KR100225467B1 - Novel acetylcholinestrase gene and method of producing asetylcholinesterase in e.coli - Google Patents

Abstract

The present invention relates to a method for mass production of neurotransmitter acetylcholinesterase (hereinafter abbreviated as AchE) in E. coli. Specifically, the present invention relates to a new AchE gene, AchE, which is modified in 5'-terminal sequence without change in amino acid sequence, an expression vector capable of producing AchE in Escherichia coli, and a method for mass production of AchE protein in Escherichia coli using the same. As the AchE protein of the present invention, it can be usefully used to develop an AchE inhibitor which is a therapeutic agent such as Alzheimer's disease.

Description

New acetylcholinesterase gene and method for producing acetylcholinesterase in E. coli using the same.

The present invention relates to a method for mass production of neurotransmitter acetylcholinesterase (hereinafter abbreviated as AchE) in E. coli.

More specifically, the present invention provides a new AchE gene, AchE, which is modified in 5'-terminal sequence without change in amino acid sequence, an expression vector capable of producing AchE in Escherichia coli, and a method of mass-producing AchE protein in Escherichia coli using the same. In relation to the present invention, the AchE protein of the present invention can be usefully used to develop an AchE inhibitor which is a therapeutic agent for Alzheimer's disease.

Acetylcholinesterase (AchE) is an enzyme that is present in many nerve tissues, and acts at the junction of cholinergic nerve endings to mediate stimulation conduction. Quickly hydrolyzes choline and breaks it down into choline and acetic acid.

Acetylcholine is mainly synthesized at the periphery of motor neurons and stored in the vesicles and then moved to the release site by electric nerve impulse delivered to the nerve terminal, and then to the synapse. Is released. The released acetylcholine crosses a narrow synapse and binds to the acetylcholine receptor to produce a physiological effect. However, only part of the released acetylcholine binds to the receptor, most of which is hydrolyzed by AchE at synapses to choline and acetic acid. In addition, acetylcholine bound to the receptor, after inducing physiological action, is hydrolyzed by AchE and inactivated by falling off the receptor. This series of reactions is extremely rapid. In other words, it is estimated that the time required for the complete hydrolysis of one acetylcholine molecule is only 80 microseconds (Kim Dong-han, medicinal chemistry focused on new drug development).

Alzheimer's disease is known to be caused by the deficiency of acetylcholine in the brain and can be treated by supplementing acetylcholine deficiency. Therefore, it is recommended to use choline, lecithin, dimethylamine, etc., which are precursors of acetylcholine, or to use AchE inhibitors (Medical Information, July 1996).

At this time, the AchE inhibitor slows down the breakdown of acetylcholine released into the synapse and compensates for the lack of acetylcholine, thereby restoring the activity of the reduced brain cells, thereby slowing Alzheimer's disease. Representative AchE inhibitors developed so far include tacrine and donepezil, which are recognized by the US FDA. While all of these drugs have the effect of improving memory and improving behavior, Warner-Lambert's tacrine is known to be toxic to the liver. On the other hand, Eisai's donepezil is rarely toxic to the liver and is much more selective and higher than tacrine (Scrip, 1996.12.3).

In order to develop a new drug by rational drug design in connection with the development of the AchE inhibitor and the like, the analysis of tertiary structures such as proteins on which they function at the molecular level should be preceded. However, the AchE inhibitors reported so far have been developed by selecting and treating AchE with the compounds one by one. Therefore, in order to develop more effective AchE inhibitors, a large amount of human AchE needed to analyze the tertiary structure of human AchE should be produced.

Accordingly, the present inventors confirmed that the AchE gene is not expressed in E. coli in a general manner in order to obtain a large amount of AchE protein used to develop an AchE inhibitor, etc., and the 5'-terminal sequence is randomly changed without changing the amino acid sequence. The AchE gene was modified by using a polymerase chain reaction (hereinafter, simply referred to as PCR) to obtain an E. coli expression vector including the same, and expressed in Escherichia coli, thereby completing the present invention by producing a large amount of AchE protein. .

