CN117264081A - Dipeptide receptor agonist and preparation method and application thereof - Google Patents
Dipeptide receptor agonist and preparation method and application thereof Download PDFInfo
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- CN117264081A CN117264081A CN202311549422.XA CN202311549422A CN117264081A CN 117264081 A CN117264081 A CN 117264081A CN 202311549422 A CN202311549422 A CN 202311549422A CN 117264081 A CN117264081 A CN 117264081A
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- C07K14/575—Hormones
- C07K14/605—Glucagons
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention discloses a double-peptide receptor agonist, a preparation method and application thereof, belongs to the field of genetic engineering, and particularly relates to preparation of protein or polypeptide medicines, in particular to preparation and application of double-target receptor (GLP-1 and GIP) agonist peptide, double-GLP-1 single-target receptor agonist and double-GIP single-target receptor agonist. The fusion protein can obtain double-target receptor (GLP-1 and GIP) agonist peptide, double-GLP-1 single-target receptor agonist and double-GIP single-target receptor agonist in one step through SpyCatcher/Tag high-efficiency self-connection without purification, and the yield is greatly improved. The method can be applied to the preparation of novel medicaments with double-target agonism activity and/or single-target agonism activity with longer half-life.
Description
Technical Field
The invention belongs to the field of genetic engineering, in particular to preparation of protein or polypeptide drugs, and particularly relates to a dipeptide receptor agonist, a preparation method and application thereof.
Background
Type 2 diabetes (type 2 diabetes mellitus,T2DM) has become a worldwide epidemic disease. Glucagon-like peptide-1 (glp-1) can promote synthesis and secretion of insulin, inhibit secretion of glucagon, and is an important choice for T2DM treatment due to its excellent hypoglycemic effect and weight-loss action (Baggio et al, gastroenterology, 2007, 132 (6): 2131-57). With the development and marketing of various glucagon-like peptide-1 analogs (gluco-like peptide-1 receptor agonists,GLP-1 RAs), a new generation of dual/multi-target receptor agonists began to become the mainstay of research and development, with tiazepatide (Tirzepatide) being approved by the U.S. food and drug administration (food and drug administration, FDA). Tirzepatide as a single molecule dual-target GLP-1/GIP (glucose-dependent insulinotropic polypeptide) agonist has been shown to be superior to the hypoglycemic and weight-reducing efficacy of semaglutin (Coskun et al, molecular metabolism, 2018, 18:3-14). However, the design of such single molecule agonists relies on the high homology of the polypeptide and it is difficult to precisely control the affinity of the molecule for different targets during the design process, resulting in difficulty in predicting the potency profile of the molecule for each target (Frias et al, new Engl J Med, 2021, 385 (6): 503-515). Furthermore, there is a limitation in the gene fusion expression strategy because GLP-1 needs to retain the natural N-terminus to ensure activity and can only be fused with peptides/proteins without this requirement.
Spy chemistry is a kind of click chemistry capable of being coded by genes, and based on two genetic coding elements, namely SpyCatcher and SpyTag, polymers can be spontaneously formed without complex post-translational modification, and the process has the advantages of no chemical reagent, rapid reaction, mild condition and wide application prospect (Zakeri et al, PNAs, 2012, 109 (12): E690-E697). Combining Spy chemistry with ELP can create a powerful protein-based framework, providing a new option for the design and preparation of multifunctional protein oligomers, polymers or networks (Sun et al, PNAs, 2014, 111 (31): 11269-11274).
In summary, there remains room for improvement in the design of GLP-1-based dual (multi) target receptor agonists, and thus there remains a need in the art to develop more efficient, simple, and widely applicable methods for preparing receptor agonists.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a dipeptide receptor agonist, and a preparation method and application thereof.
A method of preparing a dipeptide receptor agonist comprising the steps of:
(a) Constructing a fusion protein expression vector for fusing an icSAT tag and a target polypeptide of SpyTag peptide or SpyCatcher peptide and expressing the fusion protein;
(b) Mixing the two fusion proteins, and self-connecting to form a double fusion protein;
(c) And purifying by using an icSAT protein purification method and a two-step standard column purification method to obtain the dipeptide receptor agonist.
Further, the polypeptide of interest is linked to a SpyTag peptide or a SpyCatcher peptide via an ELP peptide.
Further, in step (a), the polypeptide of interest 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) are linked to the SpyTag peptide (or SpyCatcher peptide) via ELP peptide.
Further, 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.
Still further, the ELP peptide is characterized by one of the following conditions:
(a) A repeated sequence of Val-Pro-Gly-Xaa-Gly;
(b) The repeated sequence of Val-Pro-Gly-Xaa-Gly, the repeated number of Val-Pro-Gly-Xaa-Gly repeated sequence is 10-30 times;
(c) A repeated sequence of Val-Pro-Gly-Xaa-Gly, wherein Xaa is Val and Glu;
(d) A repeated sequence of Val-Pro-Gly-Xaa-Gly, wherein Xaa is Val and Glu, and the ratio of Xaa amino acids Val and Glu is 3:1-5:1;
(e) The amino acid sequence of the ELP peptide is shown as SEQ ID NO. 3.
Further, the SpyCatcher peptide and SpyTag peptide of step (a) may form an isopeptide chain; the SpyCatcher peptide comprises polypeptide of any one of the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and the SpyTag peptide comprises polypeptide of any one of the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.
Further, in the step (a), the preparation method of the fusion protein specifically comprises the following steps:
sequentially connecting the purification tag, the target polypeptide and the ELP peptide with the SpyTag peptide or the SpyCatcher peptide gene sequence to form a fusion protein gene, constructing an expression vector by the fusion protein gene, and introducing the fusion protein gene into a host cell to obtain engineering bacteria; culturing the engineering bacteria to express the fusion protein, then cracking the engineering bacteria, and capturing to obtain the fusion protein.
Further, the capturing to obtain the fusion protein comprises the steps of cracking engineering bacteria, centrifuging to obtain a supernatant, and obtaining the fusion protein.
Still further, the purification tag consists of a self-assembled peptide and a cleavage tag; the gene sequence of the self-assembled peptide is connected with the gene sequence of the cutting tag through a connector.
Preferably, the self-assembling peptide is a salinity-induced self-assembling peptide, which is a beta-sheet short peptide; the amino acid sequence of the beta-sheet short peptide is shown as SEQ ID NO. 11. Preferably, the cleavage tag is a chemical cleavage site, an enzymatic cleavage site or a self-cleavage site.
More preferably, the self-cleavage site is an intein, which isMtuΔI-CM (WT) inteins orMtuΔI-CM intein mutants, saidMtuThe ΔI-CM intein mutants includeMtuΔI-CM (m1)、MtuΔI-CM (m 2) andMtuΔI-CM (m 3); the saidMtuThe amino acid sequence of the delta I-CM (WT) is shown as SEQ ID NO. 12, whichMtuThe amino acid sequence of the delta I-CM (m 1) is shown as SEQ ID NO. 13, and theMtuThe amino acid sequence of the delta I-CM (m 2) is shown as SEQ ID NO. 14; the saidMtuThe amino acid sequence of the delta I-CM (m 3) is shown as SEQ ID NO. 15.
