Novel N-glycosidase with high expression and high activity, production method and application thereof

Abstract

The present invention relates to a novel fusion polypeptide comprising a polypeptide having N-glycosidase activity fused to an antioxidant moiety wherein the polypeptide having N-glycosidase activity is a polypeptide from a bacterium belonging to the phylum of Acidobacteria, preferably belonging to the class of Acidobacteria. The invention also relates to nucleic acid molecules, vectors and host cells encoding the fusion polypeptides, as well as compositions and kits thereof. The invention also provides methods for purification and / or for production of the fusion polypeptides, as well as in vitro uses of the fusion polypeptides or nucleic acid molecules, vectors or host cells encoding the fusion polypeptides or compositions thereof.

Description

Technical Field

[0001] This invention relates to the fields of enzymology and enzyme production, and more specifically to N-glycosidases, as well as protein analysis and deglycosylation. In particular, this invention relates to a novel fusion polypeptide comprising a polypeptide with N-glycosidase activity fused to an antioxidant moiety, wherein the N-glycosidase-active polypeptide is derived from the phylum Acidobacteria (…). Acidobacteria ) bacterial polypeptides.

[0002] The present invention also relates to nucleic acid molecules encoding the fusion polypeptide, vectors and host cells, as well as compositions thereof and kits thereof.

[0003] The present invention also provides methods for purifying and / or producing the fusion polypeptide, and in vitro uses of the fusion polypeptide or nucleic acid molecules, vectors or host cells encoding the fusion polypeptide or combinations thereof. Background Technology

[0004] N-glycosidases (also known as peptide N-glycosidases, or peptide-N4-(N-acetyl-β-glucosamine)asparagine amidases, or PNG enzymes) are a class of enzymes that catalyze the chemical or biochemical reaction of deglycosylation of peptides or polypeptides. This reaction involves cleaving the link between N-acetylglucosamine (GlcNAc) residues and asparagine residues from glycoproteins, glycosyl polypeptides, and glycopeptides. Deglycosylation produces deaminoated proteins or peptides and free polysaccharides.

[0005] PNG enzyme deficiency leads to several diseases, most of which involve the nervous system (including lysosomal storage diseases and multisystem diseases). Furthermore, glycosylation alterations are commonly acquired in cancer and inflammatory cases, which can have important functional consequences. Indeed, N-linked glycans provide structural components of the cell wall and extracellular matrix to modify protein stability and solubility to guide the transport of other glycoproteins and mediate cell signaling (cell-cell interactions and cell-matrix interactions). N-linked (de)glycosylation plays a crucial structural and functional role in various components such as antibodies, cell surface proteins, and matrix proteins.

[0006] PNG enzymes are also very useful biotechnological tools. In fact, the removal of carbohydrates from glycoproteins may be necessary for many applications, leading to the following advantages: - Simplify the analysis of the peptide portion of glycoproteins; - Simplify the analysis of polysaccharide components; - Remove heterogeneity in glycoproteins for X-ray crystallography analysis; - Remove carbohydrate epitopes from antigens; - Enhances or reduces the blood clearance rate of glycoprotein therapeutic agents; - To study the role of carbohydrates in enzyme activity and solubility; - Study ligand binding; - Allow for quality control of glycoprotein drugs; etc.

[0007] In particular, several protein analysis methods require prior deglycosylation. For example, mass spectrometry or electrophoresis analyses are often complicated by the presence of glycosylated peptides and polypeptides, and are far less efficient than their unglycosylated counterparts.

[0008] Both PNG enzymes are commercially available. Peptide: N-glycosidase F, commonly known as PNG enzyme F, is an amidase belonging to the peptide-N4-(N-acetyl-β-glucosamine)asparagine amidase class. PNG enzyme F can perform deglycosylation without denaturation, but requires long incubation and relatively large amounts of the enzyme to cleave native proteins. Additionally, PNG enzyme F cannot cleave the core α1,3-fucosylated glycan from glycoproteins.

[0009] PNG enzyme A is an amidase with properties similar to PNG enzyme F. The main difference between PNG enzyme A and PNG enzyme F is that PNG enzyme A activity is not inhibited by α1-3 fucose residues. Additionally, PNG enzyme A has difficulty cleaving large N-glycans containing sialic acid.

[0010] PNG enzymes A and F are active within a pH range of 6–9.5. The optimal pH for the activity of these PNG enzymes is approximately 7.5. Therefore, the main limitation and drawback of these commercially available PNG enzymes is that they are inactive at acidic pH conditions, thus hindering their use in protein analysis techniques that require acidic conditions, such as proteins (including antibodies) eluted by dissociation reagents in immunopurification.

[0011] Another limitation is that their buffers often contain additives that are harmful to most protein analysis techniques, including mass spectrometry.

[0012] Another PNG enzyme, PNG enzyme H+, has been described. PNG enzyme H+ is a type of enzyme derived from the bacterium *Pink Earth* (*Pink Earth*). Terriglobus roseus The N-glycosidase isolated from PNG enzymes maintains its activity under acidic conditions (Wang et al., 2014 (doi:10.1042 / BSR20140148)). However, the expression level and stability of PNG enzyme H+ are low and incompatible with large-scale production (Guo et al., 2020 (doi: 10.3389 / fbioe.2020.00741)). Furthermore, the cell lysis conditions and buffers required for the protocol used to produce and purify this enzyme are not optimal for enzyme activity and are detrimental to many protein analysis techniques.

[0013] Therefore, there is a need to provide novel enzymes that exhibit PNG enzyme activity at low pH levels to allow for industrially applicable production levels and PNG enzyme activity. Summary of the Invention

[0014] This invention addresses this need. In fact, the inventors have designed a novel enzyme capable of efficiently catalyzing deglycosylation under extremely acidic conditions. Furthermore, the inventors have demonstrated for the first time that a combination of a polypeptide with PNG enzyme activity and an antioxidant moiety leads to a significant increase in PNG enzyme expression and production yield, as well as a significant increase in PNG enzyme stability and activity, not only at low pH but also at alkaline pH. The inventors have specifically developed several fusions of PNG enzyme-active polypeptides (derived from *Acidobacterium*) with compounds possessing antioxidant properties.

[0015] The inventors have further developed a new method for optimizing PNG enzyme production, as supported by experimental data, which results in ready-to-use PNG enzymes compatible with most biotechnological applications, particularly those requiring acidic conditions, and with improved long-term stability.

[0016] Therefore, this invention provides an original, stable, multifunctional and effective tool for deglycosylation of peptides, as well as an optimized and industrially applicable production method.

[0017] Therefore, the present invention relates to a novel fusion polypeptide comprising a polypeptide with N-glycosidase activity fused to an antioxidant portion, wherein the polypeptide with N-glycosidase activity is derived from a member of the acid bacterium (Acidobacterium tumefaciens). Acidobacteria Phylum, preferably belonging to the class Acidobacteria ( Acidobacteriia ) bacterial polypeptides.

[0018] The present invention also relates to nucleic acid molecules encoding fusion polypeptides, vectors and host cells, and compositions thereof.

[0019] The present invention also provides a novel method for purifying fusion peptides, comprising the step of recovering the fusion peptides in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid.

[0020] This invention also relates to a novel method for producing fusion peptides, comprising the following steps: a) Optionally clone nucleic acid molecules as defined above in a vector; b) Introduce the vector from step a) or the vector as defined above into the host cell; c) Under suitable conditions, grow the host cells from step b) to allow for the production of the fusion peptide; and d) Optionally, the fusion peptide of step c) is purified by recovering the fusion peptide in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid.

[0021] In addition, the present invention relates to a novel method for producing fusion peptides, comprising the following steps: a) Optionally clone a nucleic acid molecule encoding a polypeptide with N-glycosidase activity as defined above in a vector; b) Introduce the vector from step a) into the host cell; c) In the presence of the antioxidant fraction, the host cells from step b) are grown under suitable conditions to allow the production of a polypeptide with N-glycosidase activity, and the antioxidant fraction is fused with the produced polypeptide with N-glycosidase activity; and d) Optionally, the fusion peptide in step c) is purified by recovering it in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid.

[0022] The present invention further relates to the in vitro use of fusion peptides or nucleic acid molecules encoding such fusion peptides, vectors or host cells or compositions thereof in any biotechnological application involving deglycosylation.

[0023] The present invention also relates to a kit comprising a fusion polypeptide or a nucleic acid molecule encoding the fusion polypeptide, a vector or a host cell or a combination thereof. Detailed Implementation

[0024] In the context of this invention, the inventors have surprisingly discovered that fusing a polypeptide with N-glycosidase activity (particularly polypeptides from bacteria belonging to the phylum Acidobacteria, preferably the class Acidobacteria) with an antioxidant moiety (e.g., an antioxidant compound) significantly enhances the N-glycosidase (PNG) activity of the polypeptide. Furthermore, the inventors have demonstrated that the fusion of the PNG enzyme polypeptide with the antioxidant moiety results in a significant expansion of the pH range in which the PNG enzyme polypeptide is active. In fact, the data disclosed herein reveal that fused polypeptides containing both PNG enzyme polypeptides and antioxidant moieties exhibit high levels of PNG enzyme activity over a wide pH range (from alkaline to highly acidic conditions, with a pH range of at least 2 to 7.5).

[0025] Therefore, through this groundbreaking work, the inventors have provided for the first time a highly stable and multifunctional PNG enzyme polypeptide that can effectively deglycosylate targets under both extremely acidic and alkaline conditions without any loss of activity.

[0026] In particular, the data obtained by the inventors demonstrates for the first time that the antioxidant component 1) improves solubility and 2) stabilizes PNG enzymes.

[0027] Further data showed a significant increase in PNG enzyme expression levels. In fact, the inventors' experimental results revealed that the fusion of the PNG enzyme peptide with a moiety possessing antioxidant properties (such as peptides or polypeptides with antioxidant properties) not only increased PNG enzyme expression levels but also promoted PNG enzyme purification. Without being bound by any theoretical framework, these improvements can be attributed to improved folding and improved solubility, which are related to the antioxidant properties of the moiety and the inventors' optimized expression system. Indeed, the data highlighted that using expression cells containing genes encoding rare transfer RNAs (such as bacterial strains engineered to contain additional copies of genes encoding rare transfer RNAs) allowed for a further significant increase in PNG enzyme peptide expression levels.

[0028] Furthermore, the inventors have surprisingly devised a novel method for producing and / or purifying peptides with N-glycosidase activity (particularly peptides from bacteria belonging to the phylum Acidobacteria) using buffers containing formic acid (FA buffer) and / or glycine-HCl buffer. Data show for the first time that eluting PNG enzyme peptides with FA buffer (supplemented with glycine-HCl or not, or glycine-HCl buffer) allows for a significant increase in the production and purification rates of PNG enzyme peptides, particularly suitable for MALDI applications. Additionally, the use of FA buffer results in the acquisition of highly pure and stable PNG enzyme peptides. PNG enzyme peptides obtained using this original method can be used in any biotechnological application involving deglycosylation. In fact, data indicate that FA buffer is compatible with any protein analysis technique, especially those requiring acidic conditions. Furthermore, data show that FA buffer also improves the storage of PNG enzyme peptides. In fact, even after long-term storage (i.e., more than one month) in FA buffer, no loss of PNG enzyme activity was detected.

[0029] definition Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry, biochemistry, cell biology, molecular biology, and medicine.

[0030] As used throughout this text, the terms “a” and “an” are meant to refer to “at least one,” “at least first,” “one or more,” or “one or more” compounds or steps, unless the context otherwise requires.

[0031] When the term “and / or” is used in this document, it includes the meanings of “and”, “or”, and “all or any other combination of elements connected by the term”.

[0032] As used herein, the term “about” or “approximately” means within 10% of a given value or range, preferably within 8%, and more preferably within 5%, and more preferably within 3%, and even more preferably within 1%.

[0033] As used herein, when used to define products, compositions, cells, uses, and methods, the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include)”), or “containing” (and any form of containing, such as “contains” and “contain)”) are open-ended and do not exclude additional, unlisted elements or method steps. Thus, a polypeptide “comprising” an amino acid sequence is defined as such when the amino acid sequence may be part of the final (and / or complete) amino acid sequence of the polypeptide. Such polypeptides may have up to several hundred additional amino acid residues (e.g., linkers and antioxidant moieties as described herein). “Comprising” indicates the exclusion of any other components or steps. “Substantially comprising” indicates the exclusion of other components or steps of any significant importance (however, it does not exclude other minor / unimportant components or steps). In this disclosure, the terms “comprising,” “consisting of,” and “substantially composed of” may be used interchangeably if necessary.

[0034] The terms “obtained from,” “originating from,” “originate from,” or “of XXX” (where XXX is any source) are used to identify the original source of a component (e.g., a polypeptide or nucleic acid molecule) but do not imply limitation on the method of preparing the component, which can be, for example, by chemical synthesis or recombination methods (including replacing codons encoding amino acid residues).

[0035] The terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably herein and should be understood as a polymeric or oligomeric macromolecule made of nucleotide monomers (preferably of at least five nucleotide monomers, also referred to as nucleotide residues). A nucleotide monomer consists of a nucleobase, a pentose sugar (e.g., but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Nucleotide monomers may be chemically modified and / or enzymatically modified. Typically, polynucleotides are formed by phosphodiester bonds or thiophosphate bonds between individual nucleotide monomers. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids (mixed polyribose-polydeoxyribonucleotides). These terms encompass single-stranded or double-stranded, linear or circular, natural or synthetic, their unmodified or modified versions (e.g., genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, and chimeric mixtures (e.g., RNA-DNA hybrids). Furthermore, polynucleotides may contain nucleotides that are not naturally occurring and may be broken down by non-nucleotide components. Exemplary DNA nucleic acids include, but are not limited to, complementary DNA (cDNA), genomic DNA, plasmid DNA, DNA vectors, viral DNA (e.g., viral genomes, viral vectors), oligonucleotides, probes, primers, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof. Exemplary RNA nucleic acids include, but are not limited to, messenger RNA (mRNA), pre-mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), RNA vectors, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc. The polynucleotides described herein can be synthesized using standard methods known in the art, such as by using automated DNA synthesizers (e.g., those commercially available from Biosearch, Applied Biosystems, etc.), or by using molecular biology techniques well known in the art (e.g., cloning, PCR, etc.) from naturally occurring sources (e.g., genomes, cDNA, etc.) or artificial sources (e.g., commercially available libraries, plasmids, etc.). Nucleic acids can be chemically synthesized, for example, via the phosphate triester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0036] The terms “peptide” and “protein” are used interchangeably herein and refer to any polymer of amino acids linked by peptide bonds, regardless of its length or post-translational modifications. These terms preferably refer to polymers containing at least six amino acid residues covalently linked by peptide bonds. Polymers can be linear, branched, or cyclic. Polymers can contain naturally occurring and / or amino acid analogs and can be broken down by non-amino acid components. There is no limit to the maximum number of amino acids contained in a polypeptide. As a general indication, the term refers to both short polymers (often referred to in the art as peptides or protein fragments) and longer polymers (often referred to in the art as polypeptides or proteins). The term specifically covers native polypeptides, modified polypeptides (also referred to as derivatives, analogs, variants, mutants), polypeptide fragments, polypeptide multimers (e.g., dimers), mutant polypeptides, engineered polypeptides, and fusion polypeptides. A polypeptide should be understood as any translational product of a polynucleotide, regardless of its size or whether it is glycosylated, and includes both peptides and proteins. Polypeptides / proteins (including protein derivatives, protein variants, protein fragments, protein domains, protein epitopes, and protein domains) available herein can be further modified by chemical or enzymatic modification or by physical modification (e.g., physisorption). These chemically or enzymatically modified peptides contain chemical groups in addition to 20 naturally occurring amino acids. Examples of such chemical or enzymatic modifications include post-translational modifications (genetically or non-genetically encoded). Chemical or enzymatic modifications of peptides can provide advantageous properties compared to the parent peptide, such as enhanced stability, increased biological half-life, improved water solubility, enhanced activity, enhanced properties, labeling, etc. These physically modified peptides can be covalently or non-covalently fixed to a support.