It is an object of the present invention to provide a new acetylcholinesterase (AchE) gene in which the 5'-terminal base sequence is modified without changing the amino acid sequence.

Specifically, the present invention provides a human AchE gene wherein the 5′-terminal nucleotide sequence is randomly modified and the 580th amino acid after its signal sequence is substituted for cysteine in serine. The present invention includes a human AchE gene having the nucleotide sequence of SEQ ID NO: 4.

It is also an object of the present invention to provide an expression vector capable of producing the AchE protein in E. coli.

Specifically, the present invention provides an expression vector comprising an AchE gene in which the 5'-terminal sequence is modified, and among these, the expression vector including the AchE gene having the nucleotide sequence of SEQ ID NO: 4 is named pET RAchE # 1. .

Another object of the present invention is to provide an E. coli transformant BL21 (DE3) / pLys S pET RAchE # 1 (KCTC 8781P, 97.1.29) transformed with the expression vector pET RAchE.

In addition, an object of the present invention is to provide a production method of culturing the E. coli transformant to induce the expression of AchE protein and then to produce AchE protein in large quantities from the crude cell eluate.

The present invention also provides an AchE protein in which a non-specific disulfide bond was inhibited by substituting a serine residue for the cysteine residue, which is the 580th amino acid after the signal sequence of AchE. Specifically, the AchE protein of the present invention has the amino acid sequence of SEQ ID NO: 5.

In addition, the AchE protein prepared in the present invention can be used to analyze its tertiary structure and select AchE inhibitors by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography.

Figure 1 shows the base sequence of the known acetylcholinesterase gene from which the N- terminal signal sequence is removed and its amino acid sequence inferred therefrom.

Figure 2a is a schematic representation of the cloning process of the acetylcholinesterase gene unmodified 5'-terminal base sequence of the present invention.

Figure 2b is a schematic representation of the cloning process of the acetylcholinesterase gene modified 5'-terminal base sequence of the present invention.

Figure 3 shows a comparison of the 5'-terminal sequence of the modified acetylcholinesterase gene and the 5'-end nucleotide sequence of the present invention unmodified acetylcholinesterase gene.

Figure 4 shows the results of the acetylcholinesterase gene of the present invention in E. coli and SDS-polyacrylamide gel electrophoresis.

Column 1 shows E. coli without the expression vector

Column 2 is E. coli containing the expression vector pET AchE

Column 3 is E. coli containing the expression vector pET RAchE

According to the above object, in the present invention, a new AchE gene randomly modified 5'-terminal sequence without change in the amino acid sequence, an expression vector comprising the same, E. coli transformed with the expression vector, and the transformed E. coli It provides a method for producing AchE comprising the step of culturing in a condition suitable for the expression of acetylcholinesterase.

Herein, it is preferable that the 5'-terminal sequence of the AchE gene substituted and modified according to the present invention is the following sequence.

ATG GAA GGA CGC GAA GAT GCT GAG CTG CTG GTA ACA GTA CGA GGA GGG CGG CTG CGT GGT

In addition, the present invention, by using the expression vector and E. coli transformants prepared above to replace the AchE protein cysteine residue after the AchE signal sequence with a serine residue to inhibit the non-specific disulfide bond (disulfide bond) AchE protein to provide. The AchE protein of the present invention has the amino acid sequence of SEQ ID NO: 5.

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

First, ribonucleic acid (RNA) is extracted from the human fetal brain and oligonucleotide (RNA) is extracted and the cDNA is generated by reverse transcriptase using d (T) primer. On the other hand, from the sequence information of AchE (Soreq, H., et al., PNAS 87: 9688, 1990), the polymerase chain reaction (PCR) corresponding to the 5'- and 3'-ends of the gene is called PCR. The primers for the present invention are designed and synthesized. At this time, the 5'-terminal primer has a nucleotide sequence of SEQ ID NO: 3 including the base sequence corresponding to the 1st to 8th amino acid and the restriction enzyme recognition site after the signal sequence of AchE, and the 3'-terminal primer Cysteine, the 580th amino acid after the signal sequence of AchE, is substituted with serine and has a nucleotide sequence of SEQ ID NO: 2 including a restriction enzyme recognition site and an end codon.

