Fusion protein and semaglutide intermediate polypeptide preparation method

Abstract

The invention discloses a fusion protein and a preparation method of semaglutide intermediate polypeptide. The fusion protein comprises a leader peptide sequence and a semaglutide intermediate polypeptide sequence (Arg34GLP-1 (9-37)) connected with the leader peptide sequence. A nucleotide sequence capable of expressing the fusion protein is designed, an escherichia coli expression system which is clear in genetic background, easy to culture and short in fermentation period is utilized to construct a recombinant strain, and then the fusion protein which is good in solubility in fermentation liquor of the recombinant strain is efficiently expressed by utilizing the recombinant strain. And then carrying out enzyme digestion on the fusion protein sequence to obtain semaglutide intermediate polypeptide. The separation and purification process of the fusion protein is simple, and the semaglutide intermediate polypeptide is high in production efficiency and purity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a fusion protein and a semaglutide intermediate polypeptide. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a hormone secreted by pancreatic α cells and intestinal neuroendocrine cells (L cells). It has the effects of promoting insulin secretion in a glucose-dependent manner, inhibiting the secretion of glucagon, promoting the regeneration of β cells, inhibiting cell apoptosis, reducing appetite and reducing weight. GLP-1 is easily degraded by dipeptidyl peptidase (DPP-IV) on the cell surface in the body and has a short half-life. Currently, a series of different methods have been used to modify GLP-1 to provide a longer duration of action in the body, such as replacing the 8th (Ala) amino acid (the action site of the DPP-IV enzyme) in the GLP-1 active fragment (7-37), chemical modification (such as acylation, glycosylation), and fusion protein to increase molecular weight.

[0003] Semaglutide is a GLP-1 analog. It uses the non-natural amino acid 2-aminoisobutyric acid to replace the alanine at position 8, uses arginine to replace the lysine at position 34, and connects the lysine at position 26 to an octadecanoic acid fatty chain, based on the GLP-1 (7-37) chain, so it has high drug stability. When semaglutide is injected subcutaneously, its absorption can be slowed down by cross-linking, and it can bind to albumin. At the same time, it has a relatively stable ability to prevent enzymes such as DPP-IV from degrading it, so it has a longer plasma half-life. Semaglutide has an excellent, safe and reliable hypoglycemic effect. At the same time, it also shows good effects in weight loss and cardiovascular system benefits, and has great market value and academic value.

[0004] The preparation methods of semaglutide in the prior art include chemical synthesis, which further includes solid phase synthesis, liquid phase synthesis or solid-liquid combination. For example, CN109627317A first solid phase synthesizes side chain protected peptides, liquid phase fragment condensation obtains fully protected semaglutide, then cleaves to obtain crude semaglutide peptide, purifies and salts to obtain semaglutide; this type of process has many steps, and a variety of organic solvents are required in the synthesis process and the amount used is large, which is not conducive to industrial scale-up and is not environmentally friendly, and there are potential amino acid racemate by-products, which brings certain risks to the long-term use of the drug.

[0005] The preparation method of Semaglutide also includes a biological method, which can prepare the intermediate polypeptide in the form of inclusion bodies in an Escherichia coli host. Since the intermediate polypeptide with a smaller molecular weight is easily degraded in the Escherichia coli host, the Escherichia coli prokaryotic expression system is not suitable for directly expressing the intermediate polypeptide. In order to enhance the stability of the intermediate polypeptide during the expression process, it is often necessary to express the intermediate polypeptide in the form of a fusion protein with a larger molecular weight to achieve co-expression of the two, thereby reducing the possibility of degradation. For example, CN111378027A discloses a method for preparing Semaglutide by tandem expression in the form of inclusion bodies. The method uses whole genome synthesis technology to integrate the tandemly expressed Semaglutide precursor gene with the plasmid pET-27b (+) to construct a recombinant expression plasmid, transform it into Escherichia coli for high-density fermentation and induced expression, and dissolve the inclusion body after collecting and breaking the thalline to obtain the inclusion body to obtain a soluble protein, and then digest and purify it by Kex2 and carboxypeptidase B to obtain Semaglutide. This method uses both Kex2 and carboxypeptidase B, and the cost of enzyme addition is high. In addition, patent CN114292338B discloses a method for preparing the intermediate polypeptide Arg 34 GLP-1(9-37) or Arg 34 The present invention discloses a method for preparing a fusion protein of GLP-1 (11-37), wherein the method expresses a fusion protein containing a semaglutide intermediate polypeptide in Escherichia coli, collects the inclusion bodies, dissolves them, and then digests them to obtain the target product. However, the maximum yield of the target product is only 3.62 g / L, which needs to be further improved. In addition, the inclusion bodies need to be dissolved, denatured, renatured, purified, etc., which is complicated to operate and has high cost. Based on the above technical problems in the prior art, it is urgently necessary to provide a semaglutide intermediate polypeptide or a preparation method thereof with a higher fusion protein expression amount, simpler process steps, and more suitable for industrial production. Summary of the invention

[0006] One object of the present invention is to provide a method for preparing a semaglutide intermediate polypeptide with higher protein expression, simpler process steps, or more suitable for industrial production.

[0007] Another object of the present invention is to provide a fusion protein for preparing a semaglutide intermediate polypeptide.

[0008] In a first aspect, the present invention provides a fusion protein, comprising:

[0009] A leader peptide sequence and a semaglutide intermediate polypeptide sequence, wherein the leader peptide sequence and the semaglutide intermediate polypeptide sequence are directly connected, or indirectly connected via a linker sequence and / or a protease cleavage site sequence;

[0010] Wherein, the leader peptide sequence is the PfKRED amino acid sequence as shown in SEQ ID NO:6 or an equivalent sequence having at least 98% identity with the PfKRED amino acid sequence, the grpE amino acid sequence as shown in SEQ ID NO:15 or an equivalent sequence having at least 98% identity with the grpE amino acid sequence, the SUMO amino acid sequence as shown in SEQ ID NO:19 or an equivalent sequence having at least 98% identity with the SUMO amino acid sequence; and

[0011] The semaglutide intermediate polypeptide sequence is Arg as shown in SEQ ID NO: 2 34 GLP-1 (9-37) amino acid sequence.

[0012] In another preferred embodiment, when a linker sequence and a protease cleavage site sequence are present, the leader peptide sequence, the linker sequence, the protease cleavage site sequence and the semaglutide intermediate polypeptide sequence are connected in sequence.

[0013] In another preferred embodiment, the linker sequence is selected from any one of GGGG, GGGGG, GGGGS, GGGSG, EAAAK, AEAAAKALEA and PAPAP.

[0014] In another preferred embodiment, the protease cleavage site sequence is selected from any one of DDDDK, KR, IEGR and IDGR.

[0015] In another preferred example, the fusion protein has an amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:17, as shown in SEQ ID NO:21, as shown in SEQ ID NO:25 or as shown in SEQ ID NO:27.

[0016] In another preferred embodiment, the fusion protein further comprises an affinity adsorption tag sequence located at the N-terminus of the leader peptide sequence, such as a histidine tag sequence (comprising at least 6 histidines).

[0017] In another preferred embodiment, the fusion protein is selected from the following group:

[0018] PfKRED or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1 (9-37);

[0019] grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1(9-37); and

[0020] SUMO or its equivalent sequence-Arg34 GLP-1(9-37).

[0021] More preferably, the fusion protein is selected from the group consisting of:

[0022] PfKRED or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1 (9-37);

[0023] 6xHis tag-grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1(9-37); and

[0024] 6xHis tag-SUMO or its equivalent sequence-Arg 34 GLP-1(9-37).

