KR101813703B1 - Method for mass producing monoacylglycerol lipase - Google Patents

Abstract

The present invention relates to a method for mass production of monoacylglycerol lipase (MGL), and more particularly, to a method for mass production of monoacylglycerol lipase (MGL), which comprises a recombinant vector containing a gene encoding a monoacylglycerol lipase protein, Culturing the transformant, and isolating and purifying the monoacylglycerol lipase protein from the transformant. The present invention also relates to a method for mass production of monoacylglycerol lipase.
The monoacylglycerol lipase protein produced by the mass production method of the present invention completely decomposes triglyceride into fatty acid and glycerol in the gastrointestinal tract, thereby delaying fat absorption and reducing blood absorption of triglyceride. When the glycerol is decomposed into monoacylglycerol lipase, it may delay the neutral fat recombination in the digestive epithelial cells or accelerate the energy consumption of the digestive epithelium even if it is absorbed. Therefore, / Or metabolic syndrome such as obesity.

Description

TECHNICAL FIELD The present invention relates to a method for mass production of monoacylglycerol lipase,

The present invention relates to a method for mass production of monoacylglycerol lipase (MGL), and more particularly, to a method for mass production of monoacylglycerol lipase (MGL), which comprises a recombinant vector containing a gene encoding a monoacylglycerol lipase protein, Culturing the transformant, and isolating and purifying the monoacylglycerol lipase protein from the transformant. The present invention also relates to a method for mass production of monoacylglycerol lipase.

Recently, metabolic syndrome-related diseases including diabetes such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, type 2 diabetes, diabetes mellitus, and nonalcoholic fatty liver have been increasing rapidly due to changes in economic development and eating habits It is a situation. These diseases often occur, but generally they are closely related to each other and are often accompanied by various symptoms.

Obesity is a disease group that most threatens the health of modern people. High-level obesity lowers insulin sensitivity and causes many metabolic changes in the body, resulting in many complications including vasculature and nervous system, resulting in death. Therefore, it is very important for the medical community to develop a lifestyle that can reduce obesity or a therapeutic drug for it.

Obesity results from an imbalance in the consumption of energy and in the use of energy. First, the energy intake is the hypothalamus of the brain, which is regulated by hormones such as leptin and ghrelin in the body. Nutrients once consumed and absorbed in the digestive tract are never excreted, used as energy or stored in the body. Therefore, in the treatment of obesity, efforts have been made to develop an obesity inhibitor as a mechanism to inhibit energy intake, and appetite suppressants such as purines and reductants are used.

Another way to reduce energy intake is to inhibit the absorption of fat in the digestive tract. The drug used in this method is Xenical, a drug that inhibits lipase, which is a lipase of interest. When Xenical is administered, the triglyceride is not decomposed and is not absorbed in the digestive tract but is discharged to the feces.

The second method is to increase the use of energy. Often, the way people cope with obesity is to "eat less and exercise a lot," so it can be said that obesity therapy is sure to increase energy consumption by exercise. Recently, efforts have been made to promote energy consumption by drugs, and studies have been conducted in order to promote energy consumption by increasing the expression of mating proteins, which are mainly present in brown fat.

However, in the case of appetite suppression, most of the drugs acting on the central nervous system are inevitably accompanied by inevitable nervous system side effects. The reduction of appetite also has a problem that accompanies depression. In the case of inhibiting Xenical, patients are reluctant to take their medicines due to the fats and odors, and the inconvenience of social life due to the control of the doctor. The method of accelerating the energy consumption using the conjugate protein is still in the development stage and it is difficult to use it for the person until the specificity of the drug is secured according to the specificity of the organ.

In addition, commercially available anti-obesity agents include thiazolidinediones (TZDs), Xenical (Korean Roche), and sibutramine. Side effects of these drugs include cardiovascular, central nervous, hepatic and renal disorders And so on. Therefore, there is an urgent need to develop high value-added, multi-functional products that prevent side effects from long-term ingestion and prevent and treat obesity.

Monoacylglycerol lipase (MGL), on the other hand, is known to cleave monoacylglycerol to form free fatty acids and glycerol. In the prior art, the expression of monoacylglycerol lipase in the intestines leads to obesity-induced obesity (Chon, et al., FASEB , 22: 807, 2008), by inhibiting monoacylglycerol lipase, has been reported to cause pain, inflammation and central nervous system (CNS) (Schlossburg et al., Nat. Neurosci . , 13 (9): 1113, 2010). These studies report that monoacylglycerol lipase acts in the body's cells, and there is no report of its action in the digestive tract and no monoacylglycerol lipase activity in the human digestive tract.

Under these circumstances, the present inventors have found that when monoacylglycerol lipase is allowed to act in the gastrointestinal tract, it completely decomposes triglyceride into fatty acid and glycerol in the gastrointestinal tract, thereby delaying fat absorption, reducing blood absorption of triglyceride, When monoacylglycerol lipase is degraded by monoacylglycerol lipase, monoacylglycerol lipase is effective in delaying triglyceride recombination in digestive epithelial cells or promoting energy consumption in the digestive epithelium. Therefore, when monoacylglycerol lipase is used for the treatment of diabetic retinopathy, nonalcoholic fatty liver, Type diabetes mellitus and / or obesity and to develop a method for mass production of monoacylglycerol lipase protein. As a result, it has been found that the monoacylglycerol lipase protein-specific Mass production methods By the present invention it has been completed.

It is an object of the present invention to provide a recombinant vector comprising a gene encoding a monoacylglycerol lipase protein.

Another object of the present invention is to provide a transformant into which said recombinant vector has been introduced.

It is another object of the present invention to provide a method for mass production of monoacylglycerol lipase.

In order to solve the above-mentioned problems, the present invention provides a recombinant vector comprising a gene encoding a monoacylglycerol lipase protein.

According to a preferred embodiment of the present invention, the monoacylglycerol lipase may be derived from any one selected from the group consisting of a human, a mouse, a rat and a pig.

According to another preferred embodiment of the present invention, the gene coding for the monoacylglycerol lipase protein may comprise the nucleotide sequence of SEQ ID NO: 5 or 6.

According to another preferred embodiment of the present invention, the monoacylglycerol lipase protein may be composed of the amino acid sequence of SEQ ID NO: 7 or 8.

According to another preferred embodiment of the present invention, the recombinant vector may be pT7-HMT.

