JPH10500291A - Thermophilic alkaline phosphatase from Thermos thermophilus - Google Patents

Abstract

(57) Abstract: A recombinant or purified thermostable alkaline phosphatase from Thermos thermophilus having an optimum pH greater than 10.5 and stable to heating at 65 ° C for 1 hour.

Description

DETAILED DESCRIPTION OF THE INVENTION          Thermophilic alkaline phosphatase from Thermos thermophilus                                Background of the Invention   This application is a co-pending application filed with Davis and Szasz in 1994. U.S. patent application Ser. No. 08/229, filed Apr. 18, 2014 and incorporated herein by reference. No. 329, issued to Thermosiph Africanus. o africanus), which is a continuation of the thermostable alkaline phosphatase. You.   Alkaline phosphatase is commonly used to remove phosphate groups from the ends of nucleic acid molecules. Commonly used in various biochemical procedures. For example, bovine intestinal alkaline phosphaters After being used to remove such phosphate groups, Is a non-thermostable enzyme. This non-heat resistance, before subsequent operation, Advantageously, there is no need to remove alkaline phosphatase from the reaction mixture. You.   Alkaline phosphatase can also be used to detect specific proteins or DNA targets Used as a non-radioactive marker for It is a protein or DNA oligo Conjugation to nucleotides helps to detect such targets. For these applications In addition, enzyme heat resistance is desired. Alkaline from various thermophiles and other microorganisms Phosphatase is known. Yeh et al., "Thermus Aquaty Inhibitory Alkaline Phospha from Camphor (Thermus aquaticus) Purification and characterization ”J. Biol. Chem.251: 3134, 1976; Hartog et al., "From the Thermus sp. Strain Rt41A. Alkaline phosphatase ",Int. J. Biochem.24: 1657, 1992; Schaffel et al., Bacillus lichenifolimi. Alkaline phosphes from Bacillus licheniformis Thatase ",Biochimica et Biophysica Acata , 526: 457, 1978; Hulett-Couring (Hulett-Co). wling) and Campbell, "Bacillus lichenifo Re Purification and Properties of Miss Alkaline Phosphatase ", 10Bioc.136 4,1971.                                 Summary of the Invention   Applicants have filed a new alkaline phosphatase with a thermophilic species, Thermus thermophile. Ruth (Thermus thermophilus) was isolated and purified. This enzyme has a very high optimum pH (pH 13 or higher) and is thermostable And its activity is low even after incubation at 65 ° C for 24 hours. It keeps at least 50%. The higher pH optimum of this enzyme is a significant advantage . This high optimum pH and therefore high pH stability, for example, allows Use of enzymes in non-radioactive detection systems when used with leptavidin Prompt. In addition, the high optimal pH of the enzyme makes the enzyme more resistant to such alkaline pH. To be used with a dioxetane substrate that is rapidly converted to the luminescent form Make it appropriate.   The thermostability of alkaline phosphatase is further dependent on the enzyme's direct response to nucleic acid probes. After intimate crosslinking, under stringent hybridization conditions, i.e. at high temperatures Hybridization and attraction of such labeled probes without loss of enzymatic activity It is advantageous in that it allows for continuous washing.   Thus, in a first aspect, the present invention provides a method for producing a composition having an optimum pH greater than 10.5, Preferably at pH 11 or greater than 11 at optimal conditions, furthermore at least 65 ° C (Ie, at least one of its activities at this temperature) 10%), heat resistance present in Thermos thermofils (Tth) It features an enzymatically active portion of alkaline phosphatase.   