CN119462955A - Fusion protein and preparation method of cable Ma Lutai intermediate polypeptide - Google Patents

Abstract

The invention discloses a preparation method of fusion protein and a cable Ma Lutai intermediate polypeptide. The fusion protein comprises a leader peptide sequence and a cable Ma Lutai intermediate polypeptide sequence (Arg ³⁴ GLP-1 (9-37)) linked to the leader peptide sequence. The invention designs a nucleotide sequence capable of expressing the fusion protein, utilizes an expression system of escherichia coli with clear genetic background, easy culture and short fermentation period to construct a recombinant strain, then utilizes the recombinant strain to efficiently express the fusion protein with good solubility in fermentation liquor of the recombinant strain, and then carries out enzyme digestion on the fusion protein sequence to obtain the cable Ma Lutai intermediate polypeptide. The invention has simple separation and purification process of fusion protein, and the production efficiency and purity of the cable Ma Lutai intermediate polypeptide are higher.

Description

Fusion protein and preparation method of cable Ma Lutai intermediate polypeptide

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of fusion protein and a cable Ma Lutai intermediate polypeptide.

Background

Glucagon-like peptide-1 (Glucagon-LIKE PEPTIDE-1, glp-1) is a hormone secreted by islet alpha cells and intestinal neuroendocrine cells (L cells), and has the effects of promoting glucose-dependent insulin secretion, inhibiting glucagon secretion, promoting beta cell regeneration, inhibiting apoptosis, reducing appetite, reducing body weight, and the like. GLP-1 is susceptible to degradation in vivo by cell surface dipeptidyl peptidase (DPP-IV) and has a short half-life, and a number of different methods have been used to modify GLP-1 to provide longer duration of action in vivo, such as substitution of amino acid 8 (Ala) in the active fragment of GLP-1 (7-37) (site of action of DPP-IV enzyme), chemical modification (e.g., acylation, glycosylation), and increase in molecular weight of fusion proteins.

The cable Ma Lutai (Semaglutide) is a GLP-1 analogue, and based on a GLP-1 (7-37) chain, the unnatural amino acid 2-aminoisobutyric acid is adopted to replace alanine at the 8 th position, arginine is adopted to replace lysine at the 34 th position, and the lysine at the 26 th position is connected with an octadecanoic acid fatty chain, so that the compound has higher drug stability. When the cable-marlutide is subcutaneously injected, the absorption effect of the cable-marlutide can be slowed down through the crosslinking effect, the cable-marlutide can be combined with albumin, and the cable-marlutide has relatively stable capability of preventing DPP-IV and other enzymes from degrading the cable-marlutide, so that the cable-marlutide has relatively long half-life in plasma. The rope Ma Lutai has excellent, safe and reliable hypoglycemic effect, and simultaneously has better effects on the aspects of weight reduction, cardiovascular system benefit and the like, and has great market value and academic value.

The current prior art methods of preparing cable Ma Lutai include chemical synthesis methods, which further include solid phase synthesis methods, liquid phase synthesis methods, or solid-liquid phase combination methods. For example, CN109627317A is prepared through solid phase synthesis of side chain protecting peptide, liquid phase segment condensation to obtain full protecting rope Ma Lutai, cleavage to obtain crude rope Ma Lutai peptide, purification and salt exchange to obtain rope Ma Lutai, and the process has several steps, needs several kinds of organic solvents in the synthesis process, and has great consumption, thus being unfavorable for industrial amplification and environment friendship, and having potential amino acid racemate by-product, and bringing certain risk to long-term use of the medicine.

The preparation method of the cable Ma Lutai also includes a biological method, which can prepare the intermediate polypeptide in the form of inclusion bodies in an E.coli host. Since intermediate polypeptides of smaller molecular weight are susceptible to degradation in E.coli hosts, E.coli prokaryotic expression systems are not suitable for direct expression of intermediate polypeptides. In order to enhance the stability of the intermediate polypeptide during expression, it is often necessary to express the intermediate polypeptide in the form of a fusion protein of greater molecular weight to achieve co-expression of the two, thereby reducing the likelihood of degradation. A method for preparing cord Ma Lutai by tandem expression in the form of inclusion bodies is disclosed in CN111378027 a. The method utilizes the whole gene combination technology to integrate the serial expressed cable-marlutide precursor gene and plasmid pET-27B (+) to construct recombinant expression plasmid, converts the recombinant expression plasmid into escherichia coli to perform high-density fermentation and induced expression, and dissolves inclusion bodies after collecting and crushing thalli to obtain inclusion bodies so as to obtain soluble proteins, and then the soluble proteins are subjected to enzyme digestion and purification by Kex2 and carboxypeptidase B to obtain the cable Ma Lutai. The method uses two enzymes, kex2 and carboxypeptidase B, and the enzyme addition cost is high. In addition, patent CN114292338B discloses a method for preparing fusion protein of cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37) or Arg ³⁴ GLP-1 (11-37), the method expresses fusion protein containing cable Ma Lutai intermediate polypeptide in escherichia coli, and the target product is obtained by enzyme digestion after collecting inclusion body for dissolution, but the yield of the target product is only 3.62g/L at most, the process of dissolution denaturation, renaturation purification and the like is required for the inclusion body, the operation is complex, and the cost is high. Based on the technical problems in the prior art, there is an urgent need to provide a cable Ma Lutai intermediate polypeptide or a preparation method thereof, which has higher expression level of fusion protein, simpler and more convenient process steps and is more suitable for industrial production.

Disclosure of Invention

The invention aims to provide a preparation method of a cable Ma Lutai intermediate polypeptide, which has higher protein expression level and simpler and more convenient process steps, or is more suitable for industrial production.

It is another object of the invention to provide a fusion protein for use in the preparation of a cable Ma Lutai intermediate polypeptide.

In a first aspect of the present invention, there is provided a fusion protein comprising:

A leader peptide sequence and a cable Ma Lutai intermediate polypeptide sequence, wherein the leader peptide sequence and the cable Ma Lutai intermediate polypeptide sequence are directly linked or indirectly linked by a linker sequence and/or a protease cleavage site sequence;

Wherein the leader peptide sequence is the PfKRED amino acid sequence shown in SEQ ID NO. 6 or an equivalent sequence having at least 98% identity to the PfKRED amino acid sequence, the grpE amino acid sequence shown in SEQ ID NO.15 or an equivalent sequence having at least 98% identity to the grpE amino acid sequence, the SUMO amino acid sequence shown in SEQ ID NO. 19 or an equivalent sequence having at least 98% identity to the SUMO amino acid sequence, and

The polypeptide sequence of the cable Ma Lutai intermediate is Arg ³⁴ GLP-1 (9-37) amino acid sequence shown in SEQ ID NO. 2.

In another preferred embodiment, when a linker sequence and a protease cleavage site sequence are present, the leader peptide sequence, linker sequence, protease cleavage site sequence and the cable Ma Lutai intermediate polypeptide sequence are sequentially linked.

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

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

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

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

In another preferred embodiment, the fusion protein is selected from the group consisting of:

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

grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg ³⁴ GLP-1 (9-37), and

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

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

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

6XHis tag-grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg ³⁴ GLP-1 (9-37), and

6XHis tag-SUMO or its equivalent-Arg ³⁴ GLP-1 (9-37).

In a second aspect of the invention, there is provided an isolated nucleic acid molecule encoding a fusion protein according to the first aspect of the invention;

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.

In a third aspect of the invention there is provided a recombinant expression vector comprising an isolated nucleic acid molecule according to the second aspect of the invention;

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

Further, the pET series plasmids comprise pET-24a (+), pET28a (+), pET-29a (+), pET-30a (+), the Duet series plasmids comprise pRSFDuet-1 and pCDFDuet-1, and the pTrc series plasmids comprise pTrc99a.

In a fourth aspect of the invention, there is provided a recombinant microbial cell expressing a fusion protein according to the first aspect of the invention, comprising an isolated nucleic acid molecule according to the second aspect of the invention, or comprising a recombinant expression vector according to the third aspect of the invention.

Further, the starting strain of the recombinant microbial cell includes, but is not limited to, E.coli JM109 (DE 3), E.coli HMS174 (DE 3), E.coli BL21 (DE 3), E.coli Rostta (DE 3), E.coli Rosttagami (DE 3), E.coli Rostta (DE 3), E.coli DH 5. Alpha., E.coli W3110 and/or E.coli K12.

In a fifth aspect of the invention there is provided the use of an isolated nucleic acid molecule according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention, or a recombinant microbial cell according to the fourth aspect of the invention, for expression of a fusion protein according to the first aspect of the invention.

In a sixth aspect, the present invention provides a method for producing a fusion protein comprising the steps of fermenting and culturing the recombinant microbial cell according to the fourth aspect of the present invention, thereby producing the fusion protein according to the first aspect of the present invention;

Preferably, the fermentation production comprises:

inoculating the seed solution of the recombinant microbial cells into a TB culture medium, culturing at 35-40 ℃, adding an inducer when the OD600 value of the culture solution reaches 0.6-0.8, and inducing the fusion protein to express at 25-27 ℃ to obtain wet thalli expressing the fusion protein after the induction is finished;

Homogenizing and crushing the wet thalli in a buffer solution, centrifuging and collecting supernatant;

purifying the supernatant to obtain the fusion protein;

further, the induction time is 16-24 hours, and/or,

The inducer is IPTG, and/or,

The recombinant microorganism cell expresses a fusion protein comprising PfKRED or an equivalent thereof, the purification comprising heating the supernatant to 100 ℃ to 120 ℃, centrifuging to obtain a secondary supernatant, extracting the fusion protein from the secondary supernatant, or

The recombinant microbial cell expresses a fusion protein bearing a histidine tag sequence and containing either grpE or an equivalent sequence thereof and SUMO or an equivalent sequence thereof, the purification comprising contacting the supernatant with an affinity resin having specific binding capacity to the histidine tag sequence of the fusion protein, thereby extracting the fusion protein;

still further, the affinity resin is a Ni-NTA resin.

In a seventh aspect, the invention provides a preparation method of a cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37), which comprises the following steps:

Performing enzyme digestion on the fusion protein provided by the first aspect of the invention or the fusion protein obtained by the preparation method according to the fifth aspect of the invention to obtain a cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37);

further, the protease cleavage site sequence of the fusion protein is DDDDK, enterokinase is adopted for the cleavage, or KR is adopted for the cleavage, kex2 protease is adopted for the cleavage, or IEGR or IDGR is adopted for the cleavage, and Factor Xa protease is adopted for the cleavage.

Further, the preparation method further comprises the step of purifying the fusion protein before the enzyme digestion;

Under the condition that the fusion protein contains PfKRED amino acid sequence shown as SEQ ID NO. 6, the purification comprises heating the solution containing the fusion protein at 100-120 ℃ to separate out the hybrid protein, thereby obtaining the purified fusion protein;

Under the condition that the fusion protein contains a histidine tag sequence, the purification comprises the step of contacting the solution containing the fusion protein with an affinity resin with specific binding force with the histidine tag sequence, thereby extracting the purified fusion protein.

In an eighth aspect of the invention there is provided the use of a fusion protein according to the first aspect of the invention, or an isolated nucleic acid molecule according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention, or a recombinant microbial cell according to the fourth aspect of the invention, in the preparation of the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37).

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) expressing strain whole-cell and supernatant electrophoresis, SDS-PAGE detection of whole-cell and supernatant of wet cell expressing PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) (theoretical molecular weight: 37.9 kDa), lane 1, pfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) whole-cell, lane 2, pfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) expressing strain supernatant, lane M, protein Marker.

FIG. 2PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) protein digestion electrophoresis, enterokinase digestion of purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) followed by SDS-PAGE detection of the non-digested purified PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) fusion protein, lane 2, purified PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) fusion protein after 8h digestion, lane 3, co-feeding of the non-digested purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein with the digested purified PfKRED-GGGSG-DDK-Arg ³⁴ -1 (9-37) fusion protein after 8h digestion, and M protein Marker.

FIG. 3 shows electrophoresis patterns of whole cell sap and supernatant of 6 XHis-tagged grpE-GGGSG-DDDDDDK-Arg ³⁴ GLP-1 (9-37) expression strain, SDS-PAGE detection of whole cell sap and supernatant of wet cell expressing 6 XHis-tagged grpE-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) (theoretical molecular weight: 28.0 kDa), lane 1, 6 XHis-tagged grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) whole cell sap, lane 2,6 XHis-tagged grpE-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) expression strain supernatant, M, protein Marker.

FIG. 4 shows electrophoresis patterns of whole bacterial liquid and supernatant of SUMO-Arg ³⁴ GLP-1 (9-37) expression strain with 6XHis tag, SDS-PAGE detection of whole bacterial liquid and supernatant of wet bacterial cells expressing SUMO-Arg ³⁴ GLP-1 (9-37) with 6XHis tag (theoretical molecular weight: 16.4 kDa), lane 1, whole bacterial liquid of SUMO-Arg ³⁴ GLP-1 (9-37) with 6XHis tag, lane 2, supernatant of SUMO-Arg ³⁴ GLP-1 (9-37) expression strain with 6XHis tag, M, protein Marker.

FIG. 5Arg ³⁴ GLP-1 (9-37) HPLC detection profile, wherein Arg ³⁴ GLP-1 (9-37) retention time is 7.679min.

FIG. 6Arg ³⁴ GLP-1 (9-37) LC-MS detection profile (H ⁺) wherein mass spectral peaks 795, 1059 are the corresponding results of GLP-1 (9-37) (molecular weight: 3175.5 Da) plus 4H ⁺, 3H ⁺, respectively.

