A dual peptide receptor agonist and its preparation method and application

Abstract

The present invention discloses a dual-peptide receptor agonist and a preparation method and application thereof, which belongs to the field of genetic engineering, especially the preparation of protein or polypeptide drugs, and specifically relates to the preparation and application of a dual-target receptor (GLP-1 and GIP) agonist peptide, a dual GLP-1 single-target receptor agonist and a dual GIP single-target receptor agonist. The fusion protein does not need to be purified, and the dual-target receptor (GLP-1 and GIP) agonist peptide, the dual GLP-1 single-target receptor agonist and the dual GIP single-target receptor agonist can be obtained in one step through the SpyCatcher / Tag efficient self-connection, which greatly improves the yield. The method can be applied to the preparation of a new drug with dual-target agonist activity and / or single-target agonist activity with a longer half-life.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, in particular to the preparation of protein or polypeptide drugs, and specifically relates to a dual-peptide receptor agonist and a preparation method and application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) has become a global epidemic. Glucagon-like peptide-1 receptor (GLP-1) can promote the synthesis and secretion of insulin and inhibit the secretion of glucagon. Due to its excellent blood sugar lowering and weight loss effects, it has become an important choice for the treatment of T2DM (Baggio et al., Gastroenterology, 2007, 132(6): 2131-57). With the development and marketing of various glucagon-like peptide-1 receptor agonists (GLP-1RAs), a new generation of dual / multi-target receptor agonists has become the mainstream of research and development. Among them, Tirzepatide has been approved for marketing by the US Food and Drug Administration (FDA). Tirzepatide, a single-molecule dual-target GLP-1 / GIP (glucose-dependent insulinotropic polypeptide) agonist, has shown superior glucose-lowering and weight-loss efficacy compared to semaglutide (Coskun et al., Molecular metabolism, 2018, 18: 3-14). However, the design of such single-molecule agonists relies on the high homology of the peptides, and it is difficult to precisely control the affinity of the molecule for different targets during the design process, making it difficult to predict the distribution of the molecule's efficacy for each target (Frias et al., New Engl J Med, 2021, 385(6): 503-515). In addition, the gene fusion expression strategy also has limitations, because GLP-1 needs to retain its natural N-terminus to ensure activity, and can only be fused with peptides / proteins that do not have this requirement.

[0003] Spy chemistry is a type of genetically encoded click chemistry based on two genetically encoded elements, SpyCatcher and SpyTag, that can spontaneously form polymers without complex post-translational modifications. This process requires no chemical reagents, is rapid, and operates under mild conditions, making it promising for broad application (Zakeri et al., PNAs, 2012, 109(12): E690-E697). Combining Spy chemistry with ELP can generate powerful protein-based frameworks, providing new options for the design and preparation of multifunctional protein oligomers, polymers, or networks (Sun et al., PNAs, 2014, 111(31): 11269-11274).

[0004] In summary, there is still room for improvement in the design of dual (multi) target receptor agonists based on GLP-1. Therefore, this field still needs to develop more efficient, simple and widely applicable methods for preparing receptor agonists. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention aims to provide a dual-peptide receptor agonist and a preparation method and application thereof.

[0006] A method for preparing a dual-peptide receptor agonist comprises the following steps:

[0007] (a) constructing a fusion protein expression vector of a target polypeptide fused with an icSAT tag and a SpyTag peptide or a SpyCatcher peptide and expressing the fusion protein;

[0008] (b) Mixing two fusion proteins and self-ligating to form a dual fusion protein;

[0009] (c) The dual-peptide receptor agonist was purified using the icSAT protein purification method and the two-step standard column purification method.

[0010] Furthermore, the target polypeptide is connected to the SpyTag peptide or the SpyCatcher peptide via an ELP peptide.

[0011] Furthermore, in step (a), the target polypeptide is glucagon-like peptide-1 (GLP-1) and / or glucose-dependent insulinotropic polypeptide (GIP); the glucagon-like peptide-1 (GLP-1) and / or glucose-dependent insulinotropic polypeptide (GIP) is connected to the SpyTag peptide (or SpyCatcher peptide) via an ELP peptide.

[0012] Furthermore, the amino acid sequence of the GLP-1 is shown as SEQ ID NO: 1, and the coding base sequence of the GLP-1 is shown as SEQ ID NO: 16; the amino acid sequence of the GIP is shown as SEQ ID NO: 2, and the coding base sequence of the GIP is shown as SEQ ID NO: 17.

[0013] Furthermore, the characteristics of the ELP peptide meet one of the following conditions:

[0014] (a) Repeating sequence of Val-Pro-Gly-Xaa-Gly;

[0015] (b) Val-Pro-Gly-Xaa-Gly repeat sequence, the number of repeats of the Val-Pro-Gly-Xaa-Gly repeat sequence is 10 to 30 times;

[0016] (c) a repeating sequence of Val-Pro-Gly-Xaa-Gly, where Xaa is Val and Glu;

[0017] (d) a repeating sequence of Val-Pro-Gly-Xaa-Gly, where Xaa is Val and Glu, and the ratio of Xaa amino acids Val and Glu is 3:1 to 5:1;

[0018] (e) The amino acid sequence of the ELP peptide is shown in SEQ ID NO: 3.

[0019] Furthermore, the SpyCatcher peptide and the SpyTag peptide in step (a) can form an isopeptide chain; the SpyCatcher peptide includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the SpyTag peptide includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0020] Furthermore, in step (a), the method for preparing the fusion protein specifically comprises the following steps:

[0021] The purification tag, target polypeptide and ELP peptide are sequentially connected with the gene sequence of SpyTag peptide or SpyCatcher peptide to form the gene of the fusion protein, the gene of the fusion protein is constructed into an expression vector and introduced into the host cell to obtain an engineered bacterium; the engineered bacterium is cultured to express the fusion protein, and then the engineered bacterium is lysed to capture the fusion protein.

[0022] Furthermore, the capturing and obtaining of the fusion protein includes lysing the engineered bacteria and then centrifuging to obtain the supernatant to obtain the fusion protein.

[0023] Furthermore, the purification tag consists of a self-assembling peptide and a cleavage tag; the gene sequence of the self-assembling peptide and the gene sequence of the cleavage tag are connected via a linker.

[0024] Preferably, the self-assembling peptide is a salinity-induced self-assembling peptide, and the salinity-induced self-assembling peptide is a β-pleated short peptide; the amino acid sequence of the β-pleated short peptide is shown in SEQ ID NO: 11. Preferably, the cleavage tag is a chemical cleavage site, an enzymatic cleavage site, or a self-cleavage site.

[0025] More preferably, the self-cleavage site is an intein, and the intein is Person ΔI-CM (WT) intein or Person ΔI-CM intein mutant, the Person ΔI-CM intein mutants include Person ΔI-CM (m1), Person ΔI-CM (m2) and Person ΔI-CM (m3); Person The amino acid sequence of ΔI-CM (WT) is shown in SEQ ID NO: 12. Person The amino acid sequence of ΔI-CM (m1) is shown in SEQ ID NO: 13. Person The amino acid sequence of ΔI-CM (m2) is shown in SEQ ID NO: 14; Person The amino acid sequence of ΔI-CM (m3) is shown in SEQ ID NO: 15.

