CN115850385A - Expression promoting peptide and application thereof - Google Patents
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Abstract
The invention relates to the technical field of genetic engineering, in particular to an expression promoting peptide suitable for recombinant expression of insulin peptide chains and GLP-1 related target agonist polypeptides and application thereof. The recombinant gene engineering bacteria constructed based on the expression promoting peptide can obviously enhance the expression quantity of insulin precursor protein, GLP-1 and related multi-target point agonist polypeptide precursors which are difficult to express or have low expression quantity, effectively reduce the production cost and have wide commercialization prospect.
Description
This application claims full priority to patent application No. 202210788609.4, filed on 7/4/2022. The entire contents of this application are incorporated herein by reference in their entirety.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an expression promoting peptide and application thereof.
Background
With the development of social economy, the living standard of people is gradually improved, the dietary structure of people is greatly changed, the incidence rate of obesity is increased, and the number of people suffering from diabetes is further increased sharply. Statistical data show that the number of diabetic patients in China exceeds 1 hundred million, and simultaneously, more than 1.5 hundred million patients with cryptomorphic diabetes mellitus are also existed in the early stage. Diabetes has become the third major chronic disease.
Diabetes mellitus is a complex chronic metabolic disease caused by long-term interaction of genetic and environmental factors, is caused by insulin secretion deficiency, is characterized by hyperglycemia, and is divided into type I diabetes mellitus and type II diabetes mellitus, wherein the type II diabetes mellitus (T2 DM) accounts for more than 95% of diabetes patients in China.
The most important drugs for treating diabetes are insulin and its derivatives, glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1 RAs) and related multi-target co-agonists. The above drugs are all protein or polypeptide drugs.
The known methods for obtaining insulin include animal tissue extraction and genetic engineering expression, and the expression of human insulin and analogues thereof by genetic engineering has long become the mainstream means in industry. The microorganisms expressing insulin in genetic engineering are mainly classified into 2 types, i.e., escherichia coli and yeast. As for prokaryotic gene expression systems, escherichia coli is used as a host bacterium, and is also the most widely used protein expression system at present. The reason is that the research on genetic background and physiological characteristics of an escherichia coli expression system is clear, and a plurality of commercial engineering bacteria can be developed and used; and the Escherichia coli is easy to culture and control, simple in transformation operation, high in expression level, low in cost, short in period and the like. When a prokaryotic system is applied to express exogenous genes, most researches utilize a fusion protein expression mode to fuse various different guide peptide sequences onto target genes to form recombinant fusion proteins. When expressed in E.coli, the leader peptide can secrete the target protein into the periplasm of cells or even outside the cells, and finally, the leader peptide is cleaved off by a protease or the like. However, for polypeptides having a part with a special structure or consisting of amino acid sequences, such as insulin precursor, a part of GLP-1 polypeptide and GLP-1 related multi-target agonist polypeptide, it is often difficult to express or the expression level is low, and thus the requirement of commercial use cannot be met. Therefore, sometimes, a segment of expression promoting peptide is required to be added, after the expression promoting peptide is connected to the leader peptide, the expression level of the fusion protein can be remarkably enhanced, and finally, the target protein segment is obtained through treatment such as enzyme digestion. At present, relatively few researches are carried out on expression promoting peptides capable of further enhancing the high expression of the proteins or polypeptides, expression vectors constructed by the expression promoting peptides and having high expression potential, and recombinant engineering bacteria thereof.
Therefore, there is still a great need to obtain expression promoting peptides suitable for various proteins or polypeptides difficult to express or low in expression level, and construct recombinant engineering bacteria with high expression capacity based on the expression promoting peptides, so as to improve the expression level of the polypeptides or proteins and effectively reduce the production cost.
Disclosure of Invention
In order to solve the technical problems, the invention provides an expression promoting peptide, and the recombinant engineering bacteria constructed based on the expression promoting peptide can efficiently express insulin peptide chain precursors and GLP-1 related target agonist polypeptides.
