CN114380903B - Insulin or analogue precursor thereof - Google Patents
Insulin or analogue precursor thereof Download PDFInfo
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- CN114380903B CN114380903B CN202111615218.4A CN202111615218A CN114380903B CN 114380903 B CN114380903 B CN 114380903B CN 202111615218 A CN202111615218 A CN 202111615218A CN 114380903 B CN114380903 B CN 114380903B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Embodiments of the present application disclose an insulin or analog precursor comprising a first peptide having the amino acid sequence MIVEF; a second peptide fragment comprising an a chain and a B chain of insulin or an analog thereof linked by a first linking peptide; wherein the first peptide segment is linked to the N-terminus of the second peptide segment via a second linking peptide. The first peptide segment in the insulin or analogue precursor of the insulin contributes to the correct folding of the precursor in the renaturation process, so that the renaturation yield of the precursor is improved, and the yield of the insulin or analogue thereof is further improved.
Description
Technical Field
The present application relates to a precursor of insulin or an analogue thereof, and a method for preparing insulin or an analogue thereof.
Background
Diabetes is one of the diseases that seriously jeopardizes human health in the world today, and insulin is an important drug currently used clinically for treating diabetes. The human natural insulin is a polypeptide consisting of an A chain formed by connecting 21 amino acids and a B chain formed by connecting 30 amino acids, wherein 1 disulfide bond is arranged in the A chain, and the A chain and the B chain are connected by two disulfide bonds.
Insulin was initially synthesized as preproinsulin on the beta cell ribosomes of islets in the pancreas. Preproinsulin is se:Sub>A molecule containing se:Sub>A leader peptide chain (SP) composed of 24 amino acids, se:Sub>A B chain (B), se:Sub>A C peptide (C) composed of 31 amino acids, and an A chain (A) aligned in this order of "SP-B-C-A". When the preproinsulin enters the endoplasmic reticulum, the leader peptide chain is cleaved off, and proinsulin (B-C-A) is formed. Proinsulin forms disulfide bonds within the endoplasmic reticulum. After formation of the three-dimensional structure, the prohormone convertase PC1/3 cleaves the proinsulin at the B-C binding site, and the convertase PC2 cleaves the C-A binding site again. Finally, the 2 basic amino acids which remain at the C-terminus of the B chain, i.e.at the N-terminus on the C-peptide, are cleaved by carboxypeptidase H to insulin when cleaved by PC 1/3.
At present, two systems are mainly adopted for producing insulin and analogues thereof, one is a yeast expression system, and the other is an escherichia coli system. US4430266 discloses that proinsulin (B-C-se:Sub>A) is directly expressed by using an escherichise:Sub>A coli host, and is subjected to renaturation in vitro, and then cleavage is performed by using Trypsin and CPB to obtain insulin with se:Sub>A correct sequence and structure.
The escherichia coli expression system has the advantages of short fermentation time, simple process, high expression level, low production cost and the like. However, the high expression of the E.coli expression system causes the expression products to gather in the cells in the form of inclusion bodies, and the inclusion bodies are required to be subjected to operations such as renaturation after the cells are broken so as to obtain correctly folded insulin and analogue precursors thereof, and the renaturation efficiency of the inclusion bodies directly influences the yield. In addition, the cleavage of leader and C peptides from precursors of insulin and its analogues to obtain mature forms typically uses proteases such as trypsins, CPB, etc. The use of Trypsin more or less leads to various miscut, various impurities are generated, difficulty is caused to subsequent fine purification, and the reduction of production cost is not facilitated.
Therefore, there is a need to improve the renaturation yield and cleavage efficiency of insulin or insulin analogue precursors in the prior art, to increase the yield and to reduce the production cost.
Disclosure of Invention
The present application aims to provide a novel insulin or insulin analogue precursor having a high renaturation yield contributing to a high yield in the production of insulin or insulin analogue, and a method for producing insulin or an insulin analogue using the precursor.
In a first aspect of the present application, there is provided a precursor of insulin or an analogue thereof, the precursor comprising a first peptide fragment and a second peptide fragment, wherein the amino acid sequence of the first peptide fragment is MIVEF and the second peptide fragment comprises an A chain and a B chain of insulin or an analogue thereof linked by a first linking peptide, the first peptide fragment being linked to the N-terminus of the second peptide fragment by a second linking peptide. It has surprisingly been found in the present application that the first peptide fragment facilitates correct folding of the precursor when the precursor of insulin or an analogue thereof is subjected to a renaturation procedure, thereby significantly improving the renaturation yield of the precursor.