[0037] As a general guideline, but not a limitation, if an amino acid polymer contains more than 50 amino acid residues, it is preferably referred to as a polypeptide or protein, and if the polymer consists of 50 or fewer amino acids, it is preferably referred to as a “peptide.” As used herein, the reading and writing conventions for the amino acid sequences of polypeptides, proteins, and peptides are conventional. The reading and writing convention for the amino acid sequences of polypeptides, proteins, and peptides is to place the amino terminus on the left and then write and read the sequence from left to right, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0038] The terms "peptide," "protein fragment," "part of a peptide or protein," or "protein domain" as used herein refer to a part of a peptide or protein, that is, a portion of the continuous amino acid sequence constituting said peptide or protein (referred to as the peptide or protein from which the fragment originates). When the fragment is a peptide, the fragment preferably contains at least 6 consecutive amino acids of the peptide or protein from which it originates; more preferably at least 8 consecutive amino acids, more preferably at least 10 consecutive amino acids, more preferably at least 12 consecutive amino acids, more preferably at least 15 consecutive amino acids, more preferably at least 20 consecutive amino acids, and more preferably at least 30 consecutive amino acids. When the fragment is a peptide or protein fragment, the fragment preferably has a three-dimensional structure under non-denaturing conditions (e.g., conditions generally non-denaturing for proteins, especially in the absence of denaturing and / or dissociating agents). When the fragment is a peptide or protein fragment, the fragment is preferably a functional fragment. "Functional fragment" means any peptide or protein fragment that has at least one original function and / or property of the peptide or protein from which said fragment originates. Preferably, the efficiency of the functional fragment in performing the function is equal to at least 30%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, preferably at least 100% efficiency. Examples of protein fragments (particularly functional fragments) include, for example, protein domains, protein epitopes, etc. Fragments available herein (including protein fragments, peptide fragments, protein domains, protein epitopes, and protein domains) can be further modified by chemical modification or enzymatic modification (e.g., one or more post-translational modifications). When the fragment is a peptide or protein fragment, the chemically modified / enzymatically modified fragment contains chemical groups other than the 20 naturally occurring amino acids (e.g., may contain one or more post-translational modifications).

[0039] The terms “amino acid,” “residue,” and “amino acid residue” are used interchangeably and cover both natural amino acids and amino acid analogs obtained by removing one molecule of water (e.g., non-natural amino acids, synthetic amino acids, and modified amino acids, including D-optical isomers or L-optical isomers).

[0040] As used herein, the term "fusion" refers to the linking (or conjugation, bonding, or coupling) of a polypeptide to a moiety. As used herein, the term "fusion polypeptide" refers to a molecule or compound comprising a polypeptide linked (or conjugated, bonded, or coupled) to a moiety. The terms "linking," "conjugation," "bonding," and "coupling" are used interchangeably herein. The moiety may be referred to as a "fusion chaperone" or a "fusion portion."

[0041] The connection can involve terminal coupling, internal coupling, lateral coupling, and any combination thereof. Furthermore, the fusion polypeptide of the present invention can comprise one or more connected portions.

[0042] The linkage can be covalent or non-covalent. It can be chemical, enzymatic, or achieved using genetic tools. Linkage can occur through any acceptable bonding mechanism. At this point, linkage can therefore occur via one or more cleavable or incleavable covalent, ionic, hydrogen, hydrophobic, or van der Waals bonds, either in the physiological medium or within the cell.

[0043] Furthermore, the linkage can occur on any reactive group of the polypeptide.

[0044] When the fusion is a genetic fusion, the fusion occurs via the covalent linkage of two or more polypeptides in a single polypeptide chain (which can be linear or non-linear (i.e., branched)) and is carried out genetically. In such cases, the linkage is performed by fusing the nucleic acid molecules encoding each said polypeptide within a frame. "In-frame fusion" means that the expression of the fused coding sequences produces a single polypeptide, and there are no translation terminators between each fused polypeptide.

[0045] The connection can be direct or indirect (i.e., via a linker). If there is no additional part or compound between the two fusion partners (peptide and part), the peptide is said to be "directly" fused to the part. For example, if there are no additional amino acid residues between the two fusion partners (peptide and part), the peptide is said to be directly fused to the part. If there is a linker between the two fusion partners (peptide and part), the peptide is said to be "indirectly" or "via a linker." Examples of linkers include, but are not limited to, affinity-based linkers (such as those derived from biotin-streptavidin interaction pairs or antibody-antigen interaction pairs) and covalent linkers (such as -SH / -NH2 groups, amine maleimide NHS-esters (such as N-hydroxysuccinimide 3-(maleimide)propionic acid), or compounds targeting the C-terminal carboxyl group of the peptide (such as N-hydroxysuccinimide), or peptides (such as peptides containing 3 to 50 (random) amino acid residues)).

[0046] The linker can be a peptide or polypeptide, for example, a peptide or polypeptide containing one or more amino acid residues, or consisting essentially of, or composed of.

[0047] When the linker is a peptide or polypeptide, its amino acid sequence can be designed to allow the fusion partner (i.e., the polypeptide and the part) of the fusion protein to fold correctly (in particular, to allow the fusion partner to retain its activity), and / or to allow cleavage between the two fusion partners (i.e., the polypeptide and the part) by a specific protease.

[0048] Technicians are able to assess whether a connector needs to be included between the two fusion couples.

[0049] The two fusion partners (i.e., the polypeptide and the moiety) can be fused in any order, such as the moiety being at the N-terminus of the polypeptide (“N-terminal fusion” or “N-terminal coupling” in this document means that the moiety is attached / linked / added at the N-terminus of the polypeptide, i.e., attached / linked / added to the N-terminal amino acid residue of the polypeptide), or the moiety being at the C-terminus of the polypeptide (“C-terminal fusion” or “C-terminal coupling” in this document means that the moiety is attached / linked / added at the C-terminus of the polypeptide, i.e., attached / linked / added to the C-terminal amino acid residue of the polypeptide), or the moiety being within the polypeptide. (In this document, "fusion within the polypeptide" or "internal fusion" or "internal coupling" means that the part is attached / linked / added within the amino acid sequence of the polypeptide, rather than at the C-terminus or N-terminus; that is, the part is inserted between two consecutive amino acid residues of the polypeptide, or the part replaces one or more consecutive amino acid residues of the polypeptide), or the part is a branch of the polypeptide (in this document, "lateral fusion" or "lateral coupling" means that the part is attached / linked / added to an amino acid residue of the polypeptide, rather than at a C-terminal amino acid residue or an N-terminal amino acid residue), and any combination thereof.

[0050] As used herein, “post-translational modification” refers to a chemical or enzymatic modification of a protein or protein fragment, whether naturally occurring or not, that occurs after or simultaneously with protein translation (e.g., biosynthesis or biochemical synthesis, e.g., using cellular mechanisms), or after or simultaneously with protein synthesis (e.g., artificial synthesis and / or chemical synthesis). This means that at least one naturally occurring amino acid of the protein or protein fragment is modified by the addition of at least one chemical group and / or by modification (including, but not limited to, removal) of at least one chemical group of the naturally occurring amino acid. Examples of such chemical or enzymatic modifications include, but are not limited to, glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, esterification, amidation, ubiquitination, sumoylation, deamination, etc. In this document, “post-translational modified protein” refers to a protein having at least one (i.e., one or more) post-translational modifications. In this document, “post-translational modified protein fragment” refers to a protein fragment having at least one (i.e., one or more) post-translational modifications.

[0051] When referring to proteins, the terms “activity,” “target activity,” or “function” refer to any biological activity of a protein screened using the methods of the present invention. Target activities include, in particular, enzyme activity, reporter activity (e.g., fluorescent activity), regulatory activity (e.g., transcription factor activity), and biological activity (e.g., pharmaceutical or antibiotic activity).

[0052] "Enzyme activity" refers to the specific catalytic activity of an enzyme.

[0053] In this article, "enzyme" refers to a protein that possesses catalytic properties (enzymatic properties). Almost all biomolecules capable of catalyzing chemical reactions in cells are enzymes; however, some catalytic biomolecules are made of RNA and are therefore different from enzymes: these are ribozymes. Enzymes function by lowering the activation energy of a chemical reaction, which increases the reaction rate. The enzyme is not modified during the reaction. The initial molecule is the enzyme's substrate, and the molecule formed from these substrates is the product of the reaction. Enzymes are characterized by their very high specificity. Furthermore, enzymes are reusable.

[0054] Enzymes are generally globular proteins that function individually or in complexes of several enzymes or subunits. Like all proteins, enzymes consist of one or more polypeptide chains that fold to form a three-dimensional structure corresponding to their native state.

[0055] Enzymes are molecules much larger than their substrates. Their size can range from about 50 residues to more than 2,000 residues. Only a small portion of an enzyme—most typically two to four residues, sometimes more—is directly involved in catalysis; this is known as the catalytic site (or catalytic domain). The catalytic site can be located near one or more binding sites, where the substrate binds and is directed to catalyze the chemical reaction. These catalytic sites and these binding sites form the active site of the enzyme.

[0056] Enzymes perform a wide variety of functions in living organisms. For example, they can participate in signal transduction and regulation of cellular processes, motility generation, active transmembrane transport, digestion, metabolism, the immune system, nucleic acid digestion or cleavage mechanisms or nucleic acid production (here referred to as "nucleic acid-acting enzymes"), and prodrug conversion mechanisms (conversion of prodrugs to drugs). Enzymes are preferably prokaryotic, eukaryotic, or viral enzymes, and more preferably derived from animals, plants, algae, microalgae, insects, microorganisms, bacteria, parasites, yeast, fungi, or viruses, and even more preferably mammalian enzymes, such as human enzymes. Various classes of enzymes are well known to those skilled in the art, and they can be referred to in particular by reference to works in the field (e.g., Schomburg D., Schomburg I., Springer Handbook of Enzymes. 2 edn. Heidelberg: Springer; 2001-2009; Liébecq C., IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN) and Nomenclature Committee of IUBMB (NC-IUBMB) Biochem. Mol. Biol. Int. 1997;43:1151-1156; IUBMB (1992), Enzyme Nomenclature 1992, Academic Press, San Diego); and specialized databases, such as those described in Schomburg D, Schomburg I. Methods Mol Biol. 2010;609:113-28. Enzymedatabases; especially the BRENDA database (which is available, in particular, at brenda-enzymes.org), for example, Chang A, Schomburg I, Placzek S, Jeske L, Ulbrich M, Xiao M, Sensen CW, Schomburg D, Nucleic Acids Res. January 2015; 43. Epub 2014 Nov 5. BRENDA in 2015: exciting developments in its 25th year of existence (as described in BRENDA in 2015: exciting developments in its 25th year of existence). The term enzyme should be considered to cover natural enzymes and their derivatives (e.g., mutant enzymes and / or engineered enzymes), provided that such derivatives are capable of enzymatic activity. As used herein, “enzyme fragment” means any part of an enzyme, preferably provided that such fragment / part is capable of enzymatic activity.In the case of proteases, the enzyme fragment preferably contains at least 6 consecutive amino acid residues of the enzyme (and preferably the enzyme catalytic site) (preferably at least 8 consecutive amino acid residues of the enzyme, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the enzyme).

[0057] In this article, "N-glycosidase," "peptide:N-glycosidase," "PNG enzyme," or "peptide-N4-(N-acetyl-β-glucosamine)asparagine amidase" refers to peptides possessing N-glycosidase activity. N-glycosidases belong to a class of enzymes that catalyze chemical or biochemical reactions involving the deglycosylation of peptides or polypeptides. As used herein, "deglycosylation" refers to a chemical or biochemical reaction involving the cleavage of the link between N-acetylglucosamine (GlcNAc) residues and asparagine residues from glycoproteins, glycopeptides, and glycopeptides. Deglycosylation reactions yield deaminated proteins or peptides and free polysaccharides.

[0058] In this document, “part” or “compound” refers to a chemical or biological entity, such as a molecule or a part / fragment thereof. The terms “part” and “compound” are considered synonyms and may be used interchangeably. Exemplary examples of parts or compounds are, but are not limited to, reagents, substrates, cosubstrates, cofactors, coenzymes, nucleic acid molecules (e.g., DNA, RNA, etc., see definition above), hormones, antigens, epitopes, ligands, antibodies, receptors, toxins, tags, etc.

[0059] As used herein, the term "antioxidant" refers to a molecule that slows down and / or prevents the oxidation of other chemicals. Oxidation is part of a redox reaction in which electrons are transferred from a substance to an oxidizing agent. This reaction can produce free radicals, which lead to destructive chain reactions. Antioxidants can stop these chain reactions by reacting with these free radicals and thus eliminating their effects. As used herein, the term "antioxidant portion" refers to the portion that has antioxidant properties (i.e., the portion that slows down and / or prevents the oxidation of other chemicals). Therefore, these terms include antioxidant compounds, antioxidant molecules, etc. Examples of antioxidant compounds include, but are not limited to: antioxidant peptides, antioxidant provitamins, antioxidant vitamins (such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), etc.), antioxidant flavonoids, compounds with thiol groups, compounds with phenolic groups, polyphenols, carotene, glutathione, lipoic acid, uric acid, tocopherols (such as α-tocopherol), panthenol (coenzyme Q), etc. Examples of antioxidant peptides include, but are not limited to: glutathione S-transferase, glutathione peroxidase, thioredoxin, catalase, superoxide dismutase, etc.

[0060] In this document, "bacteria" refers to tiny prokaryotic organisms present in the environment, belonging to the kingdom Bacteria. The term "bacteria" here specifically refers to both eubacteria and archaea (or archaea). In the context of this invention, bacteria are preferably those belonging to the phylum Acidobacteria.

[0061] In this article, "Acidobacteria phylum ( Acidobacteria The phylum Acidobacteriotaphylum is a branch of taxonomy that includes Gram-negative bacteria. This phylum includes, but is not limited to, several classes of bacteria, including: Acidobacteria, Blastocatellia, Holophagea, Polarisedimenticolia, Thermoanaerobaculi, Vicinamiibacteria, and Candidatus Acidiflorens. Acidobacteria is described in more detail on the NCBI website (e.g., using the following link: "https: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?mode=Undef&id=57723&lvl=3&lin=f&keep=1&srchmode=1&unlock").

[0062] The term “Acidobacteriia class” in this article refers to a branch of taxonomy (classification) that includes, but is not limited to, several orders of bacteria, including: Acidobacteriales, Acidoferralesorder, and Bryobacterales order.

[0063] In this article, "Acidobacteria" refers to a branch of taxonomy (classification), which includes, but is not limited to, several families of bacteria, among which is the Acidobacteriaceae family.

[0064] The term "Acidobacteriaceae" refers to a branch of taxonomy (classification) that includes, but is not limited to, several genera of bacteria, including *Acidicapsa* genus, *Acidipilia* genus, *Bryocella* genus, *Acidobacteria* genus, and *Bryocella* genus. Bryocella genus), genus *Tremella* ( Edaphobacter genus), genus *Lycium* ( Silvibacterium genus) and Earthbacteria ( Terriglobusgenus).

[0065] As used in this article, the term "Geobacterium" refers to a branch of taxonomy (classification) that includes, but is not limited to, several species of bacteria, including the pink Geobacterium species.

[0066] In this article, "Pink Earth Bacteria" ( Terriglobus roseus "Pink Earth" refers to a type of bacteria belonging to the Pink Earth species. Pink Earth ( T. roseus The bacteria are aerobic Gram-negative bacilli that lack motility.

[0067] The terms “formic acid” or “methanoic acid” are used interchangeably in this document and refer to the simplest carboxylic acid, which has the chemical formula HCOOH and the structure H−C(=O)−O−H.

[0068] In this article, the term "glycine" refers to an amino acid with a single hydrogen atom as a side chain. It is the simplest stable amino acid (carbamic acid is unstable), with the chemical formula NH₂-CH₂-COOH.

[0069] In this article, the term "hydrochloric acid" refers to an aqueous solution of hydrogen chloride (HCl).

[0070] In this article, "glycine hydrochloride" or "glycine-HCl" refers to a mixture of glycine and HCl, with the chemical formula NH2 ‐ CH2 ‐ COOH · HCl.

[0071] As used in this article, "transfer RNA" or "tRNA" refers to a linker molecule composed of RNA that acts as a physical link between the amino acid sequence of mRNA and a protein. Bacterial tRNA genes are typically (mean = 77.6 bp) shorter than those from archaea (mean = 83.1 bp) and eukaryotes (mean = 84.7 bp). Mature tRNAs follow the opposite pattern: bacterial tRNAs are typically (median = 77.6 nt) longer than those from archaea (median = 76.8 nt), while eukaryotes exhibit the shortest mature tRNAs (median = 74.5 nt).

[0072] The role of tRNA is to carry amino acids to the cell's protein synthesis mechanisms (ribosomes). The 3-nucleotide codon in messenger RNA (mRNA) is complementary to the 3-nucleotide anticodon of tRNA, leading to protein synthesis based on the mRNA codon. Therefore, the role of tRNA is to specify which sequence in the genetic code corresponds to which amino acid. A portion of tRNA matches the genetic code with a trinucleotide sequence called the anticodon. During protein biosynthesis, the anticodon forms three complementary base pairs with the codon in mRNA. The end of the tRNA is covalently attached to the amino acid corresponding to the anticodon sequence. Each type of tRNA molecule (determined by the anticodon sequence) can only attach to one type of amino acid; therefore, each organism has multiple types of tRNA. Because the genetic code contains multiple codons specifying the same amino acid, several tRNA molecules with different anticodons carry the same amino acid.

[0073] As used in this article, "rare tRNA" refers to tRNA that corresponds to rare codons (i.e., codons that are in short supply). For example, *E. coli* (… Escherichia coli The rarest codons in the spectrum are AGG and AGA, both of which encode arginine and have frequencies of only about 0.14% and 0.21%, respectively. argU The gene encodes a tRNA that reads both the AGA and AGG codons. Typically, this tRNA is produced in very small amounts in *E. coli*. If supplied on a plasmid (or integrated into the genome, for example, in several copies), this is a significant increase. argU In this case, the host *E. coli* no longer suffers from a tRNA shortage. Therefore, peptides or proteins with high levels of AGA and AGG codons (e.g., fusion peptides) are produced more efficiently. Plasmids carrying rare codon tRNAs for all genes are commercially available.

[0074] The term "antibody" ("Ab") is used in the broadest sense and encompasses both naturally occurring and engineered antibodies; including synthetic antibodies, monoclonal antibodies, polyclonal antibodies, and full-length antibodies and their fragments, variants, or fusions, provided that such fragments, variants, or fusions retain the binding property to the target protein. Such antibodies can be of any origin: human or non-human mammals (e.g., rodent or camel antibodies), or chimeric. Non-human antibodies can be humanized using recombinant methods to reduce their immunogenicity in humans. Antibodies can be derived from any well-known isotype (e.g., IgA, IgG, and IgM) and any subclass of IgG (IgG1, IgG2, IgG3, IgG4). Additionally, they can be glycosylated, partially glycosylated, or non-glycosylated. Unless the context otherwise requires, the term "antibody" also includes antigen-binding fragments of any of the above-described antibodies, including monovalent and bivalent fragments and single-chain antibodies. The term "antibody" also includes multispecific (e.g., bispecific) antibodies, provided they exhibit the same binding specificity as the parent antibody. The binding properties of candidate antibodies are within the skill level of those skilled in the art.

[0075] Various classes of antibodies are well known to those skilled in the art, who will be particularly able to refer to works in the field (e.g., Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, November 1, 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014; Bayer V., An Overview of Monoclonal Antibodies. Semin Oncol Nurs. September 2, 2019:150927; Wang W, Wang EQ, Balthasar JP. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther. 2008 Nov;84(5):548-58).

[0076] For illustrative purposes, a full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region consisting of three CH1, CH2, and CH3 domains (optionally with a hinge between CH1 and CH2). Each light chain contains a light chain variable region (VL) and a light chain constant region containing one CL domain. The VH and VL regions contain three hypervariable regions called complementarity-determining regions (CDRs), which are distributed across four conserved regions called frame regions (FRs) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and light chains are the determinants of binding specificity. As used herein, “humanized antibody” refers to a non-human (e.g., mouse, camel, rat, etc.) antibody whose protein sequence has been modified to increase its similarity to human antibodies (i.e., antibodies naturally produced in humans). Humanization methods are well known in the art and are typically carried out by replacing one or more residues in the FR region to make it resemble a human immunoglobulin sequence, while the vast majority of residues in the variable region (especially the CDR) are left unmodified and correspond to those residues in non-human immunoglobulins. A “chimeric antibody” comprises one or more elements of one species and one or more elements of another species, for example, a non-human antibody containing at least a portion of the constant region (Fc) of a human immunoglobulin.

[0077] The antibody is preferably a monoclonal antibody, and more preferably a humanized or chimeric antibody.

[0078] Representative examples of antibody fragments and / or regions are known in the art, including heavy (H) chain, light (L) chain, heavy chain variable region (VH), heavy chain constant region, CH domain, light chain variable region (VL), light chain constant region, CL domain, complementarity-determining region (CDR), constant region (Fc), Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, ds-scFv, diabody antibody, sdAb, etc.

[0079] As used herein, the terms "functional antibody fragment" or "antigen-binding fragment" refer to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments covered within the term "functional antibody fragment" are known in the art and include, but are not limited to: antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain fragment variable regions (scFv), fragment variable regions (Fv), V... H Structural domain, V LDomains, single-domain antibodies, scAbs (single-chain antibody fragments), dAbs, Fd, Fv, nanobodies, minibody, IgNARs (immunoglobulin neoantigen receptors), ds-scFv, di-scFv, bispecific T-cell binding agents (BITE), dual-affinity retargeting (DART) molecules, triple antibodies, bispecific antibodies, single-chain bispecific antibodies, alternative scaffold proteins and any combination thereof (e.g., their fusion proteins).

[0080] As used herein, the term “antigen” refers to any structure that is recognized and / or selectively bound by immune response molecules, such as antibodies, immune cell receptors (e.g., T-cell receptors (TCRs), B-cell receptors (BCRs), etc.). The term “antigen” as used herein refers to a natural or synthetic molecule that, when recognized by antibodies or cells of an organism’s immune system, can trigger an immune response in the organism. Antigens are recognized by highly variable antigen receptors (such as B-cell receptors or T-cell receptors) in the adaptive immune system and can elicit humoral or cellular immune responses. Antigens that elicit such responses are also called immunogens. Antigens can be foreign or toxic to the body, or they can be cellular molecules (e.g., proteins) associated with a specific disease. Antigens are typically peptides, proteins, carbohydrates (such as polysaccharides or polyosides), and their lipid derivatives (lipids). Antigens can also be nucleic acids or haptens (i.e., antigen fragments). Suitable antigens include, but are not limited to, biological components (e.g., peptides, polypeptides, post-translational modified polypeptides, and polynucleotides); complex components (e.g., cells, cell mixtures, living or inactivated organisms (e.g., bacteria, viruses, fungi, prions, etc.)); and combinations thereof. In the case of protein antigens, the antigenic portion recognized by the antibody or lymphocyte receptor is called an “epitope” or “antigenic determinant.” The same antigen can have several epitopes (identical or different) and thus induce different immune responses. Antigen recognition by lymphocytes depends on the nature of the epitope. B lymphocytes bind directly to conformational epitopes via their membrane immunoglobulins. T cells recognize linear epitopes presented by antigen-presenting cells. Antigens can be exogenous, i.e., foreign to the individual (in which case it can be allogeneic: from an individual of the same species; or xenogeneic: from another species), or they can be endogenous, i.e., host-specific antigens (self-antigens). Preferred antigens used herein are cancer / tumor antigens and pathogen antigens (the latter preferably selected from eukaryotic or prokaryotic pathogen antigens). The antigen is preferably a microbial, plant, algae, microalgae, bacteria, virus, parasite, yeast, fungus, insect, animal, cancer or tumor antigen; the antigen is preferably a protein, lipid or sugar antigen of bacteria, virus, parasite, yeast, fungus, cancer or tumor.Various classes of antigens are well known to those skilled in the art, who in particular may refer to works in the field (e.g., GJV Nossal, GL Ada, Antigens, Lymphoid Cells and the Immune Response, Academic Press, 1971; Marc HV VanRegenmortel, Structure of Antigens, Volume 3 CRC Press, December 20, 1995; Edouard Drouhet, Garry T. Cole, Louis De Repentigny, Jean Latge, Fungal Antitigens: Isolation, Purification, and Detection, Springer Science & BusinessMedia, November 11, 2013; Graziano DF, Finn OJ (2005) Tumor Antigens and Tumor Antigen Discovery. In: Khleif SN (ed.) Tumor Immunology and Cancer Vaccines. Cancer Treatment and Research, vol 123. Springer, Boston, MA; Wang M, Claesson MH, Methods Mol Biol.). 2014;1184:309-17. Classification of human leukocyte antigen (HLA) supertypes; and specialized databases, such as those described in Galperin, Fernández-Suárez, Rigden, The 24th annual Nucleic Acids Research database issue: a lookback and upcoming changes, NAR, Vol. 45, No. D1, January 2017, pp. D1-D11; particularly the PMAPP database, which is a database of human autoantigens (especially available at aagatlas.ncpsb.org). The term antigen should be considered to encompass natural antigens and their derivatives (e.g., mutant antigens and / or engineered antigens), provided that such derivatives can serve as targets for immune responses.As used herein, “antigen fragment” refers to any part of an antigen, preferably provided that such fragment / part can serve as a target for an immune response (e.g., epitope, immunogenic domain, etc.). In the case of protein antigens, the antigen fragment preferably comprises at least 6 consecutive amino acid residues of the antigen (preferably at least 8 consecutive amino acid residues, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues).

[0081] As used herein, “identity” or “sequence identity” refers to an exact sequence match between two polypeptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The “percentage identity” mentioned in the context of this disclosure is determined after optimal alignment of the sequences to be compared; therefore, optimal global alignment may include one or more insertions, deletions, truncations, and / or substitutions. An alignment is global, meaning it includes the entire length of the sequences to be compared. An alignment is “optimal,” meaning that the number of insertions, deletions, truncations, and / or substitutions is minimized. Optimal global alignment and percentage identity can be calculated using any sequence analysis method well known to those skilled in the art. In addition to manual comparison, global alignment can be determined using the Needleman and Wunsch algorithm (A general method applicable to the search for similarities in the amino acid sequence of two proteins, J. Mol. Biol., March 1970;48(3):443-453). For nucleotide sequences, sequence comparison can be performed using any software well known to those skilled in the art, such as Needle software. The parameters used may in particular be as follows: "Gap open" equals 10.0, "Gap extend" equals 0.5, and an EDNAFULL matrix (NCBIEMBOSS version NUC4.4). For amino acid sequences, sequence comparisons may be performed using any software well known to those skilled in the art, such as Needle software. The parameters used may in particular be as follows: "Gap open" equals 10.0, "Gap extend" equals 0.5, and a BLOSUM62 matrix. Preferably, the percentage identity as defined in the context of this invention is determined by a global sequence alignment comparing the entire length. For illustrative purposes, "at least 80% identity" herein means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0082] "Carrier" refers to a delivery system, preferably a nucleic acid molecule or viral particle, containing elements necessary to allow the administration, propagation, and / or expression of one or more nucleic acid molecules in a host cell or organism.

[0083] From a functional perspective, this term encompasses maintenance vectors (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids), or integration vectors (e.g., designed to integrate into the host cell genome and generate additional copies of the nucleic acid molecules they contain during host cell replication). The term also encompasses shuttle vectors (e.g., those that function in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., those used to transfer one or more nucleic acid molecules into the host cell genome).

[0084] From a structural perspective, the vector can be a natural, synthetic, or artificial genetic source, or a combination of natural and artificial genetic elements.

[0085] Therefore, in the context of this invention, the term "vector" should be understood broadly, including plasmids and viral vectors.

[0086] As used herein, "plasmid" refers to a replicable DNA structure. Typically, plasmid vectors contain selection marker genes that allow host cells carrying the plasmid to be identified and / or positively or negatively selected in the presence of a compound corresponding to that selection marker. Many types of positive or negative selection marker genes are known in the art. For example, antibiotic resistance genes can be used as positive selection markers, enabling selection of host cells in the presence of the corresponding antibiotic.

[0087] As used herein, the term "viral vector" refers to a nucleic acid vector, or a viral particle, that contains at least one viral genomic element and can be packaged within a viral particle. Viral vectors can be replicative or selective (e.g., designed to replicate better or selectively in one or more specific host cells), or genetically inactivated, making them replication-deficient or defective.

[0088] As used herein, the term "host cell" should be understood broadly and is not limited to a specific tissue, organ, or isolated cell. "Host cell" means a cell containing at least one polypeptide of the present invention, or at least one polypeptide obtainable by the production method of the present invention, or at least one vector of the present invention, or at least one nucleic acid molecule of the present invention, or at least one vector of the present invention, or any mixture of these substances. Advantageously, a host cell is a production cell, i.e., a cell capable of expressing one or more nucleic acid molecules (including one or more genes) encoded by the vector of the present invention and / or producing the vector of the present invention (i.e., a cell expressing one or more nucleic acid molecules (including one or more genes) encoded by the vector of the present invention and / or producing the vector of the present invention). This term also includes cells expressing polypeptides of the present invention, and cells that may be or are already recipients of nucleic acid molecules (or vectors containing said nucleic acid molecules) encoding polypeptides of the present invention, as well as progeny of such cells.

[0089] A host cell can consist of a single type of cell or a group of different types of cells. A host cell can also be a hybrid cell, that is, produced by the fusion of at least two different types of cells.

[0090] The host cell can be a cultured cell line, primary cell, stem cell, proliferating cell, or dividing cell. The term "host cell" includes prokaryotic cells, lower eukaryotic cells (such as yeast cells), and other eukaryotic cells (such as archaea, fungal cells, insect cells, plant cells, algal cells, microalgal cells, parasitic cells, animal cells, and mammalian cells (e.g., human or non-human, preferably non-human)). The host cell can be a differentiated cell, pluripotent cell, totipotent cell, stem cell, induced pluripotent stem cell (iPSC), induced totipotent stem cell, or even an embryonic cell or embryonic stem cell. In the case of embryonic cells or embryonic stem cells, the cell is preferably a non-human cell.

[0091] The term "host cell" more broadly includes cells that contain or already contain the nucleic acid molecules of the present invention, as well as descendants of such cells.

[0092] The host cell can be isolated, or organized within a tissue, organ, or even within a whole organism. In the case where the host cell is within a whole organism, the organism is not human.

[0093] Fusion peptide: PNG enzyme peptide fusion In the context of this invention, the inventors have surprisingly discovered that fusing a polypeptide with N-glycosidase activity (particularly polypeptides from bacteria belonging to the phylum Acidobacteria, preferably the class Acidobacteria) with an antioxidant moiety (e.g., an antioxidant compound) significantly enhances the N-glycosidase (PNG) activity of the polypeptide. Furthermore, the inventors have demonstrated that the fusion of the PNG enzyme polypeptide with the antioxidant moiety results in a significant expansion of the pH range in which the PNG enzyme polypeptide is active. In fact, the data disclosed herein reveal that fused polypeptides containing both PNG enzyme polypeptides and antioxidant moieties exhibit high levels of PNG enzyme activity over a wide pH range (from alkaline to highly acidic conditions, with a pH range of at least 2 to 7.5).

[0094] Therefore, through this groundbreaking work, the inventors have provided for the first time a highly stable and multifunctional PNG enzyme polypeptide that can effectively deglycosylate targets under both extremely acidic and alkaline conditions without any loss of activity.

[0095] In particular, the data obtained by the inventors demonstrates for the first time that the antioxidant component 1) improves solubility and 2) stabilizes PNG enzymes.

[0096] Further data showed a significant increase in PNG enzyme expression levels. In fact, the experimental results obtained by the inventors revealed that the fusion of this PNG enzyme peptide with a portion possessing antioxidant properties (such as peptides or polypeptides with antioxidant properties) not only increased PNG enzyme expression levels but also promoted PNG enzyme purification, thereby allowing for the acquisition of an increased amount of PNG enzyme peptide.

[0097] Therefore, this invention relates to a novel fusion polypeptide comprising, or substantially comprising, a polypeptide with N-glycosidase activity fused to an antioxidant moiety, wherein the polypeptide with N-glycosidase activity is a polypeptide derived from bacteria belonging to the phylum Acidobacteria. Therefore, this invention relates to a novel PNG enzyme polypeptide fusion.

[0098] In an advantageous embodiment, the bacteria belong to the class Acidobacteria, preferably to the order Acidobacteria, more preferably to the family Acidobacteriaceae, and even more preferably to a genus selected from the genera Acidobacterium, Oxybacterium, Linbacterium, and Teratophyte, and even more preferably to the genus Teratophyte. In a particularly preferred embodiment, the bacteria are *Geothrix pinkii*.

[0099] The polypeptide having N-glycosidase activity advantageously comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 1, preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity (or substantially constitutes thereof, or constitutes thereof). In a specific embodiment, the polypeptide having N-glycosidase activity comprises the amino acid sequence SEQ ID NO: 1 (or substantially constitutes thereof, or constitutes thereof).

[0100] Advantageously, the antioxidant portion is selected from antioxidant peptides, antioxidant provitamins, antioxidant vitamins, antioxidant flavonoids, and polyphenols.

[0101] Data obtained by the inventors show that the fusion of peptides with N-glycosidase activity and peptides with antioxidant properties leads to a significant increase in PNG enzyme expression and production yield, as well as a significant increase in PNG enzyme stability and activity, not only at low pH but also at alkaline pH. The inventors have specifically developed several fusion compounds of peptides with PNG enzyme activity (derived from *Acidobacterium*) and peptides with antioxidant properties.

[0102] Therefore, the antioxidant portion is preferably an antioxidant polypeptide, especially selected from glutathione S-transferase (GST), glutathione peroxidase, thioredoxin, catalase and superoxide dismutase, with glutathione S-transferase (GST) being the most preferred.

[0103] Antioxidant peptides can be of any origin, including those derived from any of the following groups: mammals, insects, plants, bacteria, and viruses. Antioxidant peptides are preferably derived from bacteria or mammals (such as humans), and more preferably from bacteria.

[0104] In a specific embodiment, the antioxidant polypeptide is glutathione S-transferase (GST). The GST polypeptide advantageously comprises an amino acid sequence having at least 75% identity with (or substantially consisting of, or consisting of) SEQ ID NO: 2, preferably at least 80% identity with SEQ ID NO: 2, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity. In a specific embodiment, the antioxidant polypeptide comprises the amino acid sequence SEQ ID NO: 2 (or substantially consists of, or consists of) it.

[0105] In a specific embodiment, the antioxidant polypeptide is glutathione peroxidase. The glutathione peroxidase polypeptide advantageously comprises an amino acid sequence having at least 75% identity (or substantially consisting of, or consisting of) a sequence selected from any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10; preferably having at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity. In a specific embodiment, the antioxidant polypeptide comprises the amino acid sequence SEQ ID NO: 10 (or substantially consists of, or consists of) it. The amino acid sequence is selected from any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0106] In a specific embodiment, the antioxidant polypeptide is a thioredoxin. The thioredoxin polypeptide advantageously comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 11, preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity (or substantially composed of, or composed of) the amino acid sequence SEQ ID NO: 11. In a specific embodiment, the antioxidant polypeptide comprises the amino acid sequence SEQ ID NO: 11 (or substantially composed of, or composed of) it.

[0107] In a specific embodiment, the antioxidant polypeptide is catalase. The catalase polypeptide advantageously comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 12, preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity (or substantially composed of, or composed of) the amino acid sequence SEQ ID NO: 12. In a specific embodiment, the antioxidant polypeptide comprises the amino acid sequence SEQ ID NO: 12 (or substantially composed of, or composed of) it.

[0108] In a specific embodiment, the antioxidant polypeptide is a superoxide dismutase. The superoxide dismutase polypeptide advantageously comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 13, preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity (or substantially composed of, or composed of) the amino acid sequence SEQ ID NO: 13. In a specific embodiment, the antioxidant polypeptide comprises the amino acid sequence SEQ ID NO: 13 (or substantially composed of, or composed of) it.

[0109] In a particularly advantageous embodiment, the fusion polypeptide comprises or is substantially composed of, or is composed of, the following: (i) a polypeptide having N-glycosidase activity comprising an amino acid sequence having at least 75% identity with SEQ ID NO: 1, preferably at least 80% identity with SEQ ID NO: 1, more preferably at least 85% identity, more preferably at least 90%, more preferably at least 95% identity (or substantially composed of, or composed of), still preferably comprising the amino acid sequence SEQ ID NO: 1 (or substantially composed of, or composed of), and (ii) fused with an antioxidant polypeptide as defined above, preferably a glutathione S-transferase (GST) as defined above.

[0110] In the fused peptide, the antioxidant moiety can be fused to the N-terminus of a peptide with N-glycosidase activity (N-terminal fusion), or to the C-terminus of a peptide with N-glycosidase activity (C-terminal fusion), or fused within the peptide with N-glycosidase activity (internal fusion), or fused laterally to the peptide (lateral fusion), or any combination thereof. Experimental data obtained by the inventors demonstrate that when this moiety is fused to the N-terminus of a peptide with N-glycosidase activity, the PNG enzyme peptide fusion is particularly stable and active. Therefore, the antioxidant moiety is preferably fused to the N-terminus of a peptide with N-glycosidase activity.

[0111] Furthermore, data demonstrate that when this portion is fused to the N-terminus of the peptide, both activity and stability are enhanced, regardless of the type of fusion portion with the PNG enzyme peptide.

[0112] Therefore, the present invention also relates to a fusion protein comprising a partially fused polypeptide with N-glycosidase activity, wherein the N-glycosidase-active polypeptide is derived from bacteria belonging to the class Acidobacteria, and wherein the portion is fused to the N-terminus of the N-glycosidase-active polypeptide, or fused within the N-glycosidase-active polypeptide. In such cases, particularly when the portion is not an antioxidant portion, the portion preferably is not fused to the C-terminus of the N-glycosidase-active polypeptide.

[0113] In one embodiment, the antioxidant portion is directly fused to a peptide having N-glycosidase activity or fused via a linker.

[0114] Advantageously, the linker is selected from affinity-based linkers (such as those derived from biotin-streptavidin interaction pairs or antibody-antigen interaction pairs) and covalent linkers (such as -SH / -NH2 groups, amine maleimide NHS-esters (such as N-hydroxysuccinimide ester of 3-(maleimide)propionic acid), or compounds targeting the C-terminal carboxyl group of the peptide (such as N-hydroxysuccinimide), or peptides (such as peptides containing 3 to 50 (random) amino acid residues). When the antioxidant moiety is fused to a peptide with N-glycosidase activity via the linker, the linker is preferably a peptide or polypeptide.

[0115] In a preferred embodiment, the antioxidant portion is directly fused to a polypeptide with N-glycosidase activity (there is no linker between the polypeptide and the antioxidant portion).

[0116] Therefore, in a specific implementation, the antioxidant portion is GST directly fused with a polypeptide having N-glycosidase activity.

[0117] The fusion polypeptide may comprise an amino acid sequence having at least 75% identity with SEQ ID NO: 14, preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and even more preferably at least 95% identity, or substantially constitute thereof. In a specific embodiment, the fusion polypeptide comprises the amino acid sequence SEQ ID NO: 14 (corresponding to GST directly fused with a polypeptide having N-glycosidase activity of *Geotrichum pinkis*) (or substantially constitute thereof).

[0118] The inventors have surprisingly demonstrated for the first time that PNG enzyme activity is significantly enhanced and the PNG enzyme peptides are significantly stable (particularly for short- to medium-term storage, i.e., from two days to several months) when contained in a formic acid-containing buffer (with or without glycine-HCl supplementation). PNG enzyme peptides stored in formic acid-containing buffer (FA buffer) are suitable for any biotechnological application involving deglycosylation. In fact, data indicate that FA buffer is compatible with any protein analysis technique, particularly those requiring acidic conditions. Furthermore, no loss of PNG enzyme activity can be detected even after long-term storage (i.e., more than one month) of the PNG enzyme peptides in FA buffer.

[0119] Therefore, in a preferred embodiment, the fusion peptide is provided in a buffer containing formic acid and / or glycine-HCl (or substantially composed of, or composed of) formic acid (or substantially composed of, or composed of) formic acid.

[0120] The buffer solution is preferably an aqueous buffer solution. The buffer solution preferably contains 0.01% to 10% formic acid, more preferably 0.05% to 8% formic acid, even more preferably 0.1% to 5% formic acid, and even more preferably about 2% formic acid.

[0121] Furthermore, experimental data obtained by the inventors demonstrate that adding salt (such as ammonium acetate or sodium chloride, or a combination thereof) to FA buffer (with or without glycine-HCl, or glycine-HCl buffer) further improves the production yield and activity of PNG enzyme. Therefore, in a preferred embodiment, the buffer further contains a salt, preferably selected from ammonium acetate or sodium chloride, more preferably ammonium acetate. When salt is added, the buffer preferably contains 0.01% to 10% of volume / volume of salt, more preferably 0.05% to 8% of volume / volume of salt, more preferably 0.1% to 5% of volume / volume of salt, and even more preferably about 2% of volume / volume of salt. When salt is added, the buffer preferably contains 1 mM to 20 mM of salt, more preferably 2 mM to 15 mM of salt, more preferably 3 mM to 10 mM of salt, more preferably 4 mM to 6 mM of salt, and even more preferably about 5 mM of salt.

[0122] The buffer may contain additional compounds, such as those selected from excipients, preservatives, carriers, solvents, diluents, adjuvants, dispersion media, coatings, antimicrobial and / or antifungal agents, absorbents, etc., preferably compatible with any biotechnological application involving deglycosylation (preferably compatible with deglycosylation reactions).

[0123] Examples of suitable additional compounds include, but are not limited to, surfactants (such as those having the formula C8H). 17 C6H4(OC2H4) 9-10 OH detergents (e.g., Triton) TM X-100), acidic compounds (such as hydrochloric acid (HCl) or acetic acid), amino acids (such as glycine), glycerol, glycine-HCl, ethylenediaminetetraacetic acid (EDTA), etc.

[0124] Here are some examples of suitable buffer solutions: - Buffer 1: An aqueous buffer containing 0.01% v / v to 10% v / v formic acid; - Buffer 2: Buffer 1 is supplemented with 0.01% volume / volume to 10% volume / volume of salt (such as ammonium acetate or sodium chloride, or a combination thereof), for example, supplemented with 1 mM to 20 mM salt; - Buffer 3: Buffer 1 is supplemented with 0.001% to 10% volume of acid (such as HCl or acetic acid) (more preferably 0.005% to 5% volume of acid, even more preferably 0.01% to 2% volume of acid). - Buffer 4: Buffer 2 is supplemented with 0.001% to 10% volume of acid (such as HCl or acetic acid) (more preferably 0.005% to 5% volume of acid, even more preferably 0.01% to 2% volume of acid). - Buffer 5: Buffer 1 is supplemented with 0.01% volume / volume to 10% volume / volume of surfactant (more preferably 0.05% volume / volume to 5% volume / volume of surfactant, more preferably 0.1% volume / volume to 2% volume / volume of surfactant). - Buffer 6: Buffer 2 is supplemented with 0.01% volume / volume to 10% volume / volume of surfactant (more preferably 0.05% volume / volume to 5% volume / volume of surfactant, more preferably 0.1% volume / volume to 2% volume / volume of surfactant). - Buffer 7: Buffer 3 is supplemented with 0.01% volume / volume to 10% volume / volume of surfactant (more preferably 0.05% volume / volume to 5% volume / volume of surfactant, more preferably 0.1% volume / volume to 2% volume / volume of surfactant). - Buffer 8: Buffer 4 is supplemented with 0.01% volume / volume to 10% volume / volume of surfactant (more preferably 0.05% volume / volume to 5% volume / volume of surfactant, more preferably 0.1% volume / volume to 2% volume / volume of surfactant). - Buffer 9: Buffer 1 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)). - Buffer 10: Buffer 2 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)); - Buffer 11: Buffer 3 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)). - Buffer 12: Buffer 4 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)); - Buffer 13: Buffer 5 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), even more preferably 50 mM to 100 mM glycine (especially glycine-HCl)); - Buffer 14: Buffer 6 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), even more preferably 50 mM to 100 mM glycine (especially glycine-HCl)). - Buffer 15: Buffer 7 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)); - Buffer 16: Buffer 8 is supplemented with 1 mM to 500 mM glycine (especially glycine-HCl) (more preferably 10 mM to 300 mM glycine (especially glycine-HCl), more preferably 50 mM to 100 mM glycine (especially glycine-HCl)); - Buffer 17: An aqueous buffer containing 1 mM to 500 mM glycine-HCl, more preferably 10 mM to 300 mM glycine-HCl, more preferably 50 mM to 100 mM glycine-HCl; - Buffer 18: Buffer 17 is supplemented with 0.01% volume / volume to 10% volume / volume of salt (such as ammonium acetate or sodium chloride, or combinations thereof), for example, supplemented with 1 mM to 20 mM salt.

[0125] The fusion peptides defined above can be readily obtained using standard molecular biology techniques (e.g., protein synthesis, PCR amplification, cDNA cloning, chemical synthesis) with sequence data available in the art and the information provided herein.

[0126] Nucleic acid molecules, vectors, host cells, and compositions Nucleic acid molecules The present invention also relates to a novel nucleic acid molecule encoding a fusion polypeptide as defined above, preferably wherein the antioxidant portion is an antioxidant polypeptide.

[0127] Nucleic acid molecules encoding fusion peptides as defined above can be optimized to provide high levels of expression in specific host cells or host organisms.

[0128] Indeed, it has been observed that when more than one codon is available to encode a given amino acid, the codon usage pattern in an organism is highly non-random (see, for example, Wada et al., 1992, Nucleic Acids Res. 20:2111), and codon usage can be significantly different between different hosts (see, for example, Nakamura et al., 1996, Nucleic Acids Res. 24:214).

[0129] For example, the antioxidant fraction may be of bacterial, viral, or lower eukaryotic origin, and therefore its codon usage pattern is not well-suited for efficient expression in higher eukaryotic cells (e.g., humans), and vice versa. Typically, codon optimization is performed by replacing one or more "natural" (e.g., bacterial, viral, or yeast) codons corresponding to codons that are not frequently used in the host organism with one or more more frequently used codons encoding the same amino acid. It is not necessary to replace all natural codons corresponding to infrequently used codons, as even partial replacements can achieve improved expression.

[0130] In addition, some deviations can be made from strictly adhering to optimized codon usage to accommodate the introduction of one or more restriction enzyme sites in the resulting nucleic acid molecules.

[0131] Beyond codon optimization, expression in host cells or host organisms can be further improved through additional modifications to the recombinant nucleic acid sequence(s). For example, various modifications can be envisioned to prevent the clustering of rare, suboptimal codons in concentrated regions, and / or to suppress or modify “negative” sequence elements expected to negatively impact expression levels. Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; RNA secondary structures; and / or internally hidden regulatory elements such as internal TATA boxes, chi sites, ribosome entry sites, and / or splicing donor / receptor sites.

[0132] Advantageously, nucleic acid molecules encoding fusion polypeptides as defined above can be operatively linked to suitable regulatory elements for expression in desired host cells or host organisms.

[0133] Adjustment element According to the present invention, the nucleic acid molecule of the present invention may further include regulatory elements necessary for the expression of the fusion polypeptide of the present invention in a host cell or host organism. The terms "regulatory element" or "regulatory sequence" refer to any element that allows, facilitates, or regulates expression in a given host cell or host organism. The arrangement of regulatory elements enables them to work synergistically to achieve their intended purpose; for example, a promoter enables transcription of the nucleic acid molecule from transcriptional initiation to transcriptional termination in a permitted host cell or host organism.

[0134] In a preferred embodiment, the nucleic acid molecule of the present invention comprises one or more expression cassettes, each expression cassette comprising at least one promoter located at the 5' of the nucleic acid molecule (e.g., encoding the fusion polypeptide of the present invention) and a polyadenylation signal located at the 3' of the nucleic acid molecule.

[0135] Those skilled in the art will understand that the selection of regulatory sequences can depend on a variety of factors, such as the nucleic acid molecule itself, the vector into which it is inserted, the recipient host cell or host organism, the desired expression level, etc. Promoters can be particularly important. In the context of this invention, they can be constitutive, directing the expression of the encoded product (e.g., the fusion peptide of this invention) in a variety of host cell types, or specific to certain host cells (e.g., organ-specific regulatory sequences), or regulated in response to specific events or exogenous factors (e.g., temperature, nutrient additives, hormones, etc.) or according to a stage of the viral cycle (e.g., late or early). Promoters that are inhibited in response to specific events or exogenous factors during the production steps can also be used to optimize vector production and circumvent the potential toxicity of the expressed peptide(s) in the producing cells.

[0136] In the context of this invention, various promoters known in the art can be used. Bacterial, viral (including bacteriophage), and eukaryotic (including mammalian, plant, and insect) promoters are particularly suitable. Advantageously, the promoter is derived from a species belonging to the same kingdom as the host cell in which the nucleic acid molecule of this invention is intended to be expressed. For example, if expression in a bacterial host cell is envisioned, a bacterial promoter is advantageously chosen. Bacteriophage promoters can also be used. Representative examples of bacterial promoters include, but are not limited to, TAC (trp-lac hybrid promoter), Lac (lactose operon promoter), LacUV5 promoter, constitutive promoters derived from the Escherichia coli lac operon, araBAD (promoter from the arabinose metabolism operon), trp (promoter from the Escherichia coli tryptophan operon), etc. Representative examples of bacteriophage promoters include, but are not limited to, the T7 promoter derived from T7 bacteriophage, T7lac (promoter from T7 bacteriophage plus lac operon), Sp6 (promoter from Sp6 bacteriophage), pL (promoter from λ bacteriophage), T3 (promoter from T3 bacteriophage), etc.

[0137] Those skilled in the art will understand that regulatory elements controlling nucleic acid expression may further include additional elements for: proper initiation, regulation, and / or termination of transcription (e.g., transcription termination sequences), mRNA transport (e.g., nuclear localization signal sequences, polyadenylation sequences), processing (e.g., splicing signals, self-cleaving peptides such as T2A, P2A, E2A, F2A, adapters), stability (e.g., introns, such as 16S / 19S or chimeric human β-globin / IgG, and non-coding 5' and 3' sequences), translation (e.g., initiator Met, triple leader sequences, IRES ribosome binding sites, signal peptides, etc.), targeting sequences, adapters (e.g., adapters composed of flexible residues such as glycine and serine), transport sequences, secretion signals, and sequences involved in replication or integration. Such sequences have been reported in the literature and are readily available to those skilled in the art.

[0138] carrier The present invention also relates to a carrier encoding a fusion polypeptide as defined above, such as a carrier comprising at least one nucleic acid molecule as defined above (i.e., a nucleic acid molecule encoding a fusion polypeptide as defined above, preferably wherein the antioxidant portion is an antioxidant polypeptide).

[0139] Therefore, a vector may contain one or more nucleic acid molecules as defined above, which may be identical (i.e., the vector contains one or more identical nucleic acid molecules as defined above) or different (i.e., the vector contains one or more nucleic acid molecules as defined above, each encoding a different fusion polypeptide, such as each encoding a different polypeptide with PNG enzyme activity and / or each encoding a different antioxidant moiety).

[0140] The vector can be a plasmid or a viral vector.

[0141] The vector may contain any regulatory element, preferably one or more regulatory elements as defined above with respect to nucleic acid molecules.

[0142] The vector may further contain any additional target nucleic acid molecules (e.g., encoding target polypeptides (e.g., tags (e.g., tags for immobilization, tags for detection, etc.), positive or negative selection markers, reporter proteins (including fluorescent proteins (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), etc.), genes encoding positive or negative selection markers, reporter genes, genes encoding tags, etc.).

[0143] The vectors defined above can be readily obtained using standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis) with sequence data available in the art and the information provided herein.

[0144] One or more nucleic acid molecules (such as those encoding fusion polypeptides containing nucleic acid molecules as defined above, and / or additional nucleic acid molecules encoding the target polypeptide) are inserted into the vector using any suitable technique known in the art.

[0145] When the vector is a viral vector, one or more nucleic acid molecules can be inserted into any suitable location within the viral genome, such as within the viral genome, between genes, in non-essential gene regions, or as a substitute for the viral sequence. Insertion into non-essential loci within the viral genome is preferred.

[0146] Insertion of the virus can be performed using conventional molecular biology techniques, for example, as described by Sambrook et al. (2001, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory). The inserted viral genome can be extracted via homologous recombination, as described by Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al. (2002, Cancer gene Ther. 9: 470-7), respectively.

[0147] host cells The present invention also relates to a host cell comprising a nucleic acid molecule as defined above, or a vector as defined above, or any combination thereof.

[0148] In an advantageous embodiment, the host cell has the ability to produce / express rare codons. Advantageously, the host cell contains genes encoding one or more rare tRNAs (i.e., tRNAs corresponding to codons rarely expressed by the host cell (codons in short supply in the host cell)) and / or corresponding tRNA synthetases. The host cell can be engineered to contain additional copies of the genes encoding rare tRNAs.

[0149] The host cell can be selected from prokaryotic cells, lower eukaryotic cells (such as yeast cells), and other eukaryotic cells (such as archaea cells, fungal cells, insect cells, plant cells, algal cells, microalgal cells, parasitic cells, animal cells, and mammalian cells (e.g., human or non-human, preferably non-human)). The host cell can be a differentiated cell, pluripotent cell, totipotent cell, stem cell, induced pluripotent stem cell (iPSC), induced totipotent stem cell, or even an embryonic cell or embryonic stem cell. In the case of embryonic cells or embryonic stem cells, the cell is preferably a non-human cell.

[0150] The host cell is preferably bacteria, preferably bacteria of the Enterobacteriaceae family, and more preferably Enterobacteriaceae genus. Escherichia Bacteria of the genus, more preferably Escherichia coli.

[0151] Composition The present invention also relates to a composition comprising, substantially comprising, or consisting of, a fusion polypeptide as defined above, a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, or any combination thereof.

[0152] In one embodiment, the fusion peptide, nucleic acid molecule, vector, host cell, or any combination thereof is freeze-dried. In another embodiment, the fusion peptide, nucleic acid molecule, vector, host cell, or any combination thereof is dissolved in a buffer solution.

[0153] The buffer is advantageously compatible with any biotechnological application involving deglycosylation.

[0154] The buffer solution preferably contains formic acid (or is mainly composed of it, or is composed of it). The buffer solution is preferably as defined above with respect to the fusion peptide.

[0155] The composition may contain additional compounds, such as those selected from excipients, preservatives, carriers, solvents, diluents, adjuvants, dispersion media, coatings, antimicrobial and / or antifungal agents, absorbents, etc., preferably compatible with any biotechnological application involving deglycosylation (preferably compatible with deglycosylation reactions).

[0156] The additional compound is preferably as defined above with respect to the fusion peptide.

[0157] Purification / Production Methods The inventors have unexpectedly developed a novel method for optimizing the production of PNG enzymes, supported by experimental data. This method results in ready-to-use PNG enzymes that are compatible with most biotechnological applications, particularly those requiring acidic conditions, and exhibit improved long-term stability. Specifically, the inventors have devised a novel method for producing and / or purifying peptides with N-glycosidase activity (particularly peptides from bacteria belonging to the phylum Acidobacteria, preferably the class Acidobacteria) using a formate-containing buffer (FA buffer). Data show for the first time that eluting PNG enzyme peptides with FA buffer allows for a significant increase in the production and purification rates of PNG enzyme peptides. Furthermore, the use of FA buffer results in the acquisition of highly pure and stable PNG enzyme peptides. PNG enzyme peptides obtained using this original method are ready for use in any biotechnological application involving deglycosylation. In fact, data demonstrate that FA buffer is compatible with any protein analysis technique, particularly those requiring acidic conditions. Moreover, data show that FA buffer also improves the storage of PNG enzyme peptides. Indeed, no loss of PNG enzyme activity was detected even after long-term storage (i.e., more than one month) of PNG enzyme peptides in FA buffer.

[0158] Therefore, this invention provides an original, stable, multifunctional and efficient tool for deglycosylation of peptides, as well as an optimized and industrially applicable production method.

[0159] Therefore, the present invention relates to a novel method for purifying a fusion polypeptide as defined above, comprising (or substantially comprising, or comprising) the step of recovering the fusion polypeptide in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid, more preferably in a buffer containing 0.01% v / v to 10% v / v of formic acid, more preferably in a buffer containing 0.05% v / v to 8% v / v of formic acid, and even more preferably in a buffer containing 0.1% v / v to 5% v / v of formic acid.

[0160] Advantageously, the buffer solution is as defined above with respect to the fusion peptide.

[0161] The method preferably involves a further step of directly storing the fusion peptide in a buffer solution (containing formic acid and / or glycine-HCl) for recovering the fusion peptide.

[0162] The present invention also relates to a novel method for producing fusion peptides as defined above, comprising (or substantially comprising, or consisting of) the following steps: a) Optionally clone nucleic acid molecules as defined above in a vector (preferably an expression vector, more preferably an expression plasmid); b) Introduce the vector of step a) or the vector as defined above into a host cell (e.g., a production cell), preferably a cell expressing a rare tRNA (i.e., a cell containing a gene encoding a rare tRNA). c) Under suitable conditions, allow the host cells from step b) to grow to produce the fusion peptide; and d) Optionally, the fusion peptide of step c) is purified by recovering it in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid, more preferably in a buffer containing 0.01% v / v to 10% v / v of formic acid, more preferably in a buffer containing 0.05% v / v to 8% v / v of formic acid, and even more preferably in a buffer containing 0.1% v / v to 5% v / v of formic acid.

[0163] Advantageously, the buffer solution is as defined above with respect to the fusion peptide.

[0164] The method preferably includes a further step, namely step e), whereby the fusion peptide of step d) is directly stored in a buffer for recovering the fusion peptide (the buffer of step d), the buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid.

[0165] The present invention also relates to a novel method for producing fusion peptides as defined above, comprising (or substantially comprising, or consisting of) the following steps: a) Optionally, a nucleic acid molecule encoding a polypeptide having N-glycosidase activity as described above is cloned in a vector (preferably an expression vector, more preferably an expression plasmid); b) Introduce the vector from step a) into a host cell (e.g., a production cell), preferably a cell expressing a rare tRNA (i.e., a cell containing a gene encoding a rare tRNA). c) In the presence of the antioxidant moiety as defined above, the host cells of step b) are grown under suitable conditions to allow the production of a polypeptide with N-glycosidase activity, and the antioxidant moiety is fused with the produced polypeptide with N-glycosidase activity; and d) Optionally, the fusion peptide is purified by recovering it in a buffer containing formic acid and / or glycine-HCl, preferably in a buffer containing formic acid, more preferably in a buffer containing 0.01% v / v to 10% v / v of formic acid, more preferably in a buffer containing 0.05% v / v to 8% v / v of formic acid, and even more preferably in a buffer containing 0.1% v / v to 5% v / v of formic acid.

[0166] Advantageously, the buffer solution is as defined above with respect to the fusion peptide.

[0167] The method preferably includes a further step, namely step e), whereby the fusion peptide of step d) is directly stored in a buffer solution (buffer solution of step d) for recovering the fusion peptide, the buffer solution containing formic acid and / or glycine-HCl, preferably in a buffer solution containing formic acid.

[0168] In an advantageous embodiment, the method of the present invention (as defined above) achieves (i.e., allows to achieve) a high (enhanced) expression level of the fusion polypeptide. The method of the present invention also advantageously achieves (i.e., allows to achieve) a high (enhanced) production and / or purification rate of the fusion polypeptide.

[0169] In this document, the terms “high expression level,” “high production yield,” and “high purification rate” refer to the acquisition (recovery) of a large amount of fusion peptide and / or the loss of a small amount of fusion peptide. The term “large amount of fusion peptide” means that starting from a wet weight (dry weight) of 30 ± 5 mg of host cells (i.e., the net weight of host cells after step c) as defined above), at least 0.1 mg of fusion peptide was acquired (recovered) by a net weight (dry weight). Yield can be calculated by dividing the net weight (dry weight) of the fusion peptide obtained after step d) as defined above by the net weight (dry weight) of the host cells after step c) as defined above. Therefore, in this document, “high expression level,” “high production yield,” and “high purification rate” refer to yields greater than 1 / 300.

[0170] In a preferred embodiment, the yield is greater than 1 / 300, more preferably greater than 0.004, more preferably greater than 0.01, more preferably greater than 0.02, more preferably greater than 0.1, and even more preferably greater than 0.2. In a preferred embodiment, the amount (net weight, dry weight) of the fusion polypeptide obtained (recovered) by the method of the present invention (as defined above) is greater than 0.1 mg, preferably greater than 0.2 mg, more preferably greater than 0.3 mg, more preferably greater than 0.4 mg, more preferably greater than 0.5 mg, and even more preferably greater than 1 mg.

[0171] The present invention also relates to fusion polypeptides that are obtainable, have been obtained, or are directly obtained by any of the methods defined above. Such fusion polypeptides are preferably as defined in the "Fusion Polypeptides" section above.

[0172] In vitro applications The present invention further relates to the use of fusion peptides as defined above, nucleic acid molecules as defined above, vectors as defined above, host cells as defined above, or any combination thereof in any biotechnological application involving deglycosylation, preferably in in vitro applications involving deglycosylation under acidic conditions.

[0173] Biotechnological applications involving deglycosylation can be selected from glycosylation profiling, anti-doping control, antibody characterization (such as monoclonal antibodies, chimeric antibodies, humanized antibodies, antibodies generated by phage display, etc.), hormone characterization, cell receptor characterization, and diagnosis and / or monitoring of diseases involving glycosylation disorders (such as congenital glycosylation disorder (CDG)).

[0174] Examples of applications of glycosylation profiling include, but are not limited to, glycosylation profiling of glycoprotein-based drugs (especially for verifying the correct glycosylation of such glycoproteins). Examples of glycoprotein-based drugs include, but are not limited to, those listed in Table 1 of Solá RJ, Griebenow K. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010 Feb 1;24(1):9-21. doi: 10.2165 / 11530550-000000000-00000. PMID: 20055529; PMCID: PMC2805475 (Table 1 of that literature is incorporated herein by reference).

[0175] When the biotechnology application is for the diagnosis of diseases involving glycosylation disorders, the in vitro use preferably includes glycosylation profiling (i.e., glycosylation analysis) of glycoproteins obtained from samples obtained from subjects who require it (e.g., subjects who are susceptible to or suspected of having diseases involving glycosylation disorders).

[0176] When the biotechnological application is for monitoring diseases involving glycosylation disorders, the in vitro use preferably includes glycosylation profiling (i.e., glycosylation analysis) of glycoproteins obtained from samples obtained from subjects who require it (e.g., subjects diagnosed with diseases involving glycosylation disorders). Such monitoring can be used for prognosis, diagnosis, and evaluation of treatment efficacy.

[0177] The present invention also relates to an in vitro method for glycosylation profiling, anti-doping control, antibody characterization (such as monoclonal antibodies, chimeric antibodies, humanized antibodies, phage-displayed antibodies, etc.), hormone characterization, cell receptor characterization, and diagnosis and / or monitoring of diseases involving glycosylation disorders (e.g., congenital glycosylation disorder (CDG)), the method using fusion peptides as defined above, nucleic acid molecules as defined above, vectors as defined above, host cells as defined above, or any combination thereof.

[0178] In vitro methods for diagnosing diseases involving glycosylation disorders preferably include the step of performing glycosylation profiling (i.e., glycosylation analysis) on glycoproteins obtained from samples obtained from subjects who require them (e.g., subjects who are susceptible to or suspected of having diseases involving glycosylation disorders).

[0179] In vitro methods for monitoring diseases involving glycation disorders preferably include the step of performing glycation profiling (i.e., glycation analysis) on glycoproteins obtained from samples obtained from subjects who require them (e.g., subjects diagnosed with diseases involving glycation disorders). Such monitoring can be used to evaluate the efficacy of treatments.

[0180] Reagent test kit The present invention also relates to a kit comprising (or substantially comprising, or consisting of): a) A container comprising a fusion peptide as defined above, a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, or any combination thereof (preferably, wherein the fusion peptide, nucleic acid molecule, vector, host cell, or any combination thereof is freeze-dried or dissolved in a buffer). b) Different containers optionally containing buffer solutions and / or additional compounds, wherein the buffer solutions and / or additional compounds are preferably compatible with any biotechnological application involving deglycosylation; and c) Optional, use the instruction manual.

[0181] The buffer solution preferably contains formic acid, preferably 0.01% to 10% by volume, more preferably 0.05% to 8% by volume, and even more preferably 0.1% to 5% by volume.

[0182] Advantageously, the buffer solution is as defined above with respect to the fusion peptide.

[0183] Additional compounds may be selected from excipients, preservatives, carriers, solvents, diluents, adjuvants, dispersion media, coatings, antimicrobial and / or antifungal agents, absorbents, etc., and are preferably compatible with any biotechnological application involving deglycosylation (preferably compatible with deglycosylation reactions).

[0184] The additional compound is preferably as defined above with respect to the fusion peptide. Attached Figure Description

[0185] Figure 1 A graph comparing the classic GST purification scheme with the scheme of the present invention ("new scheme").

[0186] Figure 2 The SDS-PAGE results for different protein samples are shown. Lane 1 shows the negative control, i.e., uninduced cell cultures. Lane 2 shows the sample "cultures with 1 mM IPTG". Lane 3 shows the supernatant of GST-purified and induced cultures (classical method, using GSH elution buffer). Lane 4 shows GST purification elution with 50 mM GSH. Lane 5 shows the GST-purified supernatant of induced cultures (in the method of this invention, using 1% formic acid as elution buffer).

[0187] Figure 3 LC-MS / MS results of GST purification and elution using rGSH buffer via classical methods. The gray highlighted portions correspond to the GST-PNG enzyme peptides identified in LC-MS. Amino acids 1-218 are GST tags, while 219-787 are PNG enzymes.

[0188] Figure 4 Formic acid concentration (pH) and elution efficiency. Diamonds represent elution with formic acid only. Triangles represent elution with formic acid supplemented with 5 mM ammonium acetate. Gray crosses represent elution with formic acid supplemented with 5 mM sodium chloride.

[0189] Figure 5 GST-PNG enzyme activity was measured by RNase B deglycosylation. a) SDS-PAGE results of RNase B deglycosylation: NC: Undeglycosylated RNase B; Lane 1: RNase B deglycosylated with Promega PNG enzyme F; Lane 2: GST-PNG deglycosylated RNase B eluted with GSH buffer at pH 2.6; Lane 3: GST-PNG deglycosylated RNase B eluted with 2% FA supplemented with 5 mM NaCl at approximately pH 2; Lane 4: GST-PNG deglycosylated RNase B eluted with 2% FA supplemented with 5 mM ammonium acetate at approximately pH 2; Lane 5: GST-PNG deglycosylated RNase B eluted with GSH buffer at pH 7.5; b) Gel quantification results of deglycosylation rate. Deglycosylation rate = integrated density (deglycosylated bands) / integrated density (undeglycosylated bands + deglycosylated bands).

[0190] Figure 6 MALDI MS data collection: By analyzing the spectra of glycans released from RNase B glycoprotein by the above-mentioned PNG enzyme, all glycans HexNAc(2)Hex(5-9) can be identified.

[0191] Figure 7 GST-PNG enzyme activity was determined by deglycosylation of the IgG heavy chain under strong reducing conditions. NC: Unglycosylated IgG heavy chain; Lane H1: Deglycosylated with GST-PNG enzyme at TCEP 5 mM; Lane H2: Deglycosylated with GST-PNG enzyme at TCEP 20 mM; Lane H3: Deglycosylated with GST-PNG enzyme at TCEP 50 mM; Lane H4: Deglycosylated with GST-PNG enzyme at TCEP 100 mM; Lane H5: Deglycosylated with GST-PNG enzyme at TCEP 250 mM; Lane H6: Deglycosylated with GST-PNG enzyme at DTT 20 mM; Lane H7: Deglycosylated with GST-PNG enzyme at DTT 50 mM; Lane RNB control: Deglycosylation positive control.

[0192] Figure 8 The activity of PNG enzyme (without GST tag) was determined by deglycosylation of IgG heavy chain under strong reducing conditions. NC: Undeglycosylated IgG heavy chain; Lane NH1: Deglycosylated with PNG enzyme at TCEP 40 mM; Lane NH2: Deglycosylated with PNG enzyme at TCEP 50 mM; Lane NH3: Deglycosylated with PNG enzyme at TCEP 100 mM; Lane NH4: Deglycosylated with PNG enzyme at TCEP 250 mM; Lane NH5: Deglycosylated with PNG enzyme at TCEP 300 mM; Lane NH6: Deglycosylated with PNG enzyme at DTT 50 mM.

[0193] Figure 9 GST-PNG enzyme activity and GST-PNG enzyme F activity were determined by deglycosylation of IgG heavy chains under strong reducing conditions. NC: Undeglycosylated IgG heavy chain; lane GST-PNG enzyme F: Deglycosylated with GST-PNG enzyme F at TCEP 450 mM; lane GST-PNG enzyme H+: Deglycosylated with the GST-PNG enzyme of this invention at TCEP 450 mM; lane PNG enzyme F: Commercial PNG enzyme F only.

[0194] Example Although the invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are for illustrative purposes only. Therefore, it should be understood that many modifications can be made to the illustrative embodiments, and other arrangements can be devised without departing from the spirit and scope of the invention as defined in the appended claims.

[0195] 1. Example 1: Design of a novel method for producing ready-to-use and stable N-glycosidases 1.1. Methods for producing recombinant plasmids: 1.1.1. Materials The genomic DNA of *Geotrichum pinkis* (strain DSM 18391) (Taxon ID 926566) (Gene bank CP003379.1) was purchased from DSMZ Leibniz-Institut.

[0196] Primers: PCR forward primer: 5'-Gaattc t ATG CCCCGCATCTTGTGCCGCCCT-3' (SEQ ID NO: 15); PCR reverse primer: 5'-atactcgag GCGTTTCACCGGGCAGCCTGC-3' (SEQ ID NO: 16). Primers were ordered from Eurogentec.

[0197] Plasmid: pGEX 4T3 plasmid ordered from GE Health.

[0198] Polymerase: SuperFiII high-fidelity DNA polymerase ordered from ThermoFisher.

[0199] Competent cells: Oneshot Top10 cells purchased from ThermoFisher and BL21(DE3)RIPL cells purchased from Agilent.

[0200] The amino acid sequences used in the examples are as follows: - PNG enzyme: SEQ ID NO: 1 - GST: SEQ ID NO: 2 - GST-PNG enzyme fusion protein: SEQ ID NO: 14.

[0201] 1.1.2. Methods Construction of recombinant pGEX plasmid Using SuperFiII high-fidelity DNA polymerase, the gene encoding the PNG enzyme (Tax id: 926566, between 4437827 and 4439533) was amplified by PCR from the whole genome of *Gastromyxobolus* (strain DSM 18391) according to the supplier's protocol (ThermoFisher). A three-step protocol was used, with 30 cycles. Briefly, denaturation was performed at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 55 s. Furthermore, the initial denaturation was set at 98°C for 30 s, while the final extension was performed at 72°C for 5 minutes. Primer pairs were designed based on the PNG enzyme gene and the EcoRI and XhoI restriction sites on the pGEX 4T-3 vector (as detailed in paragraph 1.1.1 above; the forward PCR primer is shown in SEQ ID NO: 15, and the reverse primer is shown in SEQ ID NO: 16).

[0202] According to the manufacturer's instructions, the amplified PCR product was inserted into the TOPO zero blunt II vector (ThermoFisher) and transformed into Oneshot TOP10 cells (ThermoFisher), then cultured on LBK plates (LB stands for LB liquid medium (Luria Broth), K stands for the antibiotic kanamycin). Colonies selected from the LBK plates were cultured in LB solution containing kanamycin, and the recombinant TOPO plasmid was further precipitated and purified for DNA sequencing. The plasmid carrying the PNG enzyme sequence was further double-digested with restriction endonucleases EcoRI (NewEngland Biolabs) and Xho I (NewEngland Biolabs), and purified using the Lonza rapid gel system (Lonza) to obtain the purified double-digested PNG enzyme gene. Simultaneously, the pGEX 4T-3 vector was also double-digested and further dephosphorylated using FastAP (ThermoFisher). The purification of the linearized pGEX 4T-3 vector was then performed in the same manner as described above. Subsequently, the two double-digested products were ligated using the ThermoFisher ligase to construct a recombinant pGEX vector carrying the PNG enzyme gene. The recombinant pGEX plasmid was then transformed into BL21(DE3)RIPL cells optimized for sequences containing rare codons and cultured on LBA plates (A represents the antibiotic ampicillin).

[0203] 1.2. Enzyme expression and purification methods: Cell culture Colonies picked from LBA plates were cultured in LB broth containing ampicillin to prepare an inoculum solution. The inoculum solution was incubated at 17°C in LB broth for 24 h until the OD value reached 2. Cell culture medium was then prepared using the LB broth inoculated with the prepared inoculum solution. When the OD value of the cell culture medium reached 0.6, cells were induced by IPTG to a final concentration of 1 mM to trigger the expression of the GST-PNG enzyme on the recombinant pGEX vector. This culture medium was incubated at 17°C for 24 h until OD > 2, resulting in high-level expression of the enzyme.

[0204] GST-PNG enzyme purification Cells from cell culture medium were precipitated at 13000 x g for 20 minutes at 4°C and stored at -20°C. For GST-PNG enzyme purification, the cell pellet from 1 mL of cell culture medium was dissolved in 250 μl of lysis buffer consisting of Tris∙HCl (125 mM, pH 7.4), lysozyme (1 mg / mL), NaCl (150 mM), DnA enzyme (2 μl), Triton X100 (0.1% v / v), and Roche Complete Protease Inhibitor (1X), and incubated at 30 rpm for 30 minutes at room temperature on a vortex mixer. The crude cell lysate was then mixed with MagneGST containing Na₂HPO₄ (4.2 mM), KH₂PO₄ (2 mM), NaCl (140 mM), and KCl (10 mM). TM MagneGST equilibrated with binding / washing buffer TM The glutathione particles were mixed and incubated at room temperature for 30 minutes. The supernatant was removed from the particles, and the particles were further washed to remove non-specific binding.

[0205] Washout: GST-PNG enzyme must be eluted from glutathione particles. This can be done as with the classic Promega MagneGST. TM As recommended by the protein purification system protocol, use TrisHCl buffer (50 mM) containing 50 mM r-GSH (r-GSH: the reduced form of glutathione) (hereinafter referred to as "rGSH buffer").

[0206] However, this elution buffer is not optimal for GST-PNG enzyme activity and subsequent use for the following reasons: 1. The classic rGSH elution buffer does not have the optimal acidic pH for GST-PNG enzymes; 2. The classic rGSH elution buffer contains Tris HCl and GSH, which is not conducive to applications such as MALDI TOF / TOF mass spectrometry analysis; 3. The GST binding capacity on the GST-PNG enzyme may be used to block eluted GSH, and cannot be further utilized; 4. rGSH classic elution buffer requires an additional cumbersome step of buffer exchange to obtain better activity and shelf life.

[0207] Therefore, a novel elution protocol and a novel elution buffer were designed to overcome these drawbacks. The inventors have demonstrated for the first time that a formic acid-containing buffer (hereinafter referred to as "FA buffer") provides optimal PNG enzyme purification rates, optimal PNG enzyme activity, and optimal PNG enzyme storage conditions, while eliminating the cumbersome step required for buffer exchange. Eliminating this step is particularly advantageous because it reduces PNG enzyme loss (caused by the aforementioned buffer exchange) and saves time. Furthermore, the inventors have shown that PNG enzymes can be stored directly in these novel formic acid-containing buffers without any loss of activity, further eliminating the buffer exchange step required for storage.

[0208] A chart comparing the classic design (rGSH design) and the new design (FA design) is shown. Figure 1 middle.

[0209] The inventors designed a novel buffer solution containing different concentrations of formic acid (including 0.1%, 0.5%, 1%, 2%, and 4%; as shown in Table 1 below). Two supplementary salts (i.e., sodium chloride (NaCl) and ammonium acetate) were added to the formic acid buffer solution to facilitate elution.

[0210] Table 1: Acidic elution buffer conditions and their pH (FA solution) The purity and sequence of the GST-PNG enzyme were estimated by SDS-PAGE and LC-MS / MS. The concentration of the eluent was estimated at UV 280 nm.

[0211] 1.3. Results 1.3.1. Expression Optimal expression and culture conditions can be developed by constructing recombinant plasmids based on pGEX vectors and selecting appropriate expression systems. Optimal conditions include preparing the inoculum solution in LB liquid medium and inoculating the culture to an OD of approximately 0.6. Induction is performed with 1 mM IPTG, followed by culture at 17°C for 24 h. Furthermore, data demonstrate that transformation into bacterial strains engineered to contain additional copies of genes encoding rare tRNAs (such as BL21-CodonPlus competent cells and BL21(DE3)RIPL) allows for a further significant increase in PNG enzyme expression levels.

[0212] Figure 2 SDS-PAGE results for protein expression are presented. Comparison of lanes 1 and 2, representing induced and uninduced cultures respectively, shows a strong overexpression band near 100 kDa in lane 2, corresponding to the GST-PNG enzyme. Lane 3 is the supernatant from the purification process, while lane 4 is the elution product purified in Tris HCl buffer (50 mM) containing 50 mM r-GSH. This result indicates excellent purification, as no other co-eluted protein bands appeared in the elution lanes (high GST-PNG enzyme purity). Lane 5 indicates purification via 1% formic acid (FA) buffer.

[0213] Furthermore, no degradation of GST-PNG enzyme was observed in FA buffer. These data demonstrate for the first time that the GST tag, with its antioxidant properties, 1) aids in the dissolution of PNG enzyme and 2) stabilizes PNG enzyme. The data further show a significant improvement in PNG enzyme expression.

[0214] 1.3.2. LC-MS / MS The eluted GST-PNG enzyme was further analyzed by LC-MS / MS. Mascot search results showed that... Figure 3 As shown, the GST-PNG enzyme protein can be identified with a high score, with protein coverage >50%. The identified trypsin-cleaved peptides are distributed throughout the entire GST-PNG enzyme sequence. The MS / MS spectra of the identified peptides from the GST-PNG enzyme are shown in the example below, exhibiting good quality. These data confirm the successful fusion of the PNG enzyme with the GST tag.

[0215] 1.3.3. Concentration 2 mL of wet precipitate yielded 60 ± 10 mg of wet weight (precipitate wet weight estimation method).

[0216] Two elution methods were used in this study. Initially, the classic elution method containing GSH elution buffer was used. After elution with GSH buffer (but before the buffer exchange step), the inventors were able to obtain 0.1 mg of GST-PNG enzyme protein from 1 mL of cell culture, with a protein concentration as high as 1.56 ± 0.41 mg∙mL⁻¹, which represents a significant improvement compared to previous reports (Wang et al., 2014).

[0217] Therefore, fusion with the antioxidant-rich portion (here, the GST tag) not only facilitates GST-PNG enzyme purification but also improves expression. Unbound by any single theory, these improvements can be attributed both to the improved folding and solubility associated with the antioxidant properties of the GST tag and to the inventors' optimized expression system. Higher enzyme concentrations mean a lower dilution effect during sample processing, which is beneficial.

[0218] However, the buffer exchange step on the eluted GST-PNG enzyme (aimed at removing GSH buffer, which is incompatible with mass spectrometry analysis) resulted in a significant loss of GST-PNG enzyme. After the buffer exchange, the amount of GST-PNG enzyme remaining in the recovery buffer was close to 0 mg.

[0219] To overcome this drawback, the inventors designed a novel elution method by altering the pH and ionic strength, primarily aimed at reducing GST-PNG enzyme loss. The experimental goal was to obtain GST-PNG enzyme in a buffer containing as little salt as possible while being directly at the enzyme's optimal pH. Initial attempts to remove GSH from GST-PNG enzyme eluted by microcone spinning columns and Zeba desalting columns failed. Furthermore, this buffer exchange step resulted in significant sample loss. Therefore, a novel scheme was devised that skips the buffer exchange step. In this original scheme, elution is performed directly with a novel formate buffer. No existing techniques were found describing how acidic conditions can disrupt GSH-GST interactions. Only one publication by Mercado-Pimentel et al. proposed using 50 mM glycine-hydrochloric acid (pH 2.5, 0.1%, with Triton X-100 added) and 150 mM NaCl to elute GST-tagged proteins (Mercado-Pimentel et al., 2002). However, such buffers are incompatible with most PNG enzyme applications. For example, volatile buffer solutions are required for compatibility with MALDI matrix and mass spectrometry applications.

[0220] Therefore, a special elution method was designed using a newly designed formate buffer (FA buffer) with or without added salt (ammonium acetate or sodium chloride).

[0221] like Figure 1 As shown, a single band between 100 and 150 kDa can be observed in lane 6, which has been verified to correspond to the GST-PNG enzyme. The data demonstrate that only 1% formic acid in the novel elution buffer, supplemented with ammonium acetate, can effectively disrupt the interaction between GSH and GST. Loading the same amount of elution solution from the same precipitate onto the gel resulted in a smaller band than the eluted GSH. Therefore, unbound GST-PNG enzyme can be clearly observed in lane 5 (the supernatant of this specific elution method), indicating that higher yields are limited by the capacity of the GSH beads used in the purification step. It should be mentioned here that the capacity of the beads used is insufficient to capture all the generated GST-PNG enzyme (therefore, a non-negligible amount of PNG enzyme is discarded in the flowthrough). As a consequence, the estimated production yield (based on the elution yield estimate) could be further improved using beads with a larger capacity.

[0222] A second elution can be performed to further improve the estimated production yield.

[0223] Other formic acid concentrations used for elution were also tested. Figure 4 The diagram shows the elution efficiency. Diamonds represent elution with formic acid only. Triangles represent elution with formic acid supplemented with ammonium acetate. As can be observed from the diamonds, elution efficiency increases with increasing formic acid concentration. At the same formic acid concentration, higher elution efficiency was achieved by adding 5 mM ammonium acetate, which may disrupt electrostatic interactions. Elution with 2% formic acid supplemented with 2% sodium chloride also resulted in higher elution efficiency than with formic acid only, although to a lesser extent than with FA buffer supplemented with ammonium acetate. Elution with 4% FA buffer supplemented with ammonium acetate eluted at least 0.14 mg of GST-PNG enzyme from 1 mL of cell culture pellet, which was significantly higher than using GSH buffer.

[0224] Overall, the data show that elution with FA buffer significantly improves the elution yield of PNG enzyme. Adding salt (ammonium acetate or sodium chloride) to the FA buffer further increases the yield (especially ammonium acetate) and PNG enzyme activity. Without being bound by any particular theory, it is believed that ammonium acetate or sodium chloride works by disrupting electrostatic interactions, thereby further improving the elution yield.

[0225] 2. Example 2: GST-PNG enzyme is highly stable and active over a wide pH range. 2.1. Activity of GST-PNG 2.1.1. On the gel Deglycosylation of RNase B was tested, and the results can be found in Figure 5a. The deglycosylation performance of GST-PNG enzyme eluted with rGSH buffer in the classical manner was compared under both acidic (pH 2.6) and alkaline (pH 7.5) conditions, using the same enzyme substrate ratio of 3.3. The results showed... Figure 5 In lanes 2 and 5 of swim 1), a significant mass shift was observed under both acidic and alkaline deglycosylation conditions compared to the unglycosylated RNase B in lane NC, consistent with the band obtained from the deglycosylation of PNG enzyme F in lane 1. This result indicates that the GST-PNG enzyme can function over a wide pH range. Significant activity loss was observed in the original PNG enzyme H+ at pH greater than 3.5, and activity almost zero at pH greater than 6 (as described by Wang et al., 2014), but this phenomenon was not observed here. Therefore, the data demonstrate that the fusion of the PNG enzyme with the GST tag significantly increases the pH range of activity. Figure 5 a) The signals in lanes 2 and 3 show the deglycosylation results eluted with 2% formic acid (NaCl and ammonium acetate as additive salts) by GST-PNG enzyme. The enzyme-substrate ratios were calculated to be 0.9 and 1.2, respectively. Both samples yielded deglycosylated bands close to 20 kDa.

[0226] Therefore, deglycosylation efficiency can be estimated by gel quantification using triplicate replicates. The final results are shown... Figure 5 In b), the bar chart shows that GST-PNG enzyme achieved complete deglycosylation of RNase B under acidic conditions. No difference was observed between GST-PNG enzyme eluted with classical elution (rGSH buffer) and FA buffer. GST-PNG enzyme retained 90% of its activity under alkaline conditions at pH 7.5. Previous work reported a significant loss of activity in PNG enzymes in 500 mM (approximately 2%) formic acid, but this was not the case with GST-PNG enzyme. PNG enzyme eluted with 2% formic acid supplemented with 5 mM sodium chloride also achieved a high deglycosylation rate, but slightly lower than that obtained by elution with 2% formic acid supplemented with ammonium acetate.

[0227] In summary, the data reveal that the GST-PNG enzyme exhibits high activity over a wide pH range, from alkaline to highly acidic conditions (pH range at least from 2 to 7.5). Therefore, through this groundbreaking work, the inventors have, for the first time, provided a highly stable and versatile PNG enzyme capable of effectively deglycosylating targets under both highly acidic and alkaline conditions without loss of activity.

[0228] 2.1.2. MALDI mass spectrometry acquisition RNase B was deglycosylated using GST-PNG enzyme in 2% formic acid, and the released glycan spectra were collected using MALDI MS with 3-aminoquinoline (3AQ) as the MALDI matrix. Figure 6 In the study, GST-PNG enzyme activity was detected, and all five glycans from RNase B glycoprotein were detected in the spectrum as sodium adducts with a 3AQ label at the reducing end, thus confirming the compatibility of the buffer with MS acquisition.

[0229] 2.2. Storage The stability of PNG enzymes after storage in classical buffer (rGSH) and FA buffer was compared. Data showed that PNG enzyme activity decreased significantly (or even disappeared) after one week of storage in classical buffer (rGSH), while no loss of activity was observed after one month of storage in FA buffer (data not shown).

[0230] 3. Example 3: GST-PNG enzyme maintains activity under strong reducing conditions The activity of GST-PNG enzyme was tested on immunoglobulin G (IgG), a pharmaceutically important glycoprotein, under a strong reducing environment.

[0231] 3.1 Experimental Conditions Glycoprotein IgG was used as a deglycosylation target. It contains approximately 50 kDa of glycosylated heavy chain and approximately 25 kDa of unglycosylated light chain. Deglycosylation of the IgG heavy chain was then evaluated.

[0232] Protein IgG I4505 was purchased from Merck (Darmstadt, Germany). 10 µg IgG was used for each condition.

[0233] IgG is first reduced under different strong reducing environments: - Denaturation environment 1: Tris(2-carboxyethyl)phosphine (TCEP) 5 mM, 20 mM, 50 mM, 100 mM or 250 mM, at room temperature on a thermomixer at 750 rpm for 1 h; - Denaturation environment 2: Dithiothreitol (DTT) 20 mM or 50 mM, at 56°C on a thermostat at 750 rpm for 30 minutes.

[0234] Therefore, glycosylation was determined at 750 rpm on a thermostat under the optimal conditions of GST-PNG enzyme (i.e., 22°C, pH=2.5) and the reaction was carried out overnight, while the reduction conditions were maintained at the same concentration as described above.

[0235] Deglycosylated products were analyzed by SDS-PAGE gel chromatography stained with Coomassie Brilliant Blue.

[0236] 3.2. Results The activity of GST-PNG enzyme was tested under two different reduction conditions generated by strong reducing reagents DTT and TCEP, both of which are stronger reducing agents than formic acid (FA).

[0237] Figure 7 The displayed gel shows the results of the deglycosylation experiment. Lanes H1-H7 represent the experimental conditions defined above: H1: TCEP 5 mM, H2: TCEP 20 mM, H3: TCEP 50 mM, H4: TCEP 100 mM, H5: TCEP 250 mM, H6: DTT 20 mM, and H7: DTT 50 mM. NC represents the negative control, where no N-glycosidase was added to the sample, and therefore the IgG heavy chain was not deglycosylated. The RNB control was used to measure the activity of the GST-PNG enzyme to ensure that positive enzyme activity was maintained during the reaction. Protein ladder bands are used and displayed on the left side of the gel to indicate the target band.

[0238] A band representing the GST-PNG enzyme, ranging from 75 to 100 kDa, was observed on the gel. For the negative control, an unglycosylated IgG heavy chain band was observed at approximately 50 kDa in lane NC. Compared to this control, it can be seen that after the deglycosylation assay, under all experimental conditions in lanes H1 to H7 (where the reaction was reduced by TCEP and DTT), the IgG heavy chain mass shifted, indicating that GST-PNG enzyme activity is maintained even in such strongly reducing environments.

[0239] 4. Example 4: GST-free PNG enzymes lose their activity under strong reducing conditions. The activity of GST-free PNG enzymes was tested on immunoglobulin G (IgG), a pharmaceutically important glycoprotein, under a strong reducing environment.

[0240] 4.1 Experimental Conditions GST label removal Thrombin T4648, purchased from Merck (Darmstadt, Germany), was used to remove the GST tag from the expressed GST-PNG enzyme.

[0241] The experiment was conducted at a ratio of 1:100 (1 unit of thrombin to 100 µg of protein IgG) at room temperature (22°C) on a thermostat at 650 rpm, and the reaction was allowed to proceed overnight (15 h 30 min).

[0242] The obtained PNG enzyme was used for subsequent deglycosylation experiments.

[0243] Deglycosylation Glycoprotein IgG was used as a deglycosylation target. It contains approximately 50 kDa of glycosylated heavy chain and approximately 25 kDa of unglycosylated light chain. Deglycosylation of the IgG heavy chain was then evaluated.

[0244] Protein IgG I4505 was purchased from Merck (Darmstadt, Germany). 10 µg IgG was used for each condition.

[0245] IgG is first reduced under different strong reducing environments: - Denaturation environment 1: Tris(2-carboxyethyl)phosphine (TCEP) 40 mM, 50 mM, 100 mM, 250 mM or 300 mM, at room temperature on a thermostat at 750 rpm for 1 h. - Degradation environment 2: DTT 50 mM, at 56°C on a thermostatic mixer at 750 rpm for 30 minutes.

[0246] Then, glycosylation was performed overnight on a thermostat at 650 rpm under the optimal conditions for PNG enzyme (i.e., 22°C, pH=2.5), while the reduction conditions were maintained at the same concentration as described above.

[0247] Deglycosylated products were analyzed by SDS-PAGE gel chromatography stained with Coomassie Brilliant Blue.

[0248] 4.2. Results The purpose of this experiment was to observe whether the PNG enzyme remained active under a strong reducing environment without any antioxidant component (GST tag).

[0249] Therefore, the inventors evaluated two different reduction conditions and tested the activity of the PNG enzyme using strong reducing reagents DTT and TCEP.

[0250] The obtained SDS-page gel was shown in Figure 8 In the middle. NC represents the negative control, in which IgG is reduced but not deglycosylated. Lanes NH1-NH6 represent the experimental conditions defined above, namely NH1: TCEP 40 mM, NH2: TCEP 50 mM, NH3: TCEP 100 mM, NH4: TCEP 250 mM, NH5: TCEP 300 mM, and NH6: DTT 50 mM.

[0251] The gel showed light bands in NH1-NH6 representing the GST-tagged PNG enzyme with a molecular weight of 60.8 kDa, between the 50 kDa and 75 kDa ladder bands, indicating successful removal of the GST tag from the GST-PNG enzyme.

[0252] Furthermore, a strong band at the 50 kDa position was observed, representing the heavy chain of IgG. No mass drift was observed in any of the NH1-NH6 lanes compared to the negative control NC lane, indicating that the activity of the GST-free PNG enzyme is insufficient to induce deglycosylation under these reducing conditions.

[0253] Therefore, these results confirm the importance of the antioxidant portion (GST tag) for enabling PNG enzymes to maintain their enzymatic activity under strong reducing conditions.

[0254] 5. Example 5: GST-PNG enzyme is active under strong reducing conditions, while GST-PNG enzyme F is not. The activity of GST-PNG enzyme against immunoglobulin G (IgG) under a strong reducing environment was compared with the activity of GST-PNG enzyme F under the same conditions.

[0255] 5.1 Experimental Conditions Glycoprotein IgG was used as a deglycosylation target. It contains approximately 50 kDa of glycosylated heavy chain and approximately 25 kDa of unglycosylated light chain. Deglycosylation of the IgG heavy chain was then evaluated.

[0256] Protein IgG I4505 was purchased from Merck (Darmstadt, Germany). 10 µg IgG was used for each condition.

[0257] IgG was first reduced under different strong reducing conditions: tris(2-carboxyethyl)phosphine (TCEP) 450 mM.

[0258] Then, under optimal conditions of GST-PNG enzyme (i.e., at 22°C, pH=2.5) and GST-PNG enzyme F (i.e., at 37°C, pH=7.8), deglycosylation was determined at 750 rpm on a thermostat and the reaction was allowed to proceed overnight.

[0259] Deglycosylated products were analyzed by SDS-PAGE gel chromatography stained with Coomassie Brilliant Blue.

[0260] 5.2. Results The results show Figure 9In the middle. NC represents the negative control in lanes 1 and 6. The commercial PNG enzyme F alone is shown near the 37 kDa position in lane 5. The product of IgG heavy chain deglycosylation by GST-PNG enzyme F is shown in lane 2, while the product of IgG heavy chain deglycosylation by GST-PNG enzyme is shown in lane 3.

[0261] By observing the tails of the two IgG heavy chain bands to compare the two results, a slight mass shift can be observed from lane 2 to lane 3, which is due to the removal of glycans from the heavy chain by the GST-PNG enzyme.

[0262] Conversely, the product quality of the heavy chain deglycosylated by GST-PNG enzyme F in lane 2 was at the same level as the two negative controls, indicating that no deglycosylation occurred when using GST-PNG enzyme-F under this strong reducing environment.

[0263] Therefore, these results show that the Bacillus PNGase fused with the antioxidant portion of the present invention maintains glycosylation activity even under strong reducing conditions (TCEP 450 mM), while non-Bacillus PNGases (such as PNGase-F) do not appear to do so even when fused with the antioxidant portion.

[0264] References

Claims

1. A fusion polypeptide comprising (or consisting essentially of, or consisting of) a polypeptide having N-glycosidase activity fused to an antioxidant moiety, wherein the polypeptide having N-glycosidase activity is a polypeptide from a bacterium belonging to the phylum Acidobacteriota. Acidobacteria phylum).

2. The fusion polypeptide according to claim 1, wherein the bacteria belong to the class Acidobacteria Acidobacteriia clas s ), preferably to the order Acidobacteriales Acidobacteriales order ), more preferably to the family Acidobacteriaceae Acidobacteriaceae family), more preferably to a genus selected from the group consisting of Acidobacterium Acidobacterium ), Geobacterium Edaphobacter ), Lindowia Silvibacterium ), and Terrestrialbacter Terriglobus .

3. The fusion polypeptide of claim 2, wherein the bacterium belongs to the genus Terriglobus.

4. The fusion polypeptide of claim 3, wherein the bacteria is Rhodopseudomonas palustris (R. palustris) Terriglobus roseus ) bacteria.

5. The fusion polypeptide of any one of claims 1 to 4, wherein the antioxidant moiety is selected from the group consisting of an antioxidant polypeptide, an antioxidant pro-vitamin, an antioxidant vitamin (such as ascorbic acid (vitamin C), an alpha-tocopherol (vitamin E)), an antioxidant flavonoid, a compound with a thiol group, a compound with a phenol group, a polyphenol, a carotinoid, glutathione, lipoic acid, uric acid, a tocopherol (such as alpha-tocopherol), and ubiquinol (coenzyme Q).

6. The fusion polypeptide of any one of the preceding claims, wherein the antioxidant moiety is an antioxidant polypeptide, preferably selected from the group consisting of glutathione S-transferase, glutathione peroxidase, thioredoxin, catalase, and superoxide dismutase.

7. The fusion polypeptide of claim 6, wherein the antioxidant moiety is selected from the group consisting of glutathione S-transferase, glutathione peroxidase, thioredoxin, catalase, and superoxide dismutase.

8. The fusion polypeptide of claim 7, wherein the antioxidant moiety is glutathione S- transferase.

9. The fusion polypeptide of any one of the preceding claims, wherein the antioxidant moiety is fused to the N-terminus of the polypeptide having N-glycosidase activity, or to the C- terminus of the polypeptide having N-glycosidase activity, or within the polypeptide having N- glycosidase activity, or any combination thereof; preferably wherein the antioxidant moiety is fused to the N-terminus of the polypeptide having N-glycosidase activity.

10. The fusion polypeptide of any one of the preceding claims, wherein the antioxidant moiety is fused directly to the polypeptide having N-glycosidase activity or via a linker, wherein the linker is preferably selected from the group consisting of an affinity-based linker (such as a linker derived from coupling biotin-streptavidin or derived from coupling antibody-antigen) and a covalent linker (such as a -SH / -NH2 group, an amine maleimide NHS ester (such as 3-(maleimidyl)propionic acid N-hydroxysuccinimidyl ester) or a compound targeting the C-terminal carboxyl group of the polypeptide (such as N-hydroxysuccinimide) or a peptide (such as a peptide comprising 3 to 50 (random) amino acid residues).

11. The fusion polypeptide of any one of the preceding claims, wherein the polypeptide having N-glycosidase activity comprises (or consists essentially of, or consists of) an amino acid sequence that is at least 85% identical to SEQ ID NO:

1.

12. The fusion polypeptide of claim 1, wherein the polypeptide having N-glycosidase activity comprises (or consists essentially of, or consists of) an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and is fused to an antioxidant polypeptide.

13. The fusion polypeptide of claim 11 or 12, wherein the polypeptide having N- glycosidase activity comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO:

1.

14. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1 to 13, wherein the antioxidant moiety is an antioxidant polypeptide.

15. A vector comprising the nucleic acid molecule of claim 14, wherein the vector is a plasmid or a viral vector.

16. A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15, preferably wherein the host cell comprises a gene encoding a rare tRNA.

17. A composition comprising the fusion polypeptide of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, or any combination thereof; preferably wherein the fusion polypeptide, the nucleic acid molecule, the vector, the host cell, or any combination thereof is lyophilized or dissolved in a buffer; wherein the composition preferably further comprises other compounds, such as compounds selected from excipients, preservatives, carriers, solvents, diluents, adjuvants, dispersion media, coating agents, antibacterial and / or antifungal agents, and absorbents; preferably wherein the buffer and / or the other compounds are preferably compatible with any biotechnological application involving deglycosylation.

18. The composition of claim 17, wherein the fusion polypeptide, the nucleic acid molecule, the vector, the host cell, or any combination thereof is dissolved in a buffer comprising formic acid and / or glycine-HCl, preferably in a buffer comprising formic acid.

19. A method for purifying a fusion polypeptide as defined in any one of claims 1 to 13, comprising the steps of: The fusion polypeptide is recovered in a buffer comprising formic acid and / or glycine-HCl, preferably in a buffer comprising formic acid at 0.01% volume / volume to 10% volume / volume, more preferably at 0.05% volume / volume to 8% volume / volume, more preferably at 0.1% volume / volume to 5% volume / volume.

20. A method for producing a fusion polypeptide as defined in any one of claims 1 to 13, comprising the steps of: a) cloning the nucleic acid molecule of claim 14, optionally in a vector; b) introducing the vector of step a) or the vector of claim 15 into a host cell, preferably a cell expressing a rare tRNA; and c) growing the host cell of step b) under suitable conditions to allow production of the fusion polypeptide.

21. The method of claim 20, further comprising a step d) of purifying the fusion polypeptide of step c) by recovering the fusion polypeptide in a buffer comprising formic acid and / or glycine-HCl, preferably in a buffer containing 0.01% to 10% formic acid, more preferably in a buffer containing 0.05% to 8% formic acid, more preferably in a buffer containing 0.1% to 5% formic acid.

22. A method for producing a fusion polypeptide as defined in any one of claims 1 to 13, comprising the following steps: a) optionally, cloning in a vector a nucleic acid molecule encoding a polypeptide having N-glycosidase activity as defined in any one of claims 1 to 13; b) introducing the vector of step a) into a host cell, preferably a cell expressing a rare tRNA; and c) growing the host cell of step b) in the presence of an antioxidant moiety as defined in any one of claims 1 to 13, under suitable conditions to allow the production of a polypeptide having N-glycosidase activity, and to allow the fusion of the antioxidant moiety to the produced polypeptide having N-glycosidase activity.

23. The method of claim 22, further comprising a step d) of purifying the fusion polypeptide of step c) by recovering the fusion polypeptide in a buffer comprising formic acid, preferably in a buffer containing 0.01% to 10% formic acid, more preferably in a buffer containing 0.05% to 8% formic acid, more preferably in a buffer containing 0.1% to 5% formic acid.

24. In vitro use of the fusion polypeptide of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, or any combination thereof, for any biotechnological application involving deglycosylation, preferably for a biotechnological application involving deglycosylation under acidic conditions, more preferably for an application selected from the group consisting of glycosylation profiling, doping control, antibody (such as monoclonal antibody, chimeric antibody, humanized antibody, phage display generated antibody, etc.) characterization, hormone characterization, cell receptor characterization, and diagnosis and / or monitoring of a disease involving a glycosylation disorder (such as a congenital disorder of glycosylation (CDG)).

25. A kit comprising: a) a container comprising the fusion polypeptide of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, or any combination thereof; b) optionally, a different container comprising a buffer, a vehicle, an excipient, and any combination thereof, wherein preferably the buffer, the vehicle, the excipient, and any combination thereof are compatible with any biotechnological application involving deglycosylation; and c) optionally, instructions for use.

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