The present invention also provides a transformant into which the aforementioned recombinant vector has been introduced.

According to a preferred embodiment of the present invention, the transformant may be E. Coli .

According to another preferred embodiment of the present invention, the E. Coli may be BL21 (DE3).

The present invention also provides a method for producing a recombinant vector comprising the steps of: a) preparing a recombinant vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 5 or 6; b) transforming the recombinant vector into E. coli and then culturing; And c) separating and purifying the monoacylglycerol lipase from the cultured E. coli transformed cells; And a method for mass production of monoacylglycerol lipase.

According to a preferred embodiment of the present invention, in step a), the gene consisting of the nucleotide sequence of SEQ ID NO: 5 is amplified into a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a pair of reverse primers consisting of the nucleotide sequence of SEQ ID NO: .

According to another preferred embodiment of the present invention, in step a), the gene consisting of the nucleotide sequence of SEQ ID NO: 6 is amplified by a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a pair of reverse primers consisting of the nucleotide sequence of SEQ ID NO: .

According to another preferred embodiment of the present invention, the recombinant vector of step a) may be pT7-HMT.

According to another preferred embodiment of the present invention, E. coli in step b) may be a BL21 (DE3) strain.

According to another preferred embodiment of the present invention, the culture in step b) may be performed by adding IPTG (Isopropyl beta-D-1-thiogalactopyranoside) for the expression of monoacylglycerol lipase protein.

According to another preferred embodiment of the present invention, the purification in step c) may be performed sequentially with Ni-NTA chromatography, protein elution and Fast protein liquid chromatography.

According to another preferred embodiment of the present invention, the elution of the protein may be performed with an ethylenediaminetetraacetic acid (EDTA) solution.

According to another preferred embodiment of the present invention, the high-speed protein liquid chromatography may be performed by sequentially performing hydrophobic interaction chromatography and ion exchange chromatography (Ion Exchange Chromatography).

The present invention provides a mass production method specialized for a monoacylglycerol lipase protein, and the monoacylglycerol lipase protein produced by the mass production method is useful for the treatment of diabetic complications such as diabetes, nonalcoholic fatty liver, hyperlipidemia, type 2 diabetes and / It has an excellent effect of preventing, ameliorating or treating metabolic syndrome, and thus can be usefully used as a raw material for medicines and health functional foods for preventing, improving or treating the diseases.

Fig. 1 is a schematic diagram showing the structure of triglycerides. Fig.
FIG. 2 is a schematic view showing the digestion and absorption process of triglyceride in the gastrointestinal tract. Two fatty acids degraded by the lipase of interest and monoacylglycerol are absorbed into digestive epithelial cells, It is released into the lymphatic system in the form of microns.
FIG. 3 is a schematic view showing the pathway of triglycerol synthesis of fat absorbed in digestive epithelial cells after digestion of triglycerides. In the presence of monoacylglycerol lipase (MGL), triglycerides are completely neutralized with three fatty acids and glycerol Because it is degraded, it consumes more energy to reform the triglyceride in digestive epithelial cells.
4 shows gene bank numbers and amino acid sequences of monoacylglycerol lipase (MGL) in human and mouse.
Figure 5 shows a bacterial expression vector system prepared for the mass production of monoacylglycerol lipase (MGL).
FIG. 6 shows the results of SDS-PAGE after separation and purification of monoacylglycerol lipase (MGL). In FIG. 6, 1 denotes dialysis pre-dialysis and 2 denotes dialysis dialysis protein.
FIG. 7 is a diagram illustrating a pure separation and purification process of monoacylglycerol lipase (MGL).
FIG. 8 shows the result of measuring the body weight change by administering monoacylglycerol lipase to ob / ob mice induced obesity for about 3 weeks.
FIG. 9 is a picture showing mouse shape, liver and epididymal fat (Epi-WAT) after monoacylglycerol lipase was administered to ob / ob mice induced obesity for about 3 weeks.
Fig. 10 shows the results of a 6-week-old mouse in which pig pancreas lipase (control) and Candida rugosa lipase (experimental group) for 7 weeks, respectively.
11 is data showing changes in blood triglyceride concentration by monoacylglycerol lipase (MGL) in a mouse to which olive oil was administered.
FIG. 12 is a graph showing changes in blood triglyceride concentration by monoacylglycerol lipase in mice administered with olive oil according to time, and is shown according to the presence or absence of tyroxapol administration.
FIG. 13 shows the results of measuring the activity of enzymes acting in the small intestine by sacrificing the mice 1 hour and 2 hours after monoacylglycerol lipase administration.
FIG. 14 is a graph showing that the ability of the small intestinal epithelial cell to recombine in the small intestine epithelial cell line Caco-2 cells is reduced by the administration of monoacylglycerol lipase.
FIG. 15 is a graph showing changes in dietary amount and lipid content of fat during administration of monoacylglycerol lipase to ob / ob mice induced obesity.
FIG. 16 is a graph showing the results of glucose tolerance test after administering monoacylglycerol lipase to ob / ob mice induced obesity for about 3 weeks.

Hereinafter, the present invention will be described in more detail.

As described above, drugs used as drugs for the treatment of metabolic syndrome such as diabetes insipidus, nonalcoholic fatty liver disease, hyperlipidemia, type 2 diabetes and / or obesity cause side effects such as heart disease, respiratory diseases, blood pressure increase and insomnia, There is a problem in that the sustainability of the system is low.

Accordingly, the inventors of the present invention confirmed that monoacylglycerol lipase exerts an excellent effect in the treatment of metabolic syndrome such as hepatic insufficiency, nonalcoholic fatty liver, hyperlipidemia, type 2 diabetes and / or obesity, As a result of intensive efforts to develop a method for mass production of glycerol lipase protein, the present invention has been completed by deriving a mass production method specialized for monoacylglycerol lipase protein.

The monoacylglycerol lipase protein produced by the mass production method of the present invention completely decomposes triglyceride into fatty acid and glycerol in the gastrointestinal tract, thereby delaying fat absorption and reducing blood absorption of triglyceride. Thus, monophosphatidylcholine, And can be usefully used in medicines and health functional foods for preventing, ameliorating or treating metabolic syndrome such as fatty liver, hyperlipidemia, type 2 diabetes and / or obesity.

