KR100220089B1 - Gene for adenylate kinase 2a isolated from human fetal liver cells, nucleic acid sequence and expression vector thereof - Google Patents

Abstract

The present invention provides an adenylate kinase gene, including its base sequence and the present gene, in Escherichia coli, which plays an important role in the energy metabolism process and induces non- globular hemolytic anemia due to a genetic defect thereof. An expression vector that can be mass produced.

Description

Adenylate kinase 2A gene isolated from human fetal hepatocytes, its nucleotide sequence and expression vector thereof

Figure 1 shows the cDNA containing the human adenylate kinase 2A (AK2A) gene, the full base sequence of clone HAK2A and the amino acid sequence of the enzymatic protein expected from this base sequence.

Figure 2 shows the comparison between the amino acid sequence of the enzyme protein expected in bovine AK2A gene (BOVAK2A) and the rat AK2A gene (RATAK2) and the amino acid sequence expected in human AK2A (HAK2A) gene.

FIG. 3 analyzes Northern blot expression of adenylate kinase in human fetal liver tissue.

Figure 4 shows the production of an expression vector capable of producing adenylate kinase by expressing human AK2A gene in E. coli.

5 shows that the human AK2A gene product expressed in E. coli has the activity of adenylate kinase.

[Use of invention]

The present invention relates to a vector that expresses human adenylate kinase gene and its nucleotide sequence and the present enzyme in E. coli.

More specifically, the present invention includes adenylate kinase genes, their base sequences and the present genes of human adenylate kinase genes, which play an important role in energy metabolic processes and induce their non-glomerular hemolytic anemia and the like by their genetic binding. It relates to an expression vector capable of producing late kinase in Escherichia coli.

[Background of invention]

Adenylate kinase (AK) catalyzes and balances the reversible transfer reactions of phosphate groups occurring between ATP, ADP and AMP, which are energy sources present in cells, and therefore has the effect of regulating the enhancement of adenine nucleotides in cells. It is an enzyme. Therefore, this AK enzyme is very important for the phosphorylation reactions related to intracellular signal transduction processes and is closely related to apoptosis, which is a phenomenon in which cells die due to energy metabolism in the human body, and carcinogenic action due to abnormal development of cells. (Hamada, et al., Adenylate kinase in "A Study of Enzymes Vol II" Florida CRC Press. 403-444. 1991)

More specifically, the in vivo function of AK enzymes discovered until recently, the AK enzyme phosphorylation as well as acts on the biosynthesis of TTP (thiamine triphosphate) with creatine kinase in muscle tissue and brain tissue It induces the production of ATP through the metastasis system and supplements the energy consumed while the cell is active. This process is very important for energy metabolism directly in the body. Therefore, when a genetic defect occurs in pyruvate kinase or a hexokinase enzyme, a phenomenon similar to that of severe hemolytic anemia occurs. When a genetic defect occurs in the present AK enzyme, congenital non-spheroidal anemia is caused. The hemolysis phenomenon caused by the genetic defect of the AK enzyme causes the genetic defect of the AK enzyme to affect the production of adenine nucleotides in the cell when the function of the corresponding system is weakened in the aged red blood cells. Red blood cells are destroyed in the process (Miwa, S. et al., Amer. J. Hematol. 14: 325, 1989).

As such, the AK enzyme is deeply related to muscle diseases such as hemolytic anemia and energy metabolism, and thus is very important in the diagnosis and treatment of the disease. In addition, many studies on AK enzymes have been conducted, and so far, it has been reported that 30 kinds of AK enzymes exist in various kinds of organisms.

In particular, in vertebrates, three types of AK genes, AK1, AK2 and AK3, are present in different tissues and have slightly different functions in the cells. Specifically, in the case of AK1, muscle cells and brain cells And in the red blood cell cytoplasm etc., the case of AK2 is present between the mitochondrial membranes in hepatocytes, kidney cells, spleen cells and heart cells (Khoo and Russell, Biochem. Biophys. Aata 268: 98-101, 1972). AK3 also acts as a phosphorylation transferase between GTP and AMP, present in the mitochondrial matrix of heart and liver cells (Tomosselli et al., Eur. J. Biochem. 93: 257-262, 1979).