Preferably, the linker is a PT linker; the PT type joint amino acid sequence is shown as SEQ ID NO. 10.
Further, in the step (b), the mixing of two fusion proteins is specifically: fusion proteins fused with SpyTag peptide and SpyCatcher peptide were prepared according to a ratio of 1: and (3) carrying out a mixing reaction according to a molar ratio of 1 to obtain a double-fusion protein solution.
Further, the temperature of the mixing reaction is 4-37 ℃, and the time of the mixing reaction is 0.5-24h.
Further, in the step (c), the icSAT protein purification method specifically includes: adding an equal volume of high-salt buffer solution into the double-fusion protein solution to aggregate the double-fusion protein, and centrifuging to leave aggregates; the pH of the solution was adjusted to cut.
Further, the high-salt buffer solution contains 1.0-1.4M Na 2 SO 4 And the pH value of the buffer solution is 8.5, the aggregation temperature is 4-37 ℃, and the aggregation time is 0.5-12 h.
Further, the cleavage is carried out by dispersing aggregates of the double-fusion protein in an acidic buffer, performing cleavage treatment, and centrifuging to obtain a supernatant. Preferably, the pH value of the acidic buffer solution is 5.5-6.8, the temperature of the cutting treatment is 4-37 ℃, and the time of the cutting treatment is 3-48 hours.
Further, in step (c), the two-step standard column purification method comprises ion exchange purification and molecular sieve purification.
The invention also provides a dipeptide receptor agonist prepared by the preparation method of any one of the above.
Further, the dual peptide receptor agonists include GLP-1 and GIP dual target receptor agonists, dual GLP-1 single target receptor agonists and dual GIP single target receptor agonists.
Further, the GLP-1 and GIP dual target receptor agonist is at a half-effective concentration EC for the in vitro agonistic activity of the GLP-1 receptor 50 Half-effective concentration EC of 0.305±0.048 nM for in vitro agonistic activity of GIP receptor 50 0.375 + -0.030 nM.
Further, 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 linked to SpyTag peptide (or SpyCatcher peptide) via ELP peptide; spyCatcher peptides and SpyTag peptides may form isopeptidic chains.
Further, the dual GLP-1 single target receptor agonist has half the effective concentration EC of GLP-1 receptor in vitro agonistic activity 50 Is 0.044+/-0.006 nM, has no in vitro agonistic activity on the GIP receptor.
Still further, the dual GIP single-target receptor agonist has a half-effective concentration EC of agonistic activity on GIP receptors in vitro 50 Is 0.187+/-0.011 nM, and has no in-vitro agonistic activity on GLP-1 receptor.
The invention provides application of the dipeptide receptor agonist in preparing a medicament for preventing or treating type II diabetes.
The present invention provides a polynucleotide comprising a nucleotide sequence encoding the fusion protein of the present invention described above or a complement thereof.
Further, 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).
Still further, the gene sequence of the purification tag comprises a self-assembled peptide, a linker and a gene sequence of the cleavage tag, wherein the gene sequence of the self-assembled peptide is connected with the gene sequence of the cleavage tag through the linker.
Preferably, the self-assembling peptide is a salinity-induced self-assembling peptide, and the linker is a PT linker.
Further, the polypeptide of interest is GLP-1 and/or GLP.
Further, the fusion protein packageIncluding but not limited to the fusion protein EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 -SpyTag、EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 -SpyCatcher(ΔN)、EFK8-MtuΔI-CM (WT)-GIP-ELP 15 SpyTag and EFK8-MtuΔI-CM (WT)-GIP-ELP 15 -SpyCatcher(ΔN)。
Further, the fusion protein EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The encoding nucleotide sequence of SpyTag is shown as 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-MtuΔI-CM (m2)-GLP-1-ELP 15 The encoding nucleotide sequence of SpyCatcher (DeltaN) is shown in SEQ ID NO. 19; the fusion protein EFK8-MtuΔI-CM (WT)-GIP-ELP 15 The encoding nucleotide sequence of the SpyTag is shown as SEQ ID NO. 20; the fusion protein EFK8-MtuΔI-CM (WT)-GIP-ELP 15 The coding nucleotide sequence of SpyCatcher (DeltaN) is shown in SEQ ID NO. 18.
The present invention provides an expression vector comprising the polynucleotide described above.
The present invention provides a host cell comprising the polynucleotide described above or the expression vector described above; the host cell is capable of expressing the fusion proteins of the invention described above.
In the recombinant expression vectors of the invention, the polynucleotide sequence encoding the fusion protein is suitably linked to expression control sequences to effect transcription as desired and ultimately production of the fusion protein in a host cell. Such expression control sequences include, but are not limited to, promoters, enhancers, ribosome binding sites, polyadenylation sites, transcriptional splice sequences, transcriptional termination sequences, and mRNA stabilizing sequences, and the like.
The recombinant expression vectors of the invention include vectors, such as plasmid vectors, which are autonomously replicable in host cells; and a vector that can be integrated into and replicated together with the host cell DNA. In a specific embodiment, the expression vector of the invention is derived from pET30a (+) from Novagen. Host cells useful for expressing the fusion proteins of the invention include prokaryotes, yeast, and higher eukaryotic cells. ExampleThe prokaryote includes Escherichia genusEscherichia) Bacillus genusBacillus) Genus PseudomonasPseudomonas) And Streptomyces genusStreptomyces) Is a bacterium of the genus (a). In a preferred embodiment, the host cell is an Escherichia cell, preferably E.coli. In a specific embodiment of the invention, the host cell used is an E.coli BL21 (DE 3) strain cell (Novagen).
In the invention, GLP-1 peptide (or GIP peptide) is fused at the C terminal of C terminal cutting intein, natural N terminal of GLP-1 peptide (or GIP peptide) is reserved, and receptor affinity reduction caused by N terminal modification is avoided; the ELP peptide is nontoxic, biodegradable, has good biocompatibility, and is very safe and reliable to use as a drug delivery material.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) According to the invention, through high-efficiency and high-specificity reaction of SpyCatcher/Tag, the fusion protein can rapidly react to obtain the target protein without purification.
(2) According to the invention, the rapid preparation of the target protein is realized by an inducible and cleavable self-aggregation tag (icSAT) protein purification method, and the natural N terminal of the target polypeptide is reserved; based on the inducible and cleavable self-aggregation tag, an experimental scheme of forming double-fusion protein by self-ligation and then purifying the icSAT protein is designed, and compared with the experimental scheme of purifying first and then self-ligation, the experimental scheme is simpler, other tags are not required to be added at the N end of the target polypeptide, the cost is saved, and the yield is obviously higher than that of purifying first and then self-ligation. (1) The two components are purified firstly and then are mixed after being connected with the first separated preparative purification, and because partial fusion proteins are not completely connected, the two components are required to be purified secondarily, and the two components are not required to be purified preparatively before being combined, and only the icSAT purification and the conventional two-step fine column purification are required to be carried out after being combined; (2) the purification and self-connection are carried out firstly, and the purification tag is lost due to the first preparative purification, so that an additional purification tag (such as His tag) is required to be added at the N or C end of the target polypeptide, the second preparative purification is carried out by adopting size exclusion chromatography and the like, the additional purification tag is required to be excised for final drug forming, the process is complex, and the cost is high; (3) the self-cleaving proteins cannot be recovered by purification followed by self-ligation, resulting in loss of intermediates and reduced yields of final receptor agonists.