[0025] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion protein as described in the first aspect of the present invention;

[0026] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:16, as shown in SEQ ID NO:20, as shown in SEQ ID NO:24 or as shown in SEQ ID NO:26.

[0027] The third aspect of the present invention provides a recombinant expression vector, which contains the isolated nucleic acid molecule as described in the second aspect of the present invention;

[0028] Further, the recombinant expression vector also includes a pET series plasmid, Duet series plasmid, pGEX series plasmid, pHY300 plasmid, pHY300PLK plasmid, pPIC3K plasmid, pPIC9K plasmid or pTrc series plasmid for integration into the isolated nucleic acid molecule;

[0029] Furthermore, the pET series plasmids include pET-24a(+), pET28a, pET-29a(+), and pET-30a(+); the Duet series plasmids include pRSFDuet-1 and pCDFDuet-1; and the pTrc series plasmids include pTrc99a.

[0030] In a fourth aspect, the present invention provides a recombinant microbial cell, which expresses the fusion protein as described in the first aspect of the present invention, contains the isolated nucleic acid molecule as described in the second aspect of the present invention, or contains the recombinant expression vector as described in the third aspect of the present invention.

[0031] Furthermore, the starting strains of the recombinant microbial cells include but are not limited to Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

[0032] The fifth aspect of the present invention provides the use of the isolated nucleic acid molecule as described in the second aspect of the present invention, or the recombinant expression vector as described in the third aspect of the present invention, or the recombinant microbial cell as described in the fourth aspect of the present invention in expressing the fusion protein as described in the first aspect of the present invention.

[0033] In a sixth aspect, the present invention provides a method for preparing a fusion protein, comprising the steps of: fermenting and culturing the recombinant microbial cell described in the fourth aspect of the present invention to produce the fusion protein described in the first aspect of the present invention;

[0034] Preferably, the fermentation production comprises:

[0035] The seed liquid of the recombinant microbial cells is inoculated into a TB culture medium, and cultured at 35-40° C., and when the OD600 value of the culture liquid reaches 0.6-0.8, an inducer is added, and the fusion protein is induced to express at 25-27° C., and wet cells expressing the fusion protein are obtained after the induction is completed;

[0036] The wet cells are homogenized and broken in a buffer solution, and the supernatant is collected after centrifugation;

[0037] purifying the supernatant to obtain the fusion protein;

[0038] Furthermore, the induction time is 16 to 24 hours; and / or,

[0039] The inducing agent is IPTG; and / or,

[0040] The recombinant microbial cell expresses a fusion protein containing PfKRED or an equivalent sequence thereof, and the purification comprises: heating the supernatant to 100° C. to 120° C., obtaining a secondary supernatant after centrifugation, and extracting the fusion protein from the secondary supernatant; or

[0041] The recombinant microbial cell expresses a fusion protein with a histidine tag sequence and containing any one of grpE or its equivalent sequence and SUMO or its equivalent sequence, and the purification comprises: contacting the supernatant with an affinity resin having a specific binding force to the histidine tag sequence of the fusion protein, thereby extracting the fusion protein;

[0042] Furthermore, the affinity resin is Ni-NTA resin.

[0043] In a seventh aspect, the present invention provides a semaglutide intermediate polypeptide Arg 34 The preparation method of GLP-1 (9-37) comprises the following steps:

[0044] The fusion protein provided in the first aspect of the present invention or the fusion protein obtained by the preparation method according to the fifth aspect of the present invention is subjected to enzymatic digestion to obtain the semaglutide intermediate polypeptide Arg 34 GLP-1 (9-37);

[0045] Furthermore, the protease cleavage site sequence of the fusion protein is DDDDK, and enterokinase is used for the cleavage; or, the protease cleavage site sequence of the fusion protein is KR, and Kex2 protease is used for the cleavage; or, the protease cleavage site sequence of the fusion protein is IEGR or IDGR, and Factor Xa protease is used for the cleavage.

[0046] Furthermore, the preparation method further comprises: a step of purifying the fusion protein before performing the enzyme cleavage;

[0047] Under the condition that the fusion protein contains the PfKRED amino acid sequence as shown in SEQ ID NO:6, the purification comprises: heating the solution containing the fusion protein at 100° C. to 120° C. to separate impurity proteins, thereby obtaining a purified fusion protein;

[0048] Under the condition that the fusion protein contains a histidine tag sequence, the purification includes: contacting an affinity resin having specific binding force to the histidine tag sequence with a solution containing the fusion protein, thereby extracting the purified fusion protein.

[0049] In an eighth aspect, the present invention provides a fusion protein as described in the first aspect of the present invention, or an isolated nucleic acid molecule as described in the second aspect of the present invention, or a recombinant expression vector as described in the third aspect of the present invention, or a recombinant microbial cell as described in the fourth aspect of the present invention in the preparation of a semaglutide intermediate polypeptide Arg 34 Application in GLP-1(9-37).

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1PfKRED-GGGSG-DDDDK-Arg 34 Electrophoresis of the whole bacterial suspension and supernatant of GLP-1(9-37) expression strain; 34 The whole cell suspension and supernatant of wet cells of GLP-1(9-37) (theoretical molecular weight: 37.9 kDa) were subjected to SDS-PAGE detection: Lane 1, PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) whole cell suspension; Lane 2, PfKRED-GGGSG-DDDDK-Arg 34 Supernatant of GLP-1(9-37) expression strain; Lane M, protein marker.

[0052] Figure 2 PfKRED-GGGSG-DDDDK-Arg 34 GLP-1 (9-37) protease cleavage electrophoresis; enterokinase was used to cleave the purified PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) was digested with enzymes and then detected by SDS-PAGE: Lane 1, purified PfKRED-GGGSG-DDDDK-Arg without enzyme digestion 34 GLP-1(9-37) fusion protein; Lane 2, purified PfKRED-GGGSG-DDDDK-Arg after 8h of restriction digestion 34 GLP-1(9-37) fusion protein; Lane 3, purified PfKRED-GGGSG-DDDDK-Arg without enzyme digestion 34 GLP-1(9-37) fusion protein and purified PfKRED-GGGSG-DDDDK-Arg after 8h of restriction digestion 34 GLP-1(9-37) fusion protein was co-injected; Lane M, protein marker.

[0053] Figure 3 grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 Electrophoresis of the whole bacterial suspension and supernatant of GLP-1(9-37) expression strain; Electrophoresis of the grpE-GGGSG-DDDDK-Arg 34 The whole cell suspension and supernatant of wet cells of GLP-1(9-37) (theoretical molecular weight: 28.0 kDa) were subjected to SDS-PAGE detection: Lane 1, grpE-GGGSG-DDDDK-Arg with 6xHis tag 34GLP-1(9-37) whole cell suspension; Lane 2, grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 Supernatant of GLP-1(9-37) expression strain; M, protein marker.

[0054] Figure 4 SUMO-Arg with 6xHis tag 34 Electrophoresis of the whole bacterial suspension and supernatant of GLP-1(9-37) expression strain; 34 SDS-PAGE analysis of the whole cell suspension and supernatant of wet cells of GLP-1(9-37) (theoretical molecular weight: 16.4 kDa): Lane 1, SUMO-Arg with 6xHis tag 34 GLP-1(9-37) whole cell suspension; Lane 2, SUMO-Arg with 6xHis tag 34 Supernatant of GLP-1(9-37) expression strain; M, protein marker.

[0055] Figure 5 Arg 34 GLP-1(9-37)HPLC detection spectrum; among them, Arg 34 GLP-1(9-37) retention time: 7.679min.

[0056] Figure 6 Arg 34 GLP-1(9-37) LC-MS detection spectrum (with H + ); Among them, mass spectrum peaks 795 and 1059 are GLP-1 (9-37) (molecular weight: 3175.5Da) plus 4 H + , 3 H + The corresponding results.

