Artificial bifunctional enzyme containing myeloperoxidase and glucose oxidase activities and its applications

Abstract

The present invention relates to the field of enzymology. More particularly, the present invention relates to artificial bifunctional enzymes and their applications.

Description

[Technical Field]

[0001] The present invention relates to the field of enzymology. More particularly, the present invention relates to artificial bifunctional enzymes and their applications. [Background technology]

[0002] Heme peroxidases are heme-containing enzymes found in all living organisms that catalyze the formation of antimicrobial compounds and may participate in innate immunity. Such peroxidases are divided into two superfamilies: the first family, found in plants, fungi, and bacteria, likely arose from gene duplication of a single common ancestral gene; the second family, found in mammals, differs from the first family in primary and tertiary structure and prosthetic group. Regardless of origin, heme peroxidases can exhibit microbicidal activity due to their ability to halogenate a wide range of organic compounds in the presence of hydrogen peroxide, which may be useful in the biomedical, biotechnology, or food industries. As a specific example, heme peroxidases may be a valuable therapy for bacterial infections, including those resistant to antibiotics. This is because such enzymes can catalyze the oxidation of halides or pseudohalides to (pseudo)hypohalous acids, which have been shown to exhibit potent bactericidal and antiviral activity.

[0003] Among heme peroxidases, mammalian peroxidases (MMPs) have been found to play a major role in the killing of invading pathogens by the innate immune system. To date, four major types of mammalian peroxidases have been discovered: myeloperoxidase (MPO) expressed in neutrophils, eosinophil peroxidase (EPO) localized in acidophilic granulocytes, lactoperoxidase (LPO) expressed in mammary, salivary, and other mucous glands, and thyroid peroxidase (TPO) found in the thyroid gland. The structure of the active site of heme peroxidases is highly conserved. In proteins, heme is maintained via two covalent bonds through the autocatalytic formation of two ester bonds with aspartic acid and glutamic acid residues. Myeloperoxidase (MPO) is distinguished from its counterparts by the presence of a third covalent sulfonium bond, which confers unique spectroscopic properties (Soret band 428 nm). Notably, this bond is responsible for the chlorinating activity of MPO.

[0004] In nature, peroxidases are commonly found co-expressed with oxidases simply because oxidases produce hydrogen peroxide, which is a substrate for peroxidases. A typical example can be found in fungi, where peroxidases that help decompose biomass are secreted together with oxidases that produce hydrogen peroxide, thereby promoting the expression of peroxidase (Abdel-Hamid et al., Adv. Appl. Microbiol. 2013, 82: 1-28; Ander et al., J. Biotechnol. 1997, 53: 115-131).

[0005] From an industrial point of view, a wide range of applications have been implemented based on the combination of peroxidase and oxidase.

[0006] For example, E-101 solution, which essentially contains porcine MPO together with glucose oxidase and sodium chloride, becomes microbicidal when contacted with glucose, allowing for disinfection of human or animal wounds (Denys et al., Infect Immun., 2019;87(7):e00261-19). LPO, when mixed with glucose oxidase in a composition, has been identified as a suitable additive for preserving food, e.g., milk, or as an oral disinfectant (WO 2008105113 A1; WO 2011116052). Human MPO, together with glucose oxidase, glucose, and halides, has also been reported as a virucidal agent against HIV (Moguilevsky et al., FEBS Lett. 1992;302(3):209-212).

[0007] Assays and biosensors based on such enzyme combinations have also been developed to apply these complementary activities, for example, to the measurement of glucose or uric acid concentrations in human serum samples (Barham et al., Analyst 1972, 97: 142-145; Chun et al., Biochip J. 2014, 8: 218-226; Mundaca-Uribe et al., Sens. Actuators B 2014, 195, 58-62).

[0008] These types of artificial systems can be relatively complex to implement, as the enzymes are free from each other but require the identification of optimal experimental conditions for both enzymes, and in the case of assays and biosensors, the co-immobilization of the two enzymes on a biocompatible surface is necessary to ensure sufficient spatial proximity for sequential catalytic reactions to occur. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2008105113 A1 [Patent Document 2] International Publication No. 2011116052 [Patent Document 3] International Publication No. 2010080424 A1 [Non-patent literature]

[0010] [Non-Patent Document 1] Abdel-Hamid et al., Adv. Appl. Microbiol. 2013, 82: 1-28 [Non-patent document 2] Ander et al., J. Biotechnol. 1997, 53: 115-131 [Non-patent document 3] Denys et al. Infect Immun., 2019;87(7):e00261-19 [Non-patent document 4] Moguilevsky et al. FEBS Lett. 1992;302(3):209-212 [Non-patent document 5] Barham et al., Analyst 1972, 97: 142-145 [Non-patent document 6] Chun et al., Biochip J. 2014, 8: 218-226 [Non-Patent Document 7] Mundaca-Uribe et al., Sens. Actuators B 2014, 195, 58-62 [Non-patent document 8] Ellis et al. ACS Catal. 2019, 9(12): 10812-10869 [Non-Patent Document 9] Tenovuo et al. Biochim Biophys Acta, 1986; 870(3): 377-84 [Non-Patent Document 10] Auer et al. J Biol Chem., 2013; 288(38): 27181-27199 [Non-Patent Document 11] Flemmig et al. J Biol Chem., 2012; 287(33): 27913-23 [Non-Patent Document 12] Roth et al. Proc Natl Acad Sci US A., 2003;100(1):62-7 [Non-Patent Document 13] Courjean et al. J Biotechnol., 2011; 151(1):122-9 [Non-Patent Document 14] Ciaurriz et al. J Colloid Interface Sci., 2014; 15;414:73-81 [Non-Patent Document 15] Chen et al. Adv Drug Deliv Rev, 2013; 65(10):1357-1369 [Non-Patent Document 16] Thermo Scientific: Bionconjugation and crosslinking technical handbook (2018) [Non-Patent Document 17] Needleman and Wunsch, Journal of Molecular Biology, 1970, 48(3): 443-53 [Non-Patent Document 18] Lehninger, 1975 [Non-Patent Document 19] Sambrook et al. in Molecular Cloning: A Laboratory Manual, 4th ed., 2012 [Non-Patent Document 20] Eggenreich et al.Biotechnology Reports, 2016, 10: 75-83 [Non-Patent Document 21] Zhang, Biotechnology Advances, 2011; 29(6): 715-725 Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, there is a need in the art to provide a simplified bifunctional peroxidase-oxidase system that does not compromise enzyme stability, selectivity, and catalysis.

[0012] The present invention addresses the above-mentioned need in the art by providing an enzyme chimera capable of catalyzing the formation of hydrogen peroxide and (pseudo)halogenated compounds in a cascade through bifunctional myeloperoxidase-glucose oxidase activity.

[0013] Indeed, we report here for the first time the successful fusion of the open reading frames of glucose oxidase (GOx from Penicillium amagasakiens) and myeloperoxidase (MPO from Rhodopirellula baltica or Homo sapiens). To this end, we engineered a range of chimeric constructs in which these enzymes were fused end-to-end to each other either directly or via a short bridging amino acid sequence. Both enzymes, expressed within a single open reading frame, remained stable and catalytically active, even in the absence of a bridging sequence. This result was rather unexpected, given that both of these proteins are very large components, and that enzymes that derive their catalytic activity from quaternary structures (MPO and GO, respectively, require dimerization for activation) can be easily deactivated by fusion with another enzyme (Ellis et al., ACS Catal. 2019, 9(12): 10812-10869). Even more surprising was the presence of a peptide bridge, referred to herein as a peptide linker, which increased the catalytic activity of the chimera to both glucose oxidase and myeloperoxidase levels. The chimeras of the present invention were also found to be microbicidal. [Means for solving the problem]

[0014] In a first aspect, the present invention relates to a non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

[0015] In a preferred embodiment, the polypeptide of the invention is a fusion polypeptide comprising myeloperoxidase linked, preferably covalently linked, to glucose oxidase.

[0016] In a preferred embodiment, the C-terminus of myeloperoxidase is linked, preferably covalently, to the N-terminus of glucose oxidase.

[0017] In a preferred embodiment, myeloperoxidase is linked, preferably covalently bound, to glucose oxidase by a linker, preferably a peptide linker.

[0018] In a preferred embodiment, the linker has the following amino acid sequence: (LX1X2X3X4X5AX6A)m A peptide linker comprising or consisting of: X1 is glutamic acid or glycine; X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty; X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, m is an integer ranging from 1 to 2, preferably 1, or a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0019] In a preferred embodiment, the peptide linker comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8), or is a peptide substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

[0020] In another preferred embodiment, the peptide linker comprises or consists of a polyglycine amino acid sequence, for example, a polyglycine amino acid sequence, including (G)m (m is an integer ranging from 2 to 10), in particular GGGGGGGG (SEQ ID NO: 9), or a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0021] In preferred embodiments, the myeloperoxidase is a microbial myeloperoxidase, such as a myeloperoxidase from Rhodopyleurola baltica, or a mammalian myeloperoxidase, such as a myeloperoxidase from human origin.

[0022] In a preferred embodiment, the glucose oxidase is a microbial glucose oxidase, for example, a glucose oxidase from Penicillium amagasakiens.

[0023] In a preferred embodiment, the polypeptide of the invention is in the form of a functional oligomer or a mixture of functional oligomers.

[0024] Another aspect relates to nucleic acids encoding the polypeptides of the invention.

[0025] A further aspect relates to a vector comprising a nucleic acid of the invention.

[0026] A further aspect is directed to a host cell comprising the vector of the invention.

[0027] In another aspect, there is provided a method for obtaining a polypeptide of the invention, comprising at least the steps of a) culturing a host cell of the invention in a culture medium under conditions suitable for expression of the polypeptide, and b) recovering said polypeptide.

[0028] In a further aspect, there is provided an antimicrobial composition comprising a non-naturally occurring polypeptide of the invention.

[0029] In a preferred embodiment, the antimicrobial composition further comprises glucose or a source of glucose and / or a halide or pseudohalide.

[0030] A further aspect relates to the in vitro use of a non-naturally occurring polypeptide or composition of the invention to halogenate non-halogenated organic compounds.

[0031] Another aspect relates to the in vitro or ex vivo use of a non-naturally occurring polypeptide or composition of the invention to kill or inhibit the growth of microorganisms.

[0032] A further aspect is directed to a non-naturally occurring polypeptide or composition of the invention for use as a medicament, preferably for the treatment of a microbial infection.

[0033] A further aspect relates to (i) a non-naturally occurring polypeptide of the invention and (ii) glucose or a glucose source and / or a halide or pseudohalide, preferably as a combined preparation for simultaneous, separate or sequential use as a medicament for the treatment of a microbial infection.

[0034] Another aspect of the invention is the amino acid sequence: (LX1X2X3X4X5AX6A)m comprising or consisting of: X1 is glutamic acid or glycine; X2 is lysine or glycine; X3 is arginine or glycine; X4 is proline or empty; X5 is glutamic acid or glycine; X6 is glutamic acid or glycine, m is an integer ranging from 1 to 2, preferably 1; or a peptide linker which is a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0035] In a preferred embodiment, the peptide linker of the present invention comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8), or is preferably a peptide substantially homologous thereto, derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a summary schematic of the reactions catalyzed by bifunctional MPO-GO polypeptides according to the invention. [Figure 2A] Figure 1 shows the catalytic efficiency of RbMPO-GO chimeras (active binding sites) according to the present invention. (A) Catalytic efficiency of the active binding sites for glucose. All chimeras catalyze glucose in vitro, although the efficiency varies depending on the nature of the peptide linker (if present) and the oligomeric state (n≧1). [Figure 2B] Figure 1 shows the catalytic efficiency of RbMPO-GO chimeras (active binding sites) according to the present invention. (A) Catalytic efficiency of the active binding sites for chloride. All chimeras catalyze NaCl in vitro, although the efficiency varies depending on the nature of the peptide linker (if present) and the oligomeric state (n≧1). [Figure 3A]Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3B] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3C] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3D]Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3E] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3F] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3G]Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3H] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3I] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3J]Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3K] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3L] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3M]Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. [Figure 3N] Figures (A-N) show the bactericidal activity and stability of RbMPO-GO chimeras according to the present invention. A620 nm measurements with different kinetics at 400 and 800 min are shown for various concentrations of chimera (as indicated in each figure) and in the presence of SCN- (25 mM) or Cl- (80 mM) with a fixed concentration of glucose (14 mM). For all experiments, kinetic analysis was performed for 16 h at 37°C. Kinetic experiments were repeated several days (d) after enzyme storage at 4°C. Data at 400 and 800 min were obtained from the kinetic curves. Bactericidal activity was measured at 37°C and evaluated in triplicate. DETAILED DESCRIPTION OF THE INVENTION

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, the nomenclature and molecular biological techniques, e.g., protein chimeric or enzymatic techniques, used herein are those well known and commonly used in the art.

[0038] The present invention may be understood more readily by reference to the following detailed description, including preferred embodiments of the invention, and the examples included therein.

[0039] The present invention provides artificial polypeptides that are capable of catalyzing the formation of hydrogen peroxide in a cascade reaction, which in turn drives the formation of (pseudo)halogenated compounds.

[0040] To this end, the present invention relates in a first aspect to a non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

[0041] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to an exact sequence of amino acids, also referred to as an amino acid sequence. Such terms therefore include polypeptides of any size, preferably at least 50, 100, 250, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 amino acids, and / or polypeptides that have undergone post-translational modifications.

[0042] A "non-naturally occurring" polypeptide, as used herein, refers to a polypeptide or protein that is not found in nature (i.e., is not a wild-caught polypeptide). Such polypeptides may typically be the product of human intervention, e.g., protein engineering.

[0043] The terms "activity," "function," "biological activity," and "biological function" are equivalent and should be understood as they are well known in the art. Preferably, such activity is an enzymatic activity. In the context of the present invention, a polypeptide is bifunctional because it exhibits at least two functions or activities, which herein are myeloperoxidase activity and glucose oxidase activity.

[0044] "Myeloperoxidase activity" is determined by the following reaction: H2O2+ X - + H + →H2O + HOX is typically characterized by the oxidation of halides or pseudohalides to (pseudo) hypohalous acids in the presence of hydrogen peroxide by In the formula, X - represents a halide or pseudohalide.

[0045] The term "halide" refers to an ion of a halogen, and as used herein, refers to chloride (Cl - ), bromide (Br - ) or iodide (I - The term "pseudohalide" refers to a polyatomic anion with acid-base and redox chemical properties similar to those of halides, and is used herein to refer to thiocyanate (SCN), thiocyanate (SCN), and any combination thereof. - Halides and pseudohalides are collectively referred to herein as (pseudo)halides.

[0046] Myeloperoxidase activity can be detected according to the protocol described in Example 1, section 1.8 below, and / or measured according to the protocols described by Tenovuo et al. (Biochim Biophys Acta, 1986; 870(3): 377-84), Auer et al. (J Biol Chem., 2013; 288(38): 27181-27199), and / or Flemmig et al. (J Biol Chem., 2012; 287(33): 27913-23).

[0047] Myeloperoxidase activity can typically be provided by myeloperoxidase. "Myeloperoxidase" refers to an enzyme that has myeloperoxidase activity, particularly when the enzyme is in the form of a haloenzyme (or holoenzyme), i.e., when the enzyme is bound or complexed with at least a cofactor and optionally an ion. Myeloperoxidase in the form of a haloenzyme is typically bound or complexed with at least heme and optionally calcium. Preferred myeloperoxidases according to the present invention are further described below.

[0048] "Glucose oxidase activity" is determined by the following reaction: Glucose (or glucose source) + O2 + H2O → D-gluconic acid + H2O2 It is typically characterized by the production of hydrogen peroxide following oxidation of glucose or a glucose source (e.g., dextrose or sucrose) by .

[0049] Glucose oxidase activity can be detected according to the protocol described in Example 1, section 1.7 below, and / or measured according to the protocols described by Roth et al. (Proc Natl Acad Sci U S A., 2003;100(1):62-7), Courjean et al. (J Biotechnol., 2011;151(1):122-9), and / or Ciaurriz et al. (J Colloid Interface Sci., 2014;15;414:73-81).

[0050] Glucose oxidase activity can typically be provided by glucose oxidase. "Glucose oxidase" refers to an enzyme that has glucose oxidase activity, particularly when the enzyme is in the form of a haloenzyme (or holoenzyme), i.e., when the enzyme is bound or complexed with at least a cofactor and optionally a sugar chain or glycan. Glucose oxidase in the form of a haloenzyme is typically bound or complexed with at least flavin adenine dinucleotide (FAD) and optionally glycosylated (e.g., at amino acid 93 with reference to the numbering of amino acid sequence SEQ ID NO: 12, the glycosylation is preferably GlucNac). Further preferred glucose oxidases according to the present invention are as follows:

[0051] In a preferred embodiment, the polypeptide according to the invention is a fusion polypeptide comprising myeloperoxidase conjugated to glucose oxidase.

[0052] As used herein, the term "fusion polypeptide" refers to a polypeptide produced by linking two or more (poly)peptides. To achieve this, two or more (poly)peptides are linked to each other directly or indirectly. Such linkage can be carried out by biological or biochemical means. For example, in biological linkage, a fusion polypeptide can be the translation product of a chimeric gene construct in which a first DNA sequence encoding a first (poly)peptide is linked to a second DNA sequence encoding a second (poly)peptide to form a single open reading frame.

[0053] Here, both myeloperoxidase and glucose oxidase fusion polypeptides according to the invention maintained catalytic activity when the C-terminus of myeloperoxidase was linked to the N-terminus of glucose oxidase.

[0054] Thus, in a preferred embodiment, the C-terminus of myeloperoxidase is linked to the N-terminus of glucose oxidase.

[0055] In the context of the present invention, such a bond between two enzymes is preferably a stable bond, typically covalent, for in vitro, ex vivo or even in vivo applications.

[0056] In a preferred embodiment, myeloperoxidase, preferably the C-terminus of myeloperoxidase, is covalently linked to glucose oxidase, preferably the N-terminus of glucose oxidase.

[0057] As used herein, the term "covalent bond" (covalently coupled, covalently bound, covalently linked, covalent coupling, covalent bonding, or covalent linkage) refers to an interatomic bond resulting from the sharing of one or more electron pairs between two atoms, for example, between two or more (poly)peptides. Typical examples of covalent bonds between two or more (poly)peptides include, but are not limited to, peptide bonds (covalent bonds that typically connect amino acids to each other) and cross-links (disulfide bridges formed between cysteine ​​chains, or cross-links between lysines and cysteines covalently linked by an oxygen atom). Two or more (poly)peptides can be covalently bonded to each other directly or indirectly. An indirect bond means that two or more (poly)peptides are connected to each other via one or more intervening bonding moieties, e.g., linkers. A direct bond means that two or more (poly)peptides are connected to each other without one or more intervening bonding moieties, e.g., linkers.

[0058] Surprisingly, the inventors have demonstrated herein that direct fusion of myeloperoxidase to glucose oxidase does not abolish the catalytic activity of each of the enzymes in the resulting polypeptide. Without being bound by theory, the inventors believe that upon such fusion, the individual enzymes retain the ability to fold independently from the rest of the polypeptide chain.

[0059] Thus, in a preferred embodiment, myeloperoxidase is directly covalently attached to glucose oxidase.

[0060] The inventors have further demonstrated herein that indirect fusion of myeloperoxidase to glucose oxidase via a linker can improve the catalytic activity of the enzyme in the resulting polypeptide, compared to direct fusion of the two enzymes. Without being bound by theory, the inventors believe that such fusion improves the ability of each enzyme to fold independently from the rest of the polypeptide chain.

[0061] Thus, in a preferred embodiment, myeloperoxidase is covalently attached to glucose oxidase by a linker.

[0062] As used herein, the term "linker" or "spacer" refers to a synthetic or natural chemical or biological moiety that can link two molecules together and provide spatial separation between the molecules. Chemical linkers are well known in the art and are typically polymer chains of various lengths that can be homo- or heterobifunctional with identical or different reactive groups and contain at least one atom, preferably at least one carbon atom. In contrast, biological linkers are typically nucleic acid and / or amino acid sequences of various lengths and contain at least one nucleic acid and / or amino acid. Methods for linking such linkers and molecules in specific proteins have been extensively described, inter alia, in Chen et al. (Adv Drug Deliv Rev, 2013; 65(10):1357-1369) and Thermo Scientific's Bioconjugation and Crosslinking Technical Handbook (2018), and can therefore be easily selected and designed by those skilled in the art.

[0063] In the context of the present invention, biological linkers, in particular peptide linkers, are particularly preferred.

