CN113773396A

CN113773396A - Insulin detemir derivative and application thereof

Info

Publication number: CN113773396A
Application number: CN202010523782.2A
Authority: CN
Inventors: 刘慧玲; 唐亚连; 杨接运
Original assignee: Ningbo Kunpeng Biotech Co Ltd
Current assignee: Ningbo Kunpeng Biotech Co Ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-10

Abstract

The invention provides a insulin detemir derivative and a preparation method thereof. Specifically, the invention provides a fusion protein comprising a green fluorescent protein folding unit and insulin detemir or an active fragment thereof. The fusion protein of the invention has obviously improved expression level, correct folding of insulin detemir protein in the fusion protein and biological activity. Moreover, the folding unit of the green fluorescent protein in the fusion protein 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 also provides a method for preparing insulin detemir by using the fusion protein and a preparation intermediate.

Description

Insulin detemir derivative and application thereof

Technical Field

The invention relates to the field of biomedicine, and more particularly relates to a insulin detemir derivative and application thereof.

Background

Diabetes is a major disease threatening human health worldwide. In China, the prevalence rate of diabetes is on a rapid rising trend along with the change of life styles and the accelerated aging process of people. Acute and chronic complications of diabetes, especially chronic complications, accumulate a plurality of organs, are disabled, have high fatality rate, seriously affect physical and psychological health of patients and bring heavy burden to individuals, families and society.

Insulin and its analogs are the most direct and effective therapeutic drugs for diabetes, and insulin has great market potential in China. Insulin detemir is a neutral, soluble, long-acting Insulin. It is an insulin analogue, which is formed by removing threonine at position 30 of B chain from prototype insulin, and combining a straight chain fatty acid with 14 carbons at lysine side chain epsilon-amino group at position B29 through acylation. The acylated fatty acid can stabilize the self-aggregation of insulin molecules, and can be reversibly combined with albumin, so that the insulin detemir is slowly absorbed from subcutaneous injection parts, the action duration is prolonged, and the risk of hypoglycemia can be reduced while the blood sugar is controlled.

Since proinsulin, insulin or insulin analogs all have a free alpha-amino group at the amino acid position a1, B1 and a free epsilon-amino group on the lysine side chain at position B29, all three free amino groups can form covalent bonds with active esters, acid halides or acid anhydrides to form monoacylated insulin, diacylated insulin or even triacylated products. From the acylation route disclosed in the various patents of the Novonid application, the N-terminal alpha-amino groups of both chains of insulin are first protected by derivatization with di-tert-butyl dicarbonate (BOC), the epsilon-amino group of lysine at position B29 is acylated with succinimidyl myristate, and finally deprotected with TFA to form insulin detemir. If the acylating agent is excessive, a large amount of di-substituted or tri-substituted derivatives can be generated, so that the process has complicated steps and many byproducts, the difficulty of downstream purification can be increased, and the overall yield is not improved. In the epsilon-amino selective acylation patent (Chinese patent CN1171742A), one-step selective acylation is adopted, and preferably activated ester (palmitic acid N-succinimidyl ester) is utilized to carry out acylation reaction under the alkaline condition of pH 10.5, and the reaction is terminated under the acidic condition.

The existing method for preparing insulin detemir or insulin analogues thereof from insulin precursors through acylation reaction has the defects of low acylation efficiency, more byproducts, high cost and the like to different degrees. Therefore, the development of a novel industrialized efficient preparation method of the insulin detemir or the analogue thereof with high acylation efficiency, few byproducts and low production cost lays an important foundation for industrial application.

Disclosure of Invention

The invention aims to provide a insulin detemir derivative and application thereof.

In a first aspect of the invention, there is provided a insulin detemir fusion protein having, from N-terminus to C-terminus, the structure shown in formula III:

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

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

a is a null or leader peptide,

FP is a folding unit of green fluorescent protein,

TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site;

r is arginine or lysine for enzyme digestion;

g is a insulin detemir backbone or an active fragment thereof;

wherein said green fluorescent protein fold units comprise 2-6 β -sheet 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, u8-u9, u9-u10-u11, u10-u11 or u4-u5-u 6.

In another preferred embodiment, G is Boc modified insulin detemir precursor having the structure shown in formula IV:

it has the structure shown in formula IV:

GB-X-GA (IV)

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

GB is a 29 th Boc modified insulin detemir B chain, the amino acid sequence is shown as SEQ ID NO. 2,

x is a linker peptide, preferably the amino acid sequence of the linker peptide is R, RR, RRR, or as shown in SEQ ID NO. 4-7 (RRGSKR, RRAAKR, RRYPGDVKR or RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR);

GA is insulin detemir A chain, and the amino acid sequence is shown in SEQ ID NO 3.

