to the third generation recombinant insulin, is long-acting insulin, has no obvious peak value and risks of hypoglycemia, sudden death and the like caused by the peak value, and is a product with the largest market share in the insulin market for years due to the characteristics of safety and long-acting property, and accounts for more than 30% of the whole insulin market.

Insulin glargine achieves the aim of long acting and maintaining time by changing amino acid of recombinant human insulin and slightly adjusting the formula. Insulin glargine is prepared by substituting charge neutral glycine for asparagine at 21 position of human insulin glargine A chain, so that hexamer is more stable. 2 arginines are added at the C end of the B chain, so that the isoelectric point is improved from 5.4 to 6.7, insulin glargine is transparent solution in weak acid environment, and the solubility is greatly reduced to precipitate in physiological environment. A small amount of zinc is added into the formula, so that crystals can be formed under the skin during subcutaneous injection, and the absorption time is delayed, thereby playing a role in reducing blood sugar for a long time.

Fusion proteins

By using the green fluorescent protein folding unit, two fusion proteins, namely the recombinant insulin glargine fusion protein containing the single-chain insulin glargine precursor according to the first aspect of the invention and the double-chain insulin glargine fusion protein containing the double-chain insulin glargine according to the third aspect of the invention, are constructed. In fact, the protective scope of the two fusion proteins of the invention may overlap, for example, the double-stranded form of insulin glargine 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 invention comprise 2-6, preferably 2-3 β -sheet units selected from the group consisting of:

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.

In another preferred embodiment, the green fluorescent protein folding unit is u3-u4-u5.

As used herein, 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 invention in monomeric and multimeric form. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention 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 invention. The polypeptide fragment, derivative or analogue of the present invention 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 invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

The invention also provides analogs of the fusion proteins of the invention. These analogs may differ from the polypeptides of the invention 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 invention are not limited to the representative polypeptides exemplified above.

In addition, the fusion proteins of the invention 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 invention" may include polynucleotides encoding a fusion protein of the invention, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The invention 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 invention. 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 invention 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 invention relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention. In the present invention, "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 invention are preferably provided in isolated form, and more preferably purified to homogeneity.

The full-length polynucleotide sequence of the present invention 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 invention, 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 invention (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 invention. 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 invention 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 invention also relates to vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors of the invention or the fusion protein coding sequences of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention 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 invention, 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 invention, 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 invention 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 invention 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 invention. 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 invention 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 insulin glargine, and the one-step yield reaches 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 enzyme digestion yield, reduce the impurity of the insulin glargine analogue and provide convenience for subsequent purification and separation.

(3) In the enzyme digestion process, the enzyme digestion yield is improved by optimizing the proportion of trypsin, controlling the enzyme digestion temperature and adding enzyme digestion auxiliary agents.

(4) In the deprotection step, boc-insulin glargine is converted into insulin glargine, 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 desalting treatment step precipitates target protein through an isoelectric point method, has a purifying effect to a certain extent, and removes part of impurity proteins. In addition, the filtering column with the pore diameter of 0.1-0.2 mu m is used for replacing the ultrafiltration membrane with small pore diameter, so that the filtering time is greatly shortened.

(6) The invention 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.

(7) The fusion protein of the invention contains insulin glargine 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 invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. 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 SpringHarbor 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 glargine-expressing Strain

Construction of insulin glargine expression plasmids, methods of construction are referred to in the art, and specific reference may be made to the description of examples in patent application No. 201910210102.9. The DNA fragment of 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.

And (3) transforming the constructed insulin glargine expression vector into an escherichia coli strain, and screening to obtain a recombinant strain for expressing the recombinant insulin glargine precursor. Wherein the amino acid sequence of the recombinant insulin glargine precursor is shown as SEQ ID NO. 1, and the 73 rd position (29 th position) of the precursor sequence is Boc-protected lysine.

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGDENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)TRRGIVEQCCTSICSLYQLENYCG(SEQ ID NO:1)

The structure of the recombinant insulin glargine precursor is as follows:

A-FP-TEV-R-G

in the method, in the process of the invention,

"-" represents a peptide bond;

a is leader peptide sequence MVSKGEELFTGV (SEQ ID NO: 2)

FP is a green fluorescent protein folding unit with a sequence of KLTLKFICTTYVQERTISFKDTYKTRAEVKFEGD (SEQ ID NO: 3);

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

r is arginine or lysine for trypsin cleavage;

g is insulin glargine modified at position 29 by Boc and has the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPK (Boc) TRRGIVEQCCTSICSLYQLENYCG (SEQ ID NO: 5).

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 150g/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 pH to 8.0-9.0 with sodium hydroxide, stirring at room temperature for 1-3h, controlling protein concentration to 10-20mg/mL, adding beta-mercaptoethanol to final concentration to 10-20mmol/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 9.0-10.5, stirring and renaturating for 10-20 h at the temperature of 2-8 ℃.