In a second aspect of the present application, the insulin or analog precursor of the present application, the amino acid sequence of the second linking peptide is [ G m S n ] x R, wherein m is an integer between 2 and 10, n is 0 or 1, and x is an integer between 1 and 10, and the second linker peptide having the amino acid sequence comprises a site capable of being recognized and cleaved by a site-specific protease, such as trypsin.
In a third aspect of the present application, there is also provided a method of preparing insulin or an insulin analogue, the method comprising the steps of:
culturing a host cell comprising a nucleic acid encoding an insulin or analog precursor thereof of the present application and/or an expression vector under culture conditions suitable for expression of the insulin or analog precursor thereof;
denaturing the insulin or analogue precursor expressed by the host cell and then renaturing to obtain correctly folded insulin or analogue precursor;
cleaving the insulin or analog precursor and collecting insulin or analog thereof.
Compared with the technical scheme of directly expressing proinsulin (B-C-A) in the prior art, in the preparation method of insulin or an analogue thereof, the first peptide segment in the precursor of the insulin or the analogue thereof is beneficial to correct folding of the precursor in the renaturation process, so that the renaturation yield of the precursor is improved, and the yield of the insulin or the analogue thereof is further improved.
In other aspects of the present application, nucleic acids encoding precursors of insulin or analogs thereof, vectors and host cells containing the nucleic acids are also provided.
Detailed Description
The present application is described in further detail below with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
It should be noted that endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and that such range or value should be understood to include values approaching such range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
According to a first aspect of the present application there is provided an insulin or analogue precursor thereof, the precursor comprising a first peptide fragment and a second peptide fragment, wherein the amino acid sequence of the first peptide fragment is MIVEF, the second peptide fragment comprises an a chain and a B chain of insulin or insulin analogue linked by a first linking peptide, and the first peptide fragment is linked to the N-terminus of the second peptide fragment by a second linking peptide. The present application found that, after expression of the insulin or analogue precursor thereof by the host cell, a precursor folded to the correct structure can be obtained by a process of denaturation followed by renaturation, and then a mature insulin polypeptide can be obtained by cleavage of the first peptide fragment and the first connecting peptide. In the process of denaturation followed by renaturation, the first peptide fragment can help the precursor to fold correctly, so that the renaturation yield of the precursor is obviously improved, and the yield of insulin or the analogues thereof is improved.
The term "insulin analogue" as used herein refers to a modified insulin in which one or several amino acid residues have been substituted, deleted and/or added in the amino acid sequence of the a and/or B chain of the insulin and still have a similar biological function as insulin.
Herein, modifications in the insulin molecule are expressed as single letter codes for chains (a or B), positions and amino acid residues substituting for amino acid residues. Terms such as "A1", "A2" and "A3" refer to amino acids at positions 1, 2 and 3, respectively, etc. in the a chain of insulin (counting from the N-terminus), respectively. Also, terms such as B1, B2, and B3 refer to amino acids at positions 1, 2, and 3, respectively, etc., in the B chain of insulin (counting from the N-terminus). Using single letter codes for amino acids, terms such as a21G, B28K and B29P refer to the amino acid at position a21 being glycine and the amino acids at positions 28 and 29 being lysine and proline, respectively.
In some embodiments, the insulin analogs of the present application comprise one or more of the following mutations: pro of B28 is substituted by Asp, lys, leu, val or Ala and/or Lys of position B29 is substituted by Pro, glu or Asp, asn of B3 is substituted by Thr, lys, gln, glu or Asp, amino acid residue Asn of A21 is substituted by Ala, gln, glu, gly, his, ile, leu, met, ser, thr, trp, tyr or Val, one or more amino acids, such as Lys, are added to the C-terminus of the A chain and/or B chain, amino acid of B1 is substituted by Glu, amino acid of B16 is substituted by Glu or His, amino acid of B30 is deleted, amino acid of B1 is deleted, amino acid of B28-30 is deleted, amino acid of B27 is deleted, the A chain and/or B chain has an N-terminal extension, and the A chain and/or B chain has a C-terminal extension, such as B chain C-terminal addition of two arginine residues. In other embodiments, the amino acid of insulin analog a14 of the present application is Asn, gln, glu, arg, asp, gly or His, the amino acid at B25 is His, and optionally further comprises one or more additional mutations. In other embodiments, the amino acid residue of insulin analog a21 of the present application is Gly, or two further arginine residues are extended at the C-terminus.
