CN113502296A - High-expression semaglutide precursor recombinant engineering bacterium and construction method thereof - Google Patents
High-expression semaglutide precursor recombinant engineering bacterium and construction method thereof Download PDFInfo
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Abstract
The invention provides recombinant escherichia coli for efficiently expressing a semaglutide precursor. The invention constructs a fusion polypeptide structure by adding two different leader peptides and enterokinase enzyme cutting sites at the front end of a semaglutide precursor, inserts an optimized encoding gene of the polypeptide structure into a pET-30a (+) expression vector to obtain a recombinant expression vector, and converts the recombinant expression vector into escherichia coli BL21(DE3) to obtain the recombinant engineering bacterium for stably and highly expressing the semaglutide precursor. The recombinant engineering bacteria not only can stably and highly express the semaglutide precursor, but also is particularly suitable for a high-density fermentation method, and can further improve the expression quantity of target protein.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for highly expressing a semaglutide precursor and a construction method 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 invisible pre-diabetic patients exist. 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.
In recent years, glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) have become the focus of research for the treatment of T2 DM. GLP-1 promotes the synthesis and secretion of insulin in a glucose-dependent manner, and plays a role in reducing blood sugar. The Chinese medicinal composition has excellent hypoglycemic effect, has the characteristics of controlling weight, regulating blood fat, improving the functions of islet beta cells and the like, and has obviously lower adverse reaction rate of hypoglycemia and the like than other medicaments for treating diabetes. At present, GLP-1 drugs on the market for the treatment of diabetes mainly include exenatide, albiglutide, dulaglutide, liraglutide and Semaglutide (also known as somaglutide).
Semaglutide (Semaglutide) is a new generation of GLP-1 analogs developed by Danish Novonide, a long-acting GLP-1 formulation (weekly formulation). The semaglutide has high homology with human GLP-1, and is obtained by replacing amino acid at position 8 (alanine is replaced by alpha-aminobutyric acid) and amino acid at position 34 (lysine is replaced by arginine) on the basis of a natural human GLP-1 (7-37) molecule, and simultaneously connecting lysine at position 26 to a C18 fatty diacid side chain through a spacer. Through the structural adjustment, the semaglutide can resist the degradation of dipeptidyl peptidase 4, is combined with albumin to prolong the half-life period in vivo, and realizes the long-acting effect of once-a-week administration. Currently, nozalode has also introduced injectable and oral formulations of semaglutide and, in addition to the treatment of diabetes, FDA approval of semaglutide for use in weight loss indications.
Because the tail end of the semaglutide main chain is provided with a dipeptide His-Aib (Aib is an unnatural amino acid), the semaglutide is prepared by adopting the recombination fermentation of the semaglutide to express the other 29 amino acids (called semaglutide precursors for short), and then connecting the dipeptide His-Aib through chemical synthesis and connecting a side chain.
At present, the research on semaglutide is mainly focused on the structure and preparation of semaglutide, and the research on expression vectors with high expression potential and recombinant engineering bacteria thereof is less. Patent CN201811634936.4 discloses a recombinant engineering bacterium of a semaglutide precursor and a construction method thereof, but the expression quantity of an inclusion body is about 145 g/L.
The prepared recombinant engineering bacteria with high expression capability and the research and development of a high-expression fermentation method thereof can improve the amount of semaglutide precursors in unit volume, remarkably reduce the cost of semaglutide raw materials, greatly reduce the price of medicines, and have great significance to enterprises and patients. The invention provides a recombinant engineering bacterium with high expression capacity of semaglutide precursor, a construction method thereof and a high-density fermentation method thereof.
