Patent Application
20230295594
The present application claims priority to Chinese patent application NO. 202010795389.9, filed on Aug. 10, 2020, entitled “Preparation and application of recombinant MANNase-GLP-1 and homologues”, the entire disclosure of which is incorporated herein by reference.
The application relates to the field of biomedical technology, and in particular, to a method for the efficient expression, purification and application of a recombinant fusion protein of MANNase and homologues thereof and GLP-1.
Diabetes is one of the prevalent diseases that seriously threaten human life and health in modern society. Diabetes is an endocrine metabolic syndrome characterized by chronic hyperglycemia. According to the National Health and Family Planning Commission, 50.1% of adults are currently in the early stage of diabetes, and they may become new diabetic patients sooner or later. More data show that the prevalence of diabetes of China has grown rapidly in recent decades: from 0.67% in the 1980s to 11.6% in 2010, an increase of 17 times with the trend in a younger age.
In recent years, Glucagon-Like peptide-1 (GLP-1) not only have excellent hypoglycemic effects, but also have the characteristics of controlling weight, regulating blood lipids, and improving the function of islet β cells. At the same time, the incidence of adverse reactions of hypoglycemia is low. The excellent effects of GLP-1 and its analogs in treating type 2 diabetes and weight loss make it have gradually occupied an important position in the diabetic therapy market in recent years. The structure of the GLP-1 is usually modified to extend its half-life. There are two types of long-term GLP-1 analogs that have been listed on the market, both of which are combined with macromolecular proteins, namely Dulaglutide and Albiglutide. The former is a combination of G4 immune albumin, the latter is a combination of serum albumin, which extend the half-life of this peptide drug. Therefore, these drugs only need to be administrated once a week. However, Albiglutide and Dulaglutide still depend on injection once a week, which cannot achieve the limitations of oral administration. After injection, there will be adverse reactions of injecting site and nausea, vomiting and abdominal pain and other gastrointestinal adverse reactions.
Mannan exists in various plants in nature. β-MANNase is the most important enzyme in the process of degradation of mannan, which is widely used in the industry. It can hydrolyze the mannan into mannan oligosaccharides. Due to the lack of corresponding enzymes in animals, mannan oligosaccharides cannot be fully degraded, but they can be metabolized by probiotics in the animal body, such as lactic acid bacteria and bifidobacteria. The products of metabolism include short-chain fatty acids (SCFA) such as acetic acid, propionic acid, and butyl. In turn, SCFA can have an important role in the body, such as inhibiting the growth of pathogenic bacteria, and supplying energy in muscles, kidneys, hearts, brains and other parts.
In order to solve the above problems existing in existing technology, this application proposes a method for the efficient expression and purification and application of a recombinant fusion protein of MANNase and homologues thereof and GLP-1. The present application uses Pichia pastoris as an engineering strain, which can efficiently express mannanase (MANNase). By constitutes a fusion protein with MANNase and GLP-1, and then through high-density fermentation culture, the fusion protein can be efficiently expressed, which has both activities of MANNase and GLP-1. During the fermentation process, it induces expression through methanol. In the clearing solution, the target protein expression is high, and the purity is high. And the purification process is a filtering concentration, optimized to have high-efficiency with a low cost. More importantly, the fusion protein, either through injection or oral administration, exerts a similar hypoglycemic effect. The technical solution used by the present invention is as follows.
The technical solution used by the present invention is as follows.
A method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1, includes the following steps.
In step (1), the plasmid is any one of pPICZαA, pPICZαB, and pPICZαC.
In step (3), the strain of Pichia pastoris is any one of X-33, GS115, KM71, SMD1168, SMD1168H.
In step (1), the sequences of the genes of encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
In step (1), this was done as follows:
The target fragments encoding GLP-1 and mannanase or homologues thereof are cloned using primer pairs. After PCR amplification and double digestion, the obtained gene sequences encoding GLP-1 and mannanase or homologues thereof are ligated to the pPICZα plasmid, which completed the construction of the recombinant expression vector.
The primer pairs include a primer pair for amplifying the gene sequence encoding GLP-1 with mannanase. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6.
