KR20000006596A - Method of mass production of beta amyloid using bacteria - Google Patents

Abstract

PURPOSE: A process for producing amyloid known to be a causative agent for Alzheimer's disease is provided for large scale production. CONSTITUTION: The process comprises the steps of: amplifying PCR by using cDNA of amyloid precursor protein gene as template and pfu polymerase as an amyloid primer containing a restriction site for easy cloning; cutting the amplified DNA fragments by a restriction enzyme; cloning the DNA into an expression vector (pGEX-4T-2) encoding glutathion S-transterase DNA; transforming bacteria with the vector; culturing the bacteria to express beta amyloid fused with glutathion S-transferase for minimizing cohesion among hydrophobic domains of amyloid.

Description

Mass production method of beta amyloid using bacteria

The present invention relates to a method for mass production of beta amyloid using bacteria.

Beta amyloid is a protein found in patients with senile dementia, or Alzheimer's disease, and is composed of 40 amino acids (beta amyloid 1-40) or 42 amino acids (beta amyloid 1-42). Beta amyloid is produced during the amyloidogenic pathway of amyloid precursor metabolism protein, and accumulates in the brain and exhibits toxicity such as neuronal degeneration and death, and destruction of synapses at the nerve cell ends.

To date, genetic Alzheimer's disease, which occurs at a relatively young age, has a toxic effect by increasing the production of beta amyloid in brain cells by accumulating abnormal genes associated with Alzheimer's disease and accumulating them around nerve cells. .

However, it is still unclear how these beta amyloids actually accumulate in the brain and how such accumulation causes various symptoms seen in senile dementia, such as neuronal degeneration and death, and the destruction of synapses at nerve cell ends. Not known

In order to develop a treatment for senile dementia, it is necessary to investigate the mechanism of action of beta amyloid, which requires a large amount of beta amyloid.

However, at present, beta amyloid synthesized by a peptide synthesizer is very expensive.

Until now, it has been difficult to produce beta amyloid by genetic engineering using bacteria, and due to the large number of hydrophobic domains, it is difficult to separate and purify each other, and a restriction enzyme site capable of obtaining desired beta amyloid is not known.

The present invention is to solve the problems of the prior art as described above, to provide a method for mass production of beta amyloid using bacterial genetic engineering.

1 shows a vector used in the present invention,

Figure 2 shows the results of Coomassie Staining and Western blot analysis of the PCR product by the method according to the present invention.

In order to achieve the above object, the method for mass production of beta amyloid according to the present invention uses cDNA of amyloid precursor metabolism protein as a template and pfu polymerase as a primer of beta amyloid containing a restriction enzyme site. PCR amplifying; And cutting the DNA fragments amplified in the above step with a restriction enzyme, cloning the vector into a vector that can be expressed in bacteria, and transforming and expressing the bacteria.

Hereinafter, a method for mass production of beta amyloid using bacteria according to the present invention will be described in detail with reference to the accompanying drawings.

Table 1 below shows the nucleotide sequence and amino acid sequence of beta amyloid.

In SEQ ID NO: 1, Aβ1-40 represents beta amyloid 1-40 consisting of 40 amino acids, and Aβ1-42 represents beta amyloid1-42 consisting of 42 amino acids. In addition, the forward primer sequence for PCR (Polymerase Chain Reaction) is 5'-AAAAAGGATCCGATGCAGAATTCCGACAT-3 'for Aβ1-40 and Aβ1-42, and is indicated as Aβ (F) in SEQ ID NO: 1. The reverse primer sequence is 5'-ACGCGTCGACTCAGACAACACCGCCCACCAT-3 'for Aβ1-40 and is represented by Aβ40 (R) in SEQ ID NO: 1, and 5'-ACGCGTCGACTCACGCTATGACAACACCGCC-3' in SEQ ID NO: 1 for Aβ1-42. It is indicated by (R).

In the present invention, after amplifying the cDNA encoding the amino acid of the beta amyloid using PCR, cloning a vector that can be expressed in bacteria, and then transformed and expressed in E. coli is used.

First, the PCR process will be described.

The cDNA of amyloid precursor protein (APP) is used as a template, and the beta amyloid primer is added as described above. In the present invention, amplification using pfu polymerase having excellent proof reading instead of taq polymerase in order to prevent errors in the PCR process.