In addition, many studies have been conducted to deeply understand AK enzymes, which play an important role in various organisms. (Kishi et al., J. Biol. Chem. 261: 2942-2945, 1988; Matsura et al., J. Biol. Chem. 264: 10148-10155, 1989). In humans, however, the gene for the AK2 enzyme has not been cloned yet, so much of its character is unknown.

The AK2 enzyme has been characterized using enzymes isolated from cows. The AK2 enzyme is divided into two enzymes, AK2A and AK2B, depending on the amino acid sequence located at the C-terminal site of the cow's tissue site and the enzyme protein. Can be. Specifically, AK2A enzyme protein is composed of 241 amino acids, AK2B enzyme protein is composed of 234 amino acids, these are the eight amino acids in the C-terminal region of the AK2A enzyme protein, Cys, Lys, Asp, Leu, Val, Met , Phe, Ile is substituted with Ser at the C-terminus of the AK2B enzyme protein. It appears that this is caused by alternating splicing mechanism that occurs during transcription of the AK2A gene, which changes the sequence of the enzyme protein at the C-terminus (Tanaka, et al., Gene, 93: 221-227, 1990).

Since the AK enzyme gene is closely related to physiological activity in human body including energy metabolism in vivo, it will contribute to molecular pathological research and utilization of muscle disease including heart disease. In humans, however, the genes for the AK2 enzyme have not been cloned yet, and despite its usefulness, little is known. Therefore, the present inventors attempted to reveal the gene of AK2 enzyme using human fetal hepatocytes, and cloned the gene of AK2A derived from the human body to reveal its sequence, and produced the expression vector pHT-AK2A to produce human AK2A having enzyme activity. The present invention has been completed by preparing enzyme proteins.

[Summary of invention]

It is an object of the present invention to provide an adenylate kinase (AK) 2A gene. The AK2A gene of the present invention is isolated from a cDNA library prepared from human fetal liver cells.

Specifically, the present invention provides each cDNA clones P05D07 and HAK2A, which comprise some and all of the human AK2A gene.

The present invention also provides Escherichia coli (Accession No .: KCTC 8735P) transformed with cDNA clone HAK2A.

It is also an object of the present invention to provide a base sequence of the AK2A gene from the cDNA clones P05D07 and HAK2A and an amino acid sequence expected therefrom.

It is also an object of the present invention to provide an expression vector capable of expressing AK2A enzyme protein in E. coli, including the AK2A gene. Specifically, the present invention provides an expression vector pHT-AK2A that effectively produces AK2A enzyme protein.

The present invention also provides a method of culturing the transformant to produce AK2A enzyme protein in E. coli.

Detailed description of the invention

Hereinafter, the present invention will be described in detail.

MRNA is isolated from human fetal liver tissue and the corresponding cDNA is synthesized using oligonucleotide primers and reverse transcriptase. The synthesized cDNA is cloned into a λZAPII XR vector or the like to prepare a cDNA library, which is a library of genes expressed in human fetal liver tissue. Partial nucleotide sequences are determined from these libraries and genes are identified by comparison with a database of genes already known using computer programs and the like (see FIG. 2 and Table 1).

Using a database to identify the nucleotide sequences of the genes, it showed 93% homology at the amino acid level with adenylate kinase 2A, a type of phosphate transferase known to exist in cattle, In terms of overall isolation, P05D07, a cDNA clone showing 97% similarity, is isolated.

To obtain a clone containing the entire structure of adenylate kinase from the cDNA library of human fetal liver tissue, cDNA clone P05D07 was used as a probe to search for and isolate a clone containing the entire structure of the present enzyme protein. At this time, the isolated clone is used to determine the cDNA total nucleotide sequence and the cDNA clone is named HAK2A (see FIG. 1). Transgenic Escherichia coli containing the HAK2A base sequence was deposited on 8 April 1996 to the Genetic Bank of Korea Institute of Science and Technology, Biotechnology Research Institute (Accession Number: KCTC 8735P).