(3) By utilizing ELP peptide sequences which do not aggregate at normal temperature, the problem that the current ELP peptide is easy to aggregate and generates toxicity when applied to the field of medicines is avoided; the ELP peptide is used for connecting the target polypeptide and the SpyTag peptide (or the SpyCatcher peptide), so that a double-peptide receptor agonist with a linear structure, namely a 'target polypeptide-ELP peptide-SpyTag peptide-SpyCatcher peptide-ELP peptide-target polypeptide', is formed, and the spatial structure is more stable; the N-terminal truncated SpyCatcher (delta N) is used, has low immunogenicity and is suitable for patent medicine.
(4) The polypeptide double agonist obtained by the invention has nanomolar in vitro agonist activity on different receptors.
(5) In a common single-molecule dual agonist (such as Tirzepatide), the change of amino acid sites can simultaneously influence the efficacy of the molecule on different targets, the changed effect is difficult to predict, and the two polypeptides are independent, so that the affinity of the dipeptide on different targets can be controlled more accurately.
Drawings
FIG. 1 is a schematic diagram of the structure of a fusion expression protein vector of the present invention, wherein A and B are each GLP-1-ELP based on the icSAT method 15 -SpyTag and GLP-1-ELP 15 Schematic structural diagram of fusion protein expression vector of Spycatcher (DeltaN).
FIG. 2 is a GLP-1-ELP based on the icSAT method 15 SDS-PAGE results map of the expression of SpyTag/Spycatcher (DeltaN) fusion proteins.
FIG. 3 is a GIP-ELP based on the icSAT method 15 Schematic structural diagram of the expression vector of the Spycatcher (DeltaN) fusion protein.
FIG. 4 is a diagram of an ICSAT based approachMtuΔI-CM(WT)、MtuΔI-CM (m1)、MtuSDS-PAGE results map of the expression of fusion proteins of ΔI-CM (m 2) intein.
FIG. 5 is a GIP-ELP based on the icSAT method 15 -schematic structural representation of the SpyTag fusion protein expression vector.
FIG. 6 is a GIP-ELP based on the icSAT method 15 SDS-PAGE results map of the expression of SpyTag fusion proteins.
FIG. 7 is a diagram showing SDS-PAGE results of the purification process of the receptor agonist of the present invention; wherein A, B, C is a SDS-PAGE result graph of a purification process of a GLP-1/GIP double-target agonist, a double GLP-1 single-target agonist and a double GIP single-target agonist based on an icSAT method respectively.
FIG. 8 is a graph of SDS-PAGE results of two-step standard column purification of a receptor agonist of the present invention; wherein A, B, C is the SDS-PAGE result of GLP-1/GIP dual-target agonist, dual GLP-1 single-target agonist and dual GIP single-target agonist purified by two standard columns, respectively.
FIG. 9 is a graph of RP-HPLC assay results for receptor agonists of the present invention; wherein A, B, C is a graph of RP-HPLC assay results for GLP-1/GIP dual-target agonist, dual GLP-1 single-target agonist and dual GIP single-target agonist, respectively.
FIG. 10 is a graph of molecular weight measurements of receptor agonists of the present invention; wherein A, B, C is a molecular weight measurement result graph of the GLP-1/GIP dual-target agonist, the dual GLP-1 single-target agonist and the dual GIP single-target agonist respectively.
FIG. 11 is a graph of in vitro agonistic activity of a receptor agonist of the present invention; wherein A, B is an in vitro agonist activity profile and an in vitro agonist activity profile, respectively.
FIG. 12 is a graph of half-life of GLP-1/GIP dual-target agonists, GLP-1 and GIP in human serum.
FIG. 13 is a flow chart of fusion protein self-ligation and icSAT protein purification.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available. The procedure used in the examples below is conventional, unless otherwise specified, and specific procedures can be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al, molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor). All the primers are biosynthesized by Shanghai workers.
Examples 1-10, ES: cell lysate supernatant; EP: cell lysate precipitation; ES1, ES2: cell lysate supernatant 1, 2, es3: a mixed solution of ES1 and ES 2; ESS: supernatant after salt aggregation of ES 3; ESP: precipitation of ES3 after salt aggregation; CP: precipitation after cutting of ESP; CS: post-cutting supernatant of ESP; wherein ES 1-CP are diluted 10 times, CS is not diluted; m: protein markers; BSA: bovine serum albumin standard.
In the recombinant expression constructs of the invention, the polynucleotide sequence encoding the fusion protein is suitably linked to an expression control sequence to effect transcription as desired and ultimately production of the fusion protein in a host cell. Such expression control sequences include, but are not limited to, promoters, enhancers, ribosome binding sites, polyadenylation sites, transcriptional splice sequences, transcriptional termination sequences, and mRNA stabilizing sequences, and the like.
Vectors for expression constructs of the invention include vectors, such as plasmid vectors, which are autonomously replicable in a host cell; and a vector that can be integrated into and replicated together with the host cell DNA. In a specific embodiment, the expression construct of the invention is derived from pET30a (+) from Novagen. Host cells useful for expressing the fusion proteins of the invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotes include Escherichia genus Escherichia) Bacillus genusBacillus) Genus PseudomonasPseudomonas) And Streptomyces genusStreptomyces) Is a bacterium of the genus (a). In a preferred embodiment, the host cell is an Escherichia cell, preferably E.coli. In a specific embodiment of the invention, the host cell used is an E.coli BL21 (DE 3) strain cell (Novagen).
Example 1: construction based on icSAT methodGLP-1-ELP of (E) 15 Expression vector of SpyTag/Spycatcher (DeltaN)
The expression vector used in the examples of the present application was pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 SpyTag and EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 Spycatcher (ΔN). A in FIG. 1 is GLP-1-ELP based on the icSAT method 15 -schematic structural representation of the SpyTag fusion protein expression vector; b in FIG. 1 is GLP-1-ELP based on icSAT method 15 Schematic structural diagram of the expression vector of the Spycatcher (DeltaN) fusion protein.
In the embodiment, the salinity-induced self-assembly peptide is EFK8, and the amino acid sequence of the salinity-induced self-assembly peptide is shown as SEQ ID NO. 11; intein isMtuThe amino acid sequence of the delta I-CM (m 2) is shown as SEQ ID NO. 14; the amino acid sequence of GLP-1 (SEQ ID NO: 1) was obtained from literature (Glaesner et al, diabetes/Metab Res Rev, 2010, 26 (4): 287-296), and compared with the native GLP-1 sequence, the GLP-1 sequence selected in this example had three mutations, each: the eighth Ala is mutated to Gly, the 22 nd Gly is mutated to Glu, the 36 th Arg is mutated to Gly, so that the stability of GLP-1 against the hydrolysis of dipeptidyl peptidase-4 (dipeptidyl peptidase-4, DPP-4) can be improved; GLP-1 sequence by ELP 15 (SEQ ID NO: 3) with SpyTag sequence (SEQ ID NO: 7) or SpyCatcher (DeltaN) (SEQ ID NO: 4). For ELP 15 SpyTag and ELP 15 E.coli host codon optimization was performed on the SpyCatcher (DeltaN) gene fragment, the optimized gene was synthesized by Guangzhou Rui Bow, and the primers involved in the plasmid construction process are shown in Table 1.