[0057] Figure 7 Arg 34 GLP-1(9-37) LC-MS detection spectrum (reduced H + ). Among them, mass spectrum peaks 1057 and 1587 are respectively GLP-1 (9-37) (molecular weight: 3175.5) losing 3 H + , 2 H + The corresponding results. DETAILED DESCRIPTION

[0058] The present inventors have conducted extensive and in-depth research, and through a large number of screening and testing, provided a fusion protein and a semaglutide intermediate polypeptide Arg 34Preparation method of GLP-1 (9-37). The fusion protein of the present invention uses a specific leader peptide sequence (PfKRED, grpE or SUMO) to promote the semaglutide intermediate polypeptide sequence (Arg 34 GLP-1 (9-37)) is correctly folded, thereby promoting the soluble expression of the fusion protein in the fermentation broth and successfully avoiding the formation of inclusion bodies. Specifically, the embodiment of the present invention first designs a nucleotide sequence capable of expressing the above-mentioned fusion protein, and uses an Escherichia coli expression system with a clear genetic background, easy cultivation, and a short fermentation cycle to construct a recombinant strain. Then, the recombinant strain is used to efficiently express a fusion protein that is soluble in the fermentation broth of the recombinant bacteria. The fusion protein is then enzymatically cleaved to obtain the semaglutide intermediate polypeptide Arg 34 GLP-1 (9-37). Since the fusion protein has good solubility, the separation and purification process is simple, so the yield of the semaglutide intermediate polypeptide produced by the preparation method of the semaglutide intermediate polypeptide of the embodiment of the present invention is very high, and the semaglutide intermediate polypeptide with high purity can also be obtained. On this basis, the present invention is completed.

[0059] the term

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0062] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0063] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0064] Leader peptide sequence

[0065] PfKRED is derived from the archaeon Pyrococcus furiosus, and its NCBI sequence number is WP_011013102.1. The inventors found that the sequence can promote the correct folding of the semaglutide intermediate polypeptide in the solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth, and making the fusion protein have good heat resistance and easy to be thermally separated and purified.

[0066] grpE is derived from Escherichia, and its NCBI sequence number is WP_001393454.1. The inventors found that the sequence can promote the correct folding of the semaglutide intermediate polypeptide in the solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth.

[0067] SUMO is derived from yeast Saccharomyces cerevisiae S288C, and its NCBI sequence number is NP_010798.1. The inventors found that the sequence can promote the correct folding of the semaglutide intermediate polypeptide in the solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth.

[0068] Experiments have shown that the above three proteins can all serve as the leading peptide sequences of the semaglutide intermediate polypeptide.

[0069] In the present invention, the leader peptide sequence is intended to include the PfKRED amino acid sequence as shown in SEQ ID NO:6 or an equivalent sequence having at least 98% identity with the PfKRED amino acid sequence, the grpE amino acid sequence as shown in SEQ ID NO:15 or an equivalent sequence having at least 98% identity with the grpE amino acid sequence, and the SUMO amino acid sequence as shown in SEQ ID NO:19 or an equivalent sequence having at least 98% identity with the SUMO amino acid sequence. Preferably, the equivalent sequence has at least 99% identity.

[0070] As used in the present invention, the term "identity" refers to the percentage of the number of identical nucleotides or amino acids in corresponding positions of two or more sequences. For amino acid sequences, the method for calculating identity is: first determine the amino acid sequence used as a comparison benchmark, and then compare the sequence consistency of another amino acid sequence with the amino acid sequence used as a comparison benchmark. Identity = the number of identical amino acids in the two amino acid sequences after sequence alignment / the total number of amino acids in the amino acid sequence used as a comparison benchmark × 100%.

[0071] As used in the present invention, the term "equivalent sequence" means that two sequences are functionally equivalent. For example, in the present invention, the equivalent sequences all have the function of promoting the correct folding of the semaglutide intermediate polypeptide in the solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth.

[0072] For example, the equivalent sequence of PfKRED differs from the amino acid sequence of PfKRED by at least L119A, and in addition thereto, may also differ by L156A and / or T93L.

[0073] The equivalent sequence of grpE differs from the grpE amino acid sequence in at least L105A, and in addition thereto, may also differ in P114F.

[0074] The equivalent sequence of SUMO differs from the SUMO amino acid sequence by at least L45A and may also differ by E59F.

[0075] The experimental results show that the equivalent sequence of PfKRED can achieve an effect similar to or equivalent to the amino acid sequence of PfKRED. The equivalent sequence of grpE can achieve an effect similar to or equivalent to the amino acid sequence of grpE. The equivalent sequence of SUMO can achieve an effect similar to or equivalent to the amino acid sequence of SUMO.

[0076] Fusion Protein

[0077] The present invention provides a fusion protein comprising:

[0078] A leader peptide sequence and a semaglutide intermediate polypeptide sequence, wherein the leader peptide sequence and the semaglutide intermediate polypeptide sequence are directly connected, or are indirectly connected through a linker sequence and / or a protease cleavage site sequence; wherein the semaglutide intermediate polypeptide sequence is Arg as shown in SEQ ID NO: 2 34 GLP-1 (9-37) amino acid sequence.

[0079] In the present invention, the leader peptide sequence is always located at the N-terminus of the semaglutide intermediate polypeptide sequence.

[0080] Furthermore, the fusion protein may also include: an affinity adsorption tag sequence located at the N-terminus of the leader peptide sequence, such as (but not limited to) a histidine tag sequence (also known as 6xHis tag, HHHHHH). The histidine tag sequence, also known as the 6xHis tag, is an amino acid sequence formed by six consecutive histidines (His). The histidine tag sequence can bind to the Ni in the Ni-NTA resin. 2+ Chelating, thereby specifically adsorbing on the Ni-NTA resin, thereby enabling the Ni-NTA resin to specifically adsorb the fusion protein containing the histidine tag sequence. Afterwards, the purified fusion protein can be obtained by elution.

[0081] In another preferred embodiment, the fusion protein is selected from the following group:

[0082] PfKRED or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1 (9-37);

[0083] grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1(9-37); and

[0084] SUMO or its equivalent sequence-Arg 34 GLP-1(9-37).

[0085] More preferably, the fusion protein is selected from the group consisting of:

[0086] PfKRED or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1 (9-37);

[0087] 6xHis tag-grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg 34 GLP-1(9-37); and

[0088] 6xHis tag-SUMO or its equivalent sequence-Arg 34 GLP-1(9-37).

[0089] Preferably, the fusion protein has an amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:17, as shown in SEQ ID NO:21, as shown in SEQ ID NO:25 or as shown in SEQ ID NO:27.

[0090] Nucleic acid molecules, recombinant expression vectors, recombinant microbial cells

[0091] The present invention also provides a nucleic acid molecule encoding the above fusion protein, especially a DNA molecule. It is understood by those skilled in the art that it is easy to obtain the sequence of the nucleic acid molecule according to the fusion protein provided by the present invention.

[0092] Further, based on the above nucleic acid molecules, the present invention also provides a recombinant expression vector comprising the nucleic acid molecule sequence. Typically, the nucleic acid molecule can be integrated into an expression vector commonly used in the art, such as pET series plasmids, Duet series plasmids, pGEX series plasmids, pHY300 plasmids, pHY300PLK plasmids, pPIC3K plasmids, pPIC9K plasmids or pTrc series plasmids.

[0093] Furthermore, the expression vector can be introduced into cells for expression. Commonly used microbial cells for expression include (but are not limited to): Escherichia coli cells, such as Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12, etc.

