CN114380903A - Insulin or its analogue precursor - Google Patents
Insulin or its analogue precursor Download PDFInfo
- Publication number
- CN114380903A CN114380903A CN202111615218.4A CN202111615218A CN114380903A CN 114380903 A CN114380903 A CN 114380903A CN 202111615218 A CN202111615218 A CN 202111615218A CN 114380903 A CN114380903 A CN 114380903A
- Authority
- CN
- China
- Prior art keywords
- insulin
- peptide
- precursor
- analogue
- analog
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Endocrinology (AREA)
- Microbiology (AREA)
- Diabetes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The embodiment of the application discloses an insulin or an analogue precursor thereof, which comprises a first peptide segment, wherein the amino acid sequence of the first peptide segment is MIVEF; a second peptide fragment comprising an a chain and a B chain of insulin or an analog thereof connected by a first connecting peptide; wherein the first peptide segment is connected to the N terminal of the second peptide segment through a second connecting peptide. The first peptide fragment in the precursor of insulin or an analogue thereof helps to correctly fold the precursor in the renaturation process, improves the renaturation yield of the precursor and further improves the yield of the insulin or the analogue thereof.
Description
Technical Field
The present application relates to an insulin or an analog precursor thereof, and a method for producing insulin or an analog thereof.
Background
Diabetes is one of the diseases seriously harming human health in the world at present, and insulin is an important medicament clinically used for treating diabetes at present. The human natural insulin is polypeptide formed by connecting an A chain formed by 21 amino acids and a B chain formed by 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 is first synthesized as preproinsulin on the beta cell ribosome of the islets of langerhans in the pancreas. Preproinsulin is a molecule comprising a leader peptide chain (SP) consisting of 24 amino acids, a B chain (B), a C peptide (C) consisting of 31 amino acids, and an A chain (A) arranged in a straight line in the order of "SP-B-C-A". When proinsulin enters the endoplasmic reticulum, the leader peptide chain is cleaved to become proinsulin (B-C-A). Proinsulin forms disulfide bonds within the endoplasmic reticulum. After formation of the three-dimensional structure, proinsulin is cleaved at the B-C binding site by prohormone converting enzyme PC1/3, which in turn cleaves the C-A binding site by converting enzyme PC 2. Finally, the 2 basic amino acids remaining at the C-terminus of the B chain when cleaved with PC1/3, i.e., the N-terminus of the C peptide, are cleaved by carboxypeptidase H to form insulin.
At present, two systems are mainly adopted for producing insulin and analogues thereof, wherein one system is a yeast expression system, and the other system is an escherichia coli system. US4430266 discloses a method for directly expressing proinsulin (B-C-A) by using an Escherichia coli host, and obtaining insulin with a correct sequence and structure by in vitro renaturation and then cutting with Trypsin and CPB.
The colibacillus 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 product to gather in the cell in the form of inclusion body, the correctly folded insulin and the analogue precursor thereof can be obtained only by the operations of renaturation and the like of the inclusion body after the cell is crushed, and the renaturation efficiency of the inclusion body directly influences the yield. In addition, the cleavage of the leader peptide and the C peptide from the precursor of insulin and its analogs to obtain the mature body generally uses proteases such as Trypsin, CPB and the like. More or less Trypsin can cause various miscut cuts and generate various impurities, thereby causing difficulty in subsequent fine purification and being not beneficial to the reduction of production cost.
Therefore, there is a need to improve the renaturation yield and the enzymatic cleavage efficiency of the insulin or insulin analog precursor in the prior art, to improve the yield, and to reduce the production cost.
Disclosure of Invention
The present application aims to provide a novel insulin or insulin analogue precursor, which has a high renaturation yield and which contributes to a high yield in the production of insulin or insulin analogues, and a process for preparing insulin or insulin analogues using the precursor.
In a first aspect of the present application, there is provided a precursor of insulin or an analog 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 a chain a and a chain B of the insulin or the analog thereof connected by a first connecting peptide, and the first peptide fragment is connected to the N-terminus of the second peptide fragment by a second connecting peptide. It has surprisingly been found in the present application that the first peptide stretch contributes to the correct folding of the precursor when the insulin or insulin analogue precursor is subjected to a renaturation procedure, thereby significantly increasing the renaturation yield of the precursor.