[0064] "Peptide linker" or "peptidic linker" is intended to mean a biological linker, as defined above, consisting of an amino acid sequence of various lengths. As a non-limiting indication, the peptide linker can be from about 2 amino acids to about 50 amino acids in length. Preferred peptide linkers according to the present invention are those whose sequences comprise a length of about 3 to about 35 amino acids, preferably about 3 to 35 amino acids, and more preferably about 8 to about 18 amino acids. Peptide linkers are well known in the art (Chen et al., Adv Drug Deliv Rev, 2013; 65(10):1357-1369, incorporated herein by reference in its entirety, especially Table 3) and can typically be identified as rigid, semi-rigid, flexible, or cleavable linkers. Rigid peptide linkers exhibit a relatively rigid structure and generally have a helical structure or are rich in proline. The amino acids proline, arginine, phenylalanine, glutamic acid, and glutamine are typically found in rigid linkers. Proline can also be useful for creating hinges within the structure. Examples of rigid peptide linkers include, but are not limited to, alpha-helix-forming peptide linkers, such as A(EAAAK)mA (where m is an integer ranging from 2 to 5) (SEQ ID NO: 1), the proline-alanine linker PAPAP (SEQ ID NO: 2), and the polyproline linker (P)m (where m is an integer ranging from 2 to 8). Semirigid linkers are peptide linkers with limited flexibility, i.e., structures that are not rigid but not completely flexible. Examples of semirigid linkers are described in WO2010080424A1, which is incorporated herein by reference in its entirety. Flexible peptide linkers allow for relatively free movement of the binding molecule, and such linkers are typically rich in small non-polar or polar amino acids, such as glycine and / or serine. Examples of flexible peptide linkers include, but are not limited to, polyglycine (G)m (where m is an integer ranging from 2 to 10) and the glycine-serine linker (GGGGS)m (where m is an integer ranging from 2 to 5) (SEQ ID NO: 3).

[0065] Rigid linkers, semi-rigid linkers, and flexible linkers are stable in vivo and do not separate the linked (poly)peptides. On the other hand, cleavable peptide linkers are susceptible to reductive or enzymatic cleavage, and examples of such linkers include disulfide bridges or protease cleavage sites. Those skilled in the art will readily understand that cleavable peptide linkers are not suitable for the present invention. In a preferred embodiment, the linker used in the present invention is not a cleavable peptide linker. Thus, the peptide linker can be a rigid, semi-rigid, or flexible peptide linker.

[0066] The following peptide linkers are particularly suitable for the fusion polypeptides according to the invention:

[0067] In a preferred embodiment, the peptide linker has the following amino acid sequence: (LX1X2X3X4X5AX6A)m comprising or consisting of: X1 is glutamic acid or glycine; X2 is lysine or glycine, X3 is arginine or glycine, X4 is proline or empty; X5 is glutamic acid or glycine, X6 is glutamic acid or glycine, m is an integer ranging from 1 to 2, preferably 1, or a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0068] Two amino acid sequences are "homologous," "substantially homologous," or "substantially similar" if the two sequences exhibit the same (or substantially the same) essential structure (e.g., tertiary, quaternary, rigid, semi-rigid, flexible, cleavable, etc.) and / or the same (or substantially the same) essential biological activity, but with one or more amino acids replaced with one or more biologically similar amino acids, or if at least about 80% of the amino acids between the two sequences are identical, or at least about 90%, preferably at least about 95%, even more preferably at least about 96% or 97%, and more preferably at least about 98% or 99% identical. In other words, two homologous amino acid sequences are said to be functional. When compared to a reference amino acid sequence, a homologous amino acid sequence may typically contain, for example, silent mutations, conservative substitutions, or small deletions of genetic material that do not affect (or substantially affect) the structure or biological activity of the reference sequence. Similar or homologous sequences can be identified by alignment using algorithms well known in the art. For example, optimal alignment of sequences can be performed by a global homology alignment algorithm, such as the algorithm described by Needleman and Wunsch (Journal of Molecular Biology, 1970, 48(3): 443-53), or by computer implementation of this algorithm. Global homology alignment may be preferred when alignment is performed using sequences of the same or similar length. The percentage of identity can be preferably calculated over the entire length of the reference sequence.

[0069] As used herein, "conservative substitution" refers to the replacement of an amino acid (or corresponding codon) with another amino acid (or corresponding codon) without altering the overall configuration and / or function of the reference (poly)peptide (or corresponding nucleic acid), and includes, but is not limited to, the replacement of an amino acid (or corresponding codon) with an amino acid (or corresponding codon) having similar properties (e.g., polarity, hydrogen bonding, acidicity, basicity, shape, hydrophobicity, aromaticity, etc.) to the amino acid (or corresponding codon). Amino acids with similar properties are well known in the art. For example, arginine, histidine, and lysine are hydrophilic, basic amino acids and may be interchangeable. Similarly, the hydrophobic amino acid isoleucine may be interchangeable with leucine, methionine, or valine. Neutral hydrophilic amino acids that may be interchangeable include asparagine, glutamine, serine, and threonine.

[0070] A "substitution" or "modification", as used herein, includes amino acids that are changed or modified from naturally occurring amino acids.

[0071] Thus, in the context of the present invention, a conservative substitution should be understood to be recognized in the art as the substitution of an amino acid for another amino acid having similar properties. Examples of conservative substitutions are provided in Table 1 below.

[0072] [Table 1]

[0073] Alternatively, conserved amino acids can be grouped as described by Lehninger, 1975, as presented in Table 2 below.

[0074] [Table 2]

[0075] As a further alternative, exemplary conservative substitutions are provided below in Table 3.

[0076] [Table 3]

[0077] In a further preferred embodiment, the peptide linker of the present invention comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7) or LEKRPEAEA (SEQ ID NO: 8), or is preferably a peptide substantially homologous thereto, derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

[0078] The peptide linkers (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7), and LEKRPEAEA (SEQ ID NO: 8) can be essentially characterized as rigid peptide linkers, while the peptide linkers LEGGEAEA (SEQ ID NO: 4) and LGKRGAGA (SEQ ID NO: 5) can essentially be characterized as semi-rigid peptide linkers.

[0079] In another preferred embodiment, the peptide linker of the invention comprises or consists of a polyglycine amino acid sequence, e.g., a polyglycine amino acid sequence, including (G)m (m is an integer ranging from 2 to 10), in particular GGGGGGGG (SEQ ID NO: 9), or a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0080] The peptide linkers (G)m (m is an integer ranging from 2 to 10) and GGGGGGGG (SEQ ID NO: 9) can essentially be characterized as flexible peptide linkers.

[0081] In the context of the present invention, rigid and semi-rigid peptide linkers are particularly preferred. Particularly preferred examples of such peptide linkers are LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), and (LEKREAEA)2 (SEQ ID NO: 6).

[0082] In preferred embodiments, the myeloperoxidase is a microbial myeloperoxidase, such as a myeloperoxidase from Rhodopyleurola baltica, or a mammalian myeloperoxidase, such as a mammalian myeloperoxidase from human origin.

[0083] Naturally occurring myeloperoxidase and polypeptides substantially homologous thereto are encompassed herein.

[0084] For example, a particularly preferred myeloperoxidase according to the present invention is a polypeptide substantially homologous thereto, comprising or consisting of the native amino acid sequence of Rhodopyleurola baltica SEQ ID NO: 10, or preferably derived from SEQ ID NO: 10 by one or more conservative substitutions.

[0085] As another example, a particularly preferred myeloperoxidase according to the invention is a polypeptide that comprises or consists of the human naturally occurring amino acid sequence SEQ ID NO:11, or is substantially homologous thereto, preferably derived from SEQ ID NO:11 by one or more conservative substitutions.

[0086] Conservative substitutions may be made particularly at non-critical amino acids or in non-critical regions.

[0087] Amino acids important for the biological activity of preferred myeloperoxidases of the present invention have indeed been identified, including amino acids 199, 202, 203, 316, 317, and 407 with reference to the numbering of amino acid sequence SEQ ID NO: 10, or amino acids 257, 260, 261, 408, and 502 with reference to the numbering of amino acid sequence SEQ ID NO: 11. Such amino acids are in particular Q199, D202, H203, E316, N317, and H407 of SEQ ID NO: 13, or Q257, D260, H261, E408, and M409 of SEQ ID NO: 11.

[0088] Thus, in a preferred embodiment, the myeloperoxidase comprises or consists of the native amino acid sequence of Rhodopyloura baltica SEQ ID NO: 10, or is a polypeptide substantially homologous thereto, preferably derived from SEQ ID NO: 10 by one or more conservative substitutions, provided that the following amino acids are conserved: Q199, D202, H203, E316, N317, and H407.

[0089] In another preferred embodiment, the myeloperoxidase comprises or consists of the human naturally occurring amino acid sequence SEQ ID NO:11, or is a polypeptide substantially homologous thereto, preferably derived from SEQ ID NO:11 by one or more conservative substitutions, provided that the following amino acids are conserved: Q257, D260, H261, E408, M409.

[0090] In a preferred embodiment, the glucose oxidase is a microbial glucose oxidase, for example, a glucose oxidase from Penicillium amagasakiens.

[0091] Naturally occurring glucose oxidase and polypeptides substantially homologous thereto are encompassed herein.

[0092] For example, a particularly preferred glucose oxidase according to the present invention is a polypeptide that comprises or consists of the native amino acid sequence SEQ ID NO: 12 of Penicillium amagasakiens, or is substantially homologous thereto, preferably derived from SEQ ID NO: 12 by one or more conservative substitutions.

[0093] As explained above, it is known in the art that myeloperoxidase and glucose oxidase each require oligomerization to be catalytically active, and therefore the inventors herein evaluated polypeptides according to the invention in various oligomeric states.

[0094] In a preferred embodiment, the polypeptide according to the invention is in the form of a functional oligomer (n≧1) or a mixture of functional oligomers (n≧1).

[0095] More precisely, the polypeptides according to the invention may be in the form of functional monomers or functional multimers or mixtures thereof.

[0096] "Oligomer" or "oligomeric state," as used herein, refers to the structural units that make up an oligomeric polypeptide. The number (n) of such structural units, also known as the degree of oligomerization, can be equal to or greater than 1 (n≧1). When n is greater than 1, the structural units are typically linked, either covalently or non-covalently. n is generally less than 100, and usually less than 30. An oligomer with n=1 is known as a monomer or single unit, while an oligomer with n>1 may be referred to as a multimer or multiunit. A monomer or single unit, as used herein, typically consists of one polypeptide (or polypeptide chain), while a multimer or multiunit typically consists of at least two polypeptides (or polypeptide chains). The growing oligomers are called dimers (n = 2), trimers (n = 3), tetramers (n = 4), pentamers (n = 5), hexamers (n = 6), heptamers (n = 7), octamers (n = 2), nonamers (n = 9), decamers (n = 10), etc.

[0097] Various oligomers of the polypeptides of the present invention may exhibit different catalytic efficiencies, but all maintain essential functionality in that they exhibit myeloperoxidase activity and glucose oxidase activity. It is within the skill of one of ordinary skill in the art to select oligomers having the desired levels of myeloperoxidase and glucose oxidase activity as needed. It may also be desirable to utilize the polypeptides of the present invention in the form of a mixture of functional oligomers to reduce production time and costs and increase production yields.

[0098] Methods for preparing the polypeptides of the present invention are as follows.

[0099] The polypeptides according to the present invention can be encoded by nucleic acids.

[0100] By "nucleic acid" or "nucleotide sequence" herein is meant an exact sequence of naturally occurring nucleotides (i.e., A, T, G, C, and U) or non-naturally occurring nucleotides. Such terms encompass single- or double-stranded DNA as well as transcription products of said DNA, e.g., RNA.