[0094] Preparation method of fusion protein

[0095] The present invention also provides a method for preparing the fusion protein, comprising the steps of: fermenting and culturing the above-mentioned recombinant microbial cells to produce the fusion protein of the present invention.

[0096] In the present invention, there is no special requirement for the fermentation culture, and the culture can be carried out using a method commonly used in the art according to the selected microbial cell type. A typical culture method includes: inoculating the seed liquid of the recombinant microbial cell into TB culture medium, culturing at 35-40°C, adding an inducer (such as IPTG) when the OD600 value of the culture liquid reaches 0.6-0.8, inducing the expression of the fusion protein at 25-27°C, and obtaining wet cells expressing the fusion protein after the induction.

[0097] Since the fusion protein of the present invention has good water solubility, the wet cells can be further homogenized and broken in a buffer solution, and the supernatant can be collected after centrifugation.

[0098] The supernatant is then purified to obtain the fusion protein. In particular, when the recombinant microbial cell expresses a fusion protein containing PfKRED or an equivalent sequence thereof, the purification may include: heating the supernatant to above 100° C. (e.g., 100-120° C.), centrifuging to obtain a secondary supernatant, and extracting the fusion protein from the secondary supernatant.

[0099] Alternatively, when the recombinant microbial cell expresses a fusion protein containing grpE or its equivalent sequence, SUMO or its equivalent sequence, an affinity adsorption tag sequence (such as a histidine tag sequence) can be designed at the N-terminus of the leader peptide sequence, and an affinity resin having a specific binding force with the affinity adsorption tag sequence of the fusion protein is contacted with the supernatant to extract the fusion protein. Those skilled in the art can select a resin having a specific binding force with the affinity adsorption tag sequence according to the affinity adsorption tag sequence, typically, such as a Ni-NTA resin with a histidine tag sequence.

[0100] Semaglutide intermediate peptide Arg 34 Preparation method of GLP-1 (9-37)

[0101] Furthermore, the other parts of the fusion protein can be removed by enzyme digestion to obtain the intermediate polypeptide Arg 34 GLP-1(9-37).

[0102] According to the weight of the protease cleavage site in the fusion protein, the corresponding protease can be selected to remove the polypeptide. In the present invention, there is no special requirement for the type of protease cleavage site, and the cleavage sites commonly used in the art can be selected, such as DDDDK, using enterokinase for the cleavage; or KR, using Kex2 protease for the cleavage; or IEGR or IDGR, using Factor Xa protease for the cleavage. In particular, when SUMO or its equivalent sequence is used as the leader peptide, there is no need to insert another cleavage site, and SUMO protease can be used directly for cleavage.

[0103] The main advantages of the present invention include:

[0104] The fusion protein of the present invention contains a leader peptide sequence, which helps the fusion protein to fold correctly after being expressed by the recombinant strain, so that it has good solubility in the fermentation broth of the recombinant strain or the supernatant after the fermentation broth is centrifuged, and is easy to extract and purify from the fermentation broth, especially from the supernatant after the centrifugation. Compared with the fusion protein that forms inclusion bodies in the fermentation broth due to incorrect folding, the fusion protein of the present invention does not need to go through the steps of denaturation and renaturation of the inclusion bodies during purification, which simplifies the purification steps, saves the cost of purification reagents (such as denaturation reagents, renaturation reagents, etc.), and improves the yield (up to 91.2%) and purity (up to 96.7%) of the finally obtained semaglutide intermediate polypeptide.

[0105] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0106] The detection methods of various embodiments of the present invention are as follows:

[0107] Arg 34 HPLC detection method of GLP-1(9-37):

[0108] Chromatographic column: Waters XBridge C8 (5 μm, 250×4.6 mm); mobile phase: 0.1% formic acid in water as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B. Gradient elution was performed according to Table 1; column temperature: 35°C; flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: 210 nm.

[0109] Table 1 HPLC gradient elution parameters

[0110]

[0111] Arg 34 The MS detection conditions of GLP-1(9-37) are as follows: mass spectrometer: Shimadzu LC-MS 2020, ion source: electrospray ionization (ESI).

[0112] The present invention uses the following reagents:

[0113] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0114] TB liquid medium: 2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17mM KH 2 PO 4 , 0.4% glycerol.

[0115] Example 1 Expression of PfKRED-GGGSG-DDDDK-Arg 34 Construction of recombinant engineering strain of GLP-1(9-37) fusion protein and inducible expression of fusion protein

[0116] This example constructs a gene capable of expressing PfKRED-GGGSG-DDDDK-Arg 34 The recombinant engineering strain of GLP-1 (9-37) fusion protein comprises the following steps:

[0117] 1.1 Design of PfKRED-GGGSG-DDDDK-Arg 34The nucleotide sequence of GLP-1 (9-37) fusion protein. Among them, the nucleotide sequence of PfKRED is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. The nucleotide sequence of the linker sequence GGGSG is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:11. The nucleotide sequence of the restriction site DDDDK is shown in SEQ ID NO:4, and the amino acid sequence is DDDDK. D represents aspartic acid (Asp), and K represents lysine (Lys). Arg 34 The nucleotide sequence of GLP-1 (9-37) is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence of the above fusion protein was submitted to Shanghai Sangon Biotechnology Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai) for synthesis and cloned into the pET28a vector. The restriction sites used were NdeⅠ and HindⅢ to obtain a recombinant plasmid containing the nucleotide sequence encoding the fusion protein. Arg in the fusion protein 34 Arg 34 The 34th amino acid of GLP-1(9-37) is arginine.

[0118] 1.2 The constructed recombinant plasmid was transformed into the expression host E.coli BL21 (DE3), spread on the LB resistance plate containing 50μg / mL kanamycin, and inverted at 37℃ for 16h. Pick a single colony and inoculate it into 5mL LB liquid culture medium containing 50μg / mL kanamycin, and culture it at 37℃ and 220rpm. When the OD600 is cultured to 0.6-0.8, the seed liquid is obtained. Then the seed liquid is inoculated into TB liquid culture medium containing 50μg / mL kanamycin at an inoculum of 1%, and cultured at 37℃ and 220rpm. When OD600 reaches 0.6, add the inducer IPTG with a final concentration of 0.1mM, and induce at 25℃ and 220rpm for 16h. Then centrifuge at 4000rpm for 20min, discard the supernatant, collect the wet bacteria, and store it in a -20℃ refrigerator for standby use. The expression of PfKRED-GGGSG-DDDDK-Arg 34 Wet cells of GLP-1(9-37) fusion protein.

[0119] Example 2 PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) fusion protein electrophoresis detection

[0120] Take 10g to express PfKRED-GGGSG-DDDDK-Arg 34Wet bacteria of GLP-1 (9-37) fusion protein were added to 50 mL of 50 mM Tris-HCl solution at pH 8.0 (the ratio of the mass of wet bacteria to the volume of Tris-HCl solution was 1 g: 5 mL) and homogenized (homogenization temperature 0-4 °C) to obtain whole bacteria solution. The whole bacteria solution was centrifuged at 10000 rpm for 10 min to obtain supernatant and precipitate. The whole bacteria solution and supernatant were tested by SDS-PAGE, and the test results were as follows: Figure 1 As shown. Figure 1 It can be seen that PfKRED-GGGSG-DDDDK-Arg 34 The band position of GLP-1(9-37) fusion protein showed that its molecular weight was consistent with the expected molecular weight (37.9 kDa), and PfKRED-GGGSG-DDDDK-Arg 34 The expression levels of GLP-1(9-37) fusion protein in the whole bacterial solution and the supernatant were basically the same, indicating that the fusion protein was basically expressed in the form of soluble protein and could be dissolved in the supernatant.

