Patent Application
20240167005
The present invention relates to field of apyrases, in particular for the degradation of organic phosphates.
The present invention also relates to analytical and diagnostic methods where contaminating nucleotides are an issue. In particular, the present invention relates to determining or quantifying the amount of ATP present in a sample that may contain contaminating nucleotides, as well as reagents for use in such methods and production of such reagents.
Adenosine triphosphate (ATP) is a molecule present in all living cells. Since the concentration of ATP is fairly constant in the cell, measurement of ATP content in a sample can be used as a proxy to determine the number of viable cells. Sensitive bioluminescent assays for measuring ATP based on luciferase/luciferin are known, see e.g., U.S. Pat. No. 3,745,090. Luciferase (e.g., from firefly) is a euglobulin protein that catalyses the oxidative decarboxylation of luciferin using ATP and molecular oxygen to yield oxyluciferin, a highly unstable, single-stage excited compound that emits light upon relaxation to its ground state. This reaction emits light proportional to ATP concentration in the reaction mixture, and by measuring the intensity of the emitted light it is possible to continuously monitor the concentration of ATP present.
In many cases, samples to be analyzed for ATP content contain contaminating ATP either extracellularly or present in a contaminating type of cells. For instance, if the number of bacteria is to be quantitated in any clinical or biological sample, any ATP contained in host cells present in the sample will interfere with the measurement. In many applications, the contaminating cells may be selectively lysed, and the ATP released, resulting in that all the contaminating ATP is in extracellular form, see e. g. U.S. Pat. No. 4,303,752 or US20110076706. In other instances, ATP analogues different from ATP may be present in a sample and interfere with ATP measurement.
The contaminating extracellular ATP, otherwise unwanted ATP or ATP analogues can be reduced or eliminated by hydrolyzing them with an enzyme called apyrase (ATP-diphosphohydrolase, E-type ATPase, ATPDase, NTDase EC 3.6.1.5). Apyrase is frequently used in methods for determining bacterial ATP in the presence of mammalian cells, where the mammalian cells are first selectively lysed, and apyrase used to degrade extracellular ATP leaving the bacterial ATP unaffected. After completion of the reaction, the apyrase can be inactivated, and the intracellular ATP of bacterial cells is released to measure bacterial ATP by the addition of luciferin/luciferase. Light emission is measured before and after the addition of a known amount of ATP standard, as internal control. The bacterial ATP (in moles) is calculated by multiplying the ratio of the light before and after adding the ATP standard with the amount of added standard. Typically, bacterial cells contain around 1 attomole of ATP per cell, making it possible to estimate the number of bacterial cells from the amount of ATP detected. It is an object of the present invention to provide improvements for such analytical and diagnostic methods.
In this context, the most commonly used apyrase is Solanum tuberosum apyrase (STA). STA exists in several isoforms and each isoform differs in ATP-degradation activity. However, the efficiency of STA in the above methods is limited by the accumulation of ADP and uncharacterized ATP-analogues in the degradation reaction when using STA. Accumulation of such contaminants inhibit the ATP degradation capability allowing some of the contaminating ATP to remain intact, which in turn limits the sensitivity of the ATP determination assays and increases the background signal. For a discussion on the limitations of STA, see WO199402816.
Further, most embodiments of the DNA sequencing method pyrosequencing (see e.g., international patent applications PCT/GB1997/002631 and PCT/GB1997/003518, or US patent applications US2013/0045876 and US2013/0189717) also rely on quantitation of ATP, usually by the luciferase/luciferin assay. At the end of every sequencing cycle, all the unincorporated nucleotides and excess ATP must be eliminated by e.g., apyrase. As detailed above, the apyrases presently used in DNA sequencing applications have problems in achieving complete degradation due to the quality of enzyme (contamination of NDP kinase), substrate specificity and batch-to-batch variations that not only results in drop off and non-linear peaks but also affects DNA sequencing of long strands.
The use of wild-type Shigella flexneri apyrase in degrading contaminating nucleoside diphosphates and/or triphosphates has been disclosed in WO 2016/071497 A1.
