KR101400725B1 - Purification method of bace-1 protein - Google Patents

Abstract

The present invention relates to a method for purifying a protein.
According to the present invention, BACE-1 (β-secretase) protein known as a cause of Alzheimer's disease is overexpressed using E. coli and then purified using refolding and Fast Protein Liquid Chromatography (FPLC) By providing a BACE-1 protein purification method and a BACE-1 (beta-secretase) protein purified by the protein purification method, an effect that can be effectively utilized for the development of a pharmaceutical composition for prevention and treatment of Alzheimer's disease have.

Description

Purification method of BACE-1 protein {PURIFICATION METHOD OF BACE-1 PROTEIN}

The present invention relates to a method for purifying a protein, and more particularly, to a method for purifying a protein by overexpressing BACE-1 (β-secretase) protein known as a cause of Alzheimer's disease using E. coli and then refolding and Fast Protein Liquid Chromatography FPLC). ≪ / RTI >

Recently, studies on various biological pathways including the onset of Alzheimer's disease have been actively conducted. A protein known as Amyloid Precursor Protein (APP) was cut to form Aβ42 peptide, and the role of BACE-1 (β-secretase) protein, which ultimately leads to the formation of amyloid plaques, was identified. Therefore, studies are underway to develop a specific inhibitor of BACE-1 protein to develop drugs for dealing with Alzheimer's disease.

Currently, as the average life span of humans increases, the population suffering from many diseases related to aging is increasing at a considerable rate. In particular, neurodegenerative diseases are on the rise, and researches for the treatment and prevention of these diseases are underway worldwide. Alzheimer's disease is one of the most common aging-related diseases caused by dementia. In the United Nations, the number of elderly people over 80 is expected to increase to 370 million by 2050, and more than 50% of elderly people over 85 years old are now suffering from Alzheimer's disease It is understood that they suffer. In addition, many people are receiving continuous treatment and considerable resources are being invested.

The onset of Alzheimer's disease is gradual, with several indications between the ages of 60 and 70, with the first sign of a decrease in cognitive function. One of the major causes of Alzheimer's disease is the amyloid beta (Aβ) peptide expressed in the brain. Amyloid beta peptide is formed by protease BACE-1 (β-secretase or Beta-site APP-cleaving enzyme) and γ-secretase. The resulting amyloid beta peptide migrates to the outer surface of the cell where it forms a lump, called a senile plaque. Amyloid beta appears in two forms, Aβ40 and Aβ42, which are determined by the cleavage site of γ-secretase. The latter form is more commonly found in geriatric plaques and is said to be more fatal. In other words, blocking the activity of BACE-1 protein and γ-secretase is the only way to prevent the formation of amyloid beta. Therefore, a new discovery of proteins or compounds that inhibit these activities is a major goal in studying Alzheimer's disease.

There have been many studies on the inhibition of BACE-1 protein and γ-secretase as a therapeutic method to prevent Alzheimer's disease. There is a growing interest in developing drugs that have no side effects to deal with these diseases. (Citron M. Beta-secretase as a target for treatment of Alzheimer's disease. J. Neurosci. Res. 2002. 70 (3): 373-9). Gene-deficient animal model studies have demonstrated that BACE-1 protein plays a key role in amyloid beta formation. A gene-deficient mouse experiment lacking the BACE-1 protein production gene failed to generate amyloid beta. However, in general mice, the opposite results were obtained (Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H , Wijsman J, Wriqht S, and McConloque L. BACE knockout mice are often associated with the promise of bate-secretase activity in the brain: implications for Alzheimer's disease therapeutics. Hum. Mol. Genet 2001. 10 (12): 1317-24). Based on these facts, the BACE-1 protein is an essential target for drug development studies to treat Alzheimer's disease.

Thus, the present inventors have over-expressed the protein in E. coli using a recombinant technology through cloning of the BACE-1 (β-secretase) gene as a target for dealing with Alzheimer's disease, and refolding and using various columns The tablet was successful. Currently, there is no patent on the pure purification of BACE-1 protein. Furthermore, it is determined that pure purification technology of BACE-1 protein can be used as basic data for treating Alzheimer's disease, and thus the present invention has been completed.

On the other hand, Korean Patent No. 10-0841599 (purification method of calcium ion-binding protein) and Korean Patent No. 10-0236768 (Ubiquin-4 protein, a specific antigen of hepatitis virus, ).

It is an object of the present invention to provide a BACE-1 (β-secretase) protein which is known to cause Alzheimer's disease and is over-expressed using E. coli and then refolded and purified using Fast Protein Liquid Chromatography (FPLC) 1 < / RTI > protein.

Another object of the present invention is to provide a BACE-1 (β-secretase) protein purified by the protein purification method.

(1) a step of inserting a BACE-1 (β-secretase) gene into an expression vector pET28-MHL; and (2) a step of introducing pET28 (3) culturing the transformant prepared in (2) above to induce production of BACE-1 protein, and (4) culturing the transformant prepared in And (5) adding a lysis buffer to the cell pellet obtained in the step (4), and then using an ultrasonic disintegrator to remove the cell pellet from the cell pellet, (6) adding a solubilization buffer to the cell pellet obtained in the step (5), mixing the cell pellet and filtering the cell debris; And (7) adding the BACE-1 protein to the solubilized buffer solution filtered by the step (6) Folding the BACE-1 protein in a concentration buffer by adding the refold buffer for refolding to induce refolding of the BACE-1 protein; and (8) concentrating the buffer containing the BACE-1 protein by the step (7) step; And (9) ion-exchange chromatography or gel-filtration chromatography of the concentrate obtained in the step (8).

The BACE-1 gene used in the above step (1) is characterized in that the 53 to 441th amino acid sequence portion or the 58th to 441st amino acid sequence portion is amplified by a polymerase chain reaction (PCR).

The solubilization buffer used in the step (6) is characterized by comprising Tris-HCl, urea and? -Mercaptoethanol.

The refolding buffer used in the step (7) is characterized by comprising Tris-HCl, oxidized glutathione, and reduced glutathione.

The ion exchange chromatography in the step (9) is characterized in that the BACE-1 protein is eluted using an elution buffer composed of Tris-HCl, urea and sodium chloride (NaCl).

In addition, the present invention provides a purified BACE-1 protein by the above protein purification method.

According to the present invention, BACE-1 (β-secretase) protein known as a cause of Alzheimer's disease is overexpressed using E. coli and then purified using refolding and Fast Protein Liquid Chromatography (FPLC) By providing a BACE-1 protein purification method and a BACE-1 (beta-secretase) protein purified by the protein purification method, an effect that can be effectively utilized for the development of a pharmaceutical composition for prevention and treatment of Alzheimer's disease have.

FIG. 1 shows the pET28-MHL vector in the DNA agarose gel for cloning of the BACE1 gene of the present invention and the DNA cut by the BseR1 restriction enzyme in the DNA ararose gel.
FIG. 2 shows the DNA agarose in which the BACE1 gene of the present invention was cloned into the vector.
Fig. 3 shows the results of SDS-PAGE of protein simple for each urea concentration by adding a refold buffer to refold the BACE1 protein of the present invention.
FIG. 4 shows the purification of the BACE1 protein of the present invention by purification using ion exchange chromatography and gel filtration chromatography, which was confirmed by SDS-PAGE.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for purifying BACE-1 protein and purification of BACE-1 protein, which can be effectively utilized for the development of pharmaceutical compositions for the prevention and treatment of Alzheimer's disease.

The BACE-1 protein purification method of the present invention provides purified BACE-1 protein purified using gene cloning and refolding, thereby providing a protein that can be used for prevention and treatment of senile Alzheimer's disease.

In addition, the BACE-1 protein according to the present invention may be used independently with a buffer contributing to the stabilization of a pharmaceutically acceptable protein according to the purpose of use, and may be any other suitable form for confirming the binding with other compounds .

The BACE-1 protein according to the present invention is useful for the treatment and / or prophylaxis of cancer, such as alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, It is stable at -80 ℃ and is not denatured.

The BACE-1 protein according to the present invention is expressed in an encapsulated body form. Protein is folded into a tertiary structure through the process of protein folding after transcription, and protein-specific features and functions are revealed in this process. In this process, the original folding is not done properly and it is formed as an inclusion body. In other words, the inclusion body refers to a state where the polypeptide tightly folds due to a false folding phenomenon, and refolding correctly restores the false folding of the protein expressed by the inclusion body. Urea and guanidine hydrogen chloride are used for protein denaturation for refolding, and glutathione, an antioxidant, can be used to eliminate the oxidation reaction that occurs during refolding. This is to prevent nonspecific disulfide bonding by oxidation during protein dissolution.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Experimental preparation

(1) Usage medium

In this experiment, LB medium (Luria-Bertani media) and TB medium (Terrific Broth media) were used as the liquid medium. LB agar solid medium was used to identify colonies of E. coli cells. Both LB medium and TB medium and LB agar solid medium contained kanamycin antibiotic at a concentration of 50 μg / ml. This is due to the presence of the kanamycin antibiotic resistance site contained in the pET28-MHL vector used in this experiment.

