THERMOSTABLE ALKALINE PHOSPHATASE

OF THERMOSIPHO AFRICANUS

Background of the Invention

Alkaline phosphatases are commonly used in routine biochemical procedures to remove phosphate groups from the terminus of a nucleic acid molecule. For example, calf intestinal alkaline phosphatase is a heat labile enzyme which is used to remove such phosphate groups, and then is inactivated by exposure to a high temperature. This thermal instability is advantageous because the alkaline phosphatase need not be removed from the reaction mixture prior to subsequent manipulations.

Alkaline phosphatase is also used as a non- radioactive marker for the detection of specific protein or DNA targets. It is conjugated to proteins or DNA oligonucleotides to aid in detection of such targets. Enzyme thermostability is desired in these applications.

Alkaline phosphatases from various thermophilic and other organisms are known. Yeh et al. , "Purification and Characterization of a Repressible Alkaline Phosphatase from Thermus aquaticus" , J. Biol . Chem. 251:3134, 1976; Hartog et al . , "An Alkaline Phosphatase from Thermus sp Strain Rt41A", Int. J. Biochem. , 24:1657, 1992; Schaffel et al. , "Alkaline Phosphatase from Bacillus licheniformis" , Biochimica et Biophvsica Acta, 526:457, 1978; Hulett-Cowling et al . , 10 Bioc. 1364, 1971.

Summary of the Invention Applicant has isolated and purified a novel alkaline phosphatase from the thermophilic species Thermosipho africanus. This enzyme has an extremely high pH optimum (around pH 11) , and is thermostable, retaining 50% of its activity even after 24 hours incubation at 65°C. The higher pH optimum of this enzyme is a significant advantage. This high pH optimum, and thus stability at high pH, enhances the use of the enzyme in non-radioactive detection systems, for example, when the enzyme is used with streptavidin. In addition, the high pH optimum of the enzyme makes it suitable for use with dioxetane substrates which undergo rapid conversion to the luminescent form at such alkaline pH. The thermostability of the alkaline phosphatase is also advantageous in that following direct cross¬ linking of the enzyme to nucleic acid probes, it allows hybridization and subsequent washes of such labelled probes under stringent hybridization conditions, that is, at elevated temperatures without loss of enzyme activity.

Thus, in a first aspect the invention features an enzymatically active portion of the thermostable alkaline phosphatase present in Thermosipho africanus (Tsa) having a pH optimum greater than 10.5, preferably an optimum at pH 11, which is also resistant to a temperature of at least 65°C (i.e. , maintains at least 10% of its activity at this temperature) .

By "alkaline phosphatase" is simply meant a protein or fragment thereof having an activity which removes a phosphate group from a molecule, such as a DNA molecule or another molecule, such as p-nitrophenyl phosphate (pNPP) . An alkaline phosphatase is one which is active at a pH greater than 7, and in the present invention has a pH optimum greater than 10.5 and preferably at pH 11.

By "thermostable" is meant that the enzyme maintains at least 10% of its activity after heating at 65°C for one hour or longer, preferably for 5 or 10 hours. While Applicant provides one example of an alkaline phosphatase of the present invention, those in the art armed with the fact that an alkaline phosphatase having a pH optimum of about 11 exists in nature and can be isolated can now readily screen portions of the enzyme to determine the presence of such an activity, and can _. use standard methodology as described herein to isolate and purify such an enzymatic portion.

In the present invention, the enzyme is preferably provided in a purified form, that is, it is isolated from the environment in which it naturally occurs. Generally, such an environment is within a bacterial cell and the protein is isolated from the cell wall and/or membranes of that cell such that it is enriched at least 10- or 100- fold compared to its presence in the cell. More preferably, it is enriched 1000- or 10,000- or more fold such that it is an essentially homogeneous preparation, that is, it is the predominant species of protein in a preparation. Even more preferably, the protein is the only species, that is, it represents at least 95% of the proteinaceous material in a sample. Such a protein may be prepared from the bacterial cells in which it naturally occurs, or may be prepared using standard recombinant DNA methodology to cause high level of expression of the protein in a bacterium or other cell in which it does not naturally occur, e.g. , E. coli. A crude extract of such recombinant protein is included within the definition of purified protein.

Using standard techniques the enzyme described below can be readily cloned, for example, by microsequencing of the protein or fragments thereof, preparation of oligonucleotides useful as probes for a

library of clones generated from the nucleic acid of a desired organism, e.g. , Thermosipho africanus, and screening of that library with such probes to isolate fragments of DNA encoding the protein. Alternatively, an antibody to the protein may be produced and an expression library screened to determine which clone expresses an antigenic determinant recognized by that antibody. Other standard techniques are well known to those of ordinary skill in the art to isolate such genes encoding . the claimed proteins. Such genes encode recombinant alkaline phosphatase.

Thus, in a second aspect the invention features recombinant alkaline phosphatase having the above properties, and cells encoding nucleic acid including such recombinant DNA. Equivalent genes encoding such phosphatases can be cloned using standard methodology.

