protease

Abstract

This invention relates to an IgM-specific protease. Specifically, the protease according to the present invention can cleave IgM, including human IgM, with high specificity. This invention further relates to a method and use of the protease, particularly for the analysis and / or modification of IgM. This invention relates to the remarkable finding that a polypeptide isolated from the genus Lachnoanaerobaculum of commensal human bacteria can cleave IgM with high specificity.

Description

【Technical Field】 【0001】 Field of the Invention The present invention relates to an IgM-specific protease. Specifically, the protease according to the present invention has high specificity and can cleave IgM including human IgM. The present invention further relates to methods and uses of the protease, particularly for the analysis and / or modification of IgM. 【Background Art】 【0002】 Background of the Invention Pathogenic bacteria have evolved numerous mechanisms to escape the host immune system, by which they can successfully colonize the desired host environment. 【0003】 One such mechanism is the ability of some bacteria to modulate the functionality of host antibodies such that the host antibodies become less active or inactive. For example, some known virulence factors encoded by bacteria have IgG-specific glycolytic or proteolytic activities (Sjoegren J., et al., Glycobiology 2020, 30(4):254-267; Brezki RJ, Jordan RE. mAbs 2010, 2(3):212-220), and other virulence factors have IgA-specific proteolytic activities (Kornfeld SJ, Plaut AG. Rev Infect Dis 1981, 3(3):521-534). 【0004】 Of the known pathogenic factors that modulate the host immune system through enzymatic modification of host proteins, the most typical origins are from zoonotic or species-specific pathogens. Indeed, IgG and IgM-specific proteases have been isolated from pathogenic species within the genus Streptococcus (Von Pawel-Rammingen U., et al., EMBO 2002, 21(7):1607-1615; Lannergard J & Guss B., IdeE, FEMS Microbiol Lett 2006, 262(2):230-5; Spoerry C., et al., J Biol Chem 2016, 291(15):7915-25; Seele J., et al., J Bacteriol 2013, 195(5)). 【0005】 The activity and importance of IgG are well known and widely utilized in biotechnology and medicine. IgM, on the other hand, is often overlooked, yet nevertheless a crucial component of the immune system. In response to microorganism infections, for example, IgM plays a vital role in maintaining the intact primary barrier, and it can neutralize microorganisms by inhibiting the binding of oral and respiratory microorganisms to their congenital cell receptors. Furthermore, IgM plays a key role in pathogen opsonization and complement activation. 【0006】 IgM has further applications in biotechnology and medicine, with applications expanding beyond responses to microorganism infections. Such applications are becoming increasingly common and attractive for therapeutic use (Samsudin F., et al., Chem Sci 2020, 11(10):2843-2854; Zhang J., et al., mAbs 2022, 14(1):2031483). 【0007】 Despite the importance of IgM in both the endogenous function of the immune system and in therapeutic and biotechnological applications, there is a lack of current tools for efficient, widely applicable, and target-specific IgM detection, analysis, and modification. 【0008】 An IgM-specific protease was identified in the zoonotic porcine pathogen Streptococcus suis (Seele J., et al., J Bacteriol 2013, 195(5)). The activity of this protease is species-restricted to substrates containing porcine IgM, but it cannot cleave human IgM. Therefore, there is a clear lack of suitable biological tools for IgM research and manipulation. In particular, the need for biological tools that specifically target human IgM remains unmet. [Prior art documents] [Non-patent literature] 【0009】 [Non-Patent Document 1] Sjoegren J., et al., Glycobiology 2020, 30(4):254-267 [Non-Patent Document 2] Brezski RJ, Jordan RE. mAbs 2010, 2(3):212-220) [Non-Patent Document 3] Kornfeld SJ, Plaut AG. Rev Infect Dis 1981, 3(3):521-534 [Non-Patent Document 4] Von Pawel-Rammingen U., et al., EMBO 2002, 21(7):1607-1615 [Non-Patent Document 5] Lannergard J & Guss B., IdeE, FEMS Microbiol Lett 2006, 262(2):230-5 [Non-Patent Document 6] Spoerry C., et al., J Biol Chem 2016, 291(15):7915-25 [Non-Patent Document 7] Seele J., et al., J Bacteriol 2013, 195(5) [Non-Patent Document 8] Samsudin F., et al., Chem Sci 2020, 11(10):2843-2854 [Non-Patent Document 9] Zhang J., et al., mAbs 2022, 14(1):2031483 [Overview of the project] [Means for solving the problem] 【0010】 Summary of the Invention This invention relates to the remarkable finding that polypeptides isolated from the commensal human bacterium Lachnoanaerobaculum can cleave IgM with high specificity. Therefore, the polypeptides of this invention enable specific and effective targeting of IgM, including human IgM. The polypeptides of this invention have utility in numerous applications as a biological tool for IgM research and manipulation. 【0011】 In the first embodiment, a polypeptide having human IgM-specific endoprotease activity is provided. 【0012】 In one embodiment, a) Amino acid sequence of Sequence ID No. 1, b) A variant of SEQ ID NO: 1 having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, or c) Either a) or b) fragment A polypeptide having endoprotease activity is provided, wherein the endoprotease activity is preferably human IgM-specific endoprotease activity. 【0013】 In another aspect, a) the amino acid sequence of SEQ ID NO: 8, b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8, or c) a fragment of either a) or b) A polypeptide having endoprotease activity is provided, preferably wherein the endoprotease activity is human IgM-specific endoprotease activity, said polypeptide comprising: 【0014】 In one aspect, a composition or combination comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide having endoprotease activity according to the invention and the second polypeptide is a polypeptide having IgG-specific endoprotease activity is provided. 【0015】 In one aspect, a polynucleotide encoding a polypeptide according to the invention is provided. 【0016】 In one aspect, a vector encoding or comprising a polynucleotide according to the invention is provided. 【0017】 In one embodiment, an expression vector encoding or comprising a polynucleotide encoding a polypeptide according to the invention, for the production of a polypeptide according to the invention, is provided. 【0018】 In one aspect, a) a polypeptide according to the invention, b) a polynucleotide according to the invention, c) a vector according to the invention, and / or d) a composition or combination according to the invention, in which the first polypeptide and the second polypeptide of the combination are provided separately, and / or e) A composition or combination according to the present invention, wherein the first polypeptide and the second polypeptide of the above combination are provided together. A kit including this will be provided. 【0019】 In one embodiment, a method for hydrolyzing IgM is provided, comprising the step of contacting a sample containing IgM with a polypeptide, composition, or combination according to the present invention. 【0020】 In one embodiment, a method for detecting the presence of IgM in a sample, a) A step of contacting a sample with the polypeptide according to the present invention, b) A step of identifying and / or isolating IgM-specific cleavage products. A method is provided in which the presence of an IgM-specific cleavage product indicates the presence of IgM in the sample. 【0021】 In one embodiment, the use of polypeptides, combinations, or kits according to the present invention is provided for the degradation, removal, or identification of IgM in a sample. 【0022】 In one embodiment, polypeptides, compositions, combinations, pharmaceutical compositions, or kits for therapeutic use are provided. [Brief explanation of the drawing] 【0023】 [Figure 1] SDS-PAGE Analysis of LU-M1: SDS-PAGE analysis of a modified version expressed by recombination of a polypeptide derived from L. umeaense, named "LU-M1," demonstrates that the polypeptide can be produced in high yield and high purity. Apparent molecular masses are indicated by referring to the molecular weight ladder (lanes 1 and 9); the molecular weights of the ladder components are shown. The "elution" lane demonstrates the ability to obtain a highly pure and homologous sample of LU-M1 after single-round affinity purification using His-tags. [Figure 2A-B]Lachnoanaerobaculum polypeptides represent the first known human IgM-specific endoprotease. The activity of different constructs was evaluated using full-length constructs from L. umeaense (LU-FL) and L. gingivalis (LG-FL) that mediated human and mouse IgG (A). LG-FL mediated to human IgG subclasses was further evaluated (B). Both LG-FL and LU-FL were mixed with human IgA and IgM (C). The activity of the LU-M1 construct was evaluated by mixing it with all human IgG subclasses as well as polyclonal IgG, IgA, and IgM (D). The general proteolytic activity of LU-M1 was investigated using a fluorescent casein substrate (E), and species specificity was investigated using purified IgM from mouse, rabbit, rat, and monkey (F). SpeB (general protease) and IdeS (IgG-specific protease) were used as controls. Figures 2A-D and 2F depict the SDS-PAGE gel, with lanes labeled as appropriate, and apparent molecular masses indicated by a molecular weight ladder. [Figure 2C-D] Same as above. [Figure 2E-F] Same as above. [Figure 3A] Lachnoanaerobaculum-derived polypeptides were treated with PNGaseF, which catalyzes site-specific cleavage of IgM, and then analyzed by liquid chromatography-mass spectrometry (LC-MS) of the reduced and denatured myeloma IgM. Total ion current chromatograms (TIC; upper panel) and deconvoluted compounds of interest (lower panel) are shown for samples that were either untreated (Figure 3A) or further treated with LU-M1 (Figure 3B). [Figure 3B] Same as above. [Figure 4] Size exclusion analysis of IgM cleavage products: Size exclusion chromatography was performed on hydrolyzed IgM samples alongside size standards consisting of thyroglobulin (1), apoferritin (2), and human IgG (3). Hydrolysis products (pentamer CH3-CH4; CH1-CH1-CH2) are indicated in the chromatogram along with intact IgM. [Figure 5A-B] Catalytic Activity Evaluation The activity of LU-M1 was evaluated under various conditions, including a series of pH (A), NaCl concentration (B), presence of divalent cations (C), temperature (D), and presence of protease inhibitors (E). Figures 5A to 5E depict the SDS-PAGE gels, with lanes labeled as appropriate, and apparent molecular masses indicated by referring to the molecular weight ladder. [Figure 5C-D] Same as above. [Figure 5E] Same as above. [Figure 6A-B] LU-M1 is a highly efficient enzyme. The ratio of LU-M1 activity to IgM was evaluated. While maintaining a constant total reaction volume, IgM was mixed with LU-M1 at various enzyme-to-substrate ratios (1:20–1:100; w:w). The reaction mixture was incubated at 37°C for 5–60 minutes. Figure 6A depicts the SDS-PAGE gel, with lanes labeled as appropriate, and apparent molecular masses indicated by a molecular weight ladder. Figures 6B and 6C depict the densitometry data obtained from the corresponding SDS-PAGE analysis. [Figure 6C] Same as above. [Figure 7] LU-M1 is active in serum. Human serum was incubated with IdeS and LU-M1 for 30 minutes at 37°C, and then analyzed by SDS-PAGE (upper panel) and Western blotting for IgG(A) and IgM(B) (lower panel). [Figure 8A-B] LU-M1 can be freeze-dried and immobilized to retain its activity. Freeze-dried LU-M1 was reconstituted with water to the same concentration, and its activity was evaluated against IgM (Figure 8A). The numbers on the SDS-PAGE indicate the freeze-drying conditions of the sample prior to reconstitution. (B) Image of freeze-dried pellet. The activity of resin-immobilized LU-M1 was evaluated against IgM (Figure 8C). The numbers on the SDS-PAGE indicate the concentration of immobilized LU-M1. Figures 8A and 8C depict the SDS-PAGE gel, with lanes labeled as appropriate, and apparent molecular masses indicated by referring to the molecular weight ladder. [Figure 8C] Same as above. [Figure 9] LU-M1 is not readily recognized by human IVIG. The presence of LU-M1-specific human IgG was assessed using Western blotting. 0.5 μg each of IdeS, Xork, and LU-M1 were separated by SDS-PAGE and stained (left panel) or transferred to nitrocellulose membranes for Western blotting (right panel). The membranes were blocked and incubated with human IVIG, and alkaline phosphatase-conjugated anti-human IgG Fc antibody was used for detection. Western blot data indicate that anti-LU-M1 antibody is little to nowhere present in IVIG. [Modes for carrying out the invention] 【0024】 Detailed description of the invention It should be understood that different applications of the disclosed products and methods may be adapted to suit specific needs in the art. It should also be understood that the terminology used herein is intended solely to describe and not to limit specific embodiments of the invention. 【0025】 All publications, patents, and patent applications cited herein are thus incorporated herein by reference in their entirety. 【0026】 Unless otherwise specified, terms are understood to have the general meaning in the art as would be considered by those skilled in the art. 【0027】 The terms “comprising,” “comprises,” and “comprised of,” as used herein, are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended, and do not exclude additional, unenumerated members, elements, or method steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.” Therefore, the term “including” is used to mean “including, but not limited to.” “Including” and “including, but not limited to” can be used interchangeably. Conversely, the term “consisting of” is intended to be exclusive or closed, and does not include additional, unenumerated members, elements, or method steps. 【0028】 As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a polypeptide” includes “polypeptides,” and so on. 【0029】 IgM IgM is an immunoglobulin (Ig) isotype typically characterized by its large size and pentameric structure. However, IgM can also exist in other structural forms, such as secretory hexamers or cell surface-representing monomers that form part of B cell receptors. 【0030】 The pentameric structure of IgM is essentially composed of five structures similar to those of IgG. IgM contains 10 potential antigen-binding sites (or paratopes), which facilitates high-avidity target binding. This high-avidity binding can enable target binding even when monovalent binding affinity is low. 【0031】 IgM is produced in the early stages of infection following antigen exposure and plays a key role in stimulating other effector functions of the immune system, such as the activation of complement and Fc receptor-mediated activity. 【0032】 IgM has been demonstrated to have advantageous therapeutic characteristics even when compared to IgG, and has been described as a promising candidate for monoclonal antibody therapy for several conditions (Samsudin F., et al., Chem Sci 2020, 11(10):2843-2854; Zhang J., et al., mAbs 2022, 14(1):2031483). 【0033】 IgM-specific endoprotease This invention relates to a polypeptide having IgM-specific endoprotease activity. 【0034】 As used herein, the term “polypeptide having IgM-specific endoprotease activity” is understood to refer to any protein, polypeptide or fragment thereof that can catalyze the cleavage of IgM. The terms endoprotease and protease may be used interchangeably herein. 【0035】 The determination of whether IgM cleavage has occurred can be readily made by those skilled in the art, for example, using common techniques in the field of protein biochemistry, such techniques include SDS-PAGE analysis, Western blotting, chromatography (e.g., size exclusion chromatography), and mass spectrometry. Such techniques are illustrated herein (see Examples), but are not intended to be limiting. 【0036】 Furthermore, it is understood that IgM-specific endoprotease activity can refer to endoprotease activity that is target (IgM) specific. This activity does not necessarily have to have a broad range of target proteins, such as trypsin. Nevertheless, IgM-specific activity encompasses activity against different IgM molecules, for example, IgM from different species or modified IgM molecules. 【0037】 Modified IgM molecules may include, for example, naturally occurring (e.g., synthetic) IgM molecules; fusion proteins containing an IgM molecule or part / sequence of an IgM molecule; and fragments of an IgM molecule. 【0038】 Those skilled in the art will fully understand that polypeptides may have residual or lower levels of activity against other targets such as IgG. 【0039】 In one embodiment, the polypeptide may have IgM-specific and IgG-specific protease activity. 【0040】 In some embodiments, the polypeptide may have low or negligible levels of detectable IgG protease activity. 【0041】 In some embodiments, the polypeptide may have a low level of detectable IgG activity, where the level of IgG activity is less than the level of IgM activity. 【0042】 In some embodiments, the polypeptide may have a low level of detectable IgG activity, where the level of IgG activity is less than 70% of the level of IgM activity, such as less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. 【0043】 In some embodiments, the IgG activity is IgG2-specific activity. 【0044】 In preferred embodiments, the polypeptide does not have detectable activity against polypeptides other than IgM. 【0045】 In another preferred embodiment, the polypeptide has no activity that allows detection of any immunoglobulin isotype other than IgM. 【0046】 In another preferred embodiment, the polypeptide does not have detectable activity against IgG. 【0047】 In a preferred embodiment of the present invention, the IgM-specific endoprotease activity is human IgM-specific endoprotease activity. 【0048】 The term "human IgM specificity" does not imply that the polypeptide is limited to human IgM. Rather, it should be understood that the endoprotease is specific to IgM, and human IgM is the recognized substrate, but not necessarily the only substrate. For example, IgM from non-human primates can also be a substrate. 【0049】 In one embodiment, polypeptide activity against IgM derived from monkeys of the genus Macaca, such as Macaca mulatta and / or Macaca fascicularis. 【0050】 In one embodiment, the polypeptide activity against IgM derived from monkeys of the genus Papio. 【0051】 Lachnoanaerobaculum Lachnoanaerobaculum is a genus of human commensal bacteria found in the human mouth and intestines. Lachnoanaerobaculum orale and Lachnoanaerobaculum umeaense were first described in 2012 after isolation from the oral cavity of healthy individuals and from the intestines of children with celiac disease, respectively (Hedberg ME, et al., J Syst Evol Microbiol 2012, 62(Pt11):2685-2690). 【0052】 Despite two very recent studies linking Lachnoanaerobaculum species L. orale and L. gingivalis to bacteremia in immunocompromised individuals (Ida Y., et al., Anaerobe 2022, 73:102506; Okada N., et al., Anaerobe 2022, 76:102610), Lachnoanaerobaculum species have not been described as causative pathogens of any pathology. 【0053】 To date, as described herein, isolates from the genus Lachnoanaerobaculum have not been demonstrated to possess proteolytic activity, nor have any specific immunoglobulin-modifying activities been identified or investigated. 【0054】 In addition, the inventors identified an IgM-specific endoprotease encoded by bacteria of the genus Lachnoanaerobaculum. 【0055】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity, which is derived from a bacterium of the genus Lachnoanaerobaculum. 【0056】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence having at least 80% sequence identity with respect to a polynucleotide sequence encoded by Lachnoanaerobaculum umeaense, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0057】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence having at least 80% sequence identity to a polynucleotide sequence encoded by Lachnoanaerobaculum gingivalis, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0058】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity and having at least 80% sequence identity with respect to the amino acid sequence encoded by Lachnoanaerobaculum umeaense, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0059】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity and having at least 80% sequence identity with respect to the amino acid sequence encoded by Lachnoanaerobaculum gingivalis, for example, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. 【0060】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity, which is derived from Lachnoanaerobaculum umeaense. 【0061】 In one embodiment, the present invention provides a polypeptide having IgM-specific endoprotease activity, which is derived from Lachnoanaerobaculum gingivalis. 【0062】 The polypeptides of the present invention may be fragments or modified versions of naturally occurring polypeptides encoded by bacteria of the genus Lachnoanaerobaculum. 【0063】 Manipulated IgM-specific endoprotease Following the identification of candidate protease genes in the Lachnoanaerobaculum species, the inventors manipulated several polypeptides. 【0064】 The polypeptide according to the present invention may contain or consist of a sequence derived from Lachnoanaerobaculum umeaense. 【0065】 Exemplary Lachnoanaerobaculum umeaense polypeptide sequences are shown in Table 1 herein. [Table 1-1] [Table 1-2] [Table 1-3] 【0066】 The polypeptide according to the present invention may contain or consist of a sequence derived from Lachnoanaerobaculum gingivalis. 【0067】 Exemplary Lachnoanaerobaculum gingivalis polypeptide and polynucleotide sequences are presented in Table 2 herein. [Table 2-1] [Table 2-2] 【0068】 Exemplary polynucleotide sequences encoding polypeptides according to the present invention are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] 【0069】 Due to the degeneracy of the genetic code, it is understood that any number of polynucleotide sequences may be used to encode a given polypeptide according to the present invention. Therefore, the polynucleotide sequences described herein are for illustrative purposes only. 【0070】 polypeptide The present invention relates to a polypeptide having IgM-specific endoprotease activity. In preferred embodiments, the IgM-specific endoprotease activity is human IgM-specific endoprotease activity. 【0071】 In one embodiment, d) Amino acid sequence of Sequence ID No. 1, e) A variant of the amino acid sequence of SEQ ID NO: 1 having at least 80% sequence identity, or f) Either a) or b) fragment A polypeptide having endoprotease activity is provided, wherein the endoprotease activity is preferably human IgM-specific endoprotease activity. 【0072】 In another embodiment, d) Amino acid sequence of Sequence ID No. 8, e) A variant of the amino acid sequence of SEQ ID NO: 8 having at least 80% sequence identity, or f) Either a) or b) fragment A polypeptide having endoprotease activity is provided, wherein the endoprotease activity is preferably human IgM-specific endoprotease activity. 【0073】 In one embodiment, the polypeptide of the present invention has IgM-specific and IgG-specific protease activity, and the polypeptide comprises or consists of any one sequence of SEQ ID NOs: 8 to 11, or a variant or fragment thereof, as defined herein. 【0074】 In some embodiments, IgG-specific activity is lower than IgM-specific activity. 【0075】 In one embodiment of the present invention, a polypeptide having IgM-specific endoprotease activity is provided, comprising or consisting of a polypeptide selected from the group consisting of SEQ ID NOs: 1 to 7. 【0076】 In one embodiment of the present invention, a polypeptide having IgM-specific endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7. 【0077】 In one embodiment of the present invention, a polypeptide having IgM-specific endoprotease activity is provided, comprising or comprising a polypeptide having at least 80% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 1 to 7, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. 【0078】 In one embodiment of the present invention, a polypeptide having IgM-specific endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11. 【0079】 In one embodiment of the present invention, a polypeptide having IgM-specific endoprotease activity is provided, comprising or comprising a polypeptide having at least 80% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 8 to 11, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. 【0080】 The present invention also encompasses other polypeptide fragments of the present invention. Such fragments should be considered polypeptides in themselves. 【0081】 In other words, the polypeptide of the present invention may comprise or consist of a fragment or a shorter section of the longer polypeptide according to the present invention, as defined herein. 【0082】 A fragment is understood to be any continuous polypeptide sequence that is identical (across the amino acids they share) but shorter than the longer sequence under the definition. For example, a polypeptide with a length of 200 amino acids may be considered a fragment of a longer polypeptide sequence with a length of 250 amino acids that shares 100% sequence identity. 【0083】 In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 7. 【0084】 In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 8 to 11. 【0085】 In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 11, wherein the fragment has a length of at least 100 amino acids, for example, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. 【0086】 In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 11, wherein the fragment has a length of at least 300 amino acids, for example, at least 310 amino acids, at least 320 amino acids, at least 330 amino acids, at least 340 amino acids, at least 350 amino acids, at least 360 amino acids, at least 370 amino acids, at least 380 amino acids, at least 390 amino acids, or at least 400 amino acids. 【0087】 Polypeptides typically have a length of at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or at least 650 amino acids. 【0088】 In a preferred embodiment, the polypeptide has a length of at least 300 amino acids, for example, at least 310 amino acids, at least 320 amino acids, at least 330 amino acids, at least 340 amino acids, at least 350 amino acids, at least 360 amino acids, at least 370 amino acids, at least 380 amino acids, at least 390 amino acids, or at least 400 amino acids. 【0089】 Polypeptides may have amino acid lengths of 700 or less, for example, 650 or less, 600 or less, 550 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less. 【0090】 It is well understood that any of the lower limits listed above can be combined with any of the upper limits listed above to provide a range for polypeptide length. For example, a polypeptide may be 75–700 amino acids long or 450–550 amino acids long. 【0091】 In one embodiment, the polypeptide has a length of 300 to 700 amino acids, for example, a length of 310 to 690 amino acids, a length of 320 to 690 amino acids, a length of 330 to 680 amino acids, a length of 330 to 670 amino acids, a length of 320 to 640 amino acids, and a length of 320 to 630 amino acids. 【0092】 In preferred embodiments, the polypeptide has a length of 300 to 700 amino acids, more preferably 320 to 690 amino acids. 【0093】 In one embodiment, a polypeptide having endoprotease activity is provided, which contains or consists of one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0094】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 11, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0095】 In one embodiment, a polypeptide fragment is provided, which is a fragment having endoprotease activity, and which includes or consists of a fragment of any one amino acid sequence of SEQ ID NOs: 1 to 11, or a fragment of an amino acid sequence having at least 80% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 11, for example, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity. 【0096】 In one embodiment, a polypeptide having endoprotease activity, a) One amino acid sequence from sequence numbers 1 to 11, b) A variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 11 that has at least 80% sequence identity, or c) Either a) or b) fragment A subsystem including this is provided. 【0097】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0098】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0099】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0100】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0101】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 98% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0102】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0103】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of the amino acid sequence of SEQ ID NO: 1. 【0104】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0105】 In one embodiment, a polypeptide fragment is provided, which is a fragment having endoprotease activity, and comprises or consists of a fragment of the amino acid sequence of SEQ ID NO: 1, or a fragment of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, for example, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity. 【0106】 In one embodiment, a polypeptide having endoprotease activity, a) Amino acid sequence of Sequence ID No. 1, b) A variant of SEQ ID NO: 1 having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, or c) Either a) or b) fragment A subsystem including this is provided. 【0107】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1. 【0108】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising the amino acid sequence of SEQ ID NO: 7. 【0109】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising the amino acid sequence of SEQ ID NO: 7. 【0110】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0111】 In one embodiment, a polypeptide fragment is provided, which is a fragment having endoprotease activity, and comprises or consists of a fragment of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0112】 In one embodiment, a polypeptide having endoprotease activity, d) Amino acid sequence of Sequence ID No. 7, e) A variant of the amino acid sequence of SEQ ID NO: 7 having at least 80% sequence identity, or f) Either a) or b) fragment A subsystem including this is provided. 【0113】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7. 【0114】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of the amino acid sequence of SEQ ID NO: 8. 【0115】 In one embodiment, a polypeptide having endoprotease activity is provided, comprising or consisting of a polypeptide having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0116】 In one embodiment, a polypeptide fragment is provided, which is a fragment having endoprotease activity, and comprises or consists of a fragment of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 8, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0117】 In one embodiment, a polypeptide having endoprotease activity, a) Amino acid sequence of Sequence ID No. 8, b) A variant of the amino acid sequence of SEQ ID NO: 8 having at least 80% sequence identity, or c) Either a) or b) fragment A subsystem including this is provided. 【0118】 In one embodiment, a polypeptide having human IgM-specific endoprotease activity is provided, comprising or consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8. 【0119】 The polypeptide according to the present invention may be provided in several states. For example, the polypeptide may be provided as an isolated polypeptide in solution, as a lyophilized solid (e.g., powder), or in a conjugated state (e.g., with other biomolecules or polymers). 【0120】 In one embodiment, the polypeptide is freeze-dried. 【0121】 In one embodiment, the polypeptide is provided in a solution. 【0122】 In one embodiment, the polypeptide is further conjugated to another molecule. 【0123】 In one embodiment, the polypeptide is immobilized on a solid (e.g., a solid support resin) or a substantially solid support (e.g., beads). 【0124】 General characteristics of polypeptides In this specification, “polypeptide” is used in its broadest sense to refer to compounds of two or more substituted amino acids, amino acid analogs, or other peptidomimetic compounds. The term “polypeptide” therefore includes short peptide sequences as well as longer polypeptides and proteins. The terms “protein,” “peptide,” and “polypeptide” can be used interchangeably. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including both D or L optical isomers, as well as amino acid analogs and peptidomimetic compounds. Thus, polypeptides according to the present invention may include entirely natural or synthetic amino acids, or mixtures of natural and synthetic amino acids. 【0125】 Polypeptides can be produced by any preferred method, including recombinant or synthetic methods. For example, polypeptides can be synthesized directly using standard techniques known in the art, such as by Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, polypeptides can be produced by transforming cells, such as bacterial cells, insect cells, or mammalian cells, with nucleic acid molecules or vectors that encode the polypeptide. 【0126】 The expression of the polypeptide of the present invention is described herein and will be even more readily achievable by those skilled in the art using standard techniques. 【0127】 Polypeptides can be derivatized or modified to aid in their production, isolation, or purification. For example, if the polypeptide of the present invention is produced by recombinant expression in a bacterial host cell, the polypeptide sequence may include an additional methionine (M) residue at the N-terminus to improve expression. Alternatively, the polypeptide of the present invention may be derivatized or modified by the addition of a ligand that can directly and specifically bind to a separation means. Alternatively, the polypeptide may be derivatized or modified by the addition of one member of a binding pair, and the separation means may include a reagent that has been derivatized or modified by the addition of the other member of the binding pair. Either preferred binding pair may be used. 【0128】 In some embodiments, the polypeptide of the present invention includes one or more tags, for example, polypeptide tags. 【0129】 In one embodiment, the tag is a detachable tag. 【0130】 In one embodiment, the tag enables specific modification of the tag or the polypeptide to which it is attached. 【0131】 In one embodiment, the tag facilitates the identification and / or isolation of polypeptides. 【0132】 In one embodiment, the tag is bound by a binding partner. In some embodiments, the binding partner is a polypeptide, e.g., an antibody or other binding protein (e.g., avidin); a metal (e.g., Ni); and / or a small organic molecule (e.g., biotin). 【0133】 The polypeptide according to the present invention may contain one or more tags selected from the group consisting of polyhistidine tags, Fc tags; FLAG tags, Rho1D4 tags, HA tags, strep tags, and Avi tags. However, any suitable tag known to those skilled in the art can be used in combination with any polypeptide of the present invention. 【0134】 In one embodiment, the tag facilitates or improves the expression and / or properties (e.g., solubility or polymer state) of the polypeptide. 【0135】 The polypeptide according to the present invention may contain one or more tags selected from the group consisting of Fc-tag, SUMO-tag, GST-tag, lysozyme-tag, MBP-tag, GFP-tag, Myc-tag, and thioredoxin-tag. 【0136】 Tags may be contained within either a C or N terminator. Furthermore, one or more tags may be separated from each other and / or other array elements by one or more linker arrays. Any number and combination of tags may be used. 【0137】 In preferred embodiments, if the polypeptide for use in the present invention is derivatized or modified by the addition of one member of a binding pair, the polypeptide is preferably tagged with histidine or biotin. Typically, the amino acid coding sequence of the histidine or biotin tag is included at the gene level, and the polypeptide is expressed by recombination in E. coli. The histidine or biotin tag is typically located at either end of the polypeptide. It may be directly linked to the polypeptide, or indirectly linked by any preferred linker sequence, such as three, four, or five glycine residues or a mixture of glycine and serine residues, e.g., a "GGS" linker. 【0138】 A histidine tag typically consists of six histidine residues, but the tag can be longer than the length of up to seven, eight, nine, ten, or twenty amino acids. Alternatively, the His tag can be shorter, for example, five or fewer, four or fewer, three or fewer, two or fewer, or even just one amino acid. 【0139】 Polypeptides may be provided in a substantially isolated or purified form; that is, isolated from most of the other components present in a cell extract from a cell expressing the polypeptide. Substantially purified is understood to mean that the polypeptide is purified to at least 50%, 60%, 70%, 80%, or preferably at least 90% homogeneity. The purity level can be assessed by any preferred means, typically involving SDS-PAGE analysis of the sample followed by Coomassie blue detection. Polypeptides may be mixed with carriers, diluents, or preservatives that do not interfere with the intended purpose of the polypeptide and may still be considered substantially isolated or purified. If a polypeptide is provided in a composition or combination with additional active components, such as another polypeptide, each of the polypeptides may be individually purified to a high level of homogeneity prior to mixing in ratios appropriate for their respective intended purposes. For example, two polypeptides may each be purified to at least 90% homogeneity prior to a 1:1 combination. Other ratios are readily identifiable to those skilled in the art. 【0140】 Polypeptides (or mixtures thereof) may be provided in a lyophilized form, which is suitable for reconstitution in aqueous solution prior to use. Lyophilized polypeptides or compositions have improved stability, thereby facilitating longer storage of the polypeptide. A method for preparing polypeptides (or mixtures thereof) in a lyophilized form is provided herein, comprising the step of freeze-drying the polypeptide (or mixture) in a suitable buffer such as Tris buffered saline (TBS). The polypeptide is typically substantially purified prior to the freeze-drying step. The resulting polypeptide (or mixture) in a lyophilized form is also provided. A method for preparing a solution of polypeptides (or mixtures) is also provided, comprising the steps of preparing polypeptides (or mixtures) in a lyophilized form and reconstituting them with a suitable carrier or diluent such as water. 【0141】 Polypeptides can be immobilized using methods known in the art, for example, as described in Datta S et al., Enzyme immobilization: an overview on techniques and support materials, 3 Biotech, 3(1):1-9 (2013). For example, polypeptides can be immobilized by adsorption, covalent bonding, affinity immobilization, or encapsulation. Materials that can be used as supports include, but are not limited to, natural supports such as agarose, collagen, gelatin, cellulose, pectin, and Sepharose, inorganic materials such as ceramics, silica, glass, activated carbon or charcoal, or synthetic polymers. For example, polypeptides can be immobilized on Sepharose or agarose, which may be provided as a resin if necessary. 【0142】 Amino acid identity can be calculated using any suitable algorithm. For example, identity can be calculated or sequences aligned using the BLAST algorithm, typically with default settings. Software for performing BLAST analysis is available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). 【0143】 The polypeptide sequence of the present invention may include a variant of any one of the amino acid sequences of Sequence IDs 1 to 11, which has been modified by adding, deleting, or substituting amino acids compared to any of the sequences of Sequence IDs 1 to 11. 【0144】 Unless otherwise specified, modifications are preferably conservative amino acid substitutions. A conservative substitution replaces an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid it replaces. Alternatively, a conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of 20 major amino acids as defined in Table 3 below. If the amino acids have similar polarity, this may be determined by reference to the hydropathic scale of the amino acid side chains (see also Table 4). 【0145】 The polypeptide sequence of the present invention may include a variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 11, which has up to 10, for example, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150, or up to 200 conservative substitutions. [Table 4] 【0146】 Polynucleotides The present invention also relates to a polynucleotide encoding a polypeptide having IgM-specific endoprotease activity. 【0147】 One aspect of the present invention provides a polynucleotide encoding a polypeptide having IgM-specific endoprotease activity. 【0148】 As used herein, the term polynucleotide is intended to encompass polymers of nucleotides or nucleotide analogs. Polynucleotides, therefore, include, for example, DNA, RNA, or DNA / RNA. 【0149】 In one embodiment, the polynucleotide is RNA. 【0150】 In one embodiment, the polynucleotide is DNA. 【0151】 In one embodiment, the polynucleotide comprises one or more modified nucleotides or nucleotide analogs. 【0152】 The polynucleotides according to the present invention may be naturally occurring, naturally derived, or synthetic. 【0153】 In one embodiment, a polynucleotide is provided that encodes a polypeptide having endoprotease activity, comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0154】 In one embodiment, a polynucleotide is provided that comprises or encodes a polypeptide having endoprotease activity, comprising a polypeptide having at least 80% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 11, for example, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 【0155】 In one embodiment, a polynucleotide is provided which is a polypeptide fragment having endoprotease activity and comprises or codes for a fragment of any one of the amino acid sequences of SEQ ID NOs: 1 to 11, or a fragment of an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 11, for example, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity. 【0156】 In one embodiment, a polynucleotide encoding a polypeptide having endoprotease activity, wherein the polypeptide is a) One amino acid sequence from sequence numbers 1 to 11, b) A variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 11 that has at least 80% sequence identity, or c) Either a) or b) fragment A polynucleotide containing the following is provided. 【0157】 In one embodiment, a polynucleotide is provided that encodes a polypeptide having human IgM-specific endoprotease activity, wherein the polypeptide contains or comprises an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 11. 【0158】 In one embodiment, a polynucleotide containing one of the sequences 12 to 22 is provided. 【0159】 In one embodiment, a polynucleotide consisting of one of the sequences 12 to 22 is provided. 【0160】 In one embodiment, a polynucleotide is provided that has at least 60% sequence identity with respect to any of the sequences of sequence numbers 12 to 22, for example, 70% sequence identity, for example, 80% sequence identity, for example, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity. 【0161】 In one embodiment, a) One of the polynucleotide sequences from sequence numbers 12 to 22, b) A variant of any of the polynucleotide sequences of SEQ ID NOs. 12-12 having at least 80% sequence identity, or c) Either a) or b) fragment A polynucleotide containing the above is provided. 【0162】 In one embodiment, a polynucleotide is provided that encodes a polypeptide having human IgM-specific endoprotease activity, wherein the polypeptide comprises or comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 11, and optionally comprises or comprises a sequence according to any one of SEQ ID NOs: 12 to 22, or a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12 to 22. 【0163】 The present invention also provides a vector comprising any of the aforementioned polynucleotides. 【0164】 The present invention also provides a vector encoding any of the aforementioned polynucleotides. 【0165】 In one embodiment, the vector is an expression vector. 【0166】 In one embodiment, the vector is a pET21a+ vector. 【0167】 General characteristics of polynucleotides The present invention therefore provides nucleic acid molecules encoding the polypeptide of the present invention, i.e., polynucleotides and vectors. 【0168】 Exemplary polynucleotide molecules encoding the polypeptides disclosed herein are provided as SEQ ID NOs: 12–22. However, it is understood that, due to the degeneracy of the gene code, any number of sequences may encode the same polypeptide. 【0169】 The polynucleotides of the present invention may include a codon for the N-terminal methionine (ATG) at the 5' end, which may be excluded as needed, as well as codons for the Gly-Ser-Gly linker and 6×His tag preceding the stop codon (TAA) at the 3' end. The optional incorporation of additional methionines and tags is described in more detail below. 【0170】 The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides or ribonucleotides or analogs thereof. Non-exclusive examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. 【0171】 The polynucleotides of the present invention encode the polypeptides of the present invention and may be provided in isolated or substantially isolated forms. Substantially isolated means that substantial, but not complete, isolation of the polypeptide from any surrounding medium may exist. The polynucleotides may be mixed with carriers or diluents that do not interfere with their intended use and may still be considered substantially isolated. 【0172】 The selected polypeptide-coding nucleic acid sequence is a nucleic acid molecule that is transcribed in vivo or in vitro (in the case of DNA) and translated into a polypeptide (in the case of mRNA) when controlled by appropriate regulatory sequences, for example, in an expression vector. The coding sequence boundary is determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA derived from viruses, prokaryotic or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' relative to the coding sequence. 【0173】 Polynucleotides can be synthesized according to methods well known in the art, as described for illustrative purposes in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention may be provided in the form of an expression cassette containing a control sequence operably linked to an inserted sequence, thereby enabling the expression of the polypeptide of the present invention in vivo (e.g., in a prokaryotic or eukaryotic expression system). Such an expression cassette is then typically provided in a vector (e.g., a plasmid or recombinant viral vector). Such an expression cassette can be administered directly to a host target. Alternatively, a vector containing the polynucleotide of the present invention can be administered to a host target. Preferably, the polynucleotide is prepared and / or administered using a gene vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and enabling the expression of the polypeptide of the present invention. 【0174】 Therefore, the present invention includes an expression vector comprising such a polynucleotide sequence. Such an expression vector is routinely constructed in the art of molecular biology and may involve, for example, plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as the use of a polyadenylation signal, which may be necessary and positioned in a precise orientation to enable the expression of the peptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further illustrative purposes in this regard, the inventors refer to Sambrook et al. 【0175】 Sequence identity between two or more polynucleotide sequences can be calculated using any suitable algorithm. For example, the BLAST algorithm can typically be used to calculate identity or align sequences, often with default settings. Software for performing BLAST analysis is available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). 【0176】 cell The present invention also provides cells that express or are capable of expressing the polypeptide of the present invention. 【0177】 In one embodiment, cells containing polynucleotides according to the present invention are provided. 【0178】 In one embodiment, cells containing the vector according to the present invention are provided. 【0179】 In another embodiment, cells containing the polypeptide according to the present invention are provided. Such cells may contain the polypeptide soluble within the cell, such as within an inclusion body, or within the cytosol; the polypeptide may be transiently present within the cell prior to secretion into the extracellular environment. 【0180】 The cells typically include prokaryotic cells, such as bacterial cells, such as E. coli. Such cells can be cultured using routine methods to produce the polypeptides of the present invention. 【0181】 In one embodiment, the cells are BL21(DE3)STAR cells. 【0182】 Furthermore, the cells according to the present invention may be eukaryotic cells, such as insect cells or mammalian cells. 【0183】 Cells according to the present invention may be modified, for example, by standard molecular biology techniques to contain a polynucleotide, vector, or polypeptide according to the present invention, and / or to express a polypeptide according to the present invention. 【0184】 Compositions and combinations The polypeptides, polynucleotides, vectors, and cells of the present invention may be provided in combination with compositions or other compounds. 【0185】 The present invention provides a composition or combination comprising the polypeptide of the present invention and another polypeptide. 【0186】 A composition is understood to mean that the components of the composition may be mixed; a combination is understood to mean that the components of the combination may not necessarily be mixed, but may be provided separately, for example, in separate containers, and can be mixed as needed. 【0187】 In one embodiment, a composition or combination comprising a first polypeptide and a second polypeptide according to the present invention is provided, wherein the second polypeptide has IgG-specific endoprotease activity. 【0188】 In one embodiment, the second polypeptide having IgG-specific endoprotease activity comprises or consists of a polypeptide having the sequence shown in any one of SEQ ID NOs. 23 to 25. 【0189】 In one embodiment, a composition or combination is provided comprising a first polypeptide and a second polypeptide according to the present invention, wherein the second polypeptide comprises a sequence or fragment thereof having IgG-specific endoprotease activity and having at least 85% sequence identity, such as at least 90%, at least 95%, at least 98%, or at least 99%, to the sequence shown in SEQ ID NO: 23. 【0190】 In particular, it is envisioned that the polypeptide of the present invention can be used in combination with a second polypeptide having IgG-specific endoprotease activity, wherein the second polypeptide contains or consists of a sequence that lacks a C-terminal polyhistidine tag compared to SEQ ID NOs. 23-25. 【0191】 In one embodiment, the second polypeptide having IgG-specific endoprotease activity is IdeS(FabRICATOR), FabALACTICA, and / or Xork. [Table 5-1] [Table 5-2] 【0192】 The present invention also encompasses compositions comprising the polypeptides, polynucleotides, vectors, and / or cells of the present invention in a mixture with aqueous components suitable for storage, such as diluents, salts, and / or buffers. 【0193】 Pharmaceutical compositions comprising polypeptides, polynucleotides, vectors, and / or cells of the present invention in a mixture with pharmaceutically acceptable diluents, salts, and / or carriers are further provided herein. 【0194】 kit Kits comprising any polypeptide, polynucleotide, composition, combination and / or cell according to the present invention are provided herein. In certain embodiments, the kit includes packaging and / or instructions for use of the kit. 【0195】 In one embodiment, a) polypeptide according to the present invention, b) Compositions according to the present invention, c) Combinations according to the present invention, d) Polynucleotide according to the present invention e) Vectors according to the present invention, and / or f) Cells according to the present invention A kit is provided that includes one or any combination of the following. 【0196】 In embodiments where the kit includes a combination, the first polypeptide and the second polypeptide may be provided separately (i.e., in different containers within the kit or in different kits) or together (i.e., in the same kit and / or in the same container within the kit). 【0197】 It is understood that any substituent portion of the kit according to the present invention may be used in the methods or uses of the present invention as defined herein. Furthermore, the kit according to the present invention may include instructions for use relating to any one of the methods or uses of the present invention. 【0198】 Method and Use The present invention also provides methods and uses relating to the polynucleotides, polypeptides, compositions, combinations, vectors, cells, and kits of the present invention. 【0199】 Any method or use described herein can be performed in vivo, in vitro, or ex vivo. 【0200】 A method for hydrolyzing IgM is provided herein, comprising the step of contacting a sample containing IgM with a polypeptide or composition or combination thereof according to the present invention. 【0201】 The present invention also provides a method for detecting the presence of IgM in a sample, a) The step of contacting a sample with the polypeptide, composition or combination of the present invention, b) A step to identify IgM-specific cleavage products and The present invention provides a method in which the presence of IgM-specific cleavage products indicates the presence of IgM in the sample. 【0202】 The present invention also provides a method for detecting the presence of IgM in a sample, a) The step of contacting a sample with the polypeptide, composition or combination of the present invention, b) A step of identifying and isolating IgM-specific cleavage products. The present invention provides a method in which the presence of IgM-specific cleavage products indicates the presence of IgM in the sample. 【0203】 In some embodiments, the method of the present invention includes an isolation step. The isolation step may include the use of gel electrophoresis and / or chromatography. 【0204】 In some embodiments, the method of the present invention includes an identification and / or analysis step. The identification and / or analysis step may include the use of gel electrophoresis, chromatography, immunoblotting, ELISA, and / or mass spectrometry. 【0205】 Therefore, in some embodiments, the method of the present invention, which includes identifying, isolating, and / or step(s), a) Gel electrophoresis, b) Immunoblotting, c) Chromatography, d) ELISA, and / or e) Mass spectrometry Including use. 【0206】 The present invention also provides the use of polypeptides, compositions, combinations, or kits according to the present invention for the degradation of IgM in a sample. 【0207】 The present invention also provides the use of polypeptides, compositions, combinations, or kits according to the present invention for the removal of IgM from a sample. 【0208】 The present invention also provides the use of polypeptides, compositions, combinations, or kits according to the present invention for the identification of IgM in a sample. 【0209】 In one embodiment of the method or use described above, IgM is human IgM. 【0210】 In the aforementioned method or one embodiment of use, the sample is not an in vivo sample. 【0211】 In one embodiment of the method or use described above, the sample is an ex vivo sample. 【0212】 In one embodiment of the method or use described above, the sample is a sample derived from a human subject. 【0213】 The polypeptides of the present invention exhibit evident activity in both simple and complex media, and the substrates can be considered substantially pure (e.g., purified IgM samples) or substantially impure (e.g., crude or complex samples). 【0214】 In one embodiment, the IgM in the sample is substantially pure. 【0215】 In one embodiment, the IgM in the sample is substantially impure. 【0216】 In one embodiment, the sample comprises or consists of a complex medium, such as a complex biological medium. 【0217】 In one embodiment, the sample comprises or consists of whole blood, a blood-derived fraction, serum, and / or plasma. 【0218】 In a preferred embodiment, the sample is a human sample. 【0219】 In some embodiments, the sample is an ex vivo sample. 【0220】 In some embodiments, the sample is a sample obtained during dialysis of a subject. 【0221】 In some embodiments, the sample is a sample obtained from a human subject who does not have a recognized disease or disorder. In some embodiments, the sample is a sample obtained from a human subject who does not have a recognized disease or disorder associated with IgM. 【0222】 In some embodiments, the sample is a sample obtained from a human subject who a) has an autoimmune disease or disorder, b) has cancer, c) has an infection by a suspected or known microorganism . 【0223】 In some embodiments, the microorganism is a bacterium, virus, or parasite. 【0224】 In some embodiments, the microorganism is a pathogenic organism. 【0225】 In some embodiments, the method described above includes the step of identifying and / or isolating IgM-specific cleavage products. 【0226】 In a further embodiment, the method additionally includes the step of contacting the sample with a polypeptide having IgG-specific endoprotease activity. 【0227】 In some embodiments, the method or use of the present invention may be for the purpose of degradation or removal of IgM from a sample. 【0228】 In some embodiments, the method or use of the present invention may be for the purpose of a diagnostic assay. 【0229】 In some embodiments, the method or use of the present invention may be for the purpose of a diagnostic assay, and the assay may be an anti-drug antibody (ADA) assay. 【0230】 Anti-drug assays are utilized to identify the presence of antibodies directed against or induced by a drug of interest. Often, such assays are performed using bridging ELISA that does not distinguish between antibody isotypes. By using the polypeptide according to the present invention that can degrade IgM, IgM in a sample can be removed or inactivated, and thus, by determining the contribution of IgM to any observed activity, it is possible to facilitate an increase in resolution in an ADA assay or other similar assays that contain IgM within a mixture of components. 【0231】 In some embodiments, the method or use of the present invention may be for the purpose of diagnostic assay quality control, such as quality control of recombinant or non-recombinant monoclonal and / or polyclonal IgM or IgM-derived molecules. 【0232】 For the development of IgM such as for therapeutic purposes, diagnostic purposes, or IgM-like drugs, it is inevitable to perform large-scale quality control to verify a drug, for example, to demonstrate the presence of its sequence and / or post-translational modifications. Often, such quality control involves the use of trypsin or a similar "bottom-up" approach that lacks specificity and thus can be improved. A polypeptide IgM protease that hydrolyzes the hinge region of IgM can facilitate such analysis. 【0233】 In some embodiments, the methods or uses of the present invention may be for the purpose of gene therapy. 【0234】 IgM has been shown to significantly (negatively) affect gene transfer by adenovirus 5, one of the most commonly used adenovirus vectors. IgM reduces vector transduction in the liver. Therefore, degradation, inactivation, or removal of IgM using the polypeptide of the present invention can assist in gene therapy applications, particularly those utilizing adenovirus 5 vectors. 【0235】 In one embodiment, a method or use according to the present invention is provided for the removal of IgM during gene therapy. "During" can refer to before, concurrently with, or after treatment of a subject or sample with the polypeptide of the present invention. 【0236】 In some embodiments, the methods or uses of the present invention may be for the treatment or prevention of cancer. 【0237】 In some embodiments, the methods or uses of the present invention may be for the treatment or prevention of diffuse large B-cell lymphoma. 【0238】 Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), affecting thousands of individuals each year, and has a poor prognosis if left untreated. Even with treatment, the overall 5-year survival rate is low. Lymphoma B cells almost always possess IgM as their B-cell receptor (BCR), and the removal or degradation of this receptor using the polypeptide of the present invention may be useful in treating cancer, for example, by inducing apoptosis and clearing lymphoma B cells. 【0239】 In some embodiments, the methods or uses of the present invention may be for the treatment or prevention of autoimmune diseases or disorders. 【0240】 In some embodiments, the methods or uses of the present invention may be for the treatment or prevention of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or Sjögren's syndrome. 【0241】 Several autoimmune diseases, including SLE, RA, and Sjögren's syndrome, are characterized by high levels of rheumatoid factor (RF), which are often used as a prognostic biomarker for the disease. High levels of RF are associated with worse outcomes and more severe pathogenesis. RF is typically an IgM-based antibody against autoIgG that stimulates the formation of disease-contributing immune complexes, for example, by causing chronic inflammation and joint destruction. Degradation or removal of IgM in such diseases using the polypeptides of the present invention may therefore be helpful in treating or preventing these diseases. 【0242】 In one embodiment, polypeptides, combinations, compositions, or pharmaceutical compositions according to the present invention are provided for use in the manufacture of pharmaceuticals. 【0243】 In one embodiment, polypeptides, combinations, compositions, or pharmaceutical compositions according to the present invention are provided for therapeutic use. 【0244】 In one embodiment, polypeptides, combinations, compositions, or pharmaceutical compositions according to the present invention are provided for use in the treatment or prevention of diseases or disorders associated with elevated IgM levels. 【0245】 In one embodiment, polypeptides, combinations, compositions, or pharmaceutical compositions according to the present invention are provided for use in the treatment or prevention of cancer. 【0246】 In one embodiment, polypeptides, combinations, compositions, or pharmaceutical compositions according to the present invention are provided for use in the treatment or prevention of diffuse large B-cell lymphoma. 【0247】 In one embodiment, there is provided a polypeptide, combination, composition or pharmaceutical composition according to the present invention for use in the treatment or prevention of an autoimmune disease or disorder. 【0248】 In one embodiment, there is provided a polypeptide, combination, composition or pharmaceutical composition according to the present invention for use in the treatment or prevention of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and / or Sjögren's syndrome. 【0249】 In one embodiment, there is provided a polypeptide, combination, composition or pharmaceutical composition according to the present invention for use in the treatment or prevention of IgM nephropathy. 【0250】 Aspects of the present invention Next, the present invention will be described as numbered aspects. Aspect 1. a) The amino acid sequence of SEQ ID NO: 1, b) A variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, or c) A fragment of either a) or b) A polypeptide having endoprotease activity, comprising Aspect 2. a) The amino acid sequence of SEQ ID NO: 8, b) A variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8, or c) A fragment of either a) or b) A polypeptide having endoprotease activity, comprising Aspect 3. The polypeptide according to Aspect 1 or Aspect 2, wherein the variant of the polypeptide has at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8. Appearance 4. a) Amino acid sequence of Sequence ID No. 2, b) A variant of the amino acid sequence of SEQ ID NO: 2 having at least 80% sequence identity, or c) Either a) or b) fragment A polypeptide according to embodiment 1 or embodiment 3, comprising or consisting of the following. Appearance 5. a) Amino acid sequence of SEQ ID NO: 9, b) A variant of the amino acid sequence of SEQ ID NO: 9 having at least 80% sequence identity, or c) Either a) or b) fragment A polypeptide according to embodiment 2 or 3, comprising or consisting of the following. Embodiment 6. The polypeptide according to any one of Embodiments 1 to 5, wherein the polypeptide fragment has a length of at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. Embodiment 7. a) Additional methionine at the N-terminus, and / or b) His tag A polypeptide according to any one of embodiments 1 to 6, including the following: Embodiment 8. The polypeptide according to Embodiment 7, wherein the His tag is located at the C-terminus. Embodiment 9. The polypeptide according to Embodiment 7, wherein the His tag is located at the N-terminus. Embodiment 10. A polypeptide according to any one of Embodiments 1 to 8, comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 6, 7, and 10. Embodiment 11. A polypeptide according to any one of Embodiments 1 to 7 and 9, comprising or consisting of sequences selected from the group consisting of SEQ ID NOs: 4 and 11. Embodiment 12. A polypeptide according to any one of Embodiments 1 to 8 and 10, comprising or consisting of Sequence ID No. 7. Embodiment 13. The polypeptide according to any one of Embodiments 1 to 12, wherein the endoprotease activity is IgM-specific endoprotease activity. Embodiment 14. A polypeptide according to any one of Embodiments 1 to 13, which cleaves human IgM. Embodiment 15. A polypeptide according to any one of Embodiments 1 to 14, provided in a solution in a lyophilized or immobilized state. Embodiment 16. A composition or combination comprising a first polypeptide according to any one of Embodiments 1 to 15 and a second polypeptide, wherein the second polypeptide has IgG-specific endoprotease activity. Embodiment 17. A polynucleotide encoding a polypeptide according to any one of Embodiments 1 to 15. Embodiment 18. A vector encoding or comprising the polynucleotide described in Embodiment 17. Embodiment 19. A cell comprising a polypeptide according to any one of Embodiments 1 to 15, a polynucleotide according to Embodiment 17, or a vector according to Embodiment 18. Embodiment 20. A cell according to Embodiment 20, which is a bacterial cell. Applicable 21. a) Polypeptide according to any one of Applicable 1 to 15, b) The composition or combination according to Embodiment 16, wherein the first polypeptide and the second polypeptide are provided separately or together. c) Polynucleotide as described in embodiment 17, d) The vector described in aspect 19, and / or e) Cells described in embodiment 20 A kit that includes this. Embodiment 22. A method for hydrolyzing IgM, comprising the step of contacting a sample containing IgM with a polypeptide according to any one of Embodiments 1 to 15 or a composition or combination according to Embodiment 16. Embodiment 23. The method according to Embodiment 22, wherein the IgM is human IgM. Embodiment 24. The method according to Embodiment 22, wherein the IgM is a non-human primate IgM, preferably a monkey of the genus Macaca, for example, Macaca mulatta and / or Macaca fascicularis and / or Papio. Embodiment 25. The sample is a) substantially pure, or b) Substantially impure The method according to any one of embodiments 22 to 24. Embodiment 26. The method according to any one of Embodiments 22 to 25, wherein the sample includes a complex biological medium. Embodiment 27. The method according to any one of Embodiments 22 to 26, wherein the sample comprises blood, serum and / or plasma. Embodiment 28. The method according to any one of Embodiments 22 to 27, wherein the sample comprises human blood, human serum and / or human plasma. Embodiment 29. The method according to any one of Embodiments 22 to 28, comprising the step of identifying and / or isolating an IgM-specific cleavage product. Embodiment 30. The method according to any one of Embodiments 22 to 29, wherein the method further comprises the step of contacting the sample with a polypeptide having IgG-specific endoprotease activity, and optionally the polypeptide having IgG-specific endoprotease activity comprises or consists of a polypeptide having the sequence shown in any one of SEQ ID NOs. 23 to 25. Embodiment 31. A method for detecting the presence of IgM in a sample, a) The step of contacting the sample with a polypeptide according to any of embodiments 1 to 15 or a composition or combination according to embodiment 16, b) A step of identifying and / or isolating IgM-specific cleavage products. A method comprising a factor wherein the presence of an IgM-specific cleavage product indicates the presence of IgM in the sample. Embodiment 32. The steps of identification and isolation are: a) Gel electrophoresis, b) Immunoblotting, c) Chromatography, d) ELISA and / or e) Mass spectrometry The method according to any one of embodiments 22 to 31, including the use of [the specified term]. Embodiment 33. Use of a polypeptide according to any of Embodiments 1 to 15, a composition or combination according to Embodiment 16, or a kit according to Embodiment 21 for the degradation, removal, or identification of IgM in a sample. Embodiment 34. The method according to any one of Embodiments 22 to 32 or the use according to Embodiment 33, wherein the sample is an in vitro, in vivo, or ex vivo sample. Embodiment 35. a) For quality control (QC) processes, and / or b) For diagnostic assays The method described in any of embodiments 22 to 32 or the use described in embodiment 33 or embodiment 34. Appearance 36. a) For the decomposition or removal of IgM during dialysis, b) For quality control of recombinant or non-recombinant monoclonal and / or polyclonal IgM or IgM-derived molecules, and / or c) For anti-drug antibody (ADA) assays The method described in any of embodiments 22 to 32 and 35, or the use described in embodiments 33 to 35. Embodiment 37. A method or use of any of Embodiments 22 to 36 that is non-therapeutic. Embodiment 38. A therapeutic method or use of any of Embodiments 22 to 36. Appearance 39. a) Kidney disease or dysfunction, b) cancer, and / or c) Autoimmune disease or disorder The method according to any one of embodiments 22-32, 35 and 38 or the use according to embodiments 33-36 and 38, for the treatment or prevention of the condition. Appearance 40. a) Diffuse large B-cell lymphoma, b) Systemic lupus erythematosus (SLE), c) Rheumatoid arthritis (RA), d) Sjögren's syndrome, and / or e) IgM nephropathy The method or use described in aspect 39, which is for the treatment or prevention of the condition. Applicable aspect 41. A polypeptide according to any one of Applicable aspects 1 to 15, a composition or combination according to Applicable aspect 16, or a kit according to Applicable aspect 21, for use in therapy. Appearance 42. A disease or disability is a) Kidney disease or dysfunction, b) cancer, and / or c) Autoimmune disease or disorder A polypeptide according to any one of embodiments 1 to 15, a composition or combination according to embodiment 16, or a kit according to embodiment 21, for use in the treatment or prevention of the aforementioned disease or disorder. Appearance 43. The disease or disorder is a) Diffuse large B-cell lymphoma, b) Systemic lupus erythematosus (SLE), c) Rheumatoid arthritis (RA), d) Sjögren's syndrome, and / or e) IgM nephropathy The polypeptide, composition, combination, or kit according to embodiment 42. Applicable aspect 44. A polypeptide according to any one of Applicable aspects 1 to 15, a composition or combination according to Applicable aspect 16, or a kit according to Applicable aspect 21, for use in the manufacture of a pharmaceutical product. Embodiment 45. The pharmaceutical product is for the treatment or prevention of a disease or disorder, and the disease or disorder is a) Kidney disease or dysfunction, b) cancer, and / or c) Autoimmune disease or disorder A polypeptide, composition, combination, or kit for use as described in Embodiment 44. Embodiment 46. The pharmaceutical product is for the treatment or prevention of a disease or disorder, and the disease or disorder is a) Diffuse large B-cell lymphoma, b) Systemic lupus erythematosus (SLE), c) Rheumatoid arthritis (RA), d) Sjögren's syndrome, and / or e) IgM nephropathy A polypeptide, composition, combination, or kit for use according to embodiment 44 or embodiment 45. 【0251】 Next, the present invention will be further described as an example. This example is intended to assist those skilled in the art in carrying out the present invention and is not intended to limit the scope of the present invention. [Examples] 【0252】 (Example 1) Identification, isolation, and purification of IgM-specific proteases from Lachnoanaerobaculum. Identification of potential proteases in Lachnoanaerobaculum The inventors used combined biological information and biochemical analysis to identify the homolog of IdeSSuis, the only known publicly known IgM-specific endoprotease (Seele J., et al., J Bacteriol 2013, 195(5)). 【0253】 Although none of the known isolates of Lachnoanaerobaculum have shown any proteolytic or specific immunoglobulin modification activity, the inventors have also found a gene containing an IdeS-like domain that shows a low level of sequence similarity to the known IgM-specific endoprotease IdeSSuis. 【0254】 The identification of genes containing IdeS-like domains was surprising for two reasons: (1) such domains had previously been identified mainly in Streptococcus, and (2) the presence of enzymes containing IdeS-like domains is associated with pathogenic bacteria rather than commensal organisms that form part of the normal human bacterial flora. 【0255】 Synthesis of candidate proteins We designed, synthesized, and used several constructs to express the desired protein in recombinant host cells. 【0256】 The structures were designed and constructed according to Table 5. [Table 5-3] [Table 5-4] 【0257】 Full-length constructs of IdeS / Mac-1-containing genes derived from both L. gingivalis and L. umeaense were expressed with either an N-terminal or C-terminal His- tag (L. gingivalis D1-N-10H, L. umeaense-D1-N-10H, L. gingivalis D1-C-6His, and L. umeaense-D1-C-6His, respectively). The full-length constructs yielded less than 5 mg / g, and the resulting polypeptides were of relatively low purity and fragmented. 【0258】 We selected constructs derived from L. umeaense and further manipulated them to express only specific fragments of the full-length protein. The L. umeaense Suis (LU-S) construct, after optimization of the purification process, expressed well and yielded high yields, but produced a protein that tended to fragment under the tested conditions. Under the tested conditions, a 38-amino acid shorter fragment (SuisΔ38; LU-d38S) was more stable but had a lower yield. 【0259】 The construct was further manipulated and optimized until a construct (L. umeaense MAC-1; further referred to as LU-M1) was found that expressed in high yield (>20 mg / g), with high purity and uniformity (Figure 1). When expressed in a bioreactor, LU-M1 expression was scalable, with a yield of approximately 10 mg / g and culture density reaching high levels (220 g cells per 600 mL batch). 【0260】 LU-M1 shares very little sequence identity with several known streptococcal pathogenic factors (Table 6). However, the construct exhibits a much higher level of sequence identity with L. gingivalis IdeS / Mac-1-containing proteins. [Table 6] 【0261】 Before transforming into BL21(DE3)STAR cells under ampicillin selection, all constructs were codon-optimized for E. coli and inserted into the pET21a+ backbone. Clones were cultured in ampicillin-supplemented LB, and protein expression was performed in baffled 2L flasks, followed by OD with the addition of 1 mM IPTG. 600 Cells were induced at a pH of 0.5–0.7. Four hours after induction at 20–37°C, cells were collected by centrifugation and maintained at -20°C until further processing. Cells were lysed by sonication, and lysates were clarified from the cell debris by centrifugation. The material was purified using His GraviTRAP, and its purity was evaluated by SDS-PAGE. 【0262】 For expression in a bioreactor system, LU-M1 was recombinantly produced using Escherichia coli BL21(DE3)STAR. Fermentation was carried out using a high-cell-density fed-batch strategy with minimal medium of known composition. The induction, collection, and purification steps were similar to those used for small-scale production. 【0263】 (Example 2) Functional characterization of IgM-specific proteases derived from Lachnoanaerobaculum To investigate the activity of the full-length constructs (LG-FL and LU-FL), purified proteins were incubated with human and mouse IgG (Figure 2A). Only LG-FL showed some activity against human serum IgG, further demonstrating its specificity for human IgG2 (Figure 2B). 【0264】 Despite homology to known IgG proteases, and due to the limited activity (LG-FL) or lack thereof (LU-FL) shown against human IgG, purified LU-FL and LG-FL were incubated with human IgA and IgM. Surprisingly, both LG-FL and LU-FL were active against IgM; no IgA-specific activity was observed (Figure 2C). 【0265】 The IgM-specific activity observed in the full-length construct was retained by the shorter, manipulated fragment LU-M1 (Figure 2D). 【0266】 Furthermore, LU-M1 was shown to lack activity against a broad range of protease substrates, including casein (Figure 2E), or IgM derived from rodent species (mouse, rabbit, rat). However, LU-M1 showed some activity against purified IgM from monkeys (Figure 2F). The purified IgM from monkeys was obtained from Rockland (017-0107) and consisted of a combination of rhesus macaque, cynomolgus macaque, and baboon IgM. 【0267】 Common protease activity was determined using the EnzCheck protease assay kit according to the manufacturer's instructions. The common protease SpeB (FabULOUS) was used as a positive control, and the IgG-specific protease IdeS (FabRICATOR) was used as a negative control. All reactants were measured using a fluorescence microplate reader after 1 and 2 hours of incubation at 37°C. 【0268】 Furthermore, to evaluate species specificity, IgM from different species was incubated with LU-M1 at 37°C for 1 hour (1:40, w:w, enzyme:substrate), and then SDS-PAGE analysis was performed. 【0269】 Myeloma IgM was deglycosylated with PNGase F and incubated with LU-M1 to investigate the site of hydrolysis (Figure 3). Only the heavy chain was hydrolyzed, while the light chain and J-chain remained intact. The sum of the intact masses of the deglycosylated heavy chain fragments (63140.6167 Da) was in good agreement with the measured mass of the reduced IgM heavy chain (63140.9971 Da). After the addition of LU-M1, the heavy chain was fragmented into m / z 37499.6142 and m / z 25659.0200. The C-terminal fragment (25659.0200 Da) could be assigned to the theoretical mass value (25657.6646 Da) of amino acids 221-453 of the human IgM constant region (UniProt commission: P01871). 【0270】 IgM myeloma (Sigma-Aldrich) was incubated at 37°C for 1 hour with and without purified recombinant LU-M1 in PBS buffer using an enzyme-to-substrate ratio of 1:40 (w:w). The mixture was denatured and reduced at 90°C for 5 minutes with 50 mM DTT (GBiosciences) and 0.5% (v / v) lauroyl sarcosine, cooled to room temperature, and then PNGase F (Genovis) was added at 2 U / μg. The N-deglycosylation reaction was carried out at 37°C for 2 hours, after which the sample was dialyzed against TBS using a Slide-A-Lyzer MINI device (ThermoFisher Scientific). The hydrolyzed material was denatured at 37°C for 30 minutes with 4 M GnHCl (Sigma-Aldrich) and 100 mM DTT. Digestion products were separated using a reversed-phase C4 column (Acquity premier BEHC4, 450 Å, 2.7 μm 2.1 × 100 mm, Waters) with an acetonitrile gradient and analyzed by ESI-QTOF-MS (Bruker Impact II). Deconvolution was performed (preformed) using Bruker Compass DataAnalysis version 5.2 and the MaxEnt algorithm. 【0271】 Similar to the porcine IgM protease IdeSSuis, digestion occurs under the CH2 region of human IgM (...VPDQDT / AIRVFA...). Due to the monomeric disulfide bond between 413 cysteine ​​residues in CH3, the native reaction leaves F(ab')2(VH-CH1-CH2) and the pentamer Fc(CH3-CH4) (Figure 4). 【0272】 (Example 3) Optimization and analysis of protease activity To further evaluate LU-M1 activity, the enzymatic activity of polypeptides was assessed in relation to pH, salt (NaCl), divalent cation, temperature, and sensitivity / resistance to protease inhibitors. 【0273】 LU-M1 can hydrolyze IgM under a wide range of pH conditions (5.5 to 9.0; Figure 5A) without a significant reduction in activity. 【0274】 Enzyme activity appears to be negatively affected by increasing NaCl concentration, with a 12.5% ​​reduction in activity observed in the presence of 150 mM NaCl, a 40% reduction at 500 mM NaCl, and a 75% reduction at 1000 mM NaCl (Figure 5B). 【0275】 LU-M1 is highly active without the addition of any divalent cations (Figure 5C). Rather, the activity is Mg 2+ and Ca 2+ It appears to be slightly reduced by the addition of ions. Zn 2+ Aggregation was observed in the presence of [the substance]. Furthermore, the addition of EDTA appears to cause a concentration-dependent reduction in enzyme activity (a 30-50% reduction depending on the concentration). 【0276】 Enzyme activity is enhanced by increasing temperature; nevertheless, LU-M1 retains some activity at 4°C (35% hydrolysis). Although slower than at 37°C, the enzyme can efficiently hydrolyze IgM at room temperature (Figure 1D). 【0277】 Next, the activity of LU-M1 was evaluated in the presence of a group of protease inhibitors (Figure 5E). Serum IgM was pre-incubated with a set of protease inhibitors (G-Biosciences) for 15 minutes, followed by the addition of LU-M1 (1:50 w:w LU-M1:IgM). The mixture was incubated at 37°C for 30 minutes, and then analyzed by SDS-PAGE. 【0278】 Based on the inhibitory capabilities of this group of protease inhibitors, LU-M1 is classified as a cysteine ​​protease; its activity is completely inhibited in the presence of iodoacetamide and strongly inhibited in the presence of AEBSF and chymostatin. None of the other protease inhibitors evaluated had a significant effect on enzyme activity. 【0279】 LU-M1 was incubated with IgM (IgM myeloma (20 μg)) at different ratios (1:20, 1:50, and 1:100; LU-M1 to IgM (w:w)) for 5–60 minutes to investigate the efficiency of hydrolysis. At low enzyme:substrate ratios (i.e., high relative enzyme concentrations), almost all IgM was hydrolyzed within 5 minutes; at 1:100, all IgM was hydrolyzed within 60 minutes (Figures 6A, 6B). Similarly, higher substrate concentrations resulted in faster hydrolysis (Figure 6C). 【0280】 Unless otherwise specified, for all activities under optimal conditions, a 1:100 enzyme-to-substrate ratio was used for a 30-minute incubation at 37°C. 【0281】 Next, the ability of the enzymes to function in the combined medium was evaluated. Undiluted serum was incubated with the IgG-specific protease IdeS, the IgM-specific protease LU-M1 as defined herein, or a combination thereof, and the resulting mixture was analyzed using SDS-PAGE and Western blotting. 【0282】 Human thermal inactivated serum (Sigma, H2667) was incubated with IdeS (1:50, w:w enzyme:IgG) and LU-M1 (1:40, w:w, enzyme:IgM) for 30 minutes at 37°C. Materials were analyzed by SDS-PAGE and Western blotting. Anti-human IgM ALP (Mabtech, 3880-9A-1000; 1:1000) and anti-human IgG ALP (Mabtech, 3310-1-1000; 1:2000) were used as primary antibodies, and the Western Breeze Kit (Invitrogen) was used for blocking, washing, and chromogenic substrate detection. 【0283】 Both IdeS and LU-M1 were found to be highly specific, targeting only IgG and IgM, respectively, in the sample. Furthermore, it was observed that both enzymes could be manipulated in the same sample without any significant effect on each other's function (Figure 7). 【0284】 These data demonstrate LU-M1's ability to catalyze the specific hydrolysis of IgM in substantially impure samples, such as complex biological samples like serum. 【0285】 (Example 4) Evaluation of the ability to manipulate LU-M1 We evaluated the ability to modify the state of LU-M1 to facilitate its handling in various situations. 【0286】 Firstly, LU-M1 was freeze-dried at various concentrations and volumes. All conditions resulted in a highly active material, thus indicating that LU-M1 is stable in freeze-dried form, can be reconstituted, and can generate active enzymes (Figure 8). 【0287】 Lyophilization was performed using Cool-Safe 10-4 Pro (ScanVac). Briefly, aluminum blocks for vials were pre-cooled to -80°C, then the sample (25-100 μL, 0.5-2 mg / mL) was resuspended in TBS, added to the block, and incubated overnight at -80°C. The sample was lyophilized for 24 hours, and the pellet was stored at -20°C. 【0288】 The activity of the freeze-dried material was evaluated by reconstituting the material in milliQ water to a concentration of 40 U / μL (1 U = 0.02 μg). The reconstituted enzyme was incubated with 1 U / μg IgM in PBS at 37°C for 1 hour, and then SDS-PAGE analysis was performed (Figure 8). 【0289】 Secondly, LU-M1 was immobilized at a working concentration of 20-30 mg / mL using NHS-activated Sepharose (Cytiva) according to the manufacturer's instructions. The activity of the immobilized protein was evaluated by mixing 50 μL of resin with 100 μg IgM (100 μL) and incubating at room temperature for 15-30 minutes by end-over-end stirring. LU-M1 was readily immobilized at high concentrations (20-30 mg / mL) with excellent coupling efficiency (92-94%) and retained activity (Figure 8C). 【0290】 These data indicate that LU-M1 is readily freeze-dried / immobilized and retains its functionality after being subjected to these processes, thus demonstrating the broad applicability of this enzyme to several scenarios and indicating its suitability for storage and / or transport in forms other than as an isolated aqueous protein. 【0291】 (Example 5) Evaluation of IVIG reactivity to LU-M1 The presence of LU-M1-specific human IgG was assessed using SDS-PAGE analysis and Western blotting (Figure 9). 【0292】 0.5 μg each of IdeS, Xork, and LU-M1 were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting. The membrane was blocked in casein solution, and then 5 μg / mL of polyclonal primary antibody mixture, IVIG, was added. The primary antibody was incubated at room temperature for 2 hours. The membrane was washed, and the secondary antibody (alkaline phosphatase conjugate α-human IgG HC, 1:2000) was added and incubated at room temperature for 1 hour. The membrane was thoroughly washed, and then the chromogenic substrate BCIP / NBT was added. 【0293】 Western blot data indicate little to no anti-LU-M1 antibodies in IVIG. The absence or low levels of anti-enzyme antibodies in human IVIG is advantageous and highly desirable. Specifically, the absence or deficiency of anti-enzyme antibodies facilitates human treatment, for example, by increasing the number of patients susceptible to the treatment and thus increasing the treatment's effectiveness, as the enzyme is not removed or does not cause adverse reactions.

Claims

[Claim 1] a) The amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8, b) A variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8, or c) A fragment of either a) or b) A polypeptide containing endoprotease activity. [Claim 2] a) The amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9, b) A variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9, or c) A fragment of either a) or b) The polypeptide according to claim 1, comprising or consisting of the following. [Claim 3] The polypeptide described above, a) Additional methionine at the N-terminus, and / or b) His tag The polypeptide according to claim 1 or claim 2, comprising, wherein the His tag is located at the C-terminus or N-terminus. [Claim 4] A polypeptide according to any one of claims 1 to 3, comprising or consisting of a sequence selected from the group consisting of sequence numbers 3, 4, 5, 6, 7, 10, and 11. [Claim 5] A polypeptide according to any one of claims 1 to 4, comprising or consisting of Sequence ID No.

7. [Claim 6] The polypeptide according to any one of claims 1 to 5, wherein the endoprotease activity is IgM-specific endoprotease activity. [Claim 7] A polypeptide according to any one of claims 1 to 6, which cleaves human IgM. [Claim 8] A polypeptide according to any one of claims 1 to 7, provided in a solution in a lyophilized or immobilized state. [Claim 9] A composition or combination comprising a first polypeptide according to any one of claims 1 to 8 and a second polypeptide, wherein the second polypeptide has IgG-specific endoprotease activity. [Claim 10] A polynucleotide encoding a polypeptide according to any one of claims 1 to 8. [Claim 11] A vector encoding or comprising the polynucleotide described in claim 10. [Claim 12] A cell comprising a polypeptide according to any one of claims 1 to 8, a polynucleotide according to claim 10, or a vector according to claim 11. [Claim 13] a) Polypeptide according to any one of claims 1 to 8, b) The composition or combination according to claim 9, wherein the first polypeptide and the second polypeptide are provided separately or together. c) The polynucleotide according to claim 10, d) The vector according to claim 11, and / or e) The cell according to claim 12 A kit that includes this. [Claim 14] A method for hydrolyzing IgM, comprising the step of contacting a sample containing IgM with a polypeptide according to any one of claims 1 to 8 or a composition or combination according to claim 10. [Claim 15] The method according to claim 14, wherein the IgM is human IgM. [Claim 16] The method according to any one of claims 13 to 15, wherein the sample comprises a complex biological medium, preferably the sample comprises blood, serum and / or plasma, and more preferably the sample comprises human blood, human serum and / or human plasma. [Claim 17] The method according to any one of claims 13 to 16, comprising the step of identifying and / or isolating an IgM-specific cleavage product. [Claim 18] The method according to any one of claims 13 to 17, further comprising the step of contacting the sample with a polypeptide having IgG-specific endoprotease activity. [Claim 19] A method for detecting the presence of IgM in a sample, a) The step of contacting the sample with the polypeptide according to any one of claims 1 to 8 or the composition or combination according to claim 9, b) The step of identifying and / or isolating the IgM-specific cleavage product A method comprising a factor wherein the presence of an IgM-specific cleavage product indicates the presence of IgM in the sample. [Claim 20] Use of a polypeptide according to any one of claims 1 to 8, a composition or combination according to claim 9, or a kit according to claim 12 for the degradation, removal, or identification of IgM in a sample.

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