(2) Cell and culture conditions

In this experiment, DH5α E. coli (Stratagene) and BL21 (DE3) E. coli (Stratagene) cells were used. The culture conditions of the cells were incubated at 37 ° C for the formation of cell colonies in the case of the solid medium and at 37 ° C and 220rpm for the liquid medium. For the mass culture of E. coli , we used our own LEX system (a system that supplies oxygen directly to the liquid medium).

(3) Preparation of BACE-1 (β-secretase) gene

The BACE-1 gene used in this experiment was amplified by Polymerase Chain Reaction (PCR) of the amino acid sequences of 53-441 and 58-441.

Example 2. Cloning of BACE-1 (β-secretase) gene

(1) Preparation of pET28-MHL vector and cleavage of vector by restriction enzyme

The pET28-MHL vector used in this experiment was prepared by adding 60 μl of DH5α E. coli cells and 2.5 μl of the vector prepared in advance. After reacting in ice for 15 minutes, the pET28-MHL vector was heat-shocked at 42 ° C for 1 minute. Then, the cells were reacted in ice for 3 minutes, 500 μl of SOC medium was added, and the cells were transformed by incubation at 37 ° C. and 650 rpm for 1 hour. The transformed E. coli cells were transformed into LB agar solid medium. Colonies were identified after culturing for 16 to 18 hours, and one colony was added to 3 ml of a liquid medium containing 50 μg / ml of kanamycin and cultured for 16 to 18 hours. The cultured cells were collected using a centrifuge and the plasmid vector was eluted. Then, 15 μl of sterilized water, 30 μl of the eluted vector, 1 μl of the restriction enzyme BseR1 and 5 μl of the buffer were added and the mixture was allowed to stand at 37 ° C. for 2 hours, and the vector was eluted with the elution buffer. (See Fig. 1)

(2) insertion of BACE-1 gene into pET28-MHL vector

To insert the BACE-1 gene amplified by the polymerase chain reaction into the vector truncated by the BseR1 restriction enzyme, 8.5 μl of the vector was mixed well with DNA ligase in a tube containing 1 μl of the BACE-1 gene prepared previously 2 μl of the vector was reacted at room temperature for 1 hour, followed by further reaction at 37 ° C for 20 minutes. BACE-1 gene inserted vector was prepared by adding 60 μl of DH5α E. coli cells and 2.5 μl of vector, and reacting in ice for 15 minutes, followed by thermal shock at 42 ° C for 1 minute. Then, the cells were reacted in ice for 3 minutes, 500 μl of SOC medium was added, and the cells were transformed by incubating the cells at 37 ° C. and 650 rpm for 1 hour. The vectors were inserted into the cells, Lt; / RTI > for a period of time to identify colonies. Ten colonies were randomly selected and amplified using the Polymerase Chain reaction. The BACE1 gene amplified by polymerase chain reaction was confirmed by electrophoresis on DNA agarose gel containing 1.5% agarose (see Figure 2).

Example 3. Purification of BACE-1 (β-secretase) protein

(1) Identification of cell colonies including BACE-1 gene

In order to cultivate large quantities of BACE-1 gene cloned cells, three colonies generated in the above procedure were arbitrarily selected and loosely dissolved in 3 ml of LB medium containing 50 μg / ml of kanamycin and cultured at 37 ° C. and 220 rpm For 16-18 hours. The cultured cells were collected using a centrifuge, and the vector was eluted using an elution buffer. To elucidate the BACE-1 gene expression, 60 ㎕ of BL21 (DE3) E. coli cells and 2.5 ㎕ of the vector were added to the BACE-1 gene for mass expression, followed by reaction in ice for 15 minutes. Lt; / RTI > for 1 minute. Then, the cells were reacted in ice for 3 minutes, and 500 μl of SOC medium was added thereto. The cells were transformed by incubating the cells at 37 ° C. and 650 rpm for one hour. The transformed cells were incubated in solid medium for 16-18 hours to identify colonies.

(2) Bulk culture of BL21 (DE3) E. coli

In order to mass-cultivate the cells, the colonies grown in the above procedure were selected and cultured in a 50 ml LB medium containing 50 μg / ml of kanamycin at 37 ° C and 220 rpm for 16-18 hours. The cultured medium was put into 2 L of TB medium containing 50 μg / ml of kanamycin and cultured at 37 ° C. in an LEX system until the OD 600 reached 0.9-1. After incubation, 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added and the production of protein was induced at 37 ° C. for 3 to 5 hours. Cells grown on TB medium were centrifuged at 4 ° C and 7,000 rpm for 10 minutes to obtain cells. The obtained cell pellet was stored at -80 ° C and used for the experiment.