In some embodiments, the insulin analog of the present application is selected from insulin glargine, insulin aspart, insulin lispro, insulin glulisine, insulin detention, or insulin deglutide; insulin glargine is preferred.
The term "insulin or analogue precursor thereof" as used herein refers to a polypeptide which can be converted to insulin or analogue thereof by one or more subsequent chemical and/or enzymatic steps.
In the insulin or analog precursor of the present application, the second peptide fragment comprises an a chain and a B chain of insulin or analog thereof linked by a first linking peptide, in some embodiments, the first linking peptide is linked to the a chain on the amino group of A1, to the B chain on the amino group of B29 or on the carboxyl group of B30.
The "first linking peptide" of the present application includes any suitable polypeptide fragment that can be cleaved enzymatically or chemically from the A and B chains without disrupting the A and B chains.
In some embodiments, the first linking peptide comprises a C peptide, e.g., comprises a native C peptide, a short C peptide, or a modified C peptide. Alternatively, the C peptide in the insulin and analog precursors of the present application is directly linked to the a and/or B chains, or the C peptide is linked to the a and/or B chains via one or two basic amino acids, respectively. In some embodiments, the C peptide is linked to the B chain by the RR and to the a chain by the KR, and during cleavage, the protease specifically cleaves from the RR and KR, allowing the a chain and the B chain to separate.
The term "C peptide" as used herein refers to the linking moiety "C" in proinsulin B-C-A and includes natural C peptides, short C peptides and modified C peptides. The natural C peptide refers to a connecting peptide for connecting a B chain and an A chain in natural human proinsulin, and the N terminal and the C terminal of the connecting peptide respectively have two basic amino acids so as to realize excision of the natural C peptide. Non-limiting examples of short C peptides include AAK, AAR, and DKAAK. The modified C peptide refers to a connecting peptide obtained by modifying the C peptide.
Numerous examples of natural C-peptides, short C-peptides and modified C-peptides for linking a-and B-chains have been disclosed in the prior art, e.g. WO90/10075 discloses insulin precursors with the C-peptide AAK. WO01/49742 discloses insulin precursors having C-peptides comprising aromatic amino acid residues. WO02/079251 discloses insulin precursors having C-peptides comprising Gly residues. WO02/079250 discloses insulin precursors having a C-peptide comprising Pro residues. WO02/100887 discloses insulin precursors having a C-peptide comprising a glycosylation site. WO2008/037735 discloses insulin precursors having a C-peptide comprising a kex2p cleavage site. It is understood that the natural C-peptides, short C-peptides and modified C-peptides disclosed in these documents can all be used as the first linking peptide of the present application.
In some embodiments, the amino acid sequence of the B chain of the insulin or analog precursor of the present application is FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID No. 14). The sequence of the A chain is GIVEQCCTSICSLYQLENYCG (SEQ ID No. 16). The first connecting peptide sequence was EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 15).
In some embodiments, the precursor of insulin or an analog thereof of the present application has a B chain sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID No. 17), a chain sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID No. 19) and a first connecting peptide sequence of RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 18).
In some embodiments, the second peptide fragment in the insulin or analog precursor of the present application is proinsulin or a proinsulin analog.
In a second aspect of the present application, the insulin or analog precursor of the present application has the amino acid sequence of [ G m S n ] x R, wherein m is an integer between 2 and 10, n is 0 or 1, and x is an integer between 1 and 10. The second connecting peptide may be recognized and cleaved by a site-specific protease, such as trypsin.
In some embodiments, n is 0 or 1 and the amino acid sequence of the second linking peptide is [ G m S] x R, wherein m is an integer between 2 and 10, x is an integer between 1 and 10, and the first peptide segment and the second connecting peptide sequence are preferably SEQ ID No. 1-7. The application surprisingly found that when certain specific second connecting peptide sequences are adopted, the efficiency of the digestion of the insulin or the precursor of the insulin analogue by protease is obviously improved, less impurities are generated in the digestion process, the subsequent fine purification is facilitated, and the production cost is reduced.
The present application also provides nucleic acids encoding precursors of the insulins or analogs thereof, vectors and host cells containing the nucleic acids.