In another preferred embodiment, the green fluorescent protein folding unit is u3-u4-u5, u8-u9, u9-u10-u11 and u10-u 11.

In another preferred embodiment, the amino acid sequence of the green fluorescent protein folding unit is shown as SEQ ID NO. 9.

In another preferred embodiment, the amino acid sequence of the insulin detemir fusion protein is shown in SEQ ID NO 1, 23, 24 and 25.

In another preferred embodiment, the 29 th lysine of GB is N ∈ - (tert-butoxycarbonyl) -lysine.

In another preferred embodiment, the insulin detemir B chain comprised in the insulin detemir backbone does not comprise a side chain.

In another preferred embodiment, the GB does not comprise side chains.

In a second aspect of the present invention, there is provided a insulin detemir backbone fusion protein having a structure represented by formula I from N-terminus to C-terminus:

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

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

a is a null or leader peptide,

FP is a green fluorescent protein folding unit;

TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site (shown as a sequence ENLYFQG, SEQ ID NO: 10);

r is arginine or lysine for enzyme digestion;

d is Boc modified insulin detemir backbone having the structure shown in formula II:

in the formula (I), the compound is shown in the specification,

"║" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

x is nothing or a connecting peptide;

GB is a 29 th Boc modified insulin detemir B chain, and the amino acid sequence is shown as SEQ ID NO. 2;

In another preferred embodiment, the R is used for trypsin enzyme digestion and carboxypeptidase enzyme digestion.

In another preferred embodiment, the C-terminus of the insulin detemir B chain is linked to the N-terminus of the insulin detemir a chain via a linking peptide.

In another preferred embodiment, the amino acid sequence of the linker peptide is R, RR, RRR, or as shown in SEQ ID NOS: 4-7 (RRGSKR, RRAAKR, RRYPGDVKR or RREAEDLQVGQVEL GGGPGAGSLQPLALEGSLQKR).

In another preferred embodiment, said Boc modified detemir insulin backbone comprises a chain a and a chain B, and the chain a and the chain B are connected by interchain disulfide bonds, preferably by two pairs.

In another preferred embodiment, the A chain comprises an intrachain disulfide bond.

In another preferred embodiment, the leader peptide has the sequence shown in SEQ ID NO:8 (sequence MVSKGEELFTGV).

In another preferred embodiment, interchain disulfide bonds are formed between the 7 th position of the B chain and the 7 th position of the A chain (A7-B7), and the 19 th position of the B chain and the 20 th position of the A chain (A20-B19) of the insulin detemir.

In another preferred embodiment, the insulin detemir forms an intrachain disulfide bond between position 6 of the A chain and position 11 of the A chain (A6-A11).

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

GB-X-GA (IV)

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

x is a connecting peptide, preferably the amino acid sequence of the connecting peptide is R, RR, RRR or shown as SE Q ID NO 4-7;

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

in the formula (I), the compound is shown in the specification,

"║" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

GB is insulin detemir B chain, the amino acid sequence is shown in SEQ ID NO. 2, and the 29 th lysine of the B chain is N epsilon- (tert-butyloxycarbonyl) -lysine.

In a fifth aspect of the present invention, there is provided a Boc-modified and Fmoc-modified insulin detemir backbone having the structure represented by formula II:

in the formula (I), the compound is shown in the specification,

"║" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

GB is insulin detemir B chain, the amino acid sequence is shown as SEQ ID NO. 2, and the 29 th lysine of the B chain is N epsilon- (tert-butyloxycarbonyl) -lysine;

and the N ends of the A chain and the B chain are both modified by Fmoc.

In another preferred embodiment, Fmoc is fluorenylmethyloxycarbonyl.

In a sixth aspect of the present invention, there is provided an Fmoc-modified insulin detemir backbone having the structure represented by formula II:

in the formula (I), the compound is shown in the specification,

"║" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

GB is insulin detemir B chain, and the amino acid sequence is shown as SEQ ID NO. 2;

and the N ends of the A chain and the B chain are both modified by Fmoc.

In another preferred embodiment, Fmoc is fluorenylmethyloxycarbonyl.

In a seventh aspect of the present invention, there is provided a method of preparing insulin detemir, the method comprising the steps of:

(A) fermenting by using recombinant bacteria to prepare the insulin detemir precursor fusion protein,

(B) preparing insulin detemir by using the insulin detemir precursor fusion protein,

wherein the insulin detemir fusion protein is as defined in the first aspect of the invention.

