JPH0331434B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a thermostable polymer derived from Thermus aquaticus.
Regarding DNA polymerase. The DNA polymerase of the present invention is suitable for temperature-cycling DNA.
It is particularly useful for copying large amounts of nucleic acid sequences from pre-existing specific nucleic acid sequences by polymerase chain reaction. [Prior art] From mesophilic microorganisms such as E. coli
Extensive research has been conducted on the isolation of DNA polymerases. For example, Bessman
et al., J. Biol. Chem. (1975), vol. 233, pp. 171-177 and Buttin and Kornberg.
-berg) J.Biol.Chem. (1966), vol. 241, 5419~
See page 5427. In contrast, from thermophilic bacteria such as Thermus aquaticus
Not much research has been done on the isolation and purification of DNA polymerase. Kaledin
et al., Biokhymiya, (1980) vol. 45, pp. 644-651, T. aquaticus
A six-step isolation and purification method for DNA polymerase from cells of the YT1 strain is disclosed. These steps include crude extract isolation, DEAE-cellulose chromatography, fractionation on hydroxyapatite, DEAE-cellulose fractionation and single-chain
It consists of chromatography on DNA-cellulose. Pools from each stage were not screened for endo- and exonuclease contamination. The molecular weight of the purified enzyme is reported to be 62,000 Daltons per monomer unit. A second purification method for polymerase from T. aquaticus was described by A. Chine et al., J. Bacteriol. (1976).
127, pp. 1550-1557. In this method, the crude extract is applied to a DEAE-Sephadex column. The dialyzed pool fraction is then subjected to treatment on a phosphocellulose column. This pooled fraction is dialyzed and bovine serum albumin (BSA) is added to prevent loss of polymerase activity. The resulting mixture is applied to a DNA-cellulose column. The molecular weight of the pooled material from the column is approximately 63,000 Daltons when dialyzed and analyzed by gel filtration and approximately 68,000 Daltons by sucrose centrifugation. However, these enzymes have problems such as operating pH range and storage stability, and cannot be used for large-scale replication and amplification of nucleic acids by temperature cycling chain reactions. [Problems to be Solved by the Invention] Therefore, the present invention provides a novel method that can be used for mass replication of nucleic acids by temperature cycling chain reaction.
It provides DNA polymerase. [Means for solving the problem] The above problem is solved by using thermostable DNA from Thermus aquaticu that catalyzes the binding of nucleotide triphosphates to form a nucleic acid strand complementary to a template strand of a nucleic acid. The solution is to provide a polymerase. This enzyme has been described in the literature by Thermus
It has a pH range wider than that of the thermostable enzyme derived from Thermus aquaticu, with a pH of 7.
50% greater activity than in case 8. Furthermore, these thermostable enzymes are stable without loss of activity over long periods of time when stored in buffers containing nonionic detergents. [Specific Description] The terms used in this specification have the following meanings. "Cell", "cell line" and "cell culture" can be used interchangeably;
All of these names include descendants. Therefore, "transformants" or "transformed cells"
includes primary cells and cultures derived from those cells without regard to the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content due to artificial or accidental mutations. Also included are mutant progeny having the same function as screened for in the originally transformed cell. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, ribosome binding sites, but can also include other less understood sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. The term "expression system" refers to a DNA containing operably linked desired coding and control sequences.
A host transformed with these sequences is capable of producing the encoded protein.
To carry out the transformation, the expression system may be contained in the vector, followed by integration of the relevant DNA into the host chromosome. The term "gene" as used herein refers to a DNA sequence that encodes a biologically active polypeptide or a precursor thereof. The polypeptide may be encoded by the complete length gene sequence or by any portion of the coding sequence as long as enzymatic activity is retained. The term "operably linked" refers to a juxtaposition in which the components are capable of performing their normal functions. For example, a coding sequence "operably linked" to a control sequence refers to an arrangement in which the coding sequence is capable of being expressed under the control of the control sequence. A "non-ionic polymeric detergent" is defined as having no ionic charge and for purposes of this invention from about 3.5 to about
Refers to a surfactant characterized in that it is able to stabilize enzymes in a PH range of 9.5, preferably in a PH range of 4 to 8.5. "Oligonucleotide" as used herein
The term is defined as a molecule consisting of two or more deoxyribonucleotides or ribonucleotides, preferably three or more. Its exact size depends on a number of factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be prepared synthetically and by cloning. As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes that each cleave double-stranded DNA at or near a specific nucleotide sequence. The term "nucleotide sequence change" as used herein refers to certain single or multiple nucleotide substitutions, deletions or insertions. Preferred thermostable enzymes for use herein are:
Thermus aquaticus
It is a DNA polymerase derived from. The various strains are also available in the American Type Culture Collection.
Rockville, Maryland, available from TDBrock, L.
Bact. (1969), vol. 98, pp. 289-297, and T. Oshima, Arch. Microbiol.
(1978), Vol. 117, pp. 189-196.
One of these preferred strains is strain YT-1. The DNA polymerase of the present invention has the following properties. (1) Action This enzyme attaches 5'-deoxyribonucleoside to the 3'-hydroxyl group of oligonucleotide or polynucleotide annealed to template DNA.
Covalent bonding of the α-phosphate of triphosphate catalyzes the template-dependent introduction of deoxyribonucleoside 5'-monophosphate into deoxyribonucleic acid. (2) Substrate specificity This enzyme requires a partially double-stranded DNA template and four types of deoxyliphonucleoside 5'-triphosphates for its sufficient activity. A partially double strand was formed here.
The DNA strand type refers to, for example, a state in which an oligonuskeotide primer having a free 3'-hydroxyl group is annealed to a single-stranded template DNA molecule. It may also show low activity against single-stranded DNA. (3) Optimal PH and stable PH range The optimal PH range is the PH at a temperature of 70℃ to 75℃.
It is 8.0-8.5. However, it has significant activity even at PH7.0. This enzyme has a pH of at least 5.0 to PH
It is relatively stable within the range of 9.4. (4) Operating temperature range It is active in the range of room temperature to 85°C, and the optimum temperature is about 75°C. The activity at 35°C is about 1% of the activity at 75°C. (5) Inactivation This enzyme has a half-life of at least 5 minutes at 97.5°C. (6) Inhibition, activation and stabilization Inhibited by dimethyl sulfoxide, dimethyl formamide, EDTA, formamide, SDS, high concentration of urea, etc. (7) Molecular weight The molecular weight of the present invention was determined by SDS-polyacrylamide gel electrophoresis using phospholidase B (molecular weight 92000), bovine serum albumin (molecular weight 66200), ovalbumin (molecular weight
45000), carbonic anhydrase (molecular weight
31,000), about 86,000 to 9,000 daltons as determined using soybean trypsin inhibitor (molecular weight 21,500) and lysozyme (molecular weight 14,000). However, recent reports indicate that phosphorylase B has a molecular weight of approximately 973,000 Daltons, and based on this molecular weight, the molecular weight of the enzyme of the present invention is approximately 94,000 Daltons. The method for measuring the activity of this enzyme and the definition of the titer are described in Example 4, and specific examples of the method for purifying this enzyme are described in Examples 1 and 4. The DNA polymerase of the present invention can be obtained by culturing Thermus aquaticus having the ability to produce the enzyme at, for example, 70° C. and collecting it from this culture. For example, cells can be grown for 20 hours and then harvested by centrifugation. After cell growth, enzyme isolation and production occurs in six steps, each step consisting of a temperature below room temperature;
Preferably it is carried out at about 4°C. In the first step or step, the cells, if frozen, are lysed and disrupted by ultrasound, with a pH of approx.
