A method for preparing proinsulin using efficient expression in Escherichia coli

Abstract

This application relates to the biomedical field, specifically to a method for efficiently preparing proinsulin using *E. coli* expression. The tandem proinsulin has the general formula leader peptide-(base cleavage site-enzyme cleavage site-proinsulin)n, wherein the leader peptide sequence is SEQ ID NO:1; the base cleavage site is a dipeptide NG; the enzyme cleavage site is selected from any one, two, or three of the following: trypsin cleavage site K, enterokinase cleavage site DDDDK, and / or Kex2 enzyme cleavage site KR or RR; n is a tandem repeat number of 3-5. The inclusion bodies obtained by this method do not require denaturation or dissolution, and the yield of inclusion bodies can reach 20 g / L. After purification, the target protein yield is approximately 6 g / L. Compared with yeast fermentation, the *E. coli* fermentation cycle is shorter, safer, and yields a higher target protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for expressing tandem proinsulin using Escherichia coli, and more specifically relating to the corresponding general formula structure of tandem proinsulin, its encoded nucleic acid, an expression vector containing said nucleic acid, Escherichia coli host cells, and inclusion body alkaline digestion and enzyme digestion methods. Background Technology

[0002] Diabetes mellitus (DM) is a common endocrine and metabolic disease characterized by hyperglycemia. According to the 10th edition of the Diabetes Atlas published by the IDF (International Diabetes Federation) in 2021, diabetes is one of the fastest-growing newly discovered diseases globally in the 21st century. It is estimated that approximately 537 million adults (aged 20-79) had diabetes in 2021, and this number is projected to reach 643 million by 2030 and 783 million by 2045. In 2021, more than 6.7 million adults died from diabetes-related diseases. The number of children and adolescents (≤19 years old) with diabetes is increasing annually. In my country, approximately 140 million people have diabetes, with a prevalence rate of 10.6%. This is projected to reach 170 million by 2045. Type 1 diabetes, type 2 diabetes, and special types of diabetes (such as gestational diabetes) are all present in the diabetic population, with type 2 diabetes accounting for nearly 95% in my country. Therefore, drug development institutions primarily focus on developing drugs for type 2 diabetes. Global diabetes-related healthcare expenditures are increasing year by year; according to IDF statistics, they were approximately $760 billion in 2019. This is projected to climb to $825 billion by 2030 and further rise to $845 billion by 2045. Clearly, the surge in the number of diabetes patients and the resulting financial burden are extremely serious issues globally.

[0003] Insulin is the only hormone in the human body that can lower blood sugar. It binds to insulin receptors on the cell surface, activating the glucose transporter GLUT4, promoting the transport of glucose transporters across the cell membrane, increasing the uptake and utilization of glucose by cells, while simultaneously inhibiting hepatic glucose production and reducing glycogenolysis and gluconeogenesis, thus effectively lowering blood sugar levels. In patients with type 1 diabetes, because insulin-producing cells are destroyed and unable to secrete insulin, they must rely on insulin injections to sustain life. For patients with type 2 diabetes, insulin is also an important treatment option when lifestyle interventions and non-insulin medications fail to control blood sugar levels.

[0004] Insulin was discovered in 1921 by Canadian scientists Banting and Best. Its discovery was a milestone in the history of diabetes treatment, and it began to be used to treat diabetes in 1922. In 1955, Sanger discovered the primary structure of insulin and was awarded the Nobel Prize in Chemistry. In 1965, Chinese scientists successfully synthesized crystalline bovine insulin. In 1969, Hodgkins determined the three-dimensional structure of insulin using X-ray diffraction and was again awarded the Nobel Prize in Chemistry. Early insulin was a crude product extracted from the pancreas of pigs, cattle, or sheep. It wasn't until 1936 that Scott obtained purified insulin crystals using recrystallization in the presence of zinc ions, laying the foundation for the development of long-acting insulin preparations.

