WO2024254620A2 - Protéase pour diagnostic - Google Patents

Protéase pour diagnostic Download PDF

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Publication number
WO2024254620A2
WO2024254620A2 PCT/US2024/040585 US2024040585W WO2024254620A2 WO 2024254620 A2 WO2024254620 A2 WO 2024254620A2 US 2024040585 W US2024040585 W US 2024040585W WO 2024254620 A2 WO2024254620 A2 WO 2024254620A2
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Prior art keywords
seq
hours
protease
biological sample
buffer
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WO2024254620A3 (fr
Inventor
Maria Tang
Stephanie MANTON
Andrea Sabine ILG
Lars Kobberoee Skov
Swapnil Surendra Mohile
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Novozymes AS
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Novozymes AS
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Publication of WO2024254620A3 publication Critical patent/WO2024254620A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the present invention pertains to methods, kits, and buffers comprising a protease for hydrolyzing proteins and extracting a non-proteinaceous component from biological samples.
  • Biological samples whether of human, animal, viral or microbial origin, often contain valuable nucleic acid information that can be used for diagnostic, research, or therapeutic purposes.
  • the extraction and purification of these nucleic acids, particularly DNA and RNA, from the myriad of other cellular components is a critical step in many molecular biology procedures.
  • Proteinase K has been a popular choice for nucleic acid extraction methods, especially in diagnostic kits. Proteinase K is effective for a range of samples, but its compatibility with some types of biological samples can vary significantly. In certain cases, Proteinase K may not efficiently break down the proteins or might adversely affect the quality of the extracted nucleic acid, leading to sub-optimal results. For instance, samples with a high fat content, calcified tissues, or certain robust cellular structures might prove challenging for Proteinase K-based extraction methods. Proteinase K is also occasionally unstable in - or inhibited by - certain components necessary for optimal extraction i.e. components present in the lysis buffer in which Proteinase K assists the nucleic acid extraction.
  • Proteinase K is sometimes replaced by QIAGEN protease, which offers a different activity and stability profile.
  • QIAGEN protease has similar limitations on in which lysis buffers the enzyme perform optimal extraction, because of the effect of lysis buffer components on the enzyme activity and stability.
  • the present invention pertains to methods, kits, and buffers comprising a protease suitable for efficient nucleic acid extraction from various biological samples. Recognizing the limitations and varied compatibility of commercial proteases, such as, Proteinase K or QIAGENTM protease with some biological samples, this invention provides methods, kits, and buffers comprising a protease enzyme with a varied activity and stability profile to serve the sample-types where nucleic acid extraction is not currently optimal.
  • the described method encompasses using a specific enzyme to hydrolyze proteins in the biological sample, thereby facilitating the extraction of nucleic acids.
  • the present method can enable extraction of high-quality nucleic acids from a diverse set of biological samples. These samples can range from those with high fat content and calcified tissues to ones with challenging cellular structures, often deemed problematic for Proteinase K-based procedures.
  • the diagnostic kit provided herein contains all necessary reagents and tools for carrying out the aforementioned method. Designed to be user-friendly, efficient, and reliable, this kit stands as an alternative or supplementary solution to traditional Proteinase K-based nucleic acid extraction kits.
  • the present disclosure provides a method for hydrolysing protein in a biological sample comprising the steps of: contacting the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: 8, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • a method for hydrolysing protein in a biological matrix comprising the steps of: contacting the biological matrix with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the present disclosure provides a method for extracting a non- proteinaceous component of a biological sample comprising the steps of: a) incubating the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and b) separating the non-proteinaceous component from the biological sample, thereby extracting the non-proteinaceous component.
  • a method for isolating and characterizing polynucleotides from a biological sample comprising: a) contacting the biological sample with a protease having at least 65% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 optionally under conditions suitable for hydrolysing protein in the biological sample; b) separating a non-proteinaceous component comprising one or more polynucleotides, from the biological sample; c) wherein the biological sample is selected from whole blood, blood plasma, tissue, urine, saliva, mucosa, and stool, and originates from an animal selected from the group consisting of: a human, or an animal; and d) wherein the one or more polynucleotides are biomarkers of a specific disease or a condition; e) wherein the method is performed at a temperature of from 20 °C to 80 °
  • a method for purifying one or more polynucleotides from a biological sample comprising at least the following steps: a. incubating the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; b. binding the one or more polynucleotides to a solid phase; c. eluting the one or more polynucleotides from the solid phase; thereby purifying one or more polynucleotides from the biological sample.
  • a method for nucleic acid extraction from a biological sample comprising: a. contacting the biological sample as defined herein with a protease comprising an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; b. incubating the biological sample under conditions sufficient for protein hydrolysis to provide an incubated sample; and c. extracting one or more polynucleotides from the incubated sample.
  • a kit comprising: a. a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and b. a material with an affinity to one or more polynucleotides.
  • a buffer comprising a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and one or more additives.
  • Figure 1 shows the proteolytic activity of SEQ ID NO: 1 and Proteinase K at different pH values between pH 2-14. Values are normalized against maximum activity of Proteinase K.
  • Figure 2 shows the proteolytic activity of SEQ ID NO: 1 , Proteinase K, and Qiagen protease at different pH values between pH 2-12. The activities are normalized to Proteinase K’s maximum activity.
  • fragment means a polypeptide, a catalytic domain, or a binding module having one or more amino acids absent from the amino and/or carboxyl terminus of the mature polypeptide, catalytic domain, or binding module, wherein the fragment has protease activity.
  • Fusion polypeptide is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a polypeptide of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.
  • Mature polypeptide means a polypeptide in its mature form following N-terminal and/or C-terminal processing (e.g., removal of signal peptide).
  • the mature polypeptide is SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6.
  • sequence difference means the percent of amino acid differences between a polypeptide and the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, and is calculated as follows:
  • variant means a polypeptide having protease activity comprising a man-made mutation, i.e., a substitution, insertion (including extension), and/or deletion (e.g., truncation), at one or more positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.
  • Wild-type in reference to an amino acid sequence or polynucleotide means that the amino acid sequence or polynucleotide is a native or naturally-occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or polynucleotides) that is found in nature.
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).
  • a method for analyzing at least one target non-proteinaceous component in a mixture derived from a biological sample.
  • This mixture comprises both non-proteinaceous and proteinaceous components.
  • the analysis comprises incubating the mixture with at least one protease.
  • the protease is characterized by an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6.
  • the term “derived” means that a biological sample is manipulated or treated in order to create a mixture of non-proteinaceous and proteinaceous components which are originally contained in the biological sample. This mixture enables the analysis, isolation, enrichment, or purification of specific non-proteinaceous components.
  • analysis shall mean that the presence or the amount of the target non- proteinaceous component is investigated, i.e. the target non-proteinaceous component is detected or determined or the amount thereof is determined.
  • Manipulation or treatment steps include chemical or physical manipulation steps which are known to the skilled person. More specifically, this can be done by lysing the biological sample.
  • a method for hydrolysing protein in a biological sample comprising the steps of: contacting the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6.
  • a method for extracting a non-proteinaceous component of a biological sample comprising the steps of: a) incubating the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, and b) separating the non-proteinaceous component from the biological sample, thereby extracting the non-proteinaceous component.
  • the method is provided wherein the protease is comprised in the kit and/or buffer as defined herein.