It is an object of the present invention to provide an alternative or improved apyrase for the applications discussed above as well as other applications.
The following terms and words have the meanings as defined below in the context of the present disclosure.
Shigella flexneri apyrase (SFA) is defined as apyrase derived from Shigella flexneri, at any suitable degree of purity. The SFA may be produced by non-recombinant means or by recombinant DNA technology. Recombinant Shigella flexneri apyrase is abbreviated rSFA herein. The native SFA has the sequence according to SEQ ID NO: 1. Apyrase activity refers to capacity to catalyse hydrolysis of nucleoside triphosphates to nucleoside diphosphates, and hydrolysis of nucleoside diphosphates to nucleoside monophosphates.
Sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook [Internet], Chapter 16), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=11, Gap extension cost=1.
ATP analogues refer to compounds with structural and functional similarity to ATP which compete with ATP for binding to enzymes that specifically interact with ATP. In the present context, the term only applies to compounds that are substrates for SFA as defined above. Preferably, the term refers to compounds that can substitute ATP as substrate for luciferases, such as firefly luciferase. In preferable embodiments, the term includes nucleoside diphosphates and nucleoside triphosphates. In more preferable embodiments, the term refers to adenosine diphosphate, deoxyadenosine alpha-thio triphosphate, adenosine tetra-phosphate, deoxyadenosine triphosphate, deoxyadenosine diphosphate, guanosine triphosphate, guanosine diphosphate, deoxyguanosine triphosphate, deoxyguanosine diphosphate, thymidine triphosphate, deoxythymidine triphosphate, thymidine diphosphate, deoxythymidine triphosphate, cytidine diphosphate, deoxycytidine triphosphate, cytidine triphosphate, deoxycytidine diphosphate, uridine diphosphate and uridine triphosphate. In most preferable embodiments, ATP analogues refers to: adenosine diphosphate, deoxyadenosine alpha-thio triphosphate, adenosine tetra-phosphate, deoxyadenosine triphosphate, deoxyadenosine diphosphate, guanosine triphosphate, guanosine diphosphate, deoxyguanosine triphosphate and deoxyguanosine diphosphate.
The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.
1. An apyrase enzyme, characterized by that:
2. The apyrase according to any of the preceding items, wherein the apyrase catalyzes the dephosphorylation of the at least one organic phosphate with at least 50-fold lower, preferably at least 100-fold lower Km compared to the apyrase of SEQ ID NO:1.
3. The apyrase according to any of the preceding items, wherein the at least one organic phosphate is para-Nitrophenylphosphate (pNPP) and/or thiamine pyrophosphate (TPP).
4. The apyrase according to any of the preceding items, wherein the apyrase exhibits a Km of less than 30 mM for dephosphorylation of para-Nitrophenylphosphate (pNPP), more preferably less than 10 mM, even more preferably less than 2 mM, most preferably less than 1 mM.
5. The apyrase according to any of the preceding items, wherein the apyrase Km for dephosphorylation of pNPP is measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
6. The apyrase according to any of the preceding items, wherein the apyrase exhibits a Km of less than 30 mM for dephosphorylation of thiamine pyrophosphate (TPP), more preferably less than 20 mM, even more preferably less than 10 mM, most preferably less than 6 mM.
7. The apyrase according to item 6, wherein the apyrase Km for dephosphorylation of TPP is measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
8. The apyrase according to any of the preceding items, wherein the apyrase also catalyses the dephosphorylation of ATP.
9. The apyrase according to any of the preceding items, wherein the apyrase catalyses the dephosphorylation of ATP with a Km differing less than 5-fold from that of the apyrase of SEQ ID NO:1, preferably less than 2-fold.
10. The apyrase according to any of the preceding items, wherein the apyrase exhibits a Km of less than 10 mM for the dephosphorylation of ATP, preferably less than 5 mM.