In another preferred embodiment, the step (B) further includes the steps of:

(ia) separating and obtaining insulin detemir precursor fusion protein inclusion bodies from the fermentation liquor of the recombinant bacteria, and obtaining insulin detemir main chain fusion protein after renaturation of the inclusion bodies;

(ib) performing enzyme digestion treatment on the insulin detemir backbone fusion protein to obtain a Boc-modified insulin detemir backbone;

(ii) performing Fmoc modification on the Boc modified insulin detemir main chain to prepare Fmoc and Boc modified insulin detemir main chains;

(iii) carrying out de-Boc treatment on the Fmoc and Boc modified insulin detemir main chain to obtain a de-Boc insulin detemir main chain;

(iv) reacting the Boc-removed detemir insulin main chain with a detemir insulin side chain to prepare Fmoc-modified detemir insulin; and

(v) and (3) carrying out Fmoc removal treatment on the Fmoc modified insulin detemir so as to prepare the insulin detemir.

In another preferred example, in step (ib), an enzymatic cleavage treatment is performed using trypsin and carboxypeptidase B.

In another preferred embodiment, said Boc modified insulin detemir backbone is according to the fourth aspect of the invention.

In another preferred embodiment, the Fmoc modification is the modification of the N-terminal of the B chain and the A chain of insulin detemir.

In another preferred embodiment, the insulin detemir side chain is as follows:

in another preferred embodiment, the side chain of insulin detemir is N-succinimidyl myristate.

In another preferred embodiment, Fmoc-Osu, DIPEA (N, N-diisopropylethylamine) and DMF (N, N-dimethylformamide) are added in step (ii) to perform Fmoc modification.

In another preferred embodiment, the molar ratio of Fmoc-Osu, DIPEA and Boc modified insulin detemir backbone added is (3-6): (10-14): (0.8-1.2), preferably (3.5-5.5): (11-13): (0.8-1.2).

In another preferred embodiment, between the step (ii) and the step (iii), a step of purifying the prepared Fmoc and Boc modified insulin detemir backbone, preferably using a methyl tert-ether/petroleum ether mixture, is further included.

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

(a) adding mixed solution of TFA (trifluoroacetic acid), stirring at low temperature, and removing Boc to obtain a Boc-removed product;

(b) the de-Boc product is subjected to a purification treatment, preferably using a methyl tert-ether/petroleum ether mixture, to obtain a solid de-Boc product, i.e. a de-Boc insulin backbone.

In another preferred embodiment, in step (iv), the reaction is carried out in the presence of DIPEA at room temperature.

In another preferred embodiment, in step (iv), the reaction is carried out in DMF.

In another preferred embodiment, the mole ratio of the Boc-depleted insulin detemir backbone, insulin detemir side chains and DIPEA is (0.8-1.2): (2-5): (4-10), preferably 1:2.5: 12.

In another preferred embodiment, in step (v), piperidine-containing DMF solution is added to perform Fmoc removal treatment, thereby preparing the insulin detemir.

In another preferred embodiment, step (v) includes a step of purifying the insulin detemir produced.

In another preferred embodiment, said Boc-modified insulin detemir backbone is prepared using genetic recombination techniques.

In another preferred embodiment, after step (ib), two purification steps are included.

In another preferred example, in step (ib), the mass ratio of the insulin detemir fusion protein to trypsin is 1:3000-1: 10000.

In another preferred example, in step (ib), the mass ratio of insulin detemir fusion protein to carboxypeptidase is 1:5000 to 1: 15000.

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

In another preferred embodiment, the method comprises the following steps:

in another preferred example, the method comprises the steps of:

(i) providing the insulin detemir backbone fusion protein of the second aspect of the invention, and performing enzyme digestion to obtain a compound 1;

(ii) subjecting compound 1 to Fmoc modification to thereby prepare compound 2;

(iii) carrying out Boc removal treatment on the compound 2 to obtain a compound 3;

(iv) reacting the compound 3 with a ditert insulin side chain to obtain a compound 4; and

(v) compound 4 was subjected to Fmoc removal treatment to obtain insulin detemir represented by compound 5.

In a seventh aspect of the invention there is provided a insulin detemir preparation prepared using the method of the sixth aspect of the invention.

In another preferred embodiment, the prepared insulin detemir has biological activity.

In an eighth aspect of the invention there is provided an isolated polynucleotide encoding a insulin detemir fusion protein according to the first aspect of the invention, an insulin detemir backbone fusion protein according to the second aspect of the invention, an insulin detemir precursor according to the third aspect of the invention, or an insulin detemir backbone according to the fourth, fifth or sixth aspects of the invention.