The nucleic acid of the present application may be a DNA molecule or an RNA molecule, or may be a nucleic acid analog. The nucleic acid molecules in the present application may comprise naturally occurring nucleic acid residues or artificially generated nucleic acid residues. The nucleic acid molecules of the present application may be single-or double-stranded, linear or circular, natural or synthetic, and without any size limitation if not otherwise indicated. The nucleic acid molecule may also comprise a promoter, which may be homologous or heterologous.
The nucleic acid molecules of the present application may be cloned into vectors. "vectors" in this application include plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In some embodiments, these vectors are suitable for use in transforming cells, eukaryotic cells such as fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a preferred embodiment, these vectors are suitable for stable transformation of bacterial cells, for example to transcribe nucleic acid molecules of the present application.
The vector of the present application may be an expression vector. Suitable expression vectors that have been widely described in the literature are all useful in this application. In one embodiment, the expression vector may contain a marker gene and an origin of replication that ensures replication in the selected host, as well as a promoter and transcription termination signal. Between the promoter and the termination signal, there is preferably at least one restriction site capable of inserting a nucleic acid sequence/molecule for which expression is desired. Preferably, the expression vector of the present application is selected from the group consisting of pET-series expression vectors, pGEX-series expression vectors, pcDNA-series expression vectors, more preferably the expression vector of the present application is a pET22b expression vector or a pET26b expression vector.
The term "host cell" herein refers to a microorganism used to express a polypeptide of interest. Host cells include any progeny of a parent cell that are not identical to the parent cell due to mutations that occur during replication.
The host cell of the present application may be a eukaryotic cell or a prokaryotic cell, preferably a prokaryotic cell. In some embodiments, the host cell of the present application is e.coli, e.g., e.coli BL21.
In some embodiments, insulin or an analog precursor thereof is expressed in a host cell, followed by isolation of the insulin or analog precursor thereof. For protein expression, the nucleic acid encoding the protein is inserted into an expression vector by standard methods. Expression is performed in a suitable stable host cell and the protein is collected from the cell (supernatant or lysed cells).
In another embodiment, the nucleic acids of the present application and/or vectors containing the nucleic acids of the present application therein may be transduced, transformed or transfected or otherwise introduced into a host cell.
In a third aspect of the present application, there is also provided a method of preparing insulin or an insulin analogue, the method comprising culturing a host cell comprising a nucleic acid encoding an insulin or an analogue precursor thereof of the present application and/or an expression vector under culture conditions suitable for expressing the insulin or an analogue precursor thereof.
In some embodiments, the methods of preparing insulin or an analog thereof of the present application further comprise the steps of: denaturing the insulin or analogue precursor expressed by the host cell and then renaturing to obtain correctly folded insulin or analogue precursor; cleaving the insulin or analog precursor and collecting insulin or analog thereof.
When expressing insulin or its analogue precursor, especially in E.coli expression systems, the insulin or its analogue precursor is usually accumulated in the form of inclusion bodies, which are then disrupted and subjected to a renaturation operation to obtain the correct insulin or its analogue precursor. Compared with the technical scheme of directly expressing proinsulin (B-C-A) in the prior art, in the preparation method of the insulin or the insulin analogue, the first peptide segment with the amino acid sequence of MIVEF in the insulin or insulin analogue precursor is helpful for the correct folding of the precursor in the renaturation process, so that the renaturation yield of the precursor is improved, and the yield of the insulin or insulin analogue is further improved.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application further comprise the steps of: construction of an expression vector comprising a nucleic acid encoding an insulin or analog precursor thereof of the present application, and construction of a host cell comprising the expression vector by transient transfection of the host cell.
Culture conditions suitable for expressing insulin or its analogue precursors should be known to those skilled in the art, who can empirically select the appropriate medium to culture under conditions suitable for host cell growth. 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 insulin or analog precursor thereof in the above method may be expressed in cells, or on cell membranes, or secreted extracellularly.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application further comprise the steps of cell lysis and collecting the expression product. The method for cell lysis and collection of the expression product is not particularly limited, and it is preferable to use 50 mM PB and 1mM EDTA solution for sterilization and collect the expression product by centrifugation. In some embodiments, the insulin or insulin analog expressed by the host cell is present as inclusion bodies.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application further comprise the step of lysing the insulin or insulin analogs expressed by the host cells. The method of solubilization is not particularly limited, and it is preferable to solubilize the collected insulin or insulin analog expressed by the host cells in a solution containing 7M urea and 20mM glycine.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application include the step of denaturing the insulin or insulin analog expressed by the host cell and then renaturating. There is no particular limitation on the method of denaturation and renaturation of the inclusion body insulin or insulin analogue. Alternatively, denaturation is performed using dithiothreitol. Alternatively, renaturation is performed with a 10-fold dilution of a renaturation solution containing 1mM oxidized glutathione, 5mM reduced glutathione and 20mM glycine.