As shown in FIG. 1, triglyceride has a form in which three fatty acids are bound to glycerol, and lipase, which is released from the human body, is a representative enzyme for degrading triglyceride in the human digestive tract. However, since the lipase is an incomplete lipase, it decomposes the fatty acids 1 and 3 but does not liberate the fatty acid bound to the 2, so that the product by the enzyme is present as two fatty acids and one monoacyl glycerol .

FIG. 2 is a schematic diagram showing the digestion and absorption process of triglyceride in the gastrointestinal tract. In FIG. 2, two fatty acids degraded by the lipase of interest and monoacylglycerol are absorbed into digestive epithelial cells and re- It is released into the lymphatic system in the form of microns.

In the process of binding the two fatty acids and the monoacylglycerol to the triglyceride, an enzyme called MGAT2 exists in the digestive epithelium. As shown in the upper part of FIG. 3, the enzyme binds monoacylglycerol with a fatty acid, Form glycerol, and the formed diacylglycerol soon becomes triglyceride. After reforming the triglyceride, it is necessary to form chylomicronous lipoprotein, but it can be circulated in the lymphatic system or vascular system while being dissolved.

As shown in the lower part of FIG. 3, assuming that monoacylglycerol lipase is used to decompose 2-monoacylglycerol into fatty acid and glycerol in the metabolic pathway, eventually the reforming pathway of triglyceride is changed. Glycerol consumes energy and needs to be phosphorylated, so that two fatty acids are attached to the phosphatidic acid, and then the phosphoric acid falls and diacylglycerol is formed to form the triglyceride. Since the above process is such that 3 to 4 ATPs are consumed more than 2-monoacylglycerol directly forms a triglyceride, energy consumption can be promoted and obesity can be prevented or treated.

The present inventors have found that the oral administration of monoacylglycerol lipase completely degrades triglycerides into fatty acids and glycerol in the gastrointestinal tract and promotes absorption of fat and delayed regeneration / energy consumption of the gastrointestinal tract, Diabetes and / or obesity (Figs. 8 to 16).

Accordingly, in the present invention, a recombinant vector containing a gene encoding a monoacylglycerol lipase protein was produced in order to mass-produce monoacylglycerol lipase.

The monoacylglycerol lipase may be derived from any one selected from the group consisting of human, mouse, white, and pig, but is not limited thereto.

In one embodiment of the present invention, an open reading frame (ORF) portion of human or mouse-derived monoacylglycerol lipase mRNA was amplified and used in the production of a recombinant vector. As shown in Fig. 4, the mRNA sequence of the human monoacylglycerol lipase may be those disclosed in GenBank Number NM001003794, and the mRNA sequence of the mouse-derived monoacylglycerol lipase may be those disclosed in GenBank Number nm_011844.

The sequence of the amplified product introduced into the recombinant vector may be a nucleotide sequence of SEQ ID NO: 5 (human MGL) and SEQ ID NO: 6 (mouse MGL), but is not limited thereto. The human monoacylglycerol lipase protein may be the amino acid sequence of SEQ ID NO: 7 and the mouse-derived monoacylglycerol lipase protein may be the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

If necessary, the amino acid sequence of the human or mouse-derived monoacylglycerol lipase shown in FIG. 4 may be modified to increase the enzyme activity, and the partial sequence of the monoacylglycerol lipase may be modified to produce monoacylglycerol from the recombinant microorganism Can be increased.

The term "recombinant vector" in the present invention means a means for efficiently expressing monoacylglycerol lipase protein in a microorganism by introducing DNA into a host cell. In the production of the recombinant vector, not only human or mouse-derived monoacylglycerol lipase, Depending on the kind of the host cell to which the monoacylglycerol lipase protein is to be produced, an expression regulatory sequence such as a promoter, a terminator, an inhancer, etc., a sequence for membrane targeting or secretion, Can be variously combined.

Examples of recombinant vectors that can be used in the present invention include but are not limited to pT7-HMT or pET system vectors such as pET-14b, pEt-15b, pEt-16b, pEt-19b, pEt- (+), pEt-29b (+), pEt-30b (+), pEt-31b (+), pEt- pEt-43.1b (+), and preferably pT7-HMT vector can be used. The pT7-HMT vector is a vector that is expressed in bacteria by fusing six His-tags and a target protein. The pT7-HMT vector can easily purify the expressed protein. The TEV protease can be used to separate the tag There are advantages.

The recombinant vector according to an embodiment of the present invention may be a bacterial expression vector system prepared for mass production of monoacylglycerol lipase (MGL), which has a cleavage map of FIG.

The present invention also provides a transformant into which the aforementioned recombinant vector has been introduced.

The term "transformed" in the present invention means that a gene can be introduced into a host cell and expressed in a host cell.

The host cell used for transformation according to the present invention may be any host cell known in the art, but it is possible to use a host having high efficiency of introduction and expression efficiency of the monoacylglycerol lipase gene. For example, , Fungal, yeast, plant or animal (e.g., mammalian or insect) cells. In one embodiment of the present invention, an E. coli system capable of expressing a monoacylglycerol lipase protein in a large amount was used, and more preferably BL21 (DE3) strain was used.

The present invention also provides a method for mass production of a monoacylglycerol lipase protein comprising the steps of:

a) preparing a recombinant vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 5 or 6;

b) transforming the recombinant vector into E. coli and then culturing; And

c) separating and purifying the monoacylglycerol lipase from the cultured E. coli .

First, as a step, a recombinant vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 5 or 6 is prepared. The construction for the recombinant vector is as described above. The production of the recombinant vector can be carried out using a commonly used cloning method. In the present invention, total RNA derived from human or mouse was extracted from adipose tissue and PCR was carried out using each primer pair shown in Table 1 as a template. The primer sequences shown in Table 1 were produced by the present inventors including restriction enzyme sites so that the amplified genes can be inserted into vectors.

Next, in step b), the recombinant vector is transformed into E. Coli and then cultured. The constitution for the transformed E. coli is the same as that for the transformants described above. Methods for introducing a recombinant vector of the present invention into a host cell and transforming the host cell include, for example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome mediated transfection known methods such as liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, and the like , But is not limited thereto. The transformed E. coli can be further cultured by adding IPTG to express the monoacylglycerol lipase protein. IPTG concentration of 0.5 ~ 1 mM was optimal for the expression of this protein, and the culture temperature was 16 ℃ for about 16 hours, and protein expression and dissolution rate were high.