In a second aspect of the present application, in the insulin or analog precursor thereof of the present application, the amino acid sequence of the second connecting peptide is [ G ]mSn]xR, wherein m is an integer between 2 and 10, n is 0 or 1, 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 an analog precursor thereof of the application and/or an expression vector under culture conditions suitable for expression of said insulin or analog precursor thereof;
carrying out denaturation and renaturation on insulin or an analogue precursor thereof expressed by host cells to obtain correctly folded insulin or an analogue precursor thereof;
cleaving said insulin or analog precursor thereof and collecting the 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 the insulin or the 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 further, the yield of the insulin or the analogue thereof is improved.
In other aspects of the application, nucleic acids encoding precursors of the insulins or analogs thereof of the application, vectors and host cells containing the nucleic acids are also provided.
Detailed Description
The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
It is noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and that such ranges or values are understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
According to a first aspect of the present application there is provided an insulin or an analogue precursor thereof, the precursor comprising a first peptide stretch and a second peptide stretch, wherein the amino acid sequence MIVEF of the first peptide stretch, the second peptide stretch comprises an a-chain and a B-chain of the insulin or insulin analogue linked by a first linking peptide, and the first peptide stretch is linked to the N-terminus of the second peptide stretch by a second linking peptide. It is found that after the insulin or insulin analogue precursor is expressed by the host cell, the precursor can be folded into a correct structure through the processes of denaturation and renaturation, and then the mature insulin polypeptide can be obtained by cutting and removing the first peptide segment and the first connecting peptide. In the process of denaturation before renaturation, the first peptide segment can help the precursor to be correctly folded, so that the renaturation yield of the precursor is obviously improved, and the yield of the 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.
Modifications in the insulin molecule are indicated herein as single letter codes for the chain (a or B), position, and amino acid residue(s) of the substituted amino acid residue(s). Terms such as "a 1", "a 2", and "A3" and the like refer to the amino acids at positions 1, 2, and 3, respectively, in the a chain of insulin (counting from the N-terminus). Similarly, terms such as B1, B2, and B3, etc., refer to the amino acids at positions 1, 2, and 3, etc., respectively, in the B chain of insulin (counting from the N-terminus). Using the one letter code for amino acids, terms such as a21G, B28K, and B29P mean that the amino acid at position a21 is glycine and the amino acids at positions 28 and 29 are lysine and proline, respectively.
In some embodiments, the insulin analogues 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 at position B29 is substituted by Pro, Glu or Asp, Asn of B3 is substituted by Thr, Lys, gin, Glu or Asp, amino acid residue Asn of a21 is substituted by Ala, gin, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val, one or more amino acids, e.g. Lys, is added to the C-terminus of the a-and/or B-chain, amino acid of B1 is substituted by Glu, amino acid of B16 is substituted by Glu or His, B30 amino acid is deleted, B1 amino acid is deleted, B28-30 amino acid is deleted, B27 amino acid is deleted, the a-and/or B-chain has an N-terminal extension, the a-and/or B-chain has a C-terminal extension, e.g. two arginine residues are added to the C-terminus of the B-chain. In other embodiments, the amino acid of insulin analog a14 of the present application is Asn, gin, Glu, Arg, Asp, Gly, or His, and the amino acid at B25 is His, and optionally further comprising one or more additional mutations. In other embodiments, the amino acid residue of insulin analog a21 of the present application is Gly, or further extended at the C-terminus by two arginine residues.
In some embodiments, the insulin analog of the present application is selected from insulin glargine, insulin aspart, insulin lispro, insulin glulisine, insulin detemir, or insulin deglutaric; preferably insulin glargine.
The term "insulin or analog precursor thereof" as used herein refers to a polypeptide that can be converted to insulin or an analog thereof by one or more subsequent chemical and/or enzymatic steps.
In the insulin or analog precursor thereof of the present application, the second peptide segment comprises an a chain and a B chain of the insulin or analog thereof linked by a first linking peptide, which in some embodiments is linked to the a chain at the amino group of a1 and linked to the B chain at the amino group of B29 or the carboxyl group of B30.
The term "first linker peptide" as used herein 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 linker 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 its analogue precursors of the present application is directly linked to the A-chain and/or B-chain, or the C-peptide is linked to the A-chain and/or B-chain via one or two basic amino acids, respectively. In some embodiments, the C peptide is linked to the B chain via an RR and to the a chain via a KR, and during the cleavage, the protease specifically cleaves from the RR and KR, allowing the a and B chains 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. Native C-peptide refers to a linker peptide linking the B-chain and the a-chain in native human proinsulin, having two basic amino acids at each of the N-and C-termini, to effect cleavage of the native C-peptide. Non-limiting examples of short C-peptides include AAK, AAR, and DKAAK. The modified C peptide is a linker peptide obtained by modifying the C peptide.