In some embodiments, the protein after the denaturation treatment may be enriched and concentrated using anion exchange chromatography on the protein prior to the renaturation treatment.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application include the step of cleaving a precursor of insulin or an analog thereof to obtain insulin or an analog thereof. Preferably, insulin or an analog thereof is obtained by cleavage of a precursor of insulin or an analog thereof by trypsin.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application further comprise the step of cleavage by carboxypeptidase. The trypsin cleaved product was digested with carboxypeptidase to remove the arginine at its C-terminus from the B-chain.
In some embodiments, the methods of preparing insulin or insulin analogs of the present application further comprise the step of purifying the mature insulin or insulin analog from the digested sample using reverse phase chromatography.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Example 1
1. Expression vector construction
Constructing genetic engineering bacteria by using an escherichia coli expression system, selecting a preferred codon of escherichia coli, taking pET22b as an expression vector, and inserting a nucleic acid sequence for encoding the insulin glargine precursor into multiple cloning sites NdeI and HindIII of the expression vector.
In this example, the first peptide sequence of the insulin glargine precursor is MIVEF, the second linker peptide sequence is GGR, i.e., the sequence of the first peptide sequence+the second linker peptide is MIVEFGGR (SEQ ID No. 1). The second peptide fragment has se:Sub>A B-C-A structure, wherein the amino acid sequence of B is FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID No. 14); c has the amino acid sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 15); a has the amino acid sequence of GIVEQCCTSICSLYQLENYCG (SEQ ID No. 16).
2. Host cell construction
BL21 (DE 3) strain was used with CaCl 2 Competent preparation was carried out by the method, the constructed expression vector was transformed into BL21 (DE 3) competent, and the mixture was spread on Amp-resistant LB agar medium and cultured overnight at 37 ℃. The monoclonal is respectively picked up and cultured in LB liquid medium added with Amp resistance, and then the seed is preserved.
3. Shake flask expression of insulin glargine precursors
The strain obtained was shake-flask cultured using SOB, and when the strain was cultured to an OD600 of about 1.0, IPTG was added to a final concentration of 0.5mM to induce expression. After 12 hours of induction, the fermentation cells were harvested. 20 μl of the fermentation cells were added to 20 μl of 5XSDSloadingbuffer, 60 μl of purified water, and the mixture was subjected to SDS-PAGE after 10-15min in boiling water to detect the expression, which showed normal expression.
4. Collection, denaturation and renaturation of insulin glargine precursor
Washing the fermentation thalli of the normal expression product with 10 mM phosphate (PB for short hereinafter) and 0.9% sodium chloride solution; breaking bacteria by adopting 50 mM PB and 1mM EDTA solution; washing thalli by adopting 20mmol/L PB, 1mM EDTA, 1.0mol/L urea and 1% sodium chloride solution, and centrifuging to obtain insulin glargine precursor inclusion bodies;
insulin glargine precursor inclusion bodies were dissolved with 7M urea, 20mM glycine (pH 10.5) and denatured with dithiothreitol at a concentration of 4mg/g (dithiothreitol/inclusion bodies) at room temperature for 1h; after denaturation, the product was diluted 10-fold with 1mM oxidized glutathione, 5mM reduced glutathione, and 20mM glycine solution (pH 10.5), and renatured at 2-8deg.C to obtain the correctly folded product.
Respectively taking a denatured liquid supernatant and a renaturation liquid supernatant for liquid phase analysis, wherein an analysis column is Agilent PLRP-S300A 5 mu M150 x 4.6mm, and the analysis conditions are 30% B and 0-2.5min; 30-60% of B,2.5-25min;100% B,25-29.5min;30% B,29.5-35min (mobile phase A:0.1% TFA, mobile phase B: 0.1% TFA+80% ACN), flow rate 1mL/min, column temperature 35 ℃, detection wavelength 214nm, 10-fold dilution of denatured liquid supernatant, sample volume 20. Mu.L of renaturation liquid supernatant, calculated renaturation rate according to the following formula:
renaturation yield = renaturation sample peak area/denaturation sample peak area x 100%.