[0121] Example 3 PfKRED-GGGSG-DDDDK-Arg 34 Purification of GLP-1(9-37) fusion protein

[0122] Because PfKRED is a thermophilic protein and will not undergo thermal denaturation and precipitation at a heating temperature of 100°C, the fusion protein can be purified by heating purification. The specific steps are as follows: Weigh 10 g of the protein expressing PfKRED-GGGSG-DDDDK-Arg 34 Wet cells of GLP-1 (9-37) fusion protein were added to 50 mL of 50 mM Tris-HCl solution at pH 8.0 (the ratio of the mass of wet cells to the volume of Tris-HCl solution was 1 g: 5 mL) for homogenization (homogenization temperature 0-4 °C), and centrifuged at 10,000 rpm for 10 min (the purpose was to remove cell fragments such as organelles and cell membranes). The supernatant (the fusion protein was dissolved in the supernatant) was taken and heated at 100 °C for 20 min (the purpose of heating was to make the impurities undergo heat denaturation and precipitate from the supernatant, while the fusion protein would not undergo heat denaturation during heating and would still be dissolved in the supernatant). Centrifuged again at 10,000 rpm for 10 min (the purpose was to remove the impurities after denaturation), and the precipitate was discarded to obtain a secondary supernatant. The secondary supernatant was dialyzed using a dialysis bag (used to remove substances with a molecular weight less than 14 kDa through the dialysis bag, United Carbon, Product No.: MD34-14), and the dialysis fluid was a 50 mM Tris-HCl solution with a pH of 8.0. After dialysis, the liquid remaining in the dialysis bag was centrifuged again and the supernatant was taken again. The supernatant contained the purified PfKRED-GGGSG-DDDDK-Arg34 GLP-1 (9-37) fusion protein. The concentration of the fusion protein was measured by Bradford method, and a total of 50.2 mL of PfKRED-GGGSG-DDDDK-Arg with a concentration of 12.1 mg / mL was obtained. 34 GLP-1 (9-37) fusion protein solution. See Table 2 for details.

[0123] The purified PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) fusion protein was tested by SDS-PAGE. The results are shown in Figure 2 Lane 1.

[0124] Example 4 Obtaining Enterokinase

[0125] Example 3 obtained the purified PfKRED-GGGSG-DDDDK-Arg 34 GLP-1 (9-37) fusion protein. Enterokinase needs to be used to perform enzymatic cleavage with DDDDK as the cleavage site to obtain the intermediate peptide Arg 34 GLP-1 (9-37). Only then can semaglutide be produced based on the semaglutide intermediate polypeptide. Therefore, this embodiment provides a method for preparing PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) fusion protein is specifically cleaved by enterokinase.

[0126] 4.1 Construction and expression of enterokinase expression strain

[0127] Enterokinase is obtained by the following method. The nucleotide sequence of enterokinase is designed, and its nucleotide sequence is shown in SEQ ID NO:9, and its amino acid sequence is shown in SEQ ID NO:10. The nucleotide sequence of enterokinase is synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai), and a recombinant plasmid containing the nucleotide sequence of enterokinase is constructed. The constructed recombinant plasmid is transferred into the expression host E.coli BL21 (DE3), coated on an LB resistance plate containing 50 μg / mL of kanamycin, and inverted at 37°C for 16h. After that, a single colony is picked and inoculated into 5mL LB liquid culture medium containing 50μg / mL kanamycin, and cultured at 37°C and 220rpm. When OD600 reaches 0.6-0.8, a seed solution is obtained, and then the seed solution is inoculated into a TB liquid culture medium containing 50μg / mL kanamycin at an inoculum of 1%, and cultured at 37°C and 220rpm. When OD600 reached 0.6, IPTG was added at a final concentration of 0.1 mM, and the mixture was induced at 25°C and 220 rpm for 16 h. The mixture was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, the wet cells were collected, and stored in a -20°C refrigerator for later use.

[0128] 4.2 Denaturation, renaturation and purification of enterokinase

[0129] Weigh 3g of wet enterokinase cells, homogenize with 50mM Tris-HCl at pH8.0 at 1:10 (g / mL), centrifuge at 10000rpm for 10min, discard the supernatant, and keep the precipitate (enterokinase is expressed in the precipitate in the form of inclusion bodies). Wash the precipitate twice with 50mMTris-HCl at pH8.0, centrifuge after washing, discard the supernatant, and keep the precipitate. Add 20mL of denaturing solution containing 50mM Tris-HCl, 20mM dithiothreitol (DTT), 8M urea and pH 8.0 to the precipitate, mix well, and incubate at 4℃ with shaking for 3h to denature the enterokinase, so that the precipitate is dissolved in the denaturing solution. Take the incubation solution, add 500mL of a refolding solution containing 50mM Tris-HCl, 1mM oxidized glutathione, 3mM reduced glutathione, 1M urea and pH 8.0, and incubate at 20°C and 100rpm for 24h to allow the denatured enterokinase to be refolded and dissolved in the refolding solution after correct folding, so that it can subsequently act specifically and efficiently on the restriction site DDDDK. Then take the incubated enzyme solution, purify and concentrate it using an anion exchange chromatography column (Q column, Boglon, catalog number: EI304203), the equilibrium solution is 50mM Tris-HCl at pH 8.0, and the eluent is a solution containing 50mMTris-HCl, 0.5M NaCl and pH 8.0. The purified and concentrated solution is dialyzed with a dialysis bag (molecular weight cutoff 7kDa, Union Carbon, catalog number: MD55-7), and the dialysate is a 50mMTris-HCl solution at pH 8.0. After dialysis, 16.6 mL of enterokinase enzyme solution was obtained. The enzyme activity of enterokinase was measured and found to be 529.3 U / mL.

[0130] Definition of enterokinase activity: Under the conditions of 25°C, 50mM Tris-HCl, pH 8.0, it can completely digest 1mg PfKRED-GGGSG-DDDDK-Arg in 8h 34 The amount of enzyme required for GLP-1(9-37) is one enzyme activity unit (U).

[0131] Example 5 Enterokinase cleavage of PfKRE-GGGSG-DDDDDK-Arg 34 GLP-1(9-37) obtains Arg 34 GLP-1(9-37)

[0132] Take 50.2mL of purified PfKRED-GGGSG-DDDDK-Arg 34 608 U of enterokinase was added to the GLP-1 (9-37) fusion protein solution (12.1 mg / ml) and the enzyme was cleaved at 25°C and 150 rpm for 8 h to obtain the protein containing Arg 34GLP-1 (9-37) protein solution. DDDDDK is the cleavage site of enterokinase, so the fusion protein can produce Arg after being cleaved by enterokinase. 34 GLP-1(9-37) and PfKRED-GGGSG-DDDDK protein (theoretical molecular weight is 34.7kDa).

[0133] The protein solution was subjected to SDS-PAGE electrophoresis detection, and the results are shown in Figure 2 . Figure 2 Lane 2 is PfKRED-GGGSG-DDDDK protein, and the band size is consistent with the theoretical value, proving that PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37) fusion protein was correctly cleaved to obtain Arg 34 GLP-1 (9-37) protein (molecular weight 3175.5Da), indicating that the enterokinase constructed in the present application example can specifically act on the restriction site DDDDDK, and the Arg 34 GLP-1(9-37) can bind to PfKRED-GGGSG-DDDDK-Arg before enzyme cleavage. 34 GLP-1(9-37) was well differentiated.

[0134] Example 6 Arg 34 Isolation and purification of GLP-1(9-37)

[0135] The Arg obtained after enzyme cleavage in Example 5 34 The GLP-1 (9-37) solution was lyophilized to obtain a lyophilized powder. The lyophilized powder was reconstituted with 10 mL of ultrapure water. The purpose of this lyophilization was to reduce the Arg 34 The total volume of GLP-1(9-37) solution was used to obtain Arg 34 GLP-1 (9-37) concentrate was prepared to facilitate the subsequent separation and purification process. Then, gel filtration chromatography (Focurose 75PG, Huiyan Bio, HN120209500M) was used with 50 mM Tris-HCl at pH 8.0 as the mobile phase to react with Arg 34 GLP-1(9-37) concentrate was separated and purified. 34 The GLP-1 (9-37) component was concentrated again using a freeze dryer (the concentrated volume was 5 ml-10 ml), and then dialyzed using a dialysis bag (molecular weight cutoff 1 kDa, Spectrum Medical, item number: 131060) to remove small molecule salts. The dialysate was a 50 mM Tris-HCl solution at pH 8.0. 34GLP-1(9-37) solution was tested by HPLC and quantified by external standard method (Arg 34 GLP-1 (9-37) gradient standard solution was used to establish a standard curve for quantification. Arg 34 The amount of GLP-1(9-37) was 46.4 mg. The Arg 34 The molecular weight of GLP-1(9-37) is consistent with the theoretical value (3175.5Da), Arg 34 The purity of GLP-1(9-37) was 96.1% and the yield was 91.2%, as shown in Table 3.

[0136] Arg 34 The calculation method of GLP-1(9-37) purity is as follows: 34 The ratio of GLP-1(9-37) peak area to total peak area was used to calculate Arg 34 Purity of GLP-1(9-37);

[0137] Arg 34 The yield of GLP-1(9-37) was calculated according to the following formula:

[0138] Yield = m Arg34GLP-1(9-37) / [(V 纯化后融合蛋白 ×C 纯化后融合蛋白 )×(MW Arg34GLP-1(9-37) / MW 融合蛋白 )].

[0139] Among them, m Arg34GLP-1(9-37) Arg 34 The mass of GLP-1(9-37) after separation and purification is 46.4 mg in this embodiment.

[0140] V 纯化后融合蛋白 and C 纯化后融合蛋白 Respectively represent the concentration and volume of the fusion protein obtained after purification. In this example, the data are from Example 3, which are 50.2 mL and 12.1 mg / mL, respectively.

[0141] MW Arg34GLP-1(9-37) and MW 融合蛋白 Respectively represent Arg 34 The theoretical molecular weight of GLP-1(9-37) and the theoretical molecular weight of the fusion protein are 3.1755 kDa and 37.9 kDa, respectively, in this embodiment.

[0142] The calculation method of purity and yield below is similar.

[0143] Example 7 Kex2 protease cleavage of PfKRED-GGGSG-KR-Arg34 GLP-1(9-37) fusion protein obtains Arg 34 GLP-1(9-37)

[0144] 7.1 PfKRED-GGGSG-KR-Arg 34 Obtaining and purifying the GLP-1 (9-37) fusion protein Referring to Example 1-3, 10 g of wet bacteria were taken to obtain 52.9 mL of PfKRED-GGGSG-KR-Arg with a concentration of 11.3 mg / mL. 34 The molecular weight of GLP-1(9-37) fusion protein (37.6 kDa) detected by SDS-PAGE was consistent with the expected molecular weight. The nucleotide sequence of KR was AAAAGA.

[0145] 7.2 Obtaining Kex2 protease

[0146] The construction and expression of Kex2 protease expression strain refer to Example 4.1; the nucleotide sequence of Kex2 protease is shown in SEQ ID NO: 12, and the amino acid sequence is shown in SEQ ID NO: 13. The restriction site of Kex2 protease is KR. K represents lysine (Lys), and R represents arginine (Arg).

[0147] Purification of Kex2 protease: Weigh 3 g of wet bacteria expressing Kex2 protease (with histidine tag sequence), homogenize and disrupt with a solution containing 20 mM Tris-HCl, 150 mM NaCl and pH 8.0 at a ratio of 1:10 (g / mL) (homogenization temperature 0-4°C), centrifuge at 10000 rpm and 4°C for 10 min, collect the supernatant, and purify using Ni-NTA resin, using a solution containing 20 mM Tris-HCl, 150 mM NaCl and pH 8.0 as the balance solution, and using a solution containing 20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole and pH 8.0 as the eluent for gradient elution (the elution can be performed according to the method recommended by the resin manufacturer); dialyze with a dialysis bag (molecular weight cutoff 7 kDa) (to remove imidazole), and the dialysate is a 50 mM Tris-HCl solution at pH 8.0; after dialysis, 7.3 mL Kex2 protease enzyme solution, the enzyme activity of Kex2 protease is 164.7U / mL.

[0148] Kex2 protease activity definition: at 25°C, 50 mM Tris-HCl, 2 mM CaCl 2 , pH 8.0, 1 mg Kex2-GGGSG-KR-Arg can be completely digested in 8 h 34 The amount of enzyme required for GLP-1(9-37) protein is one enzyme activity unit (U).

[0149] 7.3 Arg 34 Acquisition and purification of GLP-1(9-37)

[0150] Take PfKRED-GGGSG-KR-Arg obtained in (1) 34 GLP-1(9-37) fusion protein, added with CaCl at a working concentration of 2 mM 2 and 597.8U Kex2 protease, digested at 25℃, 150rmp for 8h to obtain the Arg 34 GLP-1 (9-37) protein solution. 34 The protein solution of GLP-1 (9-37) was separated and purified (see Example 6 for details). 34 The GLP-1(9-37) solution was tested by HPLC and quantified by external standard method to obtain 45.6 mg Arg 34 GLP-1(9-37), detected by LC-MS, obtained Arg 34 The molecular weight of GLP-1(9-37) is consistent with the theoretical value (3175.5Da); Arg 34 The purity of GLP-1(9-37) was 95.5%, and the yield was 90.3%.

[0151] Example 8 Enterokinase cleavage of 6xHis-tagged grpE-GGGSG-DDDDK-Arg34GLP-1(9-37) fusion protein to obtain Arg 34 GLP-1(9-37)

[0152] 8.1 grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 Construction and expression of recombinant engineering strain of GLP-1(9-37) fusion protein

[0153] Construct grpE-GGGSG-DDDDK-Arg expressing 6xHis tag 34 Recombinant engineering strain of GLP-1 (9-37) fusion protein, inducing expression of recombinant engineering strain to obtain grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 GLP-1 (9-37) fusion protein, the specific steps refer to Example 1. The nucleotide sequence of grpE is shown in SEQ ID NO: 14, and the amino acid sequence is shown in SEQ ID NO: 15. 34The whole bacterial suspension and supernatant of the recombinant engineering strain of GLP-1 (9-37) fusion protein were detected by SDS-PAGE electrophoresis. The results are shown in Figure 3 .from Figure 3 It can be seen that grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 The GLP-1(9-37) fusion protein is basically expressed in the form of soluble protein and can be dissolved in the supernatant.

[0154] 8.2 grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 Purification of GLP-1(9-37) fusion protein

[0155] Weigh 10 g of grpE-GGGSG-DDDDK-Arg expressed with a 6xHis tag (also known as 6xHis tag) 34 The wet cells of GLP-1 (9-37) were homogenized and broken with a solution containing 20mM Tris-HCl, 150mM NaCl and pH 8.0 at a ratio of 1:5 (g / mL) (homogenization temperature 0-4°C), centrifuged at 10000rpm and 4°C for 10min, and the supernatant was collected and purified using Ni-NTA resin. A solution containing 20mM Tris-HCl, 150mM NaCl and pH 8.0 was used as the equilibrium solution, and a solution containing 20mM Tris-HCl, 150mM NaCl 500mM imidazole and pH 8.0 was used as the eluent for gradient elution (the elution can be performed according to the method recommended by the resin manufacturer). Dialysis was performed using a dialysis bag (molecular weight cutoff 7kDa) to remove imidazole, and the dialysate was a 50mM Tris-HCl solution at pH 8.0. After dialysis, 12.4mL of grpE-GGGSG-DDDDK-Arg with a 6xHis tag was obtained. 34 GLP-1 (9-37) fusion protein solution; Bradford method was used to measure grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 The concentration of GLP-1(9-37) fusion protein is 23.6 mg / mL.