[0026] Preferably, the linker is a PT-type linker; the amino acid sequence of the PT-type linker is shown in SEQ ID NO: 10.

[0027] Furthermore, in step (b), the mixing of the two fusion proteins is specifically: mixing the fusion protein fused with the SpyTag peptide and the fusion protein fused with the SpyCatcher peptide at a molar ratio of 1:1 to obtain a dual fusion protein solution.

[0028] Furthermore, the temperature of the mixing reaction is 4-37° C., and the time of the mixing reaction is 0.5-24 h.

[0029] Furthermore, in step (c), the icSAT protein purification method specifically includes: adding an equal volume of high-salt buffer to the dual fusion protein solution to aggregate the dual fusion protein, and centrifuging to retain the aggregates; and adjusting the pH of the solution for cleavage.

[0030] Furthermore, the high salt buffer is a buffer containing 1.0-1.4 M Na2SO4 and a pH of 8.5, the aggregation temperature is 4-37°C, and the aggregation time is 0.5-12 h.

[0031] Furthermore, the cleavage is performed by dispersing the dual fusion protein aggregates in an acidic buffer, performing the cleavage treatment, and centrifuging to obtain the supernatant. Preferably, the pH value of the acidic buffer is 5.5-6.8, the temperature of the cleavage treatment is 4-37°C, and the cleavage treatment time is 3-48 hours.

[0032] Furthermore, in step (c), the two-step standard column purification method includes ion exchange purification and molecular sieve purification.

[0033] The present invention also provides a dual-peptide receptor agonist prepared by any of the above preparation methods.

[0034] Furthermore, the dual-peptide receptor agonist includes a GLP-1 and GIP dual-target receptor agonist, a dual GLP-1 single-target receptor agonist, and a dual GIP single-target receptor agonist.

[0035] Furthermore, the half effective concentration EC of the GLP-1 and GIP dual-target receptor agonist on the GLP-1 receptor in vitro is 50 The half effective concentration (EC) for GIP receptor agonism in vitro was 0.305 ± 0.048 nM. 50 is 0.375 ± 0.030 nM.

[0036] Furthermore, the GLP-1 and GIP dual-target receptor agonist is composed of GLP-1, SpyTag peptide, SpyCatcher peptide, GIP and two ELP peptides; GLP-1 and / or GIP are connected to the SpyTag peptide (or SpyCatcher peptide) through the ELP peptide; the SpyCatcher peptide and the SpyTag peptide can form an isopeptide chain.

[0037] Furthermore, the half effective concentration EC of the dual GLP-1 single-target receptor agonist on the GLP-1 receptor in vitro is 50 The concentration of β-actin is 0.044 ± 0.006 nM, and it has no in vitro agonist activity on GIP receptor.

[0038] Furthermore, the half effective concentration EC of the dual GIP single-target receptor agonist for the in vitro agonistic activity of the GIP receptor is 50 The concentration of β-catenin was 0.187 ± 0.011 nM, and it had no in vitro agonist activity on the GLP-1 receptor.

[0039] The present invention provides the use of the above-mentioned dual-peptide receptor agonist in the preparation of a medicine for preventing or treating type 2 diabetes.

[0040] The present invention provides a polynucleotide comprising a nucleotide sequence encoding the above-mentioned fusion protein of the present invention or a complementary sequence thereof.

[0041] Furthermore, the nucleotide sequence of the fusion protein includes the gene sequences of the purification tag, the target polypeptide, the ELP peptide and the SpyTag peptide (or the SpyCatcher peptide).

[0042] Furthermore, the gene sequence of the purification tag includes a self-assembly peptide, a linker, and a gene sequence of the cleavage tag, and the gene sequence of the self-assembly peptide and the gene sequence of the cleavage tag are connected via a linker.

[0043] Preferably, the self-assembling peptide is a salinity-induced self-assembling peptide, and the linker is a PT-type linker.

[0044] Furthermore, the target polypeptide is GLP-1 and / or GLP.

[0045] Furthermore, the fusion protein includes but is not limited to the fusion protein EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag、EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher(ΔN),EFK8- Person ΔI-CM(WT)-GIP-ELP 15 -SpyTag and EFK8- Person ΔI-CM (WT)-GIP-ELP 15 -SpyCatcher (ΔN).

[0046] Furthermore, the fusion protein EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag encoding nucleotide sequence is shown in SEQ ID NO: 18, wherein the initial ATG and the final TGA are the start codon and the stop codon, respectively; the fusion protein EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher (ΔN) encoding nucleotide sequence is shown in SEQ ID NO: 19; the fusion protein EFK8- Person ΔI-CM (WT)-GIP-ELP 15-SpyTag encoding nucleotide sequence is shown in SEQ ID NO: 20; the fusion protein EFK8- Person ΔI-CM (WT)-GIP-ELP 15 The nucleotide sequence encoding -SpyCatcher(ΔN) is shown in SEQ ID NO: 18.

[0047] The present invention provides an expression vector comprising the polynucleotide.

[0048] The present invention provides a host cell comprising the above-mentioned polynucleotide or the above-mentioned expression vector; the host cell is capable of expressing the above-mentioned fusion protein of the present invention.

[0049] In the recombinant expression vector of the present invention, the polynucleotide sequence encoding the fusion protein is appropriately linked to expression control sequences to achieve desired transcription and ultimately production of the fusion protein in host cells. The expression control sequences include, but are not limited to, promoters, enhancers, ribosome binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, and sequences that stabilize mRNA.

[0050] The recombinant expression vectors of the present invention include vectors that can replicate autonomously in host cells, such as plasmid vectors; and vectors that can integrate into host cell DNA and replicate together with the host cell DNA. In a specific embodiment, the expression vector of the present invention is derived from pET30a(+) from Novagen. Host cells used to express the fusion protein of the present invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotes include Escherichia coli ( Escherichia ), Bacillus ( Bacillus ), Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces ). In a preferred embodiment, the host cell is an Escherichia cell, preferably an Escherichia coli cell. In a specific embodiment of the present invention, the host cell used is an Escherichia coli BL21 (DE3) strain cell (Novagen).

[0051] In the present invention, the GLP-1 peptide (or GIP peptide) is fused to the C-terminus of the C-terminally cleaved intein, retaining the native N-terminus of the GLP-1 peptide (or GIP peptide) and avoiding the weakening of receptor affinity caused by N-terminal modification; the ELP peptide used is non-toxic, biodegradable, and has good biocompatibility, making it very safe and reliable to use as a drug delivery material.

[0052] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0053] (1) The present invention uses the efficient and highly specific reaction of SpyCatcher / Tag, and the fusion protein can be quickly reacted to obtain the target protein without prior purification.