The result shows that the renaturation yield of the insulin glargine precursor obtained by the method is 79.5%, and the renaturation effect is good.
Example 2
Insulin glargine was prepared as described in example 1, except that a second linker peptide sequence was used which was different from example 1, the sequence of the first peptide fragment + the second linker peptide used in this example was as follows:
| sequence numbering | Sequence(s) | Recovery yield (%) |
| SEQ ID No.2 | MIVEFGGGR | 76.9 |
| SEQ ID No.3 | MIVEFGGGGR | 75.8 |
| SEQ ID No.4 | MIVEFGGGGGGGGR | 73.2 |
| SEQ ID No.5 | MIVEFGGGGSR | 74.5 |
| SEQ ID No.6 | MIVEFGGGGSGGGGSR | 77.3 |
| SEQ ID No.7 | MIVEFGGGGSGGGGSGGGGSGGGGSGGGGSR | 76.8 |
Experimental results show that the insulin glargine precursor can be obtained through normal expression by the method of the embodiment, and the renaturation effect is good.
Example 3
1. Expression vector construction
Constructing genetic engineering bacteria by using an escherichia coli expression system, selecting a preferred codon of escherichia coli, taking pET26b as an expression vector, and inserting nucleic acid for encoding a natural human insulin precursor into multiple cloning sites NdeI and HindIII of the expression vector;
the sequence of the first peptide fragment+the second connecting peptide of the natural human insulin precursor in this example is MIVEFGGR (SEQ ID No. 1). The second peptide fragment has se:Sub>A B-C-A structure, wherein the amino acid sequence of B is FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID No. 17); c has the amino acid sequence of RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 18); a has the amino acid sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID No. 19). The constructed expression vector was designated RHI-pet26b.
2. Host cell construction
BL21 Star TM (DE 3) use of CaCl strains 2 Competent preparation is carried out by a method, and the expression vector after construction is transformed into BL21 Star TM (DE 3) competence, spread on Kan-resistant LB agar medium and cultured overnight at 37 ℃. The monoclonal is respectively picked up and cultured in LB liquid medium added with Kan resistance, and then the seed is preserved.
3. Shake flask expression
The strain obtained was shake-flask cultured using TB medium, and when the strain had been cultured to an OD600 of about 1.0, IPTG was added to a final concentration of 0.5mM to induce expression. After 12 hours of induction, the fermentation cells were harvested. 20 μl of the fermentation cells were taken and added with 5XSDSloadingbuffer20 μl of purified water and subjected to SDS-PAGE electrophoresis after 10-15min in boiling water, and the results showed that the cells were expressed normally.
4. Human insulin precursor collection, denaturation and renaturation
Washing the fermentation thalli of the normal expression product with 10 mM phosphate (PB for short hereinafter) and 0.9% sodium chloride solution; breaking bacteria by adopting 50 mM PB and 1mM EDTA solution; washing thalli by adopting 20mmol/L PB, 1mM EDTA, 1.0mol/L urea and 1% sodium chloride solution, and centrifuging to obtain a human insulin precursor inclusion body;
human insulin precursor inclusion bodies were dissolved with 7M urea, 20mM glycine (pH 10.5) and added to dithiothreitol at a concentration of 4mg/g (dithiothreitol/inclusion bodies) for denaturation at room temperature for 1h; after denaturation, the product was diluted 10-fold with 1mM oxidized glutathione, 5mM reduced glutathione, and 20mM glycine solution (pH 10.5), and renatured at 2-8deg.C to obtain the correctly folded product.
Respectively taking a denatured liquid supernatant and a renaturation liquid supernatant for liquid phase analysis, wherein an analysis column is Agilent PLRP-S300A 5 mu M150 x 4.6mm, and the analysis conditions are 30% B and 0-2.5min; 30-60% of B,2.5-25min;100% B,25-29.5min;30% B,29.5-35min (mobile phase A:0.1% TFA, mobile phase B: 0.1% TFA+80% ACN), flow rate 1mL/min, column temperature 35 ℃, detection wavelength 214nm, 10-fold dilution of denatured liquid supernatant, 20. Mu.L of sample injection volume, 20. Mu.L of renaturation liquid supernatant sample injection volume. The result shows that the renaturation yield of the human insulin precursor is 92.61 percent, and the renaturation effect is better.
Example 4
The insulin glargine precursors collected in example 1 and example 2 were subjected to isoelectric precipitation and then dissolved in 20mM sodium bicarbonate buffer, and digested with trypsin, and the enzyme amounts were determined as follows: adding trypsin into target protein 1:1000, and performing enzyme digestion under the condition of normal temperature and pH of 8.5 to obtain insulin glargine.