TABLE 1
For pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The SpyTag plasmid was first amplified by PCR with primers (S220614-ESK-F/Y200915-ter-R) to give a plasmid containing ELP 15 The SpyTag-Kan gene fragment was then used as a primer (Y200915-LPM-F/S220614-LMG-R) for the original pET30a-EFK8-PT linker for the laboratoryMtuΔI-CM (m2)-GLP-1-GS linker-SpyTagAmplifying to obtain Lac I-EFK8-PT linkerMtuΔI-CM (m2)-A gene fragment of GLP-1. And (3) carrying out Gibson assembly on the two gene fragments, transferring the obtained ligation product into escherichia coli DH5 alpha, and extracting the plasmid to transform escherichia coli expression strain BL21 (DE 3) for protein expression and subsequent experiments after screening out correct positive clones through colony PCR identification and sequencing.
pET30a-EFK8-PT linker-MtuΔI-CM (m2)-GLP-1-GS linker-SpyTag preparation process: the EFK8-Mtu DeltaI-CM (m 2) fragment was amplified with the original laboratory pET32-EFK8-Mtu DeltaI-CM (m 2) -hGH (from patent PCT/CN 2020/125054) as template, and synthesized GLP-1-ELP 15 The SpyTag fragment and pET30a vector were ligated by Gibson assembly.
For pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The SpyCatcher (DeltaN) plasmid is first amplified by PCR with primers (S220615-E-SC-F/Y200915-ter-R) to give a plasmid containing ELP 15 The fragment of the SpyCatcher (. DELTA.N) -Kan gene was then used as a primer (Y200915-LPM-F/S220614-LMG-R) for the original pET30a-EFK8-PT linker for the laboratoryMtuΔI-CM (m2)-GLP-1-GS linker-SpyTag is amplified to obtain a plasmid containing Lac I-EFK8-PT linkerMtuΔI-CM (m2)-A gene fragment of GLP-1. And (3) carrying out Gibson assembly on the two gene fragments, transferring the obtained ligation product into escherichia coli DH5 alpha, and extracting the plasmid to transform escherichia coli expression strain BL21 (DE 3) for protein expression and subsequent experiments after screening out correct positive clones through colony PCR identification and sequencing.
Example 2: GLP-1-ELP based on icSAT method 15 Expression of the SpyTag/Spycatcher (DeltaN) fusion protein
The plasmid containing pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 SpyTag and pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 BL21 (DE 3) strain of SpyCatcher (. DELTA.N)) was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin sulfate, and cultured in a shaker at 37℃to logarithmic phase (OD) 600 =0.4 to 0.6), 0.2 mM IPTG final concentration was added, and the culture conditions were: 18. collecting at 250deg.C at 250 rpm for 24 hr Obtaining cells and measuring the bacterial concentration OD 600 (OD of 1 mL will be described below 600 The cell amount of 1 is referred to as 1 OD).
The cells were treated with salt-free schizochytrium Buffer B1 (20 mM Tris-base, 1 mM Na) 2 ·EDTA·2H 2 O, pH 8.0) was resuspended to 50 OD/mL and sonicated (disruption conditions: power 200W, sonication time 3 sec, interval 3 sec, run time 8 min). At 4 ℃,15,000gThe supernatant and the pellet were collected and sampled, respectively, and SDS-PAGE was used to examine the expression of the fusion protein in the lysed supernatant and the lysed pellet.
The results are shown in FIG. 2, and the results are shown in Table 2, wherein the optical density analysis of the target bands was performed using ImageJ (National Institutes of Health) gel quantitative analysis software, and the yield and self-cleavage rate of the fusion protein in the cleavage supernatant were calculated.
TABLE 2
a Yield of protein in the protein cleavage supernatant, b self-cleavage rate (%) =icsat protein amount/(icSAT protein amount+fusion protein expression amount) ×100%.
Both fusion proteins are soluble in expression, and also have a more severe self-cleaving phenomenon. Wherein EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The molecular weight of SpyTag is 32.7 kDa, the expression quantity is 100.9mg/L LB culture medium, the self-cleavage rate is 60.4%, the molecular weight of the target protein after cleavage is 11.2kDa, but the band of the target protein after cleavage cannot be seen on a gel chart; EFK8- MtuΔI-CM (m2)-GLP-1-ELP 15 The molecular weight of Spycatcher (DeltaN) is 41.1kDa, the expression level is 136.8mg/L, the self-cleavage rate is 64.2%, and the target protein size after self-cleavage is 19.6kDa.
Example 3: construction of inteinsMtuGIP-ELP based on icSAT method for different mutant strains of ΔI-CM 15 Expression vector of Spycatcher (DeltaN)
Implementation of the present applicationThe expression vectors used in the examples were of 3 different typesMtuFusion protein expression vector of ΔI-CM mutant strain: pET30a-EFK8-MtuΔI-CM (WT/m1/m2)-GIP-ELP 15 Spycatcher (ΔN). FIG. 3 shows a GIP-ELP based on the icSAT method 15 Schematic structural diagram of the expression vector of the Spycatcher (DeltaN) fusion protein. 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), and the eighth Ala in the GIP sequence selected in this example was mutated to Gly, thereby improving the stability of the GIP against DPP-4 hydrolysis, as compared with the native GIP sequence. E.coli host codon optimization was performed on gene fragments of GIP and on-line tool DNAworks was used [ ]https://hpcwebapps.cit.nih.gov/dnaworks/) The nucleotide sequence of GIP was designed. The 4 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.
TABLE 3 Table 3
First, 4 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). Next, pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The plasmid-SpyCatcher (DeltaN) is used as a template, and PCR amplification is carried out by a primer (S220615-E-SC-F/Y200915-ter-R) to obtain the plasmid-containing ELP 15 The gene fragment of-SpyCatcher (delta N) -Kan is spliced and amplified by overlapping PCR to obtain the complete GIP-ELP 15 -SpyCatcher (Δn) -Kan fragment. With original pET30a-EFK8-PT linker of laboratoryMtuΔI-CM (WT)-hGH、pET32a-L 6 KD-PT linker-MtuDeltaI-CM (m 1) -IFN and pET32a-L 6 KD-PT linker-MtuThe ΔI-CM (m 2) -LCB3 plasmid (see pET30a-EFK8-PT linker-Mtu ΔI-CM (m 2) -GLP-1-GS linker-SpyTag for preparation methods in example 1, except for replacing the corresponding DNA fragment) was used as template by primers (D210310-Mtu-F/Y200728-LPM-R)) Respectively amplify and getMtuΔI-CM (WT)、MtuΔI-CM (m 1) andMtuthe DeltaI-CM (m 2) fragment (SEQ ID NO: 12-14). Then the original pET30a-EFK8-PT linker of the laboratory is usedMtuThe LacI-EFK8-PT linker fragment was amplified by using DeltaI-CM (WT) -hGH as template and primers (Y200915-LPM-F/Z211118-Mtu-R). The LacI-EFK8-PT linker is prepared, MtuDeltaI-CM (WT/m 1/m2/m 3) and GIP-ELP 15 The three gene fragments of-SpyCatcher (delta N) -Kan are respectively subjected to Gibson assembly, the obtained connection product is transformed into escherichia coli DH5 alpha, correct positive clones are screened out through colony PCR identification and sequencing, and the plasmid is extracted to transform escherichia coli expression strain BL21 (DE 3) for protein expression and subsequent experiments.