[0054] (2) The present invention achieves rapid preparation of the target protein and retains the natural N-terminus of the target polypeptide through an inducible and cleavable self-aggregation tag (icSAT) protein purification method; based on the inducible and cleavable self-aggregation tag, an experimental scheme is designed to first self-link to form a double fusion protein and then purify the icSAT protein. Compared with the experimental process of purification first and then self-linking, this experimental scheme is simpler and does not require the addition of other tags to the N-terminus of the target polypeptide, thus saving costs and having a significantly higher yield than purification first and then self-linking. ① Purification followed by self-ligation: After the first separate preparative purification, the two components are mixed. Since some fusion proteins are not completely connected, a second preparative purification is required. However, the present invention does not require preparative purification of the two components before combination. Only icSAT purification and conventional two-step fine column purification are required after combination; ② Purification followed by self-ligation: Since the purification tag has been lost in the first preparative purification, an additional purification tag (such as a His tag) needs to be added to the N- or C-terminus of the target polypeptide, and a second preparative purification is performed using size exclusion chromatography. The final drug product also needs to remove the additional purification tag, which is a complex process and costly. However, the present invention can directly obtain a dual-peptide receptor agonist without an additional purification tag; ③ Purification followed by self-ligation cannot recover the self-cleaved protein, resulting in loss of intermediate products and reduced yield of the final receptor agonist.

[0055] (3) By utilizing the ELP peptide sequence that does not aggregate at room temperature, the problem of toxicity caused by easy aggregation when currently used in the medical field of ELP peptides is avoided; the ELP peptide is used to connect the target polypeptide and the SpyTag peptide (or SpyCatcher peptide) to form a linear structure of "target polypeptide-ELP peptide-SpyTag peptide-SpyCatcher peptide-ELP peptide-target polypeptide" dual-peptide receptor agonist, which has a more stable spatial structure; the N-terminal truncated SpyCatcher (ΔN) is used, which has low immunogenicity and is suitable for drug development.

[0056] (4) The polypeptide dual agonist obtained in the present invention has nanomolar-level in vitro agonist activity against different receptors.

[0057] (5) In common single-molecule dual agonists (such as Tirzepatide), changes in amino acid sites will simultaneously affect the efficacy of the molecule against different targets, and the effects after the changes are difficult to predict. However, the two polypeptides of the present invention are independent, and the affinity of the dual peptides for different targets can be more accurately controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1is a schematic diagram of the structure of the fusion expression protein vector of the present invention, wherein A and B are GLP-1-ELP based on the icSAT method 15 -SpyTag and GLP-1-ELP 15 -Schematic diagram of the structure of the SpyCatcher (ΔN) fusion protein expression vector.

[0059] Figure 2 GLP-1-ELP based on the icSAT method 15 -SDS-PAGE results of the expression of SpyTag / SpyCatcher (ΔN) fusion protein.

[0060] Figure 3 GIP-ELP based on the icSAT method 15 -Schematic diagram of the structure of the SpyCatcher (ΔN) fusion protein expression vector.

[0061] Figure 4 It is based on the icSAT method. Person ΔI-CM(WT), Person ΔI-CM (m1), Person SDS-PAGE results of the expression of ΔI-CM (m2) intein fusion protein.

[0062] Figure 5 GIP-ELP based on the icSAT method 15 -Schematic diagram of the structure of the SpyTag fusion protein expression vector.

[0063] Figure 6 GIP-ELP based on the icSAT method 15 -SDS-PAGE results of the expression of SpyTag fusion protein.

[0064] Figure 7 It is an SDS-PAGE result diagram of the purification process of the receptor agonist of the present invention; wherein A, B, and C are SDS-PAGE result diagrams of the purification process of the GLP-1 / GIP dual-target agonist, dual GLP-1 single-target agonist, and dual GIP single-target agonist based on the icSAT method, respectively.

[0065] Figure 8 It is a SDS-PAGE result diagram of the receptor agonist of the present invention after two-step standard column purification; wherein A, B, and C are the SDS-PAGE results of the GLP-1 / GIP dual-target agonist, the dual GLP-1 single-target agonist, and the dual GIP single-target agonist after two-step standard column purification, respectively.

[0066] Figure 91 is a graph showing the RP-HPLC assay results of the receptor agonist of the present invention; wherein A, B, and C are graphs showing the RP-HPLC assay results of the GLP-1 / GIP dual-target agonist, the dual GLP-1 single-target agonist, and the dual GIP single-target agonist, respectively.

[0067] Figure 10 It is a graph showing the molecular weight determination results of the receptor agonist of the present invention; wherein A, B, and C are graphs showing the molecular weight determination results of the GLP-1 / GIP dual-target agonist, the dual GLP-1 single-target agonist, and the dual GIP single-target agonist, respectively.

[0068] Figure 11 Graphs A and B are graphs of in vitro agonist activity and in vitro agonist activity, respectively.

[0069] Figure 12 This is a graph of the half-lives of the GLP-1 / GIP dual-target agonist, GLP-1, and GIP in human serum.

[0070] Figure 13 This is a flow chart of fusion protein self-ligation and icSAT protein purification. DETAILED DESCRIPTION

[0071] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they are all achieved or understood by those skilled in the art with reference to the prior art. Reagents or instruments used that do not indicate the manufacturer are considered to be conventional products that can be purchased commercially. The methods used in the following examples are all conventional methods unless otherwise specified. The specific steps can be found in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The primers used are all synthesized by Shanghai Shenggong Biotechnology.

[0072] In the figures of Examples 1-10, ES: cell lysate supernatant; EP: cell lysate precipitate; ES1, ES2: cell lysate supernatants 1 and 2, ES3: a mixture of ES1 and ES2; ESS: supernatant after ES3 aggregation by salt addition; ESP: precipitate after ES3 aggregation by salt addition; CP: precipitate after ESP cleavage; CS: supernatant after ESP cleavage; ES1 to CP were all diluted 10-fold, and CS was not diluted; M: protein marker; BSA: bovine serum albumin standard.

[0073] In the recombinant expression construct of the present invention, the polynucleotide sequence encoding the fusion protein is appropriately linked to expression control sequences to achieve desired transcription and ultimately production of the fusion protein in the host cell. The expression control sequences include, but are not limited to, promoters, enhancers, ribosome binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, and sequences that stabilize mRNA.

[0074] The vectors used for expressing the constructs of the present invention include vectors that can replicate autonomously in host cells, such as plasmid vectors; and vectors that can integrate into host cell DNA and replicate together with the host cell DNA. In a specific embodiment, the expression construct of the present invention is derived from pET30a(+) from Novagen. Host cells used to express the fusion proteins of the present invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotes include Escherichia coli ( Escherichia ), Bacillus ( Bacillus ), Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces ). In a preferred embodiment, the host cell is an Escherichia cell, preferably an Escherichia coli cell. In a specific embodiment of the present invention, the host cell used is an Escherichia coli BL21 (DE3) strain cell (Novagen).