EXAMPLE 3 fusion protease cleavage

10KD ultrafiltration membrane is selected to concentrate renaturation liquid by 8-10 times. Adding dilute hydrochloric acid into the renaturation solution to adjust the pH value to 7.5-9.0. The protein concentration of the renaturated concentrate was measured by the Bradford method and the total protein amount was calculated. Adding recombinant trypsin under stirring, wherein the mass ratio of the recombinant trypsin to the total protein of the renaturation solution is 1:3000-1:10000, adding 30mmol/L succinic acid or 30mmol/L L-lysine, and the enzyme digestion temperature is 15-25 ℃ and the enzyme digestion time is 14-20 h.

After 10h of digestion, the content of Boc-insulin glargine in the digestion liquid is detected by HPLC, and when the concentration of Boc-insulin glargine detected in two continuous hours is less than 3%, the digestion is completed. Finally, the concentration of Boc-insulin glargine in the enzyme cutting liquid is 0.9-1.3 mg/mL, and the enzyme cutting rate is 30-40%.

EXAMPLE 4 deprotection

Hydrochloric acid is added into the enzyme cutting liquid, the reaction is carried out for 4 to 5 hours at the temperature of 25 to 40 ℃ to remove Boc protecting group, sodium hydroxide is added to adjust the pH value to 3.0 to 3.5, and the deprotection reaction is stopped. The deprotection yield is about 75-80%, and the purity of the insulin glargine is about 20%. EXAMPLE 5 first chromatography

The initial protein mixture contains a large amount of mycoprotein residues, and then contains enzyme digestion byproducts generated in the enzyme digestion process and hydrolyzed protein generated by deprotection. According to the difference of isoelectric points of proteins, cation exchange filler is selected to carry out crude extraction on insulin glargine. And (3) balancing 3-5 column volumes of the ion column by using 50mmol/L acetic acid and a buffer solution with the pH value of 3.0-3.5, combining insulin glargine with a cationic filler, controlling the loading capacity of the insulin glargine to be lower than 12mg/ml, eluting 20 column volumes of the chromatographic column by using a linear gradient of 1mol/L ammonium acetate containing isopropanol after loading, collecting eluted target protein peaks, and collecting elution peaks, wherein the detection result of SDS protein gel electrophoresis is shown in figure 3. The yield of the insulin glargine of the chromatography I is 80-85%, the purity is about 30%, and most mycoproteins and partial enzyme digestion byproducts can be removed in the step.

Then, desalting treatment was performed.

The collection liquid eluted by the chromatography I is added with 2mmol/L zinc acetate solution and stirred for 2 to 5min. Dropwise adding sodium hydroxide under stirring to adjust the pH to 6.0-7.0, continuously stirring for 2-10 min, standing at 2-8 ℃ for 1-5 h. Concentrating the sample to more than 10 times by using a microfiltration membrane with the pore diameter of 0.1-0.4 mu m, and replacing the sample by using 6 times of ammonium acetate solution with the pH value of 6.0-7.0.

The result shows that the yield of the insulin glargine is higher than 95%, and the purity is improved by about 40%.

EXAMPLE 6 second chromatography

The purity of insulin glargine in the initial mixed solution of the step is about 40%, wherein B is absent ₃₂ Arginine insulin glargine analogues are very similar in structure to insulin glargine and therefore difficult to remove, and high resolution cationic chromatography techniques are used to purify insulin glargine to remove some impurities based on differences in material charge.

Clarifying insulin glargine protein solution, and adding acetic acid to adjust pH to 2.5-4.5. The buffer solution with the pH value of 3.4 and the concentration of 75mmol/L glycine and 30% isopropyl alcohol is used for balancing the volume of 3-5 columns, the insulin glargine protein solution is combined with the cationic filler, the loading capacity of insulin glargine is controlled to be not more than 4mg/mL, and the insulin glargine sample is collected by linear elution with 0.3mol/L sodium chloride containing isopropyl alcohol. Finally obtaining insulin glargine with purity higher than 97 percent, and the yield reaches 75.6 percent, wherein B is absent ₃₂ The content of insulin glargine in arginine is controlled to be less than 0.5%, and the HPLC detection result is shown in figure 4

EXAMPLE 7 third chromatography

According to the difference of the hydrophobicity of substances, the reverse phase chromatographic column technology is adopted to carry out fine purification on the insulin glargine, and the hydrolysate of the insulin glargine is mainly removed. Diluting insulin glargine solution obtained by secondary chromatography with pure water for more than 4 times, and combining with C8 reversed-phase filler. Controlling the loading capacity of insulin glargine to be not higher than 5mg/mL, carrying out gradient elution by using acetonitrile solution containing 100mmol/L sodium citrate and having pH of 4.2, collecting the elution peak of insulin glargine, and finally obtaining the insulin glargine with the yield of 77.3% and the purity of 99.18%, wherein the HPLC detection result is shown in figure 5.