Human fetal brain cDNA is used as a template and the primers are used to amplify the AchE gene by PCR reaction. Gene fragments obtained by cleaving the amplified genes with restriction enzymes were replaced with an appropriate E. coli expression vector, for example, the UB A545 gene of pETUB A545 (Korean Patent Application No. 95-5096), which is an E. coli expression vector, to prepare an expression vector pET AchE. (See FIG. 2A). In addition, E. coli was transformed with the recombinant expression vector, and then cultured under conditions suitable for expression of the present gene, thereby confirming that AchE protein was not expressed (see FIG. 4).

At this time, in order to increase the expression rate of AchE according to the present invention, a 5'-terminal part of the AchE gene is prepared by performing a polymerase chain reaction of a randomly modified AchE gene with only a nucleotide sequence unchanged in the amino acid sequence. At this time, the expression vector pET AchE is used as a template and oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are synthesized and used. Specifically, the 5'-terminal primer contains a restriction enzyme recognition site for easy cloning with an E. coli expression vector, and the base sequence corresponding to the 1st to 19th amino acids after the AchE signal sequence is randomly changed without changing the amino acid sequence. And a base sequence of SEQ ID NO: 1 including a base sequence corresponding to the 20th to 26th amino acids after the signal sequence of AchE. In addition, the 3′-terminal primer has a nucleotide sequence of SEQ ID NO: 2 including a restriction enzyme recognition site and an end codon, wherein the cysteine, which is the 580th amino acid after the signal sequence of AchE, is substituted with serine.

Gene fragments obtained by cleaving the modified AchE gene with restriction enzymes were replaced with an appropriate E. coli expression vector, for example, the UB A545 gene of pETUB A545 (Korean Patent Application No. 95-5096), which is an E. coli expression vector. Prepare (see FIG. 2B). Subsequently, E. coli is transformed with the recombinant expression vector, followed by culturing under conditions suitable for expression of the present gene, thereby producing a large amount of AchE protein.

Specifically, the present invention includes the AchE gene having the nucleotide sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 5 inferred therefrom. The expression vector containing the AchE gene was named pET RAchE # 1 and transformed into Escherichia coli, and the transformant BL21 (DE3) / pLys S pET RAchE # 1 was established on January 29, 1997. Deposited to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 8781P).

Hereinafter, the present invention will be described in detail with reference to Examples.

The following examples illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Amplification of the AchE Gene

(Step 1) cDNA synthesis from human fetal brain cells

RNA extraction from fetal brain cells was performed according to the method of Cologinski et al. (Cholozynski, P., et al., Anal. Biochem. (1987) 162: 156).

To 1 g of cells, 1 ml of RNA extract (4M Guanidinium thiocyanate, 25 mM Sodium Citrate, pH7.0, 0.5% Sarcosyl, 0.1M 2-Mercaptoethanol) was added and shaken to destroy the cell membrane, followed by 0.8 ml of 2M sodium acetate (pH4). .0), 0.8 ml of phenol and 1.6 ml of chloroform-isoamyl alcohol (49: 1, v / v) were added and mixed well, followed by centrifugation at 12,000 g for 15 minutes at 4 ° C. Transferred to a new container with a volume of isopropanol, left for about 1 hour at -20 ℃ and centrifuged at 12,000g, 4 ℃ 20 minutes to obtain RNA precipitate. The precipitate was washed with 75% ethanol, then dried in vacuo and dissolved in 20ul of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). In order to synthesize cDNA disconnection, 10ul of the RNA solution obtained above was taken, 2ul of 0.1M methyl mercury ((CH ₃ ) ₂ Hg) was added, and the mixture was left at room temperature for 10 minutes to release the secondary structure of RNA. 1M 2-mercetoethanol was added and reacted for 5 minutes. It contains 5ul of 10-fold concentrated reverse transcriptase reaction solution (500mM Tris-HCl, pH8.3, 750mM KCl, 30mM MgCl ₂ , 10mM Dithiothreitol) and 2.5ul of 10mM dNTP mixed solution (dGTP, dATP, dTTP, dCTP respectively) ), Distilled water treated with 24 ul of diethylpyrocarbonate (DEPC), 1.0 ul of RNase inhibitor (RNasin, 1U / ul, Promega, USA), 1 ul of oligo (dT) _12-18 primer (dT) _12-18 primer, GibcoBRL, USA), and allowed to stand for 10 minutes at room temperature to allow RNA template and primer to adhere well, followed by 2.5ul reverse transcriptase (18U / ul, Superscript RNase H-Reverse transcriptase, BRL, USA) The reaction was carried out at 37 ° C. for 1 hour to synthesize cDNA strands.