11. The apyrase according to any of the preceding items, wherein the apyrase Km for the dephosphorylation of ATP is measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
12. The apyrase according to any of the preceding items, wherein the substitutions comprise a substitution of a residue aligning with F53.
13. The apyrase according to any of the preceding items, wherein the substitutions comprise a substitution of a residue aligning with L66.
14. The apyrase according to any of the preceding items, wherein the substitutions comprise a substitution of a residue aligning with E77.
15. The apyrase according to any of the preceding items, wherein the substitutions comprise the substitution F53V.
16. The apyrase according to any of the preceding items, wherein the substitutions comprise the substitution L66V.
17. The apyrase according to any of the preceding items, wherein the substitutions comprise the substitution E77V.
18. The apyrase according to any of the preceding items, wherein the sequence does not comprise the substitution G63R, preferably any substitution G63.
19. The apyrase according to any of the preceding items, wherein the sequence does not comprise the substitution S97P, preferably any substitution of S97.
20. The apyrase according to any of the preceding items, wherein the sequence does not comprise the substitution H116L, preferably any substitution of H166.
21. The apyrase according to any of the preceding items, wherein the apyrase comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:1, preferably at least 90%, more preferably at least 95%.
22. The apyrase according to any of the preceding items, wherein the apyrase comprises a polypeptide sequence according to SEQ ID NO: 3 residues 1-246.
23. The apyrase according to any of the preceding items, wherein the apyrase consists of the polypeptide sequence according to SEQ ID NO: 3.
24. A method for reducing the amount of contaminating nucleoside diphosphates and/or nucleoside triphosphates, comprising the steps of
25. A method for determining the amount of ATP in a sample, comprising the steps of:
26. A method for determining the amount of ATP present in a first population of cells in a sample, comprising the steps of:
27. The method according to item 26, wherein the liberation step (b) involves lysis of the first population of cells; the first population of cells comprises bacterial cells; and wherein the sample is a biological sample from an animal or a human.
28. The method according to any of items 24-27, wherein the sample is a blood sample, a plasma sample, a serum sample, a urine sample, a fecal sample, or a swab from a patient.
29. The method according to any of items 24-28, wherein at least a fraction of the contaminating ATP is present in a second population of cells, and the reduction step (a) is preceded by a step of selective liberation of ATP from the second population of cells, wherein the second population of cells are host cells from an animal from which the sample is derived.
30. A method for doing pyrosequencing comprising the steps of:
31. A use of an apyrase according to any of items 1-23 for degrading contaminating nucleoside triphosphates or nucleoside disphosphates in an analytical method.
32. A use of an apyrase according to any of items 1-23 for dephosphorylating organic phosphates.
33. A method for catalyzing the dephosphorylation of an organic phosphate, comprising contacting the organic phosphate with the apyrase of any of items 1-23.
The present invention provides a mutated variant of the Shigella flexneri apyrase (SFA, Example 1), which has similar activity towards ATP degradation (dephosphorylation) as the wild type (wt) apyrase (Example 2). However, the mutated variant exhibits considerably higher affinity and catalytic activity towards dephosphorylation of certain organic phosphates compared to the wild-type Shigella apyrase (Example 3). Experiments shown in Examples 4 and 5 further pinpoint the key mutations behind the improved activity and characterize the mutated enzyme.
The value of the Michaelis constant Km is numerically equal to the [substrate] at which the reaction rate is at half-maximum (1/2×Vmax), and is an inverse measure of the substrate's affinity for the enzyme—as a small Km indicates high affinity, meaning that the rate will approach the maximum rate (Vmax) with lower [substrate] than reactions with a higher value of Km. The Km constant is independent of the purity or concentration of the enzyme but varies between substrates and reaction conditions.
Mutated Apyrase with Broadened Substrate Specificity
Thus, in a first aspect, the present invention provides an apyrase enzyme, characterized by that:
The at least one organic phosphate may refer to para-Nitrophenylphosphate (pNPP). Alternatively, the at least one organic phosphate may refer to thiamine pyrophosphate (TPP). Preferably, the at least one organic phosphate refers to both pNPP and TPP.