An “alkaline phosphatase” is simply a molecule, such as a DNA molecule or Removes phosphate groups from other molecules such as p-nitrophenyl phosphate (pNPP) Or a fragment thereof having a certain activity. Alkaline phos Phatase is active at a pH greater than 7, and in the present invention the optimal pH is It is greater than 10.5, preferably pH 11 or higher. Such activity is , Various buffers such as CAPS, TRIS, TAPS, glycine, phosphate N a and KCl-NaOH in the presence of glycerol and divalent cations Ma Or in the absence. As shown below, the phosphine of the present invention The activity of the vatase will vary depending on such conditions. Therefore, the book The enzyme of the invention is preferably used in the presence of glycerol in 100 mM CAPS. For example, it has its optimal activity as measured in the presence of calcium ions.   "Heat resistance" means 1 hour or more at 65 ° C, preferably 5 hours or 1 hour. Means that after heating for 0 hours, the enzyme has maintained at least 10% of its activity. You. Applicants provide one example of an alkaline phosphatase of the invention, Alkaline phosphatase with a suitable pH of about 11 or greater is naturally present and The person skilled in the art, having obtained the fact that one is Can easily be screened for the portion of the enzyme that Isolating and purifying such enzymatic moieties using standard methodology as described in Can be.   In the present invention, the enzyme is preferably provided in purified form, ie The enzyme is isolated from the environment in which it naturally occurs. Generally, such an environment Is in bacterial cells, and proteins are compared to proteins present in cells Cell walls and / or cells that are at least 10- or 100-fold more abundant Or isolated from the membrane. More preferably, the protein is essentially homogeneous As is the standard, that is, the predominant species of the protein in the standard As 1000-fold or 10,000-fold or more. In addition Preferably, the protein is the only species, i.e. proteinaceous in the sample At least 95% of the material. Such a protein is naturally occurring Can be prepared from a bacterial cell that is In bacteria or other cells, such as E. coli. Prepared using standard recombinant DNA methodology causing high levels of expression of Can be. Such a crude extract of a recombinant protein is Included in the range.   Using standard techniques, the enzymes described below can be used to, for example, Desired for micro-sequencing of fragments, thermos thermofils, etc. Useful as a probe for libraries of clones created from nucleic acids of new microorganisms Of novel oligonucleotides and their libraries with such probes Clones easily by screening for DNA fragments can be isolated. Or for proteins Give rise to antibodies, and the clones develop antigenic determinants recognized by the antibodies. Expression libraries can be screened to determine You. Isolate such a gene encoding the claimed protein. Other standard techniques for doing so are well known to those skilled in the art. Such a gene is a recombinant Encodes alkaline phosphatase.   Thus, in a second aspect, the present invention provides a recombinant almin having the above characteristics. Encoding potassium phosphatase and nucleic acids comprising such recombinant DNA Cells. An equivalent encoding such a phosphatase The gene can be cloned using standard methodology.   In a third aspect, the invention relates to labeling proteins or nucleic acids. And methods for using the above enzymes in various molecular biology techniques. . For example, the enzymes of the invention can be used in standard labeling reactions and in diagnostic assays. Can be used. They are also molecular organisms where removal of phosphate groups is desired. Can be used in scientific techniques.   Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments and It will be clear from the scope of the request.                          Description of the preferred embodiment   First, the drawings are briefly described.Drawing   Figure 1 is a graph showing the pH activity of the enzyme of the present invention;   FIG. 2 shows the (most) of the enzyme of the invention in various concentrations of CAPS buffer at pH 11.0. Suitable) is a graph showing activity;   FIG. 3 is a graph showing the (optimal) activity of the enzyme of the invention in various concentrations of NaCl. Is;   FIG. 4 is a graph showing the (optimal) activity of the enzyme of the invention in various concentrations of glycerol. Is rough;   FIG. 5 is a graph showing the (optimal) activity of the enzyme of the invention at various temperatures;   FIG. 6 shows the enzyme after heating up to 24 hours at 70 ° C. in various buffers. 