Disclosure of Invention
The invention aims to solve the problem of low fermentation expression quantity in the prior art, and provides a novel recombinant engineering bacterium for stably and highly expressing the semaglutide precursor and a construction method thereof. The semaglutide is developed and developed by Novonide company, is prepared by a gene recombination technology, and has the following structure:
in the structure of semaglutide, the dipeptide of the terminal sequence is His-Aib (Aib is an unnatural amino acid) which can not be obtained by direct fermentation expression, so that a recombinant engineering bacterium for constructing a polypeptide fragment (semaglutide precursor) for expressing semaglutide except the dipeptide is generally adopted, the semaglutide precursor is obtained by fermentation and then synthesized with the dipeptide to obtain a complete semaglutide peptide sequence, and the 26 th lysine of the peptide sequence is connected with a C18 fatty diacid side chain through a spacer to obtain a semaglutide raw material. The complete peptide sequence structure of the semaglutide is H-Aib-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG; the semaglutide precursor sequence is a part without dipeptide H-Aib-, and the sequence is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 1).
Constructing recombinant engineering bacteria by taking escherichia coli as host bacteria, and further obtaining an inclusion body containing a semaglutide precursor by a high-density fermentation method; a preferred host bacterium is E.coli BL21(DE 3).
In order to realize the purpose of the invention and increase the expression amount of the semaglutide precursor in an inclusion body of a prokaryotic expression system, the inventor designs a fusion polypeptide of leader peptide-enterokinase enzyme cutting site-amino acid structure of a semaglutide precursor sequence. Wherein, the leader peptides are two kinds, namely leader peptide A1, and the amino acid sequence of the leader peptides is FEFKFEFK (SEQ ID NO. 2); and leader peptide A2, the amino acid sequence of which is FKFEFKFE (SEQ ID NO. 3); the amino acid sequence of the enterokinase enzyme cutting site is DDDDK (SEQ ID NO. 4). Thus, the amino acid sequences of the two fusion polypeptides constructed by the invention are FEFKFEFKDDDDKEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID No. 5) and FKFEFKFEDDDDKEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID No. 6), respectively. The fusion polypeptide can be digested by enterokinase to obtain a semaglutide precursor shown in SEQ ID No. 1.
Aiming at the two amino acid structures designed by the invention, in order to obtain the recombinant engineering bacteria which are suitable for high-density fermentation and stably express the target protein inclusion body, the inventor preferably designs two groups of optimized encoding gene segments of the fusion polypeptide, which are respectively shown as SEQ ID No.7 and SEQ ID No. 8. Wherein, SEQ ID No.7 is the optimized coding gene sequence of the fusion polypeptide shown in the SEQ ID No.5, and SEQ ID No.8 is the optimized coding gene sequence of the fusion polypeptide shown in the SEQ ID No. 6.
Therefore, the invention provides an optimized coding gene segment for coding the fusion polypeptide, and the nucleotide sequences of the optimized coding gene segment are respectively shown as SEQ ID No.7 and SEQ ID No. 8.
Furthermore, the invention provides a vector containing the optimized coding gene segment, wherein the vector is obtained by inserting the sequences shown in SEQ ID No.7 and SEQ ID No.8 into corresponding enzyme cutting sites of the vector. Preferably, the vector is a pET-30a (+) expression vector by adding at the 5' end of SEQ ID No.7 or SEQ ID No.8NdeI cleavage site, 3' terminal addition of stop codon andXhoafter the enzyme cutting site I, cloning to the expression vector by enzyme cuttingNdeI andXhoi is obtained between enzyme cutting sites.
Further, the invention provides a recombinant escherichia coli stably and highly expressing the semaglutide precursor, preferably, the recombinant escherichia coli is obtained by transforming the vector containing the optimized coding gene segment into escherichia coli BL21(DE 3).
On the other hand, the invention provides a construction method of the recombinant engineering bacteria for stably and highly expressing the semaglutide precursor, which comprises the following steps:
(1) synthesizing a fusion polypeptide coding gene shown as SEQ ID No.7 or SEQ ID No. 8;
(2) connecting the coding gene to an expression vector to construct a recombinant expression vector;
(3) and transforming the constructed recombinant expression vector into escherichia coli to obtain the recombinant engineering bacteria.
Preferably, the specific method in step (2) is as follows: adding the 5' end of the coding gene sequenceNdeI cleavage site, 3' terminal addition of stop codon andXhoi cleavage site, then cloning to expression vector pET-30a (+) by enzyme digestionNdeI andXhoi enzyme cutting sites, thereby obtaining a recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1.