Example 4: contains inteinMtuGIP-ELP based on icSAT method for different mutant strains of ΔI-CM 15 Expression of the Spycatcher (ΔN) fusion protein and icSAT purification
The plasmid (pET 30a-EFK8-MtuΔI-CM (WT)-GIP-ELP 15 -SpyCatcher(ΔN)、pET30a-EFK8-MtuΔI-CM (m1)-GIP-ELP 15 Spycatcher (. DELTA.N) and pET30a-EFK8-MtuΔI-CM (m2)-GIP-ELP 15 BL21 (DE 3) strain of SpyCatcher (. DELTA.N)) was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin sulfate, and cultured in a shaker at 37℃to logarithmic phase (OD) 600 =0.4 to 0.6), 0.2 mM IPTG final concentration was added, and the culture conditions were: 18. cells were harvested after 24 hours at 250 rpm at C and the bacterial concentration OD was measured 600 (OD of 1 mL will be described below 600 The cell amount of 1 is referred to as 1 OD).
The cells were treated with salt-free schizochytrium Buffer B1 (20 mM Tris-base, 1mM Na) 2 ·EDTA·2H 2 O, pH 8.0) was resuspended to 50 OD/mL and sonicated (disruption conditions: power 200W, sonication time 3 sec, interval 3 sec, run time 8 min). At 4 ℃,15,000 gThe supernatant and the pellet were collected and sampled respectively, and the results were examined by SDS-PAGE under the conditions of (C) for 20 min, as shown in FIG. 4.
To the obtained cleavage supernatant was added an equal volume of 1.4M Na 2 SO 4 Aggregation Buffer B of (a)2（1.4 M Na 2 SO 4 ，20 mM Tris-base，1mM Na 2 ·EDTA·2H 2 O, ph 8.0), at 4 ℃,12 h, to allow for sufficient aggregation of the self-assembled peptide. The suspension was then heated to 4℃and 15,000gCentrifuging for 20 min under the condition of (1) and collecting the precipitate with equal volume containing 0.7M Na 2 SO 4 Buffer B3 (0.7M Na) 2 SO 4 ，20 mM Tris-base，1mM Na 2 ·EDTA·2H 2 O, pH 8.0) after 1 washing, and the like. The volume is halved to contain 0.7M Na 2 SO 4 Buffer B4 (PBS without NaCl, 0.7M Na) 2 SO 4 ，20 mM Bis-Tris，2 mM Na 2 ·EDTA·2H 2 O, ph 6.2) was thoroughly resuspended and deposited on a rotary mixer at 25 ℃,24 h to allow adequate self-cleavage of the intein. The suspension was subjected to 15,000 at 4℃CgThe supernatant and the pellet were separated by centrifugation under conditions of 20 min. The yields, self-cleavage rates and icSAT purification effects (fusion protein aggregation efficiency and cleavage efficiency) of the three fusion proteins in this example were examined using ImageJ, and the results are shown in Table 4.
TABLE 4 Table 4
a Yield of protein in the protein cleavage supernatant, b the self-cleavage ratio (%) = icSAT protein amount/(icSAT protein amount + fusion protein expression amount) ×100%, c Aggregation efficiency (%) = reduced amount of fusion protein in supernatant after aggregation/amount of fusion protein in supernatant before aggregation x 100%, d cleavage efficiency (%) = decrease in fusion protein after cleavage/x 100% of fusion protein before cleavage.
All three fusion proteins are soluble expressed, and the intein isMtuThe expression level of the fusion protein of ΔI-CM (WT) was 216.3 mg/mL, although the intein wasMtuThe fusion protein of ΔI-CM (m 1) is low, but its aggregation efficiency and cleavage efficiency are highest among three inteins, up to 90%; intein isMtuFusion protein expression of ΔI-CM (m 1)The highest amount is 244.4 mg/mL, but the cutting efficiency is only 61.2%; intein isMtuAlthough the aggregation efficiency of the fusion protein of ΔI-CM (m 2) was high, the expression level was low, which was 160.0. 160.0 mg/mL. Thus, finally select to useMtuΔI-CM (WT) was used as an intein of the fusion protein for subsequent experiments.
Example 5: construction of EFK8-MtuΔI-CM (WT)-GIP-ELP 15 Expression vector of SpyTag
According to the test result in example 4, in this example, GIP-ELP based on the icSAT purification method 15 The same applies to the expression vector of the SpyTag fusion proteinMtuDelta I-CM (WT) as an intein, FIG. 5 shows GIP-ELP based on the icSAT method 15 -schematic structural representation of the SpyTag fusion protein expression vector. The primers involved in the plasmid construction are shown in Table 5.
TABLE 5
First, EFK8-PT linker constructed in example 3MtuΔI-CM (WT)-GIP-ELP 15 The plasmid of SpyCatcher (. DELTA.N) was used as template to amplify LacI-EFK8 using primers (Y200915-LPM-F/C220701-IMtu-R)MtuΔI-CM (WT) -GIP fragment; next, pET30a-EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 The SpyTag plasmid was used as template for the amplification of ELP using primers (C220701-IES-F/Y200915-ter-R) 15 -SpyTag-Kan fragment; finally, the obtained ligation product is transformed into escherichia coli DH5 alpha by carrying out Gibson assembly on the two gene fragments, and after correct positive clones are screened by colony PCR identification and sequencing, the plasmid is extracted to transform escherichia coli expression strain BL21 (DE 3) for protein expression and subsequent experiments.
Example 6: GIP-ELP based on icSAT method 15 Expression of the SpyTag fusion protein
The plasmid containing pET30a-EFK8-MtuΔI-CM (WT)-GIP-ELP 15 BL21 (DE 3) strain of-SpyTag) was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin sulfateIn medium, and cultured in shaking table at 37deg.C until logarithmic phase (OD 600 =0.4 to 0.6), 0.2 mM IPTG final concentration was added, and the culture conditions were: 18. cells were harvested after 24 hours at 250 rpm at C and the bacterial concentration OD was measured 600 (OD of 1 mL will be described below 600 The cell amount of 1 is referred to as 1 OD).
The cells were treated with salt-free schizochytrium Buffer B1 (20 mM Tris-base, 1mM Na) 2 ·EDTA·2H 2 O, pH 8.0) was resuspended to 50 OD/mL and sonicated (disruption conditions: power 200W, sonication time 3 sec, interval 3 sec, run time 8 min). At 4 ℃,15,000gThe supernatant and the pellet were collected and sampled respectively, and the results were examined by SDS-PAGE under the conditions of (C) for 20 min, as shown in FIG. 6. The optical density analysis of the bands of interest was performed using ImageJ (National Institutes of Health) gel quantitative analysis software according to protein quantitative standards, and the yield and self-cleavage rate of fusion proteins in the cleavage supernatants were calculated, and the results are shown in table 6.
TABLE 6
a Yield of protein in the protein cleavage supernatant, b self-cleavage rate (%) =icsat protein amount/(icSAT protein amount+fusion protein expression amount) ×100%.
The fusion protein is soluble and expressed, the expression quantity is 216.3 mg/mL, the self-cleavage rate is only 2.3%, and the fusion protein can be used for the subsequent preparation of GLP-1/GIP double agonists.
In summary, GLP-1-ELP based on icSAT purification method 15 Spycatcher (. DELTA.N)/SpyTag and GIP-ELP 15 The SpyCatcher (DeltaN)/SpyTag fusion protein expression vector can be successfully constructed and expressed in a soluble manner. It can be seen that, unlike GLP-1 fusion proteins, GIP fusion proteins have a lower self-cleavage rate, because the N-terminal residue of GIP is Tyr, which is not promoted MtuC-terminal cleavage of ΔI-CM; whereas the N-terminal residue of GLP-1 is His, it will triggerMtuThe phenomenon of self-cleavage of ΔI-CM ((Zhao et al Biotechnology journal, 2017, 12 (6): 1600656).Wherein the intein isMtuAlthough the GIP fusion protein of ΔI-CM (m 1/m 2) hardly had the self-cleavage phenomenon, the in vitro cleavage efficiency was also significantly lowered due to the changeMtuThe microenvironment of the two critical histidine residues H429 and H439 in ΔI-CM, while preserving pH dependence, prevents premature cleavage (Lin et al, aiche Journal, 2020, 66 (3): e 16806). While the intein isMtuThe GIP fusion protein of ΔI-CM (WT) had higher self-cleavage efficiency, but the in vitro cleavage efficiency was also the highest, and more target proteins could be obtained.
Example 7: preparation of GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists and dual-GIP single-target receptor agonists
To prepare GLP-1 and GIP dual target receptor agonists (GLP-1/GIP), EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 Spycatcher (. DELTA.N) and EFK8-MtuΔI-CM (WT)-GIP-ELP 15 Cell lysate supernatant of SpyTag according to 1:1, mixing the fusion proteins in a molar ratio; to prepare a dual GLP-1 single target receptor agonist (GLP-1/GLP-1), EFK8- MtuΔI-CM (m2)-GLP-1-ELP 15 Spycatcher (ΔN) and EFK8-MtuΔI-CM (m2)-GLP-1-ELP 15 Cell lysate supernatant of SpyTag according to 1:1, mixing the fusion proteins in a molar ratio; to prepare a dual GIP single target receptor agonist (GIP/GIP), EFK8-MtuΔI-CM (WT)-GIP-ELP 15 Spycatcher (. DELTA.N) and EFK8-MtuΔI-CM (WT)-GIP-ELP 15 Cell lysate supernatant of SpyTag according to 1:1, and mixing the fusion proteins according to the molar ratio.
The above mixture was allowed to react at 25℃and 80rpm for 2 hours to complete the combination. Subsequently, an equal volume of 1.4M Na was added to the mixture at completion of the reaction 2 SO 4 Buffer B2 (1.4M Na) 2 SO 4 ，20 mM Tris-base，1mM Na 2 ·EDTA·2H 2 O, ph 8.0), at 4 ℃,12 h, to allow for sufficient aggregation of the self-assembled peptide. The suspension was then heated to 4℃and 15,000gCentrifuging for 20 min under the condition of (1) and collecting the precipitate with equal volume containing 0.7M Na 2 SO 4 Buffer B3 (0.7M Na) 2 SO 4 ，20 mM Tris-base，1mM Na 2 ·EDTA·2H 2 O, pH 8.0) after 1 washing, and the like. The volume is halved to contain 0.3M Na 2 SO 4 Buffer B4 (PBS without NaCl, 0.3M Na) 2 SO 4 ，20 mM Bis-Tris，2 mM Na 2 ·EDTA·2H 2 O, ph 6.2) was thoroughly resuspended and deposited on a rotary mixer at 25 ℃,24 h to allow adequate self-cleavage of the intein. The suspension was subjected to 15,000 at 4℃CgThe supernatant and the pellet were separated by centrifugation under conditions of 20 min. SDS-PAGE was performed on the supernatant after cell lysis, the supernatant after salt aggregation and the pellet, and the supernatant after cleavage and pellet together, and the results are shown in FIG. 7. The optical density analysis of the target bands was performed using ImageJ (National Institutes of Health) gel quantitative analysis software according to protein quantitative standards, and the fusion protein aggregation efficiency, cleavage efficiency and purity were calculated, and the results are shown in table 7.
TABLE 7
a Aggregation efficiency (%) = reduced amount of fusion protein in supernatant after aggregation/amount of fusion protein in supernatant before aggregation x 100%, b cleavage efficiency (%) = decrease in fusion protein after cleavage/x 100% of fusion protein before cleavage, c purity (%) =amount of target protein/(amount of target protein+amount of hetero protein) ×100%.
Example 8: two-step standard column purification method for GLP-1 and GIP double-target receptor agonist, double GLP-1 single-target receptor agonist and double-GIP single-target receptor agonist
GLP-1 and GIP double-target receptor agonists (hereinafter referred to as GLP-1/GIP), double-GLP-1 single-target receptor agonists (hereinafter referred to as GLP-1/GLP-1) and double-GIP single-target receptor agonists (hereinafter referred to as GIP/GIP) purified in example 7 were finely purified using anion exchange columns (Unigel-30Q, 5 mL) and molecular sieve columns (HiLoad 16/600 Superdex 75 prep gradation). After loading the sample during ion exchange column purification, unbound protein was washed off with Binding buffer (20 mM Tris-HCl, pH 8.0) until UV baseline was stable. Then Elution was performed using an Elution buffer (20 mM Tris-HCl,1.0M NaCl,pH 8.0) with the following Elution procedure: 0-10% of B:0 min; 10-20% of B:10 min; 20-50% of B:0 min; 50-100% of B: and 0 min. The ion-exchanged purified protein was further purified by a molecular sieve column eluting with buffer (20mM NaCl,20mM Tris-HCl, pH 7.2) for 1 column volume. The collected elution peaks were detected by SDS-PAGE, and the detection results are shown in FIG. 8. A in FIG. 8 is the purification result of GLP-1/GIP; b in FIG. 8 is the purification result of GLP-1/GLP-1; c in FIG. 8 is the result of purification of GIP/GIP.
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 the hybrid protein, and the purity of the fusion protein can be improved.
Example 9: RP-HPLC assays for GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists and dual-GIP single-target receptor agonists
RP-HPLC assays were performed on GLP-1 and GIP dual-target receptor agonists (hereinafter GLP-1/GIP), dual GLP-1 single-target receptor agonists (hereinafter GLP-1/GLP-1) and dual-GIP single-target receptor agonists (hereinafter GIP/GIP) purified from ion exchange columns and molecular sieves in example 8. Using an Agilent 1260 system and a Acquity UPLC BEH C chromatographic column, the sample loading was 10. Mu.L, the flow rate was 0.2 mL/min, and the column temperature was 65 o And C, detection wavelength 280 nm. Mobile phase a was an aqueous solution containing 0.1% (v/v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v/v) trifluoroacetic acid. The mobile phase gradient is: eluting for 20 min by 25-85% of B. The results are shown in FIG. 9, wherein A in FIG. 9 is the RP-HPLC measurement of GLP-1/GIP; b in FIG. 9 is the RP-HPLC assay result of GLP-1/GLP-1; c in FIG. 9 is the RP-HPLC assay result of GIP/GIP.
