KR20000061545A - A Gene Coding for Thermostable Alkaline Phosphatase from Thermus caldophilus and Amino Acid Sequence Deduced Therefrom - Google Patents

Abstract

PURPOSE: A gene encoding a heat resistant alkaline phosphatase (Tca APase) derived from Thermus Caldophilus GK24 and an amino acid sequence thereof are provided. The resultant gene encoding Tca APase can produce the heat resistant alkaline phosphatase useful in enzyme immuno assay (EIA), immunostaining, ELISA, western blotting, dot blotting, hybridoma screening etc., in large scale. CONSTITUTION: An isolating and cloning method of the gene comprises the steps of: isolating Tca APase from Thermus Caldophilus GK24 and sequencing a part of the amino acids; synthesizing the oligonucleotides on the basis of the sequences; and cloning the gene encoding Tca APase using the oligonucleotides as a probe. The nucleotide sequence of the cloned gene and the amino acid sequence encoding a protein consist of 501 amino acids, in which the protein comprises a mature protein and a signal peptide consisting of approximately 27 amino acids.

Description

A Gene Coding for Thermostable Alkaline Phosphatase from Thermus caldophilus and Amino Acid Sequence Deduced Therefrom}

The present invention relates to a heat resistant alkaline phosphatase derived from Thermos caldophilus GK24. More specifically, the present invention relates to genes encoding heat resistant alkaline phosphatase derived from Summers Caldophilus GK24 and amino acid sequences inferred therefrom.

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1., Hereinafter referred to as 'APase') is an enzyme that catalyzes the hydrolysis and transphosphorylation of non-specific phosphate monoesters. . APase is an enzyme containing zinc widely distributed from microorganisms to mammals. Mostly, two zinc ions (Zn ²⁺ ) and one magnesium ion (Mg ²⁺ ) act on active sites. APase is an enzyme that is induced when phosphoric acid is deficient in the medium and is inhibited by excess phosphoric acid and secreted into intracellular periplasm or extracellular cells.

Among the various APases, Escherichia coli APase is a representative bacterial APase, a product of the phoA gene belonging to pho regulon. The electrical APase is synthesized in the form of a signal peptide attached to the cytoplasm, then translocated and cleaves the signal peptide by a peptidase, and then matures into a periplasm in a mature form. Present and active. In the cell, APase is induced to use trace amounts of inorganic phosphoric acid and phosphate-containing nutrients such as phosphorylated compounds or polyphosphoric acid from the surrounding environment when phosphoric acid is deficient.

These APases are very useful enzymes for molecular biology research. They are capable of producing soluble or insoluble pigments depending on the substrate used, thereby enzymatic immunoassay (EIA) or antibody staining of cells or tissues. Used for In addition, it is possible to use for enzyme-antibody binding fluorescence (ELISA), Western blotting, dot blotting and hybridoma screening. However, the conventional APases have a disadvantage in that the reaction rate is slow due to the weak heat resistance, and thus, there has been a continuous demand for the development of better heat resistant APases in the art.

In general, the Thermos sp. (Thermus sp.) Strains are commonly used in the production of heat-resistant enzymes, but until now, only one of the APases from the Thermos genus derived from Thermos aquaticus YT-1 is known. However, even APase derived from the electric thermos is not very heat resistant, so the need for developing APase with stronger heat resistance has been continuously raised.

Therefore, the inventors of the present invention intensively tried to separate heat-resistant alkaline phosphatase and genes encoding the same from other thermos strains, resulting in the separation of heat-resistant APase and genomic genes from Thermos caldophilus GK24. By determining the nucleotide sequence of the gene and the amino acid sequence inferred therefrom, it was confirmed that it is a novel enzyme having an amino acid sequence different from the conventional APase, to complete the present invention.

After all, the main object of the present invention is to provide a gene sequence encoding a heat resistant APase derived from Thermos caldophilus GK24.

Another object of the present invention is to provide an amino acid sequence inferred from the former gene sequence.

1 is a photograph showing the results of SDS-PAGE electrophoresis of purified Tca APase.

