CN118531030A - Expression cassette, recombinant vector, recombinant protein and application thereof - Google Patents

Abstract

The present invention relates to the field of bioengineering, and in particular to expression cassettes, recombinant vectors, recombinant proteins and their applications. The present invention provides an expression cassette, including but not limited to: a leader peptide, a restriction site, a linker and a GLP-1 receptor agonist main chain; the GLP-1 receptor agonist main chain has: an amino acid sequence as shown in SEQ ID NO: 1. Based on previous studies, the present invention has designed a multi-tandem repeat sequence, and experimentally proved the feasibility of the scheme. Through the multi-tandem repeat sequence, the biosynthesis amount of the semaglutide main chain is increased, and the ideal protein constant can be obtained after subsequent multi-step purification, providing more raw materials for subsequent experiments.

Description

Expression cassette, recombinant vector, recombinant protein and application thereof

Technical Field

The present invention relates to the field of bioengineering, and in particular to an expression box, a recombinant vector, a recombinant protein and applications thereof.

Background Art

Diabetes mellitus (DM) is a common metabolic disease, the main feature of which is organ dysfunction caused by the direct or indirect effects of chronic hyperglycemia. It can be divided into two main types: type 1 diabetes and type 2 diabetes. According to the International Diabetes Federation, the global prevalence of diabetes was estimated to be 9.3% in 2019, with approximately 463 million people suffering from the disease. It is estimated that by 2030, the prevalence of diabetes will rise to 10.2% (approximately 578 million people) and to 10.9% (approximately 700 million people) by 2045. Diabetes is one of the leading causes of mortality and disability worldwide. It not only threatens the quality of life and life expectancy of patients, but also imposes a huge economic burden on individuals and the global medical system.

Obesity is a complex chronic metabolic disease, the main feature of which is the accumulation of body fat exceeding normal levels, which has adverse effects on health. In recent years, with the change of people's lifestyles and the influence of environmental factors, the incidence of obesity has continued to rise, and has become a global health challenge. It is estimated that the global prevalence of obesity is about 14%, and in some regions it is as high as more than 40%, equivalent to about 1 billion people. This number is expected to increase to 1.5 billion by 2030.

In recent years, significant progress has been made in the field of peptide drug research and development, and the design and development of peptide drugs for specific disease targets have become more precise. This progress is attributed to the improvement of targeting, the introduction of new formulation technologies, the continuous improvement of synthesis and modification technologies, and the emergence of novel treatment strategies. These technological improvements can effectively improve the stability, solubility and biodistribution of peptide drugs, optimize the pharmacokinetic properties of peptide drugs, and prolong their plasma half-life, thereby improving their efficacy and reducing adverse reactions. In this context, semaglutide, as an emerging peptide drug, has shown good efficacy in the treatment of diabetes and adult obesity.

Semaglutide is a synthetic analog developed by Danish pharmaceutical company Novo Nordisk. It is a GLP-1 receptor agonist used to treat type 2 diabetes. GLP-1 is an insulin-releasing hormone produced by intestinal cells, which has the functions of promoting insulin secretion, inhibiting glucagon secretion and reducing appetite. In 2017, the FDA approved the injection of semaglutide under the brand name Wegovy for the treatment of adult obesity. In January 2024, the China National Medical Products Administration approved the first oral semaglutide for the treatment of type 2 diabetes, which is also the first oral GLP-1 receptor agonist approved for marketing in China.

As shown in Figure 1, semaglutide is composed of 31 amino acids with a long side chain modification. Since the half-life of the GLP-1 main chain is extremely short, only about 1-2 minutes, in order to prevent the degradation of the polypeptide, the polypeptide needs to be modified to extend the half-life of the drug and reduce the toxic side effects of the drug. Therefore, the 8th amino acid (alanine) of the polypeptide was replaced with a non-natural amino acid (a-aminoisobutyric acid) to prevent the rapid degradation of GLP-1 caused by the DPP4 enzyme to achieve the purpose of extending the half-life; the 26th lysine at the bottom was modified with a long side chain (PEG and C18 fatty diacid chain connected to glutamic acid), which increased the hydrophilicity and stability of the polypeptide, prevented the degradation of the polypeptide, and effectively extended the half-life of semaglutide.