The enzyme digestion yield of the insulin glargine precursor is calculated to be about 56% -86%.
Comparative example
Insulin glargine was prepared as described in example 1, except that a first peptide fragment sequence and/or a second linker peptide sequence was used which was different from example 1, wherein the sequence of the first peptide fragment+the second linker peptide used is as shown in the following table:
| sequence name | Amino acid sequence |
| SEQ ID No.8 | MHKSSPQGPDKLLIRLKHLIDIVESKSRSKSRASGSDVGGR |
| SEQ ID No.9 | MKKMNLAVKIATLKGTAGLKGTAVAGGR |
| SEQ ID No.10 | MGTAVAGGR |
| SEQ ID No.11 | MKWVTFLLLLSGGR |
| SEQ ID No.12 | MGWSLIILFLVATATGGR |
| SEQ ID No.13 | MEGNTREDNFKHLLGNDNVKRPSEAGR |
SDS-PAGE electrophoresis detection shows the expression conditions as shown in the following table:
| sequence(s) | Expression situation |
| SEQ ID No.8 | Normal expression |
| SEQ ID No.9 | Normal expression |
| SEQ ID No.10 | Unexpressing |
| SEQ ID No.11 | Normal expression |
| SEQ ID No.12 | Normal expression |
| SEQ ID No.13 | Normal expression |
The renaturation ratio results are shown in the following table:
| sequence name | Recovery yield/% |
| SEQ ID No.8 | Yield of renaturation<20 |
| SEQ ID No.9 | Yield of renaturation<20 |
| SEQ ID No.11 | Yield of renaturation<20 |
| SEQ ID No.12 | Yield of renaturation<20 |
| SEQ ID No.13 | 21.4 |
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the present application, and that modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the present application.
SEQUENCE LISTING
<110> Shanghai Renshi biopharmaceutical Co., ltd
<120> an insulin or analog precursor thereof
<130> 0000
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> artificial sequence
<400> 1
Met Ile Val Glu Phe Gly Gly Arg
1 5
<210> 2
<211> 9
<212> PRT
<213> artificial sequence
<400> 2
Met Ile Val Glu Phe Gly Gly Gly Arg
1 5
<210> 3
<211> 10
<212> PRT
<213> artificial sequence
<400> 3
Met Ile Val Glu Phe Gly Gly Gly Gly Arg
1 5 10
<210> 4
<211> 14
<212> PRT
<213> artificial sequence
<400> 4
Met Ile Val Glu Phe Gly Gly Gly Gly Gly Gly Gly Gly Arg
1 5 10
<210> 5
<211> 11
<212> PRT
<213> artificial sequence
<400> 5
Met Ile Val Glu Phe Gly Gly Gly Gly Ser Arg
1 5 10
<210> 6
<211> 16
<212> PRT
<213> artificial sequence
<400> 6
Met Ile Val Glu Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg
1 5 10 15
<210> 7
<211> 31
<212> PRT
<213> artificial sequence
<400> 7
Met Ile Val Glu Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg
20 25 30
<210> 8
<211> 41
<212> PRT
<213> artificial sequence
<400> 8
Met His Lys Ser Ser Pro Gln Gly Pro Asp Lys Leu Leu Ile Arg Leu
1 5 10 15
Lys His Leu Ile Asp Ile Val Glu Ser Lys Ser Arg Ser Lys Ser Arg
20 25 30
Ala Ser Gly Ser Asp Val Gly Gly Arg
35 40
<210> 9
<211> 28
<212> PRT
<213> artificial sequence
<400> 9
Met Lys Lys Met Asn Leu Ala Val Lys Ile Ala Thr Leu Lys Gly Thr
1 5 10 15
Ala Gly Leu Lys Gly Thr Ala Val Ala Gly Gly Arg
20 25
<210> 10
<211> 9
<212> PRT
<213> artificial sequence
<400> 10
Met Gly Thr Ala Val Ala Gly Gly Arg
1 5
<210> 11
<211> 14
<212> PRT
<213> artificial sequence
<400> 11
Met Lys Trp Val Thr Phe Leu Leu Leu Leu Ser Gly Gly Arg
1 5 10
<210> 12
<211> 18
<212> PRT
<213> artificial sequence
<400> 12
Met Gly Trp Ser Leu Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Gly Arg
<210> 13
<211> 27
<212> PRT
<213> artificial sequence
<400> 13
Met Glu Gly Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn
1 5 10 15
Asp Asn Val Lys Arg Pro Ser Glu Ala Gly Arg
20 25
<210> 14
<211> 32
<212> PRT
<213> artificial sequence
<400> 14
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg
20 25 30
<210> 15
<211> 33
<212> PRT
<213> artificial sequence
<400> 15
Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro
1 5 10 15
Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys
20 25 30
Arg
<210> 16
<211> 21
<212> PRT
<213> artificial sequence
<400> 16
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
1 5 10 15
Glu Asn Tyr Cys Gly
20
<210> 17
<211> 30