[0075] Example 1: Construction of GLP-1-ELP based on icSAT method 15 -SpyTag / SpyCatcher (ΔN) expression vector

[0076] The expression vector used in the examples of this application is pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag and EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher (ΔN). Figure 1 A in the equation is GLP-1-ELP based on the icSAT method. 15 -Schematic diagram of the structure of the SpyTag fusion protein expression vector; Figure 1 B is GLP-1-ELP based on icSAT method 15 -Schematic diagram of the structure of the SpyCatcher (ΔN) fusion protein expression vector.

[0077] The salinity-induced self-assembly peptide used in this example is EFK8, whose amino acid sequence is shown in SEQ ID NO: 11; the intein is PersonΔI-CM (m2), the amino acid sequence of which is shown in SEQ ID NO: 14; the amino acid sequence of GLP-1 (SEQ ID NO: 1) was obtained from the literature (Glaesner et al., Diabetes / Metab Res Rev, 2010, 26(4): 287-296). Compared with the natural GLP-1 sequence, the GLP-1 sequence selected in this example has three mutations, namely: Ala at position 8 is mutated to Gly, Gly at position 22 is mutated to Glu, and Arg at position 36 is mutated to Gly, which can improve the stability of GLP-1 against hydrolysis by dipeptidyl peptidase-4 (DPP-4); the GLP-1 sequence is mutated by ELP 15 (SEQ ID NO: 3) was linked to the SpyTag sequence (SEQ ID NO: 7) or SpyCatcher (ΔN) (SEQ ID NO: 4). 15 -SpyTag and ELP 15 The -SpyCatcher (ΔN) gene fragment was codon-optimized for the E. coli host. The optimized gene was synthesized by Guangzhou Ruibo. The primers involved in the plasmid construction process are shown in Table 1.

[0078] Table 1

[0079]

[0080] For pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag plasmid, firstly, PCR amplification was performed using primers (S220614-ESK-F / Y200915-ter-R) to obtain the ELP-containing 15 -SpyTag-Kan gene fragment, and then use primers (Y200915-LPM-F / S220614-LMG-R) to modify the original pET30a-EFK8-PT linker- Person ΔI-CM (m2) - GLP-1-GS linker-SpyTag was amplified to obtain a sample containing Lac I-EFK8-PT linker- Person ΔI-CM (m2) -GLP-1 gene fragment. The two gene fragments were subjected to Gibson assembly, and the resulting ligation product was transformed into Escherichia coli DH5α. After colony PCR identification and sequencing, the correct positive clones were screened. The plasmid was then extracted and transformed into the E. coli expression strain BL21 (DE3) for protein expression and subsequent experiments.

[0081] pET30a-EFK8-PT linker- Person ΔI-CM (m2) - Preparation of GLP-1-GS linker-SpyTag: The EFK8-Mtu ΔI-CM (m2) fragment was amplified using the existing pET32-EFK8-Mtu ΔI-CM (m2)-hGH (from patent PCT / CN2020 / 125054) in the laboratory and then synthesized with GLP-1-ELP 15 - The SpyTag fragment and pET30a vector were ligated by Gibson assembly.

[0082] For pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher (ΔN) plasmid, firstly, PCR amplification was performed using primers (S220615-E-SC-F / Y200915-ter-R) to obtain the ELP 15 -SpyCatcher(ΔN)-Kan gene fragment, and then used primers (Y200915-LPM-F / S220614-LMG-R) to modify the original pET30a-EFK8-PT linker- Person ΔI-CM (m2) - GLP-1-GS linker-SpyTag was amplified to obtain a sample containing Lac I-EFK8-PT linker- Person ΔI-CM (m2) - The two gene fragments were Gibson assembled, and the resulting ligation product was transformed into Escherichia coli DH5α. After colony PCR identification and sequencing to screen the correct positive clones, the plasmid was extracted and transformed into the Escherichia coli expression strain BL21 (DE3) for protein expression and subsequent experiments.

[0083] Example 2: GLP-1-ELP based on icSAT method 15 -Expression of SpyTag / SpyCatcher (ΔN) fusion protein

[0084] The plasmid (pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag and pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 The BL21(DE3) strain containing the WT-SpyCatcher(ΔN) was inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4~0.6), add IPTG to a final concentration of 0.2 mM, culture conditions are: 18 ℃, 250 rpm, harvest cells after 24 hours, and measure the bacterial concentration OD 600 (The following 1 mL of OD 600 The number of cells is 1, which is called OD1).

[0085] The cells were resuspended to 50 OD / mL in salt-free lysis buffer B1 (20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) and ultrasonically disrupted (the conditions were: power 200 W, ultrasonic time 3 sec, interval time 3 sec, running time 8 min). g The supernatant and precipitate were collected for sample preparation and the expression of fusion protein in the lysis supernatant and lysis precipitate was detected by SDS-PAGE.

[0086] The results are as follows Figure 2 As shown, according to the protein quantitative standard, the target band was analyzed by optical density using ImageJ (National Institutes of Health) gel quantitative analysis software to calculate the yield and self-cleavage rate of the fusion protein in the lysis supernatant. The results are shown in Table 2.

[0087] Table 2

[0088]

[0089] a Protein yield in protein lysis supernatant, b Self-cleavage rate (%) = icSAT protein amount / (icSAT protein amount + fusion protein expression amount) × 100%.

[0090] Both fusion proteins are soluble and exhibit severe self-cleavage. Person ΔI-CM (m2) - GLP-1-ELP15 -SpyTag has a molecular weight of 32.7 kDa, an expression level of 100.9 mg / L LB medium, a self-cleavage rate of 60.4%, and a molecular weight of the target protein after cleavage of 11.2 kDa. However, no band of the target protein after cleavage was visible on the gel image; EFK8- Person ΔI-CM (m2) - GLP-1-ELP 15 -SpyCatcher (ΔN) has a molecular weight of 41.1 kDa, an expression level of 136.8 mg / L, a self-cleavage rate of 64.2%, and a target protein size of 19.6 kDa after self-cleavage.

[0091] Example 3: Construction of intein-containing Person GIP-ELP of different mutants of ΔI-CM based on icSAT method 15 -SpyCatcher (ΔN) expression vector

[0092] The expression vector used in the examples of this application contains 3 different Person Fusion protein expression vector of ΔI-CM mutant: pET30a-EFK8- Person ΔI-CM (WT / m1 / m2)-GIP-ELP 15 -SpyCatcher (ΔN). Figure 3 GIP-ELP based on icSAT method 15 Schematic diagram of the structure of the SpyCatcher (ΔN) fusion protein expression vector. The GIP amino acid sequence (SEQ ID NO: 2) used in this example was obtained from the literature (Gault et al., J. Endocrinol, 2003, 176, 133-141). Compared with the native GIP sequence, the GIP sequence selected in this example has an Ala at position 8 mutated to a Gly, which improves GIP's stability against DPP-4 hydrolysis. The GIP gene fragment was codon-optimized for E. coli and analyzed using the online tool DNAworks ( https: / / hpcwebapps.cit.nih.gov / dnaworks / ) to design the nucleotide sequence of GIP. The four oligonucleotide primers (GIP-1, GIP-2, GIP-3, and GIP-4) designed by DNAworks and the primers involved in the plasmid construction are shown in Table 3.