The apyrase may exhibit a Km of less than 30 mM for dephosphorylation of para-Nitrophenylphosphate (pNPP), more preferably less than 10 mM, even more preferably less than 2 mM, most preferably less than 1 mM. The apyrase Km for dephosphorylation of pNPP is preferably measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
The apyrase may exhibit a Km of less than 30 mM for dephosphorylation of thiamine pyrophosphate (TPP), more preferably less than 20 mM, even more preferably less than 10 mM, most preferably less than 6 mM. The apyrase Km for dephosphorylation of TPP is preferably measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
Preferably, the apyrase of the first aspect is capable of catalyzing the dephosphorylation of ATP. More preferably, the apyrase catalyses the dephosphorylation of ATP with a Km differing less than 5-fold from that of the apyrase of SEQ ID NO:1, preferably less than 2-fold. The apyrase may exhibit a Km of less than 10 mM for the dephosphorylation of ATP, preferably less than 5 mM. The apyrase Km for the dephosphorylation of ATP may be measured at pH7.5 and at a temperature of 37° C., preferably in a 50 mM Tris buffer at pH7.5.
The substitutions preferably comprise a substitution of a residue aligning with F53. More preferably, the substitutions comprise the substitution F53V.
The substitutions preferably comprise a substitution of a residue aligning with L66. More preferably, the substitutions comprise the substitution L66V.
The substitutions preferably comprise a substitution of a residue aligning with E77. More preferably, the substitutions comprise the substitution E77V.
Preferably, the sequence does not comprise the substitution G63R, more preferably any substitution G63. Preferably, the sequence does not comprise the substitution of S97P, preferably any substitution of S97. Preferably, the sequence does not comprise the substitution H116L, preferably any substitution of H166. More preferably, the sequence does not comprise any substitution of any of G63, S97 or H116. Most preferably, the sequence does not comprise any of the substitutions G63R, S97P and H116L.
The apyrase of the first aspects preferably comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:1, preferably at least 90%, more preferably at least 95%. More preferably, the apyrase comprises a polypeptide sequence according to SEQ ID NO: 3. Most preferably, the apyrase consists of the polypeptide sequence according to SEQ ID NO: 3.
The International Patent Application WO2016/071497 (incorporated by reference herein) discloses methods utilizing a wild-type Shigella flexneri apyrase in reducing the amount of contaminating or unwanted nucleoside diphosphates and/or nucleoside triphosphates in several applications, including ATP determination and sequencing-by synthesis. The mutant enzymes of the first aspect described herein are useful in these methods.
In a second aspect, the present invention relates to a method for reducing the amount of contaminating nucleoside diphosphates and/or nucleoside triphosphates, comprising the steps of
In a third aspect, the present invention relates to a method for determining the amount of ATP in a sample, comprising the steps of:
In a fourth aspect, the present invention relates to a method for determining the amount of ATP present in a first population of cells in a sample, comprising the steps of:
In a preferred embodiment, the liberation step (b) involves lysis of the first population of cells; the first population of cells comprises bacterial cells; and the sample is a biological sample from an animal or a human.
The sample may be a blood sample, a plasma sample, a serum sample, a urine sample, a fecal sample, or a swab from a patient.
At least a fraction of the contaminating ATP may be present in a second population of cells, in which case the reduction step (a) may be preceded by a step of selective liberation of ATP from the second population of cells, wherein the second population of cells are host cells from an animal from which the sample is derived.
In a fifth aspect, the present invention relates to a method for doing pyrosequencing comprising the steps of:
In a sixth aspect, the present invention relates to a use of an apyrase according to the first aspect for degrading contaminating nucleoside triphosphates or nucleoside diphosphates in an analytical method.