Pfu DNA polymerase is a DNA polymerase isolated from Pyrococcus furiosus, a hyperthermic primordial bacterium that lives in the sea, and is stable at high temperatures. Therefore, enzyme activity is maintained even after long-term use in experiments requiring high temperatures such as PCR. In addition, since pfu DNA polymerase has a 5'- to 3'- DNA synthesis function and a 3'- to 5 'repair function, there is an error compared to the taq polymerase, which has been widely used in DNA synthesis. There is little advantage. pfu DNA polymerase shows the optimal temperature between 72 ℃ and 78 ℃ and maintains more than 95% of enzyme activity even after 1 hour reaction at 95 ℃.

Using a recombinant plasmid cloned with amyloid precursor metabolism protein (APP) as a template, primers (Aβ (F), Aβ40 (R), and Aβ42 (R)) capable of obtaining only beta amyloid were prepared. Amplify 120 bp and 126 bp DNA fragments.

The primers used for PCR contained artificially created restriction enzyme sites (5 '-: BamHI, 3'-: SalI). DNA fragments amplified by these primers had a BamHI site at the 5'-part, The 3'-section will have the Sal I site.

The PCR DNA fragments thus amplified were digested with titration restriction enzymes and cloned in pGEX-4T-2 (purchased from Gibco-BRL), an expression vector of E. coli. pGEX-4T-2 is a vector configured to express external DNA in a fused form with a gene called glutathione S-transferase (GST), and the GST gene immediately after the tac promoter, followed by the external Multiple Cloning Sites (MCTs) exist so that genes can be inserted. The GST fusion system consists largely of the pGEX plasmid vector, the GST purification module, and the GST detection module, which not only express a large amount of foreign genes in E. coli, but also GST and fusion proteins. Purification and GST detection modules can be used to easily and easily separate beta amyloid products.

1 shows a vector used in the present invention.

Bacterial transformation was used by modifying the CaCl ₂ method. E. coli XL1-B was incubated overnight in LB liquid medium, which was then inoculated again in fresh LB liquid medium and incubated from A ₆₀₀ to 0.6. The cells were harvested by centrifugation at 4 ° C. and 3000 rpm for 5 minutes, and then suspended by adding 100 mM MgCl ₂ at 0.2 volume ratio, followed by centrifugation at 4 ° C. and 3000 rpm for 5 minutes, and again adding 100 mM CaCl ₂ at 0.2 volume ratio, and 0 ° C. Titration cells were made by leaving for 20 minutes at.

Thereafter, the cells were harvested by centrifugation at 4 ° C. and 3000 rpm for 5 minutes, and then re-suspended in 100 mM CaCl ₂ at a 0.1 volume ratio. About 100 μl of the suspension was extracted, mixed with the beta amyloid ligation mixture, and left to stand at 0 ° C. for 30 minutes. After 90 seconds of heat shock at 42 ° C., 1 ml of fresh LB liquid medium was placed at 37 ° C. It was stabilized by incubating for 1 hour at. Then, they were plated in LB solid medium containing the antibiotic ampicillin (50 µg / ml).

Bacterial expression was first performed by selecting transformants cloned with beta amyloid and incubating overnight in LB liquid medium containing ampicillin (50 µg / ml) (containing 2% glucose), and then again using ampicillin (50 µg). / Ml) was re-inoculated with fresh LB liquid medium (including 2% glucose) to incubate for 1 hour to the total transformant 1%.

Subsequently, beta-amyloid is expressed in a fused form with GST by adding 100 mM IPGT to 1 mM and incubating for 4 hours. As a control of the beta amyloid transformant, only the host E. coli XL1-B and the vector pGEX-4T-2 were expressed as a cloned transformant, and their expression was determined by Tricine-. Confirmed by SDS PAGE analysis. In other words, the cells were collected by centrifugation of the culture medium, mixed with 5X sample buffer solution, and then bathed in boiling water for 5 minutes to prepare samples for tricine-SDS PAGE analysis. These samples are loaded onto 10% polyacrylamide gel with 0.1% SDS, followed by electrophoresis and staining.

As a control, pGEX-4T-2 is a negative control in which only a vector is transformed into a host cell to clearly confirm that beta amyloid is expressed only in the recombinant plasmid.

2 is a view showing the results of Coomassie Staining and Western blot analysis of the PCR product by the method according to the present invention. In FIG. 2, the first and sixth lanes are E. coli XL1-B as hosts as the first control group, the second and seventh lanes are pGEX-4T-2 as a vector as the second control group, and the third lane and Lane 8 is beta amyloid 1-40 (Aβ 40) protein, lanes 4 and 9 are beta amyloid 1-42 (Aβ 42) protein, and lane 5 is a protein size indicator.

The process of Kumaji dyeing is as follows. First dyeing in coloring solution (0.1% coomassie brilliant blue R-250, 50% methanol, 10% acetic acid) for about 1 hour, then decolorizing in decolorizing solution I (50% methanol, 10% acetic acid) for 1 to 2 hours, It is then added to decolorizing solution II (10% methanol, 10% acetic acid) and decolorized until the dye solution is completely removed.

Western blot analysis was performed to accurately confirm the presence or absence of beta amyloid expression. Trisine-SDS PAGE analysis was performed as described above to separate the total proteins obtained from E. coli on a 10% polyacrylamide gel and then transferred to the PVDF membrane at 200 mA for 1 hour. The protein-transported PVDF membrane was left in a blocking solution containing 5% skim milk (5% skim milk + TBST buffer) for about 1 hour and then placed in 4G8 (1: 1000), a monocolonic antibody specific for beta amyloid. The reaction is carried out at 4 ° C. for 16 to 24 hours.

Then, washed three times with TBST buffer (20mM Tris, 137mM NaCL, 0.05% Tween-20, pH 8.0) three times for 10 minutes, and then added anti-rat IgG-HRP (1: 5000), a secondary antibody, for 30 to 50 minutes. Reacted. Again washed 4 times with TSBT buffer for 15 min and detected by ECL, substrate reactant of HRP.

Finally, a method for separating beta amyloid from transformed bacteria is described.

As described above, the transformed bacteria cloned with beta amyloid were transduced for 4 hours using the induction factor IPTG, and then the cells were harvested by centrifugation at 4 ° C and 3000 rpm for 10 minutes. The harvested cells are resuspended in a cold PBS buffer, and then completely disrupted by using an ultrasonic grinder, and centrifuged at 4 ° C. and 3000 rpm for 5 minutes to recover the supernatant from which all residues of the cells are removed.

Then, 50% suspension of glutathione Sepharose (Blutathione Sepharose) 4B was added to the separated supernatant and the mixture was continuously reacted at 4 ° C. for 1 day. After washing with cold 1X PBS buffer, the Sepharose beads were centrifuged to sink, and this washing process was repeated two more times. Add glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris HCl (pH 8.0)) to the settled Sepharose beads, suspend well, incubate for 5 minutes at room temperature, and centrifuge to recover the supernatant. Beta amyloid fused with GST was eluted. Elution of the fused beta amyloid was confirmed by Tricine-SDS PAGE analysis.

In the present invention, in order to solve the problems of the prior art that a lot of hydrophobic domains of beta amyloid aggregated with each other and difficult to purify and the restriction enzyme site to obtain only the desired beta amyloid, it is easy to clone to the primer to amplify beta amyloid Restriction enzyme sites were made and put in, and the restriction enzyme sites thus prepared were put in consideration of the reading frame so that they had no effect when expressed as proteins. In addition, by attaching a relatively large protein in the front, such as GST to minimize the aggregation until separation purification.

With respect to the tertiary structure of beta amyloid, beta amyloid has no problem in the protein tertiary structure of beta amyloid produced using bacteria as in the present invention because there is no translational modification site.

Claims (6)

In the beta amyloid production method, PCR amplification using cDNA of amyloid precursor metabolite protein as a template and primers of beta amyloid containing restriction enzyme sites using pfu polymerase; And DNA fragments amplified in the above step are cut with restriction enzymes, cloned into a vector that can be expressed in bacteria, and then transformed and expressed into bacteria. Mass production of beta amyloid using bacteria Way. The method of claim 1, Beta amyloid in the method is beta amyloid 1-40 or beta amyloid1-42 method of mass production of beta amyloid using a bacteria, characterized in that. The method of claim 1, Method for mass production of beta amyloid using a bacterium, characterized in that the restriction enzyme site of the primer is Sal I of 5'- BamH I, 3'-. The method of claim 1, A method for mass production of beta amyloid using bacteria, which uses pGEX-4T-2 as the vector which can be expressed in bacteria. The method of claim 4, wherein In the method, the pGEX-4T-2 vector is a mass production method of beta amyloid using bacteria, characterized in that the DNA is expressed in a fused form with glutathione S-transferase. The method of claim 5, In the above method, the beta amyloid is expressed in the form of a fusion protein with GST and is separated using a GST purification module or a GST detection module.