In step (3), the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
In step (3), the specific steps for fermenting and culturing the recombinant engineering yeast and inducing the expression of the fusion protein MANNase-31P are as follow:
In step (S3), the fermentation culture is a high density fermentation culture.
The inducer is methanol, and the volume percentage of the inducer added is 0.2%-3%.
In step (S4), the initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4 L/min, and the pH is 5.5 when carrying out the fermentation culture.
In step (4), the specific steps for purifying, concentrating and drying the supernatant are as follows:
Application of the mannanases and homologs thereof to GLP-1 recombinant fusion proteins in the preparation of drugs for the treatment of diabetes mellitus or high-fat, high-glucose diet-induced metabolic syndrome
Pichia pastoris is a methanolotrophic yeast that grows on a medium with methanol as the only carbon source. The alcohol oxidase gene AOX1 promoter on Pichia pastoris (Saccharomyces cerevisiae) is one of the most stringent promoters known. AOX1 plays an important role in methanol oxidation, and the alcohol oxidase protein which producing account for about 30% of the total soluble protein of the yeast when methanol is the only carbon source. Placing the exogenous gene under the control of the AOX1 promoter results in a high level of expression under methanol induction.
Mannanase is the most important enzyme in the process of mannan degradation and is widely used in industry to hydrolyze mannans into mannan oligosaccharides, which, as a prebiotic, can be absorbed and metabolized by probiotic microbes present in animals, such as lactic acid bacteria and bifidobacteria, and mannanase also has the property that it is not easily degraded by digestive enzymes. The inventors of this application have innovatively recombined the protein vector of mannanase and homologue thereof with GLP-1 to construct a recombinant protein. Pichia pastoris has a strict alcohol oxidase promoter regulatory mechanism that allows it to utilize methanol as the sole carbon source, with fast cell growth and easy high-density fermentation. Therefore, the inventors of this application use the recombinant protein high-secretion expression system of Pichia pastoris to efficiently express the recombinant protein MANNase-GLP-1 and homologues thereof. Through a series of isolation and purification methods, highly efficient, stable and low-cost oral hypoglycemic peptides is obtained, breaking through the common bottleneck that existing GLP analogue fusion proteins are not resistant to gastric acid and easily degraded by various GI proteases, and realizing the oral administration of hypoglycemia.
The beneficial effects of the present invention are as follow:
The efficient expression and purification of mannanase and homologues thereof with GLP-1 recombinant fusion protein is described in the present invention. First two target fragments are cloned from existing GLP-1 synthetic sequence and plasmid containing MANNase (mannanase) or its homologues respectively, then PCR amplification, followed by double digestion. The two fragments obtained are ligated to pPICZα plasmid and constructed to obtain a recombinant expression vector. The recombinant expression vector is transformed into the receptor cells of Pichia pastoris constructed to obtain recombinant engineering yeast. And the recombinant engineering yeast are fermentation induced to express the fusion protein. The invention uses Pichia pastoris as the engineering strain, which can express mannanase efficiently. And by forming a fusion protein of mannanase and homologues thereof with GLP-1 and high-density fermentation culture, the fusion protein can be secreted and expressed efficiently and has both mannanase and GLP-1 activity. The expression was induced by methanol during the fermentation of the present invention, and high expression of the target protein in the supernatant is obtained with high purity. The purification process of the present invention is only filtration concentration, which is a cost effective preparation method. The mannanase and homologues thereof and GLP-1 recombinant fusion protein described herein is suitable for both injectable and oral administration, which have good hypoglycemic and weight-reducing effects and are of good application in obese and pre-diabetic patients, while providing a basis for further studies on the mechanism of hypoglycemia and weight reduction of mannanase and homologues thereof and GLP-1 recombinant fusion protein.
In order to more clearly illustrate the technical solutions in the embodiments or prior art of the invention, the following is a brief description of the accompanying drawings that need to be used in the description of the embodiments or prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained from them without creative effort by a person of ordinary skill in the art.
In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are described in detail below. Obviously, the described embodiments are only a part of the embodiments of the invention, and not all of them. Based on the embodiments in the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative labor fall within the scope of protection of the present invention.
The YPD medium, fermentation medium and other reagents involved in the following examples are commercially available products known to those skilled in the art.
This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1 (MANNase-GLP-1), including the steps as follow.
The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7.
Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-X-33 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.
Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5 L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4 L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 60 OD, after a 2 h starvation period, it entered the methanol induction phase. When the OD600 of the fermentation broth reached above 60, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 0.2%. During the period, the OD600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression, as shown in
The specific steps for purification, concentration and drying of the supernatant were as follows:
This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1, including the steps as follow.
The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7.
Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-GS115 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.
Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 120 OD600, after a 5 h starvation period, it entered the methanol induction phase. When the OD600 of the fermentation broth reached above 120, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 3%. During the period, the OD600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression. After 5 days of induction, the target protein was secreted into the extracellular, and the fermentation broth was collected from the tank after induction, and the supernatant was collected after centrifugation at 6000 rpm for 15 min. The content of the target protein in the supernatant was about 1-2 mg/ml.
The specific steps for purification, concentration and drying of the supernatant were as follows:
This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1, including the steps as follow.
The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7.
Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-KM71 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.
Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5 L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 90 OD600, after a 3.5 h starvation period, it entered the methanol induction phase. When the OD600 of the fermentation broth reached above 90, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 1.6%. During the period, the OD600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression, as shown in
The specific steps for purification, concentration and drying of the supernatant were as follows:
The only difference between Embodiment 4 and Embodiment 1 is that in step (3), the strain of Pichia pastoris is SMD1168, but the rest is the same as in Embodiment 1.
The only difference between Embodiment 5 and Embodiment 1 is that in step (3), the strain of Pichia pastoris is SMD1168H, but the rest is the same as in Embodiment 1.
The only difference between Embodiment 6 and Embodiment 1 is that in step (3), the steps of fermentation and culture and induction of expression of the recombinant engineering yeast are different. The specific steps for fermentation and culture of recombinant engineering yeast and induction of expression of the fusion protein MANNase-31P in this embodiment were as follow.
A single colony of the successfully constructed engineering yeast for expression of Pichia pastoris MANNase-31P-X-33 were picked and inoculated into 5 ml of YPD liquid medium and incubated overnight at 30° C. and 250 rpm in a shaker. The cultured strain was inoculated into BMGY medium at 1% and continued to shake for 24 h, after which the yeast was transferred into BMMY medium and the OD600 was adjusted to about 1.0, and the culture was continued to be induced in a shaker at 30° C. and 200 rpm, with methanol added at 0.5% by volume every 24h for 4 days. The amount of target protein in the supernatant was about 0.2 mg/ml. The protein expression by SDS-PAGE electrophoresis is shown in
1-1) Construction of a Mouse Model of Metabolic Syndrome Induced by High-Fat and High-Sugar Diet
One hundred 6-week-old (18-20 g) C57-6J mice (4-6 weeks old, male) were housed in separate cages with controlled animal room temperature of 25±2° C., humidity of 50±10%, and a 12-h light and 12-h dark cycle to acclimatize to the environment for one week. The mice were randomly divided into cages of 5-6 mice/group. Body weight and fasting blood-glucose (FBG) were measured after 12 h of fasting. The control group was fed with standard chow, and the model group was fed with high-fat, high-sugar (HFSD) chow for 24 weeks. After finishing, the body weight and FBG of each group of mice were measured.
The body weight of mice on high-fat and high-sugar diet was about 42.5 g and that of mice on normal diet was about 30 g. The fasting blood glucose of mice on high-fat and high-sugar diet was about 5.67 and that of mice on normal diet was 4.62, with statistical differences (*P<0.05, **P<0.01, ***P<0.001). As shown in
The oral glucose tolerance (OGTT) curves were obtained from regular diet mice and high-fat and high-sugar mice after overnight fasting for 12 h and gavage of D-glucose at 2 g/kg (body weight) at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 h, respectively, as shown in
HE-stained sections of liver and adipose tissue from mice on a high-fat, high-sugar diet and mice on a normal diet were analyzed. As shown in
After successful modeling, 11 animals/group were randomly grouped, plus the normal feed group, for subsequent gavage experiments.
1-2) Effect of Oral Administration of MANNase-GLP-1 on High-Fat and High-Sugar Diet-Induced Metabolic Syndrome
Mice were randomly divided into five groups, wherein fusion protein high-dose group (0.7 mg/kg-d), fusion protein-affected low-dose group (0.14 mg/kg-d), and 30 mg/kg orlistat were as positive control groups, and the normal feed control group and the high-fat, high-sugar feed as a negative control group were given the same volume of water. All mice were gavaged separately without any change in diet. Weekly weight changes were measured, and changes in fasting blood glucose were measured every two weeks. The results are shown in
2-1) Construction of a Mouse Model of Type II Diabetes Mellitus.
Fifty 6-week-old (18-20 g) BALB/C mice (males) were randomly divided into cages in groups of 8 mice each. Body weight and fasting blood-glucose (FBG) were measured after 12 h of fasting. The control group was fed with standard chow for 4 weeks and the modeling group was fed with high-fat chow for 4 weeks, and then the body weight and FBG of each group were measured. After 12 h of fasting, STZ was injected intraperitoneally at a dose of 60 mg/kg-Bw for 3 days.
The body weight and FBG of the modeled mice were measured on the 3rd, 7th, 10th and 14th days after modeling, and mice with FBG≥11.1 mmol/L and stable for one week were selected as type II diabetic model mice, and 8 mice/group were randomly divided into cages for hypoglycemia experiments.
Mice were randomly divided into three groups: diabetic negative control group (saline), diabetic positive control group (Lir), and diabetic treatment group (MANNase-GLP-1), with 8 mice in each group. All mice were gavaged with D-glucose at 1.5 g/kg (body weight) after overnight fasting for 12 h, and then subcutaneously injected with (s.c.) MANNase-GLP-1 (250 ug/50 g), and blood was collected from the tail after 0, 15, 30, 60, 120, 180 min and 300 min after injection, and the blood glucose level was measured by Roche blood glucose test paper. The results are shown in
The purpose of the present invention is to provide a method for the preparation of mannanase and homologues thereof and GLP-1 recombinant fusion protein capable of improving body weight and blood glucose in patients with high-fat and high-glucose diet-induced metabolic syndrome. Through genetic recombination technology, soluble proteins are obtained by using high density fermentation of Pichia pastoris to induce secretory expression, followed by isolation and purification by filtration and concentration to obtain high yield of target proteins. The high fermentation expression and simple isolation steps solve the limitations of the current GLP-1 analogs such as low drug yield, high cost and the need for frequent injections. More importantly, the fusion protein has hypoglycemic effect not only by injection, but also by oral administration for hypoglycemia and weight reduction, which has good application value in obese and pre-diabetic patients. It also provides a basis for further research on the mechanism of hypoglycemia and weight loss of mannanase and homologues thereof with GLP-1 recombinant fusion protein.
The above described is only a specific implementation of the present invention, but the scope of protection of the present invention is not limited to it, and any changes or substitutions that can be readily thought of by any person skilled in the art within the technical scope disclosed by the present invention shall be covered by the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the said claims.
This application proposes an efficient expression and purification method and application of MANNase and homologues thereof and GLP-1 recombinant fusion protein. Through genetic recombination technology, soluble proteins are obtained by using high density fermentation of Pichia pastoris to induce secretory expression, followed by isolation and purification by filtration and concentration to obtain high yield of target proteins. The high fermentation expression and simple isolation steps solve the limitations of the current GLP-1 analogs such as low drug yield, high cost and the need for frequent injections. More importantly, the fusion protein has hypoglycemic effect not only by injection, but also by oral administration for hypoglycemia and weight reduction, which has good application value in obese and pre-diabetic patients. It also provides a basis for further research on the mechanism of hypoglycemia and weight loss of mannanase and its homologues with GLP-1 recombinant fusion protein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010795389.9 | Aug 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/073745 | 1/26/2021 | WO |