Suspend in 7.5 buffer. In the second step, the supernatant is collected and then fractionated by adding a salt such as dry ammonium sulfate. Appropriate fractions (typically 45-75% saturation) are collected, dissolved in 0.2 potassium phosphate buffer, preferably at PH 6.5, and dialyzed against the same buffer. In the third step, the fraction from the second step, which removes nucleic acids and certain proteins, is applied to a DEAE-cellulose column equilibrated with the same buffer as above. The column is then washed with the same buffer and the protein-containing fractions are eluted, measured by absorbance at 280 nm, collected and mixed with the first buffer, preferably against a 10 mM potassium phosphate buffer. PH at
Dialyze with the same ingredients as those used in 7.5. In the fourth step, the fractions collected as described above are applied to a hydroxyapatite column equilibrated with the buffer used for dialysis in the third step. The column was then washed with a pH 7.5 solution containing 10mM 2-mercaptoethanol and 5% glycerin.
Elute the enzyme with a linear gradient of 0.01M to 0.5M potassium phosphate buffer. The pooled fractions containing the thermostable enzyme DNA polymerase activity are dialyzed against the same buffer used for dialysis in the third step. In the fifth step, the dialyzed fraction is applied to a DEAE-cellulose column equilibrated with the buffer used for dialysis in the third step. This column was then washed and 0.01
Elute the enzyme with a linear gradient of a buffer such as ~0.6M KCl. The fraction having thermostable enzyme activity is treated with deoxyribonuclease (endo- and exonuclease) using an appropriate method.
Test for contamination. For example, endonuclease activity can be determined electrophoretically from the change in molecular weight of phage λ DNA or supercoiled plasmid DNA after incubation with excess DNA polymerase. Similarly, exonuclease activity can be measured electrophoretically from changes in the molecular weight of DNA after treatment with restriction enzymes that cleave at several sites. Fractions determined to have no DNase activity are pooled and dialyzed against the same buffer used in the third step. In the sixth step, the pooled fractions are loaded onto a phosphocellulose column with constant betvolium. Wash this column and elute the enzyme with a linear gradient of a buffer such as 0.01-0.4 M KCl in potassium phosphate buffer at pH 7.5. The pooled fractions with thermostable polymerase activity and no deoxyribonuclease activity were purified at pH 8.0.
Dialyze against buffer. The molecular weight of the dialyzed product can be determined by a method such as SDS PAGE using a protein molecular weight marker. Thermus aquaticus, one of the preferred enzymes
When the molecular weight of the molecular weight standard phosphorylase B is set to 92,000 Daltons, the molecular weight of the DNA polymerase purified from A. aquaticus) is determined by the method described above.
It is determined to be approximately 86,000 to 90,000 Daltons. The thermostable enzyme of the present invention has a gene encoding this enzyme derived from Thermus aquaticus (Thermus aquaticus).
aquaticus) can also be cloned from genomic DNA and produced by recombinant DNA technology. The complete coding sequence for T. aquaticus (Taq) polymerase is contained within an approximately 18 kb (kilobase) genomic DNA insert fragment.
Bacteriophage CH35 containing the 3.5kb Bgl-Asp718 (partial) restriction fragment: Taq #4-2
It can be derived from This bacteriophage was developed as an American type on May 29, 1987.
It has been deposited with the Culture Collection (ATCC) and has deposit number 40336. This gene is also present on plasmid pFC83 (ATCC No. 67422,
A Bgl-Hind restriction fragment of approximately 750 base pairs (bp) isolated from
Plasmid pFC85 (ATCC No. 67421, May 1987)
Approximately 2.8 kb Hind− isolated from
It can be constructed by ligating to the Asp718 restriction fragment. The pFC83 restriction fragment has the portion encoding the amino terminus of the Taq polymerase gene, whereas the restriction fragment from pFC85 has the portion encoding the carboxy-terminus. Therefore, ligation of these two fragments with a correspondingly digested vector with appropriate control sequences results in translation of full-length Taq polymerase. It has also been discovered that the entire coding sequence of the Taq polymerase gene is not required to obtain a biologically active gene product with the desired enzymatic activity.
DNA with the amino terminal coding portion deleted and approximately one-third of the coding sequence absent produced a fully active gene product in polymerase analysis. In addition to the N-terminal deletion, the individual amic acid residues in the peptide chains that make up Taq polymerase are
It may be modified by oxidation, reduction, or other derivatization, and the protein may be cleaved to obtain fragments that retain activity. Such changes that do not destroy activity do not exclude the DNA sequence encoding such protein from the definition of gene. Therefore, modification of the primary structure itself by deletion, addition, or change of amino acids that are incorporated into the sequence during translation can be performed without destroying the activity of the protein. Such substitutions and other changes result in proteins having amino acid sequences encoded by DNA within the scope of the present invention. A polyclonal antiserum from a rabbit immunized with the purified polymerase of the invention was used to probe a partial genomic expression library of Thermus aquaticus to obtain the appropriate coding sequence as follows. . The cloned genomic sequence can be expressed as a fusion polypeptide, directly with its own control sequences, or by construction with control sequences appropriate for the particular host used for expression of the enzyme. can be expressed. Of course, the availability of DNA encoding these sequences provides the opportunity to modify the codon sequences to obtain mutein forms that also have DNA polymerase activity. For example, these tools provide a complete coding sequence for Taq DNA polymerase from which expression vectors can be constructed and expressed that are applicable to a variety of host systems. Furthermore, from the above,
It will be appreciated that portions of the Taq polymerase coding sequence are useful as probes, and also as probes for recovering other thermostable polymerase coding sequences in a variety of species. Therefore, a portion of the genomic DNA encoding at least 6 amino acids is replicated in E. coli and denatured and used as a probe;
Alternatively, oligonucleotides encoding at least six amino acids can be synthesized and used to search for other DNA encoding thermostable polymerases. Because the sequences of the nusrease in Thermus aquaticus and the corresponding parts of other species may not match exactly, hybridization was performed under conditions of sufficient stringency to eliminate false positives. An oligomer having approximately 18 nucleotides encoding a 6 amino acid stretch would be required to effect sorption. A sequence encoding six amino acids would provide sufficient information for such a probe. Suitable hosts, control systems and methods Generally, the production of recombinant Taq polymerase involves the following steps. First, the mature enzyme (in this case used to include all muteins), or fusions of Taq polymerase with additional sequences that do not destroy its activity, and under controlled conditions (e.g. peptidase fusion with additional sequences, which can be cleaved (by treatment with a protein) to yield an active protein. When the sequence is not interrupted by introns, it is suitable for expression in any host. This array should be in a form that is excisable and retrievable. The excised or recovered coding sequence is then preferably operably linked to suitable control sequences in a replicable expression vector. This vector is used to transform a suitable host and the transformed host is cultured under preferred conditions to produce recombinant Taq polymerase. In some cases, Taq polymerase is isolated from the culture medium or cells, but if certain impurities are tolerated, protein recovery and purification may not be necessary. Each of the above steps can be performed in a variety of ways. For example, the desired coding sequence can be obtained from a genomic fragment and used directly in a suitable host. Construction of expression vectors that can function in various hosts is carried out using appropriate replicons as described below. If suitable restriction sites are not normally available, they can be added to the ends of the coding sequence to obtain an excisable gene and inserted into these vectors. Control sequences, expression vectors and transformation methods are
It depends on the type of host cell used to express the gene. Generally, prokaryotic, yeast, insect or mammalian cells are currently useful as hosts.
Prokaryotic hosts are generally the most efficient and convenient for recombinant protein production and are therefore preferred for expression of Taq polymerase. In the specific case of Taq polymerase, there is evidence that, under both recombinant and natural conditions, significant deletions occur at the N-terminus of the protein and the activity of the protein still remains retained. exist. Isolated native proteins are the result of proteolytic degradation and not the result of incomplete gene translation. However, muteins produced from the incomplete gene of plasmid pFC85 are fully active, as are muteins produced from DNA encoding sequences with full length in DNA polymerase analysis. some kind of N
Since the truncated form is apparently active, the genetic construct or polymerase expression used may also contain the coding sequence of the corresponding truncated form. Control Sequences and Corresponding Hosts Prokaryotes are most commonly found in E. coli (E.
It is represented by various strains of coli. However, other microbial strains can also be used, such as Bacillus, for example Bacillus subtilis, various Pseudomnas or other strains. In such prokaryotic systems, plasmid vectors are used that have replication sites and control sequences derived from species compatible with the host. For example, E. coli typically
Bolivar et al., Gene, 1977, 2 volumes,
A derivative of pBR322, a plasmid derived from the E. coli species according to page 95, is used for transformation. pBR322 contains genes for ampicillin and tetracycline resistance and provides additional markers that can be retained or destroyed in constructing the desired vector. Commonly used prokaryotic control sequences described herein that promote transcription initiation and optionally have an operator and a ribosome binding site sequence include the β-lactamase (penicillinase) and lactose (1ac) promoter systems [ Chang et al., Nature (1977) vol. 198,
1056 pages], tryptophan (trp) promoter system [Goeddel et al., Nucleic Acids Res.
(1980) vol. 8, p. 4057] and the λ-derived P _L promoter [Shimatake et al.
Nature (1981) vol. 292, p. 129] and an N- _gene ribosome binding site useful as a portable control cassette, which has at least one A third DNA with restriction sites
The first is a P _L promoter operably linked to a second DNA sequence corresponding to the NRBS upstream of the sequence.
Contains DNA sequences. Also useful is the phosphatase A (phoA) system described by Chang et al., EP 196,864, published October 8, 1986, and co-inherited. However, any available promoter compatible with prokaryotes can be used. In addition to bacteria, eukaryotic microorganisms such as yeast can also be used as hosts; baker's yeast, a laboratory strain of Saccharomyces cerevisiae, is most commonly used, but many other strains are also common. is available. A vector using a 2 micron origin of replication has been shown [Broach, JR, Meth. Enz. (1983) vol. 101;
307], other plasmid vectors suitable for yeast expression are also known [e.g., Stinchcomb et al., Nature (1979) 282, 39].
P., Tschempe et al., Gene (1980)
Volume 10, page 157 and Clarke et al.
(See Meth. Enz. (1983) vol. 101, p. 300). Control sequences for yeast vectors also include promoters for the synthesis of glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg. (1968) Vol. 7, p.
Page 149, Holland et al., Biotechnology
(1978) 17 volumes, 4900 pages]. Other promoters known in the art include 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. (1980).
) and other glycolytic enzymes,
For example, glyceraldehyde-3-phosphate.
Promoters of genes for dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, crucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase There is.
Other promoters that have the added advantage of being controlled by growth conditions are the promoters of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes involved in nitrogen metabolism and enzymes involved in maltose and galactose utilization [Holland et al. Same as above]. A terminator sequence may be desirable at the 3' end of the coding sequence. Such a terminator follows the coding sequence in the yeast-derived gene.
Found in the 3′ untranslated region. Many of the exemplified vectors include the plasmid peno46 containing the enolase gene [Holland, MJ et al., J.
Biol.Chem. (1981) Vol. 256, p. 1385] or the LEU2 gene obtained from YEp13 [Broach, J. et al., Gene (1978) Vol. 8, p. 121]
However, any vector with a yeast-compatible promoter, origin of replication, and other control sequences is suitable. Of course, it is also possible to express genes encoding polypeptides in eukaryotic host cell cultures derived from multicellular organisms. For example, Tissue
Culture, Academic Press
Press), edited by Cruz and Patterson (1973). Useful host cell lines include murine myeloma N51;
There are VERO and Hela cells, as well as Chinese Hamster Ovary (CHO) cells. Expression vectors for these cells typically contain the commonly used early and late promoters (Fiers) from simian virus 40 (SV40).
et al., Natur (1978) 273, p. 113), or promoters such as those derived from polyoma, adenovirus 2, bovine papillomavirus or trisarcoma virus, and in mammalian cells such as immunoglobulin promoters and heat shock promoters. Has compatible promoter and control sequences. A DNA expression system in a mammalian system using BPV as a vector is described in US Pat.
It is disclosed in the specification of No. 4419446. A variation of this system is described in US Pat. No. 4,601,978. One general aspect of transformation of mammalian cell lines is
It is described in US Pat. No. 4,399,216.
"Enhancer" regions appear to be important for optimal expression; these regions are generally sequences found upstream of the promoter region. If desired, origins of replication can be obtained from viral sources. However, chromosomal integration is a common mechanism for DNA replication in eukaryotes. Plant cells can also be used as hosts, and control sequences compatible with plant cells, such as the nopaline synthase promoter and polyadenylation signal sequence [Depicker, A. et al., J.
Mol.Appl.Gen. (1982) vol. 1, p. 561] is available. Furthermore, expression systems using insect cells that utilize the control system provided by baculovirus vectors have recently been reported [Miller, et al.
DW) et al., Genetic Engineering (1986), edited by Setlow, JK et al., Plenum Publishing, vol. 8, 277-
297 pages]. These systems are also useful for producing Taq polymerase. Transformation Depending on the host cell used, transformation is carried out using standard techniques appropriate to that cell. Calcium treatment using calcium chloride [Cohen, SN, Proc. Natl. Acad. Sci. (USA)]
(1972) Vol. 69, p. 2110] is used for prokaryotes or other cells with substantial cell barriers. Infection with Agrobacterium tumefaciens [Shaw, CH et al., Gene (1983) 23, 315
page] is used in some plant cells. For mammalian cells that do not have a cell wall, Graham and van der Ebb
The calcium phosphate precipitation method of Eb) is preferred [Virology (1978) Vol. 52, p. 546]. Transformation into yeast was performed by Van Solingen,
P.) et al. (J.Bact., 1977, vol. 130, p. 946) and Hsiao, CL. et al. (Proc. Natl. Acad.
Sci. (USA), 1979, Vol. 76, p. 3829). Construction of λgt11 expression library DNA encoding the desired protein, e.g. Taq
A method for isolating DNA encoding a polymerase using bacteriophage λgt11 is as follows. The library can be composed of Alu fragments flanked by EcoR sites obtained by complete digestion of Thermus aquaticu DNA and insertion into the EcoR site of the λgt11 phage [Young et al. ) and Davis, Proc. Natl.
Acad.Sci.USA, 1983, vol. 80, pp. 1194-1198].