[0005] Currently, proinsulin expression mainly utilizes yeast expression systems. While yeast expression systems can achieve extracellular secretion and disulfide bond formation, their expression levels are relatively low, typically around 3 g / L. Furthermore, yeast expression media are expensive, have long fermentation cycles (usually 8-12 days), and alcohol-inducible promoters (such as AOX1) pose fire hazards and cytotoxicity. For example, Gurramkond et al. (Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microbial Cell Factories 9, 31, 2010) disclosed a typical Pichia pastoris fed-batch culture and methanol-induced process, achieving an insulin precursor yield of ~3 g / L in a 15L fermenter; the fermenter feeding cycle was 180 h, plus 3-4 days for initial seed amplification, totaling 10-12 days. Furthermore, the glycosylation pattern of yeast expression systems differs significantly from that of humans, easily triggering immune responses and affecting protein activity; high-density culture also leads to protein degradation, secretion efficiency is limited by signal peptide selection, and some proteins remain intracellularly or form inclusion bodies. These drawbacks make low-cost, large-scale mass production difficult.

[0006] The present invention aims to find an alternative to overcome the shortcomings of yeast expression systems, such as low yield, long fermentation cycle, and alcohol-induced cytotoxicity, by expressing tandem proinsulin in Escherichia coli in the form of inclusion bodies, so as to reduce insulin production costs and increase yield to meet rapidly growing demand. Summary of the Invention

[0007] This invention provides a method for expressing tandem proinsulin using Escherichia coli, the corresponding general formula of tandem proinsulin, its encoded nucleic acid, a recombinant expression vector containing said nucleic acid, Escherichia coli host cells, and methods for alkali digestion and enzyme digestion of inclusion bodies.

[0008] One aspect of this invention is a recombinant tandem proinsulin with the general formula being leader peptide-(base cleavage site-enzyme cleavage site-proinsulin)n.

[0009] The leader peptide sequence is SEQ ID NO: 1, namely MGSSHHHHHHSSGLVPRGSHMSETKPKYNYVNNKELLQAIIDWKTELANNKAPNKVVRQNDTIGLAIMLIAEGLSKRFNFSGYTQSWKQEMIADGIEASIKGLHNFDETKYKNPHAYITQACFNAFVQRGSI;

[0010] The alkaline cleavage site is a dipeptide “NG”. This design cleverly utilizes the sequence characteristic that the C-terminal amino acid of proinsulin is N, so that there are no extra amino acids at the C-terminus after alkaline cleavage, and no additional steps are needed to remove the residue.

[0011] The cleavage site is any one, two, or a combination of three of the following: trypsin cleavage site "K", enterokinase cleavage site "DDDDK", and / or Kex2 cleavage site "KR" or "RR". The trypsin recognition site is "K". The enterokinase recognition site is "DDDDK". The protease Kex2 can recognize KR or RR, or KK and RK, but its cleavage ability towards KR or RR is significantly greater than its cleavage ability towards KK and RK; therefore, KR or RR can be used in this invention. The trypsin cleavage site K is preferred. The design of the cleavage site can be complementary to the basal cleavage site to ensure no residual amino acids in the proinsulin final product.

[0012] Where n is the number of cascaded repetitions between 3 and 5.

[0013] As used in this article, the term "proinsulin" refers to the full-length or partially truncated amino acid sequence of human proinsulin, or a mutated sequence in which 1-9 amino acid residues have been substituted, inserted, or deleted relative to natural human proinsulin, including but not limited to the following sequences: FVNQHLCGSHLVEALHLVCGERGFHYTPKAMKGIVEQCCTSICSLEQLENYCN (SEQ ID NO: 2), FVNQHLCGSHLVEALYLVCGERGFFYTPKAAKGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 3), FVNQHLCGSHLVEALYLVCGERGFFYTDKAAKGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 4).

[0014] In one aspect, the present invention provides a multicopy tandem protein sequence of proinsulin, the amino acid sequence of which is shown in SEQ ID NO: 6-19.

[0015] In one aspect, the present invention provides a nucleic acid encoding the aforementioned tandemly recombinant proinsulin.