<212> PRT
<213> artificial sequence
<400> 17
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
20 25 30
<210> 18
<211> 35
<212> PRT
<213> artificial sequence
<400> 18
Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly
1 5 10 15
Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu
20 25 30
Gln Lys Arg
35
<210> 19
<211> 21
<212> PRT
<213> artificial sequence
<400> 19
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
1 5 10 15
Glu Asn Tyr Cys Asn
20
Claims (7)
1. A insulin glargine precursor or a human insulin precursor, characterized in that,
wherein, in the insulin glargine precursor, the sequence of the first peptide segment and the second connecting peptide is shown in any one of SEQ ID No. 1-7; the second peptide segment is of se:Sub>A B-C-A structure, wherein the amino acid sequence of B is shown as SEQ ID No.14, the amino acid sequence of C is shown as SEQ ID No.15, and the amino acid sequence of A is shown as SEQ ID No. 16; and the first peptide segment is connected to the N end of the second peptide segment through a second connecting peptide;
in the human insulin precursor, the sequence of the first peptide segment and the second connecting peptide is shown as SEQ ID No. 1; the second peptide is of se:Sub>A B-C-A structure, wherein the amino acid sequence of B is shown as SEQ ID No. 17; the amino acid sequence of C is shown as SEQ ID No. 18; the amino acid sequence of A is shown as SEQ ID No. 19; and the first peptide fragment is linked to the N-terminus of the second peptide fragment via a second linking peptide.
2. A nucleic acid encoding the insulin glargine precursor or the human insulin precursor of claim 1.
3. A vector comprising the nucleic acid of claim 2.
4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.
5. The host cell of claim 4, wherein the host cell is an E.coli cell.
6. A method of preparing a insulin glargine precursor or a human insulin precursor comprising the steps of:
culturing the host cell of claim 4 or 5 under culture conditions suitable for expression of the insulin glargine precursor or human insulin precursor.
7. The method of claim 6, further comprising the step of:
denaturing the insulin glargine precursor or the human insulin precursor expressed by the host cell and then renaturating to obtain correctly folded insulin glargine precursor or human insulin precursor;
cutting the insulin glargine precursor or the human insulin precursor, and collecting the insulin glargine or the human insulin.
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| CN114805610B (en) * | 2022-06-23 | 2022-10-04 | 北京惠之衡生物科技有限公司 | Recombinant genetic engineering bacterium for highly expressing insulin glargine precursor and construction method thereof |
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| CN1415019A (en) * | 1999-12-29 | 2003-04-30 | 诺沃挪第克公司 | Method for making insulin precursors and insulin precursor analogus having improved fermentation yield in yeast |
| CN102816785A (en) * | 2012-05-24 | 2012-12-12 | 上海华谊生物技术有限公司 | Preparation method of insulin glargine and analogue thereof |
| WO2013015697A1 (en) * | 2011-07-28 | 2013-01-31 | Mabion S.A. | A recombinant protein, a polynucleotide encoding it as well as a method of obtaining insulin or its an analogue |
| CN107446039A (en) * | 2016-05-31 | 2017-12-08 | 江苏恒瑞医药股份有限公司 | A kind of human insulin analogue precursor and preparation method thereof |
| CN112105635A (en) * | 2018-06-18 | 2020-12-18 | 联合化学实验室有限公司 | Leader sequences for higher expression of recombinant proteins |
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| EP2069502B1 (en) * | 2006-09-27 | 2014-02-26 | Novo Nordisk A/S | Method for making maturated insulin polypeptides |
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| WO1998001473A1 (en) * | 1996-07-05 | 1998-01-15 | Novo Nordisk A/S | Method for the production of precursors of insulin, precursors of insulin analogues, and insulin like peptides |
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