Thermolabile proteinases

Abstract

To provide a composition containing a proteinase which is inductively thermolabile under specific conditions and an enzymatically active fragment thereof, and to provide methods comprising the inactivation of such proteinases or enzymatically active fragments thereof.SOLUTION: The invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, the proteinase comprising a specific amino acid sequence or comprising an amino acid sequence which is at least about 70% identical to the specific amino acid sequence, in which 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. The invention further provides methods comprising the inactivation of such proteinases or enzymatically active fragments thereof, in which the method comprises the step of heating the sample to inactivate the proteinase or enzymatically active fragment, and in which i) the concentration of free calcium in the sample is ≤about 80 μM; or ii) the concentration of monovalent salt in the sample is ≥about 20 mM.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compositions comprising inducible heat-labile proteinases and their uses, particularly in the isolation of nucleic acids. [Background technology]

[0002] Proteinases (also called peptidases, proteases and proteolytic enzymes) are capable of hydrolyzing peptide bonds in proteins. Proteinases are widely used in a wide range of processes in industry, biotechnology and molecular biology research techniques. For example, proteinases are used in the digestion of unwanted proteins during nucleic acid purification, in the preparation of recombinant antibody fragments, in peptide sequencing in proteomics, and in the proteolytic digestion of proteins.

[0003] In order to successfully extract nucleic acids from a sample, lysis of the cell walls / membranes is necessary. Various physical or chemical methods can be employed, which can be enhanced by the addition of proteases. Membrane lipids are then removed by application of detergents or surfactants or by osmotic lysis in hypotonic solutions. The removal of proteins from the sample using proteinases is then considered the best approach. Proteinases digest contaminating, i.e. unwanted, proteins, polypeptides and peptides present in the sample by hydrolysis of peptide bonds. Proteinases also degrade nucleases and other enzymes that may be present in the sample and that may degrade nucleic acids.

[0004] Removal of proteins is particularly important during nucleic acid purification in preparation of nucleic acid samples for amplification reactions (e.g., PCR and RT-PCR). In cells, nucleic acids are generally present bound to proteins. For example, genomic DNA in eukaryotic cells is bound to histones, which can tightly package the DNA into chromatin. Many molecular biology techniques, such as PCR, require naked DNA, i.e., DNA that is not bound to histones, because the tight packaging of DNA in chromatin inhibits access to the nucleic acid for enzymes that interact with DNA, such as polymerases, nucleases, etc.

[0005] Nucleic acid purification is a multi-step process involving time, cost and sample loss, which is particularly undesirable when the starting amount of nucleic acid in the sample is low, e.g., isolated from a few hundred cells or less, e.g., from fine needle aspirated or liquid biopsies.

[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 at the carboxyl side of aromatic, aliphatic or hydrophobic amino acid residues. Due to the broad specificity of proteinase K, proteinase K can be useful in the digestion of unwanted proteins in a sample. Proteinase K also rapidly inactivates nucleases that may degrade nucleic acids present in the sample. Proteinase K is active in the presence of chemicals used in DNA extraction processes that denature other proteins, such as SDS and urea, chelating agents such as 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 is necessary to inactivate or remove it prior to the addition of downstream proteins / enzymes, such as polymerase and reverse transcriptase, without which the proteinase will degrade the downstream proteins / enzymes due to its non-specific activity.

[0008] Proteinase K can be removed from the sample, for example, by phenol extraction or CsCl isopycnic ultracentrifugation. Alternatively, the sample volume can be increased, thereby attenuating the Proteinase K activity therein. However, the act of physically removing the enzyme from the sample risks contamination and loss of the desired product. Dilution is not ideal in most cases, especially when the sample size is small.

[0009] Proteinase K inactivation procedures vary but generally involve heating to high temperatures. In many cases, however, the heat required to inactivate the protease results in degradation of one or more desired products in the sample. Proteinase K inactivation procedures include heating to 75°C for 5 minutes (BioRad procedure), heating to 95°C for 10 minutes (New England BioLabs procedure), and 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 alternative protein digestion methods that can shorten the workflow without losing desired material from the sample, particularly during nucleic acid purification procedures, and that do not involve harsh proteinase inactivation conditions.

[0011] Proteinase X (also referred to as "Serratia peptidase" and "SPRK") is a proteinase K-like proteinase isolated from Serratia species. Studies of proteinase X (Non-Patent Document 1) confirmed that, like proteinase K, it has high thermostability and further confirmed that proteinase X indeed has a higher optimum 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 to 50°C in the presence of various concentrations of SDS (a detergent commonly used in the isolation of nucleic acids from samples). According to the teachings of Larsen et al., proteinase X does not exhibit the typical heat-labile characteristics 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 Summary of the Invention Effect of the Invention

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

[0014] Such inducible thermolability 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 with inducible thermolability in a wide range of molecular biology applications. [Brief description of the drawings]

[0015] The invention will now be described by way of non-limiting examples with reference to the following figures.

[0016] [Figure 1] Figure 1 shows the proteinase activity at various temperatures for proteinases X and K. Activity is shown as % activity relative to the maximum activity observed at 65°C with 10 mM free calcium under standard assay conditions. [Diagram 2] Figure 1 shows the activity of Proteinase X at various temperatures with 0 μM, 5 μM or 10 mM free calcium in the assay buffer. Results are shown relative (%) to the standard assay conditions of 10 mM free calcium at each temperature. [Diagram 3] Figure 1 shows the activity of Proteinase K at various temperatures with 0 μM, 5 μM or 10 mM free calcium in the assay buffer. Results are shown relative (%) to the standard assay conditions of 10 mM free calcium at each temperature. [Figure 4] FIG. 1 shows the extent of inactivation of Proteinase X after heating for 15 and 30 min at 60° C. in the presence of various free calcium concentrations. Activity is shown as % remaining activity relative to the control (kept on ice, no heating step). [Diagram 5] FIG. 1 shows the extent of inactivation of proteinase K after heating for 15 and 30 min at 60° C. in the presence of various free calcium concentrations. Activity is shown as % remaining activity relative to the control (kept on ice, no heating step). [Figure 6]FIG. 1 shows the extent of inactivation of proteinase X and proteinase K after heating for 15 and 30 min at 60° C. in the presence of various free calcium concentrations. Activities are shown as % activity relative to the maximum activity observed for the same proteinases in the same buffer (containing 10 mM CaCl2) kept on ice without heat treatment. [Figure 7] Figure 1 shows the effect of NaCl concentration on the heat-labile profiles 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 (containing 10 mM CaCl2) kept on ice without heat treatment. [Figure 8] Figure 1 shows the inactivation of Proteinase X in the presence of various concentrations of NaCl at 50 and 60° C. Activity is shown as % activity relative to the maximum activity observed for Proteinase X in the same buffer (containing 0 M NaCl, 0.03 mM CaCl2) kept on ice without heat treatment. [Figure 9] Figure 1 shows the inactivation of Proteinase X in the presence of various concentrations of NaCl at 60°C. Activity is shown as % activity relative to the maximum activity observed for Proteinase X in the same buffer (containing 0M NaCl, 30 µM CaCl2) kept on ice without heat treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Thus, in one aspect, the invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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; i) the concentration of free calcium in the composition is ≦about 80 μM; or ii) the concentration of the monovalent salt in the composition is ≧20 mM.

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

[0019] The present invention relates to proteinases comprising the sequence of SEQ ID NO:1, and proteinases comprising a sequence that is at least about 70% identical to SEQ ID NO:1. Proteinases comprising a sequence that is at least about 70% identical to SEQ ID NO:1 are referred to herein as "variants of SEQ ID NO:1", "mutant proteinases" or simply "mutants". By definition, variants of SEQ ID NO:1 of the invention are also proteinases according to the invention, i.e., they have proteinase activity. References herein to a proteinase of the invention refer to a proteinase of SEQ ID NO:1 and to the variants of the invention described herein. References anywhere herein to a proteinase (or mutant proteinase) also refer to enzymatically active fragments thereof, unless otherwise indicated.

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

[0021] In a preferred embodiment, the solution of the invention is a reagent that is applied to a sample containing one or more polypeptides. Such a reagent is applied to a sample so that the proteinase in said reagent digests said 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 invention or an enzymatically active fragment thereof, and 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 may be used. It will be within the ability of the skilled artisan to identify suitable buffering agents and their range of inclusion for their intended purpose. Preferably, the buffering agent has a buffering range of pH 6.5-9.5, preferably pH 6.8-9.2, more preferably pH 7-9, more preferably pH 7.5-8.5, 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 1-250 mM, more preferably 10-200 mM, more preferably 20-150 mM, more preferably 25-100 mM.

[0023] When present, Tris-HCl is preferably present at a concentration of 25-200 mM, more preferably 50-150 mM, more preferably about 100 mM.When present, HEPES is preferably present at a concentration of 5-50 mM, more preferably 10-40 mM, more preferably 20-30 mM, 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, more preferably about 8.0.

[0025] Compositions and samples of the invention may further comprise DMSO, if present, preferably at a concentration of 0.1-5% w / w, more preferably 0.5-2.5% DMSO, more preferably about 1% DMSO.

[0026] The term "sample" refers to any composition that contains one or more polypeptides other than the proteinases of the invention.

[0027] In a further aspect, the invention provides a sample comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0028] Preferably, the free calcium concentration in said sample is ≦about 80 μM and the monovalent salt concentration in said sample is ≧about 20 mM.

[0029] Samples that do not contain the proteinases or enzymatically active fragments described herein are not an aspect of the present invention. References herein to "samples of the present invention" refer only to samples that do in fact contain the proteinases or enzymatically active fragments described herein.

[0030] The following discussion of preferred optional features and embodiments of the "sample" applies to both samples of the present invention and samples that are not part of the present invention but to which one composition of the present invention may be applied.

[0031] The proteinases of the invention are useful in methods for purification, i.e. isolation, i.e. extraction, of a biomolecule of interest from a sample containing contaminating, i.e. unwanted, polypeptides. Thus, in a preferred embodiment, the sample comprises one or more contaminating polypeptides and one or more biomolecules of interest. As used herein, the term "contaminating polypeptide" is synonymous with "unwanted polypeptide" and refers to any polypeptide in the sample other than the proteinase of the invention and any polypeptide of interest. That is, the contaminating polypeptide is one that is digested by the proteinase of the invention in order to purify or modify one or more biomolecules of interest.

[0032] The proteinases of the invention are also useful in a method for liberating a biomolecule of interest from a molecule, preferably a polypeptide, bound to it via one or more peptide bonds by hydrolyzing one or more of said peptide bonds. Thus, in a preferred embodiment, a sample comprises a biomolecule of interest bound to a molecule, preferably a polypeptide, via one or more peptide bonds. The one or more peptide bonds can be cleaved by a proteinase of the invention or an enzymatically active fragment thereof.

[0033] The term "biomolecule of interest" as used herein refers to any biomolecule in a sample, including one or more contaminating polypeptides, from which it is desired to purify the biomolecule or from which it is bound via one or more peptide bonds. Preferably, the biomolecule of interest is a nucleic acid molecule, preferably a DNA or RNA molecule. Alternatively, the biomolecule of interest is itself a polypeptide. The biomolecule of interest is not a proteinase or an enzymatically active fragment thereof that is useful in the present invention.

[0034] Preferably, the sample comprises cellular material. Preferably, the sample comprises a crude cell extract. Preferably, the sample comprises a partially purified cell extract. Preferably, the sample comprises a cell population. The cells in said 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 a fine needle biopsy. Preferably, the sample comprises encapsulated viruses. Proteinases can be used to digest the protein capsule of the virus in order to release the RNA / DNA therein 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, 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, more preferably ≦0.1 μl.

[0036] The term "polypeptide" as used herein refers to proteins, polypeptides, peptides, oligopeptides and tripeptides, i.e., any molecule containing three or more amino acids linked via peptide bonds, that can be hydrolyzed by the proteinase of the present invention. Proteinase X is known to hydrolyze peptides as short as three amino acids in length. The terms "protein", "polypeptide" and "peptide" are used interchangeably herein, and the use of each is expressly intended to refer to any and all proteins, polypeptides and peptides. Thus, the polypeptides present in the samples described herein are substrates for the proteinases of the present invention or enzymatically active fragments thereof. Proteinases and enzymatically active fragments thereof are clearly themselves polypeptides. However, the term "polypeptide" as used herein expressly excludes proteinases and enzymatically active fragments thereof that are useful in the present invention.

[0037] The terms "digest", "hydrolyze", "degrade" and "cleave" are used interchangeably herein and refer to the hydrolysis of peptide bonds within polypeptides in a sample. Digestion can be partial or complete. Proteinases useful in the present invention are non-specific and, given sufficient time, will completely digest proteins in a sample under conditions that allow enzyme function.

[0038] The compositions and samples of the invention comprise a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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.

[0039] The amino acid sequence of SEQ ID NO:1 is as follows: ADQPSPTWGIDRIDQRNLPLDNNYHTDYDGSGVTAFVIDTGVLNTHNEFGGRASSGYDFIDNDYDATDCNGHGTHVAGTIGGSTYGVAKNVNVVGVRVLNCSGSGSNSGVIAGINWVKNNASGPAVANMSLGGGASQATDDAVNAAVAAGITFVVAAGNDNSNACNYSPARAADAITVGSTTSNDSRSSFSNYGTCLDIYAPGSSITSSWYTSNSATNTISGTSMASPHVAGVAALYLDENPNLSPAQVTNLLKTRATADKVTDAKTGSPNKLLFSLANDD

[0040] The proteinase of SEQ ID NO:1 is described in Larsen et al., (2006) FEBS The proteinase is based on the amino acid sequence of proteinase X from a marine Serratia species isolated in the north of Norway, as described in the Journal 273:47-60. Proteinase X is a proteinase K-like proteinase [EC 3.4.21]. The terms "proteinase X", "protease X", "ProtX", "PRX", "peptidase X", "Serratia peptidase" and "SPRK" are used interchangeably 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 of 629 amino acids (65.5 kDa, SEQ ID NO:3).

[0042] SEQ ID NO:2:

[0043] SEQ ID NO: 3: MHKKHLIAVAVATGLAYFPVNANEYQATMVNVPQSKAIKDTYIVVFNTPSVLNLSNNNTIAEFAVQQAESLVNQYDVRVMKNFGNVLNGVLINASAQQVKALLKDPNVKYVEQDQVMSVTPMMEANADQPSPTWGIDRIDQRNLPLDNNYHTDYDGSGVTAFVIDTGVLNTHNEFGGRASSGYDFIDNDYDATDCNGHGTHVAGTIGGSTYGVAKNVNVVGVRVLNCSGSGSNSGVIAGINWVKNNASGPAVANMSLGGGASQATDDAVNAAVAAGITFVVAAGNDNSNACNYSPARAADAITVGSTTSND SRSSFSNYGTCLDIYAPGSSITSSWYTSNSATNTISGTSMASPHVAGVAALYLDENPNLSPAQVTNLLKTRATADKVTDAKTGSPNKLLFSLANDDGGCGNDCPVDETQLQNNVGIAISGATGSATYYYIDVPANAASLGINLAGGSGDADIYVSQGQK PTTTSYQCRPYQNGNNESCNFTAPTAPTAGRWYVMVQGYSNYANAQLTASYNLNGGGNCTDANCLSNGVPVTNLSGRTGTEALYKIVVPANSQLSITTSGGTGDVDLYVKAGTVPTTTSYDCRPYKNGNNESCSITVTQAGTYHVMLRGYANYSSVQLSASY

[0044] The proteinase of SEQ ID NO:3 consists of an N-terminal prepro sequence of 126 residues, a catalytic domain of 278 residues, and two C-terminal domains (repeat sequences) of a combined total of 225 residues.

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

[0046] The expressed proteinase of SEQ ID NO: 4 excludes both C-terminal domains but the first 3 residues of the first C-terminal domain and excludes the first 22 residues of the N-terminal prepro domain of SEQ ID NO: 3. This form of the 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 to a mature protein of approximately 34 kDa, 281 residues, which contains the catalytic domain and the three C-terminal amino acid residues and retains full catalytic activity (SEQ ID NO:1).

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

[0050] Thus, in each and every aspect and embodiment of the invention, the proteinase may comprise 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 precursor / 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 results in the use or presence of the proteinase of SEQ ID NO:1 for further autolytic processing. For simplicity, references anywhere in this specification to SEQ ID NO:1 are expressly intended to also represent SEQ ID NO:3 and / or SEQ ID NO:4.

[0051] The compositions and samples of the invention comprise a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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.

[0052] By "at least about 70%" it is meant that the sequence homology may be at least 69%, 69.5% or 69.9%.

[0053] In a preferred embodiment, the proteinase of the invention comprises or consists of an amino acid sequence which 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%, such as at least 98%, 99% or 99.5% identical to SEQ ID NO:1.

[0054] The sequence identity percentage according to the invention can be calculated by any of the widely available algorithms, for example using the ClustalW2 Multiple Sequence Alignment program (http: / / www.ebi.ac.uk / Tools / clustalW2) with default 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 percentage of homology 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 alignment of the variant sequence with SEQ ID NO:1 above.

[0056] Variants of SEQ ID NO:1 include amino acid sequences in which one or more amino acids of said SEQ ID NO:1 have been conservatively substituted or replaced with modified versions of said one or more amino acids or with non-naturally occurring amino acids, such as D-isomers of said one or more amino acids. Preferably, such substitutions and modifications are silent in that the modified form of the exonuclease of the invention has the same enzymatic and inactivation properties as the unmodified form.

[0057] In some embodiments, the proteinase of the invention comprises (or consists of) an amino acid sequence having single or multiple amino acid changes (additions, substitutions, insertions or deletions) compared to SEQ ID NO: 1. Such a sequence may preferably contain up to 10, such as only 1, 2, 4, 4, 5, 6, 7, 8, 9 or 10, preferably up to 5, such as 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 the variant sequence with SEQ ID NO: 1 as described above.

[0058] Preferably, the modification is silent in that the modified proteinase of the invention has the same enzymatic and inactivation properties as the unaltered form. The substitution can be with a conserved or non-conserved amino acid. Preferably, the modification is a conservative amino acid substitution. The modification can be a substitution with a modified version of one or more amino acids of SEQ ID NO: 1, or with a non-natural amino acid, for example, a D-isomer of the amino acid.

[0059] The proteinase comprising an amino acid sequence at least about 70% identical to SEQ ID NO:1 can be obtained from a prokaryotic organism found in the cold water niche. By "prokaryotic organism" is meant any organism without a cell nucleus, i.e., any organism from the domains of bacteria and archaea. Preferably, the organism is a bacterium. Preferably, the organism is not a eukaryotic organism, e.g., an organism classified in the taxonomic kingdoms Animalia, Plantae, Fungi or Protista. More preferably, the organism is selected from the genera Shewanella, Halomonas, Vibrio, Cyclomonas, Moritella and Serratia, preferably Serratia.

[0060] In some embodiments, compositions and samples of the invention comprise a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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. References elsewhere in this specification to SEQ ID NO: 1 also apply mutatis mutandis to SEQ ID NO: 3 and SEQ ID NO: 4.

[0061] Preferably, the proteinase of the present invention consists of the amino acid sequence of SEQ ID NO:1, 3 or 4, or consists of 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 consists of 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 invention may comprise further amino acids N-terminal or C-terminal to the sequence SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, these further amino acids are identical to the amino acids found at the N- or C-terminus of the sequence SEQ ID NO: 1 in the 629 amino acid sequence of SEQ ID NO: 3, respectively. Preferably, the proteinase comprises 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 amino acids N-terminal and / or C-terminal to the sequence SEQ ID NO: 1 or SEQ ID NO: 4, preferably said N- and / or C-terminal amino acids being identical to the respective amino acids found at the N- and / or C-terminus of the sequence SEQ ID NO: 1 or SEQ ID NO: 4 in the sequence SEQ ID NO: 3.

[0066] The proteinases present in the solution or sample of the present invention may be in the form of modified forms, for example fusion proteins, in which they are linked to further peptides at their N-terminus and / or C-terminus, either directly or indirectly via a peptide linker sequence. Preferably, the further N-terminal peptide, together with the linker sequence, if present, comprises a sequence of 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 amino acids in length. Preferably, the further C-terminal peptide, together with the linker sequence, if present, comprises a sequence of 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 amino acids in length.

[0067] Thus, in one embodiment, the invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 concentration of free calcium in the composition is ≦about 80 μM; or ii) the concentration of monovalent salt in the composition is ≧20 mM; 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.

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

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

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

[0071] Enzymatically active fragments of the proteinases and mutant proteinases of the invention are also provided. Enzymatically active fragments are fragments that have proteinase activity. Enzymatically active fragments can comprise 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, more preferably at least 275, 276, 277, 278, 279 or 280 amino acids of the sequence SEQ ID NO:1.

[0072] Preferably, the enzymatically active fragment of the invention comprises an N-terminal truncation of not more than 5 amino acids, preferably not more than 4, 3, 2 or 1 amino acid of the sequence SEQ ID NO: 1. Alternatively or in addition, the enzymatically active fragment of the invention preferably comprises a C-terminal truncation of not more than 30 amino acids, preferably not more than 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 amino acid of the sequence SEQ ID NO: 1.

[0073] Viewed another way, the length of an 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 SEQ ID NO:1 or the length of an amino acid sequence that is at least 70% identical to SEQ ID NO:1.

[0074] As such, the enzyme fragment of the invention is 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 SEQ ID NO: 1. Methods for determining the percentage of homology are described above.

[0075] Throughout this application, reference to a proteinase of the invention also refers to enzymatically active fragments thereof, unless otherwise specified.

[0076] Proteinases (also called peptidases or proteases) are enzymes that carry out protein catabolism by proteolysis, i.e. hydrolysis of peptide bonds. Thus, proteinases according to the invention are enzymes that have proteinase activity, i.e. protease activity, i.e. peptidase activity. The variants and enzymatically active fragments of the invention also have proteinase activity.

[0077] Proteinases of the invention that are variants or modifications 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 the proteinase of SEQ ID NO:1.

[0078] An 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 the proteinase of SEQ ID NO:1.

[0079] Suitable assays for assaying proteinase activity are known in the art, and any such assay can be used to measure the proteinase activity of a particular polypeptide. Thus, such assays can be used to measure the proteinase activity of the proteinases, mutant proteinases, and enzymatically active fragments thereof of the present invention.

[0080] A preferred assay involves the enzymatic cleavage of a substrate to a product detectable by a spectrophotometer, preferably at 410 nm (ε=8800 M -1 .cm -1Examples of suitable methods include the analysis of cleavage of Suc-Ala-Ala-Pro-Phe-pNA to 4-nitroaniline, which can be analyzed by measuring the increase in absorbance at 37° C.). It is within the ability of one skilled in the art to identify suitable substrates and equipment to generate and detect such absorbance. Preferably, absorbance is detected using a spectrophotometer or 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 also easily determine the incubation temperature and analysis time appropriate for their purpose. Those skilled in the art will know that the temperature at which the analysis is carried out should be a temperature that does not result in inactivation of the proteinase of the present invention, for example, below 40° C., preferably 25° C. Suitable analysis times can be between 30 seconds and 5 minutes, for example, 2 minutes.

[0082] The skilled artisan will also know that the appropriate concentration of enzyme to include in the assay will depend on the detection range of the spectrophotometer used. The skilled artisan will also know that a dilution step may be necessary before performing the assay in order to reach a concentration that can be detected by the spectrophotometer and an enzyme activity level in the assay sample. An activity 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 SEQ ID NO: 1 used in this example.

[0083] One of skill in the art would be readily able to formulate the remainder of the analysis mixture for their intended purpose. The analysis mixture can include a pH buffer, preferably Tris-HCl, pH 8, as described elsewhere herein.

[0084] The assay mixture preferably contains excess calcium (e.g., ≧2 mM, preferably about 10 mM) to avoid low calcium conditions that may further inactivate the proteinase. NaCl may be present at low concentrations (e.g., ≦15 mM) to avoid salt-induced thermolability of the proteinase. Also, one of skill in the art can readily dilute solutions to provide the required concentrations of components, as needed.

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

[0086] A preferred assay for measuring proteinase activity is Assay A (used in the Examples herein). Assay A is carried out 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 Incubating with 1% DMSO (optional), and 410nm (ε=8800M -1 .cm -1 ) for 2 min at 25° C. or 37° C. using a spectrophotometer (e.g. Ultrospec 2000, Pharmacia Biotec, Sweden). 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 at 25° C. or 37° C.

[0087] To measure proteinase activity, alternative methods can be readily devised by those skilled in the art.

[0088] Any suitable assay can be used to measure the relative activity of an enzymatically active fragment, variant or modification of a proteinase of the invention compared to the activity of the proteinase of SEQ ID NO: 1. The skilled artisan will be well aware that the same techniques and conditions used to assess the activity of the proteinase of SEQ ID NO: 1 should also be used to assess the activity of the fragment, variant or modification. Preferably, the assay used is Assay A described above.

[0089] According to the above preferred assay A, proteinase X has a specific activity of about 65 U / mg (65.2 U / mg). Preferably, the mutants, enzyme fragments and modifications of the present invention have a specific activity of 40-100 U / mg, more preferably 50-80 U / mg, more preferably 60-70 U / mg, more preferably 62.5-67.5 U / mg, preferably about 65 U / mg or 65.2 U / mg, as measured by the above assay A. 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 the above assay A.

[0090] The compositions and samples of the invention preferably contain ≦80 μM free calcium. As used herein, the term "free calcium" refers exclusively to calcium ions that are free, i.e., unbound, in the compositions and samples of the invention, i.e., calcium ions that are not bound to any protein or other components present in the compositions and samples of the invention, i.e., liberated calcium. References throughout to calcium concentrations in compositions and solutions of the invention refer to free calcium concentrations.

[0091] Proteinase X (SEQ ID NO:1) contains a calcium binding site formed by residues Asp11, Asp14, Gln15, Asp21 and Asn23. 2+The ion coordinates 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. Thus, the proteinases useful in the present invention naturally contain this bound calcium ion. Bound calcium ions are also present when the proteinases useful in the present invention are recombinantly produced. Any environment used to produce a proteinase will necessarily contain calcium. This means that bound ions are present in the folded proteinase structure. Such bound calcium ions are not "free calcium" as the term is used herein.

[0092] The skilled person preparing the composition or sample of the invention can ensure that the required calcium concentration is directly obtained by simply selecting the appropriate solutes and solvents to be placed in the solution or sample. It is within the capabilities of the skilled person to remove the free calcium from the solution by dialysis against a large volume of calcium-free buffer, for example 500-10000, preferably 5000 volumes of calcium-free buffer. Suitable buffers will be clear to the skilled person and will preferably be the buffers described elsewhere herein, for example 10 mM Tris-HCl, pH 7-9. Preferably, said buffer further comprises glycerol.

[0093] The concentration of free calcium in any given solution or sample can also be readily determined by one of skill in the art. Standard methods for measuring calcium are well known in the art and include, for example, titration with a standard solution of EDTA.

[0094] Bound calcium ions can be removed from proteinases by application of strong calcium chelators, such as EDTA. However, exposure of many target biomolecules to EDTA is undesirable. Furthermore, EDTA removes Mg, which is required for many nucleic acid-related enzymes (e.g., polymerases, nucleases). 2+These enzymes are used in molecular biology techniques following sample preparation with proteinases, and the presence of EDTA is particularly problematic in this context. EDTA binds free Mg ions in subsequent downstream steps. 2+ This introduces uncertainty about the concentration, which is undesirable. Mg 2+ is necessary for enzyme function, but in excess causes RNA degradation. In any method of digesting a polypeptide, it is desirable to omit the reagent, since including and then removing the reagent increases the cost, time and workflow of the process.

[0095] The inventors have surprisingly found that the mere absence of free calcium is sufficient to induce heat-labile properties in the proteinase of the present invention. The inventors have surprisingly demonstrated for the first time that the heat-labile properties of proteinase X are 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, which may bind to the proteinase and contribute to its stability and structure, for example with EDTA. On the contrary, a low concentration of free calcium ions is surprisingly sufficient to induce heat-labile properties in the enzyme. Such an inducible property is unexpected and is not observed in proteinase K, a standard proteinase used in molecular biology applications, as shown in the examples.

[0096] Thus, preferably, the proteinases and enzymatically active fragments thereof of the present invention contain calcium ions bound thereto. In other words, preferably, the compositions and samples of the present invention contain the proteinases and enzymatically active fragments thereof in their native calcium-bound state. In this scenario, calcium can be considered as a "cofactor" for the protein, and thus the proteinases are in their "holoenzyme form" in the compositions and samples of the present invention. Holoenzymes are biochemically active enzymes formed by the combination of the apoenzyme and its cofactor, in this case calcium.

[0097] Thus, preferably the compositions and samples of the present invention are essentially free of EDTA, preferably free of EDTA, and more preferably free of any calcium chelating agents. Preferably, the proteinases and enzymatically active fragments thereof present in the solutions and samples of the present invention are not exposed at any time to EDTA, and more preferably are not exposed to any calcium chelating agents.

[0098] Alternatively viewed, the invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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, i) the calcium concentration in the composition is ≦about 80 μM and the composition is essentially free of EDTA, preferably essentially free of calcium chelating agents; or ii) providing a composition, wherein the concentration of monovalent salt in said composition is ≧20 mM.

[0099] Alternatively viewed, the invention provides a sample comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA, preferably essentially free of calcium chelators; or ii) providing a sample, wherein the concentration of a monovalent salt in said sample is ≧20 mM.

[0100] Alternatively viewed, the invention provides a composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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, i) the calcium concentration in the composition is ≦about 80 μM and the composition is essentially free of EDTA, preferably essentially free of calcium chelating agents; or ii) the concentration of monovalent salt in the composition is ≧20 mM; The proteinase or enzymatically active fragment thereof further comprises an additional 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 and optional features and embodiments described elsewhere herein apply mutatis mutandis to the "alternatively viewed" aspect of the invention. In particular, the "free calcium" concentrations disclosed elsewhere herein are preferred concentrations of calcium (not defined as "free calcium") in the alternatively viewed 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 which refer to the concentration of "calcium" rather than "free calcium" and specify that the samples or compositions of the methods of the invention are essentially free of EDTA.

[0102] Preferably, the compositions and samples of all aspects 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, more preferably essentially free of calcium chelating agents.

[0103] "Essentially free" of, for example, EDTA or free calcium means that the compositions and samples are essentially free of EDTA or free calcium, but does not mean that there is a strict requirement that they be completely devoid of EDTA or free calcium. For example, due to the presence of small amounts of EDTA or free calcium in commercial products or storage solutions used to prepare the compositions or samples of the present invention, there is the possibility of minor levels of EDTA or free calcium even after steps have been taken to prevent the presence of EDTA or free calcium. Although 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 chelators means that the ratio of the concentration of Proteinase X to the concentration of calcium chelator, e.g., EDTA or EGTA, in the sample is at least 10: 1, more preferably at least 100: 1, more preferably at least 1000: 1. In cases where the sample contains more than one calcium chelator, these ratios are the ratio of the concentration of Proteinase X to the total concentration of calcium chelator 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 absent for the intended purpose, i.e., not present at levels that would substantially alter the heat sensitivity of the proteinase of the invention compared to the level or degree that would occur in the absence of free calcium.

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

[0107] Preferably, the compositions and samples of the present invention are completely free of, i.e. free of, 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, more preferably ≦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, more preferably ≦about 2.5 μM. The term "≦about X" is the same as "0 to X mM". Preferably, the composition and sample of the present invention does 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, 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, 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" being as defined elsewhere herein.

[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, 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, 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, preferably about 2 to about 10 μM.

[0113] As noted above, preferably the compositions and preparations of the present invention preferably contain free calcium in these concentrations and are essentially free of EDTA and preferably essentially free of calcium chelating agents.

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

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

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

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

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

[0119] Preferably, said further enzymes are selected from the group consisting of nucleases (preferably deoxyribonucleases, exonucleases, Bal31 nuclease, ribonuclease, mung bean 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 phosphatase, preferably thermolabile alkaline phosphatase, more preferably shrimp alkaline phosphatase), kinases, helicases, restriction enzymes and glycosylases. The composition or sample preferably comprises a combination of such further enzymes.

[0120] Preferably, the composition or sample comprises a DNA polymerase or a reverse transcriptase. If the solution or sample comprises cells or cellular material, they preferably comprise such additional enzymes that are exogenous enzymes, i.e. that are not expressed in the cells within the sample or in the cells from which the cellular material in the sample originates. In other words, the additional enzymes are applied to the sample, rather than being provided 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 interchangeably throughout. A monovalent salt is a salt that comprises 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 contain carbon. Preferably, the salt is a sodium or potassium salt, more preferably a sodium salt. Preferably, the salt is sodium chloride (NaCl) or potassium chloride (KCl), most preferably sodium chloride.

[0122] In another aspect, the compositions and samples of the present invention preferably contain monovalent counterions. 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 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 the preferred concentrations of monovalent counterions, and vice versa.

[0123] As shown in the Examples, the inventors have shown for the first time that increasing monovalent salt concentration induces heat labile proteinase X, whereas increasing monovalent salt concentration stabilizes and heat inactivates proteinase K. This result is particularly surprising. Proteinase X of SEQ ID NO: 1 is obtained from a saltwater organism 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 samples and compositions of the present invention containing a monovalent salt, 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, more preferably about 8.0.

[0125] Preferably, the monovalent salt concentration in the compositions and samples of the 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, more preferably ≧about 175 mM. Optionally, the monovalent salt concentration in the compositions and samples of the 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 1M, preferably ≦about 500mM, preferably ≦about 350mM. The monovalent salt concentration in the composition and sample of the present invention is preferably about 20mM to about 1M, preferably about 20 to about 500mM, preferably about 20 to about 400mM, preferably about 20 to about 350mM, preferably about 30 to about 350mM, preferably about 40 to about 350mM, preferably about 50 to about 350mM, preferably about 75 to about 350mM, preferably about 100 to about 350mM, preferably about 125 to about 350mM, preferably about 150 to about 350mM, preferably about 175 to about 350mM. Optionally, the monovalent salt concentration in the composition and sample of the present invention is about 200 to about 350mM, or about 300 to about 350mM.

[0127] Preferably, the monovalent salt concentration in compositions and samples of the 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, 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, 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, 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, preferably about 175 to about 200 mM.

[0131] Preferably, the monovalent salt concentration in the compositions and samples 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, 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, 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, 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, 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, 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. andThe monovalent salt concentration includes any calcium concentration value or range disclosed herein in combination with any monovalent salt concentration value or range disclosed herein for all aspects and embodiments of the invention. Preferably, the monovalent salt is NaCl or KCl, preferably NaCl.

[0135] Preferred combinations of maximum free calcium concentration and minimum monovalent salt concentration are shown below.

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

[0137] Further, preferably, the free calcium concentration in the compositions and samples of the present invention is at least about 1 μM, more preferably at least about 2 μM.Further, preferably, the monovalent salt concentration in the compositions and samples of the present invention is not more than about 500 mM, preferably not more than about 400 mM, preferably not more than about 350 mM, preferably not more than about 300 mM, preferably not more than about 250 mM, preferably not more than about 200 mM, preferably not more than about 175 mM, preferably not more than about 150 mM.

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

[0139] [Table 2]

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

[0141] [Table 3]

[0142] 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 invention comprise a proteinase that can be inactivated under particularly mild conditions. The compositions therefore have advantageous utility in a variety of molecular biology methods involving the subsequent or prior use of other enzymes. Such methods are discussed in more detail below. Thus, in a further aspect, i) the composition of the present invention; and ii) Providing a kit comprising a second composition comprising a second enzyme.

[0146] The second enzyme is not a proteinase of the invention. Optionally, the kit comprises multiple solutions containing multiple enzymes, i.e., it comprises a third solution containing a third enzyme, optionally a fourth solution containing a fourth enzyme, etc. Each solution present in the kit of the invention preferably comprises a different enzyme, each of which is not a proteinase of the invention. Preferably, the enzymes in the second subsequent solution are independently selected from the group consisting of nucleases (preferably deoxyribonucleases, exonucleases, Bal31 nuclease, ribonucleases, mung bean nuclease or S1 nuclease), polymerases (preferably DNA polymerase or RNA polymerase), reverse transcriptases, ligases, (preferably DNA ligase or RNA ligase), methylases, transferases (preferably polynucleotide adenylyltransferases), topoisomerases, guanylyltransferases, proteinases other than proteinase X, and phosphatases or combinations thereof.

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

[0148] The compositions and samples of the present invention comprise proteinases that can be inactivated by a particularly mild heat treatment step. Thus, the compositions have advantageous utility in various molecular biology methods where application of proteinases to a sample is necessary or desirable to digest one or more polypeptides in the sample, but where it is also desirable to avoid detrimentally affecting the structure or function of one or more biomolecules present. The use of standard proteinases requires i) inactivation using high temperatures for significant periods of time that can damage the biomolecules of interest present, or ii) removal / dilution of the proteinase, which can increase workflow, time and cost, and result in loss or damage of the biomolecules of interest. The present compositions enable methods that do not require such undesirable processing steps.

[0149] The present invention provides the methods discussed below. The above definitions and preferred optional features and embodiments of the compositions, samples and kits of the present invention apply mutatis mutandis to any and all methods of the present invention. In the context of all methods of the present invention, the polypeptides, samples, proteinases, enzymatically active fragments, free calcium, free calcium concentrations, monovalent salts and monovalent salt concentrations are described elsewhere herein. In particular, any of the features disclosed above, as well as concentrations of free calcium (or calcium), monovalent salts and combinations thereof, are useful in the methods of the present invention, i.e., preferably, the samples contacted or treated in the methods of the present invention comprise a concentration of free calcium, monovalent salts or any combination thereof, as disclosed elsewhere herein.

[0150] Thus, the term "sample" contacted in the method of the invention refers to any composition comprising one or more polypeptides. Preferably, the sample comprises cellular material. Preferably, the sample comprises a crude cell extract. Preferably, the sample comprises a partially purified cell extract. 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 a fine needle biopsy. Preferably, the sample comprises encapsulated viruses. Proteinases can be used to digest the protein capsule of the virus in order to release the RNA / DNA therein 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, 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.

[0152] References to proteinase anywhere in this specification also refer to enzymatically active fragments thereof, unless otherwise specified.

[0153] In all methods of the invention, preferably the sample is essentially free of EDTA, preferably of any calcium chelating agent, as this term is defined elsewhere herein. The sample is essentially free of EDTA, preferably of any calcium chelating agent, after contact with the proteinase or an enzymatically active fragment thereof.

[0154] Thus, in a further aspect, there is provided a method for digesting a polypeptide in a sample, said method comprising the steps of contacting the sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0155] Viewed another way, the present invention provides a method for digesting a polypeptide in a sample, said method comprising the steps of contacting the sample with a proteinase or an enzymatically active fragment thereof, wherein said proteinase comprises 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; i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in said sample is ≧20 mM.

[0156] In all methods of the invention, particularly those in which the sample comprises 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, more preferably about 8.0. The present inventors have confirmed for the first time that the proteinase of the invention can be inactivated under mild conditions, including at neutral and near-neutral pH.

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

[0158] The terms "digest", "hydrolyze", "degrade" and "cleave" are used interchangeably herein and refer to the hydrolysis of peptide bonds within polypeptides in a sample. Digestion can be partial or complete. Proteinases useful in the present invention are non-specific and, given sufficient time, will completely digest proteins in a sample under conditions that allow enzyme function.

[0159] The terms "contacting" / "contacting", "applying" / "applying" and "adding to" / "adding" have their ordinary meanings and are used interchangeably herein.

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

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

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

[0163] Thus, the proteinases of the invention and enzymatically active fragments thereof are used to degrade polypeptides in a sample. In particular, the method comprises contacting the sample with the proteinases of the invention under conditions that allow digestion of at least a portion of the polypeptides present in the sample. Thus, preferably, after the sample has been contacted with the proteinase or an enzymatically active fragment thereof, the method further comprises a "digestion step", i.e., a step of incubating the sample under conditions that allow digestion of the polypeptides in the sample. The amount of digestion required depends on the purpose and intent of the person performing the method, and suitable conditions to achieve the amount of digestion required can be easily determined by the skilled person.

[0164] Preferably, the digestion step involves heating the sample at a temperature between 4 and 65°C, more preferably between 20 and 55°C, most preferably between 30 and 55°C. Preferably, the incubation step has a duration between 1 second and 45 minutes, more preferably between 30 seconds and 30 minutes, more preferably between 1 and 15 minutes, more preferably between 1 and 10 minutes, even more preferably between 1 and 5 minutes. When temperatures at the upper end of these ranges are used, the duration of the incubation can be at the lower end of these ranges and vice versa. The skilled person knows that very short incubations of 1 or 2 seconds are sufficient for methods performed with microfluidic samples and for other methods where the amount of substrate present in the sample is low.

[0165] Preferably, the method comprises a subsequent "inactivation step", i.e. heating the sample to inactivate the proteinase or enzymatically active fragments thereof, which is carried out after the step of contacting the sample with the proteinase and after the step of incubating the sample under conditions allowing digestion of the polypeptides in the sample.

[0166] These steps of digestion and inactivation are generally incubations and are 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 carried out.

[0167] Thus, in a further aspect there is provided a method for digesting polypeptides in a sample, said method comprising the steps of: a) contacting the sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 at least partial digestion of polypeptides in the sample; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) a monovalent salt concentration in said sample is ≧20 mM.

[0168] In another aspect, step c) of the above method comprises: c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0169] Preferably, the free calcium (or calcium) concentration in the sample is less than about 80 μM. ,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 or potassium salt, more preferably potassium chloride or sodium chloride, most preferably sodium chloride. The digestion step b) is as described above.

[0170] As noted above, digestion can be partial or complete. Proteinases useful in the present invention are non-specific and, given sufficient time, will completely digest proteins in a sample under conditions that allow enzyme function.

[0171] Here, "digesting a polypeptide in a sample" means that the amount of full-length polypeptide in the sample is reduced to a certain extent. The degree of protein digestion can be directly analyzed by several well-known analyses. It will be within the ability of the skilled artisan to design suitable analyses for their intended purpose. For example, the residual activity of enzymes in the sample can be used as a measure of protein degradation. Changes in the proteome profile, which can be measured by mass spectrometry, can also be used to measure the degree of polypeptide degradation in a sample. A simple analysis to visualize the degree of protein degradation is to perform SDS Page and staining with a protein staining dye such as Coomassie Blue or other visual reporter molecule. Intact proteins appear as bands along the gel, which become less sharp with increasing protein degradation. The degree of degradation can be quantified by software-based image analysis.

[0172] Preferably, the sample to which the proteinase is added comprises cellular material. Preferably, the sample comprises a crude cell extract. Preferably, the sample comprises a partially purified cell extract. 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 comprises about 1 to about 1,000,000 cells. In a preferred embodiment, the sample comprises 1 to 10,000 cells, preferably 1 to 1000 cells, preferably 1 to 100 cells. In a preferred embodiment, the sample comprises a single cell. In another preferred embodiment, the sample comprises 100 to 1,000,000 cells, preferably 100 to 10,000 cells, preferably 100 to 1000 cells. Preferably, the sample is a fine needle or liquid biopsy. Proteinases can be used to lyse cellular material.

[0173] The proteinases of the invention can be used to digest any polypeptide present in a sample, preferably including capsid or scaffolding proteins, DNA or RNA binding proteins, and / or enzymes that act on DNA or RNA, such as those described elsewhere herein.

[0174] In a further aspect, the invention provides a method of 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 which is at least about 70% identical to SEQ ID NO:1, the method comprising heating the sample to inactivate the proteinase or enzymatically active fragment thereof; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0175] Viewed alternatively, the present invention provides a method for inactivating a proteinase or an enzymatically active fragment thereof in a sample, wherein said proteinase comprises 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, said method comprising the step of heating the sample to inactivate said proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA, preferably essentially free of calcium chelators; or ii) the monovalent salt concentration in said sample is ≧20 mM.

[0176] In all of the methods of the invention, the free calcium (or calcium) concentration in the sample is preferably less than about 80 μM. ,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 or potassium salt, more preferably potassium chloride or sodium chloride, most preferably sodium chloride.

[0177] As mentioned above, in the methods of the invention, the free calcium (or calcium) and monovalent salt concentrations described elsewhere herein 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 methods of the invention, preferably, the proteinase is not removed from the sample, e.g., by purification, extraction or centrifugation, and preferably the proteinase concentration in the sample is not diluted before or during the inactivation step.

[0178] The present invention is based on the surprising finding that the proteinases described herein become heat labile under mild conditions, given certain concentrations of free calcium and / or monovalent salts. These conditions must therefore 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 may increase the volume of the sample and therefore decrease the free calcium and monovalent salt concentrations therein. In use, preferably, the composition of the present invention has a small volume that does not significantly change the volume of the sample. Similarly, the proteinase of the present invention may be provided in a solution in which 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 salt in the sample to which it is added is such that the resulting sample to which the proteinase or an 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 invention comprises 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%, at least 99% compared to the activity of the proteinase in the sample before the inactivation step was performed. Viewed differently, preferably, the inactivation step results in 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%, less than 1% of residual proteinase activity. Preferably, the proteinase is completely inactivated, i.e. preferably no detectable proteinase activity remains.

[0180] When the solution containing the heat-treated proteinase is returned to a temperature below 40° C., the proteinase of the present invention does not regain activity, i.e., there is little 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. Thus, inactivation as referred to herein is irreversible.

[0181] As mentioned above, suitable assays for measuring proteinase activity are known in the art. Such assays can therefore be used to measure the proteinase activity of a proteinase or an enzymatically active fragment thereof that has been inactivated by heat treatment, compared to the proteinase activity of the same proteinase not subjected to heat treatment, thereby determining the residual activity, or the degree of inactivation achieved by the inactivation step. Any proteinase activity assay can be used to determine the relative activity of a heat-treated proteinase relative to an untreated proteinase. The skilled artisan will be well aware that the same heat-treated and untreated proteinases should be kept under the same conditions and analyzed with the same procedure. Preferably, the assay used is assay A above, and preferably the untreated proteinase is kept on ice until the proteinase activity is measured. Further preferred assays for residual activity are those disclosed in the examples.

[0182] Thus, the residual activity after the inactivation step of the method of the invention, i.e. the degree of inactivation achieved by the inactivation step of the method of the invention, can be readily determined by the skilled person by measuring the proteinase activity of a proteinase that has been subjected to an inactivation step under suitable conditions using a suitable proteinase assay and comparing it with the activity of the same proteinase that has not been subjected to such inactivation step. The activity is measured using the same assay and under the same conditions. Preferably, the assay used is assay A above or any one of the assays used in this example. Preferably, the untreated proteinase is kept on ice until the proteinase activity is measured.

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

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

[0185] It will be within the ability of one of ordinary skill in the art to heat the sample to the desired temperature for the desired time using, for example, a heat block, microwave, Joule heating device, laser heating device, or water bath.

[0186] The temperature to which the sample is heated during the inactivation step of the method, i.e. the temperature to which the proteinase is exposed, is referred to as the inactivation temperature. Preferably, said 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, preferably ≦about 58°C.

[0187] Preferably, the inactivation step comprises 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, preferably about 50 to about 58°C.

[0188] Preferably, the inactivation step comprises 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, preferably about 53 to about 58°C.

[0189] Preferably, the inactivation step comprises 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, preferably about 55 to about 60°C.

[0190] Preferably, the inactivation step comprises 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, preferably about 56 to about 60°C.

[0191] Preferably, the inactivation step comprises 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, preferably about 57 to about 60°C.

[0192] Preferably, the inactivation step comprises 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, preferably about 58 to about 60°C.

[0193] Preferably, the inactivation step comprises 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, preferably about 59 to about 60°C.

[0194] Preferably, the inactivation step comprises 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 inactivation step comprises heating at about 53°C to about 60°C, more preferably at about 53°C to about 58°C, more preferably at about 55°C.

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

[0197] Particularly preferably, the inactivation step comprises heating at about 60 to about 67°C, more preferably at about 63 to about 67°C, more preferably at about 65°C.

[0198] Preferably, the inactivation step involves heating the sample at one of the above temperatures for a period of time referred to as the "holding time." The required holding time will depend on the inactivation temperature used, the free calcium and monovalent salt concentrations in the sample, and the degree of inactivation required. Given the teachings of this application, one of skill in the art will be able to select a holding time for their particular purpose.

[0199] Preferably, the holding time is ≦about 75 min, preferably ≦about 60 min, preferably ≦about 55 min, preferably ≦about 50 min, preferably ≦about 45 min, preferably ≦about 40 min, preferably ≦about 35 min, preferably ≦about 30 min, preferably ≦about 25 min, preferably ≦about 20 min, preferably ≦about 15 min, preferably ≦about 10 min, preferably ≦about 5 min, preferably ≦about 2 min.

[0200] Preferably, the retention 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, preferably at least about 60 minutes.

[0201] Preferably, the retention 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, preferably about 2 to about 5 minutes.

[0202] Preferably, the retention 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, preferably about 5 to about 10 minutes.

[0203] Preferably, the retention 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, preferably about 10 to about 15 minutes.

[0204] Preferably, the retention 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, preferably about 15 to about 20 minutes.

[0205] Preferably, the retention 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, preferably about 20 to about 25 minutes.

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

[0207] Preferably, the retention 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, preferably about 30 to about 35 minutes.

[0208] Preferably, the retention 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, preferably about 35 to about 40 minutes.

[0209] Preferably, the retention 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, preferably about 40 to about 45 minutes.

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

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

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

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

[0214] The above retention times are 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, more preferably ≦50 μl.

[0215] Those skilled in the art will appreciate that adjustments to either the heating temperature or the hold time can be compensated for by adjusting the other. For example, an increase in the inactivation temperature may allow a shorter hold time. Conversely, an increase in the hold time may allow a lower inactivation temperature to be used.

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

[0217] In methods of the invention that include an inactivation step, any of the above inactivation temperatures can be used in combination with any of the above hold times, and any and all combinations of inactivation temperatures and hold times disclosed elsewhere herein are expressly disclosed.

[0218] Preferably, the inactivation step involves heating at a temperature of about 53° C. to about 67° C., preferably about 55° C. to about 65° C., preferably about 55 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 involves 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 involves 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] A preferred inactivation step of the present invention is as follows.

[0222] A) About 53~58℃ The mixture is heated at about 55° C. for about 45 to about 75 minutes, more preferably for about 45 to about 60 minutes, and more preferably for about 60 minutes. In such 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 is free of free calcium.

[0223] Alternatively, or in addition, in such 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 at about 60° C. for the period of time specified in any one of B1 to B4 below. B1) About 2 to about 40 minutes, more preferably about 5 to about 30 minutes. In such 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, more preferably ≦about 5 μM.

[0225] Alternatively, or in addition, in such 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, more preferably at least about 150 mM.

[0226] B2) About 5 to about 15 minutes, more preferably about 10 minutes. In such embodiments, the concentration of free calcium in the sample is preferably ≦about 10 μM, ≦about 8 μM, more preferably ≦about 5 μM, and more preferably the sample is free of free calcium.

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

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

[0229] Alternatively, or in addition, in such 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, more preferably at least about 150 mM.

[0230] B4) About 20 to 40 minutes, more preferably about 30 minutes. In such 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, more preferably ≦about 16 μM.

[0231] Alternatively, or in addition, in such 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, more preferably at least about 100 mM.

[0232] Preferably, in such 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 at about 65° C. for up to about 15 minutes, more preferably up to about 10 minutes, more preferably up to about 5 minutes. In such 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, more preferably ≦about 5 μM.

[0234] Alternatively, or in addition, in such 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.

[0235] The free calcium-dependent and monovalent salt-dependent effects on thermolability identified by the inventors also allow the inactivation of the proteinases of the invention and enzymatically active fragments thereof at high temperatures in a surprisingly short time.

[0236] D) Heating at about 65 to about 70°C, preferably about 67 to about 70°C, more preferably about 67°C or about 70°C for up to about 5 minutes, more preferably up to about 2 minutes. In such 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.

[0237] Alternatively, or in addition, in such 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.

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

[0239] One of skill in the art will readily appreciate that adjustment of one of the parameters heating time, heating temperature, free calcium concentration, and monovalent salt concentration can be compensated for by adjustment of one or more of the others.

[0240] However, and very importantly, according to the present invention, the maximum free calcium concentration is 80 μM. It is below this free calcium concentration that the heat-labile nature of the proteinase of the present invention is induced to such an extent that substantial inactivation (75% inactivation) of the proteinase can be achieved under advantageous mild conditions, in particular 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, preferably 15-30 minutes.

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

[0242] As mentioned above, typical inactivation procedures for standard proteinases used in the art 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), heating at 70°C for 15 minutes (Qiagen procedure).

[0243] In the method of the 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 is contacted with the proteinase. Viewed from another angle, 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 invention does not include a 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 earlier in the workflow or during its preparation, in which case the calcium chelating agent must be removed before applying the proteinase. Calcium chelating agents are as described elsewhere herein and it is within the capabilities of the skilled artisan to remove them from the sample before contacting it with the proteinase.

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

[0245] Preferably, the sample to which the 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, 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.

[0246] Preferably, the sample to which the proteinase or enzymatically active fragment is added contains the biomolecule of interest and one or more contaminating, ie unwanted, polypeptides.

[0247] Thus, in a further aspect, the present invention provides a method for isolating or purifying a biomolecule of interest from a sample, the sample containing one or more contaminating polypeptides, the method comprising: a) contacting the sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 at least partial digestion of polypeptides in the sample; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; and d) optionally removing the biomolecule of interest from the sample.

[0248] Viewed another way, step c) of the above method includes:

[0249] heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in the sample is ≧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 itself a polypeptide. The biomolecule of interest is not a proteinase or an enzymatically active fragment thereof.

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

[0252] Preferably, the sample comprises chromatin, the biological molecule of interest is DNA without associated histones and the contaminating proteins are 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 or comprises the product of a nuclear amplification reaction, e.g., a PCR reaction, and comprises a DNA-bound polymerase, the biomolecule of interest is DNA without bound polymerase, and the contaminating protein is bound polymerase, and step b) comprises 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 variants, 3SR, SDA, LAR or LCR, and LAMP and their variants.

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

[0255] The "product of a nucleic acid amplification reaction" is therefore considered to include essentially all of the components directly resulting from the final amplification step of the reaction. Other components may be added or some of the components may undergo 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, it may be preferred that the product of the nucleic acid amplification reaction is subjected to a treatment that results in dephosphorylation of unincorporated NTPs, for example treatment with an alkaline phosphatase, preferably a heat-labile alkaline phosphatase, such as heat-labile shrimp alkaline phosphatase (SAP), before treatment with a proteinase of the invention. An advantageous recombinant SAP is available from ArcticZymes™ AS.

[0256] Preferably, the biomolecule of interest in the sample is bound to a molecule, preferably a polypeptide, via one or more peptide bonds. In a further aspect, the present invention therefore provides a method for releasing a biomolecule of interest from a molecule, preferably a polypeptide, bound to it via one or more peptide bonds, said method comprising: a) contacting the sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 the release of a biomolecule of interest by digestion of one or more peptide bonds; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; and d) optionally removing the biomolecule of interest from the sample.

[0257] Alternatively, step c) of the above method may comprise heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in the sample is ≧20 mM.

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

[0259] In a further aspect, the present invention provides a method for producing a peptide of interest from a precursor polypeptide, the method comprising: a) contacting a polypeptide with a proteinase or an enzymatically active fragment thereof, wherein said 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 digestion of the precursor polypeptides to release the peptides of interest; c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; and d) optionally removing the biomolecule of interest from the sample.

[0260] Alternatively, step c) of the above method may comprise heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0261] Proteinases may also be 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 aspect, the present invention provides a method for separating one or more cells from other cells in a tissue or from a substrate to which said one or more cells are attached, said method comprising: a) contacting one or more cells with a proteinase or an enzymatically active fragment thereof, wherein said 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 the release of one or more cells by digestion of one or more extracellular matrix proteins; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; and d) optionally removing the biomolecule of interest from the sample.

[0262] Alternatively, step c) of the above method may comprise heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the calcium concentration in the sample is ≦about 80 μM and the sample is essentially free of EDTA; or ii) the monovalent salt concentration in the sample is ≧20 mM.

[0263] Preferably, the sample is or comprises a polyacrylamide gel containing a polypeptide, and digestion of said polypeptide is required to generate smaller fragments for analysis by mass spectrometry. Thus, the present invention provides a method for preparing a protein fragment sample, preferably for mass spectrometry, said method comprising: a) contacting a sample containing one or more polypeptides with a proteinase or an enzymatically active fragment thereof, wherein said 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 at least partial digestion of polypeptides in the sample; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof, i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; and d) optionally removing the biomolecule of interest from the sample.

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

[0265] Preferably, the sample to which the proteinase or an enzymatically active fragment thereof is added comprises one or more further enzymes. Preferably, said further enzymes are selected from the group consisting of nucleases (preferably deoxyribonucleases, exonucleases, Bal31 nuclease, ribonuclease, mung bean 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 heat-labile alkaline phosphatase, more preferably shrimp alkaline phosphatase), kinases, helicases, restriction enzymes and glycosylases. The sample preferably comprises a combination of such further enzymes. Preferably, the sample comprises a DNA polymerase or a reverse transcriptase. Preferably, such enzymes are exogenous enzymes, i.e., not expressed in cells within the sample or in cells from which the cellular material in the sample originates. The methods of the present invention provide advantageously mild proteinase inactivation conditions that can be tolerated by such additional enzymes, and thus such 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 the art, required inactivation at high temperatures that could damage the enzyme or biomolecules 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 concentration of the enzyme must be significantly diluted. Such removal or dilution steps extend the workflow, increase costs, and can result in loss or damage of materials in the sample. They are particularly unsuitable when processing small sample sizes.

[0267] Preferably, the sample to which the proteinase 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] Thus, preferably, any of the above methods comprises, following the inactivation step, a step involving enzymatic catalysis of the substrate, said subsequent step being carried out without prior removal or dilution of the proteinase or enzymatically active fragments thereof.

[0269] By "without prior removal" it is meant that the proteinase is not physically removed from the sample, for example by purification, extraction or centrifugation.

[0270] By "without prior dilution" is meant that the proteinase concentration in the sample is not significantly diluted, i.e., is not substantially inactivated by dilution. The definition of substantially inactivated is provided elsewhere herein. Preferably, the proteinase concentration in the sample is not diluted more than four-fold, more preferably more than three-fold, and more preferably more than two-fold.

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

[0272] Preferably, the sample comprises one or more nucleic acid molecules and the method comprises, following the inactivation step, a step of phosphorylation or dephosphorylation of the nucleic acid molecule without prior removal or dilution of proteinases or enzymatically active fragments thereof.

[0273] Preferably, the sample comprises one or more nucleic acid molecules and the method comprises, following the inactivation step, a step of ligation of the nucleic acid molecules without prior removal or dilution of proteinases 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 step of reverse transcription 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, following the inactivation step, a step of nucleic acid polymerization without prior removal or dilution of proteinases or enzymatically active fragments thereof.

[0276] Preferably, the sample comprises one or more nucleic acid molecules and the method comprises, following the inactivation step, a step of nucleic acid amplification without prior removal or dilution of proteinases or enzymatically active fragments thereof.

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

[0278] Preferably, the sample comprises one or more virus particles or cells, preferably bacterial cells, and the method comprises, following the inactivation step, a step of cell lysis 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 produced recombinantly in Pichia pastoris at ArcticZymes (batch 1602-1, SEQ ID NO: 1).

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

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

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

[0283] 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 (EM 8.8) with a UV spectrophotometer (Ultrospec 2000, Pharmacia Biotec, Sweden) for 2 min at 25° C. One unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per min at 25° C.

[0284] The following specific activities were measured (Table 1).

[0285] [Table 5]

[0286] Results were confirmed using a vial of proteinase K from a different supplier (Sigma, O4850, measured to 400 U / mg).

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

[0288] The cuvettes were incubated at the indicated temperatures for the duration of the analysis. The enzymatic cleavage of the substrate Suc-Ala-Ala-Pro-Phe-NA to 4-nitroaniline was monitored over time by measuring the increase in absorbance at 410 nm (EM 8.8) for 30 s in a 1.5 mL semi-microcuvette (Brand, Germany) in a UV spectrophotometer (Ultrospec 2000, Pharmacia The results were analyzed by measuring the chromatographic characteristics using a chromatographic microscope (Biotech, Sweden).

[0289] The activity was calculated as % relative activity to the maximum activity observed at 65° C. Measurements above 65° C. were technically impossible. According to the teachings of the prior art, the optimum temperature for both proteinases X and K is between 65 and 70° C.

[0290] As shown in FIG. 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. Also, 24 mU / mL proteinase (corresponding 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 temperatures in a total volume of 1000 μl in buffer (0.1 M Tris-HCl, pH 8.0, 0 mM / 0.005 mM / 10 mM CaCl2, 1% DMSO, 12 mM NaCl).

[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 (EM 8.8) over time for 30 seconds using a UV spectrophotometer.

[0293] Activity was calculated as % 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 each calcium concentration, the temperature profiles of both proteinases were similar, with maximum activity observed at 65° C. Decreasing calcium concentration appeared to decrease proteinase activity only slightly at some temperatures. When considering activity relative to the activity achieved at each particular temperature with 10 mM calcium, the activity profiles of the two proteinases differed. The results are shown in Tables 3 and 4 and Figures 2 and 3, which highlight the calcium-dependent effect on proteinase activity at various temperatures.

[0296] [Table 7]

[0297] [Table 8]

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

[0299] In contrast, according to Figure 3 and Table 4, the activity of proteinase K at each temperature was hardly affected by calcium conditions, i.e., only slight changes in activity were observed with increasing temperature, whether in the presence of high calcium concentration, low calcium concentration, or in the absence of calcium.

[0300] Taken together, the data suggest that low calcium concentrations can induce heat inactivation of proteinase X to a greater extent than proteinase K.

[0301] Example 3 :Ca of proteinase 2+ Dependent and inducible heat sensitivity The calcium-dependent thermolability of proteinases X and K was further investigated.

[0302] Heat treatment steps: The proteinase was incubated at 60°C for 15 or 30 minutes in a PCR thermocycler (Veriti, Applied Biosystems) in buffers containing various concentrations of free calcium. 0.1mg / ml proteinase X (equivalent to initial activity of 6.5U / ml proteinase X) or 0.014mg / ml proteinase K (equivalent to initial activity of 5.6U / ml proteinase K) 0.025M Tris-HCl, pH8, 300mM NaCl, 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, samples were placed back on ice. Samples used as controls were kept on ice throughout.

[0304] Analysis of residual activity After the heat treatment step, the samples were diluted 1:10 in 0.025 M Tris-HCl, pH 8, 300 mM NaCl. The dilution step was performed to reduce the enzyme activities U / ml in the samples to bring them into the range detectable by the reaction assay.

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

[0306] 0.4 μg / ml proteinase X or 0.06 μg / ml proteinase K (corresponding to 26 and 24 mU / ml initial activities, respectively) was incubated 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 NaCl) in a total volume of 250 μl.

[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 (EM 8.8) over time for 10 min, detecting the signal every 11 s using a multimode plate reader (Synergy H1, BioTek, USA).

[0308] The results are shown in Figures 4 and 5. Activity is shown as a percentage of remaining activity relative to a control sample (same proteinase and buffer kept on ice without a heating step).

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

[0310] In contrast, decreasing the free calcium concentration resulted in an increased heat sensitivity of Proteinase X, as shown in Figure 4. Substantial inactivation (>75%, i.e. less than 25% remaining activity) was achieved by heating at 60°C for 30 min at a free calcium concentration <0.063 mM, and by heating at 60°C for 15 min at a free calcium concentration <0.016 mM. >90% inactivation was preferred, which for Proteinase X was achieved by heating at 60°C for 30 min at a free calcium concentration <0.031 mM, or by heating in the absence of free calcium for 15 min.

[0311] Thus, whereas proteinase X is inducibly thermolabile in the presence of low calcium concentrations, the thermolability of proteinase K is not affected by calcium concentration.

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

[0313] Example 4: Free calcium-dependent inactivation profile of proteinases The effect of different heat treatment steps on the inactivation of proteinases X and K was evaluated. In the following experiments, an upper limit of 5 μM free calcium was used and the heat inactivation step was carried out over a range of temperatures and heating times.

[0314] Heat treatment steps: Proteinases were incubated at different temperatures (45°C, 50°C, 55°C, 60°C, 65°C and 70°C) for different times (2, 5, 10, 15, 3060 min) in buffers containing various free calcium concentrations (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, in a total volume of 50 μl.

[0315] Proteinase K was dialyzed against Ca-free storage buffer prior to use to remove any free calcium therein. After inactivation, samples were placed back on ice. Samples used as controls were kept on ice throughout.

[0316] Analysis of residual activity After the heat treatment step, the samples were diluted 1:20 in 50 mM HEPES, pH 8, 100 mM NaCl. The dilution step was performed to reduce the enzyme activities U / ml in the samples to bring them into the range detectable by the reaction assay.

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

[0318] 0.2 μg / ml proteinase X or 0.03 μg / ml proteinase K (corresponding to 13 mU and 12 mU initial activities, respectively) was incubated 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, 4 mM NaCl) in a total volume of 250 μl.

[0319] 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 (EM 8.8) over time for 10 min with a multimode plate reader (Synergy H1, BioTek, USA) detecting the signal every 11 s. Residual activity is compared to control samples that were otherwise identical to the test samples, kept on ice.

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

[0321] [Table 9]

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

[0323] ≧90% inactivation of proteinase X When heated to at least 70°C, within 2 minutes, Within 5 minutes of heating at least 65°C, Within 15 minutes of heating at least 60°C, and This is achieved within 60 minutes when heated to at least 55°C.

[0324] [Table 10]

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

[0326] ≧90% inactivation of proteinase X When heated to at least 70°C, within 5 minutes, Within 10 minutes of heating to at least 65°C, and This is achieved within 30 minutes when heated to at least 60°C.

[0327] [Table 11]

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

[0329] ≧90% inactivation of proteinase X When heated to at least 70°C, within 5 minutes, Within 10 minutes of heating to at least 65°C, and This is achieved within 30 minutes when heated to at least 60°C.

[0330] That is, at free calcium concentrations ≦5 μM, incubation at 70° C. achieved ≧95% inactivation of Proteinase X within 5 minutes, and 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 and 5 μM free calcium, incubation at 60° C. achieved ≧90% inactivation of proteinase X within 30 minutes (within 15 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 carried out using proteinase K. The results are shown in Tables 8 to 10 below.

[0334] [Table 12]

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

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

[0337] [Table 13]

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

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

[0340] [Table 14]

[0341] According to the above data, free calcium concentration < 5 μM And, ≧75% inactivation of proteinase K is When heated to at least 70°C, within 5 minutes, Heat at least 65°C for 10 minutes or less (5 minutes or less for Prot X). within 30 minutes of heating at least 60°C (within 15 minutes for Prot X), and This is achieved within 60 minutes when heated to at least 55°C.

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

[0343] Thus, the results show that at all free calcium concentrations, proteinase X requires a significantly shorter heating time to achieve the same degree of inactivation at a given temperature than proteinase K. Alternatively, for most of the heating times and temperatures tested, proteinase X inactivates more than proteinase K. This is due to the observed heat-labile nature of proteinase X, which is induced at low calcium concentrations, that is not observed for proteinase K.

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

[0345] Example 5 : Monovalent salt-dependent inducible heat-labile proteinase To determine the effect of NaCl on the heat lability 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 steps: Proteinases were incubated at various temperatures (45°C, 50°C, 55°C, 60°C, 65°C and 70°C) for 30 minutes in buffers containing 50 mM or 300 mM NaCl, which contained 0.1 mg / ml proteinase (corresponding to 6.5 U / ml proteinase X or 40 U / ml proteinase K), 25 mM HEPES, pH 8, 0 μM CaCl2 in a volume of 50 μl.

[0347] After inactivation, samples were placed back on ice. Samples used as controls were kept on ice throughout.

[0348] Residual activity analysis After the heat treatment step, samples of proteinase X or proteinase K were diluted 1:10 or 1:100, respectively, in 50 mM HEPES, pH 8, 300 mM NaCl. Residual proteinase activity was assessed 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] 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 (EM 8.8) over time for 10 min with a multimode plate reader (Synergy H1, BioTek, USA) detecting the signal every 11 s. Residual activity is compared to control samples that were otherwise identical to the test samples, kept on ice.

[0351] As shown in Figure 7, increasing NaCl concentration has the opposite effect on the thermolability of ProtX as compared to ProtK: high NaCl concentrations stabilize proteinase K at high temperatures, whereas high NaCl concentrations induce thermolability of proteinase X.

[0352] This result is particularly surprising: Proteinase X of SEQ ID NO:1 is derived from a saltwater organism and would therefore normally be expected to tolerate high salinity conditions, whereas Prot K is derived from a non-marine source, the fungus Engyodontium album (formerly Tritirachium album), and would not be expected to be stabilized by high salinity conditions.

[0353] Example 6 : Monovalent salt-dependent thermolabile profile of proteinases To further explore the effect of NaCl on the heat lability of Proteinase X, a wider range of NaCl concentrations than in Example 5 was evaluated.

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

[0355] After inactivation, samples were placed back on ice. Samples used as controls were kept on ice throughout.

[0356] Analysis of residual activity After the heat treatment step, samples were diluted 1:10 in 50 mM HEPES, pH 8, 300 mM NaCl. Residual proteinase activity was assessed as follows.

[0357] 0.4 μg / ml proteinase X (corresponding to 26 mU / ml initial activity) was incubated 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 NaCl) in a total volume of 250 μl.

[0358] 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 (EM 8.8) over time for 10 min with a multimode plate reader (Synergy H1, BioTek, USA) detecting the signal every 11 s. Residual activity is compared to control samples that were otherwise identical to the test samples, kept on ice.

[0359] The results are shown in FIG.

[0360] [Table 15]

[0361] According to the above data, at NaCl concentrations of ≧150 mM, ≧about 80% inactivation of Proteinase X is achieved by heating at 60° C. for 30 minutes or less.

[0362] Again, the above data indicates that at NaCl concentrations of ≧300 mM, ≧about 95% inactivation of Proteinase X is achieved by heating at 60° C. for ≦30 minutes, and ≧about 80% inactivation of Proteinase X is achieved by heating at 60° C. for ≦150 minutes.

[0363] FIG. 9, which is a plot of NaCl concentration (x-axis) versus residual activity after 30 / 15 min heating at 60° C. (y-axis), shows that substantial inactivation (≧75%) is achieved by 15 min heating at 60° C. for NaCl concentrations of at least about 210 mM and by 30 min heating at NaCl concentrations of at least about 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 studies, i) a decrease in the free calcium concentration or ii) an increase in the NaCl concentration induces a greater heat lability of proteinase X than that of proteinase K. The combined effect of these conditions was then examined.

[0365] Heat treatment steps: Proteinases X and K were incubated for 30 min at 60° C. in buffers containing various concentrations of NaCl (0, 25, 50, 75, 100, 125 mM) and free calcium (0, 5, 10, 20, and 20 μM CaCl2). The buffers contained 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, pH 8 in a volume of 50 μl.

[0366] Proteinase K was dialyzed against Ca-free storage buffer prior to use to remove any free calcium therein.

[0367] After inactivation, samples were placed back on ice. Samples used as controls were kept on ice throughout.

[0368] Analysis of residual activity After the heat treatment step, samples were diluted 1:20 in 50 mM HEPES, pH 8, 100 mM NaCl. Residual proteinase activity was assessed as follows.

[0369] 0.2 μg / ml proteinase X (corresponding to 13 mU / ml initial activity) or 0.03 μg / ml proteinase K (corresponding to 12 mU / ml initial activity) 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] 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 (EM 8.8) over time for 10 min with a multimode plate reader (Synergy H1, BioTek, USA) detecting the signal every 11 s. Residual activity is compared to control samples that were otherwise identical to the test samples, kept on ice.

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

[0372] [Table 16]

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

[0374] Furthermore, substantial inactivation (≧≈75%) was achieved in the presence of at least 75 mM NaCl at the highest free calcium concentration of 20 μM.

[0375] Moreover, substantial inactivation (≧≈75%) was achieved in the presence of at least 50 mM NaCl at the highest free calcium concentration of 10 μM.

[0376] Moreover, substantial inactivation (≧≈75%) was achieved in the presence of at least 50 mM NaCl at the highest free calcium concentration of 5 μM.

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

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

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

[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] In the absence of free calcium, excellent inactivation (≧90%) was achieved in the presence of at least 50 mM NaCl.

[0382] [Table 17]

[0383] The results show that Proteinase K has a very different thermolabile profile than Proteinase X. Proteinase K is increasingly thermolabile under low salinity conditions, stabilized under high salinity conditions, and is largely unaffected by calcium concentration, whereas Proteinase X is increasingly thermolabile under high salinity and low free calcium conditions.

Claims

1. A composition comprising a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 concentration of free calcium in the composition is ≦about 80 μM; or ii) A composition, wherein the concentration of a monovalent salt in said composition is ≧20 mM.

2. 2. The composition of claim 1, wherein the composition is a solution for application to a sample containing one or more polypeptides, preferably having a volume of ≦200 μl.

3. A sample comprising one or more polypeptides and a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 concentration of free calcium in the sample is ≦about 80 μM; or ii) A sample, wherein the concentration of a monovalent salt in said sample is ≧20 mM.

4. 1. A method for inactivating a proteinase or an enzymatically active fragment thereof in a sample, wherein said 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, said method comprising heating said sample to inactivate said proteinase or enzymatically active fragment; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM.

5. 1. A method for digesting polypeptides in a sample, comprising the steps of: a) contacting said sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 at least partial digestion of polypeptides in the sample; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM.

6. 1. A method for isolating or purifying a biomolecule of interest from a sample, said sample containing one or more contaminating polypeptides, said method comprising: a) contacting said sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 at least partial digestion of polypeptides in the sample; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; d) optionally removing said biomolecule of interest from said sample.

7. 1. A method for liberating a biomolecule of interest from a second molecule bound to it via one or more peptide bonds, comprising: a) contacting said sample with a proteinase or an enzymatically active fragment thereof, said proteinase comprising 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 said sample under conditions that allow for the release of said biomolecule of interest by digestion of one or more of said peptide bonds; and c) heating the sample to inactivate the proteinase or enzymatically active fragment thereof; i) the concentration of free calcium in the sample is ≦about 80 μM; or ii) the monovalent salt concentration in the sample is ≧20 mM; Optionally removing said biomolecule of interest from said sample.

8. i) the concentration of free calcium in the composition or sample is ≦about 80 μM; 8. The composition, sample or method of any one of claims 1 to 7, wherein ii) the monovalent salt concentration in the composition or sample is ≧20 mM.

9. i) the concentration of free calcium in said composition or sample is ≦about 80 μM and said composition or sample is essentially free of EDTA, preferably essentially free of calcium chelating agents; or 9. A composition, sample or method according to any one of claims 1 to 8, wherein ii) the monovalent salt concentration in the composition or sample is > about 20 mM.

10. i) the concentration of free calcium in the composition or sample is between 1 and 80 μM; and / or 10. The composition, sample or method of any one of claims 1 to 9, wherein ii) the monovalent salt concentration in the composition or sample is > about 20 mM.

11. i) the concentration of free calcium in said composition or sample is ≦about 35 μM; and / or 11. A composition, sample or method according to any one of claims 1 to 10, wherein ii) the monovalent salt concentration in the composition or sample is ≧about 40 mM.

12. A composition, sample or method according to any one of claims 1 to 11, wherein the pH of the sample is between 6.5 and 9.5, preferably between 7.5 and 8.

5.

13. 13. The method of any one of claims 4 to 12, wherein the heating step comprises heating the sample at a temperature of from about 53 to about 67°C.

14. 14. The method of any one of claims 4 to 13, wherein the heating step comprises heating the sample at a temperature of from about 53 to about 58°C, preferably for from about 45 to about 75 minutes.

15. 15. The method of claim 14, wherein the free calcium concentration in the sample is ≦about 10 μM or the monovalent salt concentration in the sample is at least about 75 mM.

16. 14. The method of any one of claims 4 to 13, wherein the heating step comprises heating the sample at a temperature of about 63 to about 67°C, preferably for up to about 10 minutes.

17. 17. The method of claim 16, wherein the free calcium concentration in the sample is ≦about 65 μM or the monovalent salt concentration in the sample is at least about 25 mM.

18. 14. The method of any one of claims 4 to 13, wherein the heating step comprises heating the sample at a temperature of from about 58 to about 63°C, preferably for from about 2 to about 40 minutes.

19. The heating step heats the sample. i) for about 5 to 15 minutes, preferably wherein the free calcium concentration in said sample is ≦about 10 μM or the monovalent salt concentration in said sample is at least about 75 mM; or ii) for about 10 to 20 minutes, preferably wherein the free calcium concentration in said sample is ≦about 35 μM or the monovalent salt concentration in said sample is at least about 50 mM; or 19. The method of claim 18, comprising iii) heating for about 20 to 40 minutes, preferably wherein the free calcium concentration in the sample is ≦about 65 μM or the monovalent salt concentration in the sample is at least about 25 mM.

20. 13. The method of any one of claims 4 to 12, wherein the heating step comprises heating the sample at a temperature of about 65 to about 70°C for up to about 5 minutes.

21. the residual proteinase activity of said proteinase or enzymatically active fragment thereof after said heating step is ≦about 25%, preferably ≦about 10%, relative to a control, and said residual activity is sufficient for the following analytical steps: 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 incubating with 1% DMSO; ii) 410nm (ε=8800M -1 .cm -1 ) for 2 minutes at a temperature of ≦40° C. using a spectrophotometer to measure the increase in absorbance, where one unit is defined as the amount of enzyme that produces 1 μmol of 4-nitroaniline per minute at the selected temperature; 21. The method of any one of claims 4 to 20, wherein the activity observed in step ii) is measured by comparing, by the same assay, the activity observed with the same amount of the same proteinase that has not been heat treated but is otherwise maintained under the same conditions as the heat treated proteinase.

22. 22. The method of any one of claims 4 to 21, wherein the sample comprises: a) 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; b) one or more nucleic acid molecules, wherein the method, following the step of heating the sample to inactivate the proteinase, comprises the steps of: i) nuclease-mediated digestion of said one or more nucleic acid molecules; ii) phosphorylation or dephosphorylation of said one or more nucleic acid molecules; or iii) linking said one or more nucleic acid molecules, without prior removal or dilution of said proteinase or enzymatically active fragment thereof, c) the sample comprises one or more RNA molecules, and the method comprises, following the step of heating the sample to inactivate the proteinase, a step of reverse transcription, without prior removal or dilution of the proteinase or enzymatically active fragments thereof; d) one or more DNA molecules, wherein the method comprises, following the step of heating the sample to inactivate the proteinase, a step of nucleic acid polymerization, preferably amplification, without prior removal or dilution of the proteinase or enzymatically active fragments thereof; or e) one or more viral particles or cells, preferably bacterial cells, wherein the method comprises a step of cell lysis following the step of heating the sample to inactivate the proteinase, without prior removal or dilution of the proteinase or enzymatically active fragments thereof.

23. i) a composition according to any one of claims 1, 2 and 8 to 12, and ii) a second composition comprising a second enzyme, preferably wherein said second enzyme is selected from the group consisting of a nuclease, a DNA polymerase, an RNA polymerase, a reverse transcriptase, a DNA ligase, an RNA ligase, a methylase, a transferase, a topoisomerase, a guanylyl transferase, a phosphatase, a transposase, a kinase, a helicase, a restriction enzyme and a glycosylase.

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