Numerous examples of natural C-peptides, short C-peptides and modified C-peptides for linking a-chain and B-chain 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 a C-peptide comprising Gly residues. WO02/079250 discloses insulin precursors having C-peptides comprising Pro residues. WO02/100887 discloses insulin precursors having a C-peptide containing a glycosylation site. WO2008/037735 discloses insulin precursors with 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 precursor insulin or analog thereof of the present application is FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID No. 14). The sequence of the A chain is GIVEQCCTSICSLYQLENYCG (SEQ ID No. 16). The first linker peptide has the sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 15).
In some embodiments, the precursor insulin or insulin analog of the present application has a B chain sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID No. 17), an a chain sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID No.19), and a first linker peptide sequence of RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 18).
In some embodiments, the second peptide segment in the precursor of insulin or an analog thereof of the present application is proinsulin or a proinsulin analog.
In a second aspect of the present application, in the insulin or analog precursor thereof of the present application, the amino acid sequence of the second connecting peptide is [ G ]mSn]xR, 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 ]mS]xR, wherein m is an integer between 2 and 10, x is an integer between 1 and 10, and the first peptide fragment and the second connecting peptide sequence are preferably SEQ ID numbers 1 to 7. The application has surprisingly found thatWhen certain specific second connecting peptide sequences are adopted, the enzyme digestion efficiency of insulin or an analogue precursor thereof by protease is obviously improved, fewer impurities are generated in the enzyme digestion process, the subsequent fine purification is facilitated, and the production cost is reduced.
The present application also provides nucleic acids encoding the insulin or analog precursors thereof of the present application, vectors and host cells comprising 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-stranded or double-stranded, linear or circular, natural or synthetic, and if not otherwise indicated, are not subject to any size limitations. The nucleic acid molecule may also comprise a promoter, which may be homologous or heterologous.
The nucleic acid molecules of the present application can be cloned into vectors. "vector" in the present application includes plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In some embodiments, these vectors are suitable for 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 the nucleic acid molecules of the present application.
The vector of the present application may be an expression vector. Suitable expression vectors which have been widely described in the literature can be used in the present application. In one embodiment, the expression vector may contain a marker gene and an origin of replication ensuring replication in the chosen host, as well as a promoter and transcription termination signals. Between the promoter and the termination signal, there is preferably at least one restriction site capable of inserting the nucleic acid sequence/molecule of which expression is desired. Preferably, the expression vector of the present application is selected from the group consisting of a pET series expression vector, a pGEX series expression vector, and a pcDNA series expression vector, and 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 BL 21.
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 carried out in suitable stable host cells and the protein is collected from the cells (supernatant or lysed cells).
In another embodiment, a nucleic acid of the present application and/or a vector containing a nucleic acid 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 for the preparation of 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 the expression of the insulin or an analogue precursor thereof.
In some embodiments, the methods of the present application for preparing insulin or an analog thereof further comprise the steps of: carrying out denaturation and renaturation on insulin or an analogue precursor thereof expressed by host cells to obtain correctly folded insulin or an analogue precursor thereof; cleaving said insulin or analog precursor thereof and collecting the insulin or analog thereof.
When the insulin or the analog precursor thereof is expressed, especially in an escherichia coli expression system, the insulin or the analog precursor thereof is usually accumulated in cells in the form of inclusion bodies, and the inclusion bodies are required to be renatured after the cells are crushed to obtain the correct insulin or the analog precursor thereof. 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 analogue thereof, the correct folding of the precursor in the renaturation process is facilitated by the fact that the amino acid sequence in the precursor of the insulin or the analogue thereof is the first peptide segment of MIVEF, the renaturation yield of the precursor is improved, and further the yield of the insulin or the analogue thereof is improved.
In some embodiments, the method of preparing insulin or insulin analogs of the present application further comprises the steps of: constructing an expression vector comprising a nucleic acid encoding a precursor of an insulin or an analog thereof of the present application, and constructing a host cell comprising the expression vector by transient transfection of the host cell.