The biosynthesis is mainly based on Escherichia coli (E.Coli) and Pichia pastoris. The peptide sequence is rationally designed and cut using special tool enzymes to finally obtain the main chain of semaglutide.

Summary of the invention

In view of this, the present invention provides an expression cassette, a recombinant vector, a recombinant protein and its application. Based on the previous research, the present invention designs a multi-tandem repeat sequence and experimentally proves the feasibility of the scheme. The multi-tandem repeat sequence increases the biosynthesis amount of the semaglutide main chain, and after subsequent purification, the ideal protein constant can be obtained, which provides a feasible scheme for the multi-tandem expression of a single structural protein, solves the problem that the multi-tandem repeat sequence of a single structural protein cannot be expressed and assembled normally, and can introduce a special protease cleavage site, and obtain a complete target protein sequence by late enzymatic cleavage.

In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

The present invention provides an expression cassette, including but not limited to: a leader peptide, an enzyme cleavage site, a linker and a GLP-1 receptor agonist backbone;

The GLP-1 receptor agonist backbone has:

(1) the amino acid sequence shown in SEQ ID NO: 1; or

(2) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (1); or

(3) A sequence having a homology of more than 90% with the amino acid sequence shown in (1).

In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 1 is: EGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

In some embodiments of the present invention, in the above expression cassette, the nucleic acid molecule encoding the GLP-1 receptor agonist backbone has a sequence as shown in any of SEQ ID NO:4 to SEQ ID NO:9;

The sequence of SEQ ID NO:4 is: GAAGGCACGTTTACCTCTGATGTGAGCTCTTATTTAGAAGGCCAGGCGGCTAAAGAATTTATTGCGTGGCTTGTGCGCGGCCGCGGC;

The sequence of SEQ ID NO:5 is: GAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAGTTTATTGCGTGGTTAGTGCGCGGTCGCGGT;

The sequence of SEQ ID NO: 6 is: GAAGGCACCTTTACGAGCGATGTGAGCAGCTATTTAGAAGGTCAGGCGGCGAAAGAATTCATTGCGTGGTTAGTGCGTGGTCGTGGT;

The sequence of SEQ ID NO:7 is: GAGGGCACCTTTACCTCCGATGTGAGCAGCTATTTGGAAGGCCAGGCGGCGAAGGAATTTATTGCGTGGCTGGTTCGTGGTCGCGGT;

The sequence of SEQ ID NO: 8 is: GAAGGCACCTTTACCTCTGATGTGAGCAGCTATCTCGAAGGCCAGGCGGCCAAAGAATTTATTGCATGGCTGGTTCGTGGCCGAGGT;

The sequence of SEQ ID NO: 9 is: GAAGGCACGTTTACCAGCGATGTGAGCTCTTACCTGGAAGGCCAGGCGGCAAAAGAATTTATCGCGTGGTTGGTGCGTGGTCGTGGC.

In some embodiments of the present invention, in the above expression cassette, the number of the GLP-1 receptor agonists is no less than 6.

In some embodiments of the present invention, in the above expression cassette, the number of the GLP-1 receptor agonists is 6, 7, 8, or 9.

In some embodiments of the present invention, in the above expression cassette, the restriction enzyme cleavage site comprises: one or more of an EK enzyme cleavage site, a KEX2 enzyme cleavage site and a CPB enzyme cleavage site.

In some embodiments of the present invention, in the above expression cassettes, the EK enzyme cleavage site is located at the N-terminus of the fourth expression cassette.

In some embodiments of the present invention, in the above expression cassette, the KEX2 enzyme cleavage site and the CPB enzyme cleavage site are located at the N-terminus of the GLP-1 receptor agonist backbone.

In some embodiments of the present invention, in the above expression cassette, the amino acid sequence of the EK enzyme cleavage site is: DDDDK.

In some embodiments of the present invention, in the above expression cassette, the sequence of the nucleic acid molecule encoding the EK enzyme cleavage site is as shown in SEQ ID NO: 10 or SEQ ID NO: 11: GATGATGATGACAAG (SEQ ID NO: 10) or GACGACGACGACAAG (SEQ ID NO: 11).

In some embodiments of the present invention, in the above expression cassette, the amino acid sequence of the KEX2 enzyme cleavage site is: RR.