[0016] In one aspect, the present invention provides a recombinant expression vector containing the nucleic acid, preferably a pET series vector.

[0017] In one aspect, the present invention provides a recombinant Escherichia coli host cell containing the expression vector, preferably the Escherichia coli host cell being BL21(DE3), BL21(DE3)pLysS, or Rosetta(DE3) cells.

[0018] Methods for transforming E. coli with recombinant expression vectors are well known in the art, with heat shock transformation being preferred, followed by selection for positive clones through antibiotic resistance. The method of inducing host protein expression by adding IPTG is a technique well known to those skilled in the art. A preferred concentration of IPTG is 0.1 mM to 1 mM, and the mixture is heated at 25°C. Induction at 37°C allows for more efficient expression of the target protein.

[0019] In one aspect, the present invention provides a method for preparing proinsulin using the aforementioned Escherichia coli host cells, comprising the following steps: (1) culturing and harvesting the Escherichia coli host cells, (2) cleaving the cells to obtain inclusion bodies, (3) alkali cleaving of the inclusion bodies, (4) refolding of the insulin precursor to form disulfide bonds, and (5) enzymatic cleavage to obtain proinsulin.

[0020] Methods for collecting *E. coli* are well-known in the art, with centrifugation being the preferred method. Methods for cell disruption are also well-known in the art, with high-pressure homogenization, freeze-thaw cycles, and ultrasonic disruption being preferred. After disruption, the cells are collected by centrifugation to collect inclusion bodies.

[0021] The inclusion bodies formed by the recombinant multicopy tandem protein sequence of proinsulin described in this invention do not require denaturation or dissolution; instead, the leader peptide can be removed by direct alkali cleavage to obtain free proinsulin.

[0022] In one aspect, the alkaline cleavage step of the present invention uses 2-6M guanidine hydrochloride, 1-2M hydroxylamine, and a pH 9.0-9.5 buffer solution for alkaline cleavage at room temperature for 6-10 hours.

[0023] In one aspect, the present invention provides a method for removing precursor peptides and other proteins after alkali cleavage, preferably using isoelectric point sedimentation. After alkali cleavage of inclusion bodies, the pH of the post-cleavage solution is adjusted to 8-9 to cause the precursor peptides and other proteins to aggregate and precipitate. Subsequent centrifugation can easily remove these other proteins, improving the purity of the target protein and greatly facilitating subsequent purification.

[0024] In one aspect, the present invention provides a method for refolding insulin precursors to form disulfide bonds. The refolding buffer is preferably 3-5 mM cysteine, 0.3-0.6 mM cysteine, pH 10.5. Ten times the volume of the refolding buffer is added to the purified supernatant, and the mixture is stirred at 2-8°C for 48 hours to refold.

[0025] After refolding, the sample is digested with enzymes to remove excess amino acids, forming an insulin precursor that can be used for subsequent side-chain acylation modifications. This invention can use trypsin, enterokinase, and / or Kex2 protease for digestion.

[0026] In a preferred embodiment, 1g of total protease, trypsin, enterokinase, and / or Kex2 enzyme are added per 100g of proinsulin for enzymatic digestion. The enzymatic digestion reaction is carried out at room temperature (20-25°C) with slow stirring and mixing for 1-3 hours.

[0027] Beneficial effects The beneficial effects of this invention are as follows: Compared to traditional yeast production systems, this invention utilizes an *E. coli* production system, avoiding cytotoxicity and fire hazards caused by alcohol induction. Furthermore, the *E. coli* fermentation cycle is 3-5 days, significantly shorter than the 8-12 day cycle of yeast. Simultaneously, due to the use of tandem expression of proinsulin, inclusion body yield can reach 20 g / L, and the target protein yield after purification is approximately 6 g / L, a substantial increase compared to the approximately 3 g / L yield of proinsulin prepared by mainstream yeast fermentation. Moreover, the obtained inclusion bodies do not require denaturation or dissolution; the leader peptide can be directly removed by alkali cleavage to obtain free proinsulin, simplifying the purification process and reducing purification costs. Attached Figure Description

[0028] Figure 1 The results show the expression of 3X proinsulin tandem protein (SEQ ID NO: 6) before and after induction by engineered bacteria. Lane 1 shows the protein expression before induction by engineered bacteria, lane 2 shows the protein expression after induction by engineered bacteria, and lane 3 is the marker.