[0156] 8.3 Enterokinase digestion of grpE-GGGSG-DDDDK-Arg with 6xHis tag 34 GLP-1(9-37), obtained Arg 34 GLP-1(9-37) and Arg 34 Isolation and purification of GLP-1(9-37)

[0157] (1)Arg 34 Obtaining GLP-1(9-37):

[0158] Take 12.4 mL of grpE-GGGSG-DDDDK-Arg with 6xHis tag obtained in Example 8.2 34 GLP-1 (9-37) protein solution (23.6 mg / mL) was added with 292.6 U enterokinase and the enzyme was cleaved at 25°C and 150 rpm for 8 h to obtain the Arg 34 GLP-1 (9-37) protein solution; the protein solution was subjected to SDS-PAGE detection, and the size of the protein band in the solution after enzyme cleavage was consistent with the theoretical value.

[0159] (2)Arg 34 Isolation and purification of GLP-1(9-37):

[0160] Take Arg obtained in (1) 34 The GLP-1 (9-37) protein solution was separated and purified. The specific operation was referred to Example 6. 34 GLP-1(9-37) solution was tested by HPLC and quantified by external standard method to obtain 29 mg Arg 34 GLP-1(9-37) protein, detected by LC-MS, obtained Arg 34 The molecular weight of GLP-1(9-37) is consistent with the theoretical value (3175.5Da); Arg 34 The purity of GLP-1(9-37) was 95.3%, and the yield was 87.4%.

[0161] Example 9 Kex2 protease cleavage of grpE-GGGSG-KR-Arg with 6xHis tag 34 GLP-1(9-37) obtains Arg 34 GLP-1(9-37), and Arg 34 Isolation and purification of GLP-1(9-37)

[0162] 9.1 Construction of grpE-GGGSG-KR-Arg expressing 6xHis tag 34 The recombinant engineering strain of GLP-1 (9-37) fusion protein is induced to express the recombinant engineering strain to obtain grpE-GGGSG-KR-Arg with a 6xHis tag 34 GLP-1 (9-37) fusion protein, the specific steps refer to Example 1-2; the concentration of the fusion protein obtained from 10g of wet bacteria is 21.8mg / mL, and the volume is 10.7mL.

[0163] 9.2 grpE-GGGSG-KR-Arg with 6xHis tag34 Purification of GLP-1(9-37) fusion protein refers to Example 8.3;

[0164] 9.3Arg 34 Acquisition and purification of GLP-1(9-37)

[0165] Take the purified grpE-GGGSG-KR-Arg with 6xHis tag 34 GLP-1(9-37) fusion protein, added with a working concentration of 2 mM CaCl 2 and 230U Kex2 protease, digested at 25℃, 150rmp for 8h. 34 GLP-1 (9-37) was separated and purified (see Example 6 for details) to obtain 23.4 mg Arg 34 GLP-1(9-37). Arg 34 The molecular weight of GLP-1(9-37) is consistent with the theoretical value (3175.5Da); Arg 34 The purity of GLP-1(9-37) was 94.5%, and the yield was 87.5%.

[0166] Example 10 SUMO protease cleavage of SUMO-Arg with 6xHis tag 34 GLP-1(9-37)

[0167] 10.1 SUMO-Arg with 6xHis tag 34 Construction and expression of recombinant engineering strain of GLP-1(9-37) fusion protein

[0168] Constructing a 6xHis-tagged SUMO-Arg 34 The recombinant engineering strain of GLP-1 (9-37) fusion protein is induced to express the recombinant engineering strain to obtain the expression of SUMO-Arg with 6xHis tag. 34 The wet bacteria of GLP-1 (9-37) fusion protein, refer to Example 1 for the specific steps.

[0169] The 6xHis-tagged SUMO-Arg 34 The whole bacterial suspension and supernatant of the recombinant engineering strain of GLP-1 (9-37) fusion protein were detected by SDS-PAGE electrophoresis. The results are shown in Figure 4 .from Figure 4 It can be seen that SUMO-Arg with 6xHis tag 34 The GLP-1(9-37) fusion protein is basically expressed in the form of soluble protein and can be dissolved in the supernatant.

[0170] The experiments revealed that the SUMO sequence directly binds to Arg 34 The fusion protein is well soluble in the supernatant rather than existing in the form of inclusion bodies, which is convenient for subsequent separation and purification.

[0171] 10.2 SUMO-Arg with 6xHis tag 34 Purification of GLP-1(9-37) fusion protein

[0172] Weigh 10 g of SUMO-Arg expressed with 6xHis tag 34 The wet bacteria of GLP-1 (9-37) fusion protein were homogenized and broken with a solution containing 20mM Tris-HCl, 150mM NaCl and pH 8.0 at a ratio of 1:5 (g / mL) (homogenization temperature 0-4°C), centrifuged at 10000rpm and 4°C for 10min, and the supernatant was collected and purified using Ni-NTA resin. A solution containing 20mMTris-HCl, 150mM NaCl and pH 8.0 was used as the equilibrium solution, and a solution containing 20mM Tris-HCl, 150mM NaCl, 500mM imidazole and pH 8.0 was used as the eluent for gradient elution. Dialysis was performed using a dialysis bag (molecular weight cutoff 7kDa, United Carbon, catalog number: MD55-7) to remove imidazole, and the dialysate was a 50mMTris-HCl solution at pH 8.0. After dialysis, 8.3mL of SUMO-Arg with a 6xHis tag was obtained. 34 The concentration of GLP-1(9-37) fusion protein was measured by Bradford method. 34 The concentration of GLP-1(9-37) fusion protein was 16.9 mg / mL.

[0173] 10.3 Acquisition and purification of SUMO protease

[0174] The steps of obtaining and purifying SUMO protease were as described in Example 7.2. 3 g of wet bacteria expressing SUMO protease were taken to obtain 6.4 mL of SUMO protease enzyme solution with an enzyme activity of 39.6 U / mL.

[0175] SUMO protease activity definition: Under the conditions of 25°C, 50mM Tris-HCl, pH8.0, it can completely cleave 1mg SUMO-Arg in 8h 34 The amount of enzyme required for GLP-1(9-37) is one enzyme activity unit (U).

[0176] 10.4 SUMO protease cleavage of SUMO-Arg with 6xHis tag34 GLP-1(9-37) obtains Arg 34 GLP-1(9-37) and Arg 34 Purification of GLP-1(9-37)

[0177] Take 8.3 mL of SUMO-Arg with 6xHis tag purified in Example 10.2 34 GLP-1 (9-37) (16.9 mg / mL) protein solution was added with 140.3 U SUMO protease and the enzyme was cleaved at 25°C and 150 rpm for 8 h to obtain the protein containing Arg 34 GLP-1 (9-37) digestion solution. The digestion solution was separated and purified (refer to Example 6 for specific steps). The purified Argo 34 GLP-1(9-37) solution was tested by HPLC and quantified by external standard method. 34 The amount of GLP-1(9-37) was 23.5 mg. The Arg 34 The molecular weight of GLP-1(9-37) is consistent with the theoretical value (3175.5Da); Arg 34 The purity of GLP-1(9-37) was 96.7%, and the yield was 86.5%.