Further preferably, the specific method of step (3) is: the recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1 is transformed and introduced into an escherichia coli expression host BL21(DE3) through a heat shock method, and the recombinant engineering bacteria are obtained through screening.
In addition, the present invention also provides a fermentation method of the semaglutide precursor, which comprises the following steps:
(1) recovering the recombinant engineering bacteria: inoculating the preserved recombinant engineering bacteria prepared by the method of the invention into a culture medium for overnight culture to obtain a resuscitation bacteria liquid;
(2) inducing expression: inoculating the resuscitation bacterial liquid cultured in the step (1) into a fermentation medium, and inducing expression;
(3) collecting thalli, carrying out ultrasonic disruption on the thalli, and washing to obtain an inclusion body containing the semaglutide precursor.
Preferably, step (1) of the above fermentation method is specifically: inoculating the preserved recombinant engineering bacteria into a sterilized LB culture medium, and culturing overnight at 37 ℃ under the condition of 200 rpm;
further preferably, the step (2) is specifically: inoculating the resuscitation bacterial liquid in the step (1) into a sterilized LB culture medium, and culturing at 37 ℃ and 220rpm until the bacteria reach OD600When the molar ratio is 0.6 to 0.8, IPTG is added for induction, and the culture is carried out overnight at 37 ℃ and 220 rpm.
The invention designs a fusion protein structure containing the semaglutide precursor by utilizing an escherichia coli prokaryotic expression system, inserts the fusion protein structure into an expression vector and expresses the fusion protein structure in a form of inclusion body, thereby realizing the stable and high expression of the semaglutide precursor; the recombinant engineering bacteria are particularly suitable for high-density fermentation, and have high industrial application value and prospect.
Drawings
FIG. 1 is SDS-PAGE electrophoresis chart of A1-GLP-1 series recombinant engineering bacteria: wherein lanes 1-2 and 4-9 correspond to recombinant strains A1-GLP-1-1 to A1-GLP-1-8, respectively, lane 3 is Marker, and lane 10 is non-induced sampling of the recombinant strain A1-GLP-1-1;
FIG. 2 is an SDS-PAGE electrophoresis chart of A2-GLP-1 series recombinant engineering bacteria: wherein, lanes 2-9 correspond to recombinant strains A2-GLP-1-1 to A2-GLP-1-8 respectively, lane 1 is Marker, and lane 10 is non-induced sampling of the recombinant strain A2-GLP-1-1;
FIG. 3 is a graph showing the results of HPLC expression level detection;
FIG. 4 is a high-density fermentation growth curve diagram of recombinant engineering bacteria A2-GLP-1-5.
Detailed Description
The following examples are presented in conjunction with the accompanying drawings for further understanding of the present invention and should not be construed as limiting the invention.
Example 1: construction of recombinant escherichia coli engineering bacteria for stably and highly expressing semaglutide precursor
(1) Respectively synthesizing fusion polypeptide coding genes shown as SEQ ID No.7 and SEQ ID No.8, and respectively adding fusion polypeptide coding genes at 5' ends of the sequencesNdeI cleavage site, 3' terminal addition of stop codon andXhoi, enzyme digestion site;
(2) cloning the gene fragments prepared in the step (1) to an expression vector pET-30a (+) through enzyme digestionNdeI andXhoi enzyme cutting sites, thereby obtaining 2 recombinant expression vectors pET-30a (+) -A1-GLP-1 and pET-30a (+) -A2-GLP-1;
(3) and (3) respectively transforming the recombinant expression vectors obtained in the step (2) into escherichia coli BL21(DE3) through a heat shock method, screening monoclonals through resistance, respectively selecting 8 clones from the two recombinant engineering bacteria, respectively naming the clones as A1-GLP-1-1 to A1-GLP-1-8 and A2-GLP-1-1 to A2-GLP-1-8, and storing the clones in glycerol.
The sequence of the genetically engineered bacteria obtained in this example was identical to the designed sequence by sequencing.
Example 2: fermentation of recombinant escherichia coli engineering bacteria for stably and highly expressing semaglutide precursor
(1) Recovery of recombinant engineering bacteria
Each of the 16 glycerol bacteria stored in example 1 was inoculated in one thousandth of the inoculum size into 16 flasks containing 20 ml of sterilized LB medium, and cultured overnight at 37 ℃ and 200rpm to obtain a resuscitative solution.
(2) Induced expression of recombinant engineering bacteria
Inoculating the resuscitating bacteria liquid obtained in the step (1) into 16 triangular flasks filled with 50 ml of sterilized LB culture medium according to the percent inoculation amount respectively, and culturing to OD under the conditions of 37 ℃ and 220rpm600When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the overnight induction expression was carried out at 37 ℃ and 220 rpm.
(3) Thallus collection and HPLC expression level detection
Collecting culture bacteria liquid, centrifuging at 8000rpm and 4 deg.C for 30min, removing supernatant to obtain thallus; the thalli is subjected to ultrasonic disruption to obtain inclusion bodies, the inclusion bodies are washed and dissolved and then subjected to expression quantity detection by HPLC (see a result in figure 3), and meanwhile, thalli retention samples are subjected to SDS-PAGE to detect the expression condition of protein (all lanes are performed under the same experimental conditions, and the results are shown in figures 1 and 2).
The SDS-PAGE detection results of 8 different monoclonals obtained from the recombinant engineering bacteria of the A1-GLP-1 series constructed by using the leader peptide A1 are shown in figure 1, and the SDS-PAGE detection results of 8 different monoclonals obtained from the recombinant engineering bacteria of the A2-GLP-1 series constructed by using the leader peptide A2 are shown in figure 2. As can be seen from the electrophoresis charts of FIG. 1 and FIG. 2, the recombinant engineered bacteria constructed by the present invention can efficiently express the inclusion body of the target protein. Meanwhile, the electrophoresis chart shows that the overall expression quantity of the series of recombinant engineering bacteria constructed by using the leader peptide A2 is higher than that of the series of recombinant engineering bacteria constructed by using the leader peptide A1.
The HPLC expression level test result is shown in figure 3, and the result graph shows that the recombinant engineering bacteria prepared by the method have higher inclusion body expression level. Meanwhile, compared with leader peptide A1, leader peptide A2 significantly increased the expression level of the fusion protein containing the semaglutide precursor, which is consistent with the SDS-PAGE result. Specifically, as can be seen from the results in FIG. 3, the expression level of the inclusion body of the target protein in the recombinant engineered bacteria constructed by using leader peptide A1 is about 8mg/g on average, and is 9.76mg/g at most; the recombinant engineering bacteria constructed by using leader peptide A2 not only have the expression quantity of all strains above 11mg/g, but also have more than half of the expression quantity exceeding 14mg/g and the highest expression quantity reaching 20.67 mg/g; compared with the recombinant engineering bacteria using the leader peptide A1, the expression amount is approximately doubled.
Example 3: high-density fermentation of recombinant escherichia coli engineering bacteria for stably and highly expressing semaglutide precursor
Respectively activating 16 recombinant engineering bacteria constructed in the embodiment 1 by secondary strains, and then performing high-density fermentation, wherein in the fermentation process, sampling every 2h after feeding is started, and measuring OD (origin-to-destination) values600Values, and drawing a growth curve; after the fermentation was completed, the cells were collected by centrifugation.
According to the fermentation result, the A1-GLP-1 series recombinant engineering bacteria and the A2-GLP-1 series recombinant engineering bacteria can be fermented to high OD600The value, especially the A2-GLP-1 series recombinant engineering bacteria, can reach OD600The value is above 240, and the biomass of the inclusion body bacteria reaches above 300 g/L. Taking A2-GLP-1-5 as an example, the fermentation OD thereof600The value can reach 259, the biomass of the inclusion body thallus reaches 320g/L, and the growth curve is shown in figure 4. Moreover, it can be expected that the recombinant engineering bacteria of the present application can obtain higher expression level through further adjustment and optimization of fermentation.
Sequence listing
<110> Beijing-Hui-Heng Biotechnology Ltd
Jilin Huisheng biopharmaceutical Co.,Ltd.
<120> high-expression semaglutide precursor recombinant engineering bacterium and construction method thereof
<141> 2021-08-19
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Claims (10)
1. An optimized gene segment for coding fusion polypeptide, the sequence of which is shown as SEQ ID No.7 or SEQ ID No. 8.
2. A recombinant expression vector comprising the gene fragment of claim 1.
3. A recombinant engineered bacterium expressing a semaglutide precursor, comprising the recombinant expression vector of claim 2.
4. The recombinant engineering bacterium of claim 3, wherein the recombinant expression vector is a recombinant pET-30a (+) expression vector, and the host engineering bacterium is selected from Escherichia coli BL21(DE 3).
5. The recombinant engineering bacterium according to claim 4, wherein the engineering bacterium is constructed according to the following method:
(1) synthesizing a fusion polypeptide coding gene shown in SEQ ID No.7 or SEQ ID No. 8;
(2) connecting the coding group to an expression vector pET-30a (+) to construct a recombinant expression vector;
(3) and (3) transforming the recombinant expression vector constructed in the step (2) into escherichia coli BL21(DE3) to obtain the recombinant engineering bacterium.
6. The recombinant engineering bacterium according to claim 5, wherein the step (2) of the method is specifically: adding the 5' end of the coding gene sequence in the step (1)NdeI cleavage site, 3' terminal addition of stop codon andXhoi cleavage site, then cloning to expression vector pET-30a (+) by enzyme digestionNdeI andXhoi enzyme cutting sites, thereby obtaining a recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1.
7. The recombinant engineering bacterium according to claim 6, wherein the step (3) of the method is specifically: the recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1 is transformed and introduced into an escherichia coli expression host BL21(DE3) through a heat shock method, and the recombinant gene engineering bacteria are obtained through screening.
8. A construction method of recombinant engineering bacteria for expressing a semaglutide precursor comprises the following steps:
(1) synthesizing a fusion polypeptide coding gene shown in SEQ ID No.7 or SEQ ID No. 8;
(2) connecting the coding group to an expression vector pET-30a (+) to construct a recombinant expression vector;
(3) and (3) transforming the recombinant expression vector constructed in the step (2) into escherichia coli BL21(DE3) to obtain the recombinant genetic engineering bacteria.
9. The construction method according to claim 8, wherein the step (2) of the method is specifically: adding the 5' end of the coding gene sequence in the step (1)NdeI cleavage site, 3' terminal addition of stop codon andXhoi cleavage site, then cloning to expression vector pET-30a (+) by enzyme digestionNdeI andXhoi enzyme cutting sites, thereby obtaining a recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1.
10. Construction method according to any one of claims 8 to 9, characterized in that step (3) of the method is in particular: the recombinant expression vector pET-30a (+) -A1-GLP-1 or pET-30a (+) -A2-GLP-1 is transformed and introduced into an escherichia coli expression host BL21(DE3) through a heat shock method, and the recombinant gene engineering bacteria are obtained through screening.
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| CN114621340A (en) * | 2022-03-11 | 2022-06-14 | 北京惠之衡生物科技有限公司 | Acylated long-acting GLP-1 derivative |
| CN114621339A (en) * | 2021-12-28 | 2022-06-14 | 北京惠之衡生物科技有限公司 | Long-acting GLP-1 derivative |
| CN114716533A (en) * | 2022-04-12 | 2022-07-08 | 北京惠之衡生物科技有限公司 | Acylated long-acting GLP-1 derivative |
| CN114790474A (en) * | 2022-06-23 | 2022-07-26 | 北京惠之衡生物科技有限公司 | Preparation method of Somalutide |
| CN114807101A (en) * | 2022-06-20 | 2022-07-29 | 北京惠之衡生物科技有限公司 | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof |
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| CN115850385A (en) * | 2022-07-04 | 2023-03-28 | 北京惠之衡生物科技有限公司 | Expression promoting peptide and application thereof |
| WO2023125178A1 (en) * | 2021-12-31 | 2023-07-06 | 南京汉欣医药科技有限公司 | Fusion protein and method for preparing semaglutide intermediate polypeptide therefrom |
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