The mutated apyrase of the first aspect has improved activity for certain substrates such as pNPP and/or TPP. In a seventh aspect, the present invention provided a use of an apyrase according to the first aspect for dephosphorylating an organic phosphate. The seventh aspect also encompasses a method for catalyzing the dephosphorylation of an organic phosphate, comprising contacting the organic phosphate with the apyrase of the first aspect. The contacting is preferably done in an aqueous solution at a temperature of 4-50° C., more preferably 20-40° C., more preferably 36-38° C. The aqueous solution preferably has a pH of about 5.5-9.5, more preferably 6.5-8.5, yet more preferably about 7.0-8.0, most preferably about 7.5.
The term “comprising” is to be interpreted as including, but not being limited to. All references are hereby incorporated by reference. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other.
The following examples are not to be regarded as limiting.
In order to improve the activity of apyrase and its tolerance to high salt conditions, the inventors mutated the apyrase gene randomly using an error prone PCR method starting from the wild-type Shigella flexneri apyrase DNA sequence (SEQ ID NO: 4) as template. The method incorporates repeated copying of the template at high MgCl2 concentration (5 mM) with Taq polymerase. After every fourth cycle, one fifth of the reaction mixture was transferred to fresh PCR tube and the same process was repeated 22 times to obtain a product that will have randomly mutated nucleotides which can be identified by sequencing. The mutated gene was cloned in pET21a vector. The mutated DNA sequence obtained is presented as SEQ ID NO: 2, containing a total of 31 mutations leading to amino-acid changes (see SEQ ID NO:3 compared to the wild-type amino-acid sequence of SEQ ID NO: 1).
The protein can be divided into 4 regions where mutations have occurred
BL21(DE3) RP codon plus cells transformed with the pET21a-mutant apyrase (SEQ ID NO: 2 as coding sequence) were grown at 37° C. until OD600 nm reaches 0.5-0.6. Expression was induced by adding 0.6 mM IPTG and incubating the culture at 18° C. for 20 hours. Cells were harvested and sonicated for checking induction. Uninduced lysate was taken as control. The induced and uninduced cells were analysed with SDS-PAGE showing effective induction (
As a crude assay for confirming expression of active enzyme, a whole cell ATP degradation activity assay was performed. 100 mM ATP was prepared in 50 mM Tris, pH 7.5. After dissolution, the pH was adjusted to 7.5 using NaOH. Pellets from 1 mL culture of induced cells were taken and washed twice with saline containing 1 mM Calcium chloride. The cells were treated with Lysozyme and EDTA to lyse the cell wall to release the periplasmic contents.
150 μL of 40 mM EDTA and 150 μL of 10 mM ATP was added to the pellet and mixed well, followed by incubation at 37° C. for 30 minutes. Thereafter, 700 μL of AMFAS reagent (equal volumes of Solution A 5% (w/v) ammonium molybdate in 5 N H2SO4 and B) 1% (w/v) ferrous ammonium sulfate in double-distilled water) was added. From the results (
Purification of his-Mutant Apyrase by Affinity Chromatography
Pellet of 500 mL culture was resuspended in 15 mL binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl and 20 mM imidazole). The suspension was subjected to ultrasonication on ice (Amplitude 38%, Pulse on—1 sec, Pulse off—1 sec for 1 minute. The sonication was repeated 6 times). The sonicated suspension was centrifuged at 14, 000 rpm for 20 minutes at 4° C.
To prepare the resin, 1.0 mL of Ni-sepharose (GE Healthcare) resin was washed with 15 mL water and then with 15 mL binding buffer. The sonicated lysate and the washed resin were mixed and incubated for 1 hour at room temperature on a rocker. After this, the lysate was drained out on a column and the beads were washed thoroughly with 200 mL binding buffer. The protein was then eluted in 1.0 mL fractions using the elution buffer (50 mM Tris, pH 7.5, 10% glycerol and 250 mM imidazole). Fractions were loaded on 12% SDS PAGE to confirm the purity of the protein (
Purified protein was quantitated by Bradford method. The yield of purified apyrase from 500 mL culture was 6.3 mg.
A titrated series of ATP solutions from 100 mM to 0.01 mM in 50 mM Tris, pH 7.5 were prepared and incubated with 700 ng of Apyrase (mutant prepared in Example 1 and wild-type Shigella flexneri apyrase as control) for 30 minutes at 37° C. (100 μL reaction). After the incubation, 100 μL AMFAS reagent was added and OD was taken at 630 nm using multimode reader (Biotek instruments). The color formation is proportional to the amount of ATP degraded (dephosphorylated to ADP or AMP). As seen in
Wild type apyrase does not hydrolyse many organic phosphates such as pNPP (Bhargava et al. Current Science 1995 vol 68:3 293-300). To test whether the mutant hydrolyses this substrate a pNPP dephosphorylation assay was performed. The reaction can be described as follows:
Substrate: para Nitro phenol phosphate (pNPP)
Products: para Nitro phenol (PNP, yellow colour, absorbance at 405 nm) and phosphate
Wild-type and mutant apyrase from Example 1 (2 μg each) were incubated with 3 mM pNPP (pNPP was dissolved in 50 mM Tris pH 7.5) in a 100 μL reaction mixture. Control reaction with pNPP lacking WT and mutant apyrase was also included. These were incubated at 37° C. for 15 minutes and absorbance was taken at 405 nm. It is clear from this assay (
To further study the capability of the mutant enzyme to hydrolyse pNPP, the substrate (pNPP) was titrated and incubated with the mutant apyrase. As controls, alkaline phosphatase (known to hydrolyse pNPP) and the wildtype enzyme were also included.
The enzymes (2 μg) were incubated with different concentrations of pNPP (dissolved in 50 mM Tris, pH 7.5) in a 100 μL reaction mixture. For alkaline phosphatase, pNPP was dissolved in carbonate buffer (pH 10.3). Control reaction with pNPP lacking WT, mutant apyrase and alkaline phosphatase was also included. These were incubated at 37° C. for 15 minutes and absorbance was taken at 405 nm. The results are shown in
In conclusion, the mutant apyrase of Example 1 is very potent in catalysing the hydrolysis pNPP while the wild-type apyrase does not have appreciable activity. It can be concluded that the mutations resulted in significantly broadened substrate selectivity, and that the mutant apyrase would be useful in applications requiring the dephosphorylation of organic phosphates.
A screening effort was undertaken to find out which of the mutations generated in the random mutagenesis experiments (Example 1) were behind the observed broadening of substrate specificity (while retaining activity on ATP).
All the mutations from the random mutagenesis were individually introduced into the Shigella flexneri apyrase sequence (SEQ ID NO: 1, termed ApiOne herein) by site directed mutagenesis by PCR using specific primers. However, not all reactions successfully generated PCR products by agarose gel electrophoresis. The successful mutations were transformed into the expression host and proteins were expressed and purified to homogeneity by affinity chromatography. Again, some mutations resulted in poor protein levels as shown by SDS-PAGE (
We screened whether the mutants could hydrolyse organic phosphates. Of the all the mutants, only 3 mutants (F53V, L66V and E77V, termed ApiTwo herein) could hydrolyse pNPP and TPP. Other substrates like 2-Glycerophosphate, Pyridoxal phosphate and phenyl phosphate were not hydrolysed by the mutants (Table 2 and
As seen from
The mutations F53V, L66V and E77V are outside the catalytic site proposed by Babu et. al (Babu, M. M., Kamalakkannan, S., Subrahmanyam, Y. V., Sankaran, K., 2002. Shigella apyrase—a novel variant of bacterial acid phosphatases? FEBS letters 512(1-3), 8-12).
Therefore, it was not surprising to see that these substrates hydrolysed ATP with the same affinity as that of the wild-type enzyme (Table 3). However, it was necessary to understand the structural basis of recognition of both pNPP and TPP, whose hydrolysis resulted due to mutations close to the loop regions outside the pocket. The role of the loop regions (AYYENFG (SEQ ID NO: 5) and TPDKDEKMAIT (SEQ ID NO: 6)) in substrate binding in acid phosphatases is very well known. Their conformational changes during binding play a key role in hydrolysis (Babu et. al, 2002). Computational modelling of ApiOne based on the template structure of acid phosphatase from Escherichia blattae clearly showed that the regions AYYENFG (SEQ ID NO: 5) and TPDKDEKMAIT (SEQ ID NO: 6) are in the loops (
When we mutated glycine 63 residue to arginine (G63R) in the ApiTwo mutant L66V (referred as double mutant from now on), it completely inhibited the ability to hydrolyse pNPP and TPP (
ApiTwo mutants were incubated at 25° C., 55° C. and 70° C. and their ability to hydrolyse different substrates were tested. ApiTwo mutants still retained significant activity (60-70%) for ATP/ADP hydrolysis. However, heating at 55° C. led to significant abrogation of pNPP and TPP hydrolysis (8-10% activity) (Table 4). Cooling the proteins to 25° C. enabled almost 85-95% activity in the case of ATP hydrolysis, but the recovery for pNPP and TPP hydrolysis was extremely poor (20-30% activity) (Table 5). This further reinstates the role of mutated regions (which are close to the crucial loop regions) and the differential mechanism of hydrolysis in ApiTwo mutants.
ApiTwo—an Enzyme with Both Pyrophosphatase and Acid Phosphatase Activity
It is well known there is high degree of homology between the protein sequences of ApiOne and acid phosphatase of Escherichia blattae. The movement of the loop regions are important during substrate binding. A pyrophosphatase like apyrase generally does not hydrolyse monophosphates, whereas acid phosphatases do. Mutation close to the loop regions caused enough conformational changes making ApiTwo behave as an acid phosphatase without compromising its pyrophosphatase activity. The thermal denaturation experiments also indicated the same. The required orientation/conformation of disordered loop regions may not be retained on cooling after denaturation. The probability of attaining the right conformation on cooling is low. Therefore, pNPP and TPP hydrolysis are severely affected after heating and activity is not recovered after cooling. Whereas, the rigid helical regions are partially denatured on heating and the probability to renature is pretty high on cooling. Hence ATP and ADP hydrolysis are not significantly affected on heating the enzyme and the activity is recovered very well after cooling (
KOD polymerase (GENEX) was used for this PCR. The composition of the mix is as follows:
As a control, a tube with all components mentioned were added except KOD polymerase.
To ensure that the template DNA (50 ng) is completely degraded since it will contain the wild type sequence, Dpnl (1 unit) was added to the remaining PCR mixtures and incubated for 1 hour at 37° C.
Transformation of Mutant PCR Mix into E. coli DH5a Strains.
A fraction (20 μL) of the Dpnl digested PCR mix was transformed into E. coli DH5a by Calcium chloride method. Colonies were inoculated in LB media supplemented with 100 μg/mL ampicillin and incubated overnight at 37° C. Plasmids were extracted using standard procedures (Takara).
ApiOne and ApiTwo mutant plasmids were transformed into E. coli RP codon plus by Calcium chloride method. Colonies were inoculated in LB media supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol, and incubated overnight at 37° C. This culture was added to 500 mL of sterile LB medium with 100 μg/mL ampicillin and 100 μg/mL chloramphenicol such that the initial OD600 nm is around 0.08. The culture was grown at 37° C. until OD600 nm reaches 0.5-0.6. Expression of ApiOne and ApiTwo mutants was induced by adding 1 mM IPTG and incubating the culture at 18° C. for 20 hours. Culture not induced by IPTG was used as a control.
Harvested cells of 500 mL culture (induced by IPTG) were resuspended in 30 mL binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl and 10 mM imidazole). The suspension was passed three times through a French press at pressure of 1000 bar.
Before binding to the resin, 1.0 mL of Ni-sepharose (GE Healthcare) resin was washed with 15 mL water. The beads were later washed with 15 mL binding buffer.
Lysates and the washed resin were mixed in 50 mL falcon tube and incubated for 1 hour at room temperature on a rocker.
After this, the lysate was drained out on a column and the beads were washed thoroughly with 200 mL binding buffer. The protein was then eluted in 1.0 mL fractions using the elution buffer (50 mM Tris, pH 7.5, 10% glycerol and 250 mM imidazole).
pNPP Assay
In a volume of 100 μL, 500 ng of pure ApiOne/ApiTwo/Double mutant proteins were incubated with 2.0 mM pNPP diluted in 50 mM Tri-HCl, pH 7.5 for 20 minutes at 37° C. The formation of product. Para-nitro phenol was monitored colorimetrically at 405 nm using multi-mode spectrophotometer (Biotek instruments).
A stock of freshly prepared TPP (50 mM) was prepared before performing every assay. In a volume of 100 μL, 500 ng of pure ApiOne/ApiTwo/Double mutant proteins were incubated with 5.0 mM TPP diluted in 50 mM Tri-HCl, pH 7.5 for 20 minutes at 37° C. Hydrolysis of Thiamine pyrophosphate (TPP) releases Thiamine, which forms a thiochrome fluorophore on addition of hydrogen peroxide and horse radish peroxidase (HRP) in alkaline conditions (NaOH-HRP-H2O2-1M NaOH, 5 U/mL HRP, 50 mM H2O2 mixed in a volumetric ratio of 3:1:1). Fluorescence was measured using multi-mode spectrophotometer (Biotek instruments) with excitation at 368 nm and emission at 450 nm.
In a volume of 100 μL, 500 ng of pure ApiOne or ApiTwo mutants were incubated with 1.0 mM ATP diluted in 50 mM Tri-HCl, pH 7.5 for 20 minutes at 37° C. To this 200 μL of Ammonium Molybdate Ferrous Ammonium Sulphate (AMFAS) reagent was added. AMFAS reagent was prepared by mixing equal volumes of reagent A and reagent B, where reagent A comprises of 5% (w/v) ammonium molybdate in 5 N H2SO4 and reagent B contains 1% (w/v) ferrous ammonium sulphate in double-distilled water. The amount of inorganic phosphate formed (product of ATP/ADP hydrolysis) is colorimetrically measured at 630 nm using multi-mode spectrophotometer (Biotek instruments).
Affinity of Recombinant Proteins Towards ATP, pNPP and TPP
In volume of 100 μL, 500 ng of pure ApiOne or ApiTwo mutants were incubated with different concentrations of substrate (ATP, pNPP and TPP). Respective colorimetric/fluorescence assays (mentioned above). The kinetic curves were plotted and the Michaelis Menten constant (Km) was calculated using GraphPad Prism 5.0 software.
ApiOne and ApiTwo (L66V) mutant proteins were incubated at different temperatures (25° C., 55° C. and 70° C.) for 2 h. Proteins stored at −20° C. were used as control. The ability of these proteins to hydrolyse ATP/ADP (1.0 mM), pNPP (2.0 mM) and TPP (5.0 mM) was studied by performing colorimetric/fluorescence assays described above. The ability of heated proteins to renature was also studied using these assays after cooling proteins to 25° C. for 1 h. Percentage activity was calculated by considering activity of controls (proteins stored at −20° C.) as 100%.
The primary sequence of ApiOne (SEQ ID NO:1) was submitted to the ITASSER server (website: zhanglab.ccmb.med.umich.edu/I-TASSER/) for the threading of the structure. The best template that was chosen by the server was the acid phosphatase (PDB entry: 1D2T) of Escherichia blattae (85% identity and 90% similarity). The structure was similar to the model reported by Babu et. al in 2002. The model was refined by loop refine tool in the Galaxy webserver (website: galaxy.seoklab.org/cgi-bin/submit.cgi?type=LOOP), ensuring that all residues fall in the desirable/allowed regions of the Ramachandran plot. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The material in the ASCII text file, named “NOVI-69001-Sequence-Listing_ST25.txt”, created Sep. 11, 2023, file size 8,192 bytes, is hereby incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21502976 | Mar 2021 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/056739 | 3/15/2022 | WO |