(Step 2) Synthesis of the primer

The following primers were synthesized using a nucleic acid synthesizer (DNA Synthesizer, Applied Biosystems Inc., U.S.A) to obtain AchE gene from cDNA obtained in step 1 using polymerase chain reaction (PCR).

At this time, the primer having the nucleotide sequence of SEQ ID NO: 3 has a restriction enzyme Nde I recognition site (underlined portion), and is designed to have a nucleotide sequence encoding amino acids 1 to 8 after the signal sequence of AchE, The primer having the nucleotide sequence of SEQ ID NO: 2 has the restriction enzyme Sal I recognition site (underlined) and the termination codon of AchE, and the 580th amino acid residue after the signal sequence of AchE to prevent nonspecific disulfide bonds. Phosphorus cysteine is designed to have a nucleotide sequence substituted with serine (in bold italic font).

(Step 3) Isolation of AchE Gene

To amplify the AchE gene, polymerase chain reaction was performed as follows. 2 μg of the primer of SEQ ID NO: 3 and 2 μg of the primer of SEQ ID NO: 2 were obtained in the reaction tube, and 1 ng of the fetal brain cell cDNA obtained in step 1 was used as a template, and 10 μg of 10-fold polymerization buffer (500 mM) was added. KCl, 100 mM Tris-HCl, pH9.0, 1% Triton X-100, 2.5 mM MgCl ₂ ), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dATP, 2 mM dCTP, 2 mM dTTP), 2.5 monomer Taq polymerase After distilled water was added and the total volume was 100 μl, PCR was performed 40 times under the conditions of 1 minute (denatured step) at 95 ° C., 40 seconds (soaked step) at 55 ° C., and 2 minutes (polymerization step) at 72 ° C. The PCR product obtained above was isolated from 7% polyacrylamide gel, and it was confirmed that about 1800 base pairs of nucleic acid was amplified, and then purified from polyacrylamide gel under the same conditions.

(Step 4) Confirming the base sequence of the amplified AchE gene

In order to confirm the nucleotide sequence of the amplified gene was performed according to the method of Sanger et al. (Sanger, F. PNAS USA, 74, 5463 (1977)) as follows. First, 1 μg of the amplified AchE gene was treated with Clenau under the conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9) to form a smooth terminal, which was then converted into phenol / chloroform. Extracted and dissolved in 20 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH8.0).

Meanwhile, 2 μg of plasmid M13mp18 was completely cleaved with restriction enzyme Sma I under conditions of NEB buffer 4 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9), and then separated by 0.7% agarose gel. 7Kb nucleic acid fragments were isolated and purified.

Put 100ng of the AchE gene fragment and 100ng of the plasmid M13 fragment into the conjugation container and add 2μl of 10-fold conjugation reaction solution (500mM Tris-HCl, 100mM MgCl ₂ , 100mM dithiothreitol, 10mM ATP, 250µg / ml bovine serum albumin, pH7). .8), distilled water was added to a total volume of 20 μl with 10 units of T4 DNA ligase, followed by reaction at 16 ° C. for 12 hours. After the reaction, it was transformed into Escherichia coli JM 105 (ATCC 47016), and 8 µl of 0.2M IPTG (Ipropyl-beta-D-thiogalactopyranoside, USB cat. No.17884, USA) solution was added to the transformed cells. 100 μl of pre-cultured helper cells (E. coli JM105), 3 ml of 2X YT supernatant (soft agar, 16 g bactotrypton per liter, 10 g yeast extract, 5 g bactoagar) and 25 μl 4% X-gal solution Mix and place a minimum plate A (Min A plate, 10.5 g K ₂ HPO ₄ per 1 l, 1 g (NH ₄ ) ₂ SO ₄ , 0.5 g sodium citrate, 12 g bactoagar, 1 ml 20% MgSO 4, 0.5 After applying evenly on ml of 1% Vit.B1, 10ml of 50% glucose), incubate at 37 ° C. for 12 hours with a toothpick, followed by pickling with transparent tooth plaque at 37 ° C. in 2 ml of 2 × YT liquid medium for 5 hours. After incubation, centrifugation was performed. A 1/4 volume PEG / NaCl (20% polyethyleneglycol, 2.5M sodium chloride) was added to the obtained supernatant, M13 phage was collected, single-stranded nucleic acid was extracted, and the nucleotide sequence of the AchE gene was analyzed. It was confirmed to have a sequence.

Example 2 Preparation of Expression Vectors of AchE Gene

2 μg of plasmid pETUB A545 (Korean Patent Application No. 95-5096, KCTC 0151BP) was added to NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9) with restriction enzymes Nde I and Sal I. After complete cleavage under the conditions of, it was separated by 1% agarose gel electrophoresis to obtain a 4.5Kb nucleic acid fragment.

Meanwhile, 2 μg of the AchE gene fragment obtained in step 3 of Example 1 was subjected to the conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9) using restriction enzymes Nde I and Sal I. After complete cleavage at 7% by acrylamide gel electrophoresis, nucleic acid fragments of about 1800 base pairs were obtained.

Put 100ng of the AchE gene fragment and 100ng of the plasmid pETUB A545 fragment obtained in the conjugation reaction tube, and then 2μl of 10-fold conjugation solution (50mM Tris-HCl, 10mM MgCl ₂ , 100mM dithiothreitol, 1mM ATP, 25 µg / Ml bovine serum albumin, pH7.8), 10 units of T4 DNA ligase and distilled water were added so that the total volume was 20 µL, and the mixture was reacted at 16 ° C for 12 hours. After the reaction, E. coli HB101 (ATCC 33694) was transformed to obtain an E. coli expression vector pET AchE containing the AchE gene.

E. coli transformants were prepared by transforming the selected expression vector pET AchE of E. coli BL21 (DE3) / pLys.S (Novagen Inc., Madison WI53711, U.S.A).

Example 3 Expression of AchE Gene in Escherichia Coli

The E. coli transformant obtained in Example 2 was a liquid Luria medium (6% bactotrypton, 0.5% yeast extract, containing 50 μg / ml of Ampicillin and 25 μg of Chloramphenicol). After shaking for 12 hours in 1% sodium chloride, 3 ml of 300 ml of M9 medium (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO 4, 0.1 mM CaCl ₂ , 0.4% Casamino Acid, 10 μg / ml Vitimin B1, 40 μg / ml Empicillin, 25 μg of Chloramphenicol) and shake incubation at 37 ° C. for about 4 hours to obtain absorbance of bacteria at a wavelength of 650 nm. IPTG was added to a final concentration of 0.4 mM IPTG at about 0.3. About 4 hours after the addition of IPTG, the absorbance of the cell culture was measured, and the bacterial cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman JA14 rotor). The collected cell precipitates were subjected to 12% polyacrylamide gel electrophoresis in the presence of SDS according to the method of Laemmli (Laemmli, 1970, Nature 227: 680) and confirmed that the AchE protein was not expressed. .

Example 4 Amplification and Expression of AchE Gene Modified with a 5′-End Base Sequence

(Step 1) Amplification of AchE Gene Modified with 5'-End Base Sequence

To amplify the AchE gene with the 5'-terminal nucleotide sequence modified, the restriction enzyme Nde I recognition site (underlined) is included, and the first to 19th amino acids after the AchE signal sequence are changed without amino acid changes. Only the nucleotide sequence was randomly modified, and after that, the primer having the nucleotide sequence of SEQ ID NO: 1 was synthesized by matching the 20th to 26th amino acid after the signal sequence of AchE.

2 μg of the primer of SEQ ID NO: 1 and 2 μg of the primer of SEQ ID NO: 2 were added to the reaction tube, followed by 1 ng of the plasmid pET AchE obtained in Example 2 as a template, followed by 10 μl of 10-fold polymerization buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2.5 mM MgCl ₂ ), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dATP, 2 mM dCTP, 2 mM dTTP), 2.5 monomer Taq polymerase and distilled water After adding the total volume to 100 µl, PCR was carried out 25 times under the conditions of 1 minute (modification step) at 95 ° C, 40 seconds (immersion step) at 55 ° C, and 2 minutes (polymerization step) at 72 ° C. The PCR product obtained above was isolated from 7% polyacrylamide gel, and it was confirmed that about 1800 base pairs of nucleic acid was amplified, and purified from polyacrylamide gel under the same conditions.

(Step 2) Preparation of expression vector of AchE gene with 5'-terminal nucleotide sequence modified

2 μg of the AchE gene fragment obtained in step 1 was completely digested with restriction enzymes Nde I and Sal I under conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9). 7% polyacrylamide gel electrophoresis was performed to obtain nucleic acid fragments of about 1800 base pairs.

Put 100ng of the AchE gene fragment obtained above and 100ng of the plasmid pETUB A545 fragment obtained in Example 2 in a conjugation reaction tube, and then 2μl of 10-fold conjugation solution (50mM Tris-HCl, 10mM MgCl ₂ , 100mM dithiothreitol, 1mM). ATP, 25 μg / ml bovine serum albumin, pH 7.8) and 10 units of T4 DNA ligase were added to distilled water to a total volume of 20 μl, followed by reaction at 16 ° C. for 12 hours. E. coli BL21 (DE) / pLys. S (Novagen Inc., Madison WI53711, USA) was transformed to obtain an E. coli expression vector pET RAchE containing the AchE gene modified 5'-end.

(Step 3) Expression of the 5'-terminally modified AchE gene in E. coli

200 colonies of Escherichia coli transformants obtained in step 2 were prepared using liquid Luria medium (6% bactotripton, 0.5%) containing 50 μg / ml of Ampicillin and 25 μg of Chloramphenicol. After incubation for 12 hours in yeast extract, 1% sodium chloride, 3 ml of 300 ml of M9 medium (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.4% casamino acid, 10 μg / ml Vitamin B1, 40 μg / ml empicillin, 25 μg of chloramphenicol) and shake culture at 37 ° C. for about 4 hours to absorb the bacteria. IPTG was added to a final concentration of 0.4 mM IPTG at about 0.3 at a wavelength of 650 nm. About 4 hours after the addition of IPTG, the absorbance of the cell culture was measured, and then bacterial cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 rotor). The collected cell precipitates were identified by 12% polyacrylamide gel electrophoresis in the presence of SDS according to the method of Lamli (Laemmli, 1970, Nature, 227: 680), and clones with increased expression compared to the control were selected. It was named pET RAchE # 1.

The transformed Escherichia coli BL21 (DE) / pLys S pET RAchE # 1 was deposited to the GenBank of the Biotechnology Research Institute of Korea Science and Technology Institute on January 29, 1997 (Accession No. KCTC 8781P).

(Step 4) Confirming the 5'-terminal sequence of the modified AchE gene

In order to identify the 5'-terminal sequence of the AchE gene from the clone obtained above, it was performed as described in Sanger et al. (Sanger, F., PNAS USA, 74, 5463 (1977)) as follows. First, 2 μg of the plasmid pET RAchE # 1 obtained in step 3 was completely cleaved with restriction enzymes Xba I and Pst I under conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9). This was then separated with a 0.7% agarose gel to separate and purify nucleic acid fragments of about 600 base pairs.