Figure 2 is a graph showing the relative activity according to the temperature of Tca APase.

3 is a graph showing the results of examining the heat resistance of Tca APase.

4 is a diagram showing the amino acid sequence of a peptide fragment obtained by cleaving Tca APase with CNBr or BNPS-skatole.

FIG. 5 shows a sequence of oligonucleotides synthesized based on the amino acid sequence of FIG. 4 above.

Figure 6 shows a restriction map of clones with 2.8 kb ApaI and 2.1 kb BamHI fragments containing the Tca APase gene.

Figure 7 shows the amino acid sequence of Tca APase compared with the amino acid sequence of APase of other species.

The inventors have described APase (hereinafter referred to as 'Tca APase') from Thermos caldophilus GK24 (Taguchi, H., et al., J. Biochem., 91, 1343 (1982)). Isolation determined some amino acid sequences, based on which oligonucleotides were synthesized, and then used as probes to clone the Tca APase gene. As a result of determining the nucleotide sequence of the electrically cloned gene and the amino acid sequence inferred therefrom, the Tca APase gene was identified as encoding a protein consisting of a total of 501 amino acids including a signal peptide of about 27 amino acids and a mature protein.

That is, the present inventors isolated Tca APase from Thermos Caldophilus GK24, and then purified by affinity chromatography to obtain Tca APase having a molecular weight of about 54,000 Daltons. As a result of examining the heat resistance of the purified Tca APase, when the 50 mM Tris-HCl (pH 8.6) buffer solution was used for 2 hours at 80 ℃ and 85 ℃ was maintained at 60% and 80% or more, respectively, 50 When mM Glycine-NaOH (pH 11.0) buffer was used, the activity was decreased by 50% after 2 hours at 80 ° C and 40% after 10 minutes at 85 ° C.

Peptide fragments were prepared from the purified Tca APase to determine the amino acid sequence of some peptides, oligonucleotides were prepared based on the previous amino acid sequence, and then used as probes in Southern blots from the Summers Caldophilus genomic gene. Gene fragments containing the Tca APase gene were selected. Cloning the entire Tca APase gene using the electric gene fragment as a probe, determining the sequencing, and analyzing it by PCGENE and NCBI-blast search to encode signal peptide and Tca APase. A reading frame of the Tca APase gene including a total of 1503 bp (1506 bp including stop codons) was determined.

The Tca APase gene had a G + C content of about 69.3%, and the GGAG sequence inferred from the ribosome binding site appeared immediately before the ATG codon, but no -10 or -40 conserved sites appeared in other species. Tca APase is first expressed as a protein consisting of a total of 501 amino acids, and then the signal peptide consisting of about 27 amino acids appears to be removed during translocation. On the other hand, the amino acid sequence of Tca APase was compared with the amino acid sequences of APases of other species. As a result, it was confirmed that Tca APase was a novel APase having an amino acid sequence different from that of APases derived from other species.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Purification of Tca APase

Seed cultures were inoculated into 500 ml of pre-culture medium [0.4% bactotrypton, 0.2% yeast extract, 1 × Castenholz salts], shaken at 72 ° C at 180 rpm, and then phosphate was removed. Inoculated 2% in 2.5L of APase optimal induction medium [0.3% sodium glutamate, 0.2% bactotryptone, 0.5% glucose, 10 mM Tris-HCl (pH 8.0), 1 × castenholz salt] and 180 ° C. at 180 ° C. Shake culture was performed for 16 hours at rpm. Subsequently, the cells were crushed by cold osmotic shock to obtain a coenzyme solution containing APase. The protein was precipitated by slowly adding 70% (w / v) saturated ammonium sulfate to the coenzyme solution, followed by dialysis using 10 mM Tris-HCl (pH 7.4) / 1 mM MgCl ₂ buffer. The coenzyme solution from which the salt was removed by dialysis was subjected to an affinity chromatography column containing 4 (4-aminophenylazo) -phenylasonic acid-Sepharose 4B as a filler. Passed to bind Tca APase. Buffer consisting of [1 M KCl / 10 mM Tris-HCl (pH 7.4)] and [2 M guanidium-HCl / 1 M KCl / 10 mM Tris-HCl (pH 7.4)] in a column bound to the electric Tca APase Was flowed while forming a concentration gradient to liberate Tca APase to obtain purified Tca APase. The results of SDS-PAGE electrophoresis are shown in FIG. 1. In FIG. 1, the first lane represents purified Tca APase, and M represents molecular weight marker, respectively. As shown in Figure 1, it was possible to obtain a pure purified APase, the molecular weight of Tca APase was about 54,000 Daltons.