[0093] Table 3

[0094]

[0095] First, the four oligonucleotide primers designed based on the GIP sequence were annealed and spliced ​​to form a complete fragment, and the complete GIP fragment was amplified using primers (S220616-MG-F / S220616-GE-R). Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher (ΔN) plasmid was used as a template and PCR amplification was performed using primers (S220615-E-SC-F / Y200915-ter-R) to obtain the ELP 15 -SpyCatcher(ΔN)-Kan gene fragment, and the two fragments were spliced ​​and amplified by overlapping PCR to obtain the complete GIP-ELP 15 -SpyCatcher (ΔN)-Kan fragment. Using the original pET30a-EFK8-PT linker- Person ΔI-CM (WT)-hGH, pET32a-L6KD-PT linker- Person ΔI-CM (m1)-IFN and pET32a-L6KD-PT linker- Person The ΔI-CM (m2)-LCB3 plasmid (preparation method refers to pET30a-EFK8-PT linker-Mtu ΔI-CM (m2)-GLP-1-GSlinker-SpyTag in Example 1, the only difference is the replacement of the corresponding DNA fragment) was used as a template, and primers (D210310-Mtu-F / Y200728-LPM-R) were used to amplify the Person ΔI-CM (WT), Person ΔI-CM (m1) and Person ΔI-CM (m2) fragment (SEQ ID NO: 12~14). Then, the original pET30a-EFK8-PT linker- Person ΔI-CM (WT)-hGH was used as a template and primers (Y200915-LPM-F / Z211118-Mtu-R) were used to amplify the LacI-EFK8-PT linker fragment. Person ΔI-CM (WT / m1 / m2 / m3) and GIP-ELP 15The three gene fragments of -SpyCatcher(ΔN)-Kan were subjected to Gibson assembly respectively, and the obtained ligation products were transformed into Escherichia coli DH5α. After the correct positive clones were screened by colony PCR identification and sequencing, the plasmids were extracted and transformed into Escherichia coli expression strain BL21(DE3) for protein expression and subsequent experiments.

[0096] Example 4: Containing intein Person GIP-ELP of different mutants of ΔI-CM based on icSAT method 15 Expression of SpyCatcher (ΔN) fusion protein and icSAT purification

[0097] The plasmids (pET30a-EFK8- Person ΔI-CM (WT)-GIP-ELP 15 -SpyCatcher(ΔN)、pET30a-EFK8- Person ΔI-CM (m1)-GIP-ELP 15 -SpyCatcher(ΔN) and pET30a-EFK8- Person ΔI-CM (m2)-GIP-ELP 15 The BL21(DE3) strain of the WT-SpyCatcher(ΔN) was inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4~0.6), add IPTG to a final concentration of 0.2 mM, culture conditions are: 18 ℃, 250 rpm, harvest cells after 24 hours, and measure the bacterial concentration OD 600 (The following 1 mL of OD 600 The number of cells is 1, which is called OD1).

[0098] The cells were resuspended to 50 OD / mL in salt-free lysis buffer B1 (20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) and ultrasonically disrupted (the conditions were: power 200 W, ultrasonic time 3 sec, interval time 3 sec, running time 8 min). g The supernatant and precipitate were collected for sample preparation and tested by SDS-PAGE. Figure 4 shown.

[0099] An equal volume of aggregation buffer B2 (1.4 M Na2SO4, 20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) containing 1.4 M Na2SO4 was added to the obtained lysis supernatant and incubated at 4°C for 12 h to allow the self-assembling peptides to fully aggregate. g The pellet was centrifuged for 20 min under the same conditions. The pellet was washed once with an equal volume of washing buffer B3 (0.7 M Na2SO4, 20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH8.0) containing 0.7 M Na2SO4 and then centrifuged under the same conditions to separate the supernatant and pellet. The pellet was fully resuspended in cleavage buffer B4 (PBS without NaCl, 0.7 M Na2SO4, 20 mM Bis-Tris, 2 mM Na2·EDTA·2H2O, pH6.2) containing 0.7 M Na2SO4 with half the volume and placed on a rotary mixer at 25°C for 24 h to allow the intein to be fully self-cleaved. The suspension was incubated at 4°C at 15,000 g The supernatant and precipitate were separated by centrifugation for 20 minutes under the conditions of 40°C. ImageJ was used to detect the yield, self-cleavage rate, and icSAT purification effect (fusion protein aggregation efficiency and cleavage efficiency) of the three fusion proteins in this example. The results are shown in Table 4.

[0100] Table 4

[0101]

[0102] a Protein yield in protein lysis supernatant, b Self-cleavage rate (%) = icSAT protein amount / (icSAT protein amount + fusion protein expression amount) × 100%, c Aggregation efficiency (%) = the amount of fusion protein reduced in the supernatant after aggregation / the amount of fusion protein in the supernatant before aggregation × 100%, d Cleavage efficiency (%) = reduction in fusion protein after cleavage / amount of fusion protein before cleavage × 100%.

[0103] These three fusion proteins are all soluble and expressed, and the intein is Person The expression level of ΔI-CM (WT) fusion protein was 216.3 mg / mL, although the intein was Person The fusion protein of ΔI-CM (m1) is low, but its aggregation efficiency and cleavage efficiency are the highest among the three inteins, reaching 90%; the intein is PersonThe fusion protein expression level of ΔI-CM (m1) was the highest, at 244.4 mg / mL, but the cleavage efficiency was only 61.2%; Person Although the aggregation efficiency of the fusion protein of ΔI-CM (m2) was high, the expression level was low, only 160.0 mg / mL. Person ΔI-CM (WT) was used as an intein of the fusion protein for subsequent experiments.

[0104] Example 5: Construction of EFK8- Person ΔI-CM (WT)-GIP-ELP 15 -SpyTag expression vector

[0105] According to the test results in Example 4, in this example, GIP-ELP based on the icSAT purification method 15 -SpyTag fusion protein expression vector also uses Person ΔI-CM (WT) as an intein, Figure 5 GIP-ELP based on icSAT method 15 Schematic diagram of the structure of the SpyTag fusion protein expression vector. The primers involved in the plasmid construction process are shown in Table 5.

[0106] Table 5

[0107]

[0108] First, the EFK8-PT linker constructed in Example 3 was used. Person ΔI-CM (WT) - GIP-ELP 15 -SpyCatcher (ΔN) plasmid was used as a template and primers (Y200915-LPM-F / C220701-IMtu-R) were used to amplify LacI-EFK8- Person ΔI-CM (WT)-GIP fragment; then, the pET30a-EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag plasmid was used as template and primers (C220701-IES-F / Y200915-ter-R) were used to amplify ELP. 15 -SpyTag-Kan fragment; finally, the above two gene fragments were subjected to Gibson assembly, and the obtained ligation product was transformed into Escherichia coli DH5α. After the correct positive clones were screened by colony PCR identification and sequencing, the plasmid was extracted and transformed into Escherichia coli expression strain BL21 (DE3) for protein expression and subsequent experiments.