The monoacylglycerol lipase is isolated and purified from the transformed E. coli cultured as the next step c). In the present invention, the purification of the monoacylglycerol lipase protein was facilitated by using His-tag. Specifically, the separation and purification method can be performed by sequentially performing Ni-NTA chromatography, protein elution, and fast protein liquid chromatography. In the process of preparing homogeneous solution of bacterial protein for Ni-NTA chromatography, the solubility of the protein was found to vary greatly depending on the composition of the homogeneous solution. Thus, the present inventors found the following conditions for optimal protein expression and optimal solubility. The protein was dissolved in a solution containing protease inhibitor in a solution containing 50 mM Tris-Cl, pH 8.0, 500 mM NaCl, 5 mM imidazole, pH 8.0 and 1 mM DTT, and 1% Triton X- After adding 100 mg of lysozyme, 2.5 mg of lysozyme was added and the bacteria was broken and the solution was frozen several times at -70 ° C and dissolved. When the nucleic acid was degraded by the ultrasonic pulverization method and centrifuged, the monoacylglycerol lipase protein was present in over 99% of the supernatant solution, and the insoluble protein called the inclusion body was hardly seen.

After the protein homogeneous solution is bound to the Ni-NTA column, the column is washed. The composition of the washing solution for the endotoxin removal is as follows. That is, a washing solution containing 50 column volumes of 0.1% Triton X-114 was sparged, and then the washing solution not containing it was shed with 10 column volumes for the removal of Triton X-114. It has been shown that endotoxin is reduced more than 1,000 times in this process.

As for the elution of the protein, it was most effective to use EDTA which is not imidazole in various tests. In the case of purification using His-tag, the protein is eluted with imidazole. In the case of monoacylglycerol lipase, elution can be carried out by using imidazole. However, when the concentration of imidazole is decreased by dialysis or dilution method, Of precipitation occurred. This phenomenon has been reported to occur in the case of some proteins at the time of dissolution of the imidazole, but in such cases, the effective elution method of the protein differs from protein to protein. In the case of the present monoacylglycerol lipase, it was eluted from high concentration EDTA (about 150 mM), and it was confirmed that the eluted protein was the most effective leaching method since no precipitation occurred upon removal of EDTA.

FIG. 6 is a graph showing the result of SDS-PAGE of "crude MGL" after dialysis of the protein obtained after protein elution in the above-mentioned procedure. The mouse monoacylglycerol lipase (MGL ) Was found to be about 33 kDa, and it was confirmed that it had the same size as that of mouse monoacylglycerol. This indicates that the isolation of monoacylglycerol lipase (MGL) has been successfully performed.

The separated monoacylglycerol lipase protein was able to remove EDTA or reduce the concentration by dialysis or dilution method.

That is, high-speed protein liquid chromatography can be performed to purify crude MGL more purely obtained above. The high-speed protein liquid chromatography can be carried out by hydrophobic interaction chromatography and ion exchange chromatography (Ion Exchange Chromatography) ). ≪ / RTI > In one embodiment of the present invention, purification was performed more purely by sequentially applying HiTrap Phenyl HP, HiTrap SP, and HiTrapQ columns.

The mass production process of the proteins specific to the protein was elucidated through a column purification process for pure separation of the monoacylglycerol lipase as described above, This model and the sequential column application for the above described culture conditions, bacterial equal solution and crushing method, composition of protein equal solution, separation and purification of proteins, washing, elution and pure purification are conditions that are specific to monoacylglycerol lipase protein, This is a method to obtain the maximum active protein with the highest probability through trial and error. If it is not applied, it will affect the structure of the protein and become an inclusion body in which the folding structure of the protein is not formed Or may be insoluble, bound to the column, aggregated, not eluted from the column, precipitated upon buffer exchange, reduced protein activity, lack of endotoxin removal, unwanted other proteins, The probability of water reduction may or may not be.

Various experiments were conducted to confirm whether the monoacylglycerol lipase protein produced by the mass production method of the present invention is effective for the treatment of metabolic syndrome such as hepaticzation, nonalcoholic fatty liver, hyperlipidemia, type 2 diabetes and / or obesity (Figs. 8 to 16).

FIGS. 8 and 9 are the results of measuring the body weight change by administering the monoacylglycerol lipase produced by the mass production method of the present invention to the ob / ob mice induced obesity for about 3 weeks, Decrease in fatty liver, and decrease in visceral fat. As a result, it has been confirmed that the long-term administration of monoacylglycerol lipase is effective for alleviating diabetic retinopathy or non-alcoholic fatty liver disease and for reducing weight, and it can be used as a therapeutic agent for hepaticopenia or nonalcoholic fatty liver disease and for treating obesity.

Fig. 10 is a graph showing the distribution rugosa- derived lipase was administered for a total of 6 weeks. When directly compared with the Figure 8 and 10, 8 is 2-position-specific lipase of the mono acyl glycerol administering a lipase to 20 unit / day, and will present the weight loss, Figure 10 is a non-specific lipase Candida one type of position The rugosa- derived lipase was administered at a dose of 2,000 units / day, and then the weight loss effect was observed. In both of the above examples, the effect was similar to about 3 g of weight loss effect. This suggests that the activity of 2-position-specific lipase is higher than that of nonspecific lipase in (1) monoacylglycerol as a substrate, (2) the affinity of 2-monoacylglycerol for non-specific lipase (3) 2-position specific lipase does not affect the degradation of triglycerides by physiological digestive fluids and rather decomposes triglycerides, which may not be absorbed. Suggesting that it does not happen. This fact implies that the 2-position specific lipase can exhibit a much better therapeutic effect than the nonspecific lipase.

The term " 2-position specific lipase "in the present invention means a lipase exhibiting reaction specificity only in the fatty acyl group at the 2-position of triglyceride, and the term" Quot; non-specific lipase "refers to a lipase that reacts with all three fatty acyl groups of triglycerides.

FIG. 11 is a graph showing changes in blood triglyceride concentration by monoacylglycerol lipase (MGL) in a mouse to which olive oil is administered, and FIG. 12 is a graph showing changes in blood triglyceride concentration by monoacylglycerol lipase As shown in Fig. As a result, it was confirmed that monoacylglycerol lipase was effective for the reduction of triglyceride in blood and it could be used as a therapeutic agent for hyperlipidemia.

FIGS. 13 and 14 show that monoacylglycerol lipase acts in the small intestine after administration and shows a delayed effect of fat recombination in Caco-2 cells, a human small intestinal epithelial cell line.

FIG. 15 is a graph showing changes in dietary amount and lipid contents during the administration of monoacylglycerol lipase. It was confirmed that the administration of monoacylglycerol lipase did not cause any change in dietary amounts and showed no side effects such as lipids. It has been confirmed that monoacylglycerol lipase of the present invention exhibits excellent efficacy as a therapeutic agent for obesity because it exhibits excellent weight loss effect without exhibiting such side effects at all, which is a serious side effect of Xenical, which is one of conventional therapeutic agents for obesity.

FIG. 16 is a graph showing the result of glucose tolerance test after administering monoacylglycerol lipase to ob / ob mice induced obesity for about 3 weeks. As a result of the administration of monoacylglycerol lipase, obesity was alleviated and thus the body Metabolism was improved and glucose processing ability was improved. These results show that the administration of the monoacylglycerol lipase of the present invention can be linked to the treatment of metabolic syndrome such as type 2 diabetes.

Therefore, the monoacylglycerol lipase protein produced by the mass production method of the present invention completely decomposes the triglyceride into fatty acid and glycerol in the gastrointestinal tract, thereby delaying the fat absorption and reducing the blood absorption of triglyceride. Therefore, , Nonalcoholic fatty liver disease, hyperlipidemia, type 2 diabetes, and / or obesity, as a raw material for medicines and health functional foods, which prevent, ameliorate or treat metabolic syndrome.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Monoacylglycerol  Production and purification of lipase proteins

1-1: Preparation of recombinant vector and recombinant microorganism

In the present invention, in order to produce a monoacylglycerol lipase protein, E. coli System and a His-tag were used. An open reading frame (ORF) portion of human-derived monoacylglycerol lipase mRNA (Genebank Number: NM_001003794) and mouse-derived monoacylglycerol lipase mRNA (Genebank Number: NM_011844) -HMT (His-Myc-TEVprotease) vector (Geisbrecht BV et al., Protein Expression Purif 46: 23-32, 2006).

The pT7-HMT vector is a vector that is expressed in bacteria by fusing six His-tags and a target protein. The pT7-HMT vector is characterized in that it can easily purify the expressed protein. TEV protease can be used to separate the tag There is an advantage.

First, a primer capable of amplifying the ORF portion (except the ATG start codon) of human or mouse-derived monoacylglycerol lipase mRNA was prepared as shown in Table 1, and a restriction enzyme site was included so that the amplified gene could be inserted into the vector Respectively.

Primer sequence gene Primer sequence limit
enzyme order
number Person
MGL forward 5'-GC CATATG ccagaggaaagttccccagg-3 ' NdeI SEQ ID NO: 1 reverse 5'-CG CTCGAG tcagggtggggacgcagttc-3 ' XhoI SEQ ID NO: 2 Mouse MGL forward 5'-GC GTCGAC cctgaggcaagttcacccagg-3 ' SalI SEQ ID NO: 3 reverse 5'-CG CTCGAG tcagggtggacacccagctc-3 ' XhoI SEQ ID NO: 4

(The underlined part indicates the part where the restriction enzyme acts)

Total RNA derived from human or mouse was extracted from adipose tissue and subjected to PCR (Polymerase chain reaction) using each of the primer pairs shown in Table 1 above as a template. As a result of confirming the sequence of the PCR amplification product, it was confirmed that the human monoacylglycerol lipase gene was represented by SEQ ID NO: 5 and the mouse-derived monoacylglycerol lipase gene was represented by SEQ ID NO: 6 (Table 2).

Gene sequence information gene Gene sequence SEQ ID NO: People MGL GC CATATG CTCGAG CG SEQ ID NO: 5 Mouse MGL GC GTCGAC CTCGAG CG SEQ ID NO: 6

The amplified human or mouse-derived monoacylglycerol lipase gene was digested with NdeI (Cat. No. R0111S, New England BioLabs, USA) or SalI (Cat. No. R0138S (New England BioLabs, USA) and XhoI (Cat. No. R0146S ; New England BioLabs, USA) and then introduced into the pT7-HMT vector carrying the same restriction sites to produce a recombinant vector. In this case, six His-tags can be used for protein purification and Myc-tag can be used for protein detection by Western analysis in the future. In addition, TEV protease was designed to remove His-Myc-tag if necessary.

1-2: Monoacylglycerol  Production and purification of lipase proteins

The recombinant vector was then transformed into E. coli BL21 (DE3) pCodon plus strain (Cat. No. 230245; Agilent, USA). This strain was inoculated into 500 ml of LB medium containing kanamycin and chloramphenicol and 2% ethanol, and incubated at an absorbance of 600 to 0.5 to 0.6. Then, IPTG was added to 1 mM And further cultured at 16 DEG C for 16 hours.

Bacteria were harvested by centrifugation and suspended in Lysis buffer (50 mM Tris-Cl, pH 8.0, 500 mM NaCl, 5 mM imidazole, pH 8.0), added with 1% Triton X-100 and then added with lysozyme A frozen-thawed method was used to break the germs into a protein equivalent solution. The His-Myc-MGL fusion protein was bound to the column using Ni-NTA agarose bead (Cat. No. 30210, Qiagen, USA) after digestion of the nucleic acid by ultrasonication and centrifugation. To remove endotoxin due to bacterial dissolution, the column was washed with a washing buffer containing 0.1% Triton X-114 to a volume of 50 column, and then washed with a washing buffer without Triton X-114 Washed with 10 column volumes.

The elution of the protein attached to the column was eluted using EDTA rather than imidazole in various tests. The protein harvested using elution solution (150 mM EDTA, pH 8.0, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0) was dialyzed with 200 times 50 mM Tris-Cl, "Was used for the experiment.

FIG. 6 shows the results of SDS-PAGE after separation and purification of mouse-derived monoacylglycerol lipase (MGL). It was confirmed that the size of mouse-derived monoacylglycerol lipase (MGL) purified in the present invention was about 33 kDa , And it was confirmed that it had the same size as the actual mouse monoacylglycerol.

The monoacylglycerol lipase to be administered to the animal was further purified by applying the crude MGL to the HiTrap Phenyl HP, HiTrap SP, and HiTrapQ columns sequentially through the same process as shown in FIG.