[0101] Thus, in a further aspect, the present invention relates to nucleic acids encoding the polypeptides described herein.

[0102] Nucleic acids, like the polypeptides they encode, are non-naturally occurring.

[0103] The nucleic acids of the present invention can be prepared by methods well known in the art, including, but not limited to, any synthetic and / or recombinant methods. It is within the skill of one of ordinary skill in the art to design the nucleotide sequence of a nucleic acid, for example, by codon optimization based on the desired expression system (e.g., host cell), to enable efficient production of a functional polypeptide.

[0104] The nucleic acid according to the invention can be advantageously included in a vector for amplifying the nucleic acid or for expressing the polypeptide of the invention in a host cell.

[0105] Thus, a further aspect of the present invention provides a vector comprising a nucleic acid as disclosed herein.

[0106] The vector may be advantageously contained in a host cell, such as a prokaryotic or eukaryotic cell. Thus, the vector may be a prokaryotic or eukaryotic vector.

[0107] Thus, the present invention also relates to a host cell comprising a vector of the invention.

[0108] The term "vector" generally refers to tools useful in the practice of molecular biology and recombinant genetic procedures. Such tools are commonly used and very well known in the art. This term encompasses vectors that are capable of replicating either to amplify a nucleic acid of interest (i.e., a cloning vector) or to express a polypeptide encoded by said nucleic acid in a host cell (i.e., an expression vector). Such types of vectors are publicly available and include, but are not limited to, plasmids, cosmids, YACs, BACs, viral vectors (adenovirus, AAV, retrovirus, e.g., lentivirus, EBV episome, etc.), and phage vectors. A vector is referred to herein as recombinant in that it is not naturally found in combination with a nucleic acid of the invention (i.e., it is not naturally occurring).

[0109] Methods for inserting nucleic acids into vectors are known to skilled practitioners. Generally, nucleic acids can be inserted into one or more restriction endonuclease sites using techniques well known in the art (see, for example, the techniques described by Sambrook et al. in Molecular Cloning: A Laboratory Manual, 4th Edition, 2012). Nucleotide sequences that allow transcription of the nucleic acid, expression of the protein encoded by the nucleic acid, and / or purification are also preferably included in the vector. Such sequences generally include, but are not limited to, at least one sequence selected from one or more signal peptide sequences, an origin of replication, one or more selectable marker genes, an enhancer element, a promoter, a transcription terminator, and, optionally, a sequence that allows protein purification. Insertion of such sequences into the vector can be carried out using standard ligation techniques known to those skilled in the art, such as those described above. In addition, it is known to those skilled in the art that such nucleotide sequences can be selected based on the host cell in which the vector is intended to replicate and / or in which the polypeptide encoded by the nucleic acid is intended to be expressed.

[0110] For example, depending on the origin of replication selected, a vector may replicate in one or more host cells: the pBR322 plasmid origin of replication is typically compatible with most gram-negative bacteria, the 2μ plasmid origin of replication is generally yeast-specific, and various viral origins of replication (SV40, polyoma, adenovirus, VSV, or BPV) are particularly useful for cloning vectors in mammalian cells.

[0111] For another example, depending on the promoter selected, the nucleic acid can be transcribed and the corresponding polypeptide expressed in one or more host cells: T7, Lac, trp, tac, λPL promoters are typically specific for E. coli / Escherichia coli, PHO5, GAP, TPI1, ADH promoters are generally adapted to yeast, polyhedrin and P10 promoters and their equivalents are conventionally used in insect cells, and finally, CMV, MT1, SV40, SRα, retroviral promoters, and heat shock protein gene promoters are particularly adapted to mammalian cells.

[0112] Non-exhaustive examples of selectable marker genes that are typically included in vectors are genes that confer resistance to antibiotics or toxins (e.g., ampicillin, neomycin, zeocin, hygromycin, kanamycin, tetracycline, chloramphenicol, or combinations thereof) and genes that allow for compensation of auxotrophic deficiencies (e.g., the gene encoding dihydrofolate reductase DHFR, which confers resistance to methotrexate, or even the TPI gene of S. pombe).

[0113] Non-exhaustive examples of nucleotide sequences that allow for the purification of the polypeptide are histidine sequences (histidine tag or Hisx6), FLAG sequences, and GST sequences. A protease cleavage sequence, e.g., VTE, may additionally be present for subsequent removal of the purification sequence.

[0114] Non-exhaustive examples of prokaryotic vectors are pET (Novagen), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, and pRIT5 (Pharmacia).

[0115] Non-exhaustive examples of eukaryotic vectors are pWLNEO, pSV2CAT, pPICZ, pcDNA3.1(+)Hyg (Invitrogen), pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pCI-neo (Stratagene), pMSG, pSVL (Pharmacia), and pQE-30 (QLAexpress).

[0116] In a preferred embodiment, the vector of the present invention is a prokaryotic vector, preferably a pET vector, for example a pET21a vector.

[0117] As used herein, the terms "host cell," "cell," and "cell line" can be used interchangeably and refer to prokaryotic or eukaryotic cells into which a vector of the invention can be introduced, e.g., to amplify the nucleic acid and / or express the polypeptide encoded by the nucleic acid. To accomplish this, host cells can be "transfected" or "transformed" by processes known in the art for transferring or introducing the vector into the host cell. Examples of such methods include, but are not limited to, electroporation, lipofection, calcium phosphate transfection, transfection using DEAE-dextran, microinjection, and biolistic methods.

[0118] The choice of host cell typically depends on the selected use, i.e., cloning of a nucleic acid or expression of a polypeptide encoded by said nucleic acid. One skilled in the art can select an appropriate host cell from among the many cell lines publicly available through, inter alia, the American Type Culture Collection (ATCC).

[0119] Examples of prokaryotic cells include bacteria, such as Escherichia (e.g., E. coli BL21, C41, RR1, LE392, B, X1776, W3110, DH5 alpha, JM109, KC8), Serratia Pseudomonas, Erwinia Methylobacterium, and the like. Gram-negative bacteria of the genera Methylobacterium, Rhodobacter, Salmonella, and Zymomonas, and Gram-positive bacteria of the genera Corynebacterium, Brevibacterium, Bacillus, Arthrobacter, and Streptomyces, but are not limited to these.

[0120] Examples of eukaryotic cells include, but are not limited to, cells isolated from fungi, plants, and animals. Such cells include, inter alia, yeast, such as yeasts of the genus Saccharomyces, cells derived from fungi, such as cells of the genera Aspergillus, Neurospora, Fusarium, or Trichoderma, animal cells, such as HEK293 cells, NIH3T3, Jurkat, MEF, Vero, HeLa, CHO, W138, BHK, COS, COS-7, MDCK, C127, Saos, PC12, HKG, and insect cells, such as Sf9, Sf21, Hi Five™, or silkworm (Bombyx mori) cells.

[0121] In a preferred embodiment, the host cell of the invention is a prokaryotic cell, preferably of the genus Escherichia, more preferably of E. coli, for example, E. coli BL21 (eg, BL21 Star(DE3)).

[0122] The present inventors have herein recombinantly produced the polypeptides of the present invention in high yield, while still allowing for proper refolding of the polypeptides.

[0123] According to a further aspect, the present invention provides a method for obtaining a polypeptide of the invention, comprising the steps of: a) culturing a host cell of the invention in a culture medium under conditions suitable for expression of the polypeptide; b) recovering the polypeptide; The present invention relates to a method comprising at least

[0124] The host cells used in the method are preferably as described above.

[0125] In a preferred embodiment, the host cell is a prokaryotic cell, preferably of the genus Escherichia, more preferably of E. coli, for example, E. coli BL21 (eg, BL21 Star(DE3)).

[0126] In step b), the polypeptide can be recovered from the host cell if said polypeptide is expressed intracellularly and / or from the culture medium in which the host cell is cultured if said polypeptide is expressed extracellularly.

[0127] Those skilled in the art can use any conventional method that allows the recovery of the polypeptide. For example, if the polypeptide is expressed in a dissolved form in the host cells, the latter can be recovered by centrifugation and suspended in a buffer solution, and then the cells can be disrupted, for example, by an ultrasonic homogenizer (sonication) or a cell disrupter, optionally in combination with urea treatment, to obtain a cell-free extract.

[0128] In the context of the present invention, a reconstitution solution may be required to ensure that the recovered polypeptide is fully folded and functional, i.e., in the form of a haloenzyme. For this purpose, the recovered polypeptide in step b) can be advantageously solubilized in a solution containing heme and FAD (or a functional derivative of heme or FAD) and, optionally, calcium. Heme and FAD are, in fact, cofactors for myeloperoxidase and oxidase, respectively.

[0129] The polypeptide recovered in step b) can be advantageously purified in a further step of the method, defined as step c), which preferably makes it possible to obtain a 100% purified or nearly 100% purified polypeptide.

[0130] Those skilled in the art can use any conventional method that allows for the purification of the polypeptide. For example, if the polypeptide is recovered in a cell-free extract as described above, a purified sample can be obtained from the supernatant obtained by centrifugation of this extract using a conventional method or a combination of conventional methods for isolating and purifying the polypeptide of the present invention. Such methods include, but are not limited to, solvent extraction, salting out with ammonium sulfate, desalting, precipitation by dialysis, filtration, ultrafiltration, organic solvents, preparative electrophoresis, isoelectric focusing, various chromatographic methods, such as ion exchange chromatography (anionic, e.g., using a resin such as diethylaminoethyl (DEAE) Sepharose, or cationic, e.g., using a resin such as S-Sepharose (Pharmacia)), hydrophobic chromatography (e.g., using a resin such as butyl Sepharose or phenyl Sepharose), size exclusion chromatography, affinity chromatography using antibodies, adsorption chromatography, chromatofocusing, high-performance liquid chromatography (HPLC), and reverse-phase HPLC, and any combination thereof.

[0131] When a chromatographic method is used in step c), one or more substeps may be performed, including but not limited to binding of the resulting polypeptide onto a solid support, e.g., a chromatographic column, washing steps, and elution steps, which may be repeated as many times as necessary to obtain the desired degree of purification of the polypeptide.

[0132] A size exclusion chromatography column may be preferred to isolate the various oligomeric forms of the polypeptide, whereas if the skilled practitioner desires to reduce production time and costs, a desalting column may be used instead to ensure isolation of the polypeptide in the form of a mixture of all functional oligomers.

[0133] The polypeptide recovered in step b) or purified in step c) can then be solubilized in a suitable buffer. A particularly preferred solubilization buffer according to the present invention is Tris buffer at pH 7.5, preferably Tris-CaCl buffer at pH 7.5.

[0134] Examples of methods by which the polypeptides of the present invention can be obtained are described in Section 1.4 of Example 1 below and in Eggenreich et al. (Biotechnology Reports, 2016, 10: 75-83). Such methods may, inter alia, enable the production of high yields of polypeptides. As reported in the subsequent Examples, the present inventors obtained about 40 mg to about 600 mg of the polypeptides of the present invention per liter of cultured host cells.

[0135] Due to their ability to catalyze the cascade formation of (pseudo)hypohalous acids from hydrogen peroxide and (pseudo)halides, which have been shown to exhibit potent bactericidal and antiviral activity, the polypeptides of the invention can be used as antimicrobial agents.

[0136] Accordingly, a further aspect of the present invention is to provide antimicrobial compositions comprising the non-naturally occurring polypeptides described herein.

[0137] The term "antimicrobial," as used herein, refers to the killing or inhibition of the growth of microorganisms, and thus encompasses the terms "bactericidal," "bacteriostatic," "virucidal," "virustatic," "fungicidal," "fungistatic," "parasiticidal," and "parasiticidal." Examples of microorganisms that may be killed or the growth of which may be inhibited in accordance with the present invention are described in further detail below.

[0138] Additional components may be included as desired. Such components may be provided in a single composition, as may be required for a particular application, or may be separated into two-component compositions and subsequently mixed prior to use. The skilled practitioner will understand that one of such components may be retained and provided separately from the polypeptide (and thus as a two-component composition) to prevent premature reaction and component depletion.

[0139] Those skilled in the art will readily appreciate that the antimicrobial composition, whether single or dual component, preferably includes a suitable substrate for glucose oxidase, e.g., glucose or a glucose source (e.g., dextrose or saccharose), and / or a suitable substrate for myeloperoxidase, e.g., a (pseudo)halide.

[0140] Thus, in preferred embodiments, the antimicrobial composition, whether single or dual, further comprises glucose or a glucose source and / or a halide or pseudohalide.

[0141] The halide or pseudohalide may be selected from the group consisting of iodide, thiocyanate, bromide, chloride, and any combination thereof, especially when the myeloperoxidase is myeloperoxidase from Rhodopyleurola baltica or myeloperoxidase from human origin. Particularly preferred (pseudo)halides according to the present invention are thiocyanate and chloride, especially thiocyanate.

[0142] For purposes of the present invention, the antimicrobial composition may be in a form suitable for in vitro, ex vivo, or in vivo use, depending on the location of the target microorganism. The form of the composition is preferably selected so as to obtain direct contact with the target microorganism. For example, if the antimicrobial composition is intended for administration to a subject, the composition may be in a form suitable for oral, nasal, topical, transdermal, or parenteral administration, depending on the location of the target microorganism.

[0143] The antimicrobial compositions of the present invention may additionally comprise pharmaceutically acceptable excipients.

[0144] As used herein, the term "pharmaceutically acceptable excipient" refers to a pharmaceutical-grade compound that is inert or chemically inert, and therefore non-toxic, but lacks pharmacological activity per se. Such excipients can be used to improve the properties of the composition, such as shelf life, retention time at the application site, consumer acceptance, etc. This includes, but is not limited to, physiologically compatible surfactants (cationic, anionic, or neutral), surface stabilizers, other enhancers, such as preservatives, wetting or emulsifying agents, solvents, buffers, saline, dispersion media, isotonic agents, and absorption delaying agents.

[0145] Those skilled in the art may also desire to combine the antimicrobial compositions of the present invention with one or more therapeutic agents, either in the composition (single composition) or separately (two-component composition). Such therapeutic agents include, for example, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, and any combination thereof. By way of illustration, examples of therapeutic agents suitable for the present invention include, but are not limited to, penicillin, cephalosporin, carbacephem, cephamycin, carbapenem, monobactam, aminoglycoside, glycopeptide, quinolone, tetracycline, macrolide, fluoroquinolone, silver, copper, chlorhexidine, polyhexanide, biguanide, chitosan, and / or acetic acid.

[0146] The polypeptides or compositions of the invention can be used in a wide range of industrial, pharmaceutical, medical, cosmetic and ecological applications, as well as in the food industry, and in particular for any purpose for which the free combination of myeloperoxidase and glucose oxidase has been reported.

[0147] For example, by virtue of its myeloperoxidase activity, a polypeptide or composition of the present invention may be useful for obtaining halogenated organic compounds of interest.

[0148] Accordingly, a further aspect of the present invention provides an in vitro use of a non-naturally occurring polypeptide or composition described herein to halogenate a non-halogenated organic compound.

[0149] In other words, the present invention relates to an in vitro method for halogenating a non-halogenated organic compound, comprising the step of contacting a polypeptide or composition described herein with the non-halogenated organic compound in vitro.

[0150] As used herein, the term "organic compound" refers to a gaseous, liquid, or solid compound whose molecules contain carbon.

[0151] An example of the halogenation of a non-halogenated organic compound (RH) using a polypeptide of the invention is as follows: X - + H2O2+ RH + H + →RX+ 2H2O In the formula, RX represents a halogenated organic compound; H2O2 is produced by the oxidation of glucose (or a glucose source) by glucose oxidase.

[0152] Particularly preferred halogenated organic compounds of interest include, but are not limited to, active organic compounds and chemical intermediates used in organic chemical synthesis, such as disinfectants, nutrients, insecticides, drugs, antibiotics, advantageously plant antibiotics, antioxidants, adhesives, and radiocontrast materials.

[0153] For illustrative purposes, when the halide is iodide, iodo compounds of interest include, but are not limited to, phenolic compounds (e.g., mono-, di-, tri-, and tetra-iodophlroglucinol, dibromoiodophenol, polymers thereof, and iodinated phlorotannins such as iodinated fuhalol, phlorethol, fucol, fucophlorethol, equol, and kalmarol), volatile hydrocarbon compounds (e.g., iodoform, iodomethane, diiodomethane, bromoiodomethane, iodoethane, iodopropane, iodobutane, etc.), terpenes, amino acid derivatives (e.g., monoiodotyrosine and diiodotyrosine, which are thyroxine precursors), and fatty acid derivatives (e.g., Eisenia iodide). Iodomethane, diiodomethane, and iodoform can be used as disinfectants or insecticides. Additionally, iodomethane, also known as methyl iodide, can be used as a chemical intermediate in organic chemical synthesis, inter alia, to methylate other compounds, such as phenols, carboxylic acids, ammonia, and produced amines, and for the industrial-scale production of acetic acid and acetic anhydride.

[0154] As another example, when the halide is iodide, radiocontrast materials obtainable by the present invention may include, but are not limited to, 1,3,5-triiodobenzene and its derivatives, such as the ionic materials diatrizoic acid, metrizoic acid, and ioxaglic acid, and the nonionic materials ioversol, iopamidol, iohexol, ioxilan, iopromide, and iodixanol. Such materials can be used for X-ray imaging, e.g., fluoroscopy.

[0155] Due to their myeloperoxidase activity, the polypeptides or compositions of the present invention may also be useful in inhibiting the growth of a wide range of microorganisms, particularly pathological microorganisms, e.g., microorganisms that are resistant to conventional therapies, by in vitro, ex vivo or in vivo applications.

[0156] Thus, a further aspect of the present invention provides the in vitro or ex vivo use of a non-naturally occurring polypeptide or composition as described herein for killing or inhibiting the growth of a microorganism.

[0157] In other words, the present invention relates to an in vitro or ex vivo method for killing or inhibiting the growth of microorganisms, comprising the step of contacting a polypeptide or composition as described herein with a substance or surface that is contaminated or at risk of being contaminated with microorganisms, in vitro or ex vivo.

[0158] Such applications may in fact be particularly suitable for treating materials or surfaces that are contaminated or susceptible to contamination by microorganisms, for example to disinfect them before or after their use. The material or surface may be the surface of any instrument, laboratory material, surgical material, etc., such as medical equipment, contact lenses, etc., particularly those intended for use in contact with a subject (e.g. sutures, bandages, gauze, staples, zippers, etc.).

[0159] "Microorganism," as used herein, refers to bacteria, as well as viruses, fungi, and parasites. For illustrative purposes, examples of bacteria that can be effectively inhibited by embodiments of the present invention include, but are not limited to, a wide range of Gram-negative or Gram-positive bacteria, such as Escherichia species (e.g., E. coli), Enterococcus species, Staphylococcus species, Streptococcus / Streptococci species, Citrobacter species, Enterobacter species, Klebsiella species, Proteus species, Acinetobacter species, Pseudomonas species, Aeromonas species, and Pasteurella species, as well as Bacillus species, Clostridium species, and the like, to name just a few. Examples of fungi that can be effectively inhibited by embodiments of the present invention include, but are not limited to, Aspergillus species, Fusarium species, Trichophyton species, etc. Particularly preferred microorganisms according to the present invention are Escherichia species, e.g., E. coli.

[0160] Additionally, because the myeloperoxidase within the polypeptide acts via a different overall mechanism of action than molecules involved in conventional therapies, e.g., antibiotics, in some cases this aspect of the invention may be useful for eliminating drug-resistant, multi-drug-resistant, or antibiotic-resistant microorganisms. By way of illustration, examples of drug-resistant microorganisms include, but are not limited to, the pathogenic bacteria MRSA (methicillin-resistant Staphylococcus aureus), VRSA (vancomycin-resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus), penicillin-resistant Enterococcus, PRSP (penicillin-resistant Streptococcus pneumoniae), isoniazid / rifampin-resistant Mycobacterium tuberculosis, as well as other antibiotic-resistant strains of Escherichia coli, Salmonella, Campylobacter, and Streptococcus.

[0161] In another aspect, the invention relates to a non-naturally occurring polypeptide or composition as described herein for use as a medicament, preferably for the treatment of a microbial infection.

[0162] In particular, the present invention is directed to the use of a polypeptide or composition as described herein for the preparation of a medicament, preferably for the treatment of a microbial infection.

[0163] The present invention also provides a method for treating a microbial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide or composition described herein.

[0164] The present invention further relates to (i) a non-naturally occurring polypeptide of the invention and (ii) glucose or a glucose source and / or a halide or pseudohalide as described above, preferably as a combined preparation for simultaneous, separate or sequential use as a medicament for the treatment of a microbial infection.

[0165] Generally speaking, the term "treatment" or "treating" means obtaining a desired physiological or pharmacological effect according to the severity of or risk for the symptom or disorder of interest, i.e., as used herein, the severity of a microbial infection or the risk for developing such a symptom or disorder. The effect may be prophylactic, with respect to partial or complete prevention of the symptom or disorder, and / or therapeutic, with respect to partial or complete cure of the symptom or disorder. The term "prophylactic" is characterized by the ability to avoid or minimize the onset or development of a symptom or disorder before its onset (e.g., after exposure to a microorganism, but before the onset of associated symptoms). The term "therapeutic" refers to the ability to inhibit (i.e., prevent the onset of) a symptom or disorder and / or alleviate (i.e., regression leading to improvement) said symptom or disorder. In the context of the present invention, a preventive effect is generally said to be achieved, for example, when an asymptomatic subject exposed to a microorganism remains asymptomatic or quasi-asymptomatic after treatment according to the present invention (e.g., no development of infection), whereas a therapeutic effect is typically said to be achieved, for example, when a symptomatic subject infected with a microorganism recovers after treatment according to the present invention (e.g., partial or complete alleviation of infection).

[0166] Due to their broad spectrum of activity, the polypeptides or compositions of the present invention may be advantageously used in the treatment of polymicrobial infections. Polymicrobial diseases involve multiple infectious agents and may include complex, combined, mixed, dual, secondary, synergistic, simultaneous, polymicrobial, or superinfections. Polymicrobial diseases include, for example, infections associated with abscesses, AIDS-related opportunistic infections, conjunctivitis, gastroenteritis, hepatitis, multiple sclerosis, otitis media, periodontal disease, respiratory diseases, and genital infections.

[0167] The polypeptides or compositions of the present invention may also be advantageously used in the treatment of microbial infections that are resistant to conventional therapies. Examples of such infections include those associated with drug-resistant microorganisms, as described above.

[0168] As indicated above, treatment according to the present invention can be achieved by administering a therapeutically effective amount of a polypeptide or composition described herein to a subject in need thereof using any suitable administration regimen. By way of example, the administration can be carried out orally, intranasally, topically, transdermally, parenterally, or any combination thereof, depending on the type of infection the subject is suffering from. The route of administration is preferably designed to provide direct contact between the polypeptide or composition and the infecting microorganism. The dose and / or administration regimen can be readily determined and adapted by a skilled practitioner depending on the age, weight, and / or severity of the infection the subject is suffering from.

[0169] It is further to be understood that the "subject" to be treated in accordance with the present invention is preferably a human or animal, more preferably a human.

[0170] In a further aspect, the present invention provides a method for the treatment of a leukemia comprising administering to a subject the amino acid sequence: (LX1X2X3X4X5AX6A)m comprising or consisting of: X1 is glutamic acid or glycine; X2 is lysine or glycine; X3 is arginine or glycine; X4 is proline or empty; X5 is glutamic acid or glycine; X6 is glutamic acid or glycine, m is an integer ranging from 1 to 2, preferably 1; or a peptide linker which is a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

[0171] In a preferred embodiment, the peptide linker of the present invention comprises or consists of any one of the following amino acid sequences: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA)2 (SEQ ID NO: 6), LEKREAEA (SEQ ID NO: 7), or LEKRPEAEA (SEQ ID NO: 8), or is a polypeptide substantially homologous thereto, preferably derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions. Particularly preferred peptide linkers of the present invention are LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), and (LEKREAEA)2 (SEQ ID NO: 6).

[0172] Conservative substitutions are as described above.

[0173] The peptide linkers described above can be used in vitro to link molecules together, preferably by covalent bonds. Examples of such molecules are myeloperoxidase and glucose oxidase, as described herein.

[0174] The present invention will be better understood in view of the following detailed experiments. Nevertheless, those skilled in the art will understand that the examples are not limiting and that various modifications, substitutions, omissions, and changes can be made without departing from the scope of the present invention. [Example]

[0175] The goal of this study was to generate an artificial bifunctional enzyme capable of catalyzing the formation of (pseudo)underhalogenated compounds from glucose and (pseudo)halides. To achieve this, we selected the open reading frames of Rhodopyleurola baltica (Example 1) or human myeloperoxidase (Example 2) fused to glucose oxidase from Penicillium amagasakiens. Genetic engineering yielded two chimeras: one with a native peptide linker between the two enzymes and one without such a linker. The results demonstrate that the presence of this native linker is key to facilitating the activation of glucose oxidase in the chimera, allowing the formation of a microbicidal compound sufficient to kill the Escherichia coli strain used for antimicrobial resistance analysis. To understand the key properties of this native linker, several mutations were generated, and the sequence and length of the linker appeared to be important for the topology, oligomerization, kinetics, and microbicidal properties of the chimera.

[0176] Example 1 1. Materials and Methods 1.1. Construction of chimeric RbMPO-GOx lacking a peptide linker pPiczα-GOx penag GOx by vector penag PCR amplification of the ORF (Zhang, Biotechnology Advances, 2011; 29(6): 715-725) was performed to introduce two restriction sites, namely, NdeI and XhoI. The amplified DNA was then digested at these two sites to generate GOx. penag The digested fragment containing the ORF was ligated into the pET21a plasmid (digested with NdeI and XhoI).

[0177] The pEt21a-RbMPO-6His vector was ligated with XhoI to penag The two linearized vectors were used as megaprimers, and the following two external primers were inserted: RbMPO C-Ter and GOx penag(Table 4). The PCR product was then digested with DpnI for 2 hours at 37°C and purified. After a final ligation step, the new construct was transformed into DH5α. To confirm the validity of the chimeric construct, clones were evaluated by colony PCR using two primers that amplify the fusion region: GOx65rc and RbMPOH407A (Table 4).

[0178] The pET21a-RbMPO-GOx plasmid was obtained.

[0179] [Table 4]

[0180] 1.2. Construction of chimeric RbMPO-GOx with a peptide linker pPiczαGOx penag The vector was digested with the restriction enzyme XhoI, which is present at two positions on either side of the enzyme ORF.

[0181] The pET21aRbMPO-6His plasmid was digested with XhoI located at the C-Ter site just before the 6His tag. The linearized plasmid was then used for GOx penag The final chimeric construct was obtained by ligation with the ORF of RbMPO. In this construct, a nucleic acid sequence encoding the peptide linker LEKREAEA (SEQ ID NO: 7) is located directly 5' of the ORF of RbMPO. This construct was verified after transformation of DH5α bacteria by performing colony PCR using the oligonucleotides GOx65rc and RbMPOH407A.

[0182] The pET21a-RbMPO-LEKREAEA-GOx plasmid was obtained.

[0183] 1.3. Construction of chimeric RbMPO-GOx with alternative peptide linkers The chimeras were modified using Stratagene's Quick-Change kit or NEB Biolabs' Q5 site-directed mutagenesis kit using the primers in Table 5.

[0184] The pET21a-RbMPO-GOx vector (linker-free) was used as a matrix and sequences encoding 8 Gly (SEQ ID NO: 9) or LGKRGAGA (SEQ ID NO: 5) were added.

[0185] Using the pET21a-RbMPO-LEKREAEA-GOx vector as a matrix, the linker was exchanged for the sequence i) LEKREAEALEKREAEA (SEQ ID NO: 6), ii) LEGGEAEA (SEQ ID NO: 4) or iii) LEKRPEAEA (SEQ ID NO: 8).

[0186] As a result, the following five plasmids were constructed using modified linkers: pET21a-RbMPO-GGGGGGGG-GOx, pET21a-RbMPO-LGKRGAGA-GOx, pET21a-RbMPO-LEKREAEALEKREAEA-GOx, pET21a-RbMPO-LEGGEAEA-GOx, and pET21a-RbMPO-LEKRPEAEA-GOx was obtained.

[0187] [Table 5]

[0188] 1.4. Generation and Purification of Chimeras Each pET21a vector expressing the chimera was transformed into E. coli BL21Star(DE3), and bacterial clones were grown on LB agar ampicillin and chloramphenicol nutrient medium. 1 L of each was incubated with 10 mL of a pre-culture performed the previous day and grown at an OD of 0.5-0.8 until bacterial growth reached the logarithmic phase. 600nmThe activity of the chimera was measured. Induction with 500 μM IPTG was used to induce transcription and translation of the chimera. After 24 hours at 22°C, the culture was precipitated. The bacteria were disrupted at 2200 bar (4°C), and the bacterial pellet was washed with a solution of 2 M urea, 50 mM Tris, 5.5 mM CaCl2, pH 7.5. Treatment of the pellet with a solution of 8 M urea, 50 mM Tris, 5.5 mM CaCl2, pH 7.5 at 4°C for 4 hours was required to resolubilize all the protein, resulting in the production of a chimera with a size of 142088.88 Da in bacterial inclusion bodies. The supernatant collected after centrifugation was titrated into reconstitution solution (20 mM Tris pH 7.5, 5.5 mM CaCl, 5 μM hemin, 200 μM FAD, 1 mM oxidized glutathione, 1 mM reduced glutathione, and 10% v / v glycerol) for 5, 8, or 15 days. The mixture was first concentrated in a concentration cassette (Sartorius PES Cassettes Vivaflow 200 with a 30 kDa cutoff) and then in a 30 kDa Amicon (Merck-Millipore Amicon Ultra 15, PLHK, membrane Ultracel-PL) to obtain a volume of less than 13 mL (first experimental setup) or 7.5 mL (second experimental setup).

[0189] In the first experimental setup, the resulting protein solution was injected onto a size-exclusion column (Hiload 26 / 600 200pn Cytiva: fractionation range 10,000–600,000 Da). After isocratic elution (50 mM Tris, 5.5 mM CaCl pH 7.5), the peak containing the chimera was concentrated. The column was calibrated with Biorad gel filtration standards. This step allowed for the separation of each chimera in various oligomeric states (n≧1) and further characterization of each oligomeric state.

[0190] In a second experimental setup, the resulting protein solution was desalted on a PD10 column equilibrated with Tris 50 mM pH 7.5, then lyophilized and stored in lyophilized form at 4° C. This allowed the isolation of all the chimeras in their various oligomeric states in a single mixture, allowing further characterization of said mixture.

[0191] 1.5.UV Spectrum The UV spectrum of each chimera was evaluated using a spectrophotometer scanning protein samples (100-fold diluted) from 800 to 200 nm. A theoretical epsilon of 144285 M / cm and the Beer-Lambert law allowed for the determination of the enzyme concentration after purification.

[0192] 1.6.SDS-PAGE Mini-PROTEAN® Precast Gels (Bio-Rad) were used. These gels were characterized by a 4-15% acrylamide gradient. Protein samples to be analyzed were homogenized with Filler Blue 4x (containing SDS and β-mercaptoethanol) and then heated to 95°C for 5 minutes. Proteins were run according to molecular weight using a denaturing running buffer (SDS) and an amperage of 35 mM per gel. After running, the gels were stained in Coomassie Blue solution for 3 hours and then subjected to a time-dependent bleaching step using a 10% acetic acid / 30% ethanol solution.

[0193] 1.7. Glucose oxidase activity of chimeras The glucose oxidase activity of the chimera was evaluated using a D-gluconic acid / D-glucono-δ-lactone kit from Megazyme. The previous day, a solution containing 5 μM chimera, 250 mM glucose, and 50 mM NaPi pH 6 was incubated at 37°C. The next day, 15.7 μL of this solution was taken and the GOX activity of the chimera was analyzed. penag The activity of the enzyme was measured at 340 nm, which corresponds to the wavelength of the final product of the kit, i.e., NADPH. The concentration of D-gluconic acid produced by the chimera after overnight reaction was calculated using the following relationship:

[0194]

number

[0195] In the formula, ε グルコン酸 =6300M-1 .cm -1 and the molecular weight of the same compound was 196.1 g / mol.

[0196] 1.8. Myeloperoxidase activity of chimeras Use chloride as a substrate The fluorescent probe aminophenylfluorescein (APF) (λ 励起 = 485 nm and λ 発光 = 525 nm) was used to measure HOCl production by the chimera as a measure of chlorination activity. Three tests were performed: (1) a fixed concentration of chimera (1 μM) and NaCl (20 mM) at various glucose concentrations (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, and 20 mM); (2) a fixed concentration of chimera (1 μM) and glucose (20 mM) at various NaCl concentrations (0, 10, 20, 50, 100, 250, 500, 750 μM, and 1 mM); and (3) a fixed concentration of chimera (1 μM) and NaCl (20 mM) at various HO concentrations (0, 0.01, 0.02, 0.05, 0.079, 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, 200, and 500 mM). In all experiments, 10 μM APF was used in each assay with 20 μL of 50 mM NaPi buffer, pH 7.5, in a 384-well plate. Each experiment was performed in triplicate. Kinetic analysis was performed for each range of values ​​from 10 to 30 minutes, and the rate of increase in fluorescence intensity was compared to the standard fluorescein range. Thus, the measured fluorescence per second was proportional to the amount of fluorescein, which in turn was proportional to the amount of HOCl produced by the chimera.

[0197] Steady-state kinetic parameters were determined by fitting the experimental data to the following equation according to the appearance of the saturation curve:

[0198]

number

[0199]

number

[0200] Eq.3: k ss =k2*[S]+b where k corresponds to the steady-state rate constant (initial rate divided by chimera concentration), kcat is the catalytic constant (maximum velocity divided by chimeric concentration) [S] is the concentration of the substrate (glucose or NaCl), K M is the Michaelis constant corresponding to the concentration of substrate (glucose or NaCl) at which 50% of the maximum velocity is observed, Ki is the inhibition constant, by which inhibition by excess substrate (glucose or NaCl) is observed; k2 is the slope where no saturation is observed within the concentration range of the substrate tested.

[0201] Uses thiocyanate as a substrate Initial velocity measurements under steady-state conditions were performed using a BioLogic SFM400 stopped-flow centrifuge in single-mixing mode with a Xe / Hg lamp and TC100 / 10 cells. One syringe was filled with the chimera, and the other syringes were filled with glucose and NaSCN. The concentrations of the chimera and glucose were fixed (300 nM and 50 mM, respectively), and measurements were performed at 37 °C and 240 nm. - The apparent concentration of OSCN (hypothiocyanite) was measured at 240 nm and 37°C in 50 mM sodium phosphate buffer, pH 6. For each value of the NaSCN substrate range, at least five 120 s exposures were taken with 2000 points per interval using the following sample intervals: 20 μs, 20 ms, and 20 m. The slope was calculated from the average exposures for each concentration and expressed as k ss (s -1 ) was determined using the slope according to the following formula:

[0202]

number

[0203] - The molar extinction coefficient of OSCN is ε -OSCN =951M -1 .s -1 The collected data was analyzed using Origin software. ss The values ​​were fitted with the Michaelis-Menten equation.

[0204] 1.9. Microbicidal Activity and Stability of Chimeras A glycerol stock of E. coli ATCC 25922, the recommended strain for antibiogram / antimicrobial activity evaluation of compounds of interest, was deposited on a Petri dish containing tryptic soy broth (TSB) agar medium for 16 hours at 37°C. 10 mL of TSB medium was inoculated with an isolated bacterial colony, and the preculture was stirred overnight at 190 rpm and 37°C. 25 mL of TSB medium was inoculated into the preculture in a volume required to obtain an OD620nm of 0.09. When the latter reached the logarithmic phase, a bacterial dilution (in TSB) of 10 mL was performed to obtain a bacterial load of 2.10. 6 CFU / mL yielded 1 OD620nm of 1.10 8 Microbicide tests were performed in triplicate for each experimental condition in 96-well microplates (Greiner Bio-One™, Cellstar™ μclear™, white, flat bottom) with a final volume of 100 μL per well. In each well, 50 μL of bacteria were added at a concentration of 1.10 for all conditions. 6 The plates were inoculated to give CFU / mL. OD620nm was measured every 15 minutes for 16 hours using a Wallac Victor2 1420 microplate reader or a SpectraMax® Paradigm® (Molecular Devices) in a 37°C constant temperature atmosphere with shaking between readings. The stability of the chimeras was also assessed by repeating these experiments after several days of enzyme storage under identical conditions at 4°C.

[0205] 2.Results 2.1. Construction of RbMPO-GOx chimera After the bacterial transformation step, several clones were selected and examined by colony PCR to verify the presence of the chimeric construct using primers GOx65rc and RbMPOH407A for chimeras without a peptide linker or the primers listed in Table 5 for chimeras with a peptide linker. Sequencing confirmed the presence of the complete chimeric sequence of each construct.

[0206] 2.2. Generation and Purification of RbMPO-GOx Chimera In the first experimental setup, all chimeras were purified 8 days after reconstitution with heme and FAD, cofactors for RbMPO and GOx, respectively. Approximately 40 mg of protein was obtained per liter of bacterial culture for each chimera. Depending on the linker peptide sequence, one or more peaks were detected during the purification process. Size-exclusion chromatography was performed, so the first peak corresponded to molecules with a larger molecular weight than those contained in later peaks. Free FAD molecules and other components of the reconstitution solution were eluted with the total volume of the S200 column. Calibration with standard proteins allowed the determination of the oligomeric state (n ≥ 1) of the chimera based on the elution volume used to calculate the partition coefficient. A total of seven chimeras were purified with or without peptide linkers, and these chimeras were analyzed and tested in various oligomeric states according to their elution volume and purity. These oligomeric states are summarized in Table 6 below. In parallel, SDS-PAGE analysis indicated that the various peaks corresponded to pure chimeras, as indicated by a band with a molecular weight of approximately 150 kDa.

[0207] UV spectra were obtained for each chimera, including that of free heme (data not shown). The spectrum of free heme (RbMPO cofactor) exhibits two major peaks, one at 385 nm and the other at 615 nm. In the chimera, the fixation of heme in the active site of RbMPO can be evidenced by the shift of these two peaks at 412 nm and 637 nm, respectively. The peak at 280 nm is characteristic of the amino acids tyrosine and tryptophan, components of RbMPO and GOx.

[0208] The following information is available in the spectrum: z

[0209]

number

[0210] ratio

[0211]

number

[0212] The mass percentage of each chimera in each oligomeric state could be estimated from the ratio. Rz indicates the presence of heme in the chimera.

[0213]

number

[0214] can indicate the purity of the chimera relative to the DNA and / or the presence of FAD in the active site of GOx. These parameters are also summarized in Table 6.

[0215] The apparent molecular weight (MWapp) of the chimeras was calculated by the ratio of their elution volumes (and then their partition coefficients, K AV ) and the calibration curve of the S200 column. These apparent oligomeric states were then determined by MW app The MW of the monomer (MW 単量体 The relative amounts obtained for each chimera in each oligomeric state were determined using the concentration of each chimera, then the Beer-Lambert function from the UV-visible spectrum, the dilution factor of the chimera, and the molar extinction coefficient. See also Table 6.

[0216] [Table 6]

[0217] The most active chimeras have the monomeric state, the best Rz, and the highest ratio compared to other chimeras.

[0218]

number

[0219] For these reasons, it was thought to be RbMPO-LEGGEAEA-GOx.

[0220] In a second experimental setup, the chimera RbMPO-LEGGEAEA-GOx was produced and purified as a mixture (i.e., without separation of the oligomeric states). Approximately 151 mg of protein was obtained from 250 ml of bacterial culture. This is a significant production yield compared to the first experimental setup, which was performed using the chimera in its individual oligomeric states.

[0221] 2.3. Enzyme Characterization of Chimeras in Individual Activity States Two enzyme assays were performed to determine the activities of the two active sites independently of each other.

[0222] 2.3.1. Glucose oxidase activity GOx using the Megazyme kit penag The specific glucose oxidase activity of the chimera was independently characterized by measuring the production of D-gluconic acid by GOx (Figure 1). An increase in absorbance at 340 nm indicates the presence of NADPH, which provides indirect evidence of D-gluconic acid production. penag The incubation was carried out at pH 6, which is known to be optimal for A. 340nm A blank was generated by measuring the amount of NADPH produced. 340nm As shown in Table 7 below, an increase in ΔA 340nm Based on these values, it was possible to calculate the concentration of D-gluconic acid produced by the chimeras and determine their specific glucose oxidase activity.

[0223] [Table 7]

[0224] All chimeras exhibited glucose oxidase activity, although with varying efficiencies.

[0225] The most active chimera was RbMPO-LEKREAEA-GOx (2.463 ± 0.246 IU / mg), followed by monomeric RbMPO-LEGGEAEA-GOx (0.731 ± 0.031 IU / mg), trimeric RbMPO-GGGGGGGG-GOx (0.639 ± 0.155 IU / mg), trimeric RbMPO-LGKRGAGA-GOx (0.443 ± 0.01 IU / mg), and finally, trimeric RbMPO-LEKRPEAEA-GOx (0.268 ± 0.03 IU / mg).

[0226] Similarly, the chimeric RbMPO-LEGGEAEA-GOx produced as a mixture exhibited glucose oxidase activity, as production of D-gluconic acid from glucose was detected by the Megazyme kit at a level of 0.298±0.049 IU / mg.

[0227] 2.3.2. Myeloperoxidase activity Use chloride as a substrate To determine the RbMPO activity of the chimeras, a range of HO concentrations was performed with a fixed concentration of NaCl. HOCl reacted with the APF probe, and the appearance of fluorescein was monitored. The steady-state kinetic parameters are summarized in Table 8 below.

[0228] [Table 8]

[0229] All chimeras exhibited chlorination activity, although with varying efficiencies.

[0230] The most active chimera was the dimeric / monomer form of RbMPO-LEKREAEA-GOx, followed by the trimeric form. The dimeric / monomer form of this chimera indeed had a higher specificity constant (1508 ± 19.4 M) compared to the other chimeras. -1 .s -1 The catalytic efficiency of this chimera was significantly higher than that of RbMPO-LEGGEAEA-GOx (34.2 ± 19.4 M). -1 .s -1 ), and RbMPO-LEKRPEAEA-GOx (excluded) (6.82 ± 4.27 M -1 .s -1 ), whose inhibition by H2O2 was slightly higher (Ki = 17.3 ± 10.6 mM). RbMPO-GGGGGGGG-GOx (excepted) had a lower specificity constant (1 ± 0.6 mM). -1 .s -1 ), whereas inhibition by H2O2 was very low (Ki = 140.5 ± 65.6 mM). Finally, the chimeric RbMPO-GOx lacking the linker showed an average specificity constant (4.44 ± 3.15 mM). -1 .s -1 ).

[0231] Furthermore, the chimeric RbMPO-LEGGEAEA-GOx produced as a mixture of oligomers exhibited myeloperoxidase activity, as evidenced by the detection of HOCl with APF. With 15 mM glucose and 500 mM NaCl, the kss was approximately 10 -3 s -1 However, due to the heterogeneity of this protein, the individual kinetic parameters kcat, K M , and K i It was not possible to determine.

[0232] 2.4. Enzymatic Characterization of the Chimeric Binding Active Site Uses chloride and glucose as substrates The activity of the two active sites coupled together was then evaluated (Figure 1).

[0233] Briefly, the objective was to measure the production of HOCl from glucose and NaCl as the enzyme's respective substrates using the APF probe. Two types of kinetic analysis were performed: i) Kinetic analysis with various concentrations of glucose and a fixed concentration of NaCl; and ii) Kinetic analysis with various concentrations of NaCl and a fixed concentration of glucose was carried out.

[0234] The steady-state kinetic parameters are summarized in Tables 9 and 10 below.

[0235] [Table 9A]

[0236] [Table 9B]

[0237] [Table 10A]

[0238] [Table 10B]

[0239] All chimeras exhibited glucose and chlorination activity, albeit with varying efficiencies. The catalytic efficiencies of these oligomeric states toward glucose and NaCl are summarized in Figures 2A and 2B.

[0240] Briefly, on glucose, RbMPO-LEGGEAEA-GOx monomer 2 had the highest catalytic efficiency (2.73 ± 1.5 M -1 .s -1 This chimera was then followed by the RbMPO-LEKREAEA-GOx dimer / monomer (2.47 ± 1.58 M -1 .s -1), RbMPO-LEKREAEALEKREAEAGOx trimer (1.7 ± 1.34 M -1 .s -1 ), and the trimer of RbMPO-LGKRGAGA-GOx (1.1 ± 0.8 M -1 .s -1 ), but this inhibition was potent (Ki = 6.1 ± 1.2 mM). For all other chimeras, kcat / KM was 1M. -1 .s -1 Overall, the excluded peak appeared to have the lowest glucose oxidase activity in each chimera. Most importantly, the presence of the linker improved the catalytic efficiency towards glucose.

[0241] In chloride, the dimer / monomer of RbMPO-LGKRGAGA-GOx (2.4 ± 0.9 M -1 .s -1 ) had the highest catalytic efficiency, followed by the dimer / monomer of RbMPO-LEKREAEALEKREAEA-GOx (0.139 ± 0.076 M -1 .s -1 ) followed by the excised form of RbMPO-LGKRGAGA-GOx (0.020 ± 0.009 M -1 .s -1 ) followed. Other chimeras were also active, with 0.02M -1 .s -1 The catalytic efficiency of the RbMPO-GGGGGGGG-GOx dimer / monomer and trimer was inconclusive due to the large error bars. It is further noteworthy that the presence of the linker improved the catalytic efficiency toward chloride.

[0242] The chimeric RbMPO-LEGGEAEA-GOx produced as a mixture also exhibited glucose oxidase activity in addition to myeloperoxidase activity, and the k of this protein was 1.07 when initial substrate concentrations of 15 mM glucose and 500 mM NaCl were used. ss / [glucose] value is 10 -3 M -1 .s -1 It was.

[0243] Taken together, these results confirm the positive impact of peptide linkers on enzyme catalytic efficiency. Moreover, it is possible to use mixtures of such oligomeric states to avoid the variations in catalytic efficiency observed with chimeras of different oligomeric states.

[0244] Uses thiocyanate and glucose as substrates Finally, although RbMPO can catalyze a wide range of (pseudo)halides, including not only chloride but also iodide, bromide, and thiocyanate (publication in preparation), the myeloperoxidase activity of some chimeras was assessed using thiocyanate as a substrate in the presence of a fixed concentration of glucose.

[0245] [Table 11]

[0246] These results indicate that the peptide linker is also suitable for efficient catalysis of thiocyanate and glucose.

[0247] 2.5. Microbicidal Activity and Stability of the Chimeras Microbicidal experiments were then performed using the chimeras that appeared to exhibit the most efficient catalytic activity toward glucose and chloride: RbMPO-LEGGEAEA-GOx, RbMPO-LGKRGAGA-GOx, and RbMPO-LEKREAEALEKREAEA-GOx. RbMPO-LEGGEAEA-GOx and RbMPO-LGKRGAGA-GOx were also of interest because they were less inhibited by HO. Finally, various oligomeric states of these chimeras, as well as a mixture of oligomeric states (RbMPO-LEGGEAEA-GOx alone), were evaluated.

[0248] As shown in Figures 3A-3N, all of these chimeras inhibited bacterial growth in the presence of either NaSCN or NaCl. Interestingly, this microbicidal effect was observed after the chimeras were stored at 4°C for 4 to 21 days, demonstrating the stability of these chimeras.

[0249] 3. Conclusion In this study, various fusions of the GOx and MPO enzymes were constructed and produced in suitable bacterial strains. To achieve this, an 8-day reconstitution step in the presence of FAD and heme prosthetic groups was required. Purification yields were high, ranging from approximately 40 mg (individual oligomers) to approximately 604 mg (mixture of oligomers) of pure chimeric enzyme per liter of bacterial culture.

[0250] The first chimeric protein was generated by directly fusing the C-terminus of the MPO enzyme to the N-terminus of the GOx enzyme.

[0251] Next, we genetically engineered various peptide linkers between the two enzymes. These linkers exhibited different properties regarding amino acid charge, amino acid length, degree of amino acid flexibility, and / or tertiary structure. To achieve this, we generated chimeras containing the peptide linker LEKREAEA or its mutant variants (introduction of proline into the middle of the peptide, mutation of one or more positively or negatively charged amino acids to neutral glycines, mutation of every amino acid to neutral glycines, or repeats of the peptide linker). Several oligomeric states (n≧1) of these chimeras were identified and purified for further analysis. Additionally, a single chimera was produced as a mixture of oligomeric states to reduce production costs and time.

[0252] The presence of the peptide linker improved the enzymatic activity compared to the chimera lacking the linker, particularly at the level of GOx activity and also towards NaCl (chlorinating activity, which is myeloperoxidase activity).

[0253] The microbicidal properties of the most active chimeras were then investigated, and this confirmed their microbicidal activity either in the individual oligomeric states or in the form of mixtures thereof, as well as their stability.

[0254] Example 2 Experiments similar to those performed in Example 1 were performed to generate chimeric constructs in which myeloperoxidase from human origin was fused to glucose oxidase from Penicillium amagasakiens. Again, genetic engineering yielded two chimeras: one with the native peptide linker between the two enzymes and one without such a linker.

Claims

1. A non-naturally occurring polypeptide having myeloperoxidase activity and glucose oxidase activity.

2. 2. The polypeptide of claim 1, which is a fusion polypeptide comprising myeloperoxidase linked, preferably covalently linked, to glucose oxidase.

3. 3. The polypeptide of claim 2, wherein the C-terminus of myeloperoxidase is linked, preferably covalently, to the N-terminus of glucose oxidase.

4. 4. The polypeptide of claim 2 or 3, wherein the myeloperoxidase is linked, preferably covalently, to the glucose oxidase by a linker, preferably a peptide linker.

5. The linker has the following amino acid sequence: (LX 1 X 2 X 3 X 4 X 5 AX 6 A)m comprising or consisting of: X 1 is glutamic acid or glycine, X 2 is lysine or glycine, X 3 is arginine or glycine, X 4 is proline or empty, X 5 is glutamic acid or glycine, X 6 is glutamic acid or glycine, m is an integer between 1 and 2, or or a peptide linker which is substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

6. The peptide linker has the following amino acid sequence: LEGGEAEA (SEQ ID NO: 4), LGKRGAGA (SEQ ID NO: 5), (LEKREAEA) 2 6. The polypeptide of claim 5, comprising or consisting of any one of SEQ ID NOs: 4 to 8, preferably a peptide substantially homologous thereto, derived from any one of SEQ ID NOs: 4 to 8 by one or more conservative substitutions.

7. 5. The polypeptide of claim 4, wherein the linker is a peptide linker comprising or consisting of a polyglycine amino acid sequence or a peptide substantially homologous thereto, preferably derived from said sequence by one or more conservative substitutions.

8. 8. The polypeptide according to any one of claims 1 to 7, wherein the myeloperoxidase is a microbial myeloperoxidase, preferably a myeloperoxidase from Rhodopyrella baltica, or a mammalian myeloperoxidase, preferably a myeloperoxidase from human origin.

9. 9. The polypeptide according to any one of claims 1 to 8, wherein the glucose oxidase is a microbial glucose oxidase enzyme, preferably a glucose oxidase from Penicillium amagasakiens.

10. 10. The polypeptide of any one of claims 1 to 9, in the form of a functional oligomer or a mixture of functional oligomers.

11. A nucleic acid encoding a polypeptide according to any one of claims 1 to 10.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the vector of claim 12.

14. A method for obtaining a polypeptide according to any one of claims 1 to 10, comprising: a) culturing the host cell of claim 13 in a culture medium under conditions suitable for expression of the polypeptide; b) recovering the polypeptide; The method includes at least

15. 15. The method of claim 14, wherein the polypeptide recovered in step b) is further solubilized in a solution containing heme and flavin adenine dinucleotide (FAD) and optionally calcium.

16. 11. An antibacterial composition comprising the non-naturally occurring polypeptide of any one of claims 1 to 10.

17. 17. The composition of claim 16, further comprising glucose or a source of glucose and / or a halide or pseudohalide.

18. 18. In vitro use of a non-naturally occurring polypeptide according to any one of claims 1 to 10 or a composition according to claim 16 or 17 to halogenate a non-halogenated organic compound.

19. 18. In vitro or ex vivo use of a non-naturally occurring polypeptide according to any one of claims 1 to 10 or a composition according to claim 16 or 17 for killing or inhibiting the growth of microorganisms.

20. 18. A non-naturally occurring polypeptide according to any one of claims 1 to 10 or a composition according to claim 16 or 17 for use as a medicament, preferably for the treatment of a microbial infection.

21. 18. A method for treating a microbial infection comprising administering to a subject a non-naturally occurring polypeptide comprising administering to said subject a composition comprising: (i) a non-naturally occurring polypeptide according to any one of claims 1 to 10 or a composition according to claim 16 or 17; and (ii) glucose or a source of glucose and / or a halide or pseudohalide, preferably as a combined preparation for simultaneous, separate or sequential use as a medicament for the treatment of a microbial infection.

22. 7. A peptide linker as defined in claim 5 or 6.

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