FIG. 7Arg ³⁴ GLP-1 (9-37) LC-MS detection profile (minus H ⁺). Wherein, the mass spectrum peaks 1057, 1587 are the corresponding results of GLP-1 (9-37) (molecular weight: 3175.5) losing 3H ⁺, 2H ⁺, respectively.

Detailed Description

Through extensive and intensive studies, the present inventors have provided a method for producing a fusion protein and the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37) by a large number of screening and testing. The fusion protein of the invention adopts a specific leader peptide sequence (PfKRED, grpE or SUMO) to promote the correct folding of a cable Ma Lutai intermediate polypeptide sequence (Arg ³⁴ GLP-1 (9-37)) so as to promote the soluble expression of the fusion protein in fermentation liquor and successfully avoid the formation of inclusion bodies. Specifically, the embodiment of the invention firstly designs a nucleotide sequence capable of expressing the fusion protein, and constructs a recombinant strain by using an escherichia coli expression system with clear genetic background, easy culture and short fermentation period. And then the recombinant strain is utilized to efficiently express the fusion protein with good solubility in fermentation liquor of recombinant bacteria. The fusion protein is then digested to obtain the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37). Because the fusion protein has good solubility, the separation and purification process is simple, so that the yield of the cable Ma Lutai intermediate polypeptide produced by adopting the preparation method of the cable Ma Lutai intermediate polypeptide is high, and the cable Ma Lutai intermediate polypeptide with higher purity can be obtained. The present invention has been completed on the basis of this finding.

Terminology

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.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of, or" consisting of.

As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40 ℃, preferably 25±5 ℃.

Leader peptide sequences

PfKRED is derived from archaebacteria Pyrococcus furiosus, NCBI accession number WP_011013102.1. The inventor discovers that the sequence can play a role in promoting the correct folding of the cable Ma Lutai intermediate polypeptide in a solution, so that the soluble expression of the fusion protein in fermentation liquor is promoted, and the fusion protein has good heat resistance and is easy to carry out thermal separation and purification.

The grpE is derived from Escherichia and has NCBI sequence number WP_001393454.1. The inventors have found that this sequence can act to promote the correct folding of the cable Ma Lutai intermediate polypeptide in solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth.

SUMO is derived from the yeast Saccharomyces CEREVISIAE S288C, which NCBI accession number NP-010798.1. The inventors have found that this sequence can act to promote the correct folding of the cable Ma Lutai intermediate polypeptide in solution, thereby promoting the soluble expression of the fusion protein in the fermentation broth.

Experiments prove that all three proteins can be used as leader peptide sequences of the cable Ma Lutai intermediate polypeptides.

In the present invention, the leader peptide sequence is intended to include the PfKRED amino acid sequence shown as SEQ ID NO. 6 or an equivalent sequence having at least 98% identity to the PfKRED amino acid sequence, the grpE amino acid sequence shown as SEQ ID NO. 15 or an equivalent sequence having at least 98% identity to the grpE amino acid sequence, the SUMO amino acid sequence shown as SEQ ID NO.19 or an equivalent sequence having at least 98% identity to the SUMO amino acid sequence. Preferably, the equivalent sequences have at least 99% identity.

As used herein, the term "identity" refers to the percentage of the number of nucleotides or amino acids that two or more sequences have identical at corresponding positions. The method for calculating the identity of the amino acid sequences comprises the steps of determining the amino acid sequence serving as a comparison standard, and comparing the sequence identity of another amino acid sequence with the amino acid sequence serving as the comparison standard. Identity = the number of identical amino acids after sequence alignment of two amino acid sequences/total number of amino acids in the amino acid sequence as reference for comparison x 100%.

As used herein, the term "equivalent sequence" means that both sequences are functionally equivalent, e.g., in the present invention, the equivalent sequences both have the effect of promoting proper folding of the cable Ma Lutai intermediate polypeptide in solution, thereby promoting soluble expression of the fusion protein in fermentation broth.

For example, the equivalent sequence PfKRED may differ by at least L119A relative to the PfKRED amino acid sequence, and in addition, there may be differences in L156A and/or T93L.

The equivalent sequence of grpE may differ from the amino acid sequence of grpE by at least L105A, but may also differ from the amino acid sequence of grpE by P114F.

In addition to the difference in equivalent sequence of SUMO relative to the SUMO amino acid sequence of at least L45A, there may be a difference in E59F.

The experimental result proves that the equivalent sequence PfKRED can obtain the effect similar to or equivalent to the PfKRED amino acid sequence. The equivalent sequence of grpE can obtain 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.

Fusion proteins

The present invention provides a fusion protein comprising:

The leader peptide sequence and the cable Ma Lutai intermediate polypeptide sequence are directly connected or indirectly connected through a linker sequence and/or a protease cleavage site sequence, wherein the cable Ma Lutai intermediate polypeptide sequence is an Arg ³⁴ GLP-1 (9-37) amino acid sequence shown as SEQ ID NO. 2.

In the present invention, the leader peptide sequence is always located at the N-terminus of the cable Ma Lutai intermediate polypeptide sequence.

Further, 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, HHHHH). The histidine tag sequence, also known as a 6xHis tag, is an amino acid sequence formed of six consecutive histidines (His). The histidine tag sequence can be chelated with Ni ²⁺ in the Ni-NTA resin, so that the histidine tag sequence is specifically adsorbed on the Ni-NTA resin, and the specific adsorption of the fusion protein containing the histidine tag sequence by the Ni-NTA resin can be realized. And eluting to obtain purified fusion protein.

In another preferred embodiment, the fusion protein is selected from the group consisting of:

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

grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg ³⁴ GLP-1 (9-37), and

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

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

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

6XHis tag-grpE or its equivalent sequence-GGGSG-DDDDK or KR-Arg ³⁴ GLP-1 (9-37), and

6XHis tag-SUMO or its equivalent-Arg ³⁴ GLP-1 (9-37).

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

Nucleic acid molecules, recombinant expression vectors, recombinant microbial cells

The invention also provides nucleic acid molecules, in particular DNA molecules, encoding the fusion proteins. It will be appreciated by those skilled in the art that it is easy to obtain the sequence of the nucleic acid molecule according to the fusion proteins provided herein.

Further, based on the above nucleic acid molecules, the present invention also provides recombinant expression vectors comprising the nucleic acid molecule sequences. Typically, the nucleic acid molecule may be incorporated into expression vectors commonly used in the art, such as pET series plasmids, duet series plasmids, pGEX series plasmids, pHY300PLK plasmids, pPIC3K plasmids, pPIC9K plasmids or pTrc series plasmids, and the like.

Further, the expression vector may be introduced into cells for expression, and commonly used microbial cells for expression include, but are not limited to, E.coli cells such as E.coli JM109 (DE 3), E.coli HMS174 (DE 3), E.coli BL21 (DE 3), E.coli Rostta (DE 3), E.coli Rosttagami (DE 3), E.coli Rostta (DE 3), E.coli DH 5. Alpha., E.coli W3110 and/or E.coli K12, etc.

Fusion protein preparation method

The invention also provides a preparation method of the fusion protein, which comprises the step of fermenting and culturing the recombinant microorganism cells so as to produce the fusion protein.

In the present invention, the fermentation culture is not particularly limited, and the culture may be carried out by a method commonly used in the art depending on the kind of microorganism cells selected. A typical culture method comprises inoculating seed solution of the recombinant microbial cells into a TB culture medium, culturing at 35-40 ℃, adding an inducer (such as IPTG) when the OD600 value of the culture solution reaches 0.6-0.8, inducing the fusion protein to express at 25-27 ℃, and obtaining wet thalli expressing the fusion protein after the induction is finished.

Because the fusion protein has good water solubility, the wet thalli can be further homogenized and crushed in a buffer solution, and supernatant is collected after centrifugation.

And purifying the supernatant to obtain the fusion protein. In particular, when the recombinant microbial cell expresses a fusion protein comprising PfKRED or an equivalent thereof, the purification may comprise heating the supernatant to above 100 ℃ (e.g., 100-120 ℃), centrifuging to obtain a secondary supernatant, and extracting the fusion protein from the secondary supernatant.

Alternatively, when the recombinant microorganism cell expresses a fusion protein comprising grpE or an equivalent sequence thereof, SUMO or an equivalent sequence thereof, the fusion protein can be extracted by designing an affinity adsorption tag sequence (e.g., a histidine tag sequence) at the N-terminus of the leader peptide sequence, and contacting the supernatant with an affinity resin having a specific binding force to the affinity adsorption tag sequence of the fusion protein. The selection of resins with specific binding capacity for the affinity adsorption tag sequence is within the skill of the art, typically as a histidine tag sequence and a Ni-NTA resin.

Preparation method of cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37)

Further, the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37) can be obtained by cleavage of other parts of the fusion protein.

And selecting a corresponding protease according to the weight of the protease cleavage site in the fusion protein, so that the polypeptide can be excised. In the present invention, the type of the cleavage site for protease is not particularly limited, and cleavage sites commonly used in the art, such as DDDDK, may be selected, and enterokinase may be used for the cleavage, KR, kex2 protease may be used for the cleavage, IEGR or IDGR, and Factor Xa protease may be used for the cleavage. In particular, when SUMO or its equivalent is used as a leader peptide, cleavage with SUMO protease can be performed directly without inserting an additional cleavage site.

The main advantages of the invention include:

The fusion protein of the invention contains a leader peptide sequence which is helpful for the fusion protein to fold correctly after being expressed by a recombinant strain, so that the fusion protein has good solubility in fermentation liquor of the recombinant strain or supernatant fluid after centrifugation of the fermentation liquor, and is easy to extract and purify from the fermentation liquor, especially from the supernatant fluid after centrifugation of the fermentation liquor. Compared with fusion proteins which form inclusion bodies in fermentation broth due to incorrect folding, the fusion protein does not need to undergo steps of denaturation and renaturation of the inclusion bodies during purification, which simplifies the purification steps, saves the cost of purification reagents (such as denaturing reagents, renaturation reagents and the like), and improves the yield (up to 91.2%) and purity (up to 96.7%) of finally prepared cable Ma Lutai intermediate polypeptides.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The detection method of each embodiment of the invention is as follows:

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

column chromatography, waters XB ridge C8 (5 μm, 250X 4.6 mm), mobile phase, 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. Gradient elution was performed as in Table 1 below, column temperature at 35℃and flow rate at 1.0mL/min, sample injection amount at 10. Mu.L, and detection wavelength at 210nm.

Gradient elution parameters of Table 1 HPLC

MS detection conditions of Arg ³⁴ GLP-1 (9-37) are shown in the following, mass spectrometer Shimadzu LC-MS 2020, ion source is electrospray (Electrospray ionization, ESI).

The embodiment of the invention adopts the following reagents:

LB liquid medium, 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride.

TB liquid medium 2% tryptone, 2.4% yeast extract, 72mM K ₂HPO₄、17mM KH₂PO₄, 0.4% glycerol.

EXAMPLE 1 construction of recombinant engineering Strain expressing PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein and inducible expression of the fusion protein

The embodiment constructs a recombinant engineering strain capable of expressing PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein, and the method comprises the following steps:

1.1 designing the nucleotide sequence of PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein. Wherein, 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 as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 11. The nucleotide sequence of the enzyme cutting 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). The nucleotide sequence of Arg ³⁴ GLP-1 (9-37) is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. The nucleotide sequence of the fusion protein is submitted to the biological engineering (Shanghai) stock limited company (Shanghai city, pingjiang region Minghu 698) to be synthesized and cloned into a pET28a vector, and the used enzyme cutting sites are NdeI and HindIII, so that the recombinant plasmid containing the nucleotide sequence for encoding the fusion protein is obtained. Arg ³⁴ in the fusion protein indicates Arg ³⁴ GLP-1 (9-37) with arginine at amino acid 34.

1.2 The recombinant plasmid which has been constructed is transformed into the expression host E.coli BL21 (DE 3), spread on LB-resistant plates containing 50. Mu.g/mL kanamycin, and cultivated upside down at 37℃for 16h. Single colonies were picked and inoculated into 5mL LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃and 220 rpm. When the culture is carried out until the OD600 is between 0.6 and 0.8, seed liquid is obtained. The seed solution was inoculated into TB liquid medium containing 50. Mu.g/mL kanamycin at an inoculum size of 1%, and cultured at 37℃and 220 rpm. Inducer IPTG was added at a final concentration of 0.1mM at OD600 to 0.6 and induced at 220rpm at 25℃for 16h. And centrifuging at 4000rpm for 20min, discarding supernatant, collecting wet thallus, and storing in a refrigerator at-20deg.C for use. Thus, wet cells expressing PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein were obtained.

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

10G of wet cells expressing PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein are taken and added into 50mL of 50mM Tris-HCl solution with pH of 8.0 (the ratio of the mass of the wet cells to the volume of Tris-HCl solution is 1g:5 mL) to carry out homogenization and crushing (the homogenization temperature is 0-4 ℃), so as to obtain whole bacterial liquid. Centrifuging the whole bacterial liquid at 10000rpm for 10min to obtain supernatant and precipitate. SDS-PAGE was performed on the whole bacterial liquid and the supernatant, and the detection results are shown in FIG. 1. As can be seen from FIG. 1, the band position of PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein showed that the molecular weight thereof was in accordance with the expected molecular weight (37.9 kDa), and the expression level of PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) fusion protein in whole bacterial liquid and supernatant was substantially uniform, indicating that the fusion protein was expressed in the form of a soluble protein substantially entirely, so as to be able to be dissolved in the supernatant.

Example 3 PfKRED-GGGSG purification of a DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein

Since PfKRED is a thermophilic protein, it is not thermally denatured and precipitated at a heating temperature of 100 ℃, and therefore, the fusion protein can be purified by a heat purification method, which comprises the steps of weighing 10g of wet cell expressing PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) fusion protein, adding 50mL of 50mM Tris-HCl solution of pH8.0 (the ratio of the mass of the wet cell to the volume of Tris-HCl solution is 1g:5 mL), homogenizing and disrupting (homogenizing temperature is 0-4 ℃) and centrifuging at 10000rpm for 10min (the purpose of removing cell debris such as organelles and cell membranes). The supernatant (in which the fusion protein was dissolved) was taken and heated at 100℃for 20 minutes (the purpose of heating was to cause thermal denaturation of the hetero protein to precipitate out of the supernatant, whereas the fusion protein remained dissolved in the supernatant since thermal denaturation did not occur upon heating). The mixture was centrifuged again at 10000rpm for 10min (for removing denatured foreign proteins), and the precipitate was discarded to obtain a secondary supernatant. The secondary supernatant was dialyzed against a dialysis bag (for removing substances having a molecular weight of less than 14kDa by passing through the dialysis bag, combined with carbonization, cat# MD 34-14), and the dialysate was 50mM Tris-HCl solution at pH 8.0. After dialysis, the liquid remaining in the dialysis bag is centrifuged again and the supernatant is taken again. The supernatant contained purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein. The concentration of the fusion protein was measured by the Bradford method to obtain a total of 50.2mL of PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein solution having a concentration of 12.1 mg/mL. As shown in table 2.

SDS-PAGE detection is carried out on the purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein, and the result is shown in lane 1 of FIG. 2.

EXAMPLE 4 enterokinase availability

Example 3 gave a purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein. The enterokinase is adopted to carry out enzyme digestion by taking DDDDK as an enzyme digestion site, and then the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37) can be obtained. It was only subsequently possible to produce cable Ma Lutai based on the intermediate polypeptide of this cable Ma Lutai. Thus, this example provides enterokinase capable of specifically cleaving PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein.

4.1 Construction and expression of enterokinase expression Strain

Enterokinase is obtained by the following method. The nucleotide sequence of enterokinase is designed, and the nucleotide sequence is shown as SEQ ID NO. 9, and the amino acid sequence is shown as SEQ ID NO. 10. The nucleotide sequence of the enterokinase is synthesized by submitting to biological engineering (Shanghai) stock limited company (Shanghai city Min's pipeline 698 of Pingjiang region), and a recombinant plasmid containing the nucleotide sequence of the enterokinase is constructed. The constructed recombinant plasmid was transferred into an expression host E.coli BL21 (DE 3), spread on LB-resistant plates containing 50. Mu.g/mL kanamycin, and cultured for 16h at 37℃in an inverted manner. Thereafter, single colonies were picked up and inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL of kanamycin, and cultured at 37℃and 220 rpm. Seed solution was obtained when OD600 was 0.6-0.8, and the seed solution was inoculated into TB liquid medium containing 50. Mu.g/mL kanamycin at an inoculum size of 1%, and cultured at 220rpm at 37 ℃. When OD600 was 0.6, IPTG was added at a final concentration of 0.1mM, induced at 25℃and 220rpm for 16h, centrifuged at 4000rpm for 20min, the supernatant was discarded, and wet cells were collected and stored in a-20℃refrigerator for further use.

4.2 Denaturation, renaturation and purification of enterokinase

The wet enterokinase cells (3 g) were weighed, homogenized with 50mM Tris-HCl (pH 8.0) at 1:10 (g/mL), centrifuged at 10000rpm for 10min, and the supernatant was discarded to leave a precipitate (enterokinase expressed in the form of inclusion bodies in the precipitate). The precipitate was washed twice with 50mM Tris-HCl, pH8.0, and after washing, centrifuged, the supernatant was discarded, leaving a precipitate. To the pellet was added 20mL of a denaturing solution containing 50mM Tris-HCl, 20mM Dithiothreitol (DTT), 8M urea and having a pH of 8.0, and the pellet was dissolved in the denaturing solution by shaking at 4℃for 3 hours. The incubation solution was taken, 500mL of a renaturation solution containing 50mM Tris-HCl, 1mM oxidized glutathione, 3mM reduced glutathione, 1M urea and having a pH of 8.0 was added, and incubated at 20℃at 100rpm for 24 hours to renature the denatured enterokinase and to fold correctly and then dissolve in the renaturation solution, so that the subsequent enzyme cleavage site DDDDK could be specifically and efficiently acted on. The incubated enzyme solution was then purified and concentrated using an anion exchange chromatography column (Q column, boglycopyrrolate, cat# EI 3041203) with an equilibration solution of 50mM Tris-HCl, pH8.0 and an eluate of 50mM Tris-HCl, 0.5M NaCl and pH 8.0. The purified and concentrated solution was dialyzed against a dialysis bag (molecular weight cut-off 7kDa, co-carbonization, cat# MD 55-7) and the dialysate was 50mM Tris-HCl solution at pH 8.0. After dialysis, 16.6mL of enterokinase enzyme solution was obtained. The enzyme activity of enterokinase was measured and found to be 529.3U/mL.

Enterokinase enzyme activity is defined as the enzyme amount required for complete enzyme digestion of 1mg PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) at 25 ℃ and 50mM Tris-HCl, pH8.0 for 8 hours as one enzyme activity unit (U).

EXAMPLE 5 enterokinase cleavage PfKRE-GGGSG-DDDDDK-Arg ³⁴ GLP-1 (9-37) Arg ³⁴ GLP-1 (9-37) was obtained

50.2ML of purified PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein solution (12.1 mg/mL) is added into 608U of enterokinase, and the mixture is subjected to shaking enzyme digestion at 25 ℃ and 150rpm for 8 hours to obtain a protein solution containing Arg ³⁴ GLP-1 (9-37). DDDDDK is the cleavage site of enterokinase, and thus, the fusion protein is capable of producing Arg ³⁴ GLP-1 (9-37) and PfKRED-GGGSG-DDDDK protein (theoretical molecular weight of 34.7 kDa) after cleavage by enterokinase.

SDS-PAGE electrophoresis of the protein solution was performed, and the results are shown in FIG. 2. FIG. 2 shows that lane 2 is PfKRED-GGGSG-DDK protein, the band size is consistent with the theoretical value, and the PfKRED-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein is correctly digested to obtain Arg ³⁴ GLP-1 (9-37) protein (the molecular weight is 3175.5 Da), which indicates that enterokinase constructed by the embodiment of the application can specifically act on the digestion site DDDDDK, and Arg ³⁴ GLP-1 (9-37) generated after digestion can be well distinguished from PfKRED-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) before digestion.

EXAMPLE 6 Arg ³⁴ isolation and purification of GLP-1 (9-37)

The Arg ³⁴ GLP-1 (9-37) solution obtained by the enzyme digestion in example 5 is freeze-dried to obtain freeze-dried powder. The lyophilized powder was reconstituted with 10mL of ultrapure water. The purpose of this lyophilization is to reduce the total volume of Arg ³⁴ GLP-1 (9-37) solution obtained after cleavage in example 5, resulting in Arg ³⁴ GLP-1 (9-37) concentrate for the subsequent isolation and purification process. Then, gel filtration chromatography (Focurose PG, huiyang, HN 120209500M) was used to isolate and purify the Arg ³⁴ GLP-1 (9-37) concentrate by flow against 50mM Tris-HCl, pH 8.0. The purified Arg ³⁴ GLP-1 (9-37) fraction was concentrated again with a lyophilizer (the volume after concentration was 5ml-10 ml), and dialyzed with a dialysis bag (molecular weight cut-off 1kDa, spectroscopic medical, cat# 131060) to remove small molecule salts, the dialysate being 50mM Tris-HCl solution pH 8.0. The Arg ³⁴ GLP-1 (9-37) solution obtained was subjected to HPLC detection, and the external standard method was used for quantification (the Arg ³⁴ GLP-1 (9-37) gradient standard solution was prepared and a standard curve was established for quantification), so that the Arg ³⁴ GLP-1 (9-37) amount was 46.4mg. The molecular weight of Arg ³⁴ GLP-1 (9-37) obtained by LC-MS detection is consistent with the theoretical value (3175.5 Da), the purity of Arg ³⁴ GLP-1 (9-37) is 96.1%, and the yield is 91.2%. Specifically, the results are shown in Table 3.

The calculation method of Arg ³⁴ GLP-1 (9-37) purity comprises calculating Arg ³⁴ GLP-1 (9-37) purity according to the ratio of Arg ³⁴ GLP-1 (9-37) peak area to total peak area in HPLC chart;

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

yield = m _{Arg34GLP-1(9-37)}/[(V _{purified fusion proteins} ×C _{purified fusion proteins} )×(MW_{Arg34GLP-1(9-37)}/MW _{Fusion proteins} ) ].

Wherein m _{Arg34GLP-1(9-37)} represents the mass of Arg ³⁴ GLP-1 (9-37) after separation and purification, which data is 46.4mg in this example.

V _{purified fusion proteins} and C _{purified fusion proteins} represent the concentration and volume, respectively, of the fusion protein obtained after purification, and this data is from example 3, 50.2mL and 12.1mg/mL, respectively.

MW _{Arg34GLP-1(9-37)} and MW _{Fusion proteins} represent the theoretical molecular weight of Arg ³⁴ GLP-1 (9-37) and the theoretical molecular weight of the fusion protein, respectively, which in this example are 3.1755kDa and 37.9kDa, respectively.

The purity and yield were calculated in the same manner as described below.

EXAMPLE 7 Kex2 protease cleavage of PfKRED-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein Arg ³⁴ GLP-1 (9-37)

7.1 PfKRED-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein and purification reference examples 1-3, 10g of wet cells were taken to obtain 52.9mL of PfKRED-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein with a concentration of 11.3mg/mL, and the molecular weight (37.6 kDa) of the protein was detected to be consistent with the expected molecular weight by SDS-PAGE. Wherein the nucleotide sequence of KR is AAAAGA.

7.2 Kex2 protease acquisition

Construction and expression of Kex2 protease expression Strain reference 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 cleavage site of Kex2 protease is KR. K represents lysine (Lys), and R represents arginine (Arg).

Kex2 protease purification by weighing 3g of wet bacterial cells expressing Kex2 protease (with histidine tag sequence), homogenizing and crushing the cells with a solution containing 20mM Tris-HCl, 150mM NaCl and having pH of 8.0 (homogenizing temperature 0-4 ℃) at 1:10 (homogenizing temperature 0-4 ℃), centrifuging the cells at 10000rpm and 4 ℃) for 10min, collecting the supernatant, purifying the cells with Ni-NTA resin, taking the solution containing 20mM Tris-HCl, 150mM NaCl and having pH of 8.0 as an equilibrium solution, and carrying out gradient elution (elution according to the method recommended by resin manufacturer) taking the solution containing 20mM Tris-HCl, 150mM NaCl, 500mM imidazole and having pH of 8.0 as an eluent, dialyzing the cells with a dialysis bag (molecular weight cut-off 7 kDa) to remove imidazole, obtaining 7.3mL Kex2 protease solution after dialysis, and the Kex2 protease activity of 164.7U/mL.

Kex2 protease Activity is defined as the amount of enzyme required to cleave 1mg of Kex2-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) protein at 25℃in 50mM Tris-HCl, 2mM CaCl ₂, pH8.0 for 8 hours as one enzyme activity unit (U).

7.3 Obtaining and purifying Arg ³⁴ GLP-1 (9-37)

The PfKRED-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein obtained in the step (1) is taken, caCl ₂ with the working concentration of 2mM and 597.8U Kex2 protease are added, and the protease is subjected to enzyme digestion for 8 hours at 25 ℃ and 150rmp, so that a protein solution containing Arg ³⁴ GLP-1 (9-37) is obtained. The protein solution of Arg ³⁴ GLP-1 (9-37) obtained after cleavage was isolated and purified (specific reference example 6). The Arg ³⁴ GLP-1 (9-37) solution obtained was subjected to HPLC detection and external standard method quantification to obtain 45.6mg Arg ³⁴ GLP-1 (9-37), the molecular weight of Arg ³⁴ GLP-1 (9-37) obtained by LC-MS detection was consistent with the theoretical value (3175.5 Da), the purity of Arg ³⁴ GLP-1 (9-37) was 95.5%, and the yield was 90.3%.

EXAMPLE 8 enterokinase cleavage of the 6 XHis-tagged grpE-GGGSG-DDDDK-Arg34GLP-1 (9-37) fusion protein to Arg ³⁴ GLP-1 (9-37)

8.1 Construction and expression of recombinant engineering Strain of grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein with 6XHis tag

Recombinant engineering strains capable of expressing grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion proteins with 6xHis labels are constructed, and the recombinant engineering strains are induced to be expressed so as to obtain grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion proteins with 6xHis labels, wherein the specific steps are described in example 1. The nucleotide sequence of grpE is shown as SEQ ID NO. 14, and the amino acid sequence is shown as SEQ ID NO. 15. SDS-PAGE electrophoresis of whole bacterial liquid and supernatant of recombinant engineering strain expressing grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein with 6XHis tag is carried out, and the result is shown in figure 3. As can be seen from FIG. 3, the 6 XHis-tagged grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein is expressed substantially entirely in the form of a soluble protein, which is soluble in the supernatant.

8.2 Purification of 6 XHis-tagged grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein

10G of wet cells expressing grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) with a 6XHis tag (also called 6XHis tag) were weighed, homogenized and broken (homogenization temperature 0-4 ℃) at 1:5 (g/mL) with a solution containing 20mM Tris-HCl, 150mM NaCl and pH8.0, centrifuged at 10000rpm at 4 ℃) for 10min, the supernatant was collected, purified using Ni-NTA resin, and eluted with a gradient elution (which can be eluted according to the resin manufacturer's recommended) with a solution containing 20mM Tris-HCl, 150mM NaCl and pH8.0 as an equilibration solution and a solution containing 20mM Tris-HCl, 150mM NaCl 500mM imidazole and pH8.0 as an elution solution. Dialysis was performed with a dialysis bag (molecular weight cut-off 7 kDa) to remove imidazole, and the dialysate was 50mM Tris-HCl solution pH 8.0. After dialysis, 12.4mL of a 6 XHis-tagged grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) fusion protein solution was obtained, and the concentration of the 6 XHis-tagged grpE-GGGSG-DDK-Arg ³⁴ GLP-1 (9-37) fusion protein was measured by the Bradford method and was 23.6mg/mL.

8.3 Enterokinase cleavage of 6 XHis-tagged grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) to obtain separation and purification of Arg ³⁴ GLP-1 (9-37) and Arg ³⁴ GLP-1 (9-37)

(1) Obtaining Arg ³⁴ GLP-1 (9-37):

12.4mL of grpE-GGGSG-DDDDK-Arg ³⁴ GLP-1 (9-37) protein solution (23.6 mg/mL) with a 6xHis tag obtained in example 8.2 is taken, 292.6U enterokinase is added, the enzyme digestion is carried out at 25 ℃ and 150rpm, the Arg ³⁴ GLP-1 (9-37) protein solution after the enzyme digestion is obtained, SDS-PAGE detection is carried out on the protein solution, and the protein band size in the solution after the enzyme digestion is consistent with a theoretical value.

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

The Arg ³⁴ GLP-1 (9-37) protein solution obtained in the step (1) is taken for separation and purification, and the specific operation is as described in reference example 6. The Arg ³⁴ GLP-1 (9-37) solution obtained is subjected to HPLC detection, and the external standard method is used for quantification, so that 29mg Arg ³⁴ GLP-1 (9-37) protein is obtained, the molecular weight of Arg ³⁴ GLP-1 (9-37) obtained by LC-MS detection and LC-MS detection is consistent with the theoretical value (3175.5 Da), the purity of Arg ³⁴ GLP-1 (9-37) is 95.3%, and the yield is 87.4%.

EXAMPLE 9 isolation and purification of 6 XHis-tagged grpE-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) by Kex2 protease cleavage to Arg ³⁴ GLP-1 (9-37) and Arg ³⁴ GLP-1 (9-37)

9.1 Constructing a recombinant engineering strain capable of expressing grpE-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein with a 6XHis tag, and inducing the recombinant engineering strain to express so as to obtain the grpE-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein with the 6XHis tag, wherein the specific steps are as described in reference example 1-2, and the concentration of the fusion protein obtained by taking 10g of wet thalli is 21.8mg/mL, and the volume is 10.7mL.

9.2 Purification of 6 XHis-tagged grpE-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) fusion protein reference example 8.3;

9.3Arg ³⁴ GLP-1 (9-37) acquisition and purification

The purified fusion protein grpE-GGGSG-KR-Arg ³⁴ GLP-1 (9-37) with the 6XHis tag is taken, 2mM CaCl ₂ and 230U Kex2 protease with working concentration are added, and the fusion protein is subjected to cleavage for 8 hours at 25 ℃ and 150rmp, and Arg ³⁴ GLP-1 (9-37) is separated and purified (specific reference example 6) to obtain 23.4mg Arg ³⁴ GLP-1 (9-37). The molecular weight of Arg ³⁴ GLP-1 (9-37) obtained by LC-MS detection is consistent with the theoretical value (3175.5 Da), the purity of Arg ³⁴ GLP-1 (9-37) is 94.5%, and the yield is 87.5%.

EXAMPLE 10 SUMO protease cleavage of 6 XHis-tagged SUMO-Arg ³⁴ GLP-1 (9-37)

10.1 Construction and expression of recombinant engineering Strain of SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein with 6XHis tag

Recombinant engineering strains capable of expressing SUMO-Arg ³⁴ GLP-1 (9-37) fusion proteins with 6xHis tags are constructed, and induced to express the recombinant engineering strains so as to obtain wet thalli expressing SUMO-Arg ³⁴ GLP-1 (9-37) fusion proteins with 6xHis tags, wherein the specific steps are as described in example 1.

SDS-PAGE electrophoresis of whole bacterial liquid and supernatant of recombinant engineering strain expressing SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein with 6XHis tag is carried out, and the result is shown in figure 4. As can be seen from FIG. 4, the 6 XHis-tagged SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein is expressed substantially entirely in the form of a soluble protein, which is soluble in the supernatant.

Experiments show that the SUMO sequence is directly connected with Arg ³⁴ GLP-1 (9-37) sequence, but not connected with GGGSG sequence, and the obtained fusion protein can be well dissolved in supernatant, but not exists in the form of inclusion bodies, so that subsequent separation and purification are facilitated.

10.2 Purification of 6 XHis-tagged SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein

10G of wet cells expressing a 6 XHis-tagged SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein were weighed, homogenized and broken up (homogenization temperature 0-4 ℃) at 1:5 (g/mL) with a solution containing 20mM Tris-HCl, 150mM NaCl and pH8.0, centrifuged at 10000rpm, 4℃for 10min, the supernatant was collected, purified using Ni-NTA resin, and gradient eluted with a solution containing 20mM Tris-HCl, 150mM NaCl and pH8.0 as an equilibrium solution and a solution containing 20mM Tris-HCl, 150mM NaCl, 500mM imidazole and pH8.0 as an eluent. Dialysis was performed with a dialysis bag (molecular weight cut-off 7kDa, co-carbonization, cat# MD 55-7) to remove imidazole, and the dialysate was 50mM Tris-HCl solution at pH 8.0. After dialysis 8.3mL of a solution containing the SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein with a 6XHis tag was obtained. The concentration of the fusion protein was determined by the Bradford method, and the concentration of the SUMO-Arg ³⁴ GLP-1 (9-37) fusion protein was 16.9mg/mL.

10.3 SUMO protease acquisition and purification

SUMO protease obtaining and purification procedure referring to example 7.2, 3g of wet cell expressing SUMO protease was taken to obtain 6.4mL of SUMO protease enzyme solution with an enzyme activity of 39.6U/mL.

SUMO protease Activity is defined as the amount of enzyme required to cleave 1mg of SUMO-Arg ³⁴ GLP-1 (9-37) at 25℃in 50mM Tris-HCl, pH8.0 for 8h as one enzyme activity unit (U).

10.4 Cleavage of SUMO-Arg ³⁴ GLP-1 (9-37) bearing a 6XHis tag by SUMO protease to obtain Arg ³⁴ GLP-1 (9-37) and purification of Arg ³⁴ GLP-1 (9-37)

8.3ML of the purified 6 XHis-tagged SUMO-Arg ³⁴ GLP-1 (9-37) (16.9 mg/mL) protein solution of example 10.2 was taken, 140.3U of SUMO protease was added, and the mixture was digested with shaking at 150rpm at 25℃for 8 hours to obtain a digested solution containing Arg ³⁴ GLP-1 (9-37). The digested solution was isolated and purified (for specific procedures, see example 6). HPLC detection and external standard quantification were performed on the Argo ³⁴ GLP-1 (9-37) solution obtained after purification, and the Arg ³⁴ GLP-1 (9-37) amount was found to be 23.5mg. The molecular weight of Arg ³⁴ GLP-1 (9-37) obtained by LC-MS detection is consistent with the theoretical value (3175.5 Da), the purity of Arg ³⁴ GLP-1 (9-37) is 96.7%, and the yield is 86.5%.

In this example, the SUMO sequence was cleaved using SUMO protease to yield Arg ³⁴ GLP-1 (9-37). Thus, the SUMO sequence can serve as a signal peptide to allow proper folding of the fusion protein, so that it can be dissolved in the fermentation broth or supernatant, rather than in inclusion bodies, to simplify subsequent protein purification. In addition, the SUMO sequence can be directly used as an enzyme cutting site of SUMO protease, and the SUMO protease has strong recognition specificity on the SUMO sequence. Therefore, GGGSG and additional enzyme cutting sequences are not needed to be added additionally, and fusion proteins with longer sequences are avoided to be obtained, so that the situation that the fusion proteins are misfolded due to overlong sequences and exist in an inclusion body form is avoided, and the subsequent purification process is facilitated. And the molecular weight of the SUMO sequence is smaller, and the ratio of Arg ³⁴ GLP-1 (9-37) in the whole fusion protein is relatively higher.

TABLE 2 parameter Table of fusion proteins in various examples

TABLE 3 parameter Table of Soxhlet Ma Lutai intermediate polypeptides in various examples

The sequence numbers related to the above examples are shown in Table 4.

Table 4 sequence numbering for the various embodiments

Sequence name	Examples numbering	Nucleotide sequence	Amino acid sequence
				Arg34GLP-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
				PfKRED-GGGSG-DDDDK-Arg34GLP-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
				grpE	Example 8	SEQ ID NO:14	SEQ ID NO:15
SUMO	Example 10	SEQ ID NO:18	SEQ ID NO:19
				SUMO-Arg34GLP-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
				PfKRED-GGGSG-KR-Arg34GLP-1(9-37)	Example 7	SEQ ID NO:24	SEQ ID NO:25
grpE-GGGSG-DDDDK-Arg34GLP-1(9-37)	Example 8	SEQ ID NO:16	SEQ ID NO:17
				grpE-GGGSG-KR-Arg34GLP-1(9-37)	Example 9	SEQ ID NO:26	SEQ ID NO:27

In table 4, the example numbers refer to the example numbers that first appear.

In addition, the inventors tested TrxA, GST, dsbC et al as leader peptides during the course of the study, but fusion proteins prepared from these leader peptides were not used because they were poorly soluble and had low expression levels.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (11)

1. A fusion protein, the fusion protein comprising:

A leader peptide sequence and a cable Ma Lutai intermediate polypeptide sequence, wherein the leader peptide sequence and the cable Ma Lutai intermediate polypeptide sequence are directly linked or indirectly linked by a linker sequence and/or a protease cleavage site sequence;

Wherein the leader peptide sequence is the PfKRED amino acid sequence shown in SEQ ID NO. 6 or an equivalent sequence having at least 98% identity to the PfKRED amino acid sequence, the grpE amino acid sequence shown in SEQ ID NO.15 or an equivalent sequence having at least 98% identity to the grpE amino acid sequence, the SUMO amino acid sequence shown in SEQ ID NO. 19 or an equivalent sequence having at least 98% identity to the SUMO amino acid sequence, and

The polypeptide sequence of the cable Ma Lutai intermediate is Arg ³⁴ GLP-1 (9-37) amino acid sequence shown in SEQ ID NO. 2.

2. The fusion protein of claim 1, wherein the leader peptide sequence, linker sequence, protease cleavage site sequence, and the cable Ma Lutai intermediate polypeptide sequence are linked in sequence when the linker sequence and protease cleavage site sequence are present;

Further, the linker sequence is selected from any one of GGGG, GGGGG, GGGGS, GGGSG, EAAAK, AEAAAKALEA and PAPAPAAP, and/or

The protease cleavage site sequence is selected from any one of DDDDK, KR, IEGR and IDGR.

3. The fusion protein of claim 1 or 2, wherein 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;

Further, the fusion protein further comprises an affinity adsorption tag sequence, such as a histidine tag sequence, preferably comprising at least 6 histidines, located at the N-terminus of the leader peptide sequence.

4. An isolated nucleic acid molecule encoding the fusion protein of 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 comprising the isolated nucleic acid molecule of claim 4;

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

Further, the pET series plasmids comprise pET-24a (+), pET28a (+), pET-29a (+), pET-30a (+), the Duet series plasmids comprise pRSFDuet-1 and pCDFDuet-1, and the pTrc series plasmids comprise pTrc99a.

6. A recombinant microbial cell expressing the fusion protein of any one of claims 1 to 3, comprising the isolated nucleic acid molecule of claim 4, or comprising the recombinant expression vector of claim 5;

Further, the starting strain of the recombinant microbial cell includes, but is not limited to, E.coli JM109 (DE 3), E.coli HMS174 (DE 3), E.coli BL21 (DE 3), E.coli Rostta (DE 3), E.coli Rosttagami (DE 3), E.coli Rostta (DE 3), E.coli DH 5. Alpha., E.coli W3110 and/or E.coli K12.

7. Use of an isolated nucleic acid molecule according to claim 4, a recombinant expression vector according to claim 5, a recombinant microbial cell according to claim 6 for expression of a fusion protein according to any one of claims 1 to 3.

8. A process for producing a fusion protein, comprising the step of fermenting and culturing the recombinant microorganism cell of claim 6, thereby producing the fusion protein of any one of claims 1 to 3.

9. A method for preparing a cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37), which is characterized by comprising the following steps:

The fusion protein of any one of claims 1 to 3, which is digested to obtain the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37).

10. The method according to claim 9, further comprising the step of purifying the fusion protein before the cleavage is performed;

Further, under the condition that the fusion protein contains PfKRED amino acid sequence as shown in SEQ ID NO. 6, the purification comprises heating the solution containing the fusion protein at 100 ℃ to 120 ℃ to separate out the hybrid protein, thereby obtaining the purified fusion protein;

Under the condition that the fusion protein contains a histidine tag sequence, the purification comprises the step of contacting the solution containing the fusion protein with an affinity resin with specific binding force with the histidine tag sequence, thereby extracting the purified fusion protein.

11. Use of a fusion protein according to any one of claims 1-3, or an isolated nucleic acid molecule according to claim 4, or a recombinant expression vector according to claim 5, or a recombinant microbial cell according to claim 6, for the preparation of the cable Ma Lutai intermediate polypeptide Arg ³⁴ GLP-1 (9-37).