To confirm the actual expression of human AK2A gene in cells, DNA fragments obtained from the complementary cDNA clone P05D07 by isolating mRNA from human fetal liver tissue were examined using Northern blot using a hybridization probe. Sambrook, et al., In "DNA Cloning: A Laboratory Manual". As shown in Figure 3, the analysis of the mRNA of the human AK2A gene, it is confirmed that there is a different transcript having a molecular weight of about 3.4kb, 2.1kb and 1.0kb, respectively (see Figure 3). This suggests that alternating splicing of mRNA also occurs in the transcription of human AK2A and AK2B genes, as seen in the transcription of bovine AK2A genes.

In addition, in order to produce recombinant protein of human AK2A enzyme in Escherichia coli, an expression vector pHT-AK2A comprising the HAK2A gene of the present invention is prepared. It is prepared by performing a polymerase chain reaction using a HAK2A clone containing the entire nucleotide sequence of the AK2A gene to obtain an AK2A gene and inserting it into a vector (see FIG. 4). In this case, the amplification station used for the polymerase chain reaction is a chemical synthesis of a sequence encoding the start and end portions of the open reading frame with reference to the entire nucleotide sequence of AK2A.

In addition, in order to produce adenylate kinase in Escherichia coli, E. coli DH5α is transformed with the expression vector pHT-AK2A to culture these transformants. In order to induce the expression of the AK2A gene in these transformants, IPTG was used in the logarithmic proliferative phase of the cultured cells, and the cultured cells were disrupted and examined for adenylate kinase activity in the crude cell solution.

At this time, adenylate kinase activity is measured by spectroscopic analysis using a chain enzyme reaction or the like (Kim, et al., Biochemistry, 29: 1107-1111, 1990). As a result, it was confirmed that the activity of the AK2A enzyme was significantly increased in E. coli transformed with the present vector compared to the control group (see FIG. 5).

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

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1

[Preparation of cDNA Library]

Total RNA contained in cells was extracted from human fetal liver tissue by using guanidine thiocyanate and acid phenol. The extracted total RNA was then passed through an oligo-dT cellulose column to separate poly (A) -RNA. To prepare the cDNA library, the first strand cDNA was synthesized by reacting 5 μg of poly (A) -RNA with 17 (dT) linked oligonucleotide primers and a reverse transcriptase of the Moroni leuchemia virus. Next, a second strand of cDNA was synthesized using RNA degrading enzyme and E. coli DNA synthase.

To clone the synthesized cDNA into a vector, the carrier, a linking chain of restriction enzyme Eco RI, which provides a base sequence complementary to both ends of the vector at both ends of the cDNA, was cut with restriction enzyme Xho I. At this time, in order to increase the length of the base included in the cDNA library and to increase the cloning efficiency, DNA fragments cut by restriction enzymes and small cDNAs of 200 bp or less were removed using a separcral column. The cDNA was then linked to the vector λZAPII XR digested with the restriction enzymes Eco RI and Xho I and phages were prepared by in vivo packaging to prepare a cDNA library of human fetal liver tissue.

Example 2

[Partial sequencing]

To determine the partial nucleotide sequence of each clone, E. coli was infected with the phage of the cDNA library and the ExAssist helper phage together to obtain the f1 phage containing only the pBluescript plasmid portion. The phages were again infected with Escherichia coli, and clones containing two strands of replicate plasmids were selected using a medium containing empicillin. E. coli containing the plasmid was then infected with M13 helper phage, and one strand of M13 DNA phage was extracted and used as template DNA for partial sequencing.

The nucleotide sequence binds the SK primer of SEQ ID NO: 1 to the running DNA, and then reacts the dNTP necessary for the reaction with ³⁵ S-dATP for displaying the reaction product on the X-ray film together with a DNA polymerase sequence. Decided. The reaction was dispensed into ddNTP solution to stop DNA polymerization. The reaction product was separated by electrophoresis on a 6% acrylamide gel containing 7M urea, and the gel was dried to read the photos from which the reactants were separated by X-ray film. As a result, a partial nucleotide sequence of SEQ ID NO: 2 was obtained.