  • the protease comprises or consists of the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, and: a. the buffer comprises TRIS, pH8, such as 30 mM TRIS, pH8, and SDS, such as 1% SDS, and MgCh, such as 40 mM MgCh, wherein the biological sample is human whole blood; or b. the buffer comprisies TRIS, pH8, such as 30 mM TRIS, pH8, and UREA, such as 0.25 mM UREA, and MgCh, such as 40 mM MgCh, wherein the biological sample is tissue.
  • TRIS, pH8, such as 30 mM TRIS, pH8, and SREA, such as 0.25 mM UREA
  • MgCh such as 40 mM MgCh
  • the present invention relates to polypeptides having protease activity referred to herein as proteases.
  • the invention relates to polypeptides having protease activity (proteases), selected from the group consisting of:
  • polypeptide consisting of the sequence of SEQ ID NO: 1 is also known as Alcalase or ProteoX.
  • the protease of the present disclosure has a sequence identity of at least 65%, such as at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
  • SEQ ID NO: 4 SEQ ID NO: 5 OR SEQ ID NO: 6, or a mature polypeptide of SEQ ID NO: 1 , SEQ ID NO:
  • the polypeptide has a sequence identity of at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO:
  • the polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 or a mature polypeptide thereof.
  • the polypeptide may have an N-terminal and/or C-terminal extension of one or more amino acids, e.g., 1-5 amino acids.
  • polypeptide is a fragment containing at least 85% amino acid residues, at least 90% amino acid residues, or at least 95% amino acid residues of the polypeptide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6.
  • the polypeptide has at most 2% sequence differences to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6, wherein the polypeptide has protease activity.
  • polypeptide is derived from SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 by substitution, deletion or addition of one or several amino acids.
  • polypeptide is derived from a mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 by substitution, deletion or addition of one or several amino acids.
  • the polypeptide is a variant of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion at one or more positions.
  • the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 is up to 15, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding module.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity.
  • protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and/or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the polypeptide has protease activity, determined using e.g. a SAAPF assay as described in Example 1 .
  • the polypeptide is a fusion polypeptide. In one embodiment, the polypeptide is isolated. In one embodiment, the polypeptide is purified.
  • the protease of the present disclosure may be formulated as a liquid protease formulation, which is generally a pourable composition, though it may also have a high viscosity.
  • the physical appearance and properties of the liquid protease formulation may vary.
  • the formulation may have different viscosities (gel to water-like), be colored, not colored, clear, hazy, and may comprise solid particles, for example as in slurries and suspensions.
  • the minimum ingredients in the formulation are the protease of the present disclosure and a solvent system to make it a liquid.
  • liquid protease formulation may also comprise other enzyme activities, such as further protease, amylase, lipase, cellulase, and/or nuclease (e.g., DNase, RNase) activities.
  • enzyme activities such as further protease, amylase, lipase, cellulase, and/or nuclease (e.g., DNase, RNase) activities.
  • the solvent system may comprise water, polyols (such as glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, sugar alcohol (e.g. sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol or adonitol), polypropylene glycol, and/or polyethylene glycol), ethanol, sugars, and salts.
  • polyols such as glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol
  • sugar alcohol e.g. sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol or adonitol
  • polypropylene glycol e.g. sorbitol, mannitol, erythrito
  • a liquid protease formulation may be prepared by mixing together a solvent system with a protease concentrate (at a desired degree of purity), or with protease particles to obtain a slurry or suspension.
  • the liquid protease composition comprises:
  • the protease of the present disclosure in the liquid composition may be stabilized using conventional stabilizing agents.
  • stabilizing agents include, but are not limited to, sugars like glucose, fructose, sucrose, or trehalose; salt to increase the ionic strength; divalent cations (e.g., Ca 2+ or Mg 2+ ); and enzyme inhibitors, enzyme substrates, or various polymers (e.g., PVP).
  • Selecting the optimal pH for the formulation may be very important for protease stability.
  • surfactants such as a nonionic surfactant (e.g., alcohol ethoxylates) can improve the physical stability of the protease formulations.
  • composition comprising a protease of the present disclosure, wherein the composition further comprises:
  • a polyol preferably selected from glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, polyethylene glycol, sugar alcohols, sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol and adonitol;
  • an additional enzyme preferably selected from protease, amylase, or lipase
  • a surfactant preferably selected from anionic and nonionic surfactants
  • Slurries or dispersions of the protease are typically prepared by dispersing small particles of proteases (e.g., spray-dried particles) in a liquid medium in which the protease is sparingly soluble, e.g., a liquid nonionic surfactant or a liquid polyethylene glycol. Powder can also be added to aqueous systems in an amount so not all go into solution (above the solubility limit).
  • Another format is crystal suspensions which can also be aqueous liquids (see for example WO20 19/002356).
  • Another way to prepare such dispersion is by preparing water-in-oil emulsions, where the protease is in the water phase, and evaporate the water from the droplets.
  • Such slurries/suspension can be physically stabilized (to reduce or avoid sedimentation) by addition of rheology modifiers, such as fumed silica or xanthan gum, typically to get a shear thinning rheology.
  • the protease of the present disclosure may also be formulated as a solid/granular enzyme formulation.
  • Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661 ,452, and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol
  • the protease of the present disclosure may be formulated as a granule for example as a co-granule that combines the protease with one or more enzymes or benefit agents (such as MnTACN or other bleaching components).
  • additional enzymes include proteases, amylases, lipases, cellulases, and/or nucleases (e.g., DNase, RNase).
  • Each enzyme will then be present in more granules securing a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes.
  • Methods for producing multi-enzyme co-granulate for the detergent industry are disclosed in the IP.com disclosure IPCGM000200739D.
  • An embodiment of the disclosure relates to a protease granule/particle comprising a protease of the present disclosure.
  • the granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include binders, such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may comprise a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core may consist of an inert particle with the protease absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. These methods are well-known in the art and have also been described in international patent application WO2015/028567, pages 3-5, which is incorporated by reference.
  • the core of the protease granule/particle may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MH PC) and polyvinyl alcohol (PVA). Examples of protease granules with multiple coatings are shown in WO 93/07263 and WO 97/23606.
  • Such coatings are well-known in the art, and have earlier been described in, for example, WO00/01793, W02001/025412, and WO2015/028567, which are incorporated by reference.
  • the present invention provides a granule, which comprises:
  • Another aspect of the invention relates to a layered granule, comprising:
  • the protease of the present disclosure may also be formulated as an encapsulated protease formulation (an ‘encapsulate’). This is particularly useful for separating the protease from other ingredients when the protease is added into, for example, a (liquid) cleaning composition, such as the detergent compositions described herein.
  • a (liquid) cleaning composition such as the detergent compositions described herein.
  • Physical separation can be used to solve incompatibility between the protease and other components. Incompatibility can arise if the other components are either reactive against the protease, or if the other components are substrates of the protease.
  • the protease may be encapsulated in a matrix, preferably a water-soluble or water dispersible matrix (e.g., water-soluble polymer particles), for example as described in WO 2016/023685.
  • a water-soluble polymeric matrix is a matrix composition comprising polyvinyl alcohol. Such compositions are also used for encapsulating detergent compositions in unit-dose formats.
  • the protease may also be encapsulated in core-shell microcapsules, for example as described in WO 2015/144784, or as described in the IP.com disclosure IPCOM000239419D.
  • Such core-shell capsules can be prepared using a number of technologies known in the art, e.g., by interfacial polymerization using either a water-in-oil or an oil-in-water emulsion, where polymers are crosslinked at the surface of the droplets in the emulsion (the interface between water and oil), thus forming a wall/membrane around each droplet/capsule. Purity of protease in formulations
  • the protease of the present disclosure used in the above-mentioned protease formulations may be purified to any desired degree of purity. This includes high levels of purification, as achieved for example by using methods of crystallization, and also none or low levels of purification, as achieved for example by using crude fermentation broth, as described in WO 2001/025411 , or in WO 2009/152176.
  • the present disclosure provides a method for hydrolysing protein in a biological matrix comprising the steps of: contacting the biological matrix with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6.
  • a “biological matrix” encompasses, for example a diverse range of substances in which biological processes occur or are contained, including, but not limited to, a biological sample comprising for example bodily fluids (such as blood, serum, plasma, urine, saliva, and cerebrospinal fluid), tissues (from organs like the liver, heart, and brain), cells (including stem cells, immune cells, and tumor cells), mucosa swabs, stool, semen, vaginal swabs, environmental samples (such as soil, water, and air microbe collections), and specialized media like fermentation broth (used in industrial microbiology and biotechnology for cultivating microorganisms and producing biochemical products).
  • bodily fluids such as blood, serum, plasma, urine, saliva, and cerebrospinal fluid
  • tissues from organs like the liver, heart, and brain
  • cells including stem cells, immune cells, and tumor cells
  • mucosa swabs such as soil, water, and air microbe collections
  • environmental samples such as soil, water, and air microbe collections
  • biological matrices can include experimental setups like cell culture media and hydrogels for tissue engineering
  • the protease having the amino acid sequence of SEQ ID NO: 1 is compatible with a range of different DNA & RNA extraction kits optimized for Proteinase K or QIAGEN protease, leading to similar or higher yields of DNA and RNA from different human and animal samples as compared to Proteinase K or Qiagen protease.
  • biological sample covers without limitation blood (e.g., whole blood, serum, plasma, blood cells), tissue (e.g., biopsy specimens, surgically removed tissues) as well as FFPE tissue, urine, saliva, mucosa (e.g., nasal, oral, throat, genital swabs), stool, semen, vaginal swabs, urogenital swabs, cerebrospinal fluid (CSF), synovial fluid, pleural fluid, peritoneal fluid, amniotic fluid, bile, sweat, tears, breast milk, earwax, skin swabs/scrapings, nail clippings, hair, bone marrow, sputum, aspirates, washes, and exhaled breath condensate.
  • blood e.g., whole blood, serum, plasma, blood cells
  • tissue e.g., biopsy specimens, surgically removed tissues
  • FFPE tissue e.g., FFPE tissue
  • urine e.g., saliva, mu
  • the present disclosure provides a method wherein the biological sample is a fluid or solid from a human or animal body.
  • the biological sample is selected from the group consisting of: whole blood, blood plasma, blood serum, tissue, urine, saliva, and stool.
  • the biological sample is selected from the group consisting of: blood and tissue.
  • the biological sample comprises bacterial cells, eukaryotic cells, viruses, or mixtures thereof.
  • the biological sample comprises one or more polynucleotides.
  • the non-proteinaceous component comprises one or more polynucleotides.
  • Formalin-Fixed, Paraffin-Embedded (FFPE) tissues represent a specialized category of biological samples that are pivotal in clinical diagnostics and biomedical research.
  • the biological sample is a FFPE sample.
  • FFPE processing involves the preservation of tissue samples by fixing them in formalin to prevent degradation, followed by embedding in paraffin wax. This method preserves the structural and molecular integrity of the tissue, allowing for long-term storage and retrospective analysis. Despite their widespread use, FFPE tissues pose unique challenges for nucleic acid extraction due to the crosslinking of proteins and nucleic acids induced by formalin fixation. [0074] The present methods of the disclosure address the efficient extraction of nucleic acids from FFPE tissues by employing a protease with enhanced activity towards crosslinked proteins.
  • This protease having at least 65% sequence identity to the amino acid sequence of Alcalase as denoted by SEQ ID NO: 1 , exhibits high performance in hydrolyzing the protein-nucleic acid complexes commonly found in FFPE samples.
  • the enzymatic activity facilitates the release of nucleic acids from the fixed tissue matrix, enabling the extraction of high-quality DNA and RNA suitable for downstream applications such as sequencing, PCR, NGS or other analyses.
  • the method includes one or more deparaffinization steps to remove the paraffin wax, followed by rehydration of the tissue.
  • the protease is then applied to the rehydrated tissue under conditions optimized for effective digestion of the crosslinked proteins without compromising the integrity of the extracted non-proteinaceous component. This process is particularly beneficial for FFPE tissues that have been stored for extended periods, where the degree of crosslinking may present additional challenges for nucleic acid recovery.
  • the present disclosure concerns the use of this method in conjunction with existing nucleic acid extraction kits designed for FFPE tissues, enhancing their efficiency and yield.
  • the compatibility of the protease of the present disclosure with a broad range of extraction buffers and conditions underscores its versatility and potential to improve nucleic acid recovery from FFPE samples across diverse research and diagnostic settings.
  • the method of the present disclosure extends the utility of FFPE tissues as valuable resources for molecular analysis by ensuring the recovery of non-proteinaceous components in sufficient quantity and quality, thereby expanding the possibilities for advancing medical research and enhancing diagnostic accuracy.
  • the biological sample originates from an animal or a human.
  • the biological sample originates from an animal selected from the group consisting of: Cattle, such as beef cattle and dairy cows; Pigs or swine; Poultry, such as chickens, turkeys, ducks, and geese; Small ruminants, such as sheep and goats; Aquatic species, such as salmon, trout, catfish, and shrimp; Equine, primarily horses; Bees, for honey production; and Other animals, such as rabbits, deer, and bison.
  • the biological sample originates from Cattle, such as beef or dairy cows
  • the one or more polynucleotides are markers of bovine spongiform encephalopathy (BSE), foot-and-mouth disease, bovine tuberculosis, and/or mastitis.
  • BSE bovine spongiform encephalopathy
  • the biological sample originates from Pigs or swine
  • the one or more polynucleotides are markers of African swine fever, porcine reproductive and respiratory syndrome (PRRS), and/or classical swine fever.
  • the biological sample originates from Poultry, such as chickens, turkeys, ducks, or geese
  • the one or more polynucleotides are markers of avian influenza, Newcastle disease, and/or Marek's disease.
  • the biological sample originates from Small ruminants, such as sheep or goats, and the one or more polynucleotides are markers of scrapie in sheep and/or contagious agalactia in goats.
  • the biological sample originates from Aquatic species, such as salmon, trout, catfish, or shrimp, and the one or more polynucleotides are markers of white spot disease in shrimp and/or infectious salmon anemia in salmon.
  • Aquatic species such as salmon, trout, catfish, or shrimp
  • the one or more polynucleotides are markers of white spot disease in shrimp and/or infectious salmon anemia in salmon.
  • the one or more polynucleotides comprise DNA, RNA, or a mixture thereof.
  • the present method, kits, and buffers enable high extraction yields for both DNA and RNA from various sources.
  • the one or more polynucleotides are derived from a virus or a microorganism.
  • the nature virus or microorganism may be identified.
  • the one or more polynucleotides are derived from a virus.
  • the virus is an enveloped virus or a non-enveloped virus.
  • the virus is a filamentous virus, icosahedral virus, or a complex virus.