In some embodiments of the present invention, in the above expression cassette, the sequence of the nucleic acid molecule encoding the KEX2 enzyme cleavage site is shown in any of SEQ ID NO:12 to SEQ ID NO:15: CGCCGT (SEQ ID NO:12), AGACGT (SEQ ID NO:13), GCCGCT (SEQ ID NO:14) or CGTCGC (SEQ ID NO:15).

In some embodiments of the present invention, in the above expression cassette, the amino acid sequence of the CPB enzyme cleavage site is: R.

In some embodiments of the present invention, in the above expression cassette, the sequence of the nucleic acid molecule encoding the CPB enzyme cleavage site is shown in any of SEQ ID NO:16 to SEQ ID NO:18: CGC (SEQ ID NO:16), CGT (SEQ ID NO:17) or AGA (SEQ ID NO:18).

In some embodiments of the present invention, in the above expression cassette, the leader peptide has:

(4) the amino acid sequence shown in SEQ ID NO: 2; or

(5) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (4); or

(6) A sequence having a homology of more than 90% with the amino acid sequence shown in (4).

In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 2 is: LVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSMRLNSA.

In some embodiments of the present invention, in the above-mentioned expression cassette, the nucleic acid molecule encoding the leader peptide has a sequence as shown in SEQ ID NO:22: CTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGCCATGGCTGATATCGGATCCATGCGCCTGAACAGCGCG.

In some embodiments of the present invention, in the above expression cassette, the Linker has:

(7) the amino acid sequence shown in SEQ ID NO: 3; or

(8) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (7); or

(9) A sequence having a homology of more than 90% with the amino acid sequence shown in (7).

In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 3 is: GSGSEEGSGS.

In some embodiments of the present invention, in the above expression cassette, the nucleotide sequence of the nucleic acid molecule encoding the Linker is shown in SEQ ID NO: 19: GGTAGCGGTTCTGAGGAAGGTTCTGGAAGC.

In some embodiments of the present invention, the above expression cassette further comprises: a Trx-6x His tag.

In some embodiments of the present invention, in the above expression cassette, the amino acid sequence of the Trx-6x His tag is as shown in SEQ ID NO: 20: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSG.

In some embodiments of the present invention, in the above expression cassette, the nucleotide sequence encoding the Trx-6x His tag is as shown in SEQ ID NO:21: ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGT.

In some embodiments of the present invention, in the above expression cassette, the Trx-6x His tag is located at the N-terminus of the leader peptide.

The present invention also provides a recombinant vector, comprising: the above expression cassette.

In some embodiments of the present invention, in the above-mentioned recombinant vector, the number of the expression cassette is greater than or equal to one.

In some embodiments of the present invention, in the above-mentioned recombinant vector, the number of the expression cassettes is 1, 2 or 3.

The present invention also provides a host for transforming and/or transfecting the above recombinant vector.

The present invention also provides a recombinant protein obtained after being expressed and renatured by the above host.

The present invention also provides the use of the above expression cassette, the above recombinant vector, the above host and/or the above protein in increasing the production and/or expression level of the GLP-1 receptor agonist.

The present invention provides an expression cassette, including but not limited to: a leader peptide, an enzyme cleavage site, a linker and a GLP-1 receptor agonist backbone;

The GLP-1 receptor agonist backbone has:

(1) the amino acid sequence shown in SEQ ID NO: 1; or

(2) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (1); or

(3) A sequence having a homology of more than 90% with the amino acid sequence shown in (1).

The present invention optimizes the design of multiple tandem repeat sequences and greatly increases the number of tandem repeats on the basis of existing patents, up to 24 tandem repeat sequences, providing a feasible solution for the multiple tandem expression of single structural proteins, solving the problem that multiple tandem repeat sequences of single structural proteins cannot be expressed and assembled normally, and by introducing special protease cleavage sites, the complete target protein sequence can be obtained by later enzymatic cleavage.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

Figure 1 shows a schematic diagram of the drug structure of semaglutide;

Figure 2 shows the design principle of multiple tandem repeat sequences;

FIG3 shows the number of multiple series sequences designed by the present invention;

Fig. 4 shows the SDS-PAGE image of protein purification;

FIG. 5 shows the SDS-PAGE image of the protein purification of the multiple expression cassette.

DETAILED DESCRIPTION

The invention discloses an expression box, a recombinant vector, a recombinant protein and applications thereof.

It should be understood that the expression "one or more of..." includes individually each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

In Examples 1 to 6 of the present invention and the effect examples, the formulas of different culture media and buffers used in the experiments are as follows:

LB medium (1 L): 10 g peptone, 10 g sodium chloride, 5 g yeast extract;

TB medium/fermentation medium (1 L): 18 g peptone, 12 g yeast extract, 10 g sodium chloride;

Feed medium (1 L): 25-65% glucose, 20 g peptone, 10 g yeast extract;

Buffer A: 50 mM Tirs·HCl pH 6.5-8.5, 300 mM NaCl;

Buffer B: 50 mM Tirs·HCl pH 6.5-8.5, 300 mM NaCl, 2 M urea;

Buffer C: 50 mM Tirs·HCl pH 6.5-8.5, 300 mM NaCl, 8 M urea;

Buffer D: 50 mM Tirs·HCl pH 6.5~8.5, 300 mM sodium chloride, gradient urea (4-2-1-0 )M.

In Examples 1 to 6 and the Effect Examples of the present invention, the raw materials and reagents used can be purchased from the market.

The present invention will be further described below in conjunction with embodiments:

Example 1 Tandem repeat sequence design principles and routes

The design principle of this patent is shown in Figure 2. pQLinkN/pET-32a is used as the expression vector. The principle of sequence design is to repeat multiple semaglutide backbones in tandem. As shown in Figure 2, a Trx-tag is added to the N-terminus. This sequence is to promote protein translation; then an EK enzyme (enterokinase) cleavage site (DDDDK-, Asp-Asp-Asp-Asp-Lys-) is added to repeat multiple copies of the sequence in tandem, and each two semaglutide backbone tandem repeats are connected by a linker with the sequence "-RR-GSGSEEGSGS-RR-" or "-RR-GSGSEEGSGS-DDDDK-".

Among them, the sequence "DDDDK-" can be specifically recognized and cut by the EK enzyme (enterokinase) from the C-terminus; the "RR-" sequence can be cut by the KEX2 enzyme (serine protease), because the KEX2 enzyme can specifically recognize the two basic amino acid residues of RR or KR, and cut from the C-terminus of these two residues; "-R-R" is cut by the CPB enzyme (carboxypeptidase B), because the CPB enzyme has the exonuclease activity of specifically recognizing the two basic amino acids of K or R, and sequentially removes the two redundant amino acids from the C-terminus, and finally forms a complete semaglutide backbone. Figure 2 shows the design principle of the multiple tandem repeat sequence, and Figure 3 shows the multiple tandem sequence involved in the present invention.

Specifically, the steps include: starting from the start codon (ATG) of the expression cassette reading frame, which is the N-terminus; ending at the termination codon (TGA) of the expression cassette reading frame, which is the C-terminus; unless otherwise specified, the expression design sequence of this patent starts from the N-terminus and ends at the C-terminus.

Example 2 Vector selection and construction technology

pQLinkN was selected as the expression vector. The characteristic of this vector is that it can connect multiple independently running multi-copy clone expression cassettes in series through recombination on a single vector. Theoretically, this vector can have N multi-clone expression cassettes. Previous studies have confirmed that there are no less than 5 multi-clone expression cassettes that can independently complete protein expression, that is, one vector can express 5 proteins.

Therefore, in the present invention, while constructing the multiple tandem repeat sequence of the semaglutide backbone, the tandem sequence is used as a copy unit to construct a polyclonal gene expression cassette again. At this time, the theoretical protein expression value of the backbone of the protein is a multiple increase of the polyclonal repeat sequence. Taking 5 tandem repeat sequences as an example: 5× repeat sequences are a copy unit, and when 2 gene expression cassettes are established, the number of backbones is 2 times, that is, 10 repeat sequences (5×2), and when 3 gene expression cassettes are established, the number of backbones is 3 times, that is, 15 repeat sequences (5×3), and this is repeated.

The vector selected by the present invention is the commercial vector pET-32a, and Nco1 (CCATGG) and Xho1 (CTCGA) are selected for sequence insertion. First, 6-9 tandem repeat sequences of a single expression cassette are designed, which are hereinafter marked as 6 X - 9 X; then multiple expression cassette repeats are designed to increase the tandem repeat sequence greater than 10 X. The present invention designs a single expression cassette with a maximum of 9 multiple tandem expression sequences and a multiple expression cassette with a maximum of 3, that is, 24 multiple tandem expression sequences.

The present invention relates to the following combination:

6 X: N-leading peptide-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB; (the order of the CPB-GLP-1 backbone sequence: SEQ ID NO:4-SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:7-SEQ ID NO:8-SEQ ID NO:9)

7 X: N-leading peptide-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB; (the order of the CPB-GLP-1 backbone sequence: SEQ ID NO: 5-SEQ ID NO: 6-SEQ ID NO: 7-SEQ ID NO: 4-SEQ ID NO: 6-SEQ ID NO: 9-SEQ ID NO: 5)

8 X: N-leading peptide-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB; (the order of the CPB-GLP-1 backbone sequence: SEQ ID NO: 5-SEQ ID NO: 6-SEQ ID NO: 7-SEQ ID NO: 8-SEQ ID NO: 9-SEQ ID NO: 10-SEQ ID NO: 11-SEQ ID NO: 12-SEQ ID NO: 13-SEQ ID NO: 14-SEQ ID NO: 15 NO:6-SEQ ID NO:9-SEQ ID NO:5-SEQ ID NO:6)

9 X：N-leading peptide-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-EK-KEX2/CPB-linker-EK-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB-linker-KEX2/CPB-GLP-1 backbone-KEX2/CPB; (sequence of CPB-GLP-1 backbone: SEQ ID NO: 5-SEQ ID NO: 6). NO:6-SEQ ID NO:7-SEQ ID NO:4-SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:7-SEQ ID NO:8-SEQ ID NO:9)

16 X: 1 8 X is used as a repeating unit, and 2 repeating units are connected in series to obtain 16 X;

24 X: 1 8 X is used as a repeating unit, and 3 repeating units are connected in series to obtain 24 X;

After obtaining the sequence design, we synthesized the optimized codon sequence of the tandem protein by gene synthesis and recombined it into the expression vector pET-32a. After correct sequencing, the protein was expressed.

Finally, we designed 6-9 tandem repeat sequences to increase the expression level and yield of semaglutide backbone protein by increasing the number of tandem repeats in trans.

Example 3 Biosynthesis technology of recombinant protein

Unless otherwise specified, transformation of E. coli, common culture medium (LB\TB, fermentation medium), feed medium, fermentation protocol, and protein purification protocol were performed using conventional laboratory methods.

The frozen plasmid was transformed and plated using BL-21 (DE3), cultured overnight at 37°C, and several single colonies were picked and cultured overnight in LB medium. This was the primary seed solution. The primary seed solution cultured overnight was added to an appropriate amount of TB medium and cultured to an OD ₆₀₀ of 2-7. This was the secondary seed solution, which could be used to inoculate the fermenter.

The secondary seed liquid was inoculated at an inoculation rate of 0.1-1%. The fermentation temperature was 37°C, the speed was set at 300-1200r/min, the ventilation was set at 500 mL/min, and the pH was 6.5-7.5 for fermentation culture. When the substrate sugar in the fermentation broth was exhausted, feeding began, and the feeding was regulated according to the fermentation parameters (such as dissolved oxygen, pH, etc.) until the fermentation was completed. When the OD ₆₀₀ of the fermentation broth reached 100, 0.1-1.5 mM IPTG was added for induction, and the fermentation temperature was appropriately lowered according to the characteristics of the protein. The induction temperature was set in the range of 15-35, which can be determined during the early small test. During the fermentation process, OD ₆₀₀ needs to be tested every two hours. When the fermentation OD enters the plateau phase, the fermentation can be terminated, and the tank can be started. The bacteria are enriched by a high-speed centrifuge, and the subsequent crushing treatment or temporary storage in a -80°C refrigerator is carried out according to the experimental arrangement.

The semaglutide main chain multi-tandem repeat sequence proteins involved in the present invention all exist in the form of inclusion bodies, so it is necessary to perform a denaturation experiment of the inclusion bodies. Take appropriate bacteria and resuspend them with buffer A at 1:5~20 (W/V), perform high-pressure crushing (large volume) or ultrasonic crushing (small volume), use a pre-cooled high-speed centrifuge at 18000 rpm, centrifuge for 40-60 min and collect the precipitate, which is the crude inclusion body. Resuspend the crude inclusion body with buffer B at 1:10 (W/V), centrifuge at 18000 rpm, centrifuge for 40-60 min and collect the precipitate, repeat this process 3 times, this is the inclusion body after washing, which can be used for subsequent denaturation experiments.

Take the inclusion bodies after appropriate washing and resuspend them with buffer C at 1:20 (W/V), incubate at room temperature, fully dissolve the inclusion bodies (denature), use a pre-cooled high-speed centrifuge at 18000 rpm, centrifuge for 40-60 min and discard the precipitate, collect the supernatant, which is the denatured inclusion body protein. The renaturation of the inclusion body protein can be implemented according to the specific conditions of the experiment, and dilution renaturation, dialysis renaturation and on-column renaturation of the affinity column can be selected. The present invention adopts dialysis renaturation. According to the molecular weight of the protein, a dialysis bag with a suitable molecular weight pore size is selected, and an appropriate amount of denatured protein is added to perform a renaturation experiment. Use buffer D for the renaturation experiment, change the liquid every 6-8 hours, and finally slowly remove the denaturant (urea or guanidine hydrochloride) to obtain the renatured protein, which is the main chain multi-tandem repeat sequence protein of semaglutide.

Example 4

The vector of 8X single expression cassette was selected for protein purification and expression. The specific experimental details are described in "Example 3 Biosynthesis Technology of Recombinant Protein". After the protein expression cassette inclusion body renaturation experiment, a protein with good purity was obtained.

Example 5

The vector of 8×2 expression cassette was selected for protein purification and expression, and the details are as in Examples 1 to 3.

Example 6

The vector of 8×3 expression cassette was selected for protein purification and expression, and the details are as in Examples 1 to 3.

Effect example

The present invention optimizes the design cassette of the protein expression and purification method, and the multi-tandem repeat sequence of the designed polypeptide can obtain the target protein. Among them, the multi-tandem repeat sequence protein of all designed polypeptides exists in the form of inclusion bodies in prokaryotic cells (E.coli), and the soluble target protein needs to be obtained through inclusion body renaturation experiment; as the number of expression cassettes increases, the protein yield also increases. Compared with 8 X, the protein yield of 8 X 3 expression cassettes increases by 64%, and the results are shown in Table 1:

Table 1

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. An expression cassette, characterized by including but not limited to: a leader peptide, a restriction site, a linker and a GLP-1 receptor agonist backbone; The GLP-1 receptor agonist backbone has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (1); or (3) A sequence having a homology of more than 90% with the amino acid sequence shown in (1). 2. The expression cassette of claim 1, wherein the number of the GLP-1 receptor agonists is no less than 6. 3. The expression cassette according to claim 1 or 2, characterized in that the restriction sites include: one or more of an EK enzyme restriction site, a KEX2 enzyme restriction site and a CPB enzyme restriction site. 4. The expression cassette according to any one of claims 1 to 3, wherein the leader peptide has: (4) the amino acid sequence shown in SEQ ID NO: 2; or (5) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (4); or (6) A sequence having a homology of more than 90% with the amino acid sequence shown in (4). 5. The expression cassette according to any one of claims 1 to 4, wherein the Linker has: (7) the amino acid sequence shown in SEQ ID NO: 3; or (8) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in (7); or (9) A sequence having a homology of more than 90% with the amino acid sequence shown in (7). 6. A recombinant vector, characterized in that it comprises: an expression cassette according to any one of claims 1 to 5. 7. The recombinant vector according to claim 6, wherein the number of the expression cassette is greater than or equal to 1. 8. A host, characterized in that it is transformed and/or transfected with the recombinant vector according to claim 6 or 7. 9. The recombinant protein is characterized in that it is obtained after being expressed and renatured by the host as described in claim 8. 10. Use of the expression cassette according to any one of claims 1 to 5, the recombinant vector according to claim 6 or 7, the host according to claim 8 and/or the protein according to claim 9 in increasing the production and/or expression of a GLP-1 receptor agonist.