(3) Lysis of BL21 (DE3) E. coli cells

To refold the BACE-1 protein, 150 ml of dissolution buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl), 4 μl of RNase and PMSF · Benzamidine solution were added to the cell pellet. Cell pellets were allowed to loosen well, and then the cell wall was broken using an ultrasonic disintegrator, followed by centrifugation at 4 ° C and 15,500 rpm for 40 minutes. The cell pellet was dissolved in 150 ml of dissolution buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl) and centrifuged at 4 ° C and 15,500 rpm for 40 minutes. The cell pellet was added to 150 ml of dissolution buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl), and 0.5% TritonX-100 was added thereto. The cell pellet was centrifuged at 4 ° C and 15,500 rpm for 40 minutes Respectively. Finally, cell pellets were obtained under the same conditions without addition of Triton X-100 alone.

(4) refolding of BACE-1 protein

To refold the BACE-1 protein, the cell pellet was placed in 75 ml of Solubilization buffer (50 mM Tris-HCl, pH 9.0, 8 M urea (sigma), 10 mM β-mercaptoethanol) For a day. They were screened with sterile gauze to filter cellular debris. The refolding of the BACE-1 protein was induced by adding 75 ml of refolding buffer (20 mM Tris-HCl, pH 9.0, 0.5 mM oxidized glutathione, 1.25 mM reduced glutathione) to the filtered solubilization buffer using gauze. Since the initial urea concentration was 8M, the refolding buffer was diluted to a concentration of 6M, 4M, 2M, 1M, and 0.5M. A buffer containing 1.2 L of BACE-1 protein was obtained and concentrated to 800 mL using a concentrated tube (see Figure 3.)

(5) Purification of BACE-1 protein using ion-exchange chromatography and gel-filtration chromatography

Purification of BACE-1 protein was performed using FPLC instrument. First, the column was equilibrated with a full equilibration buffer (20 mM Tris-HCl, pH 7.5, 0.4 M urea) for purification using an ion exchange chromatography column (GE_HiTrap ™ 5 ml Q-Sepharose HP) And eluted with buffer (20 mM Tris-HCl, pH 7.5, 0.4 M urea, 0.5 M NaCl). The eluted fractions were confirmed by SDS-PAGE, and the fractions in which the BACE-1 protein was identified were collected and concentrated to an amount of 5 ml using a concentrated tube. The column was equilibrated using a buffer (20 mM Tris-HCl, pH 7.5, 0.4 M urea) for gel-filtration chromatography and the protein was purified. Each fraction was analyzed for protein using SDS-PAGE, and about 1.2 ml of BACE1 protein was purified by using a concentrated tube (see FIG. 4).

The test results of the present invention were expressed as mean ± SE. The statistical significance between the groups was analyzed by Duncan's multiple comparison method at p <0.05 level using SPSS program (ver. 14.0, SPSS Inc., Chicago, IL, USA) Respectively.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

(1) inserting a BACE-1 (β-secretase) gene into an expression vector pET28-MHL using a BseR1 restriction enzyme;
(2) preparing E. coli transformed with the pET28-MHL vector produced by the step (1);
(3) culturing the transformant prepared in (2) above to induce production of BACE-1 protein;
(4) obtaining a cell pellet by centrifugation of the medium in which the transformant has grown by the above step (3) at 4 DEG C and 7000 rpm for 10 minutes;
(5) The lysate buffer was added to the cell pellet obtained in the above step (4), and then the cell wall was disrupted using an ultrasonic mill, followed by centrifugation at 4 ° C and 15500 rpm for 40 minutes to obtain a cell pellet ;
(6) The cell pellet obtained in the above step (5) was added with a solubilization buffer of pH 9.0 consisting of Tris-HCl, urea and? -Mercaptoethanol, Filtering the post cell debris;
(7) The pH of the solubilized buffer solution filtered by the above step (6) is adjusted to a pH comprised of Tris-HCl, oxidized glutathione and reduced glutathione for refolding BACE-1 protein 9.0 refold buffer to induce refolding of the BACE-1 protein;
(8) concentrating the buffer containing the BACE-1 protein by using the concentration tube by the step (7); And
(9) ion exchange chromatography using an elution buffer having a pH of 7.5 consisting of Tris-HCl, urea and sodium chloride (NaCl), and concentrating the concentrate obtained in the step (8) A method for purifying a protein.













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