Suitable culture conditions for the expression of insulin or an analog precursor thereof will be known to those skilled in the art and one skilled in the art can empirically select a suitable medium for culturing under conditions suitable for the 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 precursor of insulin or its analog in the above method may be expressed intracellularly or on the cell membrane, 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 collection of the expression product. The method for lysing the cells and collecting the expression product is not particularly limited, and it is preferable to disrupt the cells using a 50 mM PB, 1mM EDTA solution 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 method of preparing insulin or insulin analogs of the present application further comprises the step of solubilizing the insulin or insulin analog expressed by the host cell. The method of dissolution is not particularly limited, and insulin or an insulin analog expressed by the collected host cells is preferably dissolved 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 a step of renaturation after denaturation of the insulin or insulin analog expressed by the host cell. There is no particular limitation on the method of denaturation and renaturation of inclusion body insulin or insulin analogues. Optionally, the denaturation is performed with dithiothreitol. Optionally, renaturation is performed using a 10-fold dilution of a renaturation solution containing 1mM oxidized glutathione, 5mM reduced glutathione and 20mM glycine.
In some embodiments, the denatured proteins may be enriched and concentrated using anion exchange chromatography prior to renaturation.
In some embodiments, the method of preparing insulin or an insulin analogue of the present application comprises the step of cleaving insulin or an analogue precursor thereof to obtain insulin or an analogue thereof. Preferably, the insulin or analogue precursor thereof is cleaved by trypsin to obtain insulin or an analogue thereof.
In some embodiments, the method of preparing insulin or an insulin analog of the present application further comprises a step of enzymatic cleavage by a carboxypeptidase. The product after trypsin cleavage was digested with carboxypeptidase to remove the C-terminal arginine from the B chain.
In some embodiments, the method of preparing insulin or insulin analogs of the present application further comprises the step of purifying the mature insulin or insulin analog from the digested sample using reverse phase chromatography.
Additional aspects and advantages of the present 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 present application.
Example 1
First, expression vector construction
Constructing a genetic engineering bacterium by using an escherichia coli expression system, selecting an escherichia coli preferred codon, taking pET22b as an expression vector, and inserting a nucleic acid sequence for coding the insulin glargine precursor into a multiple cloning site NdeI and Hind III of the expression vector.
In this embodiment, the first peptide fragment of the insulin glargine precursor has the sequence MIVEF, and the second connecting peptide has the sequence GGR, i.e., the sequence of the first peptide fragment + the second connecting peptide is MIVEFGGR (SEQ ID No. 1). The second peptide segment is of a B-C-A structure, wherein the amino acid sequence of B is FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID No. 14); the amino acid sequence of C is EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 15); the amino acid sequence of A is GIVEQCCTSICSLYQLENYCG (SEQ ID No. 16).
Second, host cell construction
BL21 (DE 3) strain was inoculated with CaCl2The method is used for preparing competence, and the constructed expression vector is transformed into BL21 (DE 3) competence and spread on Amp resistant LB agar culture medium for overnight culture at 37 ℃. The single clones were individually selected and cultured in LB liquid medium supplemented with Amp resistance, and then the seeds were preserved.
Third, shake flask expression of insulin glargine precursor
The obtained strain was subjected to shake flask culture using SOB, and when the strain was cultured to OD600 of about 1.0, IPTG was added to a final concentration of 0.5mM for induction of expression. After 12 hours of induction, the fermentation cells were harvested. Adding 20 μ l of fermentation thallus into 20 μ l of 5XSDSloadingbuffer, adding 60 μ l of purified water, performing SDS-PAGE electrophoresis after boiling water bath for 10-15min to detect expression, and displaying normal expression.
Collection, denaturation and renaturation of insulin glargine precursor
Washing the fermentation thallus of the normal expression product with 10 mM phosphate (hereinafter referred to as PB) and 0.9% sodium chloride solution; breaking the bacteria by adopting a 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;
dissolving the inclusion body of the insulin glargine precursor by using 7M urea and 20mM glycine (pH10.5), and adding dithiothreitol with the concentration of 4mg/g (dithiothreitol/inclusion body) for denaturation at room temperature for 1 h; after denaturation, the product was diluted 10-fold with 1mM oxidized glutathione, 5mM reduced glutathione, 20mM glycine solution (pH 10.5) and renatured at 2-8 ℃ to give the correctly folded product.