  • the virus is a dsDNA virus, a ssDNA virus, a dsRNA virus, a +ssRNA virus, a -ssRNA virus, a ssRNA retrovirus or a dsRNA retrovirus.
  • the one or more polynucleotides are markers of one or more of: viral infections, bacterial infections, parasitic infections, fungal infections, other pathogens, or a combination thereof.
  • the one or more polynucleotides are markers of a virus of a family selected from the group consisting of: Retroviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Hepeviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Pneumoviridae, Papillomaviridae, Herpesviridae, Filoviridae, Caliciviridae, Polyomaviridae, Rhabdoviridae, Reoviridae, Bunyaviridae, Arenaviridae, Astroviridae, and Togaviridae.
  • the one or more polynucleotides are markers of a virus of a genus selected from the group consisting of: Lentivirus, Enterovirus, Hepatovirus, Rhinovirus, Orthohepadnavirus, Hepacivirus, Flavivirus, Orthohepevirus, Influenzavirus A, Influenzavirus B, Influenzavirus C, Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, Betacoronavirus, Orthopneumovirus, Papillomavirus, Simplexvirus, Varicellovirus, Lymphocryptovirus, Cytomegalovirus, Roseolovirus, Rhadinovirus, Marburgvirus, Ebolavirus, Norovirus, Orthopolyomavirus, Lyssavirus, Rotavirus, Rubivirus, Rubulavirus, Orthopoxvirus, Hantavirus, Mastadenovirus, Mamastrovirus, Arenavirus, and Deltaretrovirus.
  • Lentivirus Enterovirus
  • Hepatovirus Rhinovirus
  • the one or more polynucleotides are markers of a virus selected from the group consisting of: Human Immunodeficiency virus (HIV), Human Hepatitis A, B and C viruses (HAV, HBV, HCV), Human Influenza A, B & C viruses (IAV, IBV, ICV), Human parainfluenzavirus 1 , 2, 3 and 4 (HPIV-1 , HPIV-2, HPIV-3, HPIV-4), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS-CoV-1 , SARS-CoV-2), MERS Coronavirus (MERS-CoV), Human coronavirus (HCoV), Respiratory Syncytial virus (RSV), Human Papillomavirus (HPV), Hepatitis E virus (HEV), West-Nile-virus (WNV), Human Poliovirus, Human Herpes virus; for example Herpes simplex virus, Epstein-Barr virus (EBV), Varizella zoster virus, Human Cyclona virus, HBs
  • the one or more polynucleotides are markers of one or more of: Human Immunodeficiency Virus (HIV), Hepatitis B and C viruses (HBV and HCV), Influenza A and B viruses, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Respiratory Syncytial Virus (RSV), Human Papillomavirus (HPV), Epstein-Barr Virus (EBV), Mycobacterium tuberculosis, Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella spp., Escherichia coli (E.
  • HCV Human Immunodeficiency Virus
  • HCV Hepatitis B and C viruses
  • Influenza A and B viruses Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Respiratory Syncytial Virus (RSV), Human Papillo
  • the one or more polynucleotides are markers of a virus selected from the group consisting of: Rift valley fever virus (RVFV), African swine fever Virus (ASFV), Foot-and-mouth disease Virus (FMDV), Avian influenza Virus (AIV), Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Bovine viral diarrhea virus (BVDV), Infectious bovine rhinotracheitis virus (IBRV), Equine herpesvirus (EHV), Newcastle disease virus (NDV), Transmissible gastroenteritis virus (TGEV), Bluetongue virus (BTV), Porcine circovirus (PCV), Porcine epidemic diarrhea virus (PEDV), Porcine deltacoronavirus (PDCoV), Infectious bronchitis virus (IBV), Porcine teschovirus (PTV), Hepatitis E virus (HEV), Coronavirus.
  • RVFV Rift valley fever virus
  • ASFV African swine fever Virus
  • FMDV Foot
  • the one or more polynucleotides are markers of one or more of: Viral infections, such as Foot-and-mouth disease virus (FMDV), Porcine epidemic diarrhea virus (PEDV), and African swine fever virus (ASFV); Bacterial infections, such as Salmonella spp., Escherichia coli (E. coli), Campylobacter jejuni, Brucella spp., and Mycobacterium avium subsp.
  • Viral infections such as Foot-and-mouth disease virus (FMDV), Porcine epidemic diarrhea virus (PEDV), and African swine fever virus (ASFV)
  • Bacterial infections such as Salmonella spp., Escherichia coli (E. coli), Campylobacter jejuni, Brucella spp., and Mycobacterium avium subsp.
  • paratuberculosis paratuberculosis
  • Parasitic infections such as Cryptosporidium parvum, Eimeria spp., and Fasciola hepatica
  • Fungal infections primarily related to mycotoxins in feed
  • Other pathogens such as Coronaviruses and Clostridium perfringens.
  • SEQ ID NO:1 has a similar pH profile to Proteinase K and Qiagen Protease. All three enzymes have their maximum activity between pH 8-10 (Fig. 1) and show activity of minimum 10% of their respective maximum activity in the range between pH 6-11. However, as shown in Fig. 2, the activity of SEQ ID NO:1 is superior to Proteinase K at pH 8-10, and superior to Qiagen Protease in the broad range of pH 6-11 , even after 2 hours of incubation at the respective pH value.
  • SEQ ID NO:1 The superior activity profile for SEQ ID NO:1 across a broad range of pH values, even after prolonged incubation times, suggests that SEQ ID NO: 1 can be used at lower concentrations than Proteinase K and Qiagen Protease to achieve the same proteolytic performance, which is highly desirable for applications in diagnostics.
  • the step of contacting the biological sample with the protease comprises incubating the protease with the biological sample.
  • the protease is incubated with the biological sample for at from 20 °C to 80 °C, for example 37 °C.
  • the protease is incubated with the biological sample for from 0.5 minutes to 60 minutes, for example 30 minutes.
  • the biological sample is contacted with the protease at a pH of from 5 to 12, such as from 5 to 6, such as from 6 to 7, such as from 7 to 8, such as from 8 to 9, such as from 9 to 10, such as from 10 to 11 , such as from 11 to 12, for example wherein the pH is 8.
  • the one or more polynucleotides comprise DNA and the biological sample is whole blood.
  • the one or more polynucleotides comprise DNA and the biological sample comprises a virus.
  • the one or more polynucleotides comprise RNA and the biological sample comprises a virus.
  • the one or more polynucleotides comprise DNA and the biological sample is tissue, optionally cancer tissue, such as bladder cancer tissue.
  • the one or more polynucleotides comprise RNA and the biological sample is tissue, optionally cancer tissue, such as human head & neck cancer tissue.
  • a method for isolating and characterizing polynucleotides from a biological sample comprising: a) contacting the biological sample with a protease having at least 65% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6 optionally under conditions suitable for hydrolysing protein in the biological sample; b) separating a non-proteinaceous component comprising one or more polynucleotides, from the biological sample; c) wherein the biological sample is selected from blood, tissue, urine, saliva, and stool, and originates from an animal selected from the group consisting of: a human, or an animal; d) wherein the one or more polynucleotides are biomarkers of a specific disease or a condition; e) and wherein the method is performed at a temperature ranging from 20 °C to 80 °C and a pH of 5
  • the present disclosure provides a method for purifying one or more polynucleotides from a biological sample, said method comprising at least the following steps: a. incubating the biological sample with a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; b. binding the one or more polynucleotides to a solid phase; c. eluting the one or more polynucleotides from the solid phase; thereby purifying one or more polynucleotides from the biological sample.
  • the present disclosure provides a method for nucleic acid extraction from a biological sample, said method comprising: a. contacting the biological sample as defined herein with a protease comprising an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; b. incubating the biological sample under conditions sufficient for protein hydrolysis to provide an incubated sample; and c. extracting one or more polynucleotides from the incubated sample.
  • the method further comprises purifying one or more of the polynucleotides by: a) incubating the biological sample with the protease; b) binding the one or more polynucleotides to a solid phase; and c) eluting the one or more polynucleotide from the solid phase; thereby purifying one or more polynucleotide from the biological sample.
  • the present disclosure concerns a step of analysis.
  • the step of analysis comprises in combination with any of the method steps disclosed herein that the non-proteinaceous component which has been extracted from the biological sample disclosed herein is subjected to analysis without significantly compromising the quality or quantity of the non-proteinaceous component.
  • the protease of the present disclosure does not interfere with the non- proteinaceous component after extraction and influences the analytical output.
  • the protease of the present disclosure can reliably be inactivated by subjecting the biological sample comprising the protease to a at least a predefined temperature which is lower than temperatures which would impact the quality and/or quantity of the non-proteinaceous component. This property renders the present protease highly suitable for extracting a non-proteinaceous component for further analysis.
  • nucleic acids such as RNA and DNA
  • Analysis of the non-proteinaceous component, specifically nucleic acids such as RNA and DNA, extracted from a biological sample is important for a wide range of applications, including but not limited to, diagnostics, research, and therapeutic development.
  • nucleic acids such as RNA and DNA
  • several analytical methods are employed, each designed to assess various aspects of the nucleic acids' quality, quantity, and specific characteristics. The choice of analytical method may depend on the specific requirements of the subsequent application for which the nucleic acids are intended.
  • Quantitative PCR is one of the primary methods for analyzing extracted nucleic acids, offering both quantification and assessment of purity. This technique allows for the amplification and detection of specific DNA sequences, providing insights into the amount and quality of DNA present in the biological sample.
  • RT-qPCR reverse transcription qPCR
  • cDNA complementary DNA
  • the methods of the present disclosure further comprise a step of analysis of the non-proteinaceous component using Quantitative PCR (qPCR) and/or reverse transcription qPCR (RT-qPCR).
  • qPCR Quantitative PCR
  • RT-qPCR reverse transcription qPCR
  • NGS Next-generation sequencing
  • the methods of the present disclosure further comprise a step of analysis of the non-proteinaceous component using Next-generation sequencing (NGS).
  • NGS Next-generation sequencing
  • the method further comprises analysis of the one or more polynucleotides using molecular diagnostics, such as molecular diagnostics selected from the group consisting of: qualitative PCT (qPCR), and reverse transcription qualitative PCT (RT- qPCR), and Next-generation sequencing (NGS).
  • molecular diagnostics selected from the group consisting of: qualitative PCT (qPCR), and reverse transcription qualitative PCT (RT- qPCR), and Next-generation sequencing (NGS).
  • the step of analysis of the one or more polynucleotides using molecular diagnostics occurs subsequently to the step specified herein for isolating, characterizing, and/or purifying the one or more polynucleotides.
  • the present disclosure provides a kit comprising: a. a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; and b. a material with an affinity to one or more polynucleotides.
  • the material with an affinity to one or more polynucleotides comprises silica or glass particles. [0122] In some embodiments, the material with an affinity to one or more polynucleotides comprises magnetic beads.
  • the magnetic beads are coated with a polymer to enhance nucleic acid binding.
  • the kit further comprising a chaotropic salt to facilitate binding of the one or more polynucleotides to the material.
  • the kit comprises the protease in a buffered solution optimized for DNA or RNA extraction.
  • the kit is provided further comprising a buffer, such as a nucleic acid extraction buffer.
  • the kit is provided further comprising a buffer selected from the group consisting of: a Tris buffer with or without EDTA, a guanidine isothiocyanate buffer, a sodium dodecyl sulfate (SDS) buffer, a cetrimonium bromide (CTAB) buffer, a sodium phosphate buffer, a lysis buffer comprising guanidine hydrochloride, a guanidine thiocyanate buffer, or a mixture thereof.
  • a buffer selected from the group consisting of: a Tris buffer with or without EDTA, a guanidine isothiocyanate buffer, a sodium dodecyl sulfate (SDS) buffer, a cetrimonium bromide (CTAB) buffer, a sodium phosphate buffer, a lysis buffer comprising guanidine hydrochloride, a guanidine thiocyanate buffer, or a mixture thereof.
  • the kit comprising a buffer comprising: a. 10mM Tris-CI pH 8; 25mM EDTA; 100mM NaCI; 0.5% SDS; b. 10mM Tris-CI pH 8; 100mM EDTA; 0.5% SDS; c. 30mM TRIS HCI pH8, 1% SDS, 40mM MgCI 2 ; d. 30mM TRIS HCI pH8, 0.25M urea, 40mM MgCI 2 ; e. 30mM Tris Ci pH 8; f. SmartCut buffer; or g. P1 buffer, QIAGEN miniprep kit.
  • the kit comprising Tris-CI, such as 30mM Tris Cl pH 8 and one or more additives selected from the group consisting of: a. 0.25M UREA; b. 4.5M UREA; c. 0.8mM TCEP; d. 100mM TCEP; e. 1 % Tween-20; f. 1 % Triton-X; g. I M GuHCI; h. I M GuSCN; i. 100mM EDTA;
  • Tris-CI such as 30mM Tris Cl pH 8 and one or more additives selected from the group consisting of: a. 0.25M UREA; b. 4.5M UREA; c. 0.8mM TCEP; d. 100mM TCEP; e. 1 % Tween-20; f. 1 % Triton-X; g. I M GuHCI; h. I M GuSCN; i. 100mM EDTA;
  • the kit is provided further comprising a wash buffer to remove contaminants from the material with an affinity to one or more polynucleotides after binding.
  • the kit is provided further comprising an elution buffer, for example configured to release the one or more polynucleotides from the material.
  • the kit comprises a buffer selected from the group consisting of: a. 30mM Tris Cl pH 8; b. SmartCut buffer; and c. P1 buffer, QIAGEN miniprep kit.
  • the material with an affinity to one or more polynucleotides is affixed to a solid surface within a column or chamber.
  • the kit comprises one or more additives selected from the group consisting of: a. Tris Ci, such as 30mM Tris Cl pH 8; b. 0.25M UREA; c. 4.5M UREA; d. 0.8mM TCEP; e. 100mM TCEP; f. 1% Tween-20; g. 1% Triton-X; h. 0.02mM PMSF; i. 1M GuHCI; j. 1M GuSCN; k. 100mM EDTA; l. 100mM NaCI; m. 100mM KCI; n. 40mM MgC ; and o. 40mM CaCh.
  • the kit comprises Tris Ci, such as 30mM Tris Cl pH 8 and one or more additives selected from the group consisting of: a. 0.25M UREA; b. 4.5M UREA; c. 0.8mM TCEP; d. 100mM TCEP; e. 1% Tween-20; f. 1% Triton-X; g. 0.02mM PMSF; h. 1M GuHCI; i. 1M GuSCN; j. 100mM EDTA; k. 100mM NaCI; l. 100mM KCI; m. 40mM MgCI2; and n. 40mM CaCI2.
  • Tris Ci such as 30mM Tris Cl pH 8 and one or more additives selected from the group consisting of: a. 0.25M UREA; b. 4.5M UREA; c. 0.8mM TCEP; d. 100mM TCEP; e. 1% Tween-20; f. 1% Triton
  • a buffer comprising a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; and one or more additives.
  • the buffer is provided wherein the one or more additives are selected from the group consisting of: a. UREA; b. TCEP; c. Tween-20; d. Triton-X; e. GuHCI; f. GuSCN; g. EDTA; h. NaCI; i. KOI; j. MgCh; and
  • the one or more additives are selected from the group consisting of: a. 0.25M UREA; b. 4.5M UREA; c. 0.8mM TCEP; d. 100mM TCEP; e. 1% Tween-20; f. 1% Triton-X; h. 1M GuHCI; i. 1M GuSCN; j. 100mM EDTA; k. 100mM NaCI; l. 100mM KCI; m. 40mM MgCh; and n. 40mM CaCh.
  • a buffer comprising a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; and one or more additives selected from the group consisting of: a. UREA, such as 0.25M UREA or 4.5M UREA; b. TCEP, 0.8mM TCEP or 100mM TCEP; c. Tween-20, such as 1% Tween-20; d. Triton-X, such as 1 % Triton-X; e.
  • GuHCI such as 1M GuHCI
  • f. GuSCN such as 1 M GuSCN
  • g. EDTA such as 100mM EDTA
  • h. NaCI such as 100mM NaCI
  • i. KOI such as 100mM KOI
  • j. MgCh such as 40mM MgCh
  • k. CaCh such as 40mM CaCI 2 .
  • the buffer comprises a Tris buffer with or without EDTA , a guanidine isothiocyanate buffer, a sodium dodecyl sulfate (SDS) buffer, a cetrimonium bromide (CTAB) buffer, a sodium phosphate buffer, a lysis buffer comprising guanidine hydrochloride, a guanidine thiocyanate buffer, or a mixture thereof.
  • the buffer comprises: a) 10mM Tris- Cl pH 8; 25mM EDTA; 100mM NaCI; 0.5% SDS; b) 10mM Tris- Cl pH 8; 100mM EDTA; 0.5% SDS; c) 30mM TRIS HCI pH8, 1% SDS, 40mM MgCI 2 ; d) 30mM TRIS HCI pH8, 0.25M urea, 40mM MgCI 2 ; e) 30mM Tris Ci pH 8; f) SmartCut buffer; or g) P1 buffer, QIAGEN miniprep kit.
  • a buffer comprising the protease disclosed herein and TRIS, such as 30 mM TRIS, pH 8.0, 1% SDS, and 40 mM MgCI 2 .
  • the buffer disclosed herein further comprises TRIS, such as 30 mM TRIS, pH 8.0.
  • a buffer comprising a protease comprising an amino acid sequence which has at least 65% sequence identity to the amino acid sequence of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; and TRIS, such as 30 mM TRIS pH 8.0, and one or more additives selected from the group consisting of: a. UREA, such as 0.25M UREA or 4.5M UREA; b. TCEP, 0.8mM TCEP or 100mM TCEP; c. Tween-20, such as 1% Tween-20; d.
  • UREA such as 0.25M UREA or 4.5M UREA
  • TCEP 0.8mM TCEP or 100mM TCEP
  • Tween-20 such as 1% Tween-20
  • d Tween-20
  • Triton-X such as 1 % Triton-X
  • e. GuHCI such as 1M GuHCI
  • f. GuSCN such as 1 M GuSCN
  • g. EDTA such as 100mM EDTA
  • h. NaCI such as 100mM NaCI
  • i. KCI such as 100mM KCI
  • j. MgCh such as 40mM MgCh
  • CaCh such as 40mM CaCh.
  • the method dsclosed herein is provided wherein the protease is comprised in a buffer comprising TRIS, pH8, such as 30 mM TRIS, pH8, and SDS, such as 1% SDS, and MgCh, such as 40 mM MgCh wherein the biological sample is human whole blood.
  • the method dsclosed herein is provided wherein the protease is comprised in a buffer comprising TRIS, pH8, such as 30 mM TRIS, pH8, and UREA, such as 0.25 mM UREA, and MgCh, such as 40 mM MgCh wherein the biological sample is tissue.
  • Chemicals used in the examples herein e.g. for buffers and substrates are commercial products of at least reagent grade.
  • Table X overview of protease sequences used in the present application.
  • Example 1 Activity of a protease (SEQ ID NO: 1) at varying pH
  • the protease activity was measured using a SAAPF assay (description see below) in universal buffers of pH 2-12. The activities were tested using 0.1 pg/mL of the specified protease. Enzyme solutions were mixed with universal buffers at the respective pH values and incubated for 2h at 37°C. Subsequently the substrate was added and absorption was measured as described below.
  • Suc-AAPF-pNA is an abbreviation for N-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can be cleaved by endo-proteases. Following proteolytic cleavage, a free pNA molecule having a yellow color is liberated and can be measured by visible spectrophotometry at wavelength 405 nm.
  • the Suc-AAPF-PNA substrate is manufactured, e.g., by Bachem (cat. no. L1400, dissolved in DMSO).
  • a sample containing the polypeptide to be analyzed is diluted in activity buffer.
  • the assay is performed by transferring diluted enzyme samples to 96 well microtiter plate and adding substrate working solution. The solution is mixed at room temperature and absorption is measured every 20 sec. over 5 minutes at 405 nm. The sample is diluted to a level where the slope is linear. The slope (absorbance per minute) of the time-dependent absorption curve is directly proportional to the proteolytic activity of the polypeptide under the given conditions.
  • SEQ ID NO: 1 has a similar pH profile to Proteinase K and Qiagen Protease. All three enzymes have their maximum activity between pH 8-10 (Fig. 1) and show activity of minimum 10% of their respective maximum activity in the range between pH 6-11 (Alcalase and Proteinase K) and pH 6-12 (Qiagen Protease). As shown in Fig. 2, the activity of SEQ ID NO: 1 is superior to Proteinase K at pH 8-10, and Qiagen Protease in the broad range of pH 6-11 , even after 2 hours of incubation at the respective pH value.
  • SEQ ID NO: 1 The superior activity profile for SEQ ID NO: 1 across a broad range of pH values, even after prolonged incubation times, suggests that SEQ ID NO: 1 can be used at lower concentrations than Proteinase K and Qiagen Protease to achive the same proteolytic performance, which is highly desirable for applications in diagnostics.
  • Example 2 Activity of the protease (SEQ ID NO: 1) with common nucleic acid extraction additives
  • protease activity of selected proteases was measured using a SAAPF assay as described in Example 1 in 30mM Tris Cl buffer at pH 8 with common nucleic acid extraction additives. The proterase activity was tested with 0.1 pg/mL protease. All samples were incubated at 37 °C for 30 minutes prior to addition of substrate and measurement. The activity was indexed at 100% for each protease studied in 30 mM Tris Cl at pH 8 and protease activities measured in other additive systems were reported relative to the activity determined in 30 mM Tris- Cl at pH 8 (table 1) and normalized to the respective sample of Proteinase K (table 2) and Qiagen Protease (table 3).
  • the activity of SEQ ID NO: 1 is normalized to the respective sample of Qiagen Protease. Numbers in bold indicate that a result is on par with or superior to Proteinase K.
  • GuHCI guanidinium hydrochloride
  • TCEP Tris(2-Carboxyethyl) phosphine
  • PMSF phenylmethylsulfonyl fluoride. *PMSF is a protease inhibitor, therefore low activity is preferred.
  • SEQ ID NO: 1 exhibits high protease activity in presence of several commonly used additives with a comparable profile to Proteinase K and Qiagen Protease (table 1), while showing a higher level of activity for most tested additives (tables 2 and 3). For example for 1% Tween 20, where SEQ ID NO: 1 exhibits 191% activity relative to 100% activity for Proteinase K, and 512% activity relative to 100% activity for Qiagen Protease.
  • Table 1 The results summarized in tables 1 , 2 and 3 show that SEQ ID NO: 1 is highly compatibile with additives commonly used in nucleic acid extraction buffers and has a higher activity than Proteinase K and Qiagen Protease for almost all additives.
  • SEQ ID NO: 1 For the protease inhibitor PMSF, SEQ ID NO: 1 had lower activity than Proteinase K and Qiagen Protease, as a result of more effective inhibition of SEQ ID NO: 1 compared to the other tested proteases. Taken together, this shows that SEQ ID NO: 1 may be used in diagnostic kits at lower concentrations and with shorter sample incubation times than other tested proteases, making Alcalse a more efficient and cost-effective kit component.
  • protease activity of SEQ ID NO: 1 was measured using a SAAPF assay in different nucleic acid extraction buffers (see table 4).
  • the SAAPF assay used is described in Example 1. All the tests were performed at pH 8 and 0.1 pg/mL of each protease was used. Each sample was incubated at 37 °C for 30 minutes prior to measurement. The activity was normalized to the activity of Proteinase K and Qiagen Protease for each condition, respectively.
  • SEQ ID NO: 1 is applicable in known buffers used in nucleic acid extraction, and consistently outperforms Proteinase K and Qiagen Protease.
  • Example 4 Protease activity (SEQ ID NO: 1) in buffers used for nucleic acid restriction & elution
  • the protease activity of SEQ ID NO: 1 was measured using a SAAPF assay in a restriction buffer (CutSmart® buffer, NEB, #B6004) and a DNA resuspension (buffer P1 , Qiagen, # 19051).
  • the SAAPF assay used is described in example 1. All the tests were performed with 0.1 pg/mL of each protease. Each sample was incubated at 37 °C for 30 minutes prior to measurement. The activity was normalized to the activity of Proteinase K and Qiagen Protease for each condition, respectively.
  • Table 6 Protease activity of SEQ ID NO: 1 in selected buffers used for nucleic acid restriction and elution, respectively.
  • the activity of SEQ ID NO: 1 is normalized to the activity of Proteinase K.
  • CutSmart buffer 50mM potassium acetate; 20mM TRIS acetate; 10mM magnesium acetate; 100 pg/ml BSA; pH 7.9; P1 buffer: 50mM TRIS pH 8; 10mM EDTA; 100pg/ml RNAse A. Numbers in bold indicate that a result is superior to Proteinase K.
  • Table 7 Protease activity of SEQ ID NO: 1 in selected buffers used for nucleic acid restriction and elution, respectively.
  • the activity of SEQ ID NO: 1 is normalized to the activity of Qiagen Protease.
  • CutSmart buffer 50mM potassium acetate; 20mM TRIS acetate; 10mM magnesium acetate; 100 pg/ml BSA; pH 7.9; P1 buffer: 50mM TRIS pH 8; 10mM EDTA; 100pg/ml RNAse A. Numbers in bold indicate that a result is superior to Proteinase K.
  • SEQ ID NO: 1 is applicable for use in known buffers used in nucleic acid modification and elution, showing higher activity than Proteinase K and Qiagen Protease.
  • SEQ ID NO: 1 is a highly versatile protease that is suited for a broad range of nucleic acid applications.
  • Example 5 Nucleic acid extraction from human whole blood, mouse tissue, virus and FFPE-sections
  • Extraction and kits Separately supplied Proteinase K or protease from various commercially available kits was replaced by SEQ ID NO: 1. The same buffer for reconstituting proteinase K or protease recommended by each kit was used to dissolve SEQ I D NO: 1 whenever applicable. Proteases were first compared at constant protease concentration while varying the incubation time of protease with the sample to be extracted. In cases where the recommended incubation time was too short or too long to detect a difference by time variation, the incubation time was kept constant, and a dilution series of the protease was set up and tested for extraction efficiency.
  • the viral DNA and RNA was extracted using the QIAamp MiniElute Virus Spin Kit (cat # 57704). Viral RNA extraction was performed using nasal nares samples collected in UTM from SARS-Cov-2 positive patients. Viral DNA extraction was performed using contrived samples by spiking AAV2 virus genome into human normal plasma.
  • the FFPE DNA was extracted using the FormaPure XL RNA Reagent Kit (Beckman Coulter #C35996).
  • the FFPE RNA was extracted using the PureLinkTM FFPE RNA Isolation Kit (#K156002). FFPE DNA and RNA extraction was performed using human head & neck cancer and human bladder cancer samples.
  • Quantification and qualification of DNA and RNA after extraction Quality and quantity of extracted nucleic acids was determined using a Nanodrop spectrophotometer or Qubit fluorometer. qPCR was also used to access the quality of the extracted DNA and RNA. TaqManTM qPCR was chosen for qPCR qualification. For nucleic acids extracted from virus particles, a relative quantification was done using Taqman qPCR/ RT-PCR due to very low concentrations. Reagents information is shown below in Table 8, and information on kits used is shown in Table 9: Table 8. Overview of reagents used.
  • the extraction yield was 0.17 pg/mL for Proteinase K and 0.29 pg/mL for SEQ ID NO: 1.
  • the Ct value was 2.44 points lower for SEQ ID NO: 1 than for Qiagen protease. iv. RNA extraction from virus: QIAamp MinElute Virus Spin Kit (Qiagen #57704; due to low yields, extracted amounts of RNA were compared using Taqman RT-PCR. The lower the Ct value, the higher the DNA concentration)
  • RNA extraction from FFPE sections - human head & neck cancer PureLinkTM FFPE RNA Isolation Kit (Thermo #K156002)
  • the extraction yield was 793.23 ng/ mm 3 tissue for Proteinase K and 1119.18 ng/ mm 3 tissue for SEQ ID NO: 1 .
  • nucleic acid samples were further qualified using Taqman qPCR (DNA) or Taqman RT- PCR (RNA); target genes and reagents used are listed in table 5. Each reaction showed a uniform melting curve with one clear peak (data not shown) corresponding to the formation of only one specific product. Furthermore, PCR/RT-PCR Ct values for each nucleic acid samples generated using SEQ ID NO: 1 fulfilled the criterium of ACtfAlcalase-Proteinase K or Qiagen protease) ⁇ 1. This shows that nucleic acid samples extracted using SEQ ID NO: 1 are highly suitable for subsequent qPCR/ RT-PCR analysis.
  • SEQ ID NO: 1 is compatible with a range of different DNA & RNA extraction kits optimized for Proteinase K or QIAGEN protease, leading to similar or higher yields of DNA and RNA from different human and animal samples compared to Proteinase K or Qiagen protease.
  • the extracted DNA yield from FFPE sections from human bladder cancer was 321.94 ng/ mm 3 tissue for Proteinase K, and 390.46 ng/ mm 3 tissue for SEQ ID NO: 1 using Beckmann Coulter Kit #035996.
  • cfDNA Cell-free DNA
  • QIAamp Circulating Nucleic Acid Kit Qiagen, cat# 55114.
  • Proteinase K was replaced by SEQ ID NO: 1 , and stock concentrations of enzymes were set to 18 mg/ml.
  • Incubation of enzyme-sample mixtures were carried out at 56°C, as pretests had shown higher yields for SeqID No. 1 (Alcalase) at 56°C as compared to 60°C (data not shown). Yields of extracted cfDNA were quantified using Tapestation (Agilent).
  • SEQ ID NO: 1 is well suited for extraction of cfDNA from blood plasma, and outperforms Proteinase K.
  • Example 7 Nucleic acid extraction yield under optimized conditions compared to recommended conditions from kit.
  • Extractions were carried out using QIAamp DNA mini kit (Qiagen, cat 51304). Proteinase K supplied with the kit was replaced by SEQ ID NO: 1. The kit lysis buffer was replaced by different buffers (buffer 1 : 30mM TRIS HCI pH8, 1 % SDS, 40mM MgCh; buffer 6: 30 mM TRIS HCI pH8, 0.25M urea, 40mM MgCh). Human whole blood and mouse heart tissue were used as samples for extraction. For the blood samples, 20 pl of a 20 mg/ml solution of Proteinase K or SEQ ID NO: 1 was added to a sample volume of 100 pl.
  • Pre-tests had shown increased yields for SEQ ID NO: 1 when incubated at 37°C as compared to 56°C (data not shown), therefore incubation temperature for Alcalse was set to 37°C, and to 56°C for Proteinase K according to the instructions of the kit. Samples were incubated for 1h. The tissue samples were disrupted using TissueRuptor II device (Qiagen) prior to enzymatic treatment. 20 pl of a 20 mg/ml solution of Proteinase K or SEQ ID NO: 1 was added to 5 mg of disrupted tissue together with the lysis buffer, followed by 2h of incubation at 56°C (Proteinase K) or 37°C (Alcalase).
  • Table 10 DNA yield in ng/ ml blood, extracted from mouse heart tissue using proteinase K or SEQ ID NO: 1 under different conditions. Numbers in bold indicate that a result is superior to Proteinase K.
  • Table 11 DNA yield in g/ ml blood, extracted from human whole blood using proteinase K or SEQ ID NO: 1 under different conditions. Numbers in bold indicate that a result is superior to Proteinase K.
  • SEQ ID NO: 1 can be easily integrated into existing commercial DNA extraction kits and greatly increases DNA yields as compared to using Proteinase K with very few protocol modifications such as buffer and incubation temperature.
  • DNA extraction from mouse heart tissue led to yields of 191.2 ng DNA/mg tissue using SEQ ID NO: 1 (incubation at 37°C) and 248.1 ng DNA/mg tissue for Proteinase K (incubation at 56°C), when compared against each other using a Proteinase K-based commercially available kit.
  • SEQ ID NO: 1 incubation at 37°C
  • 248.1 ng DNA/mg tissue for Proteinase K incubation at 56°C
  • Example 8 Nucleic acid extraction carried out with and without SEQ ID NO: 1
  • Extraction and kits Extractions of DNA from human whole blood samples were carried out using DNeasy Blood & Tissue Kit (Qiagen #69504), thereby replacing Proteinase K with SEQ ID NO: 1. Extraction were carried out with SEQ ID NO: 1 (adding 20 pl of a 20 mg/ml solution of SEQ ID NO: 1 to a sample volume of 100 pl), or without addition of SEQ ID NO: 1. The incubation temperature was set to 37°C.
  • Table 12 DNA yield in g/ ml blood, extracted from human whole blood with and without SEQ ID NO: 1.
  • the present example shows that nearly no DNA was extracted when SEQ ID NO: 1 was not added during extraction, while significant yields were obtained from extraction when SEQ ID NO: 1 was added (30x increase from 1 to 35 ng/ml blood). This shows that the proteolytic activity of SEQ ID NO: 1 is required for DNA extraction.
  • Genomic DNA of a Bacillus licheniformis strain was isolated from 1 mL of overnight culture using the DNeasy Blood and Tissue kit (Qiagen #69504) in the QIAcube Connect instrument per manufacturer’s instructions. Genomic DNA was isolated per manufacturer’s instructions, thereby substituting the Proteinase K solution provided in the kit with SEQ ID NO: 1 solution at 18 mg/mL. [0177] The isolated genomic DNA was sent for whole genome DNA sequencing using Illumina short read (instrument: Illumina NextSeq 1000).
  • Phred quality score is an indicator for the probability of an incorrect base call.
  • Quality scores in next-generation sequencing (NGS) provide a metric to assess the accuracy of sequencing data. These scores, often referred to as Phred quality scores (Q scores), are used to determine the probability that a given base in the sequence is called incorrectly by the sequencer.
  • the Phred score is calculated using the formula jT> , where P is the probability of an incorrect base call. For example, a Q score of 30 corresponds to a 1 in 1000 chance of error, indicating 99.9% accuracy in base calling.
  • Q scores are useful for evaluating sequencing accuracy in NGS. High Q scores ensure the reliability of sequencing results and reduce false-positive variant calls.
  • a phred quality score of 30 is considered as benchmark for quality and means that the base call accuracy is 99.9% (the higher the score, the higher the base call accuracy as explained above).
  • Table 13 Results of next generation whole genomic sequencing of a Bacillus licheniformis strains. DNA used for sequencing was extracted using SEQ ID NO: 1.
  • Example 10 Activity of different proteases (SEQ ID NOs: 2-6) with common nucleic acid extraction additives
  • protease activity of selected proteases was measured using a SAAPF assay as described in Example 1 in 30mM Tris Cl buffer at pH 8 with common nucleic acid extraction additives. The proterase activity was tested with 0.25 pg/mL protease. All samples were incubated at 37 °C for 30 minutes prior to addition of substrate and measurement. The activity was indexed at 100% for each protease studied in 30 mM Tris- Cl at pH 8 and protease activities measured in other additive systems were reported relative to the activity determined in 30 mM Tris- Cl at pH 8.
  • Table 14 Protease activity of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and Proteinase K in presence of selected additives.
  • the activity of each protease is normalized to its respective activity in 30mM TRIS- Cl pH 8.
  • GuHCI guanidinium hydrochloride
  • TCEP Tris(2-Carboxyethyl) phosphine
  • SEQ ID NOs: 2-6 are highly compatible with additives commonly used in nucleic acid extraction buffers. While SEQ ID NOs: 4, -5 and -6 showed similar profiles as compared to Proteinase K, SEQ ID NOs: 2 and-3 showed even higher compatibility with some additives as compared to Proteinase K. For example, when incubated with 1 M guanidinium hydrochloride, SEQ ID NO: 2 retained 117% of its activity relative to 30 mM TRIS pH8, while Proteinase K only retained 34% of its activity relative to 30 mM TRIS pH8. This indicates that SEQ ID NOs: 2-6 are equally or better suited for use nucleic acid extraction as compared to Proteinase K.

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Abstract

La présente invention concerne des procédés, des kits et des tampons comprenant une protéase pour hydrolyser des protéines et extraire un composant non protéique d'échantillons biologiques.
PCT/US2024/040585 2024-08-01 2024-08-01 Protéase pour diagnostic Pending WO2024254620A2 (fr)

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