Meanwhile, 2 μg of plasmid M13mp18 was completely digested with restriction enzymes Xba I and Pst I under conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9), followed by 0.7% agar. About 7 Kb nucleic acid fragments were separated and purified by an osgel.

Into each conjugation vessel, 100 ng of the AchE gene fragment and 100 ng of the plasmid M13mp18 fragment were placed and 2 μl of 10-fold conjugation solution (500 mM Tris-HCl, 100 mM MgCl ₂ , 100 mM dithiothreitol, 10 mM ATP, 250 μg / ml Bovine serum albumin, pH7.8), 10 units of T4 DNA ligase and distilled water were added to a total volume of 20 μl, and then reacted at 16 ° C. for 12 hours. After the reaction, it was transformed into Escherichia coli JM 105 (ATCC 47016), and 8 µl of 0.2M IPTG (Isopropyl-beta-D-thiogalactopyranoside, USB cat. No.17884, USA) solution was added to the transformed cells. 100 μl of pre-cultured helper cells (E. coli JM105), 3 ml of 2X YT supernatant (soft agar, 16 g bactotrypton per liter, 10 g yeast extract, 5 g bactoagar) and 25 μl 4% X-gal solution Mix and place a minimum plate A (Min A plate, 10.5 g K ₂ HPO ₄ per 1 l, 1 g (NH ₄ ) ₂ SO ₄ , 0.5 g sodium citrate, 12 g bactoagar, 1 ml 20% MgSO ₄ , 0.5 ml of 1% Vitamin B1, 10 ml of 50% glucose), and then incubated at 37 ° C. for 12 hours with a toothpick to produce a transparent plaque with 37 ml of 2 × YT liquid medium at 37 ° C. for 5 hours. After incubation, centrifugation was performed. Into the obtained supernatant, 1/4 volume of PEG / NaCl (20% polyethyleneglycol, 2.5M sodium chloride) was added, M13 phage was collected, single-stranded nucleic acid was extracted, and the 5'-terminal sequence of the AchE gene was extracted. Confirmed. (See Figure 3)

The present invention is very useful for mass production of AchE protein in E. coli by modifying the 5'-terminal sequence without changing the amino acid sequence of the AchE gene. The AchE protein thus obtained can be widely used to develop AchE inhibitors that can treat Alzheimer's disease caused by abnormalities of the neurotransmitter acetylcholine. Specifically, the AchE protein of the present invention is not only suitable for evaluating the inhibitory ability of the AchE inhibitor to search for an AchE inhibitor, but also rationally reveals the tertiary structure of the AchE protein by nuclear magnetic resonance spectra and X-ray crystallography. Useful for designing

In particular, the present invention is expected to contribute significantly to the development of AchE inhibitors by providing a protein suitable for analyzing the tertiary structure of AchE by expressing a large amount of human AchE protein in Escherichia coli, which has not yet been reported in the tertiary structure.

(Sequence list)

SEQ ID NO: 1

Sequence length: 88

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: oligonucleotide

(Sequence list)

SEQ ID NO: 2

Sequence length: 40

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: oligonucleotide

(Sequence list)

SEQ ID NO: 3

Sequence length: 34

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: oligonucleotide

(Sequence list)

SEQ ID NO: 4

Length of sequence: 1755

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: DNA

(Sequence list)

SEQ ID NO: 5

Sequence length: 584

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (7)

A human acetylcholine esterase (AchE) gene having a nucleotide sequence of SEQ ID NO: 4, wherein the nucleotide sequence of the 5 'terminal is modified without changing the amino acid sequence. 2. The AchE gene according to claim 1, wherein the cysteine, the 580th amino acid after the signal sequence of AchE, has a nucleotide sequence substituted with serine. An expression vector comprising the AchE gene of claim 1. The expression vector pET RAchE # 1 according to claim 3. E. coli transformed with the expression vector of claim 3. 6. Escherichia coli BL21 (DE3) / pLys S pET RAchE # 1 (Accession No .: KCTC 8781P, 1997.1.29). A method for producing an AchE protein comprising the step of culturing E. coli of claim 5 under conditions suitable for the expression of AchE.

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