Respectively taking the supernatant of the denatured liquid and the supernatant of the renaturation liquid for liquid phase analysis, and analyzing the samples with Agilent PLRP-S300A 5 μ M150 x 4.6mm under the analysis condition of 30% B for 0-2.5 min; 30-60% of B, 2.5-25 min; 100% B, 25-29.5 min; 30% B, 29.5-35min (mobile phase A: 0.1% TFA, mobile phase B: 0.1% TFA +80% ACN), flow rate of 1mL/min, column temperature of 35 ℃, detection wavelength of 214nm, denaturation supernatant dilution 10 times, sample volume of 20 μ L, renaturation supernatant sample volume of 20 μ L, according to the following formula to calculate renaturation rate:
renaturation yield = renaturation sample peak area/denatured sample peak area × 100%.
The result shows that the renaturation yield of the insulin glargine precursor obtained by the method of the embodiment is 79.5 percent, and the renaturation effect is better.
Example 2
Glargine was prepared as described in example 1, except that a second linker peptide sequence different from that of example 1 was used, and the sequence of the first peptide fragment + the second linker peptide used in this example is as follows:
| sequence numbering | Sequence of | Renaturation 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 method can normally express and obtain the insulin glargine precursor, and has a good renaturation effect.
Example 3
First, expression vector construction
Constructing a genetic engineering bacterium by using an escherichia coli expression system, selecting an escherichia coli preferred codon, taking pET26b as an expression vector, and inserting nucleic acid for encoding a natural human insulin precursor into multiple cloning sites NdeI and Hind III of the expression vector;
the sequence of the first stretch of peptide + the second linker peptide of the natural human insulin precursor in this example is MIVEFGGR (SEQ ID No. 1). The second peptide segment is of a B-C-A structure, wherein the amino acid sequence of B is FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID No. 17); the amino acid sequence of C is RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID No. 18); the amino acid sequence of A is GIVEQCCTSICSLYQLENYCN (SEQ ID No. 19). The constructed expression vector is named RHI-pet26 b.
Second, host cell construction
Mixing BL21 StarTM(DE 3) Strain with CaCl2Preparing competence, transforming the constructed expression vector into BL21 StarTM(DE 3) in the competent state, spread on Kan-resistant LB agar medium and cultured overnight at 37 ℃. The single clones were selected and cultured in LB liquid medium supplemented with Kan resistance, respectively, and then the seeds were preserved.
Third, Shake flask expression
The obtained strain was subjected to shake flask culture using TB medium, and when the strain was cultured to OD600 of about 1.0, IPTG was added to the final concentration of 0.5mM for induction of expression. After 12 hours of induction, the fermentation cells were harvested. 20 mul of the fermentation thallus is added into 20 mul of 5XSDSloadingbuffer, 60 mul of purified water is added, SDS-PAGE electrophoresis is carried out after boiling water bath for 10-15min to detect the expression condition, and the result shows that the fermentation thallus can be normally expressed.
Fourthly, human insulin precursor collection, denaturation and renaturation
Washing the fermentation thallus of the normal expression product with 10 mM phosphate (hereinafter referred to as PB) and 0.9% sodium chloride solution; breaking the bacteria by adopting a 50 mM PB and 1mM EDTA solution; washing the 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;
dissolving human insulin precursor inclusion body with 7M urea and 20mM glycine (pH10.5), and adding dithiothreitol with concentration of 4mg/g (dithiothreitol/inclusion body) for denaturation at room temperature for 1 h; after denaturation, the product was diluted 10-fold with 1mM oxidized glutathione, 5mM reduced glutathione, 20mM glycine solution (pH 10.5) and renatured at 2-8 ℃ to give the correctly folded product.
Respectively taking the supernatant of the denatured liquid and the supernatant of the renaturation liquid for liquid phase analysis, and analyzing the samples with Agilent PLRP-S300A 5 μ M150 x 4.6mm under the analysis condition of 30% B for 0-2.5 min; 30-60% of B, 2.5-25 min; 100% B, 25-29.5 min; 30% B, 29.5-35min (mobile phase A: 0.1% TFA, mobile phase B: 0.1% TFA +80% ACN), flow rate of 1mL/min, column temperature of 35 ℃, detection wavelength of 214nm, dilution of denatured liquid supernatant by 10 times, injection volume of 20 muL, renaturation of supernatant of 20 muL. The result shows that the renaturation yield of the human insulin precursor is 92.61%, and the renaturation effect is good.