3 is a graph showing the stability of the present invention.Example: Alkaline phosphatase from Thermus thermophilus   Thermus thermophilus strain HB8 is transferred to the alkaline phosphatase in this microorganism. In defined media containing limited amounts of inorganic phosphate that reduces Trela; 251J. Biol. Chem.3134, 1976 Modified under aerobic conditions at 75 ° C. Medium is 1 liter. The following salts were contained per torr. Nitrilotriacetic acid 100mg, CaSO_Four ・ 2H_TwoO 60mg, MgSO_Four・ 7H_TwoO 100 mg, NaCl 8 mg, KNO_Three105 mg, ZnSO_Four・ 7H_TwoO 5mg, H_ThreeBO_Three5 mg, CuSO_Four ・ 5H_TwoO 0.16 mg, Na_TwoMoO_Four・ 2H_TwoO 0.25 mg, CoCl_Two ・ 6H_TwoO 0.4mg, MnSO_Four・ H_TwoO 22mg, FeCl_Three・ 6H_TwoO 0.28 mg. Vitamins were added per liter as follows. Biotin 0 . 1 mg, thiamine 0.1 mg and niacin 0.05 mg. The medium is . 3% L-glutamic acid, 0.004% L-lysine, 0.1% glycerol and And 0.1% glucose. Sodium glycerophosphate (40 μM) Served as a phosphate source. The pH of the medium was adjusted to 7.2. Continuous cells Collected by dynamic flow centrifugation and stored frozen at -80 ° C.   Alkaline phosphatase activity is determined by the enzyme at 37 ° C. Absorption due to release of p-nitrophenol from phenylphosphoric acid (pNPP) Spectrophotometry was measured at 405 nm by tracking the increase in power. Assay buffer Liquids were 6 mM p-nitrophenyl phosphate, 100 mM C, unless otherwise noted. It contained APS (pH 11) and 15% glycerol.   Thaw frozen cells, 10 mM Tris-HCl (pH 8), 1 M MgCl_Two And 1 mM CaCl_TwoResuspended in and dissolved by sonication . Lysates were freed of cell debris by centrifugation and 20 mM Tris, pH8. 0,25 mM MgCl_Two, 1 mM CaCl_TwoAnd 0.1% Triton X10 ( DE52 anion exchange dialyzed against buffer A) and equilibrated in buffer A Exchange Applied to the column. Most of the alkaline phosphatase activity is 25 mM M Seen in a stream adjusted to pH 6.0 by addition of ES (free acid), followed by Heparin Sepharose was applied to a CL-6B cation exchange column. The column is 0-800 mM NaCl (MgCl_TwoAnd Triton from this high salt buffer (Omitted) developed by a linear gradient.   Pool the fraction containing alkaline phosphatase activity (about 300 mM NaCl) And washed thoroughly with 20 mM Tris pH 7.4 After applying directly to the apatite column, it is added to a 20-500 mM Na phosphate pH Developed with a linear gradient of 7.0. Most of alkaline phosphatase activity Eluted with approximately 100 mM Na phosphate.   Preliminary analysis of the sample by SDS-PAGE showed that the peak of the enzyme activity was apparent. Corresponded to the major protein band run at a molecular weight of about 49,000 daltons Suggested that. Further purification and analysis are needed to confirm this point. is there. The final product is approximately 80% homologous to the 49 kd polypeptide and has a specific The crude extract showed about 10-fold purification as determined by activity experiments.   Tth alkaline phosphatase can be measured over a fairly wide range of pH values Although it shows potent activity, it appears to have an unusually high pH optimum, with 13.0 being the highest assayed. It is high (Fig. 1). Characterization of rare extreme applications at such extreme pH Was performed at pH 11.0. Optimal conditions (100 mM CAPS pH1 1.0, 15% glycerol), the enzyme is ≧ 250 units / mg at 37 ° C. Shows the specific activity of Enzyme activity was determined using buffer (FIGS. 1 and 5), ionic strength (FIGS. And 3), glycerol (FIG. 4) and temperature (FIG. 5). Is affected. The enzyme can also be inhibited because it is inhibited by 1 mM EDTA. (Data not shown), may require divalent cations. Only While the Ca for the assay mixture was⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Cu⁺⁺Or Zn⁺⁺Addition could not stimulate or inhibit the activity I understood. More routine experimentation can easily