[0109] Example 6: GIP-ELP based on icSAT method 15 Expression of SpyTag fusion proteins

[0110] The plasmid (pET30a-EFK8- Person ΔI-CM (WT)-GIP-ELP 15 The BL21(DE3) strain carrying the WT-SpyTag was inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4~0.6), add IPTG to a final concentration of 0.2 mM, culture conditions are: 18 ℃, 250 rpm, harvest cells after 24 hours, and measure the bacterial concentration OD 600 (The following 1 mL of OD 600 The number of cells is 1, which is called OD1).

[0111] The cells were resuspended to 50 OD / mL in salt-free lysis buffer B1 (20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) and ultrasonically disrupted (the conditions were: power 200 W, ultrasonic time 3 sec, interval time 3 sec, running time 8 min). g The supernatant and precipitate were collected for sample preparation and tested by SDS-PAGE. Figure 6 According to the protein quantitative standard, the target band was analyzed by optical density using ImageJ (National Institutes of Health) gel quantitative analysis software to calculate the yield and self-cleavage rate of the fusion protein in the lysis supernatant. The results are shown in Table 6.

[0112] Table 6

[0113]

[0114] a Protein yield in protein lysis supernatant, b Self-cleavage rate (%) = icSAT protein amount / (icSAT protein amount + fusion protein expression amount) × 100%.

[0115] The fusion protein was expressed soluble at a level of 216.3 mg / mL and a self-cleavage rate of only 2.3%, and can be used for the subsequent preparation of GLP-1 / GIP dual agonists.

[0116] In summary, GLP-1-ELP based on icSAT purification method 15 -SpyCatcher(ΔN) / SpyTag and GIP-ELP 15 -SpyCatcher (ΔN) / SpyTag fusion protein expression vector was successfully constructed and soluble expression was achieved. It can be seen that, unlike GLP-1 fusion protein, GIP fusion protein has a lower self-cleavage rate. This is because the N-terminal residue of GIP is Tyr, which does not promote Person The C-terminal cleavage of ΔI-CM; while the N-terminal residue of GLP-1 is His, which will trigger Person The self-cleavage phenomenon of ΔI-CM (Zhao et al., Biotechnology journal, 2017, 12(6): 1600656). Among them, the intein is Person Although the GIP fusion protein of ΔI-CM (m1 / m2) hardly undergoes self-cleavage, its in vitro cleavage efficiency is greatly reduced. This is due to the change of Person The microenvironment of the two key histidine residues H429 and H439 in ΔI-CM prevents its premature cleavage while retaining pH dependence (Lin et al., Aiche Journal, 2020, 66(3): e16806). Person Although the GIP fusion protein of ΔI-CM (WT) has a higher self-cleavage rate, its in vitro cleavage efficiency is also the highest, allowing more target protein to be obtained.

[0117] Example 7: Preparation of GLP-1 and GIP Dual-Target Receptor Agonist, Dual GLP-1 Single-Target Receptor Agonist, and Dual GIP Single-Target Receptor Agonist

[0118] To prepare a dual-target receptor agonist of GLP-1 and GIP (GLP-1 / GIP), the EFK8- Person ΔI-CM(m2)-GLP-1-ELP 15 -SpyCatcher (ΔN) and EFK8 in Example 4- Person ΔI-CM (WT)-GIP-ELP 15 -SpyTag cell lysate supernatant, according to the fusion protein molar ratio of 1:1; to prepare dual GLP-1 single target receptor agonist (GLP-1 / GLP-1), EFK8- Person ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher(ΔN) and EFK8- Person ΔI-CM (m2)-GLP-1-ELP15 -SpyTag cell lysate supernatant, according to the fusion protein molar ratio of 1:1; to prepare the dual GIP single target receptor agonist (GIP / GIP), EFK8- Person ΔI-CM(WT)-GIP-ELP 15 -SpyCatcher (ΔN) and EFK8 in Example 6- Person ΔI-CM (WT)-GIP-ELP 15 -SpyTag cell lysate supernatant was mixed at a 1:1 fusion protein molar ratio.

[0119] The above mixture was reacted at 25 °C and 80 rpm for 2 h to allow for complete binding. Subsequently, an equal volume of aggregation buffer B2 (1.4 M Na2SO4, 20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) containing 1.4 M Na2SO4 was added to the reaction mixture and incubated at 4 °C for 12 h to allow the self-assembling peptides to fully aggregate. The suspension was then incubated at 4 °C and 15,000 g The pellet was centrifuged for 20 min under the same conditions. The pellet was washed once with an equal volume of washing buffer B3 (0.7M Na2SO4, 20 mM Tris-base, 1mM Na2·EDTA·2H2O, pH8.0) containing 0.7 M Na2SO4 and then centrifuged under the same conditions to separate the supernatant and pellet. The pellet was fully resuspended in cleavage buffer B4 (PBS without NaCl, 0.3MNa2SO4, 20 mM Bis-Tris, 2 mM Na2·EDTA·2H2O, pH6.2) containing 0.3 M Na2SO4 with half the volume and placed on a rotary mixer at 25°C for 24 h to allow the intein to be fully self-cleaved. The suspension was incubated at 4°C at 15,000 g The supernatant and precipitate were separated by centrifugation for 20 min under the conditions of 50 °C. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, and the supernatant and precipitate after cutting were subjected to SDS-PAGE detection together. The results are as follows. Figure 7 According to the protein quantitative standards, the optical density of the target band was analyzed using ImageJ (National Institutes of Health) gel quantitative analysis software to calculate the aggregation efficiency, cleavage efficiency and purity of the fusion protein. The results are shown in Table 7.

[0120] Table 7

[0121]

[0122] aAggregation efficiency (%) = the amount of fusion protein reduced in the supernatant after aggregation / the amount of fusion protein in the supernatant before aggregation × 100%, b Cleavage efficiency (%) = the amount of fusion protein reduced after cleavage / the amount of fusion protein before cleavage × 100%, c Purity (%) = amount of target protein / (amount of target protein + amount of impurity protein) × 100%.

[0123] Example 8: Two-step standard column purification method for GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists, and dual GIP single-target receptor agonists

[0124] The GLP-1 and GIP dual-target receptor agonist (hereafter referred to as GLP-1 / GIP), dual GLP-1 single-target receptor agonist (hereafter referred to as GLP-1 / GLP-1), and dual GIP single-target receptor agonist (hereafter referred to as GIP / GIP), purified via icSAT in Example 7, were finely purified using an anion exchange column (UniGel-30Q, 5 mL) and a molecular sieve column (HiLoad® 16 / 600 Superdex® 75prep grade). After loading the sample onto the ion exchange column, unbound proteins were washed away with binding buffer (20 mM Tris-HCl, pH 8.0) until the UV baseline stabilized. The protein was then eluted using elution buffer (20 mMTris-HCl, 1.0 M NaCl, pH 8.0) with the following elution schedule: 0-10% B: 0 min; 10-20% B: 10 min; 20-50% B: 0 min; 50-100% B: 0 min. The ion exchange purified protein was further purified using a molecular sieve column, eluting with 1 column volume of buffer (20 mM NaCl, 20 mM Tris-HCl, pH 7.2). The collected elution peaks were analyzed by SDS-PAGE. The results are shown in the figure below. Figure 8 shown. Figure 8 A in the figure is the purification result of GLP-1 / GIP; Figure 8 B in the figure is the purification result of GLP-1 / GLP-1; Figure 8 C in the figure is the purification result of GIP / GIP.

[0125] Through the two-step standard column purification method of ion exchange column purification and molecular sieve, GLP-1 / GIP, GLP-1 / GLP-1 and GIP / GIP can be separated from impurities and the purity of the fusion protein can be improved.

[0126] Example 9: RP-HPLC Determination of GLP-1 and GIP Dual-Target Receptor Agonists, Dual GLP-1 Single-Target Receptor Agonists, and Dual GIP Single-Target Receptor Agonists

[0127] RP-HPLC analysis was performed on the GLP-1 and GIP dual-target receptor agonist (hereinafter referred to as GLP-1 / GIP), dual GLP-1 single-target receptor agonist (hereinafter referred to as GLP-1 / GLP-1), and dual GIP single-target receptor agonist (hereinafter referred to as GIP / GIP) obtained by ion exchange column and molecular sieve purification in Example 8. An Agilent 1260 system and an Acquity UPLC BEH C4 column were used with an injection volume of 10 μL, a flow rate of 0.2 mL / min, and a column temperature of 65 °C. o C, detection wavelength 280 nm. Mobile phase A is an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. The mobile phase gradient is: 25-85% B elution for 20 min. The results are shown in Figure 9 As shown, Figure 9 A in the figure is the RP-HPLC determination result of GLP-1 / GIP; Figure 9 B in the figure is the RP-HPLC determination result of GLP-1 / GLP-1; Figure 9 C in the figure is the result of RP-HPLC determination of GIP / GIP.

[0128] The results show that the peak elution times of GLP-1 / GIP are 13.922 min, GLP-1 / GLP-1 are 14.250 min, and GIP / GIP are 13.464 min. The peak area ratios indicate that the purity of both GLP-1 / GIP and GLP-1 / GLP-1 reaches 99%, while the purity of GIP / GIP is slightly lower at 98.1%.

[0129] Example 10: Molecular Weight Determination of GLP-1 and GIP Dual-Target Receptor Agonists, Dual GLP-1 Single-Target Receptor Agonists, and Dual GIP Single-Target Receptor Agonists

[0130] The molecular weights of the GLP-1 and GIP dual-target receptor agonist (hereinafter referred to as GLP-1 / GIP), dual GLP-1 single-target receptor agonist (hereinafter referred to as GLP-1 / GLP-1), and dual GIP single-target receptor agonist (hereinafter referred to as GIP / GIP) obtained by purification using an ion exchange column and molecular sieves in Example 8 were determined. Ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF) and a Biozen™ C4 column were used to determine the molecular weights of the samples. The conditions were: injection volume 10 μL, flow rate 0.5 mL / min, column temperature 75°C, and column pressure 75°C. oC, detection wavelength 214 nm. Mobile phase A is an aqueous solution containing 0.1% (v / v) TFA, mobile phase B is an acetonitrile solution containing 0.1% (v / v) TFA, the mobile phase gradient is set as: 5-60% B, elution time 3 min. The results are shown in Figure 10 As shown, Figure 10 A in the table is the molecular weight determination result of GLP-1 / GIP; Figure 10 B in the figure is the molecular weight determination result of GLP-1 / GLP-1; Figure 10 C in the figure is the molecular weight determination result of GIP / GIP.

[0131] From the results, it can be seen that the actual molecular weight of GLP-1 / GIP is 32457.60 Da, which is consistent with the theoretical molecular weight (32457.60 Da); the actual molecular weight of GLP-1 / GLP-1 is 30802.79 Da, which is consistent with the theoretical molecular weight (30802.79 Da); the actual molecular weight of GIP / GIP is 34111.82 Da, which is consistent with the theoretical molecular weight (34112.69 Da).

[0132] Example 11: Verification of in vitro agonist activity of GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists, and dual GIP single-target receptor agonists

[0133] To verify the biological activity of the GLP-1 and GIP dual-target receptor agonist (hereinafter referred to as GLP-1 / GIP), dual GLP-1 single-target receptor agonist (hereinafter referred to as GLP-1 / GLP-1), and dual GIP single-target receptor agonist (hereinafter referred to as GIP / GIP) obtained by purification via ion exchange columns and molecular sieves in Example 8, in vitro activity assays were performed. HEK293 / GLP-1R-Luciferase and HEK293 / GIPR-Luciferase cells stably expressing the reporter gene luciferase regulated by the GLP-1R (or GIPR) and the NFAT response component were used. GLP-1 or GIP specifically binds to the GLP-1R or GIPR on the cell membrane, activating adenylate cyclase and transmitting a signal, resulting in expression of the luciferase reporter gene. After adding luciferase substrate, luciferase expression can be assessed by measuring chemiluminescence, thereby evaluating the biological activity of GLP-1 / GIP, GLP-1 / GLP-1, and GIP / GIP. The experimental results were expressed using the median effective concentration (EC 50 ) indicates that EC 50 The lower the value, the stronger the drug effect.

[0134] To determine the in vitro agonist activity of samples against HEK293 / GLP-1R-Luciferase cells, dilute GLP-1 / GIP, GLP-1 / GLP-1, GIP / GIP, the control GLP-1 (SEQ ID NO: 1), and the control GIP (SEQ ID NO: 2) to 8 nM in assay buffer (DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin) as a starting concentration, followed by serial dilutions 3-fold. To determine the in vitro agonist activity of samples against HEK293 / GIPR-Luciferase cells, dilute the above samples to 200 nM in assay buffer as a starting concentration, followed by serial dilutions 4-fold. Digest HEK293 / GLP-1R-Luciferase (or HEK293 / GIPR-Luciferase) cells and dilute the cells to a suspension of 1 million cells / mL in assay buffer. Transfer the cell suspension and diluted sample to a 384-well plate (white bottom transparent) at 20 μL / well, with 3 replicates. o Incubate in a 5% CO2 incubator for 6 h. After incubation, add Luciferase Assay Buffer (30 μL / well). Wait 3 min for cells to fully lyse before reading using a microplate reader.

[0135] The results of in vitro agonist activity assays of GLP-1 / GIP, GLP-1 / GLP-1, and GIP / GIP on HEK293 / GLP-1R-Luciferase cells are shown in Figure 11 As shown in A, the results of in vitro agonist activity assay on HEK293 / GIPR-Luciferase cells are shown in Figure 11 The data were analyzed using GraphPad Prism 8 software to obtain the EC values ​​of GLP-1 / GIP, GLP-1 / GLP-1, and GIP / GIP on HEK293 / GLP-1R-Luciferase and HEK293 / GIPR-Luciferase cells. 50 , as shown in Table 8.