[0029] Figure 2 The HPLC chromatogram of the triple proinsulin tandem protein shown in SEQ ID NO: 6 after the final enzymatic digestion step is shown as an example.

[0030] Figure 3This is the mass spectrometry spectrum of the target protein, proinsulin, after HPLC purification. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] The gene synthesis and sequencing of the nucleotide sequence encoding tandem proinsulin involved in the examples were performed by Jiangsu Saisofe Biotechnology Co., Ltd. Unless otherwise specified, all other raw materials, excipients, or reagents are commercially available products. Unless otherwise specified, the methods used in the examples are conventional methods in the art.

[0033] Example 1: Sequence design of tandem proinsulin To increase the proportion of proinsulin in the recombinant expressed protein and simplify subsequent protein purification, a 3-5 tandem design was employed for the proinsulin protein sequence. A leader peptide sequence was added to its N-terminus to enhance the expression level of the recombinant protein. A double-layer protection mechanism, consisting of a base cleavage site "NG" and an enzyme cleavage site, was added in the middle of the tandem sequence to facilitate the subsequent acquisition of the free insulin backbone. The enzyme cleavage site can be any one, two, or a combination of three of the following: trypsin cleavage site "K", enterokinase cleavage site "DDDDK", and / or Kex2 enzyme cleavage sites "KR" or "RR". The specific sequence designed in this invention is shown in Table 1 below.

[0034] Table 1. Design of tandem proinsulin sequences

[0035] Example 2: Construction of tandem proinsulin recombinant plasmid and engineered bacteria The amino acid sequences designed in Example 1 were optimized for codon preference in *E. coli* expression (SEQ ID NO: 5 is an example of the optimized DNA). The optimized genes were synthesized by Jiangsu Saisofe Biotechnology Co., Ltd., and integrated into pET28a to complete the construction of recombinant expression plasmids. Enzyme digestion and sequencing verified that the plasmids were consistent with the target sequences. The recombinant plasmids were introduced into BL21(DE3) competent cells via heat shock transformation, and single clones were obtained by plating on resistance plates to construct the tandem protein expression recombinant engineered bacteria.

[0036] Example 3: Induction of tandem proinsulin expression 3.1 Culture medium preparation LB medium: Tryptone 10g / L, Yeast Extract 5g / L, Sodium Chloride 10g / L (1-2% agar powder added to solid medium), sterilized at 115℃ for 30min.

[0037] TB liquid culture medium: Tryptone 11.8 g / L, Yeast Extract 23.6 g / L, Glycerol 5 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, sterilized at 115℃ for 30 min.

[0038] 3.2 Resuscitation of engineered bacteria Take one recombinant engineered glycerol bacterium and inoculate it into LB liquid medium (50ml / 500ml shake flask) at an inoculation rate of 0.1%. Add 25μg / ml Kana to each flask and incubate overnight (about 16h) at 37℃ and 220rpm in a full-temperature shaking incubator.

[0039] 3.3 Amplification and Culture of Engineered Bacteria and Induction of Recombinant Protein Expression Remove the seed culture medium and transfer 2 ml of the seed culture to a bottle of TB liquid culture medium (150 ml / 500 ml baffled shaker flask). After transfer, return the flask to a full-temperature shaking incubator at 37°C and 220 rpm for 2-3 hours. Remove the culture medium and add 150 μl of 1M IPTG (final concentration 1 mM) to each flask. Return the flask to a full-temperature shaking incubator at 30°C and 220 rpm overnight to induce target protein expression.