determine the metal ion requirement of this enzyme. Can be After 24 hours of incubation at 70 ° C, the protein is (Fig. 6) is considered to be extremely heat resistant because it retains almost 90% of the activity of You. This enzyme is more active in CAPS buffer but more stable in Tris. It is thought that. Activity in Tris is due to pH action or buffer action It is not clear that the enzyme activity is stimulated by the high concentration of this buffer. Is done.Use   Thermus thermophilus alkaline phosphatase is a protein and nuclear It may be a potentially useful enzyme for non-isotopic detection of acids. For example, the heat resistance of this enzyme Is that the enzyme is directly crosslinked to the DNA probe and then subjected to stringent conditions (ie, Sufficient candidate to hybridize to a specific target under high temperature Can be Furthermore, the wide temperature activity range of this enzyme (FIG. 5) Gives flexibility in choice. Finally, the ultimate in Tth alkaline phosphatase The high optimum pH makes it very suitable for high pH applications.   Specifically, the alkaline phosphatase of the present invention is used for detecting proteins and nucleic acids. There are several possible uses for the many non-isotopic methods for exit. For example, this enzyme The optimal pH is high enough to convert rapidly to the luminescent form at alkaline pH. Make the enzyme suitable for the xetane substrate. Furthermore, this alkaline phosphatase The high heat resistance makes it useful for cross-linking directly to nucleic acid probes. C Hybridization and subsequent washing is performed under stringent conditions without losing enzyme activity. That is, it can be performed under high temperature). Streptavidin-bound alkaline phos Positively charged membranes, such as in the case of nucleic acid hybridization of phatases PH greater than 9.5 gives a reduced background when used as Is preferred.   Alkaline phosphatase from different microorganisms behaves similarly during purification (Or maybe not). High pH, optimal for activity, is used for purification itself. However, for screening see below. High optimal The temperature is such that after cloning into a host such as E. It may be useful when purifying enzymes. Heat-treat the extract from E. coli All denaturing proteins could be sedimented.   If enzymes stable at 65 ° C to 75 ° C are desired, grow well at those temperatures A machine to discover such enzymes by trying to isolate new microorganisms It is possible to increase the association. In addition, select microorganisms that can tolerate high pH. I was able to. Further, after alkaline phosphatase is found to be desirable, the compound By using 5-bromo-4-chloro-3-indolyl phosphoric acid (X-Phos) Of microorganisms or recombinant clones for alkaline phosphatase activity The library can be screened. The phosphate group is removed from this compound. If obtained, a blue color is obtained, which is very favorable for screening for activity. Make it convenient. Next, the pH activity profile is such that the phosphate removal activity is It would be prepared to check if it was a fatase.   Other embodiments are within the following claims.

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Claims (1)

[Claims]   1. PH optimum is greater than 10.5 and safe for heating at 65 ° C for 1 hour. Regular Thermos thermophilus (Thermus thermophilus) A purified enzymatically active portion of a thermostable alkaline phosphatase present in.   2. PH optimum is greater than 10.5 and safe for heating at 65 ° C for 1 hour. A recombinant DNA encoding a stable thermostable alkaline phosphatase.   3. A cell comprising the recombinant DNA according to claim 2.   4. PH optimum is greater than 10.5 and safe for heating at 65 ° C for 1 hour. Constant recombinant alkaline phosphatase.   5. A method for detecting a nucleic acid or protein in a sample, wherein the optimal pH is 1 Heat resistant alkalinity greater than 0.5 and stable to heating at 65 ° C for 1 hour Providing a nucleic acid, protein or antibody labeled with a phosphatase The above method comprising: