Heat-labile proteinase

Abstract

To provide a composition containing a proteinase with inducible thermolability, and to provide uses thereof, particularly in the isolation of nucleic acids.SOLUTION: Provided is a composition containing a proteinase or an enzymatically active fragment thereof, the proteinase containing a specific amino acid sequence or an amino acid sequence which is at least about 70% identical to the sequence, wherein i) the concentration of free calcium in the composition is ≤about 80 μM; or ii) the concentration of monovalent salt in the composition is ≥about 20 mM. Under such conditions, the proteinases and the enzymatically active fragment thereof are inducibly thermolabile.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a proteinase having inducible heat-gathering properties, and in particular to its use in the isolation of nucleic acids. [Background technology]

[0002] Proteinases (also known as peptidases, proteases, and protein-degrading enzymes) can hydrolyze peptide bonds in proteins. Proteinases are widely used in a broad range of processes in industrial, biotechnology, and molecular biology research techniques. For example, proteinases are used for digesting unwanted proteins during nucleic acid purification, preparing recombinant antibody fragments, peptide sequencing in proteomics, and protein-digestive digestion of proteins.

[0003] To successfully extract nucleic acids from a sample, cell wall / membrane lysis is necessary. Various physical or chemical methods can be employed, and these can be enhanced by the addition of proteases. Subsequently, membrane lipids are removed by the application of detergents or surfactants, or by osmotic lysis in hypotonic solution. Then, the removal of proteins from the sample using proteinases is considered the best method. Proteinases digest contaminants present in the sample, i.e., unwanted proteins, polypeptides, and peptides, by hydrolysis of peptide bonds. Proteinases also degrade nucleases and other enzymes that may be present in the sample and could potentially degrade nucleic acids.

[0004] Protein removal is particularly important during nucleic acid purification in the preparation of nucleic acid samples for amplification reactions (e.g., PCR and RT-PCR). In cells, nucleic acids generally exist bound to proteins. For example, genomic DNA in eukaryotic cells is bound to histones, thereby allowing the DNA to be densely packaged in chromatin. Many molecular biology techniques, such as PCR, require naked DNA, i.e., DNA not bound to histones, because the dense packaging of DNA in chromatin inhibits enzymes that interact with DNA, such as polymerases and nucleases, from approaching the nucleic acid.

[0005] Nucleic acid purification is a multi-step process that involves time, cost, and sample loss. Sample loss is particularly undesirable when the initial amount of nucleic acid in the sample is small, for example, when isolated from hundreds of cells or from biopsy material aspirated with a fine needle or from liquid biopsy material.

[0006] The most commonly used proteolytic enzyme in nucleic acid purification is proteinase K (EC 3.4.21.64). This enzyme was first discovered in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is a nonspecific serine endopeptidase that catalyzes the cleavage of peptide bonds on the carboxyl side of aromatic, aliphatic, or hydrophobic amino acid residues. Due to its broad specificity, proteinase K can be useful in digesting unwanted proteins in a sample. Proteinase K also rapidly inactivates nucleases that can degrade nucleic acids present in the sample. Proteinase K is active in the presence of other chemicals used in DNA extraction processes that denature proteins, such as SDS and chelating agents like urea and EDTA, sulfhydryl reagents, trypsin inhibitors, and chymotrypsin inhibitors. Proteinase K has optimal activity in the range of 50–65°C, generally around 55°C.

[0007] When proteinase K is used for sample purification, it must be inactivated or removed before adding downstream proteins / enzymes, such as polymerase and reverse transcriptase. Without such inactivation or removal, the proteinase will degrade downstream proteins / enzymes due to its nonspecific activity.

[0008] Proteinase K can be removed from a sample, for example, by phenol extraction or isodense ultracentrifugation of CsCl. Alternatively, the sample volume can be increased to weaken the proteinase K activity within it. However, physically removing the enzyme from a sample carries the risk of contamination and loss of the desired product. Dilution is often not ideal, especially when the sample size is small.

[0009] Procedures for inactivating proteinase K vary, but generally involve heating to high temperatures. However, in many cases, the heat required to inactivate the protease results in the decomposition of one or more desired products in the sample. Procedures for inactivating proteinase K sometimes involve heating to 75°C for 5 minutes (BioRad procedure), heating to 95°C for 10 minutes (New England BioLabs procedure), or heating to 70°C for 15 minutes (Qiagen procedure), sometimes in combination with reagents. The use of such high temperatures is relatively harsh on the sample of interest.

[0010] Therefore, there is a need for an alternative proteolysis method that can shorten the workflow without losing desired material from the sample, particularly during nucleic acid purification procedures, and does not involve harsh proteinase inactivation conditions.

[0011] Proteinase X (also known as "Serratia peptidase" and "SPRK") is a proteinase K-like proteinase isolated from the Serratia species. Studies on proteinase X (Non-Patent Literature 1) have confirmed that it possesses high thermal stability, similar to proteinase K, and further confirmed that proteinase X actually has a higher optimal temperature (70°C) than proteinase K (55°C). Larsen et al. showed that proteinase X retains sufficient enzymatic activity after heating at 50°C for 30 minutes and retains much higher activity than proteinase K after heating at 50°C in the presence of various concentrations of SDS (a surfactant commonly used for the isolation of nucleic acids from samples). According to Larsen et al., proteinase X does not exhibit the typical thermotonicity characteristic of enzymes isolated from cold-adapted organisms. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Larsen et al.,(2006)FEBS Journal 273:47-60 [Overview of the Initiative] [Effects of the Invention]

[0013] The inventors have, surprisingly, revealed for the first time that the heat-gaining properties of proteinase X are induced when proteinase is present in a composition with a low concentration of free calcium ions. The inventors have confirmed that it is not necessary to remove calcium ions that can bind to proteinase and contribute to its stability and structure, for example, using EDTA. On the contrary, the mere absence or low concentration of free calcium ions in the composition is, surprisingly, sufficient to induce the heat-gaining properties of proteinase X. The inventors have also, surprisingly, revealed for the first time that the heat-gaining properties of proteinase X are induced when proteinase is present in a composition having a specific monovalent salt concentration.

[0014] This inducible heat-gathering property is unexpected and is not observed in proteinase K, a standard proteinase used in molecular biology applications. Our findings enable the advantageous use of proteinase X, which possesses inducible heat-gathering properties, in a wide range of molecular biology applications. [Brief explanation of the drawing]

[0015] Herein, the present invention will be described by non-limiting embodiments with reference to the following drawings.

[0016] [Figure 1] This figure shows the proteinase activity of proteinases X and K at various temperatures. Activity is expressed as % activity relative to the maximum activity observed at 65°C using 10 mM free calcium under standard analytical conditions. [Figure 2] This figure shows the activity of proteinase X at various temperatures using 0 μM, 5 μM, or 10 mM free calcium in the analytical buffer. The results are shown as relative (%) to the standard analytical condition of 10 mM free calcium at each temperature. [Figure 3] This figure shows the activity of proteinase K at various temperatures using 0 μM, 5 μM, or 10 mM free calcium in the analytical buffer. The results are shown as relative (%) to the standard analytical condition of 10 mM free calcium at each temperature. [Figure 4] This figure shows the degree of inactivation of proteinase X after heating at 60°C for 15 and 30 minutes in the presence of various free calcium concentrations. Activity is shown as % residual activity compared to the control (maintained on ice, no heating step). [Figure 5] This figure shows the degree of inactivation of proteinase K after heating at 60°C for 15 and 30 minutes in the presence of various free calcium concentrations. Activity is shown as % residual activity compared to the control (maintained on ice, no heating step). [Figure 6]Figure showing the degree of inactivation of Proteinase X and Proteinase K after heating at 60 °C for 15 and 30 minutes in the presence of various free calcium concentrations. Activity is shown as % activity relative to the maximum activity observed for the same proteinase in the same buffer maintained on ice without heat treatment (containing 10 mM CaCl2). [Figure 7] Figure showing the effect of NaCl concentration on the thermolability profile of Proteinase X and Proteinase K. Incubation for 30 minutes at the indicated temperatures in the absence of free calcium and in the presence of 50 mM or 300 mM NaCl. Activity is shown as % activity relative to the maximum activity observed for the same proteinase in the same buffer maintained on ice without heat treatment (containing 10 mM CaCl2). [Figure 8] Figure showing the inactivation of Proteinase X at 50 and 60 °C in the presence of various concentrations of NaCl. Activity is shown as % activity relative to the maximum activity observed for Proteinase X in the same buffer maintained on ice without heat treatment (containing 0 M NaCl, 0.03 mM CaCl2). [Figure 9] Figure showing the inactivation of Proteinase X at 60 °C in the presence of various concentrations of NaCl. Activity is shown as % activity relative to the maximum activity observed for Proteinase X in the same buffer maintained on ice without heat treatment (containing 0 M NaCl, 30 μM CaCl2).

Mode for Carrying Out the Invention

[0017] Thus, in one aspect, the present invention provides a composition comprising a proteinase or an enzymatically active fragment thereof. The proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, i) the free calcium concentration in the composition is ≦ about 80 μM, or ii) the concentration of monovalent salt in the composition is ≧ about 20 mM.

[0018] Preferably, the free calcium concentration in the composition is ≤ about 80 μM, and the monovalent salt concentration in the composition is ≥ about 20 mM.

[0019] The present invention relates to a proteinase comprising the sequence of SEQ ID NO: 1, and a proteinase comprising a sequence that is at least about 70% identical to SEQ ID NO: 1. A proteinase comprising a sequence that is at least about 70% identical to SEQ ID NO: 1 is referred to herein as a "mutant of SEQ ID NO: 1," a "mutant proteinase," or simply a "mutant." By definition, the mutants of SEQ ID NO: 1 of the present invention are also proteinases according to the present invention, i.e., they possess proteinase activity. The term "proteinase of the present invention" herein refers to the proteinase of SEQ ID NO: 1 and the mutants of the present invention described herein. Wherever herein the term "proteinase" (or "mutant proteinase") refers to its enzymatically active fragment unless otherwise specified.

[0020] Preferably, the composition is a solution, preferably an aqueous solution. In this specification, the term "solution" means a liquid mixture in which one or more minor components (solutes) are uniformly distributed within a major component (solvent). Generally, the minor components (solutes) of a solution are soluble in the major component (solvent). However, in this specification, the term "solution" also includes mixtures in which the minor components (solutes) are not soluble in the major component (solvent). That is, in this specification, the term "solution" also includes mixtures, i.e., dispersions, in which the major component is in the liquid phase and the minor components are particles insoluble in the liquid phase. Preferably, the major component, i.e., the solvent, i.e., the liquid phase, is water. Preferably, the solution contains water. The solution of the present invention contains at least the proteinase of the present invention or an enzymatically active fragment thereof as a minor component.

[0021] In one preferred embodiment, the solution of the present invention is a reagent applied to a sample containing one or more polypeptides. Such a reagent is applied to the sample so that the proteinase in the reagent digests the one or more polypeptides present in the sample. Preferably, the sample contains multiple polypeptides. In this embodiment, the solution preferably contains the proteinase of the present invention or an enzymatically active fragment thereof, but does not contain any further enzymes.

[0022] Preferably, the composition also includes a buffering agent. Suitable buffering agents are well known in the art, and any such buffering agent can be used. It will be within the capabilities of those skilled in the art to determine suitable buffering agents and the range in which they are included for their intended purpose. Preferably, the buffering agent has a buffering range of pH 6.5 to 9.5, preferably pH 6.8 to 9.2, more preferably pH 7 to 9, more preferably pH 7.5 to 8.5, and more preferably about pH 8. Preferably, the buffering agent is Tris or HEPES. Preferably, the buffering agent is present in the composition at a concentration of 1 to 250 mM, more preferably 10 to 200 mM, more preferably 20 to 150 mM, and more preferably 25 to 100 mM.

[0023] If present, Tris-HCl is preferably present at a concentration of 25-200 mM, more preferably 50-150 mM, and more preferably about 100 mM. If present, HEPES is preferably present at a concentration of 5-50 mM, more preferably 10-40 mM, more preferably 20-30 mM, and more preferably about 25 mM.

[0024] Preferably, the compositions and samples of the present invention have a pH of 6.5 to 9.5, preferably 6.8 to 9.2, more preferably 7 to 9, more preferably 7.5 to 8.5, and more preferably about 8.0.

[0025] The compositions and samples of the present invention may further contain DMSO. If present, DMSO is preferably present at a concentration of 0.1 to 5% w / w, more preferably 0.5 to 2.5% DMSO, and more preferably about 1% DMSO.

[0026] The term "sample" refers to any composition comprising one or more polypeptides other than the proteinase of the present invention.

[0027] In a further embodiment, the present invention provides a sample comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof. The proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0028] Preferably, the free calcium concentration in the sample is ≤ approximately 80 μM, and the monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0029] Samples that do not contain the proteinase or enzymatically active fragment described herein are not embodiments of the present invention. The term "sample of the present invention" herein refers only to samples that truly contain the proteinase or enzymatically active fragment described herein.

[0030] The following considerations of preferred optional characteristics and embodiments of the "sample" apply to both the sample of the present invention and the sample that is not part of the present invention but to which one composition of the present invention can be applied.

[0031] The proteinase of the present invention is useful for methods of purifying, i.e., isolating, i.e., extracting, a biomolecule of interest from a sample containing contaminating, i.e., unwanted, polypeptides. Therefore, in one preferred embodiment, the sample comprises one or more contaminating polypeptides and one or more biomolecules of interest. In this specification, the term “contaminating polypeptide” is synonymous with “unwanted polypeptide” and refers to any polypeptide in the sample other than the proteinase of the present invention and any of the target polypeptides. That is, the contaminating polypeptides are digested by the proteinase of the present invention in order to purify or modify one or more biomolecules of interest.

[0032] The proteinase of the present invention is also useful in a method for releasing a target biomolecule from a molecule, preferably a polypeptide, to which it is bound via one or more peptide bonds, by hydrolyzing one or more of the peptide bonds. Therefore, in a preferred embodiment, the sample contains a target biomolecule bound to a molecule, preferably a polypeptide, via one or more peptide bonds. One or more peptide bonds can be cleaved by the proteinase of the present invention or an enzymatically active fragment thereof.

[0033] In this specification, the term “target biomolecule” refers to any biomolecule in a sample, including one or more contaminating polypeptides, for which purification of the biomolecule from the sample is desired, or release of the biomolecule from molecules bound to it via one or more peptide bonds is desired. Preferably, the target biomolecule is a nucleic acid molecule, preferably a DNA or RNA molecule. Alternatively, the target biomolecule is a polypeptide itself. The target biomolecule is neither a proteinase nor an enzymatically active fragment thereof useful for the present invention.

[0034] Preferably, the sample contains cellular material. Preferably, the sample contains crude cell extract. Preferably, the sample contains partially purified cell extract. Preferably, the sample contains a cell population. The cells in the sample may be intact or lysed, preferably lysed. Preferably, the sample contains a tissue sample or one or more body fluids. Preferably, the sample is fine-needle biopsy material. Preferably, the sample contains an inclusion virus. Proteinases can be used to digest the protein capsules of the virus to release the RNA / DNA in the protein capsules for identification, quantification, and / or amplification.

[0035] Preferably, the sample of the present invention has a volume of ≥10 μl. Preferably, the sample of the present invention has a volume of ≤1000 μl, more preferably ≤500 μl, more preferably ≤300 μl, more preferably ≤250 μl, more preferably ≤200 μl, more preferably ≤150 μl, more preferably ≤100 μl, more preferably ≤75 μl, and more preferably ≤50 μl. Alternatively, the sample is a microfluidic sample. Preferably, the microfluidic sample of the present invention has a volume of ≥0.01 μl. Preferably, the microfluidic sample of the present invention has a volume of ≤10 μl, preferably ≤5 μl, more preferably ≤1 μl, more preferably ≤0.5 μl, and more preferably ≤0.1 μl.

[0036] In this specification, the term “polypeptide” refers to any molecule containing three or more amino acids linked via peptide bonds, including proteins, polypeptides, peptides, oligopeptides, and tripeptides, that can be hydrolyzed by the proteinase of the present invention. Proteinase X is known to hydrolyze peptides with a length of three amino acids and shorter peptides. The terms “protein,” “polypeptide,” and “peptide” are used herein without distinction, and each use is explicitly intended to refer to any of proteins, polypeptides, and peptides. Therefore, polypeptides present in the samples described herein are substrates for the proteinase or its enzymatically active fragments of the present invention. It is obvious that the proteinase and its enzymatically active fragments are polypeptides themselves. However, in this specification, the term “polypeptide” explicitly excludes the proteinase and its enzymatically active fragments that are useful for the present invention.

[0037] The terms “digest,” “hydrolyze,” “decompose,” and “cleave” are used without distinction herein and refer to the hydrolysis of peptide bonds within polypeptides in a sample. Digestion may be partial or complete. Proteinases useful in the present invention are nonspecific and, given sufficient time, completely digest proteins in a sample under conditions that allow for enzymatic function.

[0038] The compositions and samples of the present invention comprise a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1.

[0039] The amino acid sequence of Sequence ID No. 1 is as follows: ADQPSPTWGIDRIDQRNLPLDNNYHTDYDGSGVTAFVIDTGVVLNTHNEFGGRASSGYDFIDNDYDATDCNGHGTHVAGTIGGSTYGVAKNVNVVGVRVLNCSGSGSNSGVIAGINWVKNNASGPAVANMSLGGGASQATDDAVNAAVAAGITFVVAAGNDNSNACNYSPARAADAITVGSTTSNDSRSSFSNYGTCLDIYAPGSSITSSWYTSNSATNTISGTSMASPHVAGVAALYLDENPNLSPAQVTNLLKTRATADKVTDAKTGSPNKLLFSLANDD

[0040] The proteinase of sequence number 1 was identified by Larsen et al., (2006) FEBS. The present invention is based on the amino acid sequence of proteinase X derived from a marine Serratia species isolated in northern Norway, as described in Journal 273:47-60. Proteinase X is a proteinase K-like proteinase [EC3.4.21]. The terms “proteinase X,” “protease X,” “ProtX,” “PRX,” “peptidase X,” “Serratia peptidase,” and “SPRK” are used without distinction herein. Preferably, the proteinase of the present invention is derived from Serratia proteamaculans. Proteinase X is a serine peptidase.

[0041] The gene encoding proteinase X has 1890 base pairs (SEQ ID NO: 2) and encodes a precursor protein with 629 amino acids (65.5 kDa, SEQ ID NO: 3).

[0042] Sequence ID 2:

[0043] Sequence ID 3:MHKKHLIAVAVATGLAYFPVNANEYQATMVNVPQSKAIKDTYIVVFNTPSVLNLSNNNTIAEFAVQQAESLVNQYDVRVMKNFGNVLNGVLINASAQQVKALLKDPNVKYVEQDQVMSVTPMMEANADQPSPTWGIDRIDQRNLPLDNNYHTDYDGSGVTAFVIDTGVLNTHNEFGGRASSGYDFIDNDYDATDCNGHGTHVAGTIGGSTYGVAKNVNVVGVRVLNCSGSGSNSGVIAGINWVKNNASGPAVANMSLGGGASQATDDAVNAAVAAGITFVVAAGNDNSNACNYSPARAADAITVGSTTSND SRSSFSNYGTCLDIYAPGSSITSSWYTSNSATNTISGTSMASPHVAGVAALYLDENPNLSPAQVTNLLKTRATADKVTDAKTGSPNKLLFSLANDDGGCGNDCPVDETQLQNNVGIAISGATGSATYYYIDVPANAASLGINLAGGSGDADIYVSQGQK PTTTSYQCRPYQNGNNESCNFTAPTAPTAGRWYVMVQGYSNYANAQLTASYNLNGGGNCTDANCLSNGVPVTNLSGRTGTEALYKIVVPANSQLSITTSGGTGDVDLYVKAGTVPTTTSYDCRPYKNGNNESCSITVTQAGTYHVMLRGYANYSSVQLSASY

[0044] The proteinase of Sequence ID No. 3 consists of a 126-residue N-terminal prepro sequence, a 278-residue catalytic domain, and two C-terminal domains (repetitive sequences) totaling 225 residues.

[0045] The enzyme is recombinantly expressed in Pichia pastoris as an active peptidase of approximately 40.2 kDa, consisting of 385 amino acids, having the sequence shown in Sequence ID No. 4 below. NEYQATMVNVPQSKAIKDTYIVVFNTPSVLNLSNNNNTIAEFAVQQAESLVNQYDVRVMKNFGNVLNGVLINASAQQVKALLKDPNVKYVEQDQVMSVTPMMEANADQPSPTWGIDRIDQRNLPLDNNYHTDYDGSGVTAFVIDTGVLNTHNEFGGRASSGYDFIDNDYDATDCNGHGTHVAGTIGGSTYGVA KNVNVVGVRVLNCSGSGSNSGVIAGINWVKNNASGPAVANMSLGGGASQATDDAVNAAVAAGITFVVAAGNDNSNACNYSPARAADAITVGSTTSNDSRSSFSNYGTCLDIYAPGSSITSSWYTSNSATNTISGTSMASPHVAGVAALYLDENPNLSPAQVTNLLKTRATADKVTDAKTGSPNKLLFSLANDD

[0046] The proteinase expressed by SEQ ID NO: 4 has both C-terminal domains excluded except for the first three residues of the first C-terminal domain, and the first 22 residues of the N-terminal preprodomain of SEQ ID NO: 3. This form of protein can be purified.

[0047] Recombinant expression of proteinase X in E. coli is described in Larsen et al., (2006) FEBS Journal 273:47-60.

[0048] Following expression, the enzyme is converted by autolysis into a mature protein of approximately 34 kDa and 281 residues. This protein contains a catalytic domain and three C-terminal amino acid residues and retains sufficient catalytic activity (SEQ ID NO: 1).

[0049] Residues 1-104 of SEQ ID NO: 4 correspond to residues 23-126 of SEQ ID NO: 3. Residues 1-281 of SEQ ID NO: 1 correspond to residues 105-385 of SEQ ID NO: 4 and to residues 127-407 of SEQ ID NO: 3.

[0050] Accordingly, in each and all aspects and embodiments of the present invention, the proteinase may include the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4, preferably SEQ ID NO: 1. SEQ ID NOs: 3 and 4 are precursors / immature forms of the proteinase of SEQ ID NO: 1. Those skilled in the art will understand that the use or presence of the proteinase of SEQ ID NO: 3 or 4 leads to the use or presence of the proteinase of SEQ ID NO: 1 for further autodegradation processing. For simplicity, anywhere herein the notation SEQ ID NO: 1 is expressly intended to also represent SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0051] The compositions and samples of the present invention comprise a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1.

[0052] "At least about 70%" means that the sequence homology may be at least 69%, 69.5%, or 69.9%.

[0053] In a preferred embodiment, the proteinase of the present invention comprises or consists of an amino acid sequence that is at least 71%, at least 72%, at least 73%, at least 74%, or at least 75%, preferably at least 80%, 85%, 90%, or 95%, for example, at least 98%, 99%, or 99.5%, identical to SEQ ID NO: 1.

[0054] The sequence homology ratio according to the present invention can be calculated using any of the widely available algorithms, for example, using the ClustalW2 Multiple Sequence Alignment program (http: / / www.ebi.ac.uk / Tools / clustalW2) and initial settings parameters (DNA Gap Open Penalty=15.0; DNA Gap Extension Penalty=6.66; DNA Matrix=Identity; Protein Gap Open Penalty=10.0; Protein Gap Extension Penalty=0.2; Protein matrix=Gonnet; Protein / DNA ENDGAP=-1; Protein / DNA GAPDIST=4).

[0055] The homology ratio is preferably determined between the N-terminal Ala residue (residue 1) and the C-terminal Asp residue (residue 281) of SEQ ID NO: 1 after the alignment of SEQ ID NO: 1 and the mutant sequence.

[0056] A variant of Sequence ID No. 1 includes an amino acid sequence in which one or more amino acids of Sequence ID No. 1 are conserved substitutions, or are substituted with modified versions of the one or more amino acids or non-native amino acids, such as D isomers of the one or more amino acids. Preferably, such substitutions and modifications are silent substitutions and modifications in that the modified exonuclease of the present invention has the same enzymatic and inactivating properties as the unmodified exonuclease.

[0057] In some embodiments, the proteinase of the present invention comprises (or comprises) an amino acid sequence having one or more amino acid changes (additions, substitutions, insertions, or deletions) compared to SEQ ID NO: 1. Such sequences may preferably contain up to 10 modified amino acids, e.g., only 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10; preferably up to 5, e.g., only 1, 2, 3, 4, or 5; preferably 1, 2, or 3; more preferably 1 or 2 modified amino acids. Preferably, the number of modifications is determined between the N-terminal Ala residue (residue 1) and the C-terminal Asp residue (residue 281) of SEQ ID NO: 1 after alignment of SEQ ID NO: 1 and the variant sequence.

[0058] Preferably, the modification is silent, in that the modified proteinase of the present invention has the same enzymatic and inactivating properties as the invariant form. Substitutions may be by conserved or non-conserved amino acids. Preferably, the modification is a conserved amino acid substitution. The modification may be a substitution by a modified version of one or more amino acids of SEQ ID NO: 1, or a substitution by a non-natural amino acid, such as a D isomer of an amino acid.

[0059] Proteinases containing an amino acid sequence at least about 70% identical to Sequence ID No. 1 can be obtained from prokaryotes found in the cold water niche. “Prokaryotes” means any organism without a cell nucleus, i.e., any organism originating from the realm of bacteria and archaea. Preferably, the organism is a bacterium. Preferably, the organism is not a eukaryote, for example, an organism classified in the taxonomic kingdoms of Animalia, Plantae, Fungi, or Protistia. More preferably, the organism is selected from the genera Shewanella, Halomonas, Vibrio, Cyclomonas, Moritera, and Serratia, preferably Serratia.

[0060] In some embodiments, the compositions and samples of the present invention comprise a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 3 or 4. The portion of this specification that refers to SEQ ID NO: 1 also applies to SEQ ID NO: 3 and SEQ ID NO: 4 with the necessary modifications.

[0061] Preferably, the proteinase of the present invention consists of the amino acid sequence of SEQ ID NO: 1, 3, or 4, or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, 3, or 4.

[0062] Preferably, the proteinase of the present invention comprises the amino acid sequence of SEQ ID NO: 1, 3, or 4. In some embodiments, the proteinase of the present invention consists of the amino acid sequence of SEQ ID NO: 1, 3, or 4.

[0063] Preferably, the proteinase of the present invention consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1.

[0064] Preferably, the proteinase of the present invention comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the proteinase of the present invention consists of the amino acid sequence of SEQ ID NO: 1.

[0065] The proteinase of the present invention may include further amino acid N-terminuses or C-terminuses of the sequence of SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, these further amino acids are identical to the amino acids at their respective positions found at the N-terminus or C-terminus of the sequence of SEQ ID NO: 1 within the 629 amino acid sequence of SEQ ID NO: 3. Preferably, the proteinase includes amino acids 1 to 50, more preferably 1 to 40, more preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 at the N-terminus and / or C-terminus of the sequence of SEQ ID NO: 1 or SEQ ID NO: 4, preferably the N and / or C-terminal amino acids are identical to the respective amino acids found at the N and / or C-terminus of the sequence of SEQ ID NO: 1 or SEQ ID NO: 4 within the sequence of SEQ ID NO: 3.

[0066] The proteinases present in the solution or sample of the present invention can be modified, for example, in the form of fusion proteins in which they are directly or indirectly bound to further peptides at the N-terminus and / or C-terminus via a peptide linker sequence. Preferably, the additional N-terminal peptide, and together with the linker sequence if present, comprises sequences 1-50, more preferably 1-40, more preferably 1-30, more preferably 1-20, more preferably 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid lengths. Preferably, the additional C-terminal peptide, and together with the linker sequence if present, comprises sequences 1-50, more preferably 1-40, more preferably 1-30, more preferably 1-20, more preferably 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid lengths.

[0067] Accordingly, in one embodiment, the present invention provides a composition comprising a proteinase or an enzymatically active fragment thereof. The proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The free calcium concentration in the composition is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the composition is ≥ approximately 20 mM, The proteinase or enzymatically active fragment thereof further comprises a further peptide sequence which is the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which is at least about 70% identical to SEQ ID NO: 1.

[0068] Further one or more peptide sequences may be useful in processes of secretion, isolation, solubilization and / or purification or identification of proteinases, or for tethering proteinases to a solid support. Suitable peptide sequences are well known in the art, and any such sequences can be used. Suitable N and C-terminal sequences include, for example, histidine tags, preferably containing 1 to 20, more preferably 5 to 15, more preferably 6, 7, 8, 9, 10, 11, or 12 histidine residues, most preferably 6 or 12 histidine residues.

[0069] Therefore, the proteinase of the present invention can be a modified proteinase. Further modifications include the introduction of small chemical groups to available atoms of the polypeptide, for example, protecting groups to the N and C termini or to the R groups of non-essential amino acid residues within the polypeptide. In another embodiment, the proteinase of the present invention can be provided immobilized on a solid support, such as particles, pellets, beads, sheets, gels, filters, membranes, fibers, capillaries, tips, microtiter strips, slides, tubes, plates, wells, etc. Preferably, the support is magnetic (preferably paramagnetic or superparamagnetic), for example, magnetic particles, for example, magnetic beads and pellets. Further modifications include dimers or trimers of the proteinase of the invention. Such entities may have a homogeneous or heterogeneous monomer composition.

[0070] Preferably, such modifications are silent modifications in that the modified proteinase of the present invention has the same enzymatic and inactivating properties as the unmodified proteinase.

[0071] Enzymatically active fragments of the proteinase and mutant proteinase of the present invention are also provided. The enzymatically active fragment is a fragment having proteinase activity. The enzymatically active fragment may contain at least 225, preferably at least 235, preferably at least 250, more preferably at least 260, at least 270, at least 271, 272, 273 or 274, and more preferably at least 275, 276, 277, 278, 279 or 280 amino acids of the sequence of Sequence ID No. 1.

[0072] Preferably, the enzymatically active fragment of the present invention comprises an N-terminal truncation of 5 or fewer amino acids in the sequence of SEQ ID NO: 1, preferably 4, 3, 2, or 1 or fewer amino acids. Alternatively, or in addition to the above, the enzymatically active fragment of the present invention preferably comprises a C-terminal truncation of 30 or fewer amino acids in the sequence of SEQ ID NO: 1, preferably 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acids.

[0073] Alternatively, the length of the enzyme fragment of the present invention is preferably at least 80%, preferably at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the length of the amino acid sequence that is at least 70% identical to SEQ ID NO: 1.

[0074] The enzyme fragment of the present invention is itself preferably at least 70%, preferably at least 80%, at least 85%, or at least 90%, more preferably at least 95% (e.g., at least 98%, 99%, or 99.5%), or 100% identical to the corresponding portion of Sequence ID No. 1. The method for determining the homology ratio is described above.

[0075] Throughout this application, the term "proteinase" in this invention also refers to its enzymatically active fragment, unless otherwise specified.

[0076] Proteinases (also called peptidases or proteases) are enzymes that perform protein catabolism by hydrolysis of peptide bonds, i.e., protein degradation. Therefore, the proteinase according to the present invention is an enzyme that has proteinase activity, i.e., protease activity, i.e., peptidase activity. The mutants and enzymatically active fragments of the present invention also have proteinase activity.

[0077] The proteinases of the present invention, which are mutants or modified versions of SEQ ID NO: 1, exhibit at least 70%, preferably at least 80%, more preferably at least 85%, at least 90%, or at least 95%, even more preferably at least 99%, and most preferably at least 100% of the proteinase activity of SEQ ID NO: 1.

[0078] The enzymatically active fragment of the present invention exhibits at least 70%, preferably at least 80%, more preferably at least 85%, at least 90%, or at least 95%, even more preferably at least 99%, and most preferably at least 100% of the proteinase activity of SEQ ID NO: 1.

[0079] Appropriate analyses for analyzing proteinase activity are known in the art, and any such analysis can be used to measure the proteinase activity of a particular polypeptide. Therefore, such analyses can be used to measure the proteinase activity of the proteinase, mutant proteinase, and enzymatically active fragments thereof of the present invention.

[0080] A preferred analysis involves enzymatic cleavage of the substrate into a product detectable by a spectrophotometer, preferably at 410 nm (ε=8800M). -1 .cm -1This includes the analysis of the cleavage of Suc-Ala-Ala-Pro-Phe-pNA to 4-nitroaniline, which can be analyzed by measuring the increase in absorbance in ). Identifying suitable substrates and apparatus for producing and detecting such absorbance is within the capabilities of those skilled in the art. Preferably, the absorbance is detected using a spectrophotometer or a microplate reader. Many spectrophotometers utilize cuvettes with a volume of 1000 μl. Many microplate readers utilize wells with a volume of 250 μl.

[0081] Those skilled in the art can easily determine the appropriate incubation temperature and analysis time for these purposes. Those skilled in the art will know that the temperature at which the analysis is performed should be such that it does not result in the inactivation of the proteinase of the present invention, for example, below 40°C, preferably 25°C. The appropriate analysis time can be 30 seconds to 5 minutes, for example, 2 minutes.

[0082] Those skilled in the art will also know that the appropriate concentration of the enzyme to be included in the analysis depends on the detection range of the spectrophotometer used. Those skilled in the art will also know that a dilution step may be necessary before performing the analysis to bring the concentration to a level detectable by the spectrophotometer and the enzyme activity level in the analyte sample. Activities of 10–50 mU / mL, preferably 13–26 mU / mL, can be used, which is equivalent to 0.2–0.4 μg / mL for proteinase X of Sequence ID No. 1 used in this embodiment.

[0083] Those skilled in the art will be able to readily formulate the remainder of the analytical mixture for their intended purposes. The analytical mixture may contain a pH buffer, preferably Tris-HCl, pH 8, as described elsewhere herein.

[0084] The analytical mixture preferably contains excess calcium (e.g., ≥2 mM, preferably about 10 mM) to avoid low calcium conditions that could further inactivate the proteinase. NaCl may be present at a low concentration (e.g., ≤15 mM) so as not to induce the heat-extracting properties of the proteinase induced by the salt. Furthermore, those skilled in the art can easily dilute the solution to provide the required concentrations of the components as needed.

[0085] Another component, such as DMSO, can be added to the reaction mixture as desired, or another component, such as DMSO, can be present because it was added earlier in the workflow and is acceptable for activity analysis.

[0086] The preferred analysis for measuring proteinase activity is Analysis A (used in the examples herein). Analysis A is performed in a 1000 μl or 250 μl cuvette. 13 or 26 mU / mL proteinase (corresponding to 0.2 or 0.4 μg of proteinase X of SEQ ID NO: 1 used in this example) 1mM substrate Suc-Ala-Ala-Pro-Phe-pNA ≤15 mM NaCl (e.g., 12 mM NaCl or 4 mM NaCl) 0.1 mM Tris-HCl pH 8, 10 mM CaCl2, and Incubate 1% DMSO (optional), and 410nm (ε=8800M) -1 .cm -1 This includes analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at ) using a spectrophotometer (e.g., Ultraspec 2000, Pharmacia Biotec, Sweden) for 2 minutes at 25°C or 37°C. One unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a temperature below 40°C, preferably 25°C or 37°C.

[0087] Those skilled in the art can devise alternative methods to measure proteinase activity.

[0088] Using any suitable analysis, the relative activity of an enzymatically active fragment, variant, or modified version of the proteinase of the present invention can be measured by comparing it to the activity of the proteinase of SEQ ID NO: 1. Those skilled in the art will know that the same techniques and conditions used to evaluate the activity of the proteinase of SEQ ID NO: 1 should also be used to evaluate the activity of the fragment, variant, or modified version. Preferably, the analysis used is Analysis A described above.

[0089] According to the preferred analysis A described above, proteinase X has a specific activity of approximately 65 U / mg (65.2 U / mg). Preferably, the mutants, enzyme fragments, and modifications of the present invention have a specific activity of 40 to 100 U / mg, more preferably 50 to 80 U / mg, more preferably 60 to 70 U / mg, more preferably 62.5 to 67.5 U / mg, preferably about 65 U / mg or 65.2 U / mg, as measured by analysis A above. Preferably, the mutant proteinases and enzyme fragments of the present invention have the same specific activity as proteinase X of SEQ ID NO: 1, as measured by analysis A above.

[0090] The compositions and samples of the present invention preferably contain ≤80 μM free calcium. In this specification, the term "free calcium" refers exclusively to free, i.e., unbound, calcium ions in the compositions and samples of the present invention, i.e., calcium ions that are not bound to any proteins or other components present in the compositions and samples of the present invention, i.e., free calcium. The overall notation of calcium concentration in the compositions and solutions of the present invention refers to the free calcium concentration.

[0091] Proteinase X (SEQ ID NO: 1) contains a calcium-binding site formed by residues Asp11, Asp14, Gln15, Asp21, and Asn23. 2+The ions coordinate to the carboxyl oxygen atoms of the side chains of Asp11, Asp14, and Asp21, the amide oxygen atom of Gln15, and the carbonyl oxygen atoms of Asp11 and Asn23. This bound calcium ion is necessary for correct protein folding. Therefore, the proteinases useful in this invention naturally contain this bound calcium ion. The bound calcium ion is also present when the proteinases useful in this invention are recombinantly produced. Any environment used in the production of proteinases necessarily contains calcium. This means that the bound ion is present in the folded proteinase structure. These bound calcium ions are not "free calcium" in the terms used herein.

[0092] Those skilled in the art who prepare the compositions or samples of the present invention can directly and reliably obtain the required calcium concentration simply by selecting appropriate solutes and solvents to be added to the solution or sample. Removing free calcium from the solution by dialysis against a large volume of calcium-free buffer, for example, 500 to 10000, preferably 5000 volumes of calcium-free buffer, is within the capabilities of those skilled in the art. A suitable buffer will be obvious to those skilled in the art and will preferably be a buffer described elsewhere herein, for example, 10 mM Tris-HCl, pH 7 to 9. Preferably, the buffer further contains glycerol.

[0093] The concentration of free calcium in any given solution or sample can also be easily measured by those skilled in the art. Standard methods for measuring calcium are well known in the art, and include, for example, titration using a standard solution of EDTA.

[0094] Bound calcium ions can be removed from proteinases by applying a strong calcium chelating agent, such as EDTA. However, exposing numerous target biomolecules to EDTA is undesirable. Furthermore, EDTA is necessary for many nucleic acid-related enzymes (e.g., polymerases, nucleases), which require Mg. 2+It binds to ions. These enzymes are used in molecular biology techniques following sample preparation using proteinases, and the presence of EDTA is particularly problematic in this context. EDTA binds to free Mg in subsequent downstream steps. 2+ Introducing uncertainty regarding concentration is undesirable. Mg 2+ While necessary for enzyme function, excess amounts can lead to RNA degradation. Any method of polypeptide digestion that involves reagents and subsequently requires their removal increases process cost, time, and workflow; therefore, it is desirable to omit reagents.

[0095] The inventors have surprisingly found that the mere absence of free calcium is sufficient to induce thermotipation in the proteinase of the present invention. Surprisingly, the inventors have revealed for the first time that the thermotipation of proteinase X is induced when the proteinase is present in an environment with a low concentration of free calcium ions. The inventors have confirmed that it is not necessary to remove calcium ions that can bind to the proteinase and contribute to its stability and structure, for example, using EDTA. On the contrary, simply lowering the concentration of free calcium ions is surprisingly sufficient to induce thermotipation in the enzyme. This induceable property is unexpected and is not observed in proteinase K, a standard proteinase used in molecular biology applications, as shown in the examples.

[0096] Therefore, preferably, the proteinase and its enzymatically active fragment of the present invention contain calcium ions bound thereto. In other words, preferably, the compositions and samples of the present invention contain the proteinase and its enzymatically active fragment in their natural calcium-bound state. In this scenario, calcium can be considered a "cofactor" of the protein, and therefore the proteinase is their "holoenzyme form" in the compositions and samples of the present invention. A holoenzyme is a biochemically active enzyme formed by a combination of an apoenzyme and its cofactor (calcium in this case).

[0097] Therefore, preferably, the compositions and samples of the present invention are essentially EDTA-free, preferably EDTA-free, and more preferably free of any calcium chelating agent. Preferably, the proteinase and enzymatically active fragments present in the solutions and samples of the present invention are not exposed to EDTA at any point, and more preferably not exposed to any calcium chelating agent.

[0098] From another perspective, the present invention relates to a composition comprising a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The calcium concentration in the composition is ≤ approximately 80 μM, and the composition is essentially free of EDTA, preferably essentially free of calcium chelating agents, ii) To provide a composition in which the monovalent salt concentration in the composition is ≥ approximately 20 mM.

[0099] From another perspective, the present invention relates to a sample comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample is essentially free of EDTA, preferably essentially free of calcium chelating agents, ii) Provide a sample in which the monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0100] From another perspective, the present invention relates to a composition comprising a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The calcium concentration in the composition is ≤ approximately 80 μM, and the composition is essentially free of EDTA, preferably essentially free of calcium chelating agents, ii) The monovalent salt concentration in the composition is ≥ approximately 20 mM, The present invention provides a composition wherein the proteinase or enzymatically active fragment thereof further comprises a further peptide sequence that is N-terminal and / or C-terminal to the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1.

[0101] Preferred optional features and embodiments described elsewhere in this specification also apply to “alternative views” of the Invention, with necessary modifications. In particular, the “free calcium” concentrations disclosed elsewhere in this specification are preferred concentrations of calcium (not defined as “free calcium”) in alternative views compositions and samples of the Invention that are essentially free of EDTA, and more preferably, essentially free of any calcium chelating agent. The same applies to the following methods of the Invention that refer to the concentration of “calcium” rather than “free calcium” and explicitly state that the samples or compositions of the methods of the Invention are essentially free of EDTA.

[0102] Preferably, the compositions and samples of all embodiments of the present invention are essentially free of EDTA and EGTA, more preferably, essentially free of calcium chelating agents capable of removing bound calcium from the proteinase X structure, and more preferably, essentially free of calcium chelating agents.

[0103] For example, "essentially free" of EDTA or free calcium means that the composition and sample are essentially free of EDTA or free calcium, but it does not mean that there is a strict requirement that they be completely free of EDTA or free calcium. For example, due to the presence of small amounts of EDTA or free calcium in the commercially available product or storage solution used in the preparation of the composition or sample of the present invention, there is still a possibility of minute levels of EDTA or free calcium even after steps have been taken to prevent the presence of EDTA or free calcium. While detailed examination may reveal the presence of some EDTA, it is present in such small amounts that it can be considered absent for the intended purpose, i.e., not at a level that substantially alters the free calcium level or the degree of calcium binding to the proteinase of the present invention compared to the level or degree that would occur in the absence of EDTA.

[0104] "Essentially free" in relation to calcium chelating agents means that the ratio of the proteinase X concentration to the concentration of the calcium chelating agent, such as EDTA or EGTA, in the sample is at least 10:1, more preferably at least 100:1, and more preferably at least 1000:1. If the sample contains more than 1 calcium chelating agent, these ratios are the ratio of the proteinase X concentration to the total concentration of the calcium chelating agent in the sample.

[0105] Although detailed examination may reveal the presence of some free calcium, it is present in such small amounts that it can be considered negligible for the intended purpose; that is, it is not present at a level that substantially alters the heat-vitality of the proteinase of the present invention compared to the level or degree that would occur in the absence of free calcium.

[0106] Preferably, the compositions and samples of the present invention are completely free of EDTA, more preferably free of calcium chelating agents capable of removing bound calcium from the proteinase X structure, and even more preferably free of calcium chelating agents.

[0107] Preferably, the compositions and samples of the present invention are completely free of free calcium, i.e., they do not contain free calcium.

[0108] The free calcium concentration in the composition and sample of the present invention is preferably ≤ about 80 μM, preferably ≤ about 65 μM, more preferably ≤ about 40 μM, more preferably ≤ about 35 μM, ≤ about 32 μM or ≤ about 30 μM, more preferably ≤ about 25 μM or ≤ about 20 μM, more preferably ≤ about 16 μM or ≤ about 15 μM, more preferably ≤ about 10 μM, more preferably ≤ about 8 μM or ≤ about 5 μM, and more preferably ≤ about 2.5 μM. The term "≤ about X" is the same as "0 to X μM". Preferably, the composition and sample of the present invention do not contain free calcium.

[0109] Preferably, the free calcium concentration in the composition and sample of the present invention is at least about 1 μM. That is, the free calcium concentration in the composition and sample of the present invention is preferably about 1 to about 80 μM, more preferably about 1 to about 65 μM, preferably about 1 to about 40 μM, preferably about 1 to about 35 μM, preferably about 1 to about 32 μM, preferably about 1 to about 30 μM, preferably about 1 to about 25 μM, preferably about 1 to about 20 μM, preferably about 1 to about 16 μM, preferably about 1 to about 15 μM, preferably about 1 to about 10 μM, preferably about 1 to about 8 μM, preferably about 1 to about 5 μM, and preferably about 1 to about 2.5 μM.

[0110] Preferably, the free calcium concentration in the composition and sample of the present invention is at least about 2 μM. That is, the free calcium concentration in the composition and sample of the present invention is preferably about 2 to about 80 μM, more preferably about 2 to about 65 μM, preferably about 2 to about 40 μM, preferably about 2 to about 35 μM, preferably about 2 to about 32 μM, preferably about 2 to about 30 μM, preferably about 2 to about 25 μM, preferably about 2 to about 20 μM, preferably about 2 to about 16 μM, preferably about 2 to about 15 μM, preferably about 2 to about 10 μM, preferably about 2 to about 8 μM, preferably about 2 to about 5 μM, and preferably about 2 to about 2.5 μM.

[0111] Preferably, the compositions and samples of the present invention are essentially free of free calcium, more preferably free of free calcium. The term "essentially free" is defined elsewhere in this specification.

[0112] Particularly preferably, the free calcium concentration in the composition and sample of the present invention is ≤ about 35 μM, ≤ about 32 μM, preferably ≤ about 16 μM, and preferably ≤ about 10 μM. Particularly preferably, the free calcium concentration in the composition and sample of the present invention is at least about 1 μM, and preferably at least about 2 μM. That is, particularly preferably, the free calcium concentration in the composition and sample of the present invention is about 1 to about 35 μM, preferably about 1 to about 32 μM, preferably about 2 to about 35 μM, preferably about 2 to about 32 μM, preferably about 1 to about 16 μM, preferably about 2 to about 16 μM, preferably about 1 to about 10 μM, and preferably about 2 to about 10 μM.

[0113] As described above, preferably, the compositions and samples of the present invention contain such concentrations of free calcium, are essentially free of EDTA, and are essentially free of calcium chelating agents.

[0114] Preferably, the concentration of proteinase or an enzymatically active fragment thereof in the composition of the present invention is 0.1 mg / ml to 20 mg / ml, more preferably 0.5 mg / ml to 10 mg / ml, and most preferably 2 mg / ml to 5 mg / ml.

[0115] Using the above preferred analysis A, the inventors confirmed that the specific activity of proteinase X is approximately 65 U / mg. Preferably, the activity of the proteinase or an enzymatically active fragment thereof in the composition of the present invention is measured by analysis A above and is 0.0015 U / μl to 0.30 U / μl, more preferably 0.008 U / μl to 0.15 U / μl, and most preferably about 0.03 U / μl to 0.08 U / μl.

[0116] Preferably, the concentration of proteinase or an enzymatically active fragment thereof in the sample of the present invention is 0.001 mg / ml to 5 mg / ml, more preferably 0.05 mg / ml to 0.5 mg / ml, and most preferably 0.015 mg / ml to 0.1 mg / ml.

[0117] Preferably, the activity of the proteinase or enzymatically active fragment thereof in the sample of the present invention is measured by analysis A above and is 0.07 U / ml to 325 U / ml, more preferably 3.25 U / ml to 32.5 U / ml, and most preferably 1.0 U / μl to 6.5 U / ml.

[0118] Optionally, the composition or sample of the present invention may include one or more further functional proteins selected from the group consisting of antibodies, single-stranded DNA-binding proteins (SSBs), and enzymes.

[0119] Preferably, the further enzymes are selected from the group consisting of nucleases (preferably deoxyribonuclease, exonuclease, Bal31 nuclease, ribonuclease, mangubean nuclease, or S1 nuclease), polymerases (preferably DNA polymerase or RNA polymerase), reverse transcriptases, transposases, ligases (preferably DNA ligase or RNA ligase), methylases, polynucleotide adenylyltransferases, topoisomerases, guanylyltransferases, phosphatases (preferably alkaline phosphatases, preferably heat-labile alkaline phosphatases, more preferably shrimp alkaline phosphatases), kinases, helicases, restriction enzymes, and glycosylases. The composition or sample preferably contains a combination of these further enzymes.

[0120] Preferably, the composition or sample comprises DNA polymerase or reverse transcriptase. If the solution or sample contains cells or cellular material, they preferably include further enzymes that are exogenous enzymes, i.e., not expressed in cells within the sample or in cells from which the cellular material in the sample originates. In other words, the further enzymes are applied to the sample rather than being supplied by the cells or cellular material in the sample. In these embodiments, the additional presence of endogenously produced enzymes is not excluded.

[0121] The compositions and samples of the present invention preferably comprise a monovalent salt, preferably a monovalent inorganic salt. The terms “salt” and “monovalent salt” are used synonymously throughout. A monovalent salt is a salt containing a monovalent counterion. A divalent salt is a salt in which at least one of the counterions is divalent, such as MgCl2. An inorganic salt is a salt in which none of the counterions are carbon. Preferably, the salt is a sodium salt or a potassium salt, more preferably a sodium salt. Preferably, the salt is sodium chloride (NaCl) or potassium chloride (KCl), most preferably sodium chloride.

[0122] Alternatively, the compositions and samples of the present invention preferably contain monovalent pairions. Preferably, the compositions and samples of the present invention also contain monovalent cations and preferably monovalent anions. Preferably, the monovalent ions are inorganic. Preferably, the cations are sodium ions or potassium ions, preferably sodium ions. Preferably, the compositions and samples contain sodium and chloride ions or potassium and chloride ions, most preferably sodium and chloride ions. The preferred concentrations of monovalent salts disclosed herein are essentially preferred concentrations of monovalent pairions, and vice versa.

[0123] As shown in the examples, the inventors have for the first time demonstrated that while an increase in monovalent salt concentration induces the heat-loosening of proteinase X, an increase in monovalent salt concentration stabilizes and thermally inactivates proteinase K. This result is particularly surprising. Proteinase X of SEQ ID NO: 1 is obtained from saline organisms and is therefore normally expected to tolerate high-salinity conditions. In contrast, proteinase K is obtained from a non-marine source, the fungus Engyodontium album (formerly Tritirachium album), and is not expected to be stabilized by high-salinity conditions.

[0124] In the samples and compositions of the present invention containing monovalent salts, the composition and sample preferably have a pH of 6.5 to 9.5, preferably 6.8 to 9.2, more preferably 7 to 9, more preferably 7.5 to 8.5, and more preferably about 8.0.

[0125] Preferably, the monovalent salt concentration in the composition and sample of the present invention is ≥ about 20 mM, preferably ≥ about 25 mM, more preferably ≥ about 30 mM, more preferably ≥ about 40 mM, more preferably ≥ about 50 mM, more preferably ≥ about 75 mM, more preferably ≥ about 100 mM, more preferably ≥ about 125 mM, more preferably ≥ about 150 mM, and more preferably ≥ about 175 mM. Optionally, the monovalent salt concentration in the composition and sample of the present invention is ≥ about 200 mM, ≥ about 250 mM, ≥ about 300 mM, ≥ about 400 mM, or ≥ 500 mM.

[0126] Preferably, the monovalent salt concentration in the composition and sample of the present invention is ≤ about 1 M, preferably ≤ about 500 mM, and preferably ≤ about 350 mM. Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 mM to about 1 M, preferably about 20 to about 500 mM, preferably about 20 to about 400 mM, preferably about 20 to about 350 mM, preferably about 30 to about 350 mM, preferably about 40 to about 350 mM, preferably about 50 to about 350 mM, preferably about 75 to about 350 mM, preferably about 100 to about 350 mM, preferably about 125 to about 350 mM, preferably about 150 to about 350 mM, and preferably about 175 to about 350 mM. Optionally, the monovalent salt concentration in the composition and sample of the present invention is about 200 to about 350 mM, or about 300 to about 350 mM.

[0127] Preferably, the monovalent salt concentration in the composition and sample of the present invention is ≤ about 500 mM, preferably ≤ about 400 mM, preferably ≤ about 300 mM, preferably ≤ about 250 mM, preferably ≤ about 200 mM, preferably ≤ about 175 mM, and preferably ≤ about 150 mM.

[0128] Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 300 mM, preferably about 30 to about 300 mM, preferably about 40 to about 300 mM, preferably about 50 to about 300 mM, preferably about 75 to about 300 mM, preferably about 100 to about 300 mM, preferably about 125 to about 300 mM, preferably about 150 to about 300 mM, and preferably about 175 to about 300 mM. Optionally, the monovalent salt concentration in the composition and sample of the present invention is about 200 to about 300 mM.

[0129] Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 250 mM, preferably about 30 to about 250 mM, preferably about 40 to about 250 mM, preferably about 50 to about 250 mM, preferably about 75 to about 250 mM, preferably about 100 to about 250 mM, preferably about 125 to about 250 mM, preferably about 150 to about 250 mM, and preferably about 175 to about 250 mM. Optionally, the monovalent salt concentration in the composition and sample of the present invention is about 200 to about 250 mM.

[0130] Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 200 mM, preferably about 30 to about 200 mM, preferably about 40 to about 200 mM, preferably about 50 to about 200 mM, preferably about 75 to about 200 mM, preferably about 100 to about 200 mM, preferably about 125 to about 200 mM, preferably about 150 to about 200 mM, and preferably about 175 to about 200 mM.

[0131] Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 175 mM, preferably about 30 to about 175 mM, preferably about 40 to about 175 mM, preferably about 50 to about 175 mM, preferably about 75 to about 175 mM, preferably about 100 to about 175 mM, preferably about 125 to about 175 mM, and preferably about 150 to about 175 mM.

[0132] Preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 150 mM, preferably about 30 to about 150 mM, preferably about 40 to about 150 mM, preferably about 50 to about 150 mM, preferably about 75 to about 150 mM, preferably about 100 to about 150 mM, and preferably about 125 to about 150 mM.

[0133] Particularly preferably, the monovalent salt concentration in the composition and sample of the present invention is about 20 to about 175 mM, preferably about 20 to about 150 mM, and preferably about 50 to about 150 mM. Particularly preferably, the monovalent salt concentration in the composition and sample of the present invention is about 30 to about 175 mM, preferably about 30 to about 150 mM, and preferably about 30 to about 150 mM. Particularly preferably, the monovalent salt concentration in the composition and sample of the present invention is about 40 to about 175 mM, preferably about 40 to about 150 mM, and preferably about 40 to about 150 mM.

[0134] Preferably, the compositions and samples of the present invention have a free calcium concentration as defined elsewhere herein. andThis includes monovalent salt concentrations. This clearly indicates any combination of any calcium concentration value or range disclosed herein with respect to all aspects and embodiments of the present invention with respect to any monovalent salt concentration value or range disclosed herein. Preferably, the monovalent salt is NaCl or KCl, preferably NaCl.

[0135] The preferred combinations of the highest free calcium concentration and the lowest monovalent salt concentration are shown below.

[0136] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0137] Furthermore, preferably, the free calcium concentration in the composition and sample of the present invention is at least about 1 μM, more preferably at least about 2 μM. Furthermore, preferably, the monovalent salt concentration in the composition and sample of the present invention is about 500 mM or less, preferably about 400 mM or less, preferably about 350 mM or less, preferably 300 mM or less, preferably about 250 mM or less, preferably about 200 mM or less, preferably about 175 mM or less, and preferably about 150 mM or less.

[0138] The preferred combinations of maximum free calcium concentration and monovalent salt concentration are shown below.

[0139] [Table 2]

[0140] The preferred combinations of free calcium concentration and monovalent salt concentration are shown below.

[0141] [Table 3]

[0142] The preferred combinations of free calcium concentration and monovalent salt concentration are shown below.

[0143] [Table 4]

[0144] Particularly preferably, the free calcium concentration in the composition and sample of the present invention is ≤ about 5 μM, preferably ≤ about 2 μM, more preferably ≤ about 1 μM, and most preferably about 0 mM. Particularly preferably, the monovalent salt concentration in the composition and sample is 20 to 125 mM, preferably 20 to 100 mM, and most preferably 20 to 50 mM. Particularly preferably, the monovalent salt concentration in the composition and sample is 30 to 125 mM, preferably 30 to 100 mM, and most preferably 30 to 50 mM.

[0145] The compositions and samples of the present invention contain proteinases that can be inactivated under particularly mild conditions. Therefore, the compositions have advantageous utility in various molecular biology methods, including subsequent or prior use of other enzymes. Such methods are discussed in more detail below. That is, one further embodiment is: i) Compositions of the present invention and ii) A kit is provided that includes a second composition containing a second enzyme.

[0146] The second enzyme is not the proteinase of the present invention. Optionally, the kit includes multiple solutions containing multiple enzymes, i.e., it includes a third solution containing a third enzyme, and optionally a fourth solution containing a fourth enzyme, and so on. Each solution present in the kit of the present invention preferably contains a different enzyme, each of which is not the proteinase of the present invention. Preferably, the enzyme in the second subsequent solution is independently selected from the group consisting of nucleases (preferably deoxyribonuclease, exonuclease, Bal31 nuclease, ribonuclease, mangubean nuclease, or S1 nuclease), polymerases (preferably DNA polymerase or RNA polymerase), reverse transcriptases, ligases (preferably DNA ligase or RNA ligase), methylases, transferases (preferably polynucleotide adenylyltransferase), topoisomerases, guanylyltransferases, proteinases other than proteinase X, and phosphatases or combinations thereof.

[0147] Preferably, the kit of the present invention comprises the composition of the present invention and a second composition comprising DNA polymerase or reverse transcriptase.

[0148] The compositions and samples of the present invention contain proteinases that can be inactivated, in particular, by a mild heat treatment step. Therefore, the compositions have a usefulness that is advantageous to various molecular biology methods where the application of a proteinase to a sample is necessary or desirable to digest one or more polypeptides in the sample, but it is also desirable that this does not adversely affect the structure or function of one or more biomolecules present. The use of standard proteinases requires either i) inactivation using high temperatures for a considerable period of time, which may damage the target biomolecule present, or ii) removal / dilution of the proteinase, which increases workflow, time, and cost, and may result in the loss or damage of the target biomolecule. The compositions enable methods that do not require these undesirable processing steps.

[0149] The present invention provides methods discussed below. The above definitions relating to the compositions, samples and kits of the present invention, as well as preferred optional features and embodiments, apply to all methods of the present invention with necessary modifications. In relation to all methods of the present invention, polypeptides, samples, proteinases, enzymatically active fragments, free calcium, free calcium concentrations, monovalent salts and monovalent salt concentrations are described elsewhere in this specification. In particular, any of the features disclosed above, as well as the concentrations of free calcium (or calcium), monovalent salts and combinations thereof, are useful in the methods of the present invention, i.e., preferably, the sample to be contacted or treated in the methods of the present invention contains a certain concentration of free calcium, monovalent salts or any combination thereof, as disclosed elsewhere in this specification.

[0150] Accordingly, the term “sample” as used in the method of the present invention refers to any composition comprising one or more polypeptides. Preferably, the sample comprises cellular material. Preferably, the sample comprises crude cell extracts. Preferably, the sample comprises partially purified cell extracts. Preferably, the sample comprises a cell population. The cells in the sample may be intact or lysed, preferably lysed. Preferably, the sample comprises a tissue sample or one or more body fluids. Preferably, the sample is fine-needle biopsy material. Preferably, the sample comprises an inclusion virus. Proteinases can be used to digest the protein capsules of the virus to release RNA / DNA from the protein capsules for identification, quantification, and / or amplification.

[0151] Preferably, the sample has a volume of ≥10 μl. Preferably, the sample has a volume of ≤1000 μl, more preferably ≤500 μl, more preferably ≤300 μl, more preferably ≤250 μl, more preferably ≤200 μl, more preferably ≤150 μl, more preferably ≤100 μl, more preferably ≤75 μl, and more preferably ≤50 μl. Alternatively, the sample is a microfluidic sample. Preferably, the microfluidic sample has a volume of ≥0.01 μl. Preferably, the microfluidic sample has a volume of ≤10 μl, more preferably ≤5 μl, more preferably ≤1 μl, more preferably ≤0.5 μl, and more preferably ≤0.1 μl.

[0152] Throughout this specification, unless otherwise specified, the term "proteinase" also refers to its enzymatically active fragment.

[0153] In all methods of the present invention, the sample preferably contains essentially no EDTA, preferably any calcium chelating agent. The meaning of this term is defined elsewhere in this specification. The sample, after contact with a proteinase or an enzymatically active fragment thereof, contains essentially no EDTA, preferably any calcium chelating agent.

[0154] Therefore, in a further embodiment, a method for digesting polypeptides in a sample is provided. The method comprises the step of contacting the sample with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0155] In other words, the present invention is a method for digesting a polypeptide in a sample, the method comprising the step of contacting the sample with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) To provide a method in which the monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0156] In all methods of the present invention, particularly in methods in which the sample contains a monovalent salt, the sample preferably has a pH of 6.5 to 9.5, preferably 6.8 to 9.2, more preferably 7 to 9, more preferably 7.5 to 8.5, and more preferably about 8.0. The inventors have for the first time confirmed that the proteinase of the present invention can be inactivated under mild conditions, including neutral and near-neutral pH.

[0157] Preferably, the free calcium (or calcium) concentration in the sample is approximately 80 μM or less. ,and, The monovalent salt concentration in the sample is at least about 20 mM. Preferably, the monovalent salt concentration in the sample is at least about 30 mM, more preferably at least about 40 mM, and more preferably at least about 50 mM.

[0158] The terms “digest,” “hydrolyze,” “decompose,” and “cleave” are used without distinction herein and refer to the hydrolysis of peptide bonds within polypeptides in a sample. Digestion may be partial or complete. Proteinases useful in the present invention are nonspecific and, given sufficient time, completely digest proteins in a sample under conditions that allow for enzymatic function.

[0159] The terms “to bring into contact,” “to apply,” and “to add to” have their usual meanings and are used interchangeably herein.

[0160] In the method of the present invention, preferably, the proteinase or an enzymatically active fragment thereof is provided in the form of one of the compositions of the present invention as described above.

[0161] Preferably, a proteinase or an enzymatically active fragment thereof is added to the sample at a concentration of 0.001 mg / ml to 5 mg / ml, more preferably 0.05 mg / ml to 0.5 mg / ml, and most preferably 0.015 mg / ml to 0.1 mg / ml. These concentrations represent the proteinase concentration in the sample.

[0162] Preferably, the activity of the proteinase or its enzymatically active fragment in the sample after application, i.e., in the sample, is measured by analysis A above and is 0.07 U / ml to 325 U / ml, more preferably 3.25 U / ml to 32.5 U / ml, and most preferably 1.0 U / μl to 6.5 U / ml.

[0163] Therefore, the proteinase and its enzymatically active fragments of the present invention are used to degrade polypeptides in a sample. In particular, the method involves contacting the sample with the proteinase of the present invention under conditions that allow for the digestion of at least a portion of the polypeptides present in the sample. Therefore, preferably, after the sample has come into contact with the proteinase or its enzymatically active fragments, the method further includes a “digestion step,” i.e., a step of incubating the sample under conditions that allow for the digestion of polypeptides in the sample. The amount of digestion required depends on the purpose and intention of the person performing the method, and suitable conditions for achieving the required amount of digestion can be easily determined by those skilled in the art.

[0164] Preferably, the digestion step includes heating the sample at a temperature of 4 to 65°C, more preferably 20 to 55°C, and most preferably 30 to 55°C. Preferably, the incubation step has a duration of 1 second to 45 minutes, more preferably 30 seconds to 30 minutes, more preferably 1 to 15 minutes, more preferably 1 to 10 minutes, and even more preferably 1 to 5 minutes. If the upper limit of these temperatures is used, the incubation duration can be the lower limit of these temperatures, and vice versa. Those skilled in the art know that very short incubations of 1 or 2 seconds are sufficient in methods using microfluidic samples and in other methods where the amount of substrate present in the sample is small.

[0165] Preferably, the above method includes a subsequent "inactivation step," i.e., a step of heating the sample to inactivate the proteinase or its enzymatically active fragments. Such an inactivation step is performed after the step of contacting the sample with the proteinase and after the step of incubating the sample under conditions that allow for the digestion of polypeptides in the sample.

[0166] These digestion and inactivation steps are generally incubations, as described herein, particularly in the examples. The above characteristics and embodiments regarding pH and (free) calcium, monovalent salt, and EDTA concentrations are the conditions in the sample at the time the inactivation step is performed.

[0167] Therefore, in a further embodiment, a method for digesting polypeptides in a sample, wherein the method is a) A step of contacting a sample with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) A step of incubating the sample under conditions that allow at least partial digestion of polypeptides in the sample, and c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) A method is provided which includes the step of having a monovalent salt concentration in the sample ≥ approximately 20 mM.

[0168] From another perspective, step c) of the above method is, c) Inactivating the proteinase or its enzymatically active fragment by heating the sample, where, i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0169] Preferably, the free calcium (or calcium) concentration in the sample is approximately 80 μM or less. ,and, The monovalent salt concentration in the sample is at least about 20 mM. Preferably, the monovalent salt is a monovalent inorganic salt, preferably a sodium salt or a potassium salt, more preferably potassium chloride or sodium chloride, and most preferably sodium chloride. Digestion step b) is as described above.

[0170] As described above, digestion can be partial or complete. The proteinase useful in this invention is nonspecific and, given sufficient time, completely digests the proteins in the sample under conditions that allow for enzymatic function.

[0171] Here, "digestion of polypeptides in a sample" means that the amount of full-length polypeptides in the sample is reduced to a certain extent. The degree of protein digestion can be directly analyzed by several well-known analyses. Designing appropriate analyses for their intended purposes will be within the capabilities of those skilled in the art. For example, the residual activity of enzymes in the sample can be used as a measure of protein degradation. The degree of polypeptide degradation in a sample can also be measured using changes in the proteome profile, which can be measured, for example, by mass spectrometry. A simple analysis to visualize the degree of protein degradation is to perform staining with protein staining dyes such as SDS-Page and Coomassie blue or other visual reporter molecules. Intact proteins will appear as bands along the gel, and the bands will become less sharp as protein degradation increases. The degree of degradation can be quantified by software-based image analysis.

[0172] Preferably, the sample to which proteinase has been added contains cellular material. Preferably, the sample contains crude cell extract. Preferably, the sample contains partially purified cell extract. Preferably, the sample contains a cell population. The cells in the sample may be intact or lysed, preferably lysed. Preferably, the sample contains a tissue sample or one or more body fluids. Preferably, the sample contains about 1 to about 1,000,000 cells. In one preferred embodiment, the sample contains 1 to 10,000 cells, preferably 1 to 1,000 cells, preferably 1 to 100 cells. In one preferred embodiment, the sample contains single cells. In another preferred embodiment, the sample contains 100 to 1,000,000 cells, preferably 100 to 10,000 cells, preferably 100 to 1,000 cells. Preferably, the sample is a fine needle or liquid biopsy material. Proteinase can be used to lyse cellular material.

[0173] The proteinase of the present invention can be used to digest any polypeptide present in a sample. Preferably, the polypeptide in the sample is a capsid or scaffolding protein, a DNA or RNA-binding protein, and / or an enzyme that acts on DNA or RNA, as described elsewhere in this specification.

[0174] In a further embodiment, the present invention provides a method for inactivating a proteinase or an enzymatically active fragment thereof in a sample. The proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and the method comprises the step of heating the sample to inactivate the proteinase or the enzymatically active fragment. i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0175] In other words, the present invention is a method for inactivating a proteinase or an enzymatically active fragment thereof in a sample, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and the method comprises the step of heating the sample to inactivate the proteinase or the enzymatically active fragment. i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample is essentially free of EDTA, preferably essentially free of calcium chelating agents, ii) To provide a method in which the monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0176] In all of the methods of the present invention, preferably, the free calcium (or calcium) concentration in the sample is about 80 μM or less. ,and, The monovalent salt concentration in the sample is at least about 20 mM. Preferably, the monovalent salt is a monovalent inorganic salt, preferably a sodium salt or a potassium salt, more preferably potassium chloride or sodium chloride, and most preferably sodium chloride.

[0177] As described above, in the method of the present invention, the free calcium (or calcium) and monovalent salt concentrations described elsewhere in this specification are the concentrations in the sample at the start of the inactivation step. Preferably, the concentrations are also the concentrations in the sample after contact with the proteinase or an enzymatically active fragment thereof. In the method of the present invention, preferably, the proteinase is not removed from the sample, for example, by purification, extraction or centrifugation, and preferably, the proteinase concentration in the sample is not diluted before or during the inactivation step.

[0178] This invention is based on the remarkable finding that the proteinases described herein become heat-convertible under mild conditions when given a certain concentration of free calcium and / or monovalent salts. Therefore, these conditions must be present during the inactivation step of the method of the present invention. Those skilled in the art will readily understand that contacting a sample with a proteinase or an enzymatically active fragment thereof can increase the volume of the sample and, consequently, decrease the concentrations of free calcium and monovalent salts within it. In use, preferably, the composition of the present invention has a small volume that does not significantly alter the volume of the sample. Similarly, according to the present invention, the proteinases of the present invention can be provided in a solution such that the free calcium concentration in the solution is greater than about 80 μM and / or the monovalent salt concentration is less than about 20 mM, but the volume and concentration of free calcium and / or monovalent salts in the sample to which it is added can be provided in a solution such that the resulting sample to which the proteinase or enzymatically active fragment thereof is applied contains a free calcium concentration of about 80 μM or less and a monovalent salt concentration of at least about 20 mM.

[0179] Preferably, the inactivation step in the method of the present invention includes reducing the activity of the proteinase in the sample by at least 75%, more preferably at least 80% or at least 85%, more preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the activity of the proteinase in the sample before the inactivation step is performed. Alternatively, preferably, the inactivation step results in a residual proteinase activity of less than 25%, more preferably less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Preferably, the proteinase is completely inactivated, i.e., preferably, no detectable proteinase activity remains.

[0180] Even when the solution containing the heat-treated proteinase returns to a temperature below 40°C, the proteinase of the present invention does not regain its activity; that is, there is almost no residual activity, specifically less than 10%, preferably less than 5%, 2%, 1%, 0.5%, or 0.1%, and most preferably no detectable proteinase activity remains. Therefore, the inactivation as described herein is irreversible.

[0181] As described above, suitable analyses for measuring proteinase activity are known in the art. Therefore, these analyses can be used to measure the proteinase activity of a heat-inactivated proteinase or an enzymatically active fragment thereof compared to the proteinase activity of the same proteinase that has not been heat-treated, thereby determining the residual activity or the degree of inactivation achieved by the inactivation step. Any proteinase activity analysis can be used to determine the relative activity of a heat-treated proteinase to an untreated proteinase. Those skilled in the art will know that the same heat-treated and untreated proteinases should be maintained under the same conditions and analyzed using the same procedure. Preferably, the analysis used is analysis A described above, and preferably, the untreated proteinase is maintained on ice until the proteinase activity is measured. A more preferred analysis of residual activity is disclosed in the examples.

[0182] Therefore, the residual activity after the inactivation step of the method of the present invention, i.e., the degree of inactivation achieved by the inactivation step of the method of the present invention, can be easily measured by those skilled in the art by measuring the proteinase activity of the proteinase that has undergone the inactivation step under appropriate conditions using an appropriate proteinase analysis and comparing it with the activity of the same proteinase that has not undergone such an inactivation step. The activity is measured using the same analysis under the same conditions. Preferably, the analysis used is analysis A above, or any one of the analyses used in this embodiment. Preferably, the untreated proteinase is kept on ice until the proteinase activity is measured.

[0183] The degree of inactivation can be expressed as the inactivation rate or residual activity rate relative to the activity of the untreated proteinase.

[0184] Since the free calcium concentration in the sample is approximately 80 μM or less, and / or the monovalent salt concentration in the sample is at least approximately 20 mM, proteinases and their enzymatically active fragments in the sample can be inactivated under mild heating conditions.

[0185] Heating a sample to a desired temperature for a desired time, for example, using a heat block, microwave, Joule heating device, laser heating device, or water bath, would be within the capabilities of those skilled in the art.

[0186] The temperature at which the sample is heated during the inactivation step of this method, i.e., the temperature at which the proteinase is exposed, is referred to as the inactivation temperature. Preferably, the inactivation temperature is ≤ about 70°C, preferably ≤ about 67°C, preferably ≤ about 65°C, preferably ≤ about 64°C, preferably ≤ about 63°C, preferably ≤ about 62°C, preferably ≤ about 61°C, preferably ≤ about 60°C, and preferably ≤ about 58°C.

[0187] Preferably, the inactivation step involves heating the sample containing the proteinase to a temperature of about 50 to about 67°C, preferably about 50 to about 65°C, preferably about 50 to about 64°C, preferably about 50 to about 63°C, preferably about 50 to about 62°C, preferably about 50 to about 61°C, preferably about 50 to about 60°C, and preferably about 50 to about 58°C.

[0188] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 53 to about 67°C, preferably about 53 to about 65°C, preferably about 53 to about 64°C, preferably about 53 to about 63°C, preferably about 53 to about 62°C, preferably about 53 to about 61°C, preferably about 53 to about 60°C, and preferably about 53 to about 58°C.

[0189] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 55 to about 67°C, preferably about 55 to about 65°C, preferably about 55 to about 64°C, preferably about 55 to about 63°C, preferably about 55 to about 62°C, preferably about 55 to about 61°C, and preferably about 55 to about 60°C.

[0190] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 56 to about 67°C, preferably about 56 to about 65°C, preferably about 56 to about 64°C, preferably about 56 to about 63°C, preferably about 56 to about 62°C, preferably about 56 to about 61°C, and preferably about 56 to about 60°C.

[0191] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 57 to about 67°C, preferably about 57 to about 65°C, preferably about 57 to about 64°C, preferably about 57 to about 63°C, preferably about 57 to about 62°C, preferably about 57 to about 61°C, and preferably about 57 to about 60°C.

[0192] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 58 to about 67°C, preferably about 58 to about 65°C, preferably about 58 to about 64°C, preferably about 58 to about 63°C, preferably about 58 to about 62°C, preferably about 58 to about 61°C, and preferably about 58 to about 60°C.

[0193] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 59 to about 67°C, preferably about 59 to about 65°C, preferably about 59 to about 64°C, preferably about 59 to about 63°C, preferably about 59 to about 62°C, preferably about 59 to about 61°C, and preferably about 59 to about 60°C.

[0194] Preferably, the inactivation step includes heating the sample containing the proteinase to a temperature of about 55°C to about 65°C, preferably about 60°C to about 65°C, more preferably about 55°C to about 60°C.

[0195] Particularly preferably, the deactivation step includes heating at about 53°C to about 60°C, more preferably about 53°C to about 58°C, and more preferably about 55°C.

[0196] Particularly preferably, the deactivation step includes heating at about 58°C to about 67°C, more preferably about 58°C to about 63°C, and more preferably about 60°C.

[0197] Particularly preferably, the deactivation step includes heating to about 60 to about 67°C, more preferably about 63 to about 67°C, and more preferably about 65°C.

[0198] Preferably, the inactivation step includes heating the sample at one of the above temperatures for a time referred to as the “holding time.” The required holding time depends on the inactivation temperature used, the concentrations of free calcium and monovalent salts in the sample, and the desired degree of inactivation. Taking into account the teachings of this application, those skilled in the art will be able to select holding times for their particular purposes.

[0199] Preferably, the holding time is ≤ approximately 75 minutes, preferably ≤ approximately 60 minutes, preferably ≤ approximately 55 minutes, preferably ≤ approximately 50 minutes, preferably ≤ approximately 45 minutes, preferably ≤ approximately 40 minutes, preferably ≤ approximately 35 minutes, preferably ≤ approximately 30 minutes, preferably ≤ approximately 25 minutes, preferably ≤ approximately 20 minutes, preferably ≤ approximately 15 minutes, preferably ≤ approximately 10 minutes, preferably ≤ approximately 5 minutes, and preferably ≤ approximately 2 minutes.

[0200] Preferably, the holding time is at least about 1 minute, preferably at least about 2 minutes, preferably at least about 5 minutes, preferably at least about 10 minutes, preferably at least about 15 minutes, preferably at least about 20 minutes, preferably at least about 25 minutes, preferably at least about 30 minutes, preferably at least about 35 minutes, preferably at least about 40 minutes, preferably at least about 45 minutes, preferably at least about 50 minutes, and preferably at least about 60 minutes.

[0201] Preferably, the holding time is about 2 to about 75 minutes, preferably about 2 to about 60 minutes, preferably about 2 to about 55 minutes, preferably about 2 to about 50 minutes, preferably about 2 to about 45 minutes, preferably about 2 to about 40 minutes, preferably about 2 to about 35 minutes, preferably about 2 to about 30 minutes, preferably about 2 to about 25 minutes, preferably about 2 to about 20 minutes, preferably about 2 to about 15 minutes, preferably about 2 to about 10 minutes, and preferably about 2 to about 5 minutes.

[0202] Preferably, the holding time is about 5 to about 75 minutes, preferably about 5 to about 60 minutes, preferably about 5 to about 55 minutes, preferably about 5 to about 50 minutes, preferably about 5 to about 45 minutes, preferably about 5 to about 40 minutes, preferably about 5 to about 35 minutes, preferably about 5 to about 30 minutes, preferably about 5 to about 25 minutes, preferably about 5 to about 20 minutes, preferably about 5 to about 15 minutes, and preferably about 5 to about 10 minutes.

[0203] Preferably, the holding time is about 10 to about 75 minutes, preferably about 10 to about 60 minutes, preferably about 10 to about 55 minutes, preferably about 10 to about 50 minutes, preferably about 10 to about 45 minutes, preferably about 10 to about 40 minutes, preferably about 10 to about 35 minutes, preferably about 10 to about 30 minutes, preferably about 10 to about 25 minutes, preferably about 10 to about 20 minutes, and preferably about 10 to about 15 minutes.

[0204] Preferably, the holding time is about 15 to about 75 minutes, preferably about 15 to about 60 minutes, preferably about 15 to about 55 minutes, preferably about 15 to about 50 minutes, preferably about 15 to about 45 minutes, preferably about 15 to about 40 minutes, preferably about 15 to about 35 minutes, preferably about 15 to about 30 minutes, preferably about 15 to about 25 minutes, and preferably about 15 to about 20 minutes.

[0205] Preferably, the holding time is about 20 to about 75 minutes, preferably about 20 to about 60 minutes, preferably about 20 to about 55 minutes, preferably about 20 to about 50 minutes, preferably about 20 to about 45 minutes, preferably about 20 to about 40 minutes, preferably about 20 to about 35 minutes, preferably about 20 to about 30 minutes, and preferably about 20 to about 25 minutes.

[0206] Preferably, the holding time is about 25 to 75 minutes, preferably about 25 to 60 minutes, preferably about 25 to 55 minutes, preferably about 25 to 50 minutes, preferably about 25 to 45 minutes, preferably about 25 to 40 minutes, preferably about 25 to 35 minutes, and preferably about 25 to 30 minutes.

[0207] Preferably, the holding time is about 30 to about 75 minutes, preferably about 30 to about 60 minutes, preferably about 30 to about 55 minutes, preferably about 30 to about 50 minutes, preferably about 30 to about 45 minutes, preferably about 30 to about 40 minutes, and preferably about 30 to about 35 minutes.

[0208] Preferably, the holding time is about 35 to about 75 minutes, preferably about 35 to about 60 minutes, preferably about 35 to about 55 minutes, preferably about 35 to about 50 minutes, preferably about 35 to about 45 minutes, and preferably about 35 to about 40 minutes.

[0209] Preferably, the holding time is about 40 to about 75 minutes, preferably about 40 to about 60 minutes, preferably about 40 to about 55 minutes, preferably about 40 to about 50 minutes, and preferably about 40 to about 45 minutes.

[0210] Preferably, the holding time is about 45 to about 75 minutes, preferably about 45 to about 60 minutes, preferably about 45 to about 55 minutes, and preferably about 45 to about 50 minutes.

[0211] Preferably, the holding time is about 50 to 75 minutes, preferably about 50 to 60 minutes, and preferably about 50 to 55 minutes.

[0212] Preferably, the holding time is about 55 to 75 minutes, more preferably about 55 to 60 minutes.

[0213] Preferably, the holding time is about 5 minutes to about 40 minutes, preferably about 10 to about 35 minutes, and preferably about 15 to about 30 minutes. Particularly preferably, the holding time is about 5 to about 15 minutes, or about 10 to about 20 minutes, or about 20 to about 40 minutes, preferably about 25 to about 35 minutes.

[0214] The above retention time is particularly suitable for samples with a volume of ≤1000 μl, preferably ≤500 μl, more preferably ≤300 μl, more preferably ≤250 μl, more preferably ≤200 μl, more preferably ≤150 μl, more preferably ≤100 μl, more preferably ≤75 μl, and more preferably ≤50 μl.

[0215] Those skilled in the art know that adjusting one of the heating temperature or holding time can be compensated for by adjusting the other. For example, increasing the inactivation temperature may allow for a reduction in the holding time. Conversely, extending the holding time may allow for the use of a lower inactivation temperature.

[0216] Furthermore, those skilled in the art know that when the amount of proteinase present is small, for example in the case of a microfluidic sample, sufficient inactivation can occur within a very short time frame, for example, within 1 to 30 seconds, preferably 1 to 20 seconds, preferably 1 to 10 seconds, preferably 1 to 5 seconds, and potentially even within just 1 or 2 seconds. Any of the above inactivation temperatures can be used with these short retention times. For such short retention times to be effective, the sample containing the proteinase to be inactivated preferably has a volume of ≤10 μl, preferably ≤5 μl, more preferably ≤1 μl, more preferably ≤0.5 μl, and more preferably ≤0.1 μl.

[0217] In the method of the present invention, which includes an inactivation step, any of the above inactivation temperatures may be used in combination with any of the above holding times. Any combination of inactivation temperatures and holding times disclosed elsewhere in this specification is explicitly disclosed.

[0218] Preferably, the inactivation step includes heating at a temperature of about 53°C to about 67°C, preferably about 55°C to about 65°C, preferably about 55°C to about 63°C for a holding time of about 2 to about 75 minutes, preferably about 5 to about 40 minutes, more preferably about 10 to about 30 minutes, for example, about 10, about 15, or about 30 minutes.

[0219] Preferably, the inactivation step includes heating at a temperature of about 55 to about 60°C for a holding time of about 2 to about 75 minutes, preferably about 5 to about 40 minutes, more preferably about 10 to about 30 minutes, for example, about 10, about 15, or about 30 minutes.

[0220] Preferably, the inactivation step includes heating at a temperature of about 60 to about 65°C for a holding time of about 2 to about 75 minutes, preferably about 5 to about 40 minutes, more preferably about 10 to about 20 minutes, for example, about 10 or about 15 minutes.

[0221] The preferred deactivation step of the present invention is as follows:

[0222] A) Approximately 53~58℃ Preferably, heating at about 55°C for about 45 to about 75 minutes, more preferably about 45 to about 60 minutes, and more preferably about 60 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 10 μM, more preferably ≤ about 8 μM, more preferably ≤ 5 μM, and more preferably the sample does not contain free calcium.

[0223] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 50 mM, more preferably at least about 75 mM, more preferably at least about 100 mM, or at least about 150 mM.

[0224] B) approx. 58~63℃ Preferably, heating at approximately 60°C for one of the following times as described in B1 to B4 below. B1) Approximately 2 to 40 minutes, more preferably 5 to 30 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 80 μM, more preferably ≤ about 65 μM, more preferably ≤ about 35 μM, more preferably ≤ about 20 μM, more preferably ≤ about 10 μM, and more preferably ≤ about 5 μM.

[0225] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 20 mM, more preferably at least about 25 mM, more preferably at least about 30 mM, more preferably at least about 40 mM, more preferably at least about 50 mM, more preferably at least about 75 mM, more preferably at least about 100 mM, and more preferably at least about 150 mM.

[0226] B2) Approximately 5 to 15 minutes, preferably about 10 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 10 μM, ≤ about 8 μM, more preferably ≤ about 5 μM, and more preferably the sample does not contain free calcium.

[0227] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 75 mM, more preferably at least about 100 mM, and more preferably at least about 150 mM.

[0228] B3) Approximately 10 to 20 minutes, more preferably about 15 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 35 μM, more preferably ≤ about 16 μM, more preferably ≤ about 8 μM, and more preferably ≤ about 5 μM, and the sample does not contain free calcium.

[0229] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 50 mM, more preferably at least about 75 mM, more preferably at least about 100 mM, and more preferably at least about 150 mM.

[0230] B4) Approximately 20-40 minutes, preferably about 30 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 80 μM, ≤ about 65 μM, more preferably ≤ about 35 μM, more preferably ≤ about 30 μM, and more preferably ≤ about 16 μM.

[0231] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 25 mM, more preferably at least about 30 mM, more preferably at least about 40 mM, more preferably at least about 50 mM, more preferably at least about 75 mM, and more preferably at least about 100 mM.

[0232] Preferably, in these embodiments, when the monovalent salt concentration is 100 mM or less, the calcium concentration is 30 μM or less. Preferably, when the monovalent salt concentration is 75 mM or less, the calcium concentration is 20 μM or less. Preferably, when the monovalent salt concentration is 50 mM or less, the calcium concentration is 10 μM or less.

[0233] C) approx. 63~about 67℃ Preferably, heat at approximately 65°C for a maximum of approximately 15 minutes, more preferably for a maximum of approximately 10 minutes, and more preferably for a maximum of approximately 5 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 80 μM, more preferably ≤ about 65 μM, more preferably ≤ about 35 μM, more preferably ≤ about 20 μM, more preferably ≤ about 10 μM, and more preferably ≤ about 5 μM.

[0234] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 20 mM, more preferably at least about 25 mM, more preferably at least about 30 mM, more preferably at least about 40 mM, more preferably at least about 50 mM, more preferably at least about 75 mM, and more preferably at least about 100 mM.

[0235] The free calcium-dependent and monovalent salt-dependent effects on heat-loosening properties, as confirmed by the inventors, also enable the inactivation of the proteinase and its enzymatically active fragments at high temperatures in a remarkably short time. Therefore, in another preferred embodiment, the inactivation step includes the following:

[0236] D) Heating at approximately 65 to 70°C, preferably approximately 67 to 70°C, more preferably approximately 67°C or 70°C for a maximum of approximately 5 minutes, more preferably for a maximum of approximately 2 minutes. In these embodiments, the free calcium concentration in the sample is preferably ≤ about 80 μM, more preferably ≤ about 65 μM, more preferably ≤ about 35 μM, and more preferably ≤ about 20 μM.

[0237] Alternatively, or in addition, in these embodiments, the monovalent salt concentration in the sample is preferably at least about 20 mM, more preferably at least about 25 mM, more preferably at least about 30 mM, more preferably at least about 40 mM, more preferably at least about 50 mM, more preferably at least about 75 mM, and more preferably at least about 100 mM.

[0238] Most preferably, the heating / inactivation step c) includes heating the sample at a temperature of 55-60°C for 15-30 minutes.

[0239] Those skilled in the art will readily understand that adjustment of one of the parameters—heating time, heating temperature, free calcium concentration, and monovalent salt concentration—can be compensated for by one or more other adjustments.

[0240] However, very importantly, according to the present invention, the maximum free calcium concentration is 80 μM. Below this free calcium concentration, the thermosensitivity of the proteinase of the present invention is induced to such an extent that substantial inactivation (75% inactivation) of the proteinase can be achieved, particularly at an inactivation temperature of 53-67°C and a holding time of 2-75 minutes, preferably 5-60 minutes, more preferably 10-40 minutes, and preferably 15-30 minutes, under favorable mild conditions.

[0241] Similarly, according to the present invention, the minimum monovalent salt concentration is 20 mM. At or above this monovalent salt concentration, the thermosensitivity of the proteinase of the present invention is induced to such an extent that substantial inactivation (75% inactivation) of the proteinase can be achieved, particularly at an inactivation temperature of 53-67°C and a holding time of 2-75 minutes, preferably 5-60 minutes, more preferably 10-40 minutes, and preferably 15-30 minutes, under favorable mild conditions.

[0242] As mentioned above, typical inactivation procedures for standard proteinases used in this field require much harsher conditions, such as heating at 75°C for 5 minutes (BioRad procedure), heating at 95°C for 10 minutes (New England BioLabs procedure), or heating at 70°C for 15 minutes (Qiagen procedure).

[0243] In the method of the present invention, preferably, the sample is essentially free of EDTA, more preferably free of EDTA, and more preferably free of any calcium chelating agent at the time it comes into contact with the proteinase. In other words, preferably, the sample to which the proteinase is applied is essentially free of EDTA, more preferably free of EDTA, and preferably free of any calcium chelating agent. Preferably, the method of the present invention does not include the step of applying EDTA, preferably any calcium chelating agent, to the sample after the addition of the proteinase. The sample may have come into contact with a calcium chelating agent at some point in the early part of the workflow or during its preparation, in which case the calcium chelating agent must be removed before the application of the proteinase. Calcium chelating agents are as described elsewhere in this specification, and removing them from the sample before contacting the sample with the proteinase is within the capabilities of those skilled in the art.

[0244] As described above, in all methods of the present invention, particularly in methods in which the sample contains a monovalent salt, the sample preferably has a pH of 6.5 to 9.5, preferably 6.8 to 9.2, more preferably 7 to 9, more preferably 7.5 to 8.5, and more preferably about 8.0. The inventors have for the first time confirmed that the proteinase of the present invention can be inactivated under mild conditions, including neutral and near-neutral pH. Therefore, preferably, the method further includes a step of adjusting the pH of the sample to 6.5 to 9.5, preferably 6.8 to 9.2, more preferably 7 to 9, more preferably 7.5 to 8.5, and more preferably about 8.0 before the inactivation step. The step of adjusting the pH of the sample is well known to those skilled in the art, and any such step can be used in the methods of the present invention.

[0245] Preferably, the sample to which proteinase is added has a volume of ≥10 μl. Preferably, the sample has a volume of ≤1000 μl, more preferably ≤500 μl, more preferably ≤300 μl, more preferably ≤250 μl, more preferably ≤200 μl, more preferably ≤150 μl, more preferably ≤100 μl, more preferably ≤75 μl, and more preferably ≤50 μl. Alternatively, the sample is a microfluidic sample. Preferably, the microfluidic sample has a volume of ≥0.01 μl. Preferably, the microfluidic sample has a volume of ≤10 μl, more preferably ≤5 μl, more preferably ≤1 μl, more preferably ≤0.5 μl, and more preferably ≤0.1 μl.

[0246] Preferably, the sample to which the proteinase or enzymatically active fragment is added contains the target biomolecule and one or more contaminants, i.e., unwanted polypeptides.

[0247] Therefore, in a further embodiment, the present invention provides a method for isolating or purifying a target biomolecule from a sample. The sample comprises one or more contaminating polypeptides, and the method is: a) A step of contacting a sample with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) A step of incubating the sample under conditions that allow at least partial digestion of polypeptides in the sample, and c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM, and d) The procedure includes the optional step of removing the target biomolecule from the sample.

[0248] Alternatively, step c) of the above method includes the following:

[0249] A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0250] Preferably, the “biomolecule of interest” is a nucleic acid molecule, preferably a DNA or RNA molecule. Preferably, the biomolecule of interest is a polypeptide itself. The biomolecule of interest is neither a proteinase nor an enzymatically active fragment thereof.

[0251] The proteinase of the present invention can be used to digest a viral protein capsule in order to release RNA / DNA from the viral protein capsule for identification, quantification, and / or amplification. Therefore, preferably, the biological sample comprises one or more inclusion viruses, the biomolecule of interest is the nucleic acid molecule of the virus, preferably RNA or DNA, and the contaminating polypeptide is of the viral protein capsule, and step b) comprises incubating the sample under conditions that allow for at least partial digestion of the one or more viral protein capsules, i.e., sufficient digestion to release the nucleic acid molecule from the capsule.

[0252] Preferably, the sample comprises chromatin, the biomolecule of interest is DNA without bound histones, the contaminating protein is histones bound thereto, and step b) comprises incubating the sample under conditions that allow at least partial digestion of the histones in the sample.

[0253] Preferably, the sample is the product of a nuclear amplification reaction, such as a PCR reaction, or contains the product, contains a DNA-binding polymerase, the target biomolecule is DNA that does not contain the binding polymerase, the contaminating protein is the binding polymerase, and step b) includes incubating the sample under conditions that allow at least partial digestion of the polymerase in the sample. Amplification methods include, but are not limited to, PCR and its variations, 3SR, SDA, LAR or LCR and LAMP and their variations.

[0254] The term "nucleic acid amplification reaction" refers to any in vitro means for increasing the copy number of a target sequence of a nucleic acid or its complementary sequence.

[0255] The "product of a nucleic acid amplification reaction" is thus considered to include essentially all of the components directly obtained from the final amplification step of the reaction. Other components can be added, or some of the components can be subjected to some modification or processing, but essentially none of the components, or at least none of the nucleic acid components, are removed. Preferably, the product of the nucleic acid amplification reaction is the direct product of the final amplification step. However, the product of the nucleic acid amplification reaction may preferably be subjected to a treatment that causes dephosphorylation of unincorporated NTPs, for example, treatment with alkaline phosphatase, preferably thermolabile alkaline phosphatase, such as thermolabile shrimp alkaline phosphatase (SAP), prior to treatment with the proteinase of the present invention. A preferred recombinant SAP is available from ArcticZymes™ AS.

[0256] Preferably, the target biomolecule in the sample binds to a molecule, preferably a polypeptide, via one or more peptide bonds. In a further aspect, the present invention thus provides a method for releasing a target biomolecule from a molecule, preferably a polypeptide, bound thereto via one or more peptide bonds. The method is a) A step of contacting a sample with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) A step of incubating a sample under conditions that enable the release of the target biomolecule by digestion of one or more peptide bonds, and c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM, and d) The procedure includes the optional step of removing the target biomolecule from the sample.

[0257] Alternatively, step c) of the above method includes heating the sample to inactivate the proteinase or its enzymatically active fragments, i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0258] Preferably, the biomolecule of interest is a polypeptide or protein, and the molecule to which it binds is a polypeptide signal sequence or fusion tag, preferably a his tag (e.g., a hexahistidine tag), a FLAG tag, a maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), a small molecule ubiquitin-like modifier (SUMO), ubiquitin (Ub), or green fluorescent protein (GFP).

[0259] In a further embodiment, the present invention provides a method for producing a target peptide from a precursor polypeptide. The method is as follows: a) A step of contacting a polypeptide with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) Incubating the sample under conditions that allow for the digestion of the precursor polypeptide in order to release the target peptide, c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM, and d) The procedure includes the optional step of removing the target biomolecule from the sample.

[0260] Alternatively, step c) of the above method includes heating the sample to inactivate the proteinase or its enzymatically active fragments, i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0261] Proteinases are also used to separate one or more cells from other cells in a tissue, or from a substrate to which they are attached, by digestion of extracellular matrix proteins. In a further embodiment, the present invention provides a method for separating one or more cells from other cells in a tissue, or from a substrate to which the one or more cells are attached. The method is a) A step of contacting one or more cells with a proteinase or an enzymatically active fragment thereof, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) A step of incubating a sample under conditions that allow the release of one or more cells by digestion of one or more extracellular matrix proteins, and c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM, and d) The procedure includes the optional step of removing the target biomolecule from the sample.

[0262] Alternatively, step c) of the above method includes heating the sample to inactivate the proteinase or its enzymatically active fragments, i) The calcium concentration in the sample is ≤ approximately 80 μM, and the sample does not essentially contain EDTA, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM.

[0263] Preferably, the sample is a polyacrylamide gel containing a polypeptide, or a polyacrylamide gel comprising the polypeptide, wherein digestion of the polypeptide is required to produce small fragments for analysis by mass spectrometry. Accordingly, the present invention provides a method for preparing a protein fragment sample, preferably for mass spectrometry. The method is as follows: a) A step of contacting a sample containing one or more polypeptides with a proteinase or an enzymatically active fragment thereof, wherein the proteinase contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1. b) A step of incubating the sample under conditions that allow at least partial digestion of polypeptides in the sample, and c) A step of heating the sample to inactivate the proteinase or its enzymatically active fragments, where, i) The free calcium concentration in the sample is ≤ approximately 80 μM, or ii) The monovalent salt concentration in the sample is ≥ approximately 20 mM, and d) The procedure includes the optional step of removing the target biomolecule from the sample.

[0264] Preferably, the sample is a polyacrylamide gel containing one or more polypeptides.

[0265] Preferably, the sample to which a proteinase or an enzymatically active fragment thereof is added comprises one or more further enzymes. Preferably, the further enzymes are selected from the group consisting of nucleases (preferably deoxyribonuclease, exonuclease, Bal31 nuclease, ribonuclease, mangubean nuclease, or S1 nuclease), polymerases (preferably DNA polymerase or RNA polymerase), reverse transcriptases, ligases (preferably DNA ligase or RNA ligase), methylases, transferases (preferably polynucleotide adenylyltransferase), topoisomerases, guanylyltransferases, phosphatases (preferably alkaline phosphatase, preferably thermolabile alkaline phosphatase, more preferably shrimp alkaline phosphatase), kinases, helicases, restriction enzymes, and glycosylases. Preferably, the sample comprises a combination of these further enzymes. Preferably, the sample comprises DNA polymerase or reverse transcriptase. Preferably, these enzymes are exogenous enzymes, i.e., not expressed in cells within the sample or in cells from which cellular material in the sample originates. The method of the present invention provides advantageous mild proteinase inactivation conditions that can be tolerated by such additional enzymes, and therefore these enzymes can be present during the proteinase inactivation step, thereby simplifying the subsequent workflow.

[0266] As mentioned above, proteinase K, the standard proteinase currently used in this field, requires inactivation at high temperatures that can damage the enzyme or the biomolecule of interest in the sample. If inactivation of proteinase K is required without high-temperature heating, the proteinase must be removed from the sample or the enzyme concentration must be significantly diluted. Such removal or dilution steps can prolong the workflow, increase costs, and result in the loss or damage of material in the sample. They are particularly unsuitable when processing small sample sizes.

[0267] Preferably, the sample to which protease is added has a volume of ≦1000 μl, more preferably ≦500 μl, more preferably ≦300 μl, more preferably ≦250 μl, more preferably ≦200 μl, more preferably ≦150 μl, more preferably ≦100 μl, more preferably ≦75 μl, more preferably ≦50 μl. Alternatively, the sample is a microfluidic sample. Preferably, the microfluidic sample has a volume of ≧0.01 μl. Preferably, the microfluidic sample has a volume of ≦10 μl, preferably ≦5 μl, more preferably ≦1 μl, more preferably ≦0.5 μl, more preferably ≦0.1 μl.

[0268] Therefore, preferably, any of the above methods includes, following the inactivation step, a step including the enzymatic action of a substrate, and the subsequent step is performed without prior removal or dilution of the protease or an enzymatically active fragment thereof.

[0269] "Without prior removal" means that the protease is not physically removed from the sample, for example, by purification, extraction or centrifugation.

[0270] "Without prior dilution" means that the protease concentration in the sample is not significantly diluted, that is, it is not substantially inactivated by dilution. The definition of substantial inactivation is described elsewhere in this specification. Preferably, the protease concentration in the sample is not diluted more than 4-fold, more preferably not more than 3-fold, more preferably not more than 2-fold.

[0271] Preferably, the sample contains one or more nucleic acid molecules, and the method includes, following the inactivation step, a step of nuclease-mediated digestion of the nucleic acid molecules without prior removal or dilution of the protease or an enzymatically active fragment thereof.

[0272] Preferably, the sample contains one or more nucleic acid molecules, and the method includes, following the inactivation step, a step of phosphorylation or dephosphorylation of the nucleic acid molecules without prior removal or dilution of the protease or an enzymatically active fragment thereof.

[0273] Preferably, the sample comprises one or more nucleic acid molecules, and the method comprises an inactivation step followed by a step of linking the nucleic acid molecules without prior removal or dilution of proteinase or enzymatically active fragments thereof.

[0274] Preferably, the sample comprises one or more RNA molecules, and the method comprises an inactivation step followed by a reverse transcription step without prior removal or dilution of proteinases or enzymatically active fragments thereof.

[0275] Preferably, the sample comprises one or more nucleic acid molecules, and the method comprises an inactivation step followed by a nucleic acid polymerization step without prior removal or dilution of proteinase or enzymatically active fragment thereof.

[0276] Preferably, the sample comprises one or more nucleic acid molecules, and the method comprises an inactivation step followed by a nucleic acid amplification step without prior removal or dilution of proteinase or enzymatically active fragment thereof.

[0277] Preferably, the sample comprises one or more nucleic acid molecules, and the method comprises an inactivation step followed by a nanopore sequencing step without prior removal or dilution of proteinase or enzymatically active fragment thereof.

[0278] Preferably, the sample comprises one or more virus particles or cells, preferably bacterial cells, and the method comprises an inactivation step followed by a cell lysis step without prior removal or dilution of proteinase or enzymatically active fragments thereof. [Examples]

[0279] Example 1: Proteinase specific activity In all examples, proteinase K was purchased from Thermo Fischer (prod. No. EO0491, 28.9 kDa), and proteinase X was generated in ArcticZymes by recombinant Pichia pastoris (batch 1602-1, SEQ ID NO: 1).

[0280] To measure the specific activity (U / mg proteinase) of the two proteinases, the proteinase concentrations in the solution were first measured using NanoDrop. NanoDrop is a spectrophotometric method that quantifies protein concentration by measuring absorbance at a wavelength of 280 nm.

[0281] Proteinase K was confirmed to be present at a concentration of 14.3 mg / ml. A 10,000-fold diluted stock solution (1.43 μg / ml) was then used. Proteinase X was confirmed to be present at a concentration of 9.2 mg / ml. A 1,000-fold diluted stock solution (9.2 μg / ml) was then used.

[0282] The activity (U / mL) of proteinases X and K was measured by standard kinetic peptide-based analysis. 0.4 μg / ml Proteinase X or 0.06 μg / ml Proteinase K 1mM substrate Suc-Ala-Ala-Pro-Phe-pNA 12mM NaCl, 0.1M Tris-HCl pH8, 10mM CaCl2, 1% DMSO was supplied in a total volume of 1000 μl.

[0283] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 410 nm (EM8.8) using a UV spectrophotometer (Ultrospec 2000, Pharmacia Biotec, Sweden) at 25°C for 2 minutes. One unit of enzyme was defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at 25°C.

[0284] The specific activity was measured as follows (Table 1).

[0285] [Table 5]

[0286] The results were confirmed using vials of proteinase K from different suppliers (Sigma, O4850, measured up to 400 U / mg).

[0287] Example 2 Effect of free calcium concentration on proteinase activity The activity of proteinases X and K was measured at various temperatures by standard peptide-based analysis. 0.37 μg / ml Proteinase X or 0.06 μg / ml Proteinase K (equivalent to 24 mU / mL Proteinase) 1mM substrate Suc-Ala-Ala-Pro-Phe-pNA 12mM NaCl, 0.1M Tris-HCl, pH8.0, 10mM CaCl2, 1% DMSO was supplied in a total volume of 1000 μl.

[0288] The cuvette was incubated at the indicated temperature during analysis. The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was observed over 30 seconds in a 1.5 mL semi-micro cuvette (Brand, Germany) using a UV spectrophotometer (Ultrospec 2000, Pharmacia) to measure the increase in absorbance at 410 nm (EM8.8). The analysis was performed by measuring using Biotech (Sweden).

[0289] Activity was calculated as a percentage relative activity to the maximum activity observed at 65°C. Measurements above 65°C were technically impossible. According to prior art teachings, the optimal temperature for both proteinases X and K is 65–70°C.

[0290] As shown in Figure 1, the two proteinases have similar temperature-activity profiles in the presence of 10 mM calcium.

[0291] The temperature-activity profiles of proteinases X and K were also measured under low-calcium (5 μM) and calcium-free (0 μM) conditions. In addition, 24 mU / mL proteinase (equivalent to 0.37 μg / mL proteinase X or 0.06 μg / mL proteinase K) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at the indicated temperature in a buffer solution (0.1 M Tris-HCl, pH 8.0, 0 mM / 0.005 mM / 10 mM CaCl2, 1% DMSO, 12 mM NaCl) in a total volume of 1000 μl.

[0292] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 410 nm (EM8.8) over 30 seconds using a UV spectrophotometer.

[0293] The activity was calculated as a percentage relative activity to the activity observed at 65°C in the presence of 10 mM CaCl2 (Table 2). The activity observed at 65°C was the maximum activity observed.

[0294] [Table 6]

[0295] At various calcium concentrations, the temperature profiles of both proteinases were similar, with maximum activity observed at 65°C. Decreasing calcium concentration appeared to slightly reduce proteinase activity at several temperatures. The activity profiles of the two proteinases differed when compared to the activity achieved at each specific temperature using 10 mM calcium. The results are shown in Tables 3 and 4 and Figures 2 and 3. These highlight the calcium-dependent effect on proteinase activity at various temperatures.

[0296] [Table 7]

[0297]

Table 8

[0298] According to FIG. 2 and Table 3, for Proteinase X, low calcium concentration or absence of calcium resulted in some loss of activity (maximum decrease < 10%) at 55 °C and 45 °C, and there was no appreciable decrease in activity at 25 °C or 35 °C. An approximately 20% decrease in activity was observed at 65 °C in the absence of calcium, and an approximately 10% decrease was observed at 65 °C with low calcium (0.005 mM).

[0299] In contrast, according to FIG. 3 and Table 4, the activity of Proteinase K at each temperature was hardly affected by calcium conditions. That is, whether in the presence of high calcium concentration, low calcium concentration, or absence of calcium, only a slight change in activity was observed with increasing temperature.

[0300] In summary, the data suggest that low calcium concentration can induce greater thermal inactivation of Proteinase X than Proteinase K.

[0301] Example 3 : Ca of Proteinase 2+ Dependent inducible heat-labile The calcium-dependent heat-lability of Proteinases X and K was further investigated.

[0302] Heat treatment step: The proteinases were incubated at 60 °C for 15 or 30 minutes in a buffer containing various concentrations of free calcium in a PCR thermocycler (Veriti, Applied Biosystems). The buffer was 0.1 mg / ml Proteinase X (equivalent to 6.5 U / ml Proteinase X initial activity) or 0.014 mg / ml Proteinase K (equivalent to 5.6 U / ml Proteinase K initial activity) 0.025M Tris-HCl, pH8, 300mM NaCl, It contains CaCl2 (1 mM / 0.25 mM / 0.125 mM / 0.063 mM / 0.031 mM / 0.016 mM / 0.008 mM / 0 mM) in a volume of 50 μl.

[0303] After inactivation, the samples were returned to ice. The control samples were kept on ice throughout the entire process.

[0304] Analysis of residual activity After the heat treatment step, the sample was diluted 1:10 with 0.025 M Tris-HCl, pH 8, 300 mM NaCl. This dilution step reduced the enzyme activity U / ml in the sample, bringing it within a range detectable by reaction analysis.

[0305] The residual activity of proteinases was evaluated as follows.

[0306] 0.4 μg / ml proteinase X or 0.06 μg / ml proteinase K (corresponding to initial activities of 26 and 24 mU / ml, respectively) were incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37°C in a total volume of 250 μl in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl2, 1% DMSO, 12 mM NaCl).

[0307] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 405 nm (EM8.8) over a 10-minute period, detecting the signal every 11 seconds using a multimode plate reader (Synergy H1, BioTek, USA).

[0308] The results are shown in Figures 4 and 5. Activity is shown as the residual activity rate compared to the control sample (the same proteinase and buffer maintained on ice without the heating step).

[0309] As shown in Figure 5, the decrease in free calcium concentration did not affect the thermal inactivation of proteinase K, and proteinase K was present in all samples incubated at 60°C. 2+ Approximately 40% of the activity is lost regardless of concentration. Substantial inactivation of proteinase K (≥75%) was not achieved under any conditions.

[0310] In contrast, as shown in Figure 4, a decrease in free calcium concentration resulted in an increase in the heat-labile properties of proteinase X. Substantial inactivation (≥75%, i.e., less than 25% residual activity) was achieved by heating at 60°C with a free calcium concentration of ≤0.063 mM for 30 minutes, and by heating at 60°C with a free calcium concentration of ≤0.016 mM for 15 minutes. ≥90% inactivation was preferred, which was achieved for proteinase X by heating at 60°C with a free calcium concentration of ≤0.031 mM for 30 minutes, or by heating in the absence of free calcium for 15 minutes.

[0311] Therefore, while proteinase X is inductively heat-labile in the presence of low calcium concentrations, the heat-labile properties of proteinase K are not affected by calcium concentration.

[0312] This difference is further illustrated in Figure 6. Figure 6 shows the inactivation profiles of two proteinases after heating at 60°C for 15 / 30 minutes at various calcium concentrations.

[0313] Example 4: Proteinase inactivation profile dependent on free calcium The effects of different heat treatment steps on the inactivation of proteinases X and K were evaluated. In the following experiments, a free calcium upper limit of 5 μM was used, and the thermal deactivation step was performed within a range of temperatures and heating times.

[0314] Heat treatment step: Proteinases were incubated at various temperatures (45°C, 50°C, 55°C, 60°C, 65°C, and 70°C) for various durations (2, 5, 10, 15, 30, and 60 minutes) in buffers containing varying concentrations of free calcium (CaCl2, 0 μM, 2.5 μM, or 5 μM). The buffers further contained 0.1 mg / ml (6.5 U / ml initial activity) proteinase X or 0.016 mg / ml (6.4 U / ml initial activity) proteinase K, 25 mM HEPES, pH 8, 100 mM NaCl, and a total volume of 50 μl.

[0315] Before use, proteinase K was dialyzed against a storage buffer that did not contain calcium to remove free calcium. After inactivation, the sample was returned to ice. A control sample was maintained on ice throughout the entire process.

[0316] Analysis of residual activity After the heat treatment step, the sample was diluted 1:20 with 50 mM HEPES, pH 8, and 100 mM NaCl. This dilution step reduced the enzyme activity U / ml in the sample, bringing it within a range detectable by reaction analysis.

[0317] The residual activity of proteinases was evaluated as follows.

[0318] 0.2 μg / ml proteinase X or 0.03 μg / ml proteinase K (corresponding to initial activities of 13 mU and 12 mU, respectively) were incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37°C in a total volume of 250 μl in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl2, 1% DMSO, 4 mM NaCl).

[0319] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 405 nm (EM8.8) over 10 minutes using a multimode plate reader (Synergy H1, BioTek, USA), detecting the signal every 11 seconds. The residual activity was compared with a control sample, which was identical to the test sample except that it was maintained on ice.

[0320] The results are shown in Tables 5-7.

[0321] [Table 9]

[0322] According to the data above, in the absence of free calcium, ≥75% inactivation of proteinase X is achieved. At least 70°C, within 2 minutes of heating, At least 65°C, within 5 minutes of heating, At least 60°C, within 10 minutes of heating, and This can be achieved within 60 minutes when heated to at least 55°C.

[0323] Proteinase X ≥ approximately 90% inactivation is At least 70°C, within 2 minutes of heating, At least 65°C, within 5 minutes of heating, At least 60°C, within 15 minutes when heating, and This can be achieved within 60 minutes when heated to at least 55°C.

[0324] [Table 10]

[0325] According to the data above, free calcium concentration Less than 2.5 μM So, ≥75% inactivation of proteinase X is, At least 70°C, within 5 minutes of heating, At least 65°C, within 5 minutes of heating, At least 60°C, within 15 minutes when heating, and This can be achieved within 60 minutes when heated to at least 55°C.

[0326] Proteinase X ≥ approximately 90% inactivation is At least 70°C, within 5 minutes of heating, At least 65°C, within 10 minutes of heating, and This is achieved within 30 minutes when heated to at least 60°C.

[0327] [Table 11]

[0328] According to the data above, free calcium concentration Less than 5 μM So, ≥75% inactivation of proteinase X is, At least 70°C, within 5 minutes of heating, At least 65°C, within 5 minutes of heating, At least 60°C, within 15 minutes when heating, and This can be achieved within 60 minutes when heated to at least 55°C.

[0329] Proteinase X ≥ approximately 90% inactivation is At least 70°C, within 5 minutes of heating, At least 65°C, within 10 minutes of heating, and This is achieved within 30 minutes when heated to at least 60°C.

[0330] Specifically, at a free calcium concentration of ≤5 μM, incubation at 70°C achieved ≥95% inactivation of proteinase X within 5 minutes, while incubation at 65°C achieved ≥95% inactivation of proteinase X within 10 minutes (within 5 minutes in the absence of free calcium).

[0331] In the presence of both 2.5 μM and 5 μM free calcium, incubation at 60°C achieved ≥90% inactivation of proteinase X within 30 minutes (compared to within 15 minutes in the absence of free calcium).

[0332] Incubation at ≥55°C for 60 minutes achieved 80% inactivation of proteinase X in the presence of 5 μM free calcium, 85% inactivation in the presence of ≤2.5 μM free calcium, and approximately 90% inactivation in the absence of free calcium.

[0333] For comparison, the same study was conducted using proteinase K. The results are shown in Tables 8-10 below.

[0334] [Table 12]

[0335] According to the data above, in the absence of free calcium, ≥75% inactivation of proteinase K is achieved. At least 70°C, within 2 minutes of heating, At least 65°C, within 10 minutes of heating (within 5 minutes for Prot X), At least 60°C, within 30 minutes of heating (within 10 minutes for Prot X), This is achieved within 60 minutes when heated at least 55°C (less inactivation than with Prot X).

[0336] Proteinase K inactivation of ≥90% is At least 70°C, within 2 minutes of heating, At least 65°C, within 10 minutes of heating (within 5 minutes for Prot X), and This is achieved within 60 minutes (within 15 minutes for Prot X) when heated to at least 60°C.

[0337] [Table 13]

[0338] According to the data above, free calcium concentration Less than 2.5 μM So, ≥75% inactivation of proteinase K is, At least 70°C, within 5 minutes of heating, At least 65°C, within 10 minutes of heating (within 5 minutes for Prot X), At least 60°C, within 30 minutes when heating (within 15 minutes for Prot X), and This is achieved within 60 minutes when heated at least 55°C (less inactivation than with Prot X).

[0339] Proteinase K inactivation of ≥90% is At least 70°C, within 5 minutes of heating, At least 65°C, within 15 minutes (within 10 minutes for Prot X), and This is achieved within 60 minutes (30 minutes for Prot X) when heated to at least 60°C.

[0340] [Table 14]

[0341] According to the data above, free calcium concentration Less than 5 μM So, ≥75% inactivation of proteinase K is, At least 70°C, within 5 minutes of heating, At least 65°C, within 10 minutes of heating (within 5 minutes for Prot X), At least 60°C, within 30 minutes when heating (within 15 minutes for Prot X), and This can be achieved within 60 minutes when heated to at least 55°C.

[0342] Approximately 90% inactivation of proteinase K is achieved by the following heating steps: At least 70°C, within 5 minutes of heating, At least 65°C, within 15 minutes (within 10 minutes for Prot X), and This is achieved within 60 minutes (30 minutes for Prot X) when heated to at least 60°C.

[0343] Therefore, the results indicate that, at all free calcium concentrations, the heating time required to achieve a similar degree of inactivation at a given temperature is considerably shorter for proteinase X than for proteinase K. Alternatively, at most of the tested heating times and temperatures, proteinase X exhibits greater inactivation than proteinase K. This is because the observed heat-gathering ability of proteinase X is induced at low calcium concentrations, which is not observed with proteinase K.

[0344] Therefore, the effect of calcium on the ability of proteinase K to be inactivated by heat treatment is considerably less pronounced than its effect on proteinase X.

[0345] Example 5 :Monovalent salt-dependent inducible thermotipation of proteinases To determine the effect of NaCl on the heat-looping properties of proteinases X and K, the inactivation profiles of both proteinases were measured at various temperatures in solutions containing i) 50 mM (low-salt conditions) or ii) 300 mM NaCl (high-salt conditions).

[0346] Heat treatment step: Proteinases were incubated for 30 minutes at various temperatures (45°C, 50°C, 55°C, 60°C, 65°C, and 70°C) in a buffer containing 50 mM or 300 mM NaCl. The buffer contained 0.1 mg / ml proteinase (equivalent to 6.5 U / ml proteinase X or 40 U / ml proteinase K), 25 mM HEPES, pH 8, and 0 μM CaCl2 in 50 μl volume.

[0347] After inactivation, the samples were returned to ice. The control samples were kept on ice throughout the entire process.

[0348] Analysis of residual activity After the heat treatment step, samples of proteinase X or proteinase K were diluted 1:10 or 1:100 with 50 mM HEPES, pH 8, and 300 mM NaCl, respectively. The residual activity of the proteinases was evaluated as follows.

[0349] 0.4 μg / ml proteinase X (corresponding to an initial activity of 26 mU / ml) or 0.04 μg / ml proteinase K (corresponding to an initial activity of 16 mU / ml) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37°C in a total volume of 250 μl in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl2, 1% DMSO, 12 mM NaCl).

[0350] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 405 nm (EM8.8) over 10 minutes using a multimode plate reader (Synergy H1, BioTek, USA), detecting the signal every 11 seconds. The residual activity was compared with a control sample, which was identical to the test sample except that it was maintained on ice.

[0351] As shown in Figure 7, increasing the NaCl concentration has the opposite effect on the heat-gaining ability of ProtX compared to ProtK. High NaCl concentrations stabilize proteinase K at high temperatures, while high NaCl concentrations induce heat-gaining ability of proteinase X.

[0352] This result is particularly surprising. Proteinase X, sequence number 1, is obtained from saltwater organisms and is therefore normally expected to tolerate high-salinity conditions. In contrast, Prot K is obtained from a non-marine source, the fungus Engyodontium album (formerly Tritirachium album), and is not expected to be stabilized by high-salinity conditions.

[0353] Example 6 : Monovalent salt-dependent thermoloyalty profile of proteinases To further investigate the effect of NaCl on the heat-looping properties of proteinase X, a wider range of NaCl concentrations than in Example 5 was evaluated.

[0354] Heat treatment step: Proteinase X was incubated at 50°C or 60°C for 15 or 30 minutes in a buffer containing 0, 50, 150, 300, or 600 mM NaCl. The buffer contained 0.1 mg / ml proteinase X (equivalent to 6.5 U / ml), 25 mM HEPES, pH 8, and 0.03 mM CaCl2 in a volume of 50 μl.

[0355] After inactivation, the samples were returned to ice. The control samples were kept on ice throughout the entire process.

[0356] Analysis of residual activity After the heat treatment step, the sample was diluted 1:10 with 50 mM HEPES, pH 8, and 300 mM NaCl. The residual proteinase activity was evaluated as follows.

[0357] 0.4 μg / ml proteinase X (corresponding to an initial activity of 26 mU / ml) was reacted with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37°C in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl2, 1% DMSO, 12 mM). The sample was incubated in NaCl at a total volume of 250 μl.

[0358] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 405 nm (EM8.8) over 10 minutes using a multimode plate reader (Synergy H1, BioTek, USA), detecting the signal every 11 seconds. The residual activity was compared with a control sample, which was identical to the test sample except that it was maintained on ice.

[0359] The results are shown in Figure 8 and Table 11.

[0360] [Table 15]

[0361] According to the above data, at an NaCl concentration of ≥150 mM, ≥80% inactivation of proteinase X can be achieved by heating at 60°C for 30 minutes or less.

[0362] As the above data shows, at an NaCl concentration of ≥300 mM, ≥95% inactivation of proteinase X can be achieved by heating at 60°C for 30 minutes or less, and ≥80% inactivation of proteinase X can be achieved by heating at 60°C for 150 minutes or less.

[0363] Figure 9, a plot of NaCl concentration (x axis) versus residual activity after heating at 60°C for 30 / 15 minutes (y axis), shows that substantial inactivation (≥75%) is achieved by heating at 60°C for 15 minutes at an NaCl concentration of at least approximately 210 mM, and by heating for 30 minutes at an NaCl concentration of at least approximately 100 mM.

[0364] Example 7 The combined effect of free calcium concentration and monovalent salt concentration on the heat-labile properties of proteinases. According to the above study, i) a decrease in free calcium concentration or ii) an increase in NaCl concentration induces the heat-labile properties of proteinase X more significantly than proteinase K. The combined effects of these conditions were further investigated.

[0365] Heat treatment step: Proteinases X and K were incubated at 60°C for 30 minutes in buffer solutions containing various concentrations of NaCl (0, 25, 50, 75, 100, 125 mM) and free calcium (0, 5, 10, 20, and 20 μM CaCl2). The buffer solution consisted of 50 μl of 6.5 U / ml proteinase X or 6.4 U / ml proteinase K (equivalent to 0.1 mg / ml proteinase X or 0.016 mg / ml proteinase K), 25 mM HEPES, and pH 8.

[0366] Before use, proteinase K was dialyzed against a storage buffer that did not contain calcium to remove the free calcium.

[0367] After inactivation, the samples were returned to ice. The control samples were kept on ice throughout the entire process.

[0368] Analysis of residual activity After the heat treatment step, the sample was diluted 1:20 with 50 mM HEPES, pH 8, and 100 mM NaCl. The residual proteinase activity was evaluated as follows.

[0369] 0.2 μg / ml proteinase X (corresponding to an initial activity of 13 mU / ml) or 0.03 μg / ml proteinase K (corresponding to an initial activity of 12 mU / ml) was incubated with 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA at 37°C in a total volume of 250 μl in standard reaction buffer (0.1 M Tris-HCl, pH 8.0, 10 mM CaCl2, 1% DMSO, 4 mM NaCl).

[0370] The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was analyzed by measuring the increase in absorbance at 405 nm (EM8.8) over 10 minutes using a multimode plate reader (Synergy H1, BioTek, USA), detecting the signal every 11 seconds. The residual activity was compared with a control sample, which was identical to the test sample except that it was maintained on ice.

[0371] The results are shown in Tables 12 and 13.

[0372] [Table 16]

[0373] Substantial inactivation (≥75%) was achieved at all free calcium concentrations in the presence of ≥100 mM NaCl.

[0374] Furthermore, substantial inactivation (≥approximately 75%) was achieved at a maximum free calcium concentration of 20 μM in the presence of at least 75 mM NaCl.

[0375] Furthermore, substantial inactivation (≥approximately 75%) was achieved at a maximum free calcium concentration of 10 μM in the presence of at least 50 mM NaCl.

[0376] Furthermore, substantial inactivation (≥approximately 75%) was achieved at a maximum free calcium concentration of 5 μM in the presence of at least 50 mM NaCl.

[0377] Furthermore, substantial inactivation (≥approximately 75%) was achieved in the absence of free calcium and in the presence of at least 25 mM NaCl.

[0378] Excellent inactivation (≥approximately 90%) was achieved at a maximum free calcium concentration of 20 μM in the presence of at least 125 mM NaCl.

[0379] Excellent inactivation (≥90%) was achieved at a maximum free calcium concentration of 10 μM in the presence of at least 100 mM NaCl.

[0380] Excellent inactivation (≥90%) at a maximum free calcium concentration of 5 μM and at least 75 mM This was achieved in the presence of NaCl.

[0381] Excellent inactivation (≥approximately 90%) was achieved in the absence of free calcium, in the presence of at least 50 mM NaCl.

[0382] [Table 17]

[0383] The results show that proteinase K has a very different heat-gain profile than proteinase X. Proteinase K gradually becomes heat-gaining under low-salinity conditions, stabilizes under high-salinity conditions, and is hardly affected by calcium concentration. On the other hand, proteinase X gradually becomes heat-gaining under high-salinity and low-free-calcium conditions.

Claims

1. A method for inactivating a proteinase in a sample, wherein the proteinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

1. The above method includes the step of heating the sample in any of the following i) to iii) to inactivate the proteinase; i) At a temperature of 53°C to 58°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 300 mM; ii) At a temperature of 58°C to 63°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 50 mM; or iii) At a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 210 mM; Includes, The residual proteinase activity of the proteinase after the step of heating the sample to inactivate the proteinase is ≤25% of that of the control, and the residual activity is determined in the following analytical step, i.e., i) In a 1000 μl or 250 μl cuvette A step of incubation with 10-50 mU / mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl pH 8, 10 mM CaCl2, and optionally 1% DMSO. ii) A step of analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at 410 nm (ε = 8800 M - 1 cm - 1) using a spectrophotometer at a temperature of ≤ 40°C for 2 minutes, where 1 unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a selected temperature. iii) A step to compare the activity observed in step ii) with the activity observed in the same amount of the same proteinase that was not heat-treated but was maintained under the same conditions as the heat-treated proteinase, using the same analysis. A method of measurement.

2. A method for digesting polypeptides in a sample, a) A step of contacting the sample with a proteinase, wherein the proteinase includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

1. b) Incubating the sample under conditions that allow at least partial digestion of the polypeptide in the sample, and c) A step of heating the sample in any of the following i) to iii) to inactivate the proteinase; i) At a temperature of 53°C to 58°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 300 mM; ii) At a temperature of 58°C to 63°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 50 mM; or iii) At a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 210 mM; Includes, The residual proteinase activity of the proteinase after the step of heating the sample to inactivate the proteinase is ≤25% of that of the control, and the residual activity is determined in the following analytical step, i.e., i) In a 1000 μl or 250 μl cuvette A step of incubation with 10-50 mU / mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl pH 8, 10 mM CaCl2, and optionally 1% DMSO. ii) A step of analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at 410 nm (ε = 8800 M - 1 cm - 1) using a spectrophotometer at a temperature of ≤ 40°C for 2 minutes, where 1 unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a selected temperature. iii) A step to compare the activity observed in step ii) with the activity observed in the same amount of the same proteinase that was not heat-treated but was maintained under the same conditions as the heat-treated proteinase, using the same analysis. A method of measurement.

3. A method for isolating or purifying a target biomolecule from a sample, wherein the sample contains one or more contaminating polypeptides, and the method is a) A step of contacting the sample with a proteinase, wherein the proteinase includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

1. b) Incubating the sample under conditions that allow at least partial digestion of the polypeptide in the sample, c) A step of heating the sample in any of the following i) to iii) to inactivate the proteinase; i) At a temperature of 53°C to 58°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 300 mM; ii) At a temperature of 58°C to 63°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 50 mM; or iii) At a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 210 mM, and d) A step of optionally removing the target biomolecule from the sample. Includes, The residual proteinase activity of the proteinase after the step of heating the sample to inactivate the proteinase is ≤25% of that of the control, and the residual activity is determined in the following analytical step, i.e., i) In a 1000 μl or 250 μl cuvette A step of incubation with 10-50 mU / mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl pH 8, 10 mM CaCl2, and optionally 1% DMSO. ii) A step of analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at 410 nm (ε = 8800 M - 1 cm - 1) using a spectrophotometer at a temperature of ≤ 40°C for 2 minutes, where 1 unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a selected temperature. iii) A step to compare the activity observed in step ii) with the activity observed in the same amount of the same proteinase that was not heat-treated but was maintained under the same conditions as the heat-treated proteinase, using the same analysis. A method of measurement.

4. A method for releasing a target biomolecule in a sample from a second molecule bound to it via one or more peptide bonds, a) A step of contacting the sample with a proteinase, wherein the proteinase includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:

1. b) Incubating the sample under conditions that enable the release of the target biomolecule by digestion of one or more peptide bonds, c) A step of heating the sample in any of the following i) to iii) to inactivate the proteinase; i) At a temperature of 53°C to 58°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 300 mM; ii) At a temperature of 58°C to 63°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 50 mM; or iii) At a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 210 mM, and d) The step of optionally removing the target biomolecule from the sample. Includes, The residual proteinase activity of the proteinase after the step of heating the sample to inactivate the proteinase is ≤25% of that of the control, and the residual activity is determined in the following analytical step, i.e., i) In a 1000 μl or 250 μl cuvette A step of incubation with 10-50 mU / mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl pH 8, 10 mM CaCl2, and optionally 1% DMSO. ii) A step of analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at 410 nm (ε = 8800 M - 1 cm - 1) using a spectrophotometer at a temperature of ≤ 40°C for 2 minutes, where 1 unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a selected temperature. iii) A step to compare the activity observed in step ii) with the activity observed in the same amount of the same proteinase that was not heat-treated but was maintained under the same conditions as the heat-treated proteinase, using the same analysis. A method of measurement.

5. The method according to any one of claims 1 to 4, wherein step ii) heating the sample to inactivate the proteinase comprises heating the sample at a temperature of 58°C to 63°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 100 mM.

6. The method according to any one of claims 1 to 5, wherein step ii) heating the sample to inactivate the proteinase includes the step of heating the sample at a temperature of 60°C to 63°C.

7. The method according to any one of claims 1 to 4, wherein step iii) heating the sample to inactivate the proteinase comprises heating the sample at a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 250 mM.

8. The method according to any one of claims 1 to 4, wherein step i) heating the sample to inactivate the proteinase comprises heating the sample at a temperature of 54°C to 58°C for at least 30 minutes, and the monovalent salt concentration in the sample is at least 300 mM.

9. The method according to any one of claims 1 to 4, wherein step iii) heating the sample to inactivate the proteinase comprises heating the sample at a temperature of 60°C to 67°C for at least 15 minutes, and the monovalent salt concentration in the sample is at least 450 mM.

10. The residual proteinase activity of the proteinase after the step of heating the sample to inactivate the proteinase is ≤10% of that of the control, and the residual activity is determined in the following analytical step, i.e., i) In a 1000 μl or 250 μl cuvette 10-50 mU / mL heat-treated proteinase, 1 mM substrate Suc-Ala-Ala-Pro-Phe-pNA, ≤15 mM NaCl, 0.1 mM Tris-HCl pH 8, 10 mM CaCl ２ And optionally, a step of incubating 1% DMSO, ii) 410nm (ε=8800M －１ . cm －１ A step of analyzing the cleavage of the substrate to 4-nitroaniline by measuring the increase in absorbance at ) using a spectrophotometer for 2 minutes at a temperature of ≤40°C, where 1 unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at a selected temperature. iii) A step to compare the activity observed in step ii) with the activity observed in the same amount of the same proteinase that was not heat-treated but was maintained under the same conditions as the heat-treated proteinase, using the same analysis. The method according to any one of claims 6, 8, or 9, as measured by

11. The method according to any one of claims 1 to 10, wherein the pH of the sample is 6.5 to 9.

5.

12. A method according to any one of claims 1 to 11, wherein the sample is One or more further enzymes selected from the group consisting of nucleases, DNA or RNA polymerases, reverse transcriptases, DNA or RNA ligases, methylases, transferases, topoisomerases, guanylyl transferases, phosphatases, transposases, kinases, helicases, restriction enzymes, and glycosylases. A method that includes this.

13. A method according to any one of claims 1 to 12, The sample contains one or more nucleic acid molecules, The method, following the step of heating the sample to inactivate the proteinase, includes the following steps, namely: i) Nuclease-mediated digestion of one or more nucleic acid molecules, ii) Phosphorylation or dephosphorylation of one or more nucleic acid molecules, iii) Linking of one or more nucleic acid molecules A method comprising the proteinase without prior removal or dilution.

14. A method according to any one of claims 1 to 13, The aforementioned sample contains one or more RNA molecules, A method comprising, following the step of heating the sample to inactivate the proteinase, a step of reverse transcription without prior removal or dilution of the proteinase.

15. A method according to any one of claims 1 to 14, The aforementioned sample contains one or more DNA molecules, A method comprising the step of nucleic acid polymerization, following the step of heating the sample to inactivate the proteinase, without prior removal or dilution of the proteinase.

16. A method according to any one of claims 1 to 15, The sample contains one or more virus particles or cells, A method comprising the step of heating the sample to inactivate the proteinase, followed by a step of cell lysis without prior removal or dilution of the proteinase.

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