[0040] 3.4 Bacterial cell collection After approximately 21 hours of induction, the culture was removed from the flask. The fermentation broth was transferred in batches to 50ml centrifuge tubes and centrifuged at 4°C and 10,000 rpm for 5 minutes to collect the precipitated cells. The precipitate was then analyzed using SDS-PAGE. PAGE was used to examine the expression of recombinant proteins in bacterial cells.

[0041] Figure 1 The electrophoresis results of the engineered bacteria for the triple proinsulin tandem protein, as shown in SEQ ID NO: 6, are displayed before and after induction of expression. The results show that the expression level of the recombinant protein is significantly increased after induction.

[0042] Example 4: Collection of Inclusion Bodies 4.1 Preparation of cell disruption buffer Bacterial cell lysis buffer: 5 mmol / L EDTA, 25 mmol / L Tris HCl, pH 8.0. 4.2 Obtaining Inclusion Bodies Resuspend the bacterial cells in 10 mL of bacterial cell disruption buffer at a ratio of 1 g of bacterial cells. Stir thoroughly until no lumps of bacterial cells remain. The suspension is then subjected to ultrasonic disruption under the following conditions: 900 W equipment power, 45% power, φ6 probe, 3 seconds of sonication followed by a 5-second pause, for a total of 30 minutes. After sonication, centrifuge at 4°C and 10,000 rpm for 10 minutes, discard the supernatant, and collect the precipitate inclusion bodies.

[0043] According to SDS PAGE and inclusion body collection results, shake-flask fermentation verification showed that the yield of the triple proinsulin tandem protein shown in SEQ ID NO: 6 could reach 20 g / L of fermentation broth.

[0044] Example 5: Alkali cleavage of inclusion bodies This embodiment aims to demonstrate the advantages of the invented method in terms of process simplification. Specifically, the inclusion bodies obtained by this invention do not require denaturation or dissolution; instead, the lead peptide can be directly cleaved using alkali cleavage to obtain free proinsulin. This saves materials and shortens processing time.

[0045] 5.1 Preparation of alkaline shearing buffer Inclusion body cleavage buffer: 3M guanidine hydrochloride, 1M hydroxylamine, pH 9.0-9.5.

[0046] 5.2 Alkali Cutting Add 15 ml of inclusion body cleavage buffer to every 1 g of centrifuged inclusion bodies, and stir at 45-50 °C for 4-6 h. After the reaction, adjust the pH of the cleavage buffer to 8.5 to cause the cleaved leader peptide and other proteins to aggregate and precipitate. Centrifuge at 8000 rpm for 20 min to remove the other proteins and collect the supernatant.

[0047] Example 6: Refolding of insulin precursor to form disulfide bonds 6.1 Preparation of refolding buffer Refolding buffer: 3-5 mM cysteine, 0.3-0.6 mM cysteine, pH 10.5.

[0048] 6.2 Insulin precursor refolding to form disulfide bonds Add the supernatant to 10 times its volume of refolding solution and stir at 2-8°C for 48 hours to refold.

[0049] Example 7: Enzymatic cleavage to form the insulin backbone The refolded product obtained in Example 6 was subjected to enzymatic digestion by adding 1g of total protease, trypsin, enterokinase, and / or Kex2 enzyme per 100g of proinsulin. The enzymatic digestion reaction was carried out at room temperature (20-25°C) with slow stirring and mixing for 1-3 hours.

[0050] Example 8: Preparation of insulin backbone for HPLC detection and mass spectrometry confirmation The enzyme digestion reaction mixture from Example 7 was analyzed by HPLC and mass spectrometry. The detection methods are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C8 (2.1 mm) 100mm, 1.7μm) Main reagents: Mobile phase A: 0.1% formic acid-water solution Mobile phase B: 0.1% formic acid-acetonitrile solution Main experimental parameters: Column temperature: 40℃; Wavelength: 214nm; Flow rate: 0.4ml / min; Injection plate temperature: 5℃; Injection volume: 10μl; The HPLC linear elution conditions are shown in Table 2 below: Table 2. HPLC linear elution conditions

[0051] The purified proinsulin backbone HPLC chromatogram is shown below. Figure 2 As shown, the peak was observed at RT22.461 min, with a purity higher than 95% and a yield of approximately 6 g / L.

[0052] The purified proinsulin backbone was analyzed by mass spectrometry using HPLC, and the results are as follows: Figure 3 As shown, the detected molecular weight is 5633.56, which is consistent with the theoretical molecular weight.

[0053] In summary, compared to traditional yeast production systems, this invention utilizes an *E. coli* production system, avoiding safety hazards such as cytotoxicity and fire caused by alcohol induction. Furthermore, the *E. coli* fermentation cycle is 3-5 days, significantly shorter than the 8-12 day cycle of yeast. Simultaneously, due to the use of tandem expression of proinsulin, inclusion body yield can reach 20 g / L, and the purified target protein yield is approximately 6 g / L, a substantial increase compared to the approximately 3 g / L yield of proinsulin prepared by mainstream yeast fermentation. Moreover, the obtained inclusion bodies do not require denaturation or dissolution; the leader peptide can be directly removed by alkali cleavage to obtain free proinsulin, simplifying the purification process and reducing purification costs.

[0054] All technical features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can also be replaced by other features that have the same, equivalent, or similar function. Therefore, unless otherwise specified, each disclosed feature is merely an example of a series of equivalent or similar features.

[0055] From the above description, those skilled in the art can easily understand the key features of the present invention. Without departing from the spirit and scope of the present invention, many modifications can be made to adapt to various different uses and conditions. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A recombinant tandem proinsulin, characterized in that, The tandem proinsulin formula is leader peptide-(base cleavage site-enzyme cleavage site-proinsulin)n, wherein the leader peptide sequence is SEQ ID NO: 1; the base cleavage site is a dipeptide "NG"; the enzyme cleavage site is selected from any one or a combination of two or three enzyme cleavage sites selected from trypsin cleavage site "K", enterokinase cleavage site "DDDDK" and / or Kex2 enzyme cleavage site "KR" or "RR"; n is a tandem repeat number of 3-5.

2. The recombinant tandem proinsulin according to claim 1, characterized in that, The amino acid sequence of the proinsulin is selected from one of SEQ ID NO: 2-4.

3. The recombinant tandem proinsulin according to claim 1 or 2, characterized in that, The recombinant tandem proinsulin amino acid sequence is selected from one of SEQ ID NO: 6-19.

4. A nucleic acid, characterized in that, It encodes the recombinant tandem proinsulin as described in any one of claims 1-3.

5. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 4.

6. A recombinant Escherichia coli host cell, characterized in that, It comprises the recombinant expression vector as described in claim 5.

7. The recombinant Escherichia coli host cell according to claim 6, characterized in that, The host cells of the Escherichia coli mentioned therein are BL21 (DE3), HMS174 (DE3), pLysS, or Rosetta (DE3).

8. A method for preparing proinsulin using recombinant Escherichia coli host cells as described in claim 6 or 7, characterized in that, The method includes the following steps: (1) culturing and harvesting the Escherichia coli host cells, (2) cleaving the bacterial cells to obtain inclusion bodies, (3) alkaline cleavage of the inclusion bodies, (4) refolding of insulin precursor to form disulfide bonds, and (5) enzymatic cleavage to obtain proinsulin.

9. The method according to claim 8, characterized in that, The inclusion body alkaline cleavage step (2) uses 2-6M guanidine hydrochloride, 1-2M hydroxylamine, and pH 9.0-9.5 buffer solution for alkaline cleavage at room temperature for 6-10 hours.

10. The method according to claim 8, characterized in that, The insulin precursor refolding step (3) uses 3-5 mM cysteine, 0.3-0.6 mM cysteine, pH 10.5, and is stirred at 2-8℃ for 48 hours for refolding.

11. The method according to claim 8, characterized in that, In the enzymatic digestion step (4), 1g of total protease, trypsin, enterokinase, and / or Kex2 enzyme are added per 100g of proinsulin and reacted at room temperature for 1-3 hours.

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