Example 3

[Analysis of base sequence and amino acid sequence]

In order to search for the nucleotide sequence and amino acid sequence of each cDNA obtained in Example 1, a main computer connected to a local area network within the biotechnology laboratory was used. Analysis of nucleic acid nucleotide sequences was performed using the BLASTN program to search the GenBank database, and analysis of amino acid sequences using the BLASTX program was used to search the Swiss port protein database (Altschul et al., J. Mol). Biol. 215: 403-410, 1990) (see FIG. 2 and Table 1).

Example 4

[Isolation of Clones Having Partial Base Sequences of Human AK2A Gene and Determination of Total Base Sequences thereof]

The cDNA clone P05D07, which has a partial nucleotide sequence of the human adenylate kinase gene, was selected to determine the DNA base sequence of the AK2A gene included in this clone.

The cDNA clone P05D07 used for data retrieval has the partial nucleotide sequence of SEQ ID NO: 2, and the base sequence of the gene having similarity with the sequence of amino acids expected in the nucleic acid nucleotide sequence of the clone of the present invention was searched using a database. The results are shown in Table 1. In Table 1, the registration number of the AK2A gene cloned and registered in GenBank and the name of the gene are recorded, and the open reading frame (ORF) is a translation skeleton used when translating three nucleic acid sequences in pairs. Indicates. The high score is a numerical representation of the homology of the nucleotide sequences, with a value of +5 if the two corresponding bases on the two genes being compared are the same base and -4 if they are not the same base. It is calculated. Therefore, it can be judged that the higher the numerical value obtained after summing the values obtained from the entire bases searched, the higher the similarity is. Probability also means the probability that the searched sequence will coincide with data in the database.

Therefore, as shown in Table 1 and 2, it can be seen that the base sequence of the cDNA clone P05D07 has a high homology with the nucleic acid base sequence of the AK gene cloned in cows or mice, among the base sequences of the genes identified in the search process.

Example 5

[Identification of entire nucleotide sequence and amino acid sequence of human AK2A gene]

To find cDNA clones containing the entire gene of human AK2A, cDNA libraries of human fetal liver tissues were searched by the plaque hybridization method. In this case, the P05D07 clone of Example 4 was used as a probe, and five cDNA clones were isolated as a result of the search. From the isolated clones, the nucleic acid nucleotide sequence of SEQ ID NO: 5 containing the entire nucleotide sequence of the gene and the amino acid sequence of SEQ ID NO: 6 flowing out from this nucleotide sequence were identified. In addition, the cDNA clone having the entire nucleic acid base sequence was named HAK2A (see also Figure 1).

The HAK2A gene consists of 931 bases, where one open reading frame consists of 717 bases. Starting from the first methionine from these nucleic acid sequences, 239 amino acids are translated to form the enzyme protein, with a molecular weight of about 26KD.

The enzyme protein was compared with bovine AK2A and rat AK2 enzyme protein by amino acid sequence, and showed 98% similarity (95% identity and 3% similar substitution) and 96% similarity (92% identity and 4, respectively). % Substitutions), so they can be judged as proteins having the same function, and the gene of the present invention was confirmed to be a new gene that is not known at all to the world (see FIG. 2).

Example 6

[Preparation of Escherichia Coli Transformant Containing cDNA Clone HAK2A]

E. coli DH5α was transformed with the cDNA clone HAK2A obtained in Example 4 to obtain a transformant. This transformant was deposited on April 8, 1996 to the Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute (Accession Number: KCTC 8735P).

Example 7

[MRNA Analysis of Human AK2A Gene]

In order to confirm the actual expression of human AK2A gene in cells, mRNA was isolated from human fetal liver tissue and subjected to electrophoresis on formaldehyde gel. Next, they were transferred to nylon membranes and immobilized mRNA with a UV-cross linker. Here isotope DNA fragments obtained from complementary cDNA clone P05D07. Northern blot using a P-labeled hybridization probe (Sambrook, et al., In DNA Cloning: A Laboratory Manual).

Example 8

[Production of Expression Vectors Producing Human AK2A Recombinant Protein]

Clone P05D07, which has a partial nucleotide sequence of human AK2A gene, was cleaved with Sma I and Xho I and then smoothed with klenow enzyme. The plasmid pHT-AK2At1 was prepared by conjugation with the pHT12I plasmid which was previously cut and smoothed with Nco I. This was again cleaved with Nco I, and the Nco I fragment obtained by the polymerase chain reaction using the clone HAK2A containing the entire nucleotide sequence of the AK2A gene was conjugated to prepare an expression vector pHT-AK2A (see FIG. 4). .