Example 4
The insulin glargine precursors collected in example 1 and example 2 were respectively precipitated at isoelectric points, dissolved in 20mM sodium bicarbonate buffer, and digested with trypsin, according to the enzyme amounts: adding trypsin into the target protein 1:1000, and carrying out enzyme digestion at the normal temperature and the pH value of 8.5 to obtain the insulin glargine.
The enzyme digestion yield of the insulin glargine precursor is about 56-86 percent by calculation.
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 different from example 1 was used, wherein the sequences of the first peptide fragment + the second linker peptide used are 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 |
The expression of SDS-PAGE is shown in the following table:
| sequence of | Expression profiles |
| SEQ ID No.8 | Normal expression |
| SEQ ID No.9 | Normal expression |
| SEQ ID No.10 | Is not expressed |
| SEQ ID No.11 | Normal expression |
| SEQ ID No.12 | Normal expression |
| SEQ ID No.13 | Normal expression |
The renaturation results are shown in the following table:
| sequence name | Yield of renaturation/% |
| SEQ ID No.8 | Recovery yield of renaturation<20 |
| SEQ ID No.9 | Recovery yield of renaturation<20 |
| SEQ ID No.11 | Recovery yield of renaturation<20 |
| SEQ ID No.12 | Recovery yield of renaturation<20 |
| SEQ ID No.13 | 21.4 |
In the description herein, reference to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," 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 application. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and that modifications, substitutions, and alterations may be made to the above embodiments by those of ordinary skill in the art within the scope of the present application.
SEQUENCE LISTING
<110> Shanghai Rencui biopharmaceutical GmbH
<120> an insulin or an 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 (15)
1. An insulin or insulin analog precursor comprising
A first peptide segment, wherein the amino acid sequence of the first peptide segment is MIVEF;
a second peptide fragment comprising an a chain and a B chain of insulin or an analog thereof connected by a first connecting peptide;
wherein the first peptide segment is connected to the N terminal of the second peptide segment through a second connecting peptide.
2. The insulin or analog precursor of claim 1, wherein the amino acid sequence of the second connecting peptide is [ G ]mSn]xR, wherein m is an integer between 2 and 10, n is 0 or 1, and x is an integer between 1 and 10.
3. An insulin or an analogue precursor thereof as claimed in claim 1 or 2, wherein said first linker peptide is a polypeptide fragment which can be cleaved enzymatically or chemically from the a chain and the B chain.
4. An insulin or an analogue precursor thereof according to any one of claims 1 to 3, wherein the first linking peptide comprises a C-peptide.
5. An insulin or an analogue precursor thereof according to claim 4, wherein the C-peptide is selected from a natural C-peptide, a short C-peptide or a modified C-peptide.
6. An insulin or an analogue precursor thereof according to claim 1, wherein the second peptide stretch is proinsulin or an proinsulin analogue.
7. An insulin or an analogue precursor thereof according to claim 1, wherein the insulin analogue is selected from insulin glargine, insulin aspart, insulin lispro, insulin glulisine, insulin detemir or insulin degludec.
8. An insulin or an analogue precursor thereof according to claim 7, wherein the insulin analogue is insulin glargine.
9. The insulin or analog precursor thereof according to claim 1, wherein the insulin is natural human insulin.
10. A nucleic acid encoding the insulin or analog precursor thereof of any one of claims 1 to 9.
11. A vector comprising the nucleic acid of claim 10.
12. A host cell comprising the nucleic acid of claim 10 or the vector of claim 11.
13. The host cell of claim 12, wherein the host cell is an e.
14. A method of preparing insulin or an analog thereof, comprising the steps of:
culturing the host cell of claim 12 or 13 under culture conditions suitable for expression of said insulin or analog precursor thereof.