As used herein, the term "protein of interest" refers to a product of interest or its precursor protein for which high expression is desired.
As used herein, the term "leader peptide" refers to a polypeptide sequence that is linked to a polypeptide or protein of interest, directs the soluble expression, secretion, or aids in the proper folding of the polypeptide or protein of interest to achieve soluble expression, and is also referred to as a "leader peptide," leader peptide, "or" secretory peptide.
As used herein, the term "expression promoting peptide" refers to a polypeptide sequence which is linked after a leader peptide and before a protein or polypeptide of interest to further enhance the expression level of the protein or polypeptide of interest, or which can promote the expression of the protein or polypeptide of interest which is difficult to express by a conventional leader peptide.
The term "peptide" refers to a molecule comprising amino acid sequences linked by peptide bonds, whether in length, post-translational modification or function.
As used herein, the term "insulin" includes native insulin and insulin analogs.
As used herein, the term "native insulin" refers to a hormone that is a 51 amino acid residue polypeptide (5808 daltons) that plays an important role in many key cellular processes. The mature form of human insulin consists of 51 amino acids and is arranged into an A chain (GlyAl-AsnA 21) and a B chain (PheB 1-ThrB 30) with a total molecular weight of 5808 Da. The molecule is stabilized by two interchain disulfide bonds (A20-B19, A7-B7) and one intrachain disulfide bond (A6-A11). The insulins of the invention include natural, synthetically provided, or genetically engineered (e.g., recombinant) sources, and in various embodiments of the invention, the insulins may be human natural insulin or insulin analogs.
The term "insulin analog" as used herein refers to an altered form of insulin, which is a more rapidly acting or more effectively acting form of insulin. Non-limiting examples of such analogs include Insulin lispro, insulin Degludec, insulin Aspart (Insulin Aspart), and Insulin Glargine (Glargine Insulin). A "lispro" insulin analogue is essentially identical in primary structure to human insulin and differs from human insulin by the exchange of a lysine at position B28 and a proline at position B29. It is a short acting insulin analogue. "glycopyrrolate" insulin analogues differ from human insulin by replacing the glycine at a21 with asparagine and adding two arginine residues at the C-terminus of the B-chain. The insulin glargine solution was prepared and injected at pH 4.0. These modifications increase the isoelectric point to a more neutral pH, decrease solubility under physiological conditions, and cause precipitation of insulin glargine at the injection site, thereby slowing absorption. Insulin glargine is a long-acting analogue lasting from 20 to 24 hours.
The term "insulin precursor" as used herein refers to a single-chain molecule of native insulin or insulin analogue in which the insulin a and B chains are linked, in particular by C-peptide linking the insulin B and a chains.
As used herein, the term "polynucleotide fragment" refers to a nucleotide chain consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The nucleic acid fragments of the invention may be deoxyribonucleic acid fragments (DNA) or ribonucleic acid fragments (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. A nucleic acid fragment encoding insulin that can be used according to the methods provided herein can be any nucleic acid fragment encoding an insulin polypeptide or a precursor thereof (including proinsulin and preproinsulin).
The term "coding sequence" as used herein refers to a polynucleotide sequence which, when placed under the control of appropriate control sequences, is transcribed into mRNA which is translated into a polypeptide. The boundaries of the coding sequence are generally determined by a start codon, located at the beginning of the open reading frame at the 5 'end of the mRNA, and a stop codon located at the 3' end of the open reading frame of the mRNA.
The term "GLP-1 polypeptide" generally refers to GLP-1 (7-37) polypeptides and analogs thereof. GLP-1 (7-37) polypeptide refers to a polypeptide consisting of amino acids 7 to 37 of a human native GLP-1 polypeptide, and GLP-1 (7-37) polypeptide analogs refer to a polypeptide obtained by modifying human native GLP-1 (7-37) amino acids, wherein the modification comprises removing and/or substituting (replacing) and/or adding (extending) one or more amino acid residues, wherein the amino acid can be a naturally occurring amino acid or an artificially synthesized amino acid.
The term "GLP-1 related multi-target agonist polypeptide" refers to a polypeptide that possesses co-agonists that activate both the GLP-1 receptor and other diabetes related multi-target receptors, including but not limited to glucose-dependent insulinotropic polypeptide (GIP) receptor, glucagon (GCG) receptor. The co-agonist can be a GLP-1 receptor and GIP receptor double-target co-agonist (GLP-1/GIP co-agonist), a GLP-1 receptor and GCG receptor double-target co-agonist (GLP-1/GCG co-agonist), or a GLP-1 receptor, GIP receptor and GCG receptor triple-target co-agonist (GLP-1/GIP/GCG co-agonist).
The first aspect of the invention provides the use of the polypeptide as shown in SEQ ID NO. 1 as expression promoting peptide for constructing recombinant Escherichia coli engineering bacteria. The polypeptide shown as SEQ ID NO. 1 is used as an expression promoting peptide, can efficiently express some target proteins or polypeptides which are difficult to express, or remarkably enhance the target proteins or polypeptides with low expression level, and is particularly suitable for insulin precursors, GLP-1 polypeptides, GLP-1 related multi-target agonist polypeptides and the like.
The second aspect of the invention provides a fusion protein containing target protein, which is formed by sequentially connecting at least a guide peptide, an expression promoting peptide, an enzyme cutting site segment and a target protein segment, wherein the amino acid sequence of the expression promoting peptide is shown as SEQ ID NO. 1.
Wherein the amino acid sequence of the enzyme cutting site is selected from K or DDDDK (SEQ ID NO: 2). The enzyme cutting site K can be hydrolyzed by Lys-C enzyme, and the enzyme cutting site DDDDK can be hydrolyzed by enterokinase.
As a modification of the technical solution of the present invention, the leader peptide may be a leader peptide sequence that is conventional in the art, and preferably FKFEFKFE (SEQ ID NO: 7) or FEFKFEFK (SEQ ID NO: 8).
Wherein the target protein is selected from the group consisting of an insulin precursor, a GLP-1 polypeptide and a GLP-1 related multi-target agonist polypeptide, to increase the expression level thereof. As a particular embodiment of the invention, the amino acid sequence of the protein of interest is selected from the group consisting of: 3 to 6 of SEQ ID NO.
Wherein, the amino acid sequence of the protein or polypeptide related to the invention is specifically shown in the following table 1:
TABLE 1
In a third aspect, the invention provides a polynucleotide fragment for encoding the above fusion protein. As a specific embodiment of the present invention, the sequence of the polynucleotide fragment is shown in any one of SEQ ID No. 9 to SEQ ID No. 16.
The fourth aspect of the invention provides an expression vector, which comprises the polynucleotide fragment, wherein the expression vector is a recombinant pET-30a (+) expression vector.
The fifth aspect of the invention provides a recombinant escherichia coli engineering bacterium, which comprises the expression vector, and escherichia coli is preferably selected from BL21 (DE 3). Furthermore, the construction method of the recombinant escherichia coli engineering bacteria at least comprises the following steps:
(1) Synthesizing a polynucleotide fragment encoding the fusion protein of the invention;
(2) Cloning the polynucleotide fragment into an expression plasmid to construct a recombinant expression vector;
(3) And transforming the expression vector into escherichia coli to obtain the recombinant escherichia coli engineering bacteria.
Preferably, the polynucleotide sequence of the present invention is as shown in any one of SEQ ID No. 9-SEQ ID No. 16; the plasmid of the invention is preferably pET-30a (+); the Escherichia coli of the present invention is preferably BL21 (DE 3).
The sixth aspect of the present invention provides a method for preparing the above recombinant escherichia coli engineering bacteria, comprising the following steps:
(1) Synthesizing a polynucleotide encoding the fusion protein of the invention;
(2) Cloning the polynucleotide into an expression plasmid to construct a recombinant expression vector;
(3) And transforming the expression vector into escherichia coli to obtain the recombinant escherichia coli engineering bacteria.
Preferably, the polynucleotide sequence of the present invention is as shown in any one of SEQ ID No. 9 to SEQ ID No. 16; the plasmid of the invention is preferably pET-30a (+); the Escherichia coli of the present invention is preferably BL21 (DE 3).
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the invention provides an expression promoting peptide capable of obviously improving the expression quantity of a target protein in escherichia coli and a recombinant gene engineering bacterium, and can obviously enhance the expression quantity of insulin precursor protein, GLP-1 and related multi-target co-agonist polypeptide precursors thereof which are difficult to express or have low expression quantity.
Drawings
FIGS. 1 to 3 are electrophoretograms of embodiments of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
EXAMPLE 1 preparation of Long-acting acylated insulin derivatives
(1) The fusion protein sequence was designed as shown in table 2:
TABLE 2
Where "/" indicates that the sequence is not present.
(2) Plasmid construction: the coding sequences of the fusion proteins shown in Table 2 were constructed, corresponding to fusion protein numbers 1-8 in Table 3, and the specific coding polynucleotide fragments are shown in Table 3.
TABLE 3
The constructed coding nucleotide fragment is inserted into a prokaryotic expression plasmid pET-30a through Nde1 and Xho1 sites respectively, and sequencing verification is carried out to obtain a recombinant expression plasmid.
(3) Constructing recombinant engineering bacteria: adding the constructed recombinant expression plasmid into 30 mu L of BL21 (DE 3), carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 30-90 s, carrying out ice bath for 2min, adding 500 mu L of LB culture medium, carrying out shake culture at 37 ℃ for 1 h, coating on an LB solid culture plate, standing overnight at 37 ℃, selecting a monoclonal, adding the monoclonal into 5mL of LB liquid culture medium containing kanamycin, carrying out shake overnight at 37 ℃, taking 500 mu L of overnight bacterial liquid, adding 500 mu L of 50% glycerol bacteria, mixing uniformly to obtain glycerol cryopreserved bacteria, and storing at-80 ℃.
(4) Shake flask expression
The frozen strains are picked and added into 50mL LB culture medium, shaking is carried out at 37 ℃ overnight, the overnight bacterial liquid is added into the LB liquid culture medium according to the proportion of 1/100, and 0.5mM IPTG (isopropyl-beta-D-thiogalactoside) is added after 4 hours for induction expression. Shaking culture at 37 deg.C for 24 hr. Centrifuging at 7000rpm for 20 minutes to collect the bacteria.
(5) SDS-PAGE electrophoresis detection of expression results:
the obtained electrophoresis detection expression results are shown in fig. 1 to 3:
in fig. 1, lane 1 shows the expression result of the fusion protein No. 2, and lane 2 shows the expression result of the fusion protein No. 1.
As can be seen from FIG. 1, the recombinant engineered bacteria constructed using the fusion protein (No. 1) containing the peptide of the present invention can clearly see the expression of the target protein, while the recombinant engineered bacteria constructed using the fusion protein (No. 2) containing no peptide of the present invention can express no target protein or only a very low expression level.
In FIG. 2, lane 1 shows the expression result of the fusion protein of number 3; lane 2 shows the expression result of the fusion protein of reference number 4; lane 3 is a fusion protein non-induced control of number 3 (step of IPTG-induced expression was not performed in step 3).
As can be seen from FIG. 2, after the recombinant engineered bacteria constructed without the fusion protein (No. 4) of the expression promoting peptide of the invention is induced, the expression situation is similar to that of the non-induced recombinant engineered bacteria constructed with No. 3, and the target protein is not expressed basically, or the expression quantity is very low; the recombinant engineering bacteria constructed by using the fusion protein (number 3) of the expression promoting peptide can obviously express the target protein.