Example 2: Investigation of Heat Resistance of Tca APase

The optimum reaction temperature and heat resistance of Tca APase obtained in Example 1 were investigated. The composition of the reaction solution was to include 185 μl of buffer, 5 μl of Tca APase, and 100 μl of 10 mM pNPP. Tca APase activity was determined by measuring the absorbance at 410 nm for pNPs with chromophores formed by freeing phosphoric acid. In order to determine the optimum reaction temperature, the activity was measured every 10 ° C. from the reaction temperature of 40 ° C. to 100 ° C., and 50 mM Tris-HCl (pH 8.6) or 50 mM Glycine-NaOH containing 1 mM MgCl ₂ was used as the buffer solution. (pH 11.0) was used. Figure 2 is a graph showing the relative activity according to the temperature of Tca APase. As shown in Figure 2, the optimum reaction temperature was about 85 ℃.

In order to investigate the heat resistance of Tca APase, the buffer solution and the enzyme solution were mixed at 80 ° C. to 100 ° C. at 5 ° C., heat-treated for 2 hours, cooled in ice water, and substrates were added to measure activity at 80 ° C. As a buffer solution, 50 mM Tris-HCl (pH 8.6) and 50 mM Glycine-NaOH (pH 11.0) buffer were used, and the resultant was measured at 80 ° C. and 85 ° C. and the activity was measured. , 50 mM Tris-HCl (pH 8.6) buffer solution was maintained at 60% and 80% or more after 2 hours at 80 ℃ and 85 ℃, respectively, 50 mM Glycine-NaOH (pH 11.0) buffer solution When used for 2 hours at 80 ℃ was shown to decrease to 40% after 10 minutes at 50% and 85 ℃. As a result, 50 mM Tris-HCl (pH 8.6) was more preferable for the heat resistance of Tca APase than 50 mM Glycine-NaOH (pH 11.0) buffer. △ in 80 ℃, when left in Glycine-NaOH buffer solution; ◇ when it is left in Glycine-NaOH buffer solution at 85 degreeC; ○ when left in Tris-HCl buffer solution at 80 ℃; And □ represent heat resistance when left in 85 ° C. and Tris-HCl buffer solution.

Example 3: Some Amino Acid Sequencing of Tca APase

In order to partially determine the amino acid sequence of Tca APase, firstly, the purified Tca APase obtained in Example 1 was lyophilized, followed by CNBr cleaving the carboxy terminus of methionine and tryptophan, respectively. BNPS-skatole was treated. That is, in order to treat CNBr, first, carboxymethylation of cysteine residues of Tca APase was performed to completely denature the protein by the following method. After completely dissolving 1 mg of completely lyophilized Tca APase in 200 μl of buffer, nitrogen gas (N2 gas) was treated for 15 minutes in the upper layer, and further treated with nitrogen gas for 1 hour by adding 200 μl of 4 mM DTT. Wrap the aluminum sample with aluminum foil to shield it from light, add 160 μl of 500 mM idioacetic acid (pH 8.5) while stirring slowly, and then process nitrogen gas again and seal it completely. The reaction was carried out for 30 minutes at. It was then dialyzed overnight with 50 mM ammonium bicarbonate buffer and lyophilized. The lyophilized Tca APase was added to a 70% formic acid solution, and CNBr was added to 100 times the weight of the added Tca APase, followed by nitrogen gas treatment for 24 hours at 25 ° C. Distilled water was added thereto and centrifugal vacuum drying to obtain CNBr treated Tca APase.