From the results, the peak time of GLP-1/GIP was 13.922 min, the peak time of GLP-1/GLP-1 was 14.250 min, and the peak time of GIP/GIP was 13.464 min. As can be seen from the ratio of the peak areas, the purity of GLP-1/GIP and GLP-1/GLP-1 can reach 99%, while the purity of GIP/GIP is slightly lower than that of the former two, and is 98.1%.
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
Molecular weight determinations were performed on GLP-1 and GIP dual-target receptor agonists (hereinafter GLP-1/GIP), dual GLP-1 single-target receptor agonists (hereinafter GLP-1/GLP-1) and dual-GIP single-target receptor agonists (hereinafter GIP/GIP) purified by ion exchange columns and molecular sieves in example 8. The molecular weight of the samples was determined using ultra performance liquid chromatography-time of flight mass spectrometry combined with a UPLC-Q-TOF and Biozen ™ C4 chromatographic column. The conditions are as follows: sample injection amount 10. Mu.L, flow rate 0.5 mL/min, column temperature 75 o And C, detection wavelength 214 nm. Mobile phase a was an aqueous solution containing 0.1% (v/v) TFA, mobile phase B was an acetonitrile solution containing 0.1% (v/v) TFA, and the mobile phase gradient was set to: 5-60% of B, and eluting for 3 min. The results are shown in FIG. 10, wherein A in FIG. 10 is the molecular weight measurement result of GLP-1/GIP; b in FIG. 10 is the GLP-1/GLP-1 molecular weight measurement result; c in FIG. 10 is the molecular weight measurement result of GIP/GIP.
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 was 34111.82 Da, which was consistent with the theoretical molecular weight (34112.69 Da).
Example 11: in vitro agonistic activity validation of GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists and dual-GIP single-target receptor agonists
To verify the biological activities of GLP-1 and GIP dual-target receptor agonists (hereinafter referred to as GLP-1/GIP), dual GLP-1 single-target receptor agonists (hereinafter referred to as GLP-1/GLP-1) and dual-GIP single-target receptor agonists (hereinafter referred to as GIP/GIP) purified by ion exchange columns and molecular sieves in example 8, an in vitro activity assay was required. HEK 293/GLP-1R-luciferases and HEK 293/GIPR-luciferases cells, GLP-1 or NFAT response module regulated reporter Luciferase are usedAfter GIP is specifically combined with GLP-1R or GIPR on the cell membrane, adenylate cyclase can be activated and signals can be transmitted, so that a luciferase reporter gene can be expressed, and after a luciferase substrate is added, the expression level of the luciferase can be estimated by detecting a chemiluminescence value, so that the biological activities of GLP-1/GIP, GLP-1/GLP-1 and GIP/GIP can be estimated. Experimental results half effective concentration (median effective concentration, EC 50 ) Representation, EC 50 The lower the value, the stronger the drug effect.
In determining the in vitro agonist activity of the samples on HEK293/GLP-1R-Luciferase cells, GLP-1/GIP, GLP-1/GLP-1, GIP/GIP, control GLP-1 (SEQ ID NO: 1) and control GIP (SEQ ID NO: 2) were diluted to 8 nM with assay buffer (DMEM medium containing 0.5% FBS, 1% penicillin-streptomycin) as the initial concentrations, and then diluted 3-fold in sequence. When the in vitro agonistic activity of the samples on HEK293/GIPR-Luciferase cells was measured, the samples were diluted to 200 nM with assay buffer, and then were serially diluted 4-fold in gradient. HEK293/GLP-1R-Luciferase (or HEK 293/GIPR-Luciferase) cells were digested and diluted to 100 ten thousand/mL of suspension with assay buffer. The cell suspension and diluted samples were transferred to 384 well plates (white bottom) respectively, 20 μl/well, and 3 multiplex wells were set. Finally, the 384-well plate is placed at 37 o C、5% CO 2 Incubate 6 h in incubator. After the incubation was completed, luciferase Assay Buffer, 30. Mu.L/well was added. After waiting for 3 min for the cells to lyse sufficiently, the reading was performed using an enzyme-labeled instrument.
The results of the in vitro agonist activity assays of GLP-1/GIP, GLP-1/GLP-1 and GIP on HEK293/GLP-1R-Luciferase cells are shown as A in FIG. 11, and the results of the in vitro agonist activity assay of GLP-1/GIPR-Luciferase cells on HEK293/GIPR-Luciferase cells are shown as B in FIG. 11. Analysis of data using data processing software GraphPad Prism 8 to obtain GLP-1/GIP, GLP-1/GLP-1, GIP/GIP versus HEK293/GLP-1R-Luciferase and HEK293/GIPR-Luciferase cells EC 50 As shown in table 8.
TABLE 8
From the experimental results, it was found that EC of GLP-1/GIP dual agonist for GLP-1R 50 Is 0.305 + -0.048 nM, and single GLP-1 (EC 50 =0.128±0.003 nM), the activity was reduced by about 50%. While EC of GLP-1/GLP-1 agonists of similar architecture 50 At 0.044.+ -. 0.006, the activity is significantly increased compared to single GLP-1 and GLP-1/GIP dual agonists due to differences in agonistic activity resulting from the number of GLP-1 in the polymer. In addition, the agonistic activity of individual GIP and GIP/GIP agonists on GLP-1R was assayed simultaneously, and the results indicated that GIP did not agonize GLP-1R. EC for GLP-1/GIP dual agonists for GIPR 50 Is 0.375 + -0.030 nM, and a single GIP (EC) 50 =0.348±0.019 nM) is similar; is similar to the GIP/GIP agonist (EC 50 The agonistic activity of GLP-1/GIP dual agonist was reduced by about 50% corresponding to the number of GIPs compared to the activity of =0.187±0.011 nM). At the same time, the agonistic activity of individual GLP-1 and GLP-1/GLP-1 agonists on GIPR was also determined, and the results indicate that GLP-1 does not agonize GIPR.
Example 12: human serum stability validation of GLP-1 and GIP dual-target receptor agonists, dual GLP-1 single-target receptor agonists and dual-GIP single-target receptor agonists
To verify the stability in serum of GLP-1/GIP, GLP-1/GLP-1 and post-called GIP/GIP purified by ion exchange column and molecular sieve in example 8, these samples were pooled with human serum at 37, respectively o C was incubated (final sample concentration of 0.6 mg/mL, final serum concentration of 20%). The initial time was recorded and 50 μl of the mixture was taken at intervals of 0, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h. After the incubation, 2 volumes of acetonitrile was added to precipitate the protein, and the mixture was stirred at 4 o Standing at C for 30 min, and then at 20,000gCentrifuging for 5 min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting residual sample concentration in supernatant by Agilent 1260 system and Acquity UPLC BEH C4 chromatographic column. Sample injection amount 10. Mu.L, flow rate 0.2 mL/min, column temperature 65 o And C, detection wavelength 280 nm. Mobile phase A is a mixture containingWith an aqueous solution of 0.1% (v/v) trifluoroacetic acid, mobile phase B was an acetonitrile solution containing 0.1% (v/v) trifluoroacetic acid, and the elution conditions were: eluting for 30 min by 5-70% of B. The results are shown in FIG. 12. Data were analyzed using data processing software GraphPad Prism 8 to obtain half-lives of GLP-1, GIP and GLP-1/GLP-1 in human serum, and the results are shown in table 9.