In FIG. 3, lane 1 shows the expression result of the fusion protein numbered 5, lane 2 shows the expression result of the fusion protein numbered 6, and lane 3 shows the uninduced expression of the fusion protein numbered 5 (i.e., the IPTG-induced expression step is not performed in step 3); lane 4 shows the expression result of the fusion protein numbered 7, lane 5 shows the expression result of the fusion protein numbered 8, and lane 6 shows the uninduced expression of the fusion protein numbered 7 (i.e., the step of inducing expression without IPTG in step 3).
As can also be seen from FIG. 3, the recombinant engineering bacteria constructed without the fusion protein of the invention containing the expression promoting peptide have substantially no expression of the target protein or have very low expression level after induction; the recombinant engineering bacteria constructed by the fusion protein of the expression promoting peptide can obviously express the target protein. As a result, as described above, the recombinant genetically engineered bacteria constructed using the sequence of the expression promoting peptide of the present invention can observe significant target protein expression after induction, while the recombinant genetically engineered bacteria constructed without the sequence of the expression promoting peptide of the present invention and the recombinant genetically engineered bacteria constructed without the sequence of the expression promoting peptide of the present invention have substantially no target protein expression or have very low expression level. As can be seen, the effect of the expression promoting peptide is remarkable.
The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (11)
1. The polypeptide shown as SEQ ID NO. 1 is used for constructing the expression promoting peptide in the recombinant Escherichia coli engineering bacteria.
2. The fusion protein containing the target protein is characterized by being formed by sequentially connecting at least a guide peptide, an expression promoting peptide, an enzyme cutting site fragment and a target protein fragment, wherein the amino acid sequence of the expression promoting peptide is shown as SEQ ID NO. 1.
3. The fusion protein of claim 2, wherein the amino acid sequence of the cleavage site is selected from the group consisting of K, DDDDK.
4. The fusion protein of claim 2, wherein the protein of interest is selected from the group consisting of an insulin precursor, a GLP-1 polypeptide, and a GLP-1 related multi-target agonist polypeptide.
5. The fusion protein of claim 2, wherein the leader peptide has an amino acid sequence as set forth in SEQ ID NO 7 or SEQ ID NO 8.
6. A polynucleotide fragment encoding the fusion protein of any one of claims 2 to 5.
7. An expression vector comprising the polynucleotide fragment of claim 6.
8. The expression vector of claim 7, wherein the expression vector is a recombinant pET-30a (+) expression vector.
9. A recombinant engineered Escherichia coli comprising the expression vector of claim 7 or 8, wherein the Escherichia coli is selected from BL21 (DE 3).
10. The recombinant engineered escherichia coli strain as claimed in claim 9, wherein the construction method of the recombinant engineered escherichia coli strain at least comprises the following steps:
(1) Synthesizing the polynucleotide fragment of claim 6;
(2) Cloning the polynucleotide fragment into a plasmid pET-30a (+), and constructing an expression vector;
(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.
11. A method for preparing the recombinant Escherichia coli engineering bacteria of claim 9, which comprises the following steps:
(1) Synthesizing the polynucleotide fragment of claim 6;
(2) Cloning the polynucleotide fragment to a plasmid pET-30a (+), and constructing an expression vector;
(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.
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| CN116693652B (en) * | 2023-08-02 | 2024-01-05 | 北京惠之衡生物科技有限公司 | GLP-1/GIP receptor dual agonist derivative and preparation method and application thereof |
| CN117756914A (en) * | 2023-12-15 | 2024-03-26 | 瀚晖制药有限公司 | Preparation method of lithocarpic insulin |
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| CN115850385B (en) | 2023-08-11 |
| CN115947822B (en) | 2023-08-18 |
| CN115947822A (en) | 2023-04-11 |
| CN115716876A (en) | 2023-02-28 |
| CN115716876B (en) | 2023-06-23 |
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