The unique EcoR site in this bacteriophage is located at the carboxy terminus of the β-galactosidase gene, so that the inserted DNA in the proper frame and orientation is fused with the β-galactosidase under the control of the lactose operon promoter/operator. It is expressed as a protein. The genomic expression library is then screened using antibody plaque hybridization. This method is called "epitope selection" and uses antiserum against the fusion protein sequence encoded by the phage to identify hybridizing plaques. For example, if this recombinant phage library is
An antibody that recognizes the 90,000 Dalton Taq polymerase can be used to screen to identify phages that contain DNA segments encoding antigenic determinants of this protein. Approximately 2 x ¹⁰⁵ recombinant phages were added to each rabbit.
Screen using Taq polymerase antiserum. In this primary screening, a positive signal is detected and one or more of these plaques are
Candidate plaques that do not react with pre-immune serum and react with immune serum are purified and analyzed in some detail. To test the fusion protein produced by the recombinant phage, a lysogenic strain of the phage in host Y1089 is obtained. After inducing the lysogenic strains and gel electrophoresing the proteins they produce, it is possible to observe that each lysogenic strain produces new proteins not found in other lysogenic strains, or duplicate sequences. may occur. Pick out phages with a positive signal. In the example described here, a single positive plaque was picked for further identification, replated at lower density to purify the recombinants, and purified clones were analyzed by size class by digestion with EcoR restriction enzyme. do. Next, the isolated DNA
Probes were generated from the insert sequences, appropriately labeled, and these probes were prepared as described by Maniatis et al., Moleclar Cloning: A.
Laboratory Manual, 1982, conventional plaque hybridization or colony hybridization analysis methods can be used. The labeled probe was used to probe a second genomic library constructed in Charon 35 bacteriophage [Wilhelmine, AM et al., Gene.
1983, vol. 26, pp. 171-179]. This library is based on Thermus Aquatics.
aquaticus) genomic DNA was generated by partially digesting Sau3A and cloning the size-fractionated fragments (15-20 kb) into the BamH site of the Sialon 35 phage. This probe was used to isolate phages containing DNA encoding Taq polymerase. One of the resulting phages, designated CH35:Taq #4-2, was found to have the entire gene sequence. A partial sequence encoding part of this gene was also isolated. Construction of Vectors Construction of suitable vectors containing the desired coding and control sequences uses standard ligation and restriction techniques well known in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, trimmed, and religated into the desired shape. Site-specific DNA cleavage is performed by treatment with suitable restriction enzyme(s) under conditions well known in the art, and details are provided by the manufacturers of these commercially available restriction enzymes. It is stated that See, eg, New England Biolabs, Product Catalog. Typically, about 1 μg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in about 20 μg of buffer;
Typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about 1 to 2 hours at about 37°C are useful, although variations can be made. After each incubation, the proteins can be removed by extraction with phenol/chloroform followed by ether extraction, and the nucleic acids can be recovered from the aqueous fraction by ethanol precipitation. If desired, size separation of cleavage fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. For a general description of size separation, see Methods in Enzymology, 1980, vol. 65,
It is described on pages 499-560. The restriction cleaved fragment was dissolved in 50mM Tris, PH7.6, 50mM NaCl, 10mM _MgCl2 , 10
20 in mM DTT and 50-100 μM dNTPs.
E. coli in the presence of four deoxynucleotide triphosphates (dNTPs) using an incubation time of approximately 15-25 minutes at ~25°C.
Blunt ends can be made by treatment with a large fragment of DNA polymerase (Klenow). The Klenow fragment fills in the 5' cohesive ends, but even in the presence of four dNTPs, there are no overhangs. Chews back the 3′ single strands to be selectively extracted, if desired, by supplying only one or selected dNTPs within the limits dictated by the nature of the cohesive end. Repair can be performed. After treatment with Klenow, the mixture was extracted with phenol/chloroform and precipitated with ethanol. Treatment with S1 nuclease under appropriate conditions results in hydrolysis of the single-stranded portion. Synthetic oligonucleotides have been described by Matteucci et al. (J.Am.Chem.Soc., 1981).
103, pp. 3185-3191) or using an automated synthesis method. Single strand quinarzing prior to annealing or for labeling was performed using 50 mM Tris, PH 7.6,
10mM _MgCl2 , 5mM dithiothreitol,
This is carried out using an excess of polynucleotide kinase, for example about 10 units, relative to 1 nM substrate in the presence of 1-2 mM ATP. When this kinase treatment is for labeling the probe,
ATP has a high specific activity of γ- ^32P . Ligation is performed under standard conditions and temperatures listed below.
Perform in a volume of 30μ. 20mM Tris-HCl, (PH
7.5) 10mM MgCl ₂ , 10mM DTT, 33μg/
ml BSA, 10mM to 50mM NaCl, and (for "sticky end" ligation) 40μM ATP, 0.01 to
0.02 [Weiss] units of T4 DNA ligase,
0°C; or (for “blunt end” ligations) 1mM
ATP, 0.3 to 0.6 [Weiss] units
T4 DNA ligase, 14°C. Intramolecule “sticky end”
Ligations are typically performed at a total DNA concentration of 33-100 μg/ml (total end concentration of 5-100 nM). Intermolecular blunt end ligations (usually using a 10-30 fold molar excess of linker) are performed at a total end concentration of 1 μM. In constructing vectors using "vector fragments," the vector fragments are usually treated with bacterial alkaline phosphatase (BAP).
The 5' phosphate is removed to prevent vector religation. BAP digestion is carried out at 60° C. for about 1 hour at pH 8 in about 150 mM Tris in the presence of Na ⁺ and Mg ⁺² using about 1 unit of BAP per mg of vector. To recover the nucleic acid fragments, the preparation is extracted with phenol/chloroform and ethanol precipitated. Ligation can also be prevented in vectors in which the undesired fragments have been double-digested by additional restriction enzyme digestion. DNA sequence modification cDNA or genome that requires modification to the sequence
For portions of the vector derived from DNA, site-specific primer-directed mutagenesis is used. This technique is now standard in the art and is performed using synthetic oligonucleotide primers that are complementary to the single-stranded phage DNA to be mutagenized, except for limited mismatches representing the desired mutations. Briefly, a synthetic oligonucleotide is used as a primer to direct the synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. A culture solution of the transformed bacteria is spread on the upper layer of agar to form plaques from single cells containing phages. Theoretically, 50% of new plaques contain phage with the variant as a single strand, and 50% have the original sequence. The plaques were transferred to a nitrocellulose filter and the kinased synthetic compound was transferred to a nitrocellulose filter at a temperature that allowed accurate matching hybridization and that the mismatch with the original strand was sufficient to prevent hybridization. Perform "lifts" hybridization with primers. Next, plaques that hybridize with this probe are collected and cultured.
Collect DNA. Proof of Construction In the following constructions, the correct ligation of the plasmid construct was first performed using a reconstituted mixture of E. coli (E.
coli strain MM294 or other suitable host. As is understood in the art, successful transformants are selected for ampicillin resistance, tetracycline resistance, or other antibiotic resistance depending on the mode of plasmid construction. Plasmids from the transformants were then purified by Clewell, DB et al.
(Proc. Natl. Acad. Sci. USA, 196, Volume 62, 1159
Page), or in some cases by the chloramphenicol amplification method [Clewell, DB;
J. Bacteriol., 1972, vol. 110, p. 667]. The isolated DNA was analyzed by restriction treatment and/or by Sanger, F. et al. (Proc. Natl. Acad. Sci. USA, 1977, 74, 5463).
The dideoxy method of Messing et al. (Nucleic Acids Res., 1981).
(Vol. 9, p. 309) or Maxam et al. (Methods in
Enzymology, 1980, Vol. 65, p. 499). Examples of hosts The host strains used for cloning and expression in this invention are as follows. For cloning and sequencing and expression of constructs under the control of most bacterial promoters, E. coli obtained from the E. coli Genetic Stock Center GCSG #6135 was used. KK
MM294 was used as a host. Expression under the control of the P _L N _RBS promoter requires E. coli K12
strain MC1000λ lysogen strain, N ₇ N ₅₃ c1857SusP ₈₀ ,
ATCC39531 can be used. In this specification, E. coli DG116, deposited as ATCC (ATCC 53606) on April 7, 1987, is used. For M13 phage recombinants, use E. coli susceptible to phage infection, e.g. E. coli K12.
Using strain DG98. DG98 stock on July 13, 1984
Deposited with ATCC and has deposit number 39768. Expression in mammals is COS-7, COS-A2, CV-
1 and murine bacteria, and in insect cells based on expression in Spodoptera frugipeida. Stabilization of Enzyme Activity For long-term stability, enzymes are preferably stored in a buffer containing one or more nonionic polymeric surfactants. Such surfactants generally have a molecular weight in the range of about 100 to 250,000, preferably about 4,000 to 200,000 Daltons, and stabilize the enzyme at a pH of about 3.5 to about 9.5, preferably about 4 to 8.5. It is something to do. Examples of such surfactants include McCtcheon's Emulsifiers & Detergents, North American Edition (1983), published by MC Publishing Co., McCtcheon Division, 175, Rotcl.
Glenrock, New Jersey (USA), 295~
There is one described on page 298, and please refer to the above-mentioned document for details. Preferably,
Surfactants include ethoxylated fatty alcohol ethers and lauryl ethers, ethoxylated akylphenols, octylphenoxy polyethoxyethanol compounds, modified oxyethylated and/or oxypropylated linear alcohols, polyethylene glycols. selected from the group consisting of monooleate compounds, polysorbate compounds and phenolic aliphatic alcohol ethers. More specifically, Tween 20, which is preferably a polyoxyethylated (20) sorbitan monolaurate (ICI Americas Inc.);
Wilmington, DE] and Iconol® NP-40 (BASF), an ethoxylated alkylphenol (nonyl)
I&T Corporation, Parsippany, New Jersey). The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention or claims. In these examples, all percentages are by weight for solids and by volume for liquids, and all temperatures are in degrees Celsius, unless otherwise specified. Example 1 Thermus Aquatics
Thermus aquaticus strain YT1, available without any control as ATCC No. 25, 104 from American Type Culture Collection, 12301 Park Round Live, Rockville, MD, was purified in a flask.
It was grown in the following medium. Sodium citrate 1mM Potassium phosphate PH7.9 5mM Ammonium chloride 10mM Magnesium sulfate 0.2mM Calcium chloride 0.1mM Sodium chloride 1g/1 Yeast extract 1g/1 Tryptone 1g/1 Glucose 2g/1 Ferrous sulfate 0.01mM (Before autoclaving (pH adjusted to 8.0) The flask starter cultured overnight at 70°C in the above medium was inoculated into 10 fermenters. A total of 600 ml of seed from the seed flask was inoculated into 10 identical media.
The PH was adjusted to 8.0 with ammonium hydroxide, the dissolved enzyme was adjusted to 40%, the temperature was adjusted to 70°C, and the stirring speed was 400 rpm. For the first five steps after cell proliferation
Purification was carried out using the method of Kaledin et al., supra (with slight modifications) and a different method in the sixth step. All six steps were performed at 4°C. The fractionation rate on the column was 0.5 columns/hour.
The volume of the gradient during elution was 10 column volumes. An alternative preferred method is described below in Example 6. In summary, the above cultured T. aquaticus (T.
aquaticus) cells were harvested by centrifugation after 9 hours of culture at a cell concentration of 1.4 g dry weight/late exponential phase. 20g cells, 50mM
It was suspended in 80 ml of a buffer containing Tris-HCl (PH7.5) and 0.1 mM EDTA. The cells were lysed and the lysate was centrifuged for 2 hours at 35000 rpm in a 4°C rotor. Collect the supernatant (fraction A) and collect the protein fraction that precipitates between 45 and 75% saturation in ammonium sulfate, 0.2 M potassium phosphate (PH6.5), 10 m
Fraction B was obtained by dissolving in a buffer containing M2-mercaptoethanol and 5% glycerin and finally dialyzing against the same buffer. Fraction B was treated with DEAE equilibrated with the above buffer.
- Applied to a 2.2 x 30 cm column of cellulose. The column was then washed with the same buffer and the protein-containing fraction (determined by absorbance at 280 nm) was collected. The combined protein fractions were added to 0.01M potassium phosphate (PH7.5), 10mM2-
Fraction C was obtained by dialysis against a second buffer containing mercaptoethanol and 5% glycerin. Fraction C was applied to a 2.6 x 21 cm column of hydroxyapatite equilibrated with a second buffer. The column was then washed and added with 10mM 2-mercaptoethanol and 5% glycerin.
The enzyme was eluted with a linear gradient of 0.01-0.5M potassium phosphate (PH7.5) buffer. Fractions containing DNA polymerase activity (90-180mM potassium phosphate) were collected and placed in an Amicon stirring cell.
Fraction D was obtained by concentrating 4-fold using a YM10 membrane and dialyzing against a second buffer. Fraction D was applied to a 1.6 x 28 cm column of DEAE-cellulose equilibrated with a second buffer. Wash this column and add 0.01-0.5M in the second buffer.
DNA by linear gradient of potassium phosphate
The polymerase was eluted. Fractions were measured for endonuclease and exonuclease contamination. This is done by detecting by electrophoresis (for endonucleases) the change in the molecular weight of phage λ DNA or supercoiled plascid DNA after incubation with excess DNA polymerase and restriction enzymes that cleave the DNA into several fragments. (for exoclease). Only DNA polymerase fractions (65-95mM potassium phosphate) with minimal Nusrease contamination were pooled. Add autoclaved gelatin to this pool.
Fraction E was obtained by adding in an amount of 250 μg/ml and dialysis against a second buffer. Fraction E was applied to a phosphoglucose column and 0.01~
Elution was performed with a 100ml 0.4M KCl gradient.
Fractions were determined for endonuclease/exonuclease contamination as described above and also for polymerase activity (by the method of Kaledin et al.). The pooled fractions were dialyzed against the second buffer and then concentrated by dialysis against 50% glycerin and the second buffer. The molecular weight of the polymerase was determined by SDS-PAGE. Marker protein is phospholidase B
(92000), Bovine Serum Albumin (66200), Ovalbumin (45000), Kobonitsukanhydrase (31000), Soybean Trypsin Inhibitor (21500),
and lysozyme (14400). Preliminary data indicate that 62,000-63,000 Daltons, rather than the 62,000-63,000 Daltons reported in the literature (e.g. Kaledin et al.)