According to a ninth aspect of the invention, there is provided a vector comprising a polynucleotide according to the eighth aspect of the 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.

According to a tenth aspect of the present invention, there is provided a host cell comprising a vector according to the ninth aspect of the present invention or a polynucleotide according to the eighth aspect of the present invention integrated exogenously into the chromosome.

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

In an eleventh aspect of the invention, there is provided a formulation or pharmaceutical composition comprising a detemir insulin precursor fusion protein according to the first aspect of the invention, a detemir insulin backbone fusion protein according to the second aspect of the invention, a detemir insulin precursor according to the third aspect of the invention, or a detemir insulin backbone according to the fourth, fifth or sixth aspect of the invention, and a pharmaceutically acceptable carrier.

Drawings

FIG. 1 shows the plasmid pBAD-FP-TEV-R-Insulin-Detemir (Lys)²⁹Boc) pattern.

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

FIG. 3 shows SDS-PAGE of Boc-insulin backbone fusion proteins after renaturation of inclusion bodies.

FIG. 4 shows HPLC detection profile of Boc-detet insulin backbone purification.

FIG. 5 shows the HPLC detection profile of the pure final product of insulin detemir of the present invention.

Detailed Description

The present inventors have extensively and intensively studied and found a novel method for preparing insulin detemir. Specifically, the method utilizes Fmoc orthogonal protection method to carry out side chain addition step in the preparation process of insulin detemir, and optimizes the conditions of purification and synthesis in the preparation process. The method of the invention does not need expensive solid phase synthesis instruments, shortens the production period, has simple production process and improves the purity and the yield of the product.

Insulin detemir

Insulin detemir is a neutral, soluble, long-acting Insulin. It is an insulin analogue, which is formed by removing threonine at position 30 of B chain from prototype insulin, and combining a straight chain fatty acid with 14 carbons at lysine side chain epsilon-amino group at position B29 through acylation. The acylated fatty acid can stabilize the self-aggregation of insulin molecules, and can be reversibly combined with albumin, so that the insulin detemir is slowly absorbed from subcutaneous injection parts, the action duration is prolonged, and the risk of hypoglycemia can be reduced while the blood sugar is controlled.

Construction of insulin detemir expression plasmid

Expression construct FP-TEV-R-Insulin-detemir (Lys)²⁹Boc) contains Insulin-destemir (Lys²⁹Boc) fused to the C-terminus of FP-TEV-R. The sequence is subjected to codon optimization, and can realize high-level expression of foreign protein in escherichia coli. The expression vector "pBAD/His A (Kanar)" and the vector containing "FP-TEV-R-Insulin-destemir (Lys) were digested with restriction enzymes Nco I and Xho I²⁹Boc) "plasmid of the target gene, the digested product was separated by agarose electrophoresis, extracted using agarose gel DNA recovery kit, and finally two DNA fragments were ligated using T4 DNA ligase. Chemically transforming the ligation product into E.coli Top10 cells, and culturing the transformed cells in a medium containing 50. mu.gLB agar medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar) with kanamycin overnight. 3 viable colonies were picked, cultured overnight in 5mL of liquid LB medium (10g/L yeast peptone, 5g/L yeast extract powder, 10g/L NaCl) containing 50. mu.g/mL kanamycin, and plasmid extraction was performed using a plasmid miniprep kit. The extracted plasmid was then sequenced using sequencing oligonucleotide primer 5'-ATGCCATAGCATTTTTATCC-3' to confirm correct insertion. The resulting plasmid was designated "pBAD-FP-TEV-R-destemir (Lys)²⁹Boc)”。

Fusion proteins

By means of the green fluorescent protein folding unit, two fusion proteins are constructed according to the invention, namely a insulin detemir precursor fusion protein according to the first aspect of the invention comprising single-chain insulin detemir and an insulin detemir backbone fusion protein according to the second aspect of the invention comprising double-chain insulin detemir. In fact, the scope of protection of two fusion proteins of the invention may partially overlap, for example, insulin detemir in a double-chain form contained in the fusion protein, whose C-terminus of the B-chain may also be linked to the N-terminus of the a-chain by a linker peptide, or may be considered as a single chain containing intrachain disulfide bonds.

The green fluorescent protein fold unit FP comprised in the fusion protein of the invention comprises 2 to 6, preferably 2 to 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 can be selected from: u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3-u4-u5, u5-u6-u7, u8-u9-u10, u9-u 9-u 9-u 9, u9-u 9-u 9-u 9, u 9-36u 9, u 9-9, u 36u 9-36u 9, u 36u 9-9, u 9-36u 9-9, u 9-36u 9-9, u 9-9, u 9-36u 9-9, u 9-36u 9-9, u-36u-9, u 36u 9, u 9-36u 9, u 36u 9-9, u 9-36u 9, u-9, u 9-9, u-9, u 9-36u-9, u-9, u 9-9, u 9-9, u 36u-9, u 9-36u 9, u 36u-9, u-36u-9, u-9-, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6, or u10-I-u 11.

The term "fusion protein" as used herein also includes variants having the above-described activities. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which a polypeptide is fused with another compound (such as a compound for increasing the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused with the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of the present invention. These conservative variants are preferably produced 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 amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (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, modifications may be made to the fusion proteins of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a 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 increase their resistance to proteolysis or to optimize solubility.

The term "polynucleotide encoding a fusion protein of the present invention" may include a polynucleotide encoding a fusion protein of the present invention, and may also include polynucleotides that additionally include 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 present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is 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 encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or 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 sequence of the polynucleotide of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. 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.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

Methods for amplifying DNA/RNA using PCR techniques are preferably used to obtain the polynucleotides of the invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on 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, as well as genetically engineered host cells transformed with the vectors of the invention or the coding sequences of the fusion proteins of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

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

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

(2) a host cell 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 a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is 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 translation 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 present invention and appropriate transcription/translation 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 a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, 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 described above, together with 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: escherichia 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 transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.

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

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant 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 culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These 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 (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Fmoc modification

In the field of biological medicine, the use of polypeptides is increasing, amino acids are basic raw materials for polypeptide synthesis technology, and all amino acids contain alpha-amino and carboxyl, and some also contain side chain active groups, such as: hydroxyl, amino, guanidyl, heterocycle and the like, therefore, amino and side chain active groups need to be protected in a peptide grafting reaction, and the protecting groups are removed after the polypeptide is synthesized, so that amino acid misconnection and a plurality of side reactions can occur.

Fmoc is a base-sensitive protecting group and can be removed in 50% dichloromethane solution of ammonia such as concentrated ammonia water or dioxane-methanol-4N Na OH (30: 9: 1), piperidine, ethanolamine, cyclohexylamine, 1, 4-dioxane, pyrrolidone, etc.

Fmoc-protecting groups are generally introduced by Fmoc-Cl or Fmoc-OSu under weakly basic conditions such as sodium carbonate or sodium bicarbonate. Fmoc-OSu allows easier control of reaction conditions and fewer side reactions than Fmoc-Cl. Fmoc has strong ultraviolet absorption with maximum absorption wavelengths of 267nm (. epsilon.18950), 290nm (. epsilon.5280) and 301nm (. epsilon.6200), so that the detection can be realized by using the ultraviolet absorption, and a plurality of convenience is brought to the automatic polypeptide synthesis of an instrument. Moreover, the method is compatible with a wide range of solvents and reagents, has high mechanical stability, and can be used for various carriers and various activation modes, and the like. Therefore, the Fmoc protecting group is most commonly used in polypeptide synthesis today.

Fmoc-OSu (fluorenylmethoxycarbonylsuccinimides)

Insulin detemir side chain

N-Succinimidyl myristate (English name: N-Succinimidyl myristate) is the side chain of insulin detemir.

The preparation of the insulin detemir comprises the steps of firstly obtaining a main chain of the insulin detemir with 29-bit Boc protected lysine by utilizing a gene recombination technology, and then connecting a side chain Myr-OSu of the insulin detemir to obtain the insulin detemir.

Preparation of insulin detemir

The synthesis route of the insulin detemir provided by the invention is shown as follows, a Fmoc modified compound 2 is prepared from a Boc-insulin main chain (compound 1), a compound 3 is obtained after the Boc protection of the compound 2 is removed, the compound 3 reacts with an activated insulin detemir-OSu side chain to obtain a compound 4, and then the compound 5 insulin detemir is obtained through the Fmoc removal reaction.

Specifically, the present invention provides a method for preparing insulin detemir, comprising the steps of:

(i) providing a Boc modified insulin detemir backbone;

(iii) carrying out de-Boc treatment on the Fmoc and Boc modified insulin detemir main chains and reacting the main chains with insulin detemir side chains to prepare Fmoc modified insulin detemir; and

(iv) and (3) carrying out Fmoc removal treatment on the Fmoc modified insulin detemir so as to prepare the insulin detemir.

The main advantages of the invention include:

(1) the invention directly utilizes a biosynthesis mode to produce the Boc modified insulin detemir main chain without adopting methods such as dilution, ultrafiltration liquid exchange and the like to remove excessive inorganic salt in the supernatant of fermentation liquid. Second, cyanogen bromide cleavage, oxidative sulfitolysis and related purification steps are not required in the preparation of the peptide of interest.