[0136] Table 8

[0137]

[0138] The experimental results show that for GLP-1R, the EC of GLP-1 / GIP dual agonists is 50 was 0.305 ± 0.048 nM, which was comparable to that of single GLP-1 (EC 50=0.128 ± 0.003 nM), the activity decreased by about 50%. 50 The activity of single GLP-1 and GLP-1 / GIP dual agonists was 0.044 ± 0.006, which was significantly improved compared with single GLP-1 and GLP-1 / GIP dual agonists. This was due to the difference in agonist activity caused by the number of GLP-1 in the polymer. In addition, the agonist activity of single GIP and GIP / GIP agonists on GLP-1R was also measured. The results showed that GIP did not have an agonist effect on GLP-1R. For GIPR, the EC of GLP-1 / GIP dual agonists was 0.044 ± 0.006. 50 was 0.375 ± 0.030 nM, which was comparable to that of single GIP (EC 50 =0.348 ± 0.019 nM) was similar to the agonist activity of the GIP / GIP agonist of the same structure (EC 50 =0.187 ± 0.011 nM), the agonist activity of the GLP-1 / GIP dual agonist decreased by approximately 50%, corresponding to the number of GIPs present. The agonist activity of single GLP-1 and GLP-1 / GLP-1 agonists on GIPR was also measured, and the results showed that GLP-1 did not stimulate GIPR.

[0139] Example 12: Human serum stability verification of GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists, and dual GIP single-target receptor agonists

[0140] To verify the stability of GLP-1 / GIP, GLP-1 / GLP-1 and GIP / GIP purified by ion exchange column and molecular sieve in serum in Example 8, these samples were mixed with human serum at 37 o C (final sample concentration was 0.6 mg / mL, final serum concentration was 20%). The initial time was recorded, and 50 μL of the mixture was taken at time intervals of 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 h. After the incubation, 2 volumes of acetonitrile were added to precipitate the protein. The mixture was then incubated at 4 o C for 30 min, then g The supernatant was collected and filtered with a 0.22 μm filter membrane. The residual sample concentration in the supernatant was determined using an Agilent 1260 system and an Acquity UPLC BEH C4 column. The injection volume was 10 μL, the flow rate was 0.2 mL / min, and the column temperature was 65 °C. oC, detection wavelength 280 nm. Mobile phase A is an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. The elution conditions are: 5-70% B elution for 30 min. The results are shown in Figure 12 The data were analyzed using GraphPad Prism 8, and the half-lives of GLP-1, GIP, and GLP-1 / GLP-1 in human serum were obtained. The results are shown in Table 9.

[0141] Table 9

[0142]

[0143] The half-life determination results of dual GLP-1 single-target receptor agonists and dual GIP single-target receptor agonists are similar to those of GLP-1 and GIP dual-target receptor agonists, and are significantly longer than those of GLP-1 and GIP.

[0144] The above embodiments are only preferred implementation modes of the present invention and are only used to explain rather than limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a dual-peptide receptor agonist, characterized in that: The steps include: (a) constructing a fusion protein expression vector of a target polypeptide fused with an icSAT tag and a SpyTag peptide or a SpyCatcher peptide and expressing the fusion protein; the target polypeptide is glucagon-like peptide-1 and / or a glucose-dependent insulinotropic polypeptide; the glucagon-like peptide-1 and / or a glucose-dependent insulinotropic polypeptide is connected to the SpyTag peptide or the SpyCatcher peptide via an ELP peptide; the icSAT tag is composed of a self-assembling peptide and a cleavage tag, the self-assembling peptide is a salinity-induced self-assembling peptide, and the salinity-induced self-assembling peptide is a β-folded short peptide; the amino acid sequence of the β-folded short peptide is as shown in SEQ ID NO: 11; The fusion protein includes EFK8- Mt ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag, EFK8- Mt ΔI-CM(m2)-GLP-1-ELP 15 -SpyCatcher(ΔN),EFK8- Mt ΔI-CM (WT)-GIP-ELP 15 -SpyTag and EFK8- Mt ΔI-CM (WT)-GIP-ELP 15 -SpyCatcher(ΔN); fusion protein EFK8- Mt ΔI-CM (m2)-GLP-1-ELP 15 -SpyTag encoding nucleotide sequence is shown in SEQ ID NO: 18, fusion protein EFK8- Mt ΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher (ΔN) encoding nucleotide sequence is shown in SEQ ID NO: 19, fusion protein EFK8- Mt ΔI-CM (WT)-GIP-ELP 15 -SpyTag encoding nucleotide sequence is shown in SEQ ID NO: 20, fusion protein EFK8- Mt ΔI-CM (WT)-GIP-ELP 15 - The encoding nucleotide sequence of SpyCatcher (ΔN) is shown in SEQ ID NO: 21; (b) Mixing two fusion proteins and self-ligating to form a double fusion protein; (c) The dual-peptide receptor agonist was purified using the icSAT protein purification method and the two-step standard column purification method.

2. The method for preparing a dual peptide receptor agonist according to claim 1, characterized in that: In step (a), the method for preparing the fusion protein specifically comprises the following steps: The gene sequences of the purification tag, the target polypeptide and the ELP peptide are sequentially connected with the SpyTag peptide or the SpyCatcher peptide to form a fusion protein gene, and the fusion protein gene is used to construct an expression vector and introduced into a host cell to obtain an engineered bacterium; the engineered bacterium is cultured to express the fusion protein, and then the engineered bacterium is lysed to capture the fusion protein; The capturing and obtaining of the fusion protein comprises lysing the engineered bacteria and then centrifuging and obtaining the supernatant to obtain the fusion protein.

3. The method for preparing a dual peptide receptor agonist according to claim 1, characterized in that: In step (b), the two fusion proteins are mixed, specifically, the fusion protein fused with SpyTag peptide and the fusion protein fused with SpyCatcher peptide are mixed in a 1:1 molar ratio to obtain a dual fusion protein solution; the temperature of the mixing reaction is 4-37°C, and the mixing reaction time is 0.5-24 h; In step (c), the icSAT protein purification method specifically comprises: adding an equal volume of high-salt buffer to the double fusion protein solution to cause aggregation, and adjusting the pH of the solution to perform cleavage; The high salt buffer is a buffer containing 1.0~1.4 M Na2SO4 and a pH of 8.

5. The aggregation temperature is 4~37°C and the aggregation time is 0.5~12 h. The cleavage is to disperse the aggregates of the double fusion protein in an acidic buffer, perform a cleavage treatment, and centrifuge to obtain the supernatant. The pH value of the acidic buffer is 5.5~6.8, the cleavage treatment temperature is 4~37°C, and the cleavage treatment time is 3~48 h. In step (c), the two-step standard column purification method includes ion exchange purification and molecular sieve purification.

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