Freeze-dried Tca APase 1.4 mg was added to 840 μl glacial acetic acid solution containing 1.3 μg / μl BNPS-skatole and 280 μl of distilled water was added. Treated. The same amount of distilled water was added to the electrolytic solution, and the yellow precipitate formed by centrifugation was removed, BNPS-skatole was removed, the supernatant was collected, distilled water was added, and lyophilized to terminate the reaction.

The fragments obtained by the previous CNBr or BNPS-skatole treatment were subjected to peptide analysis electrophoresis and separated into several bands, and the separated bands were separated into 10 mM CAPS buffer [2.213g CAPS, 15% MeOH, pH] on PVDF membrane. 11.0], followed by electroblotting and staining with a solution containing 0.2% Ponceau S and 1% acetic acid to produce one peptide fragment (AP-1) and BNPS-skatole produced by CNBr treatment. Three peptide fragments (Trp-1, Trp-2 and Trp-3) generated by treatment were selected. The amino acid sequence of a total of four peptide fragments was determined (see FIG. 4) and a probe to be used in the Southern blot for cloning of the Tca APase gene was synthesized based on the amino acid sequence. That is, in consideration of codon usage, the amino acid sequence having the smallest number of third codon bases is selected as much as possible, and the third base characterizing the thermos strain gene codon is G or C, as shown in FIG. 5. Oligonucleotides AP-2 and AP-3 having nucleotide sequences were synthesized and used as probes in Southern blots.

Example 4: Isolation of Thermos caldophilus genomic gene

Genomic genes of Thermos caldophilus were isolated using the modified method of Marmur as follows (Marmur, J., J. Mol. Biol., 3: 208-218). (1961)): First, centrifuge the Summers Caldophilus GK24 cells shaken using 200 ml of medium [[0.4% Bactottriptone, 0.2% yeast extract, 1 × Castenholz salts] Separated and recovered, suspended with 4 ml of SETB (20% sucrose / 50 mM Trsi-HCl (pH 7.6) / 50 mM EDTA) buffer and attenuated the cell membrane by repeating five times of freezing and thawing. Thereafter, 10 mg / ml of lysozyme and 100 μg / ml of RNase A were added to react at 37 ° C. for 40 minutes. SDS was added to the reaction solution to 1% of the final volume, and 5 ml of a mixture of chloroform: isoamyl alcohol (24: 1, v / v) was added and the mixture was stirred slowly overnight. Phenol and chloroform were added to the reaction solution, and 100 mg of 10 mg / ml proteinase K was added thereto, followed by reaction at 37 ° C. for 30 minutes, further reaction at 55 ° C. for 20 minutes, followed by extraction with phenol and chloroform. Two times ethanol and 0.1 times 3 M sodium acetate (pH 5.2) were added to the supernatant to extract the genes. The extracted gene was washed twice with 70% ethanol, dried in vacuo, dissolved in TE (10 mM Tris-HCl, pH 7.6 / 1 mM EDTA, pH 8.0) buffer, and then absorbed at 260 nm. It was.

Example 5: Southern Blot

AP-2 and AP-3 oligonucleotides synthesized in Example 3 were used for the Southern blot for selecting the Tca APase gene from the genomic gene of Thermos caldophilus GK24. [Γ-32P] Labeled with dATP (3000 Ci / mmol) and T4 polynucleotide kinase and purified by Sephadex G-25 mini-column. The genomic genes of Thermos Caldophilus GK24 obtained from Example 4 were treated with restriction enzymes of BamHI, HindIII / PstI, SacI and StuI, respectively, separated by 0.7% agarose gel electrophoresis, and then electrolabeled. Southern blot using a probe showed a signal at about 2.1 kb when cut with BamHI and a signal at about 7.0 kb when cut with HindIII / PstI.

Example 6: Cloning of the Tca APase Gene

Genomic DNA digested with BamHI was electrophoresed with low melting agarose gel and only about 2.1 kb fragments were recovered and cloned into pBluescript SK + treated with the same restriction enzyme. E. coli was transformed with an electroengineered pBluescript SK + plasmid to obtain approximately 700 colonies, and colony hybridization was performed using the same probe as that used in the Southern blot of Example 5. As a result, 6 out of 700 colonies were detected. A signal emerged from the colony.