TABLE 9
The half-life measurement results of the double GLP-1 single-target receptor agonist and the double GIP single-target receptor agonist are similar to those of the GLP-1 double-target receptor agonist and the GIP double-target receptor agonist, and are obviously longer than those of the GLP-1 double-target receptor agonist and the GIP double-target receptor agonist.
The above examples are only preferred embodiments of the present invention, and are intended to be illustrative only and not to be limiting of the invention, since various changes, substitutions, modifications and the like can be made by those skilled in the art without departing from the spirit of the invention.
Claims (10)
1. A method for preparing a dipeptide receptor agonist comprising the steps of:
(a) Constructing a fusion protein expression vector for fusing an icSAT tag and a target polypeptide of SpyTag peptide or SpyCatcher peptide and expressing the fusion protein;
(b) Mixing the two fusion proteins, and self-connecting to form a double fusion protein;
(c) And purifying by using an icSAT protein purification method and a two-step standard column purification method to obtain the dipeptide receptor agonist.
2. The method of claim 1, wherein in step (a), the target polypeptide is glucagon-like peptide-1 and/or a glucose-dependent insulinotropic polypeptide; the glucagon-like peptide-1 and/or glucose-dependent insulinotropic polypeptide is linked to a SpyTag peptide or a SpyCatcher peptide via an ELP peptide; the SpyCatcher peptide and the SpyTag peptide can form an isopeptide chain, the SpyCatcher peptide comprises a polypeptide with an amino acid sequence shown in any one of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and the SpyTag peptide comprises a polypeptide with an amino acid sequence shown in any one of SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9;
the ELP peptide is characterized by one of the following conditions:
(a) A repeated sequence of Val-Pro-Gly-Xaa-Gly;
(b) The repeated sequence of Val-Pro-Gly-Xaa-Gly, the repeated number of Val-Pro-Gly-Xaa-Gly repeated sequence is 10-30 times;
(c) A repeated sequence of Val-Pro-Gly-Xaa-Gly, wherein Xaa is Val and Glu;
(d) A repeated sequence of Val-Pro-Gly-Xaa-Gly, wherein Xaa is Val and Glu, and the ratio of Xaa amino acids Val and Glu is 3:1-5:1;
(e) The amino acid sequence of the ELP peptide is shown as SEQ ID NO. 3.
3. The method for preparing a dipeptide receptor agonist according to claim 1, wherein in step (a), the method for preparing the fusion protein comprises the following steps:
sequentially connecting the purification tag, the target polypeptide and the ELP peptide with the SpyTag peptide or the SpyCatcher peptide gene sequence to form a fusion protein gene, constructing an expression vector by the fusion protein gene, and introducing the fusion protein gene into a host cell to obtain engineering bacteria; culturing the engineering bacteria to express the fusion protein, then cracking the engineering bacteria, and capturing to obtain the fusion protein;
the capturing to obtain fusion protein comprises the steps of cracking engineering bacteria, centrifuging to obtain supernatant fluid to obtain fusion protein;
the purification tag consists of a self-assembled peptide and a cleavage tag; the gene sequence of the self-assembled peptide is connected with the gene sequence of the cutting tag through a connector; the joint is a PT-type joint; the PT type joint amino acid sequence is shown as an amino acid sequence shown as SEQ ID NO. 10;
the self-assembly peptide is salinity-induced self-assembly peptide, and the salinity-induced self-assembly peptide is beta-sheet short peptide; the amino acid sequence of the beta-sheet short peptide is shown as SEQ ID NO. 11;
The cleavage tag is a chemical cleavage site, an enzymatic cleavage site or a self cleavage site, the self cleavage site is an intein, and the intein isMtu ΔI-CM (WT) inteins orMtuΔI-CM intein mutants, saidMtuThe ΔI-CM intein mutants includeMtu ΔI-CM (m1)、Mtu ΔI-CM (m 2) andMtu ΔI-CM (m 3); the saidMtuThe amino acid sequence of the delta I-CM (WT) is shown as SEQ ID NO. 12, whichMtuThe amino acid sequence of the delta I-CM (m 1) is shown as SEQ ID NO. 13, and theMtuThe amino acid sequence of the delta I-CM (m 2) is shown as SEQ ID NO. 14; the saidMtuThe amino acid sequence of the delta I-CM (m 3) is shown as SEQ ID NO. 15.
4. The method of claim 1, wherein in step (b), the two fusion proteins are mixed, specifically: fusion proteins fused with SpyTag peptide and SpyCatcher peptide were prepared according to a ratio of 1: mixing and reacting at a molar ratio of 1 to obtain a double-fusion protein solution; the temperature of the mixing reaction is 4-37 ℃, and the time of the mixing reaction is 0.5-24 h; in the step (c), the icSAT protein purification method specifically comprises the following steps: adding an equal volume of high-salt buffer solution into the double-fusion protein solution to aggregate, and adjusting the pH value of the solution to cut; the high-salt buffer solution contains 1.0-1.4M Na 2 SO 4 Buffer solution with pH of 8.5, wherein the aggregation temperature is 4-37 ℃ and the aggregation time is 0.5-12 h; the cutting is to disperse aggregates of double fusion proteins in an acidic buffer solution, perform cutting treatment, and centrifugally take supernatant; the pH value of the acidic buffer solution is 5.5-6.8, the temperature of the cutting treatment is 4-37 ℃, and the time of the cutting treatment is 3-48 hours; in step (c), the two-step standard column purification method comprises ion exchange purification and molecular sieve purification.
5. The dipeptide receptor agonist according to any one of claims 1 to 4.
6. The dual peptide receptor agonist of claim 5, wherein the dual peptide receptor agonist comprises 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;
half-effective concentration EC of the GLP-1 and GIP dual-target receptor agonist on the in vitro agonistic activity of the GLP-1 receptor 50 Half-effective concentration EC of 0.305±0.048 nM for in vitro agonistic activity of GIP receptor 50 0.375+ -0.030 nM;
half-effective concentration EC of the dual GLP-1 single target receptor agonist on the in vitro agonistic activity of the GLP-1 receptor 50 0.044.+ -. 0.006 nM, no in vitro agonistic activity at GIP receptor;
Half-effective concentration of the dual GIP single-target receptor agonist EC on the in vitro agonistic activity of the GIP receptor 50 Is 0.187+/-0.011 nM, and has no in-vitro agonistic activity on GLP-1 receptor.
7. Use of the dipeptide receptor agonist of claim 6 in the manufacture of a medicament for the prevention or treatment of type two diabetes.
8. A polynucleotide comprising a nucleotide sequence encoding the fusion protein of claim 1 or a complement thereof.
9. An expression vector comprising the polynucleotide of claim 8.
10. A host cell comprising the polynucleotide of claim 8 or the expression vector of claim 9.
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