In a third aspect, the invention features a method for use of the above enzymes in labelling of protein or nucleic acid, and in various molecular biology techniques. Thus, the enzymes of the present invention may be used in standard labelling reactions and in diagnostic assays. They may be also used in molecular biology techniques in which removal of a phosphate group is desired. The alkaline phosphatase of Thermosipho africanus

(Tsa) is distinct from that present in Thermus aguaticus (Taq) and Thermus thermophilus (Tth) . As shown in the data presented below on an SDS PAGE gel the subunit size of the Tsa alkaline phosphatase (apparent subunit molecular weight approximately 47,000 Daltons) is different from the subunit sizes of alkaline phosphatase from both Taq and Tth with apparent molecular weights of about 51,000 Daltons each.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiment The drawings will first briefly be described. Drawings

Fig. 1 is a graphical representation showing the pH optimum of an enzyme of the present invention;

Fig. 2 is a similar graph showing the temperature optimum of the enzyme;

Fig. 3 is a graph showing the stability of the enzyme after heating at 65°C for up to twenty-two hours; Fig. 4 is a copy of a 4-15% SDS polyacrylamide gel electrophoresis (PAGE) comparing molecular weights of various alkaline phosphatases; and

Fig. 5 is the Tsa alkaline phosphatase NH ₂ - terminal amino acid sequence. The following are two examples of Tsa alkaline phosphatase isolation and characterization. These are not limiting in the invention.

Example 1: Alkaline Phosphatase from Thermosipho africanus Thermosipho africanus, strain DSM 5309 was grown under anaerobic conditions at 75°C as described by Huber et al., 12 System. Appl . Microbiol . 38, pp. 32-37, 1989. Cells were harvested by continual flow centrifugation and stored at -80°C.

Alkaline phosphatase activity was measured spectrophotometrically at 405nm by following the increase in absorbance due to the release of p-nitrophenol from p- nitrophenyl phosphate by the enzyme at 37°C. The assay buffer contained 6mM p-nitrophenyl phosphate, lOOmM Glycine-NaOH (pH 10) , ImM MgCl ₂ and ImM ZnCl ₂ .

Frozen cells were thawed, resuspended in 20mM

Tris-HCl (pH8), 50mM NaCl, 10% glycerol, ImM EDTA, ImM DTT

(Buffer A) and lysed by sonication. The lysate was cleared of cellular debris by centrifugation before applying to a DE52 anionic exchange column (typically 10

grams resin per gram of cells) . The column was developed with 10 column volumes of a linear gradient from 50 to 1000 mM NaCl in Buffer A. The majority of alkaline phosphatase activity eluted at a salt concentration of about 400 mM. Appropriate fractions were pooled, dialyzed extensively against 25 mM HEPES-KOH (pH7.25), lOmM KCl, 10% glycerol, ImM EDTA, ImM DTT and applied to a Heparin Sepharose CL-6B column. Enzyme activity was found in the flow-through, which was subsequently applied to a _ Pll phosphocellulose column. The Pll flow-through was chromatographed on Affi-Gel Blue affinity resin. The enzyme did not bind to this column and therefore the flow- through was extensively dialyzed against Buffer A and applied to a Q-Sepharose anionic exchange column which was developed with a linear gradient from 50 to 800 mM NaCl. While the elution profile exhibited several minor peaks of activity, the majority of alkaline phosphatase activity appeared in the flow-through.

In light of the fact that this enzyme did not bind to most of the resins tested, the Q-Sepharose flow- through was rechromatographed on DE52 resin as described above except using a slightly shallower gradient from 50 to 800 mM NaCl. The major activity peak eluted at about 360 mM salt. The peak fractions were pooled, dialyzed extensively against 20mM KP0 ₄ , 50mM KCl, 10% glycerol, ImM DTT and chromatographed on hydroxylapatite. The column was developed with a linear gradient from 20 to 600 mM KP0 ₄ . The peak alkaline phosphatase activity eluted at about 100 mM KP0 ₄ . After pooling, the peak fraction was supplemented to a final concentration of 0.1 mM EDTA, ImM MgCl ₂ , ImM ZnCl ₂ and stored at 4°C.

Analysis of this preparation by SDS-PAGE revealed several major protein bands, therefore the apparent molecular weight of the alkaline phosphatase was not determined. While not purified to homogeneity, the final product did represent a 69-fold purification of the crude

extract as determined by specific activity studies. The enzyme preparation was further characterized by determining the pH optimum (Fig. 1) , the temperature optimum (Fig. 2) and enzyme stability at 65°C (Fig. 3) , using standard methods.

Example 2 : Purification and Characterization of a thermostable Alkaline Phosphatase from Thermosipho africanus

Thermosipho africanus. strain DSM 5309 was grown in a modified form of DSM media 141, under anaerobic conditions at 75°C as described by Huber et al., (System. Appl. Microbiol. 12:38-47 (1989)) . Cells were harvested and stored at -80°C as above.

Alkaline phosphatase activity was measured spectrophotometrically at 405 nm by following the increase in absorbance due to the release of p-nitrophenol from p- nitrophenyl phosphate (pNPP) by the enzyme at 37°C. The assay buffer was as above, or contained 100 mM CAPs (3-

(cyclohexylamino) -1-propanesulfonic acid; pH 11) , ImM MgCl ₂ and 6mM pNPP.

Alkaline phosphatase was released from bacterial cells by osmotic shock. 30 grams of frozen cells were resuspended in 75 ml of 25 mM Tris pH 7.4, 25mM NaCl and 2mM EDTA (Buffer C) and mixed on a magnetic stir plate for one hour at room temperature. The lysate was cleared of cellular debris by centrifugation before applying to a DE 52 anionic exchange column (Whatman; 0.5 grams resin per gram frozen cells) equilibrated in Buffer C. The majority of alkaline phosphatase activity appeared in the flow- through which was supplemented to ImM MgCl ₂ and subsequently applied to a Heparin Sepharose CL-6B column

(Pharmacia) which was developed with 12.5 column volumes of a linear gradient from 25 to lOOOmM NaCl in Buffer C plus ImM MgCl ₂ . Enzyme activity eluted at about 450mM NaCl. Peak fractions were pooled and applied to a hydroxylapatite column (Bio-Rad) which was washed with two

column volumes of 25mM Tris pH 7.4 followed by 13 column volumes of a linear gradient from 10 to 300mM Naphosphate pH 7.0. Enzyme activity eluted at about 150mM Na phosphate. Appropriate fractions were pooled, buffer exchanged into 25 mM Tris pH 9.0 on a Centriprep 30 apparatus (Amicon) and chromatographed on Q-Sepharose FF anionic exchange resin (Pharmacia) . The column was developed with 12 volumes of a linear gradient from 0 to 300 mM NaCl in 25mM Tris pH 9.0. The majority of alkaline phosphatase activity eluted at about 80mM NaCl.

Analysis of this preparation by SDS-PAGE revealed a single protein band which migrated at an apparent molecular weight of approximately 47,000 daltons (Fig. 4) . The gel used was purchased from BioRad as a Mini-Protein II Ready Gel, Catalogue No. 161-0902, 4-15% gradient gel and used according to manufacturer's specifications. The final product represented a greater than 1000-fold purification of the crude extract as determined by specific activity studies. The enzyme preparation was further characterized by determining the pH optimum, the temperature optimum and enzyme stability at 65°C (as above) and partial amino acid sequence (Fig. 5) . Utilities The alkaline phosphatase from Thermosipho africanus may have several potential uses in the numerous non-isotopic methods for the detection of proteins and nucleic acids. For example, the high pH optimum of this enzyme may make it highly suitable with dioxetane substrates which undergo rapid conversation to the luminescent form at alkaline pH. In addition, the high thermostability of this alkaline phosphatase may make it a good candidate for direct crosslinking to nucleic acid probes. Hybridization and subsequent washes could be carried out under stringent conditions (i.e. , elevated temperatures) without loss of enzyme activity.

Uses

Alkaline phosphatases of this invention have several potential uses in the numerous non-isotopic methods for the detection of proteins and nucleic acids. For example, the high pH optimum of this enzyme makes it suitable with dioxetane substrates which undergo rapid conversion to the luminescent form at alkaline pH. In addition, the high thermostability of this alkaline phosphatase makes it useful for direct crosslinking to nucleic acid probes. Hybridization and subsequent washes can be carried out under stringent conditions (i.e. , elevated temperatures) without loss of enzyme activity. When using streptavidin conjugated alkaline phosphatase on positively charged membranes, as in nucleic acid hybridization, pH greater than 9.5 is preferred to give decreased background.

Alkaline phosphatases from different organisms may (or may not) behave similarly during purification. The high pH optimum for activity cannot be exploited for the purification per se, but see below for screening. The high temperature optimum will be useful in purifying such enzymes after cloning into hosts that grow at a moderate temperature, such as E. coli. Extracts from E. coli could be heat treated to precipitate all proteins that denature at elevated temperatures.

If an enzyme is desired which is stable at 65°- 75°C, it is possible to enhance the chances of discovery of such an enzyme by trying to isolate novel organisms that grow well at those temperatures. One could also select for organisms that are tolerant of high pH. In addition, knowing that an alkaline phosphatase is desired, one can then screen organisms, or libraries of recombinant clones, for alkaline phosphatase activity by use of the compound 5-bromo-4-chloro-3-indolyl phosphate (X-Phos) . A blue color is obtained when the phosphate group is removed from this compound, making it very convenient to

screen for activity. A pH activity profile would then be prepared to determine whether the phosphate removing activity was an alkaline phosphatase.

Other embodiments are within the following claims.