Monoacylglycerol  Activity measurement of lipase protein

In the present invention, the separated protein and its substrate oleoyl-rac-glycerol (Cat. No. M7765, Sigma, USA) were reacted in order to measure the activity of the mouse-derived monoacylglycerol lipase purified in Example 1, The liberated glycerol was measured using the Glycerol assay kit (Cat. No. MAK117, Sigma, USA) with reference to the kit attached to the kit. 1 unit was defined as the amount by which 1 μmole of monoacylglycerol was completely decomposed for 1 hour at pH 7.4, 37. The amount of this activity measurement was calculated with reference to the glycerol standard.

Protein concentration was measured using the Pierce ™ BCA assay kit (Cat. No. 23225, ThermoFisher Scientific, USA), and the activity per mg was calculated.

The endotoxin that may remain in the protein was determined using the Pierce ™ LAL Chromogenic Endotoxin Quantitation Kit (Cat. No. 88282, ThermoFisher Scientific, USA).

The mouse-derived monoacylglycerol lipase measured as above showed the following results.

Concentration 3 to 5 mg / ml, total 300 to 500 mg / 40 liter culture

Activity 30-100 units / mg protein

Endotoxin <10 EU / ml (* EU, endotoxin unit)

Monoacylglycerol  Lipase Reduces Obesity

In the present Example, it was confirmed whether the monoacylglycerol lipase prepared in Example 1 was delayed in weight loss or weight gain in obesity ob / ob mice administered for about 3 weeks. (20 ~ 50 units) of monoacylglycerol lipase were orally administered to 11-week-old mice in an ob / ob mouse (central experimental animal, Korea) expressing obesity due to mutation of the leptin gene and a decrease in appetite suppression (Gavage) for about 3 weeks. In order to equalize the intake of monoacylglycerol lipase with the consumption of feed, a method of fasting and then feeding the diets was selected.

As a result, as shown in FIG. 8, after the administration of the monoacylglycerol lipase, the body weight was significantly decreased, and it was confirmed that the body weight difference was about 4 ~ 5 g after 3 weeks compared to the control group. In addition, as shown in FIG. 9, it was confirmed by visual examination that weight loss, hepatic manifestation, alleviation of fatty liver, and reduction of visceral fat.

2- Of monoacylglycerol  Cutting and reducing obesity

In this example, the effect of reducing the 2-monoacylglycerol-cleaving enzyme was confirmed. Specifically, lysozyme (L3126, Sigma, USA) was used as a control for the pancreatic pancreas with 1,3-specific lipase and Candida rugosa lipase (L1756, Sigma, USA) was selected and administered to ob / ob mice (Central laboratory animals, Korea) for 7 weeks. As a result, as shown in Fig. 10, the lipase of C. rugosa , a nonspecific lipase, showed a significant decrease in body weight as compared with the control group. This indicates that proteins or compounds cleaving 2-monoacylglycerol can be used for the treatment of obesity it means.

Monoacylglycerol  Reduction of triglyceride in blood by lipase administration

In the present invention, monoacylglycerol lipase was orally administered to mice to confirm whether blood mononuclear glycerol lipase produced in Example 1 was actually reduced in blood triglyceride, and on the basis of the activity measurement result of Example 2, unit).

First, C57BL6 / J mice were fasted for 4 hours or more, and then 250 올리 of olive oil was administered by gavage using a tube. At the same time, 250 쨉 l of saline was administered to the control group and 250 쨉 l of monoacylglycerol lipase protein Respectively.

After 2 hours, the mice were sacrificed and blood was collected and the amount of triglyceride in the blood was analyzed. The absorbance was measured at 500 nm according to the manual included in the kit using a Triglyceride Colorimetric Assay Kit (Cat No 10010303, Cayman, USA). As a result, it was confirmed that the blood triglyceride was significantly reduced in the mouse group to which monoacylglycerol lipase was administered as shown in Fig. Thus, it was confirmed that the monoacylglycerol lipase of the present invention has an effect of reducing the blood absorption of triglyceride by completely decomposing the triglyceride into fatty acid and glycerol in the gastrointestinal tract.

In addition, as shown in FIG. 12, when the above experiment was conducted, the absorption of fat in the monoacylglycerol lipase-administered group was much lower than that of the group administered with physiological saline (FIG. 12) (Fig. 12B).

In the small intestine Monoacylglycerol  Measurement of lipase activity

In this example, the activity of monoacylglycerol lipase administered orally was measured in the small intestine in order to demonstrate that the fat absorption reduction effect actually demonstrated in Example 5 is the result of the action of monoacylglycerol lipase.

Mice were sacrificed after 1 hour or 2 hours after administration of physiological saline or monoacylglycerol lipase, and the jejunum region in the small intestine was divided into proximal and distal portions, Was allowed to elute in PBS. The activity of the monoacylglycerol lipase present therein was measured using the method in Example 2. [

As a result, as shown in FIG. 13, the group administered with monoacylglycerol (MGL) was used as a distal portion after proximal administration 1 hour after oral administration, and the total activity of the protein remaining therein was administered orally It corresponds to 1/5 to 1/10 of one protein. This means that the administered monoacylglycerol lipase is destroyed by gastric acid and gastric juice, but at least 1/10 of the enzyme activity is in the small intestine. Therefore, it is necessary to administer more than the necessary amount in the mouse experiment. Monoacylglycerol lipase can be protected from gastric acid and gastric juice by methods such as coating agent in the future.

Monoacylglycerol  Delayed Neutral Fat Reassembly in Small Cells by Lipase

In order to demonstrate that the delayed effect of neutral fat absorption actually observed in Examples 5 and 6 was delayed by neutral fat recombination and blood freezing process in the small intestine, Caco-2 cells (human small intestine cell line) Cat No. HTB37, ATCC, USA). As shown in Fig. 11, the culture system was prepared by placing the digestive fat and monoacylglycerol lipase protein on the apical side of the small intestine and placing the blood side on the basolateral side so that the small intestinal epithelium between them And it can be beneficial to the blood. Caco-2 cells were differentiated for 21 days. After the differentiation of the two mediums, the cells were allowed to pass through the cells. After the differentiation, they were examined for resistance to intercellular junction by resistance measurement. Oleic acid and monooleoylglycerol were administered to the small intestine, Or monoacylglycerol lipase was administered. After incubation for 17 hours, the blood-side medium was recovered and the ApoB protein, a lipoprotein surface protein contained in the medium, was measured by an ELISA method.

As a result, as shown in Fig. 14, it was confirmed that the amount of ApoB detected in monoacylglycerol lipase-treated group was very low, and if it inhibited the absorption of monoacylglycerol by this enzyme, It is a direct proof that you can do it.

Combining the results of Examples 5, 6 and 7 with the results of Example 3 shows that administration of monoacylglycerol lipase in mice slows or reduces the triglyceride recombination of small intestinal epithelial cells, This indicates that the monoacylglycerol lipase can be used as a therapeutic agent for obesity by inhibiting the absorption of monoacylglycerol by the enzyme.

2- Monoacylglycerol  Administration of lipase Diet amount  And Fatty  Confirm

When the monoacylglycerol lipase is administered, the amount of the monoacylglycerol lipase may be decreased by the addition of the monoacylglycerol lipase The amount of dietary fat was measured, and the fat content was measured at random. As a result, as shown in Fig. 15, administration of monoacylglycerol lipase did not cause a change in dietary amount, and side effects such as fat were not observed. This means that the action of inhibiting fat absorption is accompanied by promoting energy consumption in the small intestine. It shows that drugs such as Xenical (ingredient orlistat) have different mechanism from inhibiting fat absorption and have no side effects of lipid.

Glucose load test

In this Example, a glucose load test was performed on a mouse to which monoglycerol lipase was administered in Example 3 above.

Monoglycerol lipase was injected intraperitoneally with 1.5 mg / g body weight of glucose and blood glucose levels were measured at 15, 30, 60 and 120 minutes after loading.

As a result, it was confirmed that the glucose load test was greatly improved as shown in FIG. These results suggest that monoacylglycerol lipase may be used for the treatment of metabolic syndrome such as diabetes.

<110> Industry-Academic Cooperation Foundation, Yonsei University          Daegu Gyeongbuk Institute of Science and Technology <120> Method for mass producing monoacylglycerol lipase <130> 1061087 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Human monoacylglycerol lipase (MGL) forward primer <400> 1 gccatatgcc agaggaaagt tccccagg 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> human MGL reverse primer <400> 2 cgctcgagtc agggtgggga cgcagttc 28 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> mouse MGL forward primer <400> 3 gcgtcgaccc tgaggcaagt tcacccagg 29 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> mouse MGL reverse primer <400> 4 cgctcgagtc agggtggaca cccagctc 28 <210> 5 <211> 925 <212> DNA <213> Artificial Sequence <220> <223> Human monoacylglycerol lipase (MGL) <400> 5 gccatatgcc agaggaaagt tcccccaggc ggaccccgca gagcattccc taccaggacc 60 tccctcacct ggtcaatgca gacggacagt acctcttctg caggtactgg aaacccacag 120 gcacacccaa ggccctcatc tttgtgtccc atggagccgg agagcacagt ggccgctatg 180 aagagctggc tcggatgctg atggggctgg acctgctggt gttcgcccac gaccatgttg 240 gccacggaca gagcgaaggg gagaggatgg tagtgtctga cttccacgtt ttcgtcaggg 300 atgtgttgca gcatgtggat tccatgcaga aagactaccc tgggcttcct gtcttccttc 360 tgggccactc catgggaggc gccatcgcca tcctcacggc cgcagagagg ccgggccact 420 tcgccggcat ggtactcatt tcgcctctgg ttcttgccaa tcctgaatct gcaacaactt 480 tcaaggtcct tgctgcgaaa gtgctcaacc ttgtgctgcc aaacttgtcc ctcgggccca 540 tcgactccag cgtgctctct cggaataaga cagaggtcga catttataac tcagaccccc 600 tgatctgccg ggcagggctg aaggtgtgct tcggcatcca actgctgaat gccgtctcac 660 gggtggagcg cgccctcccc aagctgactg tgcccttcct gctgctccag ggctctgccg 720 atcgcctatg tgacagcaaa ggggcctacc tgctcatgga gttagccaag agccaggaca 780 agactctcaa gatttatgaa ggtgcctacc atgttctcca caaggagctt cctgaagtca 840 ccaactccgt cttccatgaa ataaacatgt gggtctctca aaggacagcc acggcaggaa 900 ctgcgtcccc accctgactc gagcg 925 <210> 6 <211> 925 <212> DNA <213> Artificial Sequence <220> Mouse monoacylglycerol lipase (MGL) <400> 6 gcgtcgaccc tgaggcaagt tcacccaggc gaactccaca gaatgttccc taccaggacc 60 tgcctcacct ggtcaatgca gacggacagt acctcttttg tagatactgg aagcccagtg 120 gcacacccaa ggccctcatc tttgtgtccc atggagctgg ggaacactgt ggccgttatg 180 atgagctggc tcatatgttg aaggggctgg acatgctggt atttgcccat gaccatgttg 240 gccatgggca gagtgaggga gagaggatgg tggtgtcgga cttccaagtt tttgtcagag 300 atgtgctgca acacgtggac accatccaga aggactaccc cgacgtcccc atcttcctcc 360 tgggccactc catgggcggt gccatctcca tcctagtggc tgcagagagg ccaacctact 420 tttctggcat ggtcctgatt tcacctctgg tccttgccaa tccggaatct gcatcgactt 480 tgaaggtcct tgctgccaaa ctgctcaatt ttgtcctgcc aaatatgacc ttggggcgca 540 ttgactccag cgtgctgtct cggaacaagt cggaggttga cctgtacaac tctgacccac 600 tcgtctgccg agcagggctg aaggtgtgct ttggcataca gctgctgaat gccgtcgcaa 660 ggtggagcg agcaatgccc aggctgacac tgccattcct gctgctgcag ggttctgctg 720 accggctttg cgacagcaaa ggtgcctacc tgctcatgga atcatcccgg agtcaggaca 780 aaacactcaa gatgtatgaa ggtgcctatc acgtcctcca cagggagctt ccggaagtga 840 ccaactccgt cctccatgaa gtaaactcgt gggtgtctca caggatagca gcagcaggag 900 ctgggtgtcc accctgactc gagcg 925 <210> 7 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Human MGL protein <400> 7 Met Pro Glu Glu Ser Ser Pro Arg Arg Thr Pro Gln Ser Ile Pro Tyr   1 5 10 15 Gln Asp Leu Pro His Leu Val Asn Ala Asp Gly Gln Tyr Leu Phe Cys              20 25 30 Arg Tyr Trp Lys Pro Thr Gly Thr Pro Lys Ala Leu Ile Phe Val Ser          35 40 45 His Gly Ala Gly Glu His Ser Gly Arg Tyr Glu Glu Leu Ala Arg Met      50 55 60 Leu Met Gly Leu Asp Leu Leu Val Phe Ala His Asp His Val Gly His  65 70 75 80 Gly Gln Ser Glu Gly Glu Arg Met Val Val Ser Asp Phe His Val Phe                  85 90 95 Val Arg Asp Val Leu Gln His Val Asp Ser Met Gln Lys Asp Tyr Pro             100 105 110 Gly Leu Pro Val Phe Leu Leu Gly His Ser Met Gly Gly Ala Ile Ala         115 120 125 Ile Leu Thr Ala Ala Glu Arg Pro Gly His Phe Ala Gly Met Val Leu     130 135 140 Ile Ser Pro Leu Val Leu Ala Asn Pro Glu Ser Ala Thr Thr Phe Lys 145 150 155 160 Val Leu Ala Ala Lys Val Leu Asn Leu Val Leu Pro Asn Leu Ser Leu                 165 170 175 Gly Pro Ile Asp Ser Ser Val Leu Ser Arg Asn Lys Thr Glu Val Asp             180 185 190 Ile Tyr Asn Ser Asp Pro Leu Ile Cys Arg Ala Gly Leu Lys Val Cys         195 200 205 Phe Gly Ile Gln Leu Leu Asn Ala Val Ser Arg Val Glu Arg Ala Leu     210 215 220 Pro Lys Leu Thr Val Pro Phe Leu Leu Leu Gln Gly Ser Ala Asp Arg 225 230 235 240 Leu Cys Asp Ser Lys Gly Ala Tyr Leu Leu Met Glu Leu Ala Lys Ser                 245 250 255 Gln Asp Lys Thr Leu Lys Ile Tyr Glu Gly Ala Tyr His Val Leu His             260 265 270 Lys Glu Leu Pro Glu Val Thr Asn Ser Val Phe His Glu Ile Asn Met         275 280 285 Trp Val Ser Gln Arg Thr Ala Thr Ala Gly Thr Ala Ser Pro Pro     290 295 300 <210> 8 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Mouse MGL protein <400> 8 Met Pro Glu Ala Ser Ser Pro Arg Arg Thr Pro Gln Asn Val Pro Tyr   1 5 10 15 Gln Asp Leu Pro His Leu Val Asn Ala Asp Gly Gln Tyr Leu Phe Cys              20 25 30 Arg Tyr Trp Lys Pro Ser Gly Thr Pro Lys Ala Leu Ile Phe Val Ser          35 40 45 His Gly Ala Gly Glu His Cys Gly Arg Tyr Asp Glu Leu Ala His Met      50 55 60 Leu Lys Gly Leu Asp Met Leu Val Phe Ala His Asp His Val Gly His  65 70 75 80 Gly Gln Ser Glu Gly Glu Arg Met Val Val Ser Asp Phe Gln Val Phe                  85 90 95 Val Arg Asp Val Leu Gln His Val Asp Thr Ile Gln Lys Asp Tyr Pro             100 105 110 Asp Val Pro Ile Phe Leu Leu Gly His Ser Met Gly Gly Ala Ile Ser         115 120 125 Ile Leu Val Ala Ala Glu Arg Pro Thr Tyr Phe Ser Gly Met Val Leu     130 135 140 Ile Ser Pro Leu Val Leu Ala Asn Pro Glu Ser Ala Ser Thr Leu Lys 145 150 155 160 Val Leu Ala Ala Lys Leu Leu Asn Phe Val Leu Pro Asn Met Thr Leu                 165 170 175 Gly Arg Ile Asp Ser Ser Val Leu Ser Arg Asn Lys Ser Glu Val Asp             180 185 190 Leu Tyr Asn Ser Asp Pro Leu Val Cys Arg Ala Gly Leu Lys Val Cys         195 200 205 Phe Gly Ile Gln Leu Leu Asn Ala Val Ala Arg Val Glu Arg Ala Met     210 215 220 Pro Arg Leu Thr Leu Pro Phe Leu Leu Leu Gln Gly Ser Ala Asp Arg 225 230 235 240 Leu Cys Asp Ser Lys Gly Ala Tyr Leu Leu Met Glu Ser Ser Arg Ser                 245 250 255 Gln Asp Lys Thr Leu Lys Met Tyr Glu Gly Ala Tyr His Val Leu His             260 265 270 Arg Glu Leu Pro Glu Val Thr Asn Ser Val Leu His Glu Val Asn Ser         275 280 285 Trp Val Ser His Arg Ile Ala Ala Ala Gly Ala Gly Cys Pro Pro     290 295 300

Claims (17)

delete delete delete delete delete delete delete delete a) preparing a recombinant vector by introducing into the pT7-HMT vector a gene consisting of the nucleotide sequence of SEQ ID NO: 5 or 6;
b) transforming the recombinant vector into E. coli BL21 (DE3), adding 0.5-1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and culturing at 16 ° C;
c) The transformed E. coli BL21 (DE3) cultured in step b) was suspended in a dissolution buffer of 50 mM Tris-Cl, pH 8.0, 500 mM NaCl and 5 mM imidazole, pH 8.0, And lysozyme to produce a homogenized liquid; And
d) Separation and purification of 2-position specific monoacylglycerol lipase protein by Ni-NTA chromatography, protein elution using 150 mM EDTA (ethylenediaminetetraacetic acid) and fast protein liquid chromatography ;
Position-specific monoacylglycerol lipase protein. 10. The method according to claim 9, wherein the gene consisting of the nucleotide sequence of SEQ ID NO: 5 in step (a) is amplified into a reverse primer pair consisting of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: Mass production method. The method according to claim 9, wherein the gene consisting of the nucleotide sequence of SEQ ID NO: 6 in step a) is amplified into a reverse primer pair consisting of the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: Mass production method. delete delete delete delete delete The mass production method according to claim 9, wherein the high-speed protein liquid chromatography in step d) is performed sequentially by hydrophobic interaction chromatography and ion exchange chromatography.

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