It was about 86,000 to 90,000 Daltons. This polymerase was mixed with 25mM Tris-HCl.
(PH6.4 and PH8.0), 0.1M KCl, 10mM _MgCl2 ,
1mM 2-mercaptoethanol, 10nmole each of dGTP, dATP and TTP, and 0.5μCi ( ^3H )
dCTP, 8 μg “activated bovine” thymus DNA
and incubated in 50μ of a mixture containing 0.5-5 units of polymerase. "Activated" DNA is a natural preparation of DNA after partial hydrolysis by digestion with DNase I until 5% of the DNA is transferred to the acid-soluble fraction. The reaction was carried out for 30 min at 70°C, and 0.125M EDTA
It was stopped by the addition of approximately 50 μ of a saturated aqueous solution of sodium phosphate containing -Na ₂ . The sample
Treatments and activity were determined as described by Kaledin et al., supra. This result indicates that the polymerase has a pH of 6.4.
It was shown to be more active than half of the activity in 8.0. In contrast, Kaledin et al.
7.0 was found to have 8% of the activity at PH8.3. Therefore, the thermostable enzyme of the present invention
The PH profile is broader than that of enzymes such as Kaledin et al. Finally, when one or more nucleotide triphosphates are removed from the DNA polymerase assay reaction mixture, little activity is observed using the enzyme of the invention, and the activity is consistent with the expected values, and The enzyme showed high fidelity.
In contrast, the activity observed with Kaledin et al., supra, is inconsistent with the expected value and suggests incorrect introduction of the nucleotide triphosphate. Example 2 Gene Search A Identification of a DNA Sequence Probe for the Taq Polymerase Gene A specific DNA sequence probe for the Taq po1 gene was obtained by immunoscreening of a λgt11 expression library. DNA of T. aquaticus
Completely digested with Alu, ligated with EcoR 12-merlinker (CCGGAATTCCGG, New England Biolabs), digested with EcoR,
Then, it was ligated with EcoR-digested dephosphorylated λgt11 DNA (Progema Biotech). The ligated DNA was packaged (Jigapack Plus, Strategene) and E. coli K-
12 strain Y1090 (obtained from R. Young) was transfected. The library was initially screened with 2 x 10 ⁵ plaques using a 1:2000 dilution of rabbit polyclonal antiserum against purified Taq polymerase (see Examples 1 and 4) [Young, et al. RA and RWDavis (1983)
Science, 222:778-782]. Candidate plaques were replated at critical dilution and rescreened until uniformity (approximately 3 cycles). Phages were purified from candidate plaques that did not react with preimmune serum and reacted with immune serum. E. coli K-12 strain Y1089 using candidate phages
(R.Young) was lysogenized. Lysogens were screened for production of an IPTG-inducible fusion protein (larger than β-galactosidase) that reacts with Taq polymerase antiserum. Epitope-specific antibodies were affinity purified from total polyclonal antibodies using solid-phase size-fractionated fusion proteins [Goldstein, LSB et al. (1986) J. Cell. Biol. 102:
2076−2087]. The "raised" epitope-selected antibodies are now used in Western analysis to
We identified a λgt11 phage candidate encoding a Taq polymerase-specific DNA sequence. λgt:
One λgt11 phage candidate, designated λgt1, was used to assemble purified Taq polymerase from whole rabbit polyclonal Taq polymerase antiserum and an antibody that specifically reacts with both the crude extract containing Taq polymerase into a specific phage λgt1. :1 was used for further investigation. Approximately 115 bp of Thermus aquaticus DNA
EcoR-compatible Alu fragments were labeled (Maniatis et al., supra) to form Taq polymerase-specific probes. This probe was used in Southern analysis and to screen a T. aquaticus DNA random genomic library. B. Thermus aquaticus - Construction and screening of random genome library λ Phagesiaron 35 (Wilhelmine, AM
et al., supra) and ligated through their cohesive ends, thoroughly digested with BamH, mixed, and the annealed arms were separated from the stuffer fragments.
Calcium acetate density gradient ultracentrifugation (Maniatis et al.
Purified by the method described above). T. aquaticus
DNA was partially degraded with Sau3A and 15-20kb
Size fractions were purified by sucrose density gradient ultracentrifugation. A random genomic library was constructed by linking target DNA fragments and vector DNA fragments at a molar ratio of 1:1. The DNA was packaged and transfected into E. coli K-12 strains LE392 or K802. Contains over 99% recombinant
First Fage library of over 20000
Amplified on E. coli K-12 strain LE392. A CH35 Taq genomic phage library was screened with a radiolabeled EcoR insert at λgt11:1 (Maniatis et al., supra). Specifically hybridizing candidate phage plaques were purified and further analyzed.
One phage, designated CH35::4-2,
More than 4 T. aquaticus DNA fragments were released after digestion with Hind (approximately 8.0,
4.5, 0.8, 0.58kb). Plasmids obtained by digesting four types of Hind T. aquaticus DNA fragments with Hind.
BSM13 ⁺ (3.2kb, Vector Cloning Systems, San Diego), and each
E. coli K-12 strain DG98 was transformed and cloned. About 8.0kb Hind from CH35::4-2
DNA fragment plasmid pFC82
A 4.5 kb Hind DNA fragment from CH35::4-2 was isolated in plasmid pFC83 (7.7 kb). E. coli strain DG98 containing pFC82 was shown to contain thermostable high temperature DNA polymerase activity (Table 1). In addition, the cells synthesized a new approximately 60 kd molecular weight polypeptide that is immunologically associated with Taq DNA polymerase. The Taq polymerase coding region of the 8.0 kb HindDNA fragment was further processed and
2.8kb Hind− of 8.0kb Hind fragment
The Asp718 portion was located proximal to the 1as-promoter. This region was subcloned to obtain plasmid pFC85 (6.0 kb). After incubation with IPTG, E. coli DG98 cells containing plasmid pFC85 synthesize up to 100 times more thermostable Taq polymerase-related activity compared to the original parental clone (pFC82/DG98) (first
table). Cells containing pFC85 have a significant amount of thermostable
Synthesize DNA polymerase activity, Taq
Only a portion of the po1 DNA sequence is translated, approximately
Accumulation of a 60 kd Taq polymerase-related polypeptide occurs.

【table】

[Table] Example 3 The gene for the thermostable enzyme of this invention was expressed in various bacterial expression vectors, such as DG141 (ATCC 39588) and
It can be expressed in pP _L N _RBS ATG.
Both of these host vectors are derivatives of pBR322 and can act on a tryptophan promoter operator and a ribosome binding site (DG141) operably linked to the ATG start codon, or a sequence containing the λP _L promoter and the ATG start codon. Gene N binding site (pP _L
N _RBS ATG). Either of these host vectors can be restricted with Sac and balanced terminated with Klenow or S1 nuclease to create a convenient site for later insertion of the Taq polymerase gene. The full length Taq polymerase gene was constructed from the DNA insert fragment subcloned into plasmids pFC83 and pFC85 as follows. Vector BSM13 ⁺ (commercially available from Vector Cloning Systems, San Diego, CA) was digested at the unique Hind site and
repaired by Klenow and dNTP, and
It was ligated to the Bgl octanucleotide linker 5'-CAGATCTG-3' using T4 DNA ligase and transformed into E. coli strain DG98. Amp ^R
Plasmids were isolated from 1acZα ⁺ transformations. One of the clones was digested with Bgl and Asp718,
The large vector fragment was then purified by gel electrophoresis. Plasmid pFC83 was then digested with Bgl and Hind and a fragment of approximately 750 bp was isolated. Plasmid pFC85 was digested with Hind and Asp718 and a fragment of approximately 2.8 kb was isolated,
Then, by ligating the three pieces, about 750bp from pFC83
Bgl−Hind fragment and Bgl of BSM13 ⁺
- ligated with the Asp718 vector fragment.
This ligation mixture was used to transform E. coli DG98 strain (ATCC No. 39768, deposited on July 13, 1984),
Select the Amp ^R colony from this and approx.
A 6.75kb plasmid (pLSG1) was isolated.
DG95 induced by isopropyl-β-D-thiogalactoside (IPTG) containing pLSG1
The cells synthesized Taq DNA polymerase of a size indistinguishable from the native enzyme isolated from T. aquaticus. Plasmid pLSG1 can then be used to form a single-stranded DNA template according to methods recommended by Vector Cloning Systems. Next, oligonucleotide-directed mutagenesis [Zoller and Smith, Nuc. Acids Res. (1982) 10:
6487-6500] can be used to introduce a Sph restriction site as part of the ATG start codon (internal in the coding sequence of the Taq polymerase gene).
upstream of the Hind site). Similarly, a Bgl site was introduced after the carboxy terminus of the gene (approximately 0.7 kb upstream from the Asp718 site) and added to the expression vector.
Subcloning of the Taq polymerase gene can be facilitated. After completing the site-directed mutagenesis, the gene was extracted from the BSM13 ⁺ vector to approximately 3.2kb.
isolated on the Sph-BstE restriction fragment;
Klenow fragment and all four dNTPs
and pre-digested with Sac using T4 DNA ligase (blunt end conditions),
They can be repaired with Klenow and dNTPs and treated with calf intestinal phosphatase to form dephosphorylated blunt ends and inserted into the expression vectors described above. E. coli DG116 using this ligation mixture
and the resulting transformants were transformed into Taq
Screen for polymerase production. Enzyme expression can be confirmed by Western immunoblot analysis and activity analysis. Approximately 2.8kb Hind-Asp718 of plasmid pFC85
A larger portion of the Taq polymerase gene contained in the fragment can be transferred to, for example, the plasmid pP _L
Expression can be achieved using N _RBS ATG by linking the amino-terminal Hind restriction site encoding the Taq pol gene to the ATG start codon. The expression product of this fusion is approximately 66,000-68,000
Bring the Dalton truncated polymerase. This particular construct Hind plasmid pFC85
and treatment with Klenow fragment in the presence of dATP, dGTP and dCTP. The resulting fragment
Single-stranded extensions were removed by further treatment with S1 nuclease, and the resulting DNA was converted to Asp718
and treated with Klenow fragment in the presence of all four dNTPs. The recovered fragments were previously digested with Sac using T4 DNA ligase and digested in the presence of dGTP.
Dephosphorylated plasmid pP _L N _RBS that had been treated with Klenow fragment to form ATG blunt ends.
Can be linked to ATG. This ligation mixture is then used to transform E. coli DG116 and transformants are screened for production of Taq polymerase. Again, expression can be confirmed by Western immunoblot analysis and activity analysis. Example 4 Purification Thermostable polymerase was purified from Thermus aquaticus according to the previously described example.
recombinantly produced enzymes can be purified from bacterial cultures containing the recombinantly produced enzymes using methods for direct purification from cultures of C. aquaticus, with minor modifications necessary in the preparation of crude extracts. After collecting bacteria by centrifugation, 60g of cells were collected at 50m.
M was suspended in 75 ml of buffer containing Tris-HCl (PH 8.1) and 1 mM EDTA. Cells were lysed in a French press at 14,000-16,000 PSI after which 4 volumes (300 ml) of additional Tris-EDTA were added. Buffer A (5 m
M and 0.5% v/v each of NP-40 and Tween 20) were added and the solution was thoroughly sonicated while cooling. The resulting homogeneous suspension was further diluted with Buffer A to a final volume of 7.5-8 times the starting cell weight. This is called a fraction. The polymerase activity in the fractions and supernatant fractions was determined by
0.025M TAPS−HCl (PH9.2, 20℃), 0.002M
MgCl ₂ , 0.05M KCl, 1mM 2-mercaptoethanol, 0.2mM each of dGTP, dATP, TTP,
0.1mMdCTP [α- ^32P , 0.05Ci/mM], 12.5μ
g “activated” salmon sperm DNA and 0.01-0.2
unit polymerase (10mM Tris-HCl,
Measurements were made in a 50μ mixture consisting of pH 8, 50mM KCl, 1mg/ml autoclaved gelatin, 0.5% NP-40, 0.5% Tween 20 and 1mM 2 - diluted in mercaptoethanol).
One unit corresponds to 10 nM production in 30 minutes. "Activated" DNA is a natural preparation of DNA after it has been partially hydrolyzed by DNase until 5% of the DNA is converted to the acid-soluble fraction. The reaction was carried out at 74°C for 10 min, and then 2 m
M 50 μg/ml carrier DNA solution in EDTA
Transferred into 1.0 ml (0°C). 20 of the same volume (1.0ml)
% TCA and 2% sodium pyrophosphate were added.
After 15-20 minutes at 0°C, the sample was
Filter through GF/C disk and cool 5%
Wash thoroughly with TCA-1% pyrophosphate,
Then wash with cold 95% ethanol and dry.
I counted. Fractions in a Beckman TI45 rotor.
Centrifugation was performed at 35,000 rpm for 2 hours at 2°C, and the collected supernatant was fractionated. The minimum amount of Polymin P required to precipitate 90-95% of the activity (this amount has generally been found to be 0.25-0.3% of the final volume)
After determination of Taq polymerase activity, Taq polymerase activity was precipitated with Polymin P (BRL, Gaithelburg, MD) (10 v/v %, adjusted to PH 7.5 and autoclaved). Appropriate levels of Polymin P were slowly added to the fractions while stirring at 0° C. for 15 minutes.
The solution was centrifuged at 13000 rpm for 20 minutes in a Beckman JA14 rotor at 0°C. Measure the activity of the supernatant and reduce the pellet to 1/5 volume by 0.5
Resuspended in ×Buffer A (1:2 diluted with water). The suspension was recentrifuged and the pellet resuspended in 1/4 volume of buffer A (containing 0.4M KCl). This suspension was thoroughly homogenized and placed at 4°C overnight. The homogenate was centrifuged as above and the collected supernatant was designated fraction. The protein fraction was collected by “precipitation” with ammonium sulfate at 75% saturation and centrifuged (27000 rpm,
SW27 rotor for 30 minutes) and the floating thin film
Resuspend in 50mM Tris-HCl (PH8), 1mM EDTA. These steps were repeated and the protein suspension was diluted with P-cell buffer containing 80mM KCl [20mM _KPO4 , PH7.5, 0.5mM EDTA, 5%
mM β-mercaptoethanol, 5w/v% glycerose, 0.5v/v% NP-40 and Tween 20]
It was thoroughly dialyzed. Transfer the dialysate to a centrifuge bottle and add 80mM
All protein recovered from the broth, which had been rinsed with P-cell buffer containing KCl, was added. Centrifugation was performed at 20,000 xg and the time was reduced to 15 minutes. The supernatant was pooled and the remaining pellet was washed and extracted with P-cell buffer and 80mM KCl and recentrifuged. The supernatants were then combined and named fractions. Fractions were transferred to a 2.2 x 22 cm plate of phosphocellulose equilibrated with P-cell buffer containing 80 mM KCl.
column. The column was washed with the same buffer (2.5-3 column volumes) and P-
Proteins were eluted using a linear gradient from 80 to 400 mM KCl in cell buffer. Fractions containing DNA polymerase activity (approximately 0.18-0.20 M KCl) were pooled and concentrated 3-4 times on an Amicon stirred cell and YM30 membrane. The cell was rinsed with P-cell buffer without KCl and added to the fraction concentrate (0.15M KCl final volume adjustment) to fractionate. The fractions were applied to a 5 ml heparin-sepharose CL-6B column (Pharmacia) equilibrated with P-cell buffer and 0.15 M KCl. column
Wash with 0.15M KCl buffer (3-4 column volumes) and 0.15-0.65M in P-cell buffer.
Proteins were eluted with a linear gradient of KCl. A 1:10 dilution into a gelatin-free diluent for SDS-PAEG analysis and a subsequent 1:20 dilution into a diluent containing 1 mg/ml gelatin for use in enzyme measurements. I went there. The active fraction (eluted with approximately 0.3M KCl) was supercoiled.
Excess amount of specific and non-specific endonucleases/topoisomerases on the DNA template.
Changes in the molecular weight of supercoiled plasmid DNA after incubation with DNA polymerase were determined by electrophoretic detection. Exonuclease contamination was detected after incubation with small amounts of linear DNA fragments. An approximately 88 kd protein was found to be the main band in the peak fraction. The main fraction, termed fraction, has the highest polymerase activity with the least detectable tendonuclease activity when this pool is measured at approximately 3-5 polymerase units/600 ng DNA for 30 minutes at 55°C. Was. The fractions were mixed with 10mM _KPO4 (PH7.5), 5mM β-
Mercaptoethanol, 5% glycerol, 0.2
%NP-40 and 0.2% Tween 20 (HA buffer)
Dialyzed against. This dialyzed sample was
Apply to a 3 ml column of hydroxyapatite,
and 10-250mM _KPO4 in HA buffer (PH7.5)
The enzyme was eluted with a linear gradient of . Polymerase activity began to elute at 75mM _KPO4 and peaked at 100mM _PO4 . Active peak fractions were measured at 1:100 to 1:300 dilutions. As in the previous chromatography step, a 1:10 dilution in gelatin-free diluent was prepared for SDS-PAGE analysis. Fractions with no significant endonuclease or double-stranded exonuclease activity at 55°C with 5 polymerase units were pooled and designated fractions. The fractions were treated with 25mM sodium acetate (PH5.22),
5% glycerose, 5mM β-mercaptoethanol, 0.1mM EDTA, 0.1% NP-40 and 0.1%
Dialysis was performed against a solution of Tween 20 and the pH was adjusted to 5 at room temperature. Transfer the dialyzed sample to 2 ml of pre-equilibrated DEAE-Tris-Acryl-M.
(LKB) column and then washed with the same buffer. Fractions containing polymerase activity that did not adhere to the column were pooled and adjusted to 50 mM NaCl in the same buffer to obtain fractions. Fractions were diluted in the same buffer (25mM sodium acetate, 50mM NaCl, 5% glycerol, 0.1mlM
EDTA, 0.1% NP-40 and 0.1% Tween 20)
2 m of CM-Tris-Acryl-M equilibrated with
(LKB) column. This column is 4~
Five column volumes of the same buffer were washed and the enzyme was eluted with a linear gradient from 50 to 400 mM NaCl in sodium acetate buffer. The peak of polymerase activity eluted at approximately 0.15-0.20M NaCl. Polymerase activity was measured at dilutions of 1:300 to 1:500, first diluted 1:10 in diluent without gelatin for SDS-PAGE analysis. DNA polymerase assay salt (25mM TAPS-HCl, PH9.4, 2.0mM
Specific and non-specific endonuclease/topoisomerase measurements were performed using _MgCl2 and 50mM KCl) across the activity peaks on supercoiled DNA templates, as well as nuclease measurements on M13ssDNA and PBR322 fragments. Active fractions containing no detectable nuclease were pooled and silver-stained with SDS-
It was run on a PAGE mini gel. This result is
Approximately 88 kd with a specific activity of approximately 250,000 units/mg
shows a single bunt. Reference Example 1 Taq polymerase purified as described in Example 4 has Taq endonuclease activity and
It was found to be free of contamination with Taq exonuclease activity. Additionally, Taq polymerase preferably contains approximately
It is stored in a storage buffer containing 0.1 to about 0.5% v/v. More preferably, the storage buffer is
50v/v% glycerol, 100mM KCl, 20mM
Tirs-HCl (PH8.0), 0.1mM ethylenediaminetetraacetic acid (EDTA), 1mM dithiothreitol,
It consists of 0.5v/v% NP-40, 0.5v/v% Tween 20 and 20μg/ml gelatin and is preferably stored at -20°C. Reference Example 2 Taq polymerase purified as described in Example 1 was formulated for storage as described in the preceding example. However, no nonionic polymer detergent was used. This enzyme stock mixture was found to be inactive when the activity was measured as described in that example.
When NP-20 and Tween 20 were added to the storage buffer, sufficient enzyme activity was preserved, indicating that a non-ionic detergent was required for the stability of the enzyme preparation. The bacteriophage and bacterial strains listed below are part of the Cetus Master Culture Collection (CMCC).
1400, 53rd Street, Emeryville, California, USA, and American Type Culture
Deposited at the Collection (ATCC), 12301 Park Round Live, Rockville, Maryland, USA. These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty).

【table】 [Brief explanation of the drawing]

Figure 1 shows approximately 4.5 kb of HindT. aquaticus subcloned into plasmid BSM13 ⁺ .
Restriction map of plastid pFC83 containing the DNA insert. Figure 2 shows approximately 2.8 kb Hind− subcloned into plasmid BSM13 ⁺ .
Restriction map of plasmid pFC85 containing the Asp718T. aquaticus DNA insert.

Claims (1)

[Claims] 1. The following properties: (1) Action By covalently bonding α-phosphate of deoxyribonucleoside 5′-triphosphate to the 3′-hydroxyl group of an oligonucleotide or polynucleotide annealed to template DNA, Catalyzes the template-dependent introduction of deoxyribonucleoside 5'-monophosphate into deoxyribonucleic acid; (2) Substrate specificity The template DNA strand and the oligonucleotide or polynucleotide hybridized thereto for sufficient activity. (3) Optimum PH The optimum PH range is PH at a temperature of 70°C to 75°C.
8.0 to 8.5, and has significant activity even at PH7.0; (4) Active temperature range: room temperature to 85°C; optimal temperature is approximately 75°C; and (5) Molecular weight: SDS− Phosphoridase B (molecular weight 92000) and bovine serum albumin (molecular weight 66200) were used as molecular weight standards by polyacrylamide gel electrophoresis.
and approximately 86,000 to 90,000 daltons when determined using ovalbumin (molecular weight 45,000),
A thermostable DNA polymerase having the following: If the molecular weight of phosphorylase B is about 97,300 Daltons, it is about 94,000 Daltons.