At this time, the amplification station used in the polymerase chain reaction was prepared by chemically synthesizing to have the sequence of SEQ ID NO: 3 and SEQ ID NO: 4 to reference the entire nucleotide sequence of the AK2A gene to encode the start and end of the open reading frame.

Example 9

[Enzyme Activity of Human AK2A Recombinase Enzyme Protein Produced in Escherichia Coli]

In order to confirm that the AK2A gene of the present invention effectively expressed the present enzyme protein and exhibited adenylate kinase activity, E. coli DH5α was transformed with the expression vector pHT-AK2A and the standard control pHT12I, respectively, and these transformants were cultured. In order to induce the expression of the enzyme protein by the AK2A gene, 0.5 mM IPTG was added to the logarithmic growth of the cultured cells, followed by shaking culture. The cultured cells were recovered, disrupted by ultrasonication, and examined for adenylate kinase activity with crude cell solution.

Adenylate kinase activity was determined spectroscopically using chain enzyme reactions (Kim, et al., Biochemistry, 29: 1107-1111, 1990). Reaction with 75 mM triethanolamine (pH7.6), 1.2 mM AMP, 1.0 mM ATP, 1 mM MgSO, 120 mM KCl, 0.2 mM NADH, 0.3 mM phosphinolpyruvic acid, 16.5 units lactic acid dehydrogenase, 6 units pyruvic acid kinase The mixed solution was left at 25 ° C. for 10 minutes. Next, the sample was placed in the reaction mixture, and the progress of the reaction was measured using a spectrophotometer to the extent that the absorbance decreased with time at a wavelength of 340 nm.

As a result, the enzymatic activity of adenylate kinase in E. coli with the expression vector pHT-AK2A containing the AK2A gene was found to be 960 mU / mg. This was about twice as high as 550mU / mg in Escherichia coli with the plasmid pHT12I used as the standard control strain (see Figure 5). Therefore, it was confirmed that the human AK2A gene was expressed in E. coli transformed by the expression vector pHT-AK2A, which is not shown in the normal strain.

Therefore, the AK2A gene of the present invention can be useful for measuring the activity of adenylate kinase expressed in cells and for diagnosing and treating hemolytic anemia or muscle disease, and in view of the base sequence results of the present AK2A gene, By studying these characteristics in depth, they can greatly contribute to the molecular pathological studies of related diseases.

In addition, the present AK2A gene and its expression vector can be used to obtain a large amount of the present enzyme protein present in the body, which can be applied to the treatment of specific diseases as well as the study of AK enzymes.

[SEQUENCE TABLE]

SEQ ID NO: 1

Sequence length: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQUENCE TABLE]

SEQ ID NO: 2

Length of sequence: 332

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: cDNA

[SEQUENCE TABLE]

SEQ ID NO: 3

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQUENCE TABLE]

SEQ ID NO: 4

Sequence length: 30

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQUENCE TABLE]

SEQ ID NO: 5

Sequence length: 931

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: cDNA

[SEQUENCE TABLE]

SEQ ID NO: 6

Sequence length: 239

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (8)

An adenylate kinase 2A gene derived from human tissue comprising the nucleotide sequence of SEQ ID NO: 5. The adenylate kinase gene of claim 1, wherein the adenylate kinase gene is derived from a cDNA clone HAK2A comprising the entire adenylate kinase 2A gene. The transformant (accession number: KCTC 8735P) characterized by producing E. coli transforming with the cDNA clone HAK2A of claim 2. Adenylate kinase 2A from human tissue comprising the amino acid sequence of SEQ ID NO: 6. An expression vector comprising the human adenylate kinase 2A gene of claim 1. The expression vector of claim 5, wherein the expression vector is pHT-AK2A. A transformant characterized by producing E. coli transforming with the expression vector of claim 5. A method for producing an enzyme protein, comprising culturing the transformant of claim 7 to produce adenylate kinase in Escherichia coli.