EXAMPLE 8 precipitation and lyophilization

Adding water for injection into the three-time chromatography elution collection liquid to dilute the water until the acetonitrile content is not more than 15% (v/v), adding zinc acetate until the concentration is 2mmol/L, adjusting the pH to 6.8-7.1 by using sodium hydroxide, and standing and precipitating at 4-8 ℃. Collecting the precipitate, washing the precipitate with more than 100 times of water for injection, collecting the washed precipitate sample, and drying to obtain insulin glargine.

Comparative example

The construction and expression of the fusion protein expression strain were carried out in a similar manner to example 1, except that the amino acid sequence of the fusion protein used for expression was as shown in SEQ ID NO. 10.

MKKLLFAIPLVVPFYSHSTMELEICSWYHMGIRSFLEQKLISEEDLNSAVDRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)TRRGIVEQCCTSICSLYQLENYCG(SEQ ID NO:10)

The fusion protein comprises a B chain and an A chain of insulin glargine and also comprises a gIII signal peptide.

The results showed that the culture was carried out for 20h, OD ₆₀₀ About 140, the fermentation was completed to obtain about 3L of fermentation liquid, and the fermentation liquid was centrifuged to obtain about 105g/L of wet cell. After the fermentation broth is centrifuged, a crushing buffer solution is added, and the fermentation broth is subjected to bacteria crushing twice by a high-pressure homogenizer, and the sediment is collected by centrifugation to obtain the inclusion body. About 30g of wet weight inclusion bodies per liter of fermentation broth are finally obtained.

The results show that compared with the expression of the fusion protein with a conventional structure, the expression quantity of the fusion protein is obviously improved, and the insulin glargine protein in the fusion protein is correctly folded and has biological activity.

All documents mentioned in this application are incorporated by reference 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 claims appended hereto.

Sequence listing

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Claims

1. A recombinant insulin glargine fusion protein, which is characterized in that the structure is shown as a formula I:

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

in the method, in the process of the invention,

"-" 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 glargine or an active fragment thereof;

wherein the green fluorescent protein folding units are u2-u3, u4-u5, u1-u2-u3, u3-u4-u5 or u4-u5-u6:

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)

2. The fusion protein of claim 1, wherein G is a Boc-modified insulin glargine precursor having the structure of formula II:

GB- X-GA (II)

in the method, in the process of the invention,

x is none or a linking peptide;

3. The fusion protein of claim 1, wherein the sequence of the recombinant insulin glargine fusion protein is set forth in SEQ ID NO. 1.

4. The fusion protein of claim 1, wherein the TEV is a TEV cleavage site.

5. A double-chain insulin glargine fusion protein, which is characterized in that the structure is shown in a formula III:

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

in the method, in the process of the invention,

"-" 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;

D is Boc modified double-chain insulin glargine and has a structure shown in the following formula IV;

in the method, in the process of the invention,

"║" represents disulfide bonds;

x is none or a connecting peptide;

6. The double-stranded insulin glargine fusion protein of claim 5, wherein said TEV is a TEV cleavage site.

7. A method of preparing a Boc-modified insulin glargine precursor, the method comprising the steps of:

(i) Fermenting with recombinant bacteria to prepare the fusion protein of claim 1;

(ii) And (3) performing enzyme digestion on the fusion protein to obtain a Boc modified insulin glargine precursor, wherein the structure of the Boc modified insulin glargine precursor is shown as a formula II:

GB- X-GA (II)

in the method, in the process of the invention,

x is a NO-or-connecting peptide, and the amino acid sequence of the connecting peptide is R or is shown as SEQ ID NO. 6-9;

8. A method of preparing Boc-modified double-stranded insulin glargine, comprising the steps of:

(ii) The fusion protein is subjected to enzyme digestion, so that Boc modified double-chain insulin glargine is obtained,

the structure of the Boc modified double-chain insulin glargine is shown in a formula IV:

GA

║

GB (IV)

in the method, in the process of the invention,

"║" represents disulfide bonds;

GB is insulin glargine B chain, the amino acid sequence is shown in the 1 st-32 rd position of SEQ ID NO: 5, and lysine at the 29 th position of the B chain is N epsilon- (tert-butoxycarbonyl) -lysine.

9. An isolated polynucleotide encoding the recombinant insulin glargine fusion protein of claim 1, the double-stranded insulin glargine fusion protein of claim 5.

10. A vector comprising the polynucleotide of claim 9.

11. A host cell comprising the vector of claim 10, or the polynucleotide of claim 9 integrated into the chromosome, or expressing the recombinant insulin glargine fusion protein of claim 1, or the double-stranded insulin glargine fusion protein of claim 5.

12. A formulation or pharmaceutical composition comprising the recombinant insulin glargine fusion protein of claim 1, the double-stranded insulin glargine fusion protein of claim 5, and a pharmaceutically acceptable carrier.