Plasmid was isolated from the first 6 colonies by alkaline denaturation, and plasmid hybridization was performed using the same probe as that used in the Southern blot of Example 5. As a result, four positive clones were obtained. From this, a 2.1 kb BamHI fragment was prepared, labeled with [α-32P] dCTP (3000 Ci / mmol) using a multi-priming labeling kit, and the electrically labeled 2.1 kb BamHI fragment was probed. Southern blot for the genomic gene of Summers Caldophilus GK24 was carried out in the same manner as in Example 5. As a result, a signal appeared in a fragment of about 1.5 kb obtained by cleavage with SacI, a fragment of 1.8 kb and a fragment of about 2.8 kb obtained by cleavage with ApaI. Among them, about 1.5 kb fragments obtained by cutting with SacI and about 2.8 kb fragments obtained by cutting with ApaI were inserted into pBluescript SK + digested with the same restriction enzymes, and E. coli was transformed. Of these, five positive clones were obtained when transformed with a vector containing an Apa I fragment of about 2.8 kb. Plasmids were prepared from electropositive clones, and the entire plasmid gene of approximately 3.1 kb was then digested with restriction enzymes such as Apa I, BamH I, Sac I, Sac II, Sma I and Xho I and electrophoresed with gene markers to compare the respective sizes A map was prepared. As a result, a restriction map as shown in Fig. 6 was obtained.

Example 7: Determination of Tca APase Gene Sequence and Amino Acid Sequence Comparison

Based on the restriction map determined in Example 6 above, subcloning was performed so that the size of the inserted gene was about 0.5 kb. Using the T3 and T7 primers, the nucleotide sequence of each subclone was determined by Sanger's dideoxy chain termination method, and analyzed by PCGEN (PCGENE). Based on the sequence analysis of NCBI-blast search and the internal amino acid sequence of Tca APase determined in the previous Example 3, a Tca containing a total of 1503 bp sequences encoding signal peptides and mature proteins The reading frame base sequence of the APase gene (see SEQ ID NO: 1) and the amino acid sequence inferred therefrom (SEQ ID NO: 2) were determined.

The G + C content of the Tca APase gene is 69.3%, and as shown in SEQ ID NO: 1, the GGAG sequence inferred from the ribosome binding site appears immediately before the ATG codon, but it is -10 or -40 in the APase of other species. No conserved sites were found. Tca APase appears to be expressed first as a protein consisting of a total of 501 amino acids, including signal peptides, and, given the sequence properties of most signal peptides, it appears to be cleaved at Ala 27 during translocation. On the other hand, by comparing the amino acid sequence homology with Escherichia coli APase and Bacillus subtilis APase using NCBI-Blast search, it was confirmed that the homology was relatively low at 27% and 32%, respectively (see : Figure 7). In FIG. 7, Tca represents APase of Thermos Caldophilus GK24, Bsu3 represents APase III of Bacillus subtilis, Bsu4 represents APase IV of Bacillus subtilis, and Eco represents APase of Escherichia coli, and shaded portions represent species sequences. Indicates a preservation site.

As described and demonstrated in detail above, the present invention provides a heat-resistant alkaline phosphatase gene derived from Thermos caldophilus GK24 and an amino acid sequence inferred therefrom. The heat-resistant alkaline phosphatase gene of the present invention is enzyme immunoassay (EIA), antibody staining (immunostaining), enzyme-antibody binding fluorescence assay (ELISA), Western blotting, dot blotting and hybridoma selection ( Mass production of heat-resistant alkaline phosphatase useful for hybridoma screening, etc. may be realized.

Claims (2)

A heat resistant alkaline phosphatase gene having the same nucleotide sequence as SEQ ID NO: 1 derived from Thermos caldophilus GK24. A heat resistant alkaline phosphatase having the same amino acid sequence as SEQ ID NO: 2 derived from Thermus caldophilus GK24 derived from the base sequence of claim 1.