[0178] In this example, SUMO protease was used to cut off the SUMO sequence to obtain Arg 34 GLP-1 (9-37). Therefore, the SUMO sequence can be used as a signal peptide to enable the fusion protein to fold correctly, so that it can be dissolved in the fermentation broth or supernatant instead of being located in the inclusion body, which can simplify the subsequent protein purification process. In addition, the SUMO sequence can be directly used as a cleavage site for SUMO protease, which has a strong recognition specificity for the SUMO sequence. Therefore, there is no need to add GGGSG and additional cleavage sequences, which avoids obtaining a fusion protein with a longer sequence, thereby avoiding the situation where the fusion protein is misfolded and exists in the form of inclusion bodies due to the excessive length of the sequence, which is beneficial to the subsequent purification process. The molecular weight of the SUMO sequence is relatively small, and Arg 34 The proportion of GLP-1(9-37) in the entire fusion protein is relatively high.

[0179] Table 2 Parameters of fusion proteins in various embodiments

[0180]

[0181]

[0182] Table 3 Parameters of Semaglutide intermediate polypeptides in various embodiments

[0183]

[0184] The sequence numbers involved in the above embodiments are shown in Table 4.

[0185] Table 4 Sequence numbers involved in various embodiments

[0186] sequence name Example No. Nucleotide sequence Amino acid sequence <![CDATA[Arg 34 GLP-1(9-37)]]> Example 5 SEQ ID NO:1 SEQ ID NO:2 GGGSG Example 1 SEQ ID NO:3 SEQ ID NO:11 DDDDK Example 1 SEQ ID NO:4 SEQ ID NO:28 PfKRED Example 1 SEQ ID NO:5 SEQ ID NO:6 <![CDATA[PfKRED-GGGSG-DDDDK-Arg 34 GLP-1(9-37)]]> Example 3 SEQ ID NO:7 SEQ ID NO:8 Enterokinase Example 4 SEQ ID NO:9 SEQ ID NO:10 KR Example 7 AAAAGA KR Kex2 protease Example 7 SEQ ID NO:12 SEQ ID NO:13 gfrE Example 8 SEQ ID NO:14 SEQ ID NO:15 SUMO Example 10 SEQ ID NO:18 SEQ ID NO:19 <![CDATA[SUMO-Arg 34 GLP-1(9-37)]]> Example 10 SEQ ID NO:20 SEQ ID NO:21 SUMO protease Example 10 SEQ ID NO:22 SEQ ID NO:23 <![CDATA[PfKRED-GGGSG-KR-Arg 34 GLP-1(9-37)]]> Example 7 SEQ ID NO:24 SEQ ID NO:25 <![CDATA[grpE-GGGSG-DDDDK-Arg 34 GLP-1(9-37)]]> Example 8 SEQ ID NO:16 SEQ ID NO:17 <![CDATA[grpE-GGGSG-KR-Arg 34 GLP-1(9-37)]]> Example 9 SEQ ID NO:26 SEQ ID NO:27

[0187] In Table 4, the example number refers to the example number that first appears.

[0188] In addition, during the research process, the inventors tested TrxA, GST, DsbC, etc. as leader peptides, but the fusion proteins prepared by these leader peptides had poor solubility and low expression levels, so they were not adopted.

[0189] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A fusion protein, characterized in that The fusion protein comprises: A leader peptide sequence and a semaglutide intermediate polypeptide sequence, wherein the leader peptide sequence and the semaglutide intermediate polypeptide sequence are directly connected, or indirectly connected via a linker sequence and / or a protease cleavage site sequence; Wherein, the leader peptide sequence is the PfKRED amino acid sequence as shown in SEQ ID NO:6 or an equivalent sequence having at least 98% identity with the PfKRED amino acid sequence, the grpE amino acid sequence as shown in SEQ ID NO:15 or an equivalent sequence having at least 98% identity with the grpE amino acid sequence, the SUMO amino acid sequence as shown in SEQ ID NO:19 or an equivalent sequence having at least 98% identity with the SUMO amino acid sequence; and The semaglutide intermediate polypeptide sequence is Arg as shown in SEQ ID NO: 2 34 GLP-1 (9-37) amino acid sequence.

2. The fusion protein according to claim 1, characterized in that When a linker sequence and a protease cleavage site sequence are present, the leader peptide sequence, the linker sequence, the protease cleavage site sequence and the semaglutide intermediate polypeptide sequence are sequentially connected; Further, the linker sequence is selected from any one of GGGG, GGGGG, GGGGS, GGGSG, EAAAK, AEAAAKALEA and PAPAP: and / or The protease cleavage site sequence is selected from any one of DDDDK, KR, IEGR and IDGR.

3. The fusion protein according to claim 1 or 2, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:17, as shown in SEQ ID NO:21, as shown in SEQ ID NO:25 or as shown in SEQ ID NO:27; Furthermore, the fusion protein further comprises an affinity adsorption tag sequence located at the N-terminus of the leader peptide sequence, such as a histidine tag sequence, preferably comprising at least 6 histidines.

4. An isolated nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 3; Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:16, as shown in SEQ ID NO:20, as shown in SEQ ID NO:24 or as shown in SEQ ID NO:

26.

5. A recombinant expression vector, characterized in that: It contains the isolated nucleic acid molecule as claimed in claim 4; Further, the recombinant expression vector also includes a pET series plasmid, Duet series plasmid, pGEX series plasmid, pHY300 plasmid, pHY300PLK plasmid, pPIC3K plasmid, pPIC9K plasmid or pTrc series plasmid for integration into the isolated nucleic acid molecule; Furthermore, the pET series plasmids include pET-24a(+), pET28a, pET-29a(+), and pET-30a(+); the Duet series plasmids include pRSFDuet-1 and pCDFDuet-1; and the pTrc series plasmids include pTrc99a.

6. A recombinant microbial cell, characterized in that It expresses the fusion protein according to any one of claims 1 to 3, contains the isolated nucleic acid molecule according to claim 4, or contains the recombinant expression vector according to claim 5; Furthermore, the starting strains of the recombinant microbial cells include but are not limited to Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

7. Use of the isolated nucleic acid molecule according to claim 4, the recombinant expression vector according to claim 5, or the recombinant microbial cell according to claim 6 in expressing the fusion protein according to any one of claims 1 to 3.

8. A method for preparing a fusion protein, characterized in that: The method comprises the steps of: fermenting and culturing the recombinant microbial cell according to claim 6, thereby producing the fusion protein according to any one of claims 1 to 3.

9. A Semaglutide intermediate polypeptide Arg 34 The method for preparing GLP-1(9-37) is characterized in that: The steps include: The fusion protein according to any one of claims 1 to 3 is subjected to enzymatic digestion to obtain the semaglutide intermediate polypeptide Arg 34 GLP-1(9-37).

10. The preparation method according to claim 9, characterized in that: The preparation method further comprises: a step of purifying the fusion protein before performing the enzyme cleavage; Further, under the condition that the fusion protein contains the PfKRED amino acid sequence as shown in SEQ ID NO:6, the purification includes: heating the solution containing the fusion protein at 100° C. to 120° C. to separate impurity proteins, thereby obtaining a purified fusion protein; Under the condition that the fusion protein contains a histidine tag sequence, the purification includes: contacting an affinity resin having specific binding force to the histidine tag sequence with a solution containing the fusion protein, thereby extracting the purified fusion protein.

11. The fusion protein according to any one of claims 1 to 3, or the isolated nucleic acid molecule according to claim 4, or the recombinant expression vector according to claim 5, or the recombinant microbial cell according to claim 6 in the preparation of the intermediate polypeptide Arg of semaglutide 34 Application in GLP-1(9-37).

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