(2) In the method of the present invention, the specific gravity of the Boc-insulin backbone contained in the fusion protein is high (the fusion ratio is increased), FP or A-FP in the fusion protein contains arginine and lysine, can be digested into small fragments by protease, has a large molecular weight difference compared with the target protein, is easy to separate, and the Boc-insulin backbone or an analog precursor is separated by using a chromatographic column, with a one-step yield of 70% or more, which is 3 times higher than that of the conventional method, and the most of the pigment can be removed, and the Boc-insulin backbone yield is about 1.5-1.8 g/L.

(3) The synthetic process steps of the invention are simplified by more than two thirds, and the process time and the equipment investment cost are reduced.

(4) Due to the protection of the 29-Boc-lysine, the invention can directly use the orthogonal reaction with Fmoc protection to synthesize insulin detemir.

(5) The insulin detemir synthesized by the method disclosed by the invention has no N-terminal fatty acid acylated impurities, is beneficial to downstream purification, and reduces the cost.

(6) Compared with solid phase synthesis, the method of the invention does not produce racemized impurity polypeptide, does not need to use a large amount of modified amino acid, does not use a large amount of organic reagent, has small environmental pollution and lower cost.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1 construction and expression of insulin-expressing ditto Strain

The construction of insulin-expressing plasmid is described in the examples of patent application No. 201910210102.9. Fusion protein FP1-TEV-R-Insulin-detemir (Lys)²⁹Boc) of the DNA fragment, cloned intoExpression vector plasmid pBAD/His A (purchased from NTCC, kanamycin resistance) at NcoI-XhoI site downstream of araBAD promoter to obtain plasmid pBAD-FP-TEV-R-Insulin-Detemir (Lys)²⁹Boc). The plasmid map is shown in FIG. 1.

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

The constructed plasmid pBAD-FP-TEV-R-Insulin-Detemir (Lys)²⁹Boc) and pEvol-pylRs-pylT are jointly transformed into an Escherichia coli TOP10 strain, and the expression of Insulin-Detemir backbone fusion protein FP1-TEV-R-Insulin-Detemir (Lys)²⁹Boc) in a culture medium.

The fusion protein comprises FP1 of u3-u4-u5, and the amino acid sequence is as follows:

MVSKGEELFTGVKLTLKFICTTYVQERTISFKDTYKTRAEVKFEGD ENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)RGIVEQCCTSICSLYQLENYCN(SEQ ID NO:1)

using a similar approach, the following fusion proteins were constructed:

fusion protein FP2-TEV-R-Insulin-Detemir (Lys)²⁹Boc), comprising FP2 from u8 to u9, having the amino acid sequence:

GIKANFKIRHNVEDVQLADHYQQNTPIGENLYFQGRFVNQHLCGSHLVE ALYLVCGERGFFYTPK(Boc)RGIVEQCCTSICSLYQLENYCN(SEQ ID NO:23)

fusion protein FP3-TEV-R-Insulin-Detemir (Lys)²⁹Boc), comprising FP3 being u9-u10-u11, having the amino acid sequence:

VQLADHYQQNTPIGHYLSTQSVLSKDHMVLLEFVTAAGIENLYFQGRFVN QHLCGSHLVEALYLVCGERGFFYTPK(Boc)RGIVEQCCTSICSLYQLENYCN(SEQ ID NO:24)

fusion protein FP4-TEV-R-Insulin-Detemir (Lys)²⁹Boc), comprising FP4 from u10 to u11, having the amino acid sequence:

HYLSTQSVLSKDHMVLLEFVTAAGIENLYFQGRFVNQHLCGSHLVEALYL VCGERGFFYTPKRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:25)

constructing corresponding expression strains by using a conventional method in the field, performing shake flask fermentation, and performing electrophoresis detection on the expression quantity of the insulin detemir fusion protein in the fermentation liquor.

Selection of fusion protein FP1-TEV-R-Insulin-Detemir (Lys)²⁹Boc), preparing seed liquid culture medium, inoculating, performing two-stage culture to obtain second-stage seed liquid, culturing for 20 hr, and obtaining OD₆₀₀About 180, obtaining about 3L fermentation liquor after fermentation is finished, and obtaining about 130g/L wet thalli by centrifugation. And (3) after the fermentation liquor is centrifuged, adding a crushing buffer solution, performing bacteria crushing twice by using a high-pressure homogenizer, adding Tween 80 and EDTA-2Na with a certain concentration after centrifugation, washing once, centrifuging, and collecting precipitates to obtain the inclusion body. Approximately 40g of wet inclusion bodies, which contain insulin detemir precursor fusion protein, are finally obtained per liter of fermentation broth.

Example 2 denaturation and cleavage of Boc-insulin backbone inclusion bodies

Adding 7.5-9.0mol/L urea solution into the inclusion body obtained in example 1 according to the weight-volume ratio of 1:10(m/v), stirring and dissolving at room temperature, controlling the total protein concentration of the inclusion body solution to be 10-25mg/mL, adjusting the pH value to be 11.0-11.8 by NaOH, adding beta-mercaptoethanol, and uniformly stirring. Dripping the inclusion body dissolving solution into renaturation buffer solution containing 0.2-1.0mmol/L L-cystine, 0.3-0.5mmol/L EDTA-2Na and 5-10mmol/L glycine to dilute the inclusion body dissolving solution by 5-10 times, maintaining the pH value of the fusion protein renaturation solution at 10.5-11.8, controlling the temperature at 4-8 ℃, the renaturation time at 10-20h and the renaturation rate at more than 60% (the electrophoresis detection is shown in figure 3), and obtaining the insulin detemir main chain fusion protein containing disulfide bonds between insulin detemir A chains and insulin detemir B chains.

Taking Boc-detemir insulin main chain fusion protein renaturation liquid, adjusting the pH value to 8.0-9.5, adding recombinant trypsin according to the ratio of 1:3000-1:10000, adding recombinant carboxypeptidase B according to the ratio of 1:5000-1:15000, and carrying out enzyme digestion at 15-25 ℃ for 20-40h to finally obtain the Boc-detemir insulin main chain, wherein the enzyme digestion rate is higher than 70%.

EXAMPLE 3 Primary purification of Boc-ditert insulin backbone

The insoluble mixture in the enzyme digestion solution is separated by filtering through a membrane with the pore diameter of 300 and 750 KD. According to the difference of isoelectric points of proteins, the Boc-dete insulin main chain obtained in the example 2 is primarily purified by adopting an anion exchange chromatography technology to remove most impurities, the combined loading capacity of the Boc-dete insulin main chain and a filler is controlled to be lower than 50mg/mL, the Boc-dete insulin main chain is collected by gradient elution, the purity of the Boc-dete insulin main chain after coarse purification reaches more than 70%, and the yield is more than 85%.

EXAMPLE 4 reversed phase chromatography of Boc-ditert insulin backbone

The Boc-ditert insulin backbone solution obtained in example 3 was isolated and purified by reverse phase chromatography. Taking an aqueous solution containing 0.065% of trifluoroacetic acid as a mobile phase A; acetonitrile solution containing 0.065% trifluoroacetic acid was used as mobile phase B. And combining the Boc-detemir insulin main chain with a filler, controlling the loading amount of the Boc-detemir insulin main chain to be less than 10mg/mL, carrying out gradient elution, and collecting the Boc-detemir insulin main chain. The experimental result shows that the purity of the Boc-insulin-ditert backbone collected by reverse phase chromatography is more than or equal to 90 percent (the HPLC detection chart is shown in figure 4), and the yield is more than 60 percent.

EXAMPLE 5 preparation of insulin detemir Using Boc-insulin backbone

Taking the Boc-insulin detest main chain compound 1 dried product obtained in example 4 (in this example, the feeding molar ratio is 30 mg), adding Fmoc-Osu, DIPEA and DMF according to the molar ratio of Table 1, reacting for 8-12 hours under the condition of pH7.5-8.5 to obtain Fmoc and Boc protected insulin detest main chains, then adding cold methyl tert-ether/petroleum ether mixed solvent into the reaction system, centrifuging the solid precipitate, washing the solid with methyl tert-ether and petroleum ether mixed solvent for 2-3 times to obtain Fmoc protected compound 2: DiFmoc-Insulin-detemir (Lys)²⁹Boc)。

TABLE 1 molar ratio of the feeds

	Boc-detetine insulin backbone	Fmoc-OSu	DIPEA	DMF
					Equivalent weight or volume	1.0eq	5eq	12eq	1V

Taking the compound 2, adding TFA solution, stirring at low temperature for 0.5-2.0h, adding a cold mixed solution of methyl tert-ether and petroleum ether into the reaction mixture, settling and centrifuging. The solid was washed 2-3 times with methyl tert-ether and dried to obtain Boc-removed solid compound 3: DiFmoc-Insulin-Detemir (Lys)²⁹NH₂)。

Dissolving the compound 3 after Boc removal in DMF solution, adding 12eq of DIPEA, adding 2-5eq of side chain compound Myr-OSu under the condition of pH8.0-9.0, and stirring the reaction mixture at room temperature for 2-3 hours. And (3) after the reaction is finished, adding a cold methyl tertiary ether/n-hexane mixed solvent to precipitate a solid product, washing for 2-3 times, and drying to obtain a white compound 4: DiFmoc-Insulin-Detemir-Myr- (Lys)²⁹NH)。

Adding 20% piperidine in DMF solution to compound 4, reacting at room temperature for 0.5-2.0h, adding mixed solution of methyl tert-ether and petroleum ether to the reaction system to precipitate product, centrifuging the solid, washing with mixed solution of methyl tert-ether and petroleum ether for 2-3 times, and dryingObtaining the compound 5 after Fmoc removal: Insulin-Detemir- -Myr- (Lys)²⁹NH). And then adding a mixed solvent of cold methyl tert-ether and petroleum ether with the volume of 10-20 times of the reaction system, precipitating and centrifuging, washing the solid for more than 3 times by using the mixed solvent of methyl tert-ether and petroleum ether, and pumping to dry to finally obtain the insulin detemir.

The synthesized insulin detemir is subjected to two-step high-pressure reverse phase chromatography, and finally the insulin detemir with the purity of more than 99% is obtained (see figure 5).

Comparative example

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 shown in SEQ ID NO: 22.

MKKLLFAIPLVVPFYSHSTMELEICSWYHMGIRSFLEQKLISEEDLNSAVDRFVNQHLCGSHLVEALYLVCGERGFFYTPK(Boc)RGIVEQCCTSICSLYQLENYCN(SEQ ID NO:22)

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

The results showed OD after 20h of cultivation₆₀₀About 140 g/L, and obtaining about 3L fermentation liquor after fermentation is finished, and obtaining wet thalli of about 105g/L by centrifugation. And after the fermentation liquor is centrifuged, adding a crushing buffer solution, crushing the bacteria twice by using a high-pressure homogenizer, and centrifuging and collecting the precipitate to obtain the inclusion body. About 30g of wet inclusion bodies, in which insulin detemir fusion protein is present, can be finally obtained per liter of fermentation broth.

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

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Claims

1. A insulin detemir fusion protein, comprising, from N-terminus to C-terminus, a structure according to formula III:

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

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

a is a null or leader peptide,

FP is a folding unit of green fluorescent protein,

TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site;

r is arginine or lysine for enzyme digestion;

g is a insulin detemir backbone or an active fragment thereof;

2. The fusion protein of claim 1, wherein the green fluorescent protein folding unit is u2-u3, u4-u5, u1-u2-u3, u3-u4-u5, u8-u9, u9-u10-u11, u10-u11, or u4-u5-u 6.

3. The fusion protein of claim 1, wherein G is Boc-modified insulin detemir precursor having the structure of formula IV:

GB-X-GA (IV)

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

x is a connecting peptide;

4. The fusion protein of claim 1, wherein the amino acid sequence of the insulin detemir fusion protein is as shown in SEQ ID NO 1, 23, 24, 25.

5. A insulin detemir backbone fusion protein, characterized by having a structure represented by formula I from N-terminus to C-terminus:

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

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

a is a null or leader peptide,

FP is a green fluorescent protein folding unit;

r is arginine or lysine for enzyme digestion;

in the formula (I), the compound is shown in the specification,

"|" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

x is nothing or a connecting peptide;

6. a Boc-modified insulin detemir precursor having the structure shown in formula IV:

GB-X-GA (IV)

in the formula (I), the compound is shown in the specification,

"-" represents a peptide bond;

x is a connecting peptide, preferably, the amino acid sequence of the connecting peptide is R, RR, RRR, or shown as SEQ ID NO. 4-7;

7. A Boc-modified insulin detemir backbone having the structure shown in formula II:

in the formula (I), the compound is shown in the specification,

"|" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

8. The backbone of claim 7, wherein the N-terminus of both the A and B chains is Fmoc modified.

9. An Fmoc-modified detemir insulin backbone having the structure shown in formula II:

in the formula (I), the compound is shown in the specification,

"|" represents a disulfide bond;

GA is insulin detemir A chain, the amino acid sequence is shown as SEQ ID NO 3,

and the N ends of the A chain and the B chain are both modified by Fmoc.

10. An isolated polynucleotide encoding the insulin detemir fusion protein of claim 1, the insulin detemir backbone fusion protein of claim 5, the insulin detemir precursor of claim 6, or the insulin detemir backbone of claims 7-8.