15. The method of claim 14, further comprising the steps of:
carrying out denaturation and renaturation on insulin or an analogue precursor thereof expressed by host cells to obtain correctly folded insulin or an analogue precursor thereof;
cleaving said insulin or analog precursor thereof and collecting the insulin or analog thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111615218.4A CN114380903B (en) | 2021-12-28 | 2021-12-28 | Insulin or analogue precursor thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111615218.4A CN114380903B (en) | 2021-12-28 | 2021-12-28 | Insulin or analogue precursor thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114380903A true CN114380903A (en) | 2022-04-22 |
| CN114380903B CN114380903B (en) | 2023-07-25 |
Family
ID=81198177
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111615218.4A Active CN114380903B (en) | 2021-12-28 | 2021-12-28 | Insulin or analogue precursor thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114380903B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114805610A (en) * | 2022-06-23 | 2022-07-29 | 北京惠之衡生物科技有限公司 | Recombinant gene engineering bacterium for high expression of insulin glargine precursor and construction method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2002100887A2 (en) * | 2001-06-08 | 2002-12-19 | Novo Nordisk A/S | Method for making insulin precursors and insulin precursor analogs |
| 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 |
| US20100184133A1 (en) * | 2006-09-27 | 2010-07-22 | Novo Nordisk A/S | Method for making maturated insulin polypeptides |
| 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 |
-
2021
- 2021-12-28 CN CN202111615218.4A patent/CN114380903B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| WO2002100887A2 (en) * | 2001-06-08 | 2002-12-19 | Novo Nordisk A/S | Method for making insulin precursors and insulin precursor analogs |
| US20100184133A1 (en) * | 2006-09-27 | 2010-07-22 | Novo Nordisk A/S | Method for making maturated insulin polypeptides |
| 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 |
| 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 |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| MARCIN ZIELIŃSKI ET AL.: "Expression and purification of recombinant human insulin from E. coli 20 strain", 《 PROTEIN EXPRESSION AND PURIFICATION》, vol. 157, pages 63 - 69 * |
| 房德兴,王永山,周宗安,翟春生,顾志香,王元伦: "人胰岛素原类似物(BKRA)基因的合成与表达", 中国生物化学与分子生物学报, no. 05, pages 518 - 524 * |
| 李晓红等: "重组人胰岛素制备工艺", 《四川大学学报》, no. 4, pages 79 - 83 * |
| 马雪: "重组人胰高血糖素样肽-1前药（Pro-rhGLP-1）的表达、纯化及药效学研究", 《全国优秀博士学位论文》, pages 1 - 128 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114805610A (en) * | 2022-06-23 | 2022-07-29 | 北京惠之衡生物科技有限公司 | Recombinant gene engineering bacterium for high expression of insulin glargine precursor and construction method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114380903B (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU648670B2 (en) | A-C-B Proinsulin, method of manufacturing and using same, and intermediates in insulin production | |
| CN111072783B (en) | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence | |
| CN110724187B (en) | Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof | |
| JP4504014B2 (en) | Methods for producing insulinotropic GLP-1 (7-36) polypeptides and / or GLP-1 analogs | |
| Alexander et al. | Characterization and modelling of the hydrophobic domain of a sunflower oleosin | |
| AU610135B2 (en) | Polypeptides with activity against gram-positive and gram-negative bacteria | |
| US20160168226A1 (en) | Process for production of insulin and insulin analogues | |
| CN114380903B (en) | Insulin or analogue precursor thereof | |
| WO2020187270A1 (en) | Fusion protein containing fluorescent protein fragments and uses thereof | |
| CA2159079C (en) | Methods and dna expression systems for over-expression of proteins in host cells | |
| WO2012115638A1 (en) | Glargine proinsulin compositions and methods of producing glargine insulin analogs therefrom | |
| CN111320702A (en) | Method for efficient secretion fusion expression and recombinant preparation of bacillus prodigiosus nuclease in methanol yeast | |
| CN114933658B (en) | Short peptide element and application method thereof | |
| CA2050606C (en) | Enzymatic process for the conversion of preproinsulins into insulins | |
| CN112105635A (en) | Leader sequences for higher expression of recombinant proteins | |
| AU2019218315A1 (en) | Codon optimized precursor gene and signal peptide gene of human insulin analogue | |
| WO2019143193A1 (en) | N-terminal fusion partner for producing recombinant polypeptide, and method for producing recombinant polypeptide using same | |
| EP3344651B1 (en) | A process for obtaining insulin with correctly formed disulfide bonds | |
| CN114686504B (en) | Application of Lpp or mutant thereof as molecular chaperone in secretory expression of recombinant protein in escherichia coli | |
| CN101172996A (en) | Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation | |
| JP4386722B2 (en) | Method for producing acylated polypeptide | |
| KR102064810B1 (en) | N-terminal fusion partner for preparing recombinant polypeptide and method of preparing recombinant polypeptide using the same | |
| US20160030520A1 (en) | Liquid insulin compositions and methods of making the same | |
| US20030082671A1 (en) | Method for making acylated polypeptides | |
| CN113801235A (en) | Insulin lispro derivative and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40072579 Country of ref document: HK |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |