Enzyme compositions for glycosylation antigen removal, related methods, uses, apparatus and systems

Abstract

To provide an enzyme capable of more efficiently cleaving a carbohydrate antigen from cells.SOLUTION: The present invention provides an isolated nucleic acid sequence, encoding (a) a GalNAc deacetylase with a specific sequence or (b) Galactosaminidase with a specific sequence, and a vector comprising the nucleic acid sequence and a heterologous nucleic acid sequence. The vector may also include a heterologous nucleic acid sequence, selected from one or more of protein tags and cleavage sites.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Cross-reference to related applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 719,272, “Enzymatic Composition for Glycan Antigen Cleavage, Related Methods, Uses, Apparatus and Systems,” filed on 17 August 2018.

[0002] The present invention relates to the field of enzyme compositions. In particular, the present invention relates to enzyme compositions for cleaving antigens, and to uses, methods, apparatus and systems for cleaving antigens using such compositions. [Background technology]

[0003] Because the plasma of a person with blood type A contains antibodies against the B antigen, and vice versa, incompatible transfusions can lead to complement activation and red blood cell (RBC) lysis (Daniels 2010), accurate blood type matching is a central requirement in transfusion medicine. These cell surface antigens are glycan structures terminated with α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal) for blood types A and B, respectively. On the other hand, O-type red blood cells do not contain these terminal sugars and can be transfused in general (Garratty 2008). Therefore, it is necessary to have a sufficient supply of O-type red blood cells in blood banks in case of emergencies where a patient's blood type is unknown or unclear. However, the supply is often limited.

[0004] As a means of converting A or B RBCs to O, the concept of enzymatically removing the GalNAc or Gal structure from A or B RBCs was first proposed and demonstrated by Goldstein (Goldstein 1982; US4609627 and CA2272925). Using α-galactosidase from green coffee beans, B-type RBCs were converted to O-type and subsequently transfused successfully (Kruskall 2000). However, this approach was non-practical due to the amount of enzyme required. Conversion of A-type is more difficult, mainly because there are many subtypes with different internal linkages in the A blood type (Clausen 1989). Similarly, α-galactosidase has been used for the removal of B-type antigens (see, for example, EP2243793). A major step forward towards practical conversion, including A-type conversion, was made by screening a library of bacteria for both A and B conversion activities using a tetrasaccharide substrate. Two new families of glycosidases showing high antigen cleavage activity at neutral pH values were discovered (CAZy GH109 α-N-acetylgalactosaminidase and GH110 α-galactosidase (Liu 2007)). Both enzymes completely removed their respective antigens and converted the corresponding RBCs. However, a significant amount of enzyme is required, especially for A-type conversion (60 mg enzyme / unit of blood), limiting further development. Enzymes that more efficiently remove sugar chain antigens from cells would be useful. Summary of the Invention

[0005] The present invention is based in part on the surprising discovery that a combination of galactosaminidase and GalNAc deacetylase, as described herein, is orders of magnitude more efficient than previously identified A antigen-cleaving enzymes. For example, under certain conditions, some GalNAc deacetylase and galactosaminidase enzymes can cleave A antigen at 1 μ / ml or less. Further, the cleavage efficiency by the enzyme combination is maintained at a pH suitable for maintaining the viability of red blood cells (i.e., a pH between about 6.5 and about 7.5). Further, the enzymes are found to be active at temperatures between 4°C and 37°C, which is also suitable for blood collection, washing, and storage protocols. Further, the efficiency of the enzymes is further improved by the addition of a crowding agent (e.g., dextran). It is also understood that the same two-step removal process can be applied to donor organs. The enzymes described herein are tested mainly on samples with 10% hematocrit because they work better, and the amount required for a bag (about 220 ml) packed with red blood cells (rbc) containing a hematocrit value of about 80% was calculated.

[0006] In certain embodiments lacking a crowding agent, to remove A antigen from red blood cells, 3 μg / ml of 10% hematocrit, 1 hour at 37°C, >5.3 mg of each enzyme per bag packed with rbc may be used, and in other embodiments having a crowding agent, to cleave A antigen from red blood cells, 0.5 μg / ml of 10% hematocrit, 1 hour at 37°C, >0.9 mg of each enzyme per bag packed with rbc may be used. However, it will be understood by those skilled in the art that if the cells are incubated for longer, more enzyme can be used to shorten the time the blood can be processed, or less enzyme can be used.

[0007] According to one embodiment, a composition is provided, the composition comprising (a) a purified GalNAc deacetylase protein, and (b) a purified galactosaminidase protein.

[0008] According to one embodiment, a composition is provided which comprises (a) a purified GalNAc deacetylase protein selected from one or more of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, 32, 33, 34, and 35, and (b) a purified galactosaminidase protein selected from one or more of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0009] According to a further embodiment, a composition is provided which comprises a purified enzyme having GalNAc deacetylase activity essentially consisting of an amino acid sequence at least 90% identical to one of sequence numbers 2, 4, 5, 17, 23, 29, 31 and 32-35, and a purified enzyme having galactosaminidase activity essentially consisting of an amino acid sequence at least 90% identical to one of sequence numbers 7, 9, 10, 19, 21, 36 and 37.

[0010] According to a further embodiment, a composition is provided which comprises a purified enzyme having GalNAc deacetylase activity essentially consisting of an amino acid sequence at least 85% identical to one of sequence numbers 2, 4, 5, 17, 23, 29, 31 and 32-35, and a purified enzyme having galactosaminidase activity essentially consisting of an amino acid sequence at least 85% identical to the sequence described in one of sequence numbers 7, 9, 10, 19, 21, 36 and 37.

[0011] According to a further embodiment, a composition is provided which comprises a purified enzyme having GalNAc deacetylase activity essentially consisting of an amino acid sequence at least 80% identical to one of sequence numbers 2, 4, 5, 17, 23, 29, 31 and 32-35, and a purified enzyme having galactosaminidase activity essentially consisting of an amino acid sequence at least 80% identical to one of sequence numbers 7, 9, 10, 19, 21, 36 and 37.

[0012] According to a further embodiment, a composition is provided which comprises a purified enzyme having GalNAc deacetylase activity essentially consisting of an amino acid sequence at least 75% identical to one of sequence numbers 2, 4, 5, 17, 23, 29, 31 and 32-35, and a purified enzyme having galactosaminidase activity essentially consisting of an amino acid sequence at least 75% identical to one of sequence numbers 7, 9, 10, 19, 21, 36 and 37.

[0013] In a further embodiment, a composition is provided which comprises an enzyme selected from one or more of the following: (a) a purified GalNAc deacetylase protein, which is the purified Flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; and (b) a purified galactosaminidase protein, which is the purified Flavonifractor plautii galactosaminidase protein of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.

[0014] According to further embodiments, a composition is provided which comprises an enzyme selected from one or more of the following: (a) purified GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 17 and SEQ ID NO: 32 and (b) purified galactosaminidase protein, which is purified flavonifractor plautigalactosaminidase protein of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 36 and SEQ ID NO: 37.

[0015] In a further embodiment, a composition is provided which comprises an enzyme selected from one or more of the following: (a) a purified GalNAc deacetylase protein, which is the purified Clostridium tertium GalNAc deacetylase protein of SEQ ID NO: 17 and SEQ ID NO: 32; and (b) a purified galactosaminidase protein, which is the purified Clostridium tertium galactosaminidase protein of SEQ ID NO: 19 and SEQ ID NO: 36.

[0016] The GalNAc deacetylase and galactosaminidase composition can cleave the A antigen at concentrations of 1 μg / ml or less. The GalNAc deacetylase and galactosaminidase composition may have A antigen cleavage activity at pH levels between approximately 6.5 and 7.5. The GalNAc deacetylase and galactosaminidase composition may have A antigen cleavage activity at temperatures between 4°C and 37°C.

[0017] The composition may include (a) the purified GalNAc deacetylase and the purified galactosaminidase being immobilized, (b) the purified GalNAc deacetylase being immobilized, or (c) the purified galactosaminidase being immobilized.

[0018] The immobilized enzyme may be bound to a surface, which may be selected from one or more of the following: (a) beads or microspheres, (b) a container, (c) a tube, (d) a column, and (e) a matrix. The composition may further include a crowding agent. The crowding agent may be selected from one or more of dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll®, and inactive proteins.

[0019] According to further embodiments, a purified enzyme is provided comprising the flavonifractor plauti GalNAc deacetylase of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

[0020] According to further embodiments, a purified enzyme comprising the flavonifractor plautigalactosaminidase of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10 is provided.

[0021] According to further embodiments, a purified enzyme comprising Clostridium tertium GalNAc deacetylase of SEQ ID NO: 17 or SEQ ID NO: 32 is provided.

[0022] According to further embodiments, a purified enzyme comprising Clostridium tertium galactosaminidase of SEQ ID NO: 19 or SEQ ID NO: 36 is provided.

[0023] According to further embodiments, an isolated nucleic acid sequence encoding a GalNAc deacetylase is provided, selected from one or more of SEQ ID NOs: 1, SEQ ID NOs: 3, SEQ ID NOs: 16, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 28, and SEQ ID NOs: 30.

[0024] According to further embodiments, an isolated nucleic acid sequence encoding a galactosaminidase selected from one or more of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 18, and SEQ ID NOs: 20 is provided.

[0025] Further embodiments provide vectors comprising nucleic acids as described herein. The vector may also comprise heterologous nucleic acid sequences, wherein the heterologous nucleic acid sequence is selected from one or more protein tags and cleavage sites.

[0026] Protein tags include albumin-binding protein (ABP), alkaline phosphatase (AP), AU1 epitope, AU5 epitope, AviTag, bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxyl carrier protein (BCCP), blue tongue virus tag (B-tag), single-domain camel antibody (C-tag), calmodulin-binding peptide (CBP or calmodulin-tag), chloramphenicol acetyltransferase (CAT), and cellulose-binding. Domain (CBP), Chitin-binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase (DHFR), DogTag, E2 epitope, E-tag, FLAG epitope (FLAG-tag), Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glutathione S-transferase (GST), Human influenza hemagglutinin (HA), HaloTag (trademark), Alternating histidine and glutamine tags (HQ tag), Alternating histidine and asparagine tags ( HN tag), histidine affinity tag (HAT), horseradish peroxidase (HRP), HSV epitope, isopept tag (Isopep-tag), ketosteroid isomerase (KSI), KT3 epitope, LacZ, luciferase, maltose-binding protein (MBP), Myc epitope (Myc-tag), NE-tag, NusA, PDZ domain, PDZ ligand, polyarginine (Arg-tag), polyaspartic acid (Asp-tag), polycysteine ​​(Cys-tag), polyglutamic acid (Glu-tag), Hyhistidine (His-tag), Polyphenylalanine (Phe-tag), Profinity eXact, Protein C, Rho1D4 tag, S1-tag, S-tag, Softag 1, Softag 3, SnoopTag Jr, SnoopTag, SpotTag, SpyTag (Spy-tag), Streptavidin-binding peptide (SBP), Staphylococcus aureus protein A (Protein A), Staphylococcus aureus protein G (Protein G), StrepTag, Streptavidin (SBP-tag), StrepTag II, Sdy-tag, Low molecular weight ubiquitin-like modifier (SUMO),One or more of the following methods may be selected: tandem affinity purification (TAP), T7 epitope, tetracysteine ​​tag (TC tag), thioredoxin (Trx), TrpE, Ty tag, ubiquitin, universal, V5 tag, VSV-G or VSV- tag, and Xpress tag.

[0027] A further embodiment provides a method for enzymatically removing A antigen from blood, red blood cells, or a donor organ, comprising the steps of (a) mixing a GalNAc deacetylase protein and a galactosaminidase protein with (1) blood containing type A antigen, (2) type A or AB red blood cells, or (3) a donor organ presenting type A antigen, and (b) incubating the enzyme together with (1) blood, (2) type A or AB red blood cells, or (3) the donor organ for a period of time sufficient for the enzyme to cleave the A antigen from the blood, red blood cells, or donor organ.

[0028] GalNAc deacetylase is a purified protein selected from one or more of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, 32, 33, 34, and 35, and galactosaminidase may be a purified protein selected from one or more of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0029] The composition may also include a purified enzyme having GalNAc deacetylase activity, which essentially consists of an amino acid sequence at least 90% identical to one of sequence numbers 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity, which essentially consists of an amino acid sequence at least 90% identical to one of sequence numbers 7, 9, 10, 19, 21, 36, and 37.

[0030] GalNAc deacetylase may be the purified flavonifractor plauti GalNAc deacetylase protein of SEQ ID NO: 4 or SEQ ID NO: 5, and galactosaminidase may be the purified flavonifractor plauti galactosaminidase protein of SEQ ID NO: 9 or SEQ ID NO: 10.

[0031] The above method may further include the addition of a crowding agent. The crowding agent may be selected from one or more of the following: dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll®, hyperbranched glycerol, and inactive proteins.

[0032] The method described above may further include washing blood, red blood cells, or donor organs to remove GalNAc deacetylase, galactosaminidase, and crowding agents.

[0033] GalNAc deacetylase and galactosaminidase may be able to cleave A antigen at concentrations of 1 μg / ml or less. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.5 and 7.5. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperature between 4°C and 37°C.

[0034] According to a further embodiment, a blood collection and storage system is provided comprising (a) purified GalNAc deacetylase protein and (b) purified galactosaminidase protein.

[0035] The system may further include a surface on which the enzyme is immobilized, the surface being selected from one or more of the following: (a) beads or microspheres, (b) a container, (c) a tube, (d) a column, or (e) a matrix.

[0036] According to a further embodiment, a blood collection and storage device is provided, which comprises (a) a surface, (b) purified GalNAc deacetylase protein immobilized on the surface, and (c) purified galactosaminidase protein immobilized on the surface.

[0037] The apparatus surface on which the enzyme is immobilized may be selected from one or more of the following: (a) beads or microspheres, (b) container, (c) tube, (d) column, (e) matrix. The container may be a bag.

[0038] GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 100 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 90 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 80 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 70 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 60 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 50 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 40 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 30 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 20 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 15 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 14 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 13 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 12 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 11 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 10 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 9 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 8 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 7 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 6 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 5 μg / ml or less.GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 4 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 3 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 2 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 1 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.9 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.8 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.7 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.6 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.5 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.4 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.3 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.2 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.1 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.09 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.08 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.07 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.06 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.05 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.04 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.03 μg / ml or less.GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.02 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave A antigen at concentrations of 0.01 μg / ml or less.

[0039] GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.5 and approximately 7.5. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.0 and approximately 8.0. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.8 and approximately 7.8. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.9 and approximately 7.9. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at pH between approximately 6.4 and approximately 7.8.

[0040] GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperatures between 4°C and 37°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperatures between 3°C and 38°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperatures between 4°C and 40°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperatures between 4°C and 37°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at temperatures between 5°C and 37°C.

[0041] Purified GalNAc deacetylase and purified galactosaminidase may be immobilized. The immobilized enzymes may be attached to a surface. The surface may be selected from one or more of the following: beads or microspheres, containers, tubes, columns, or matrices. The container may be a bag.

[0042] According to another embodiment, a purified enzyme is provided comprising the flavonifractor plouti GalNAc deacetylase of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

[0043] According to another embodiment, a purified enzyme comprising the flavonifractor plautigalactosaminidase of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10 is provided.

[0044] According to another embodiment, a purified enzyme is provided comprising purified Clostridium tertium GalNAc deacetylase and the galactosaminidase fusion protein of SEQ ID NO: 14.

[0045] According to another embodiment, a vector comprising the nucleic acid described in this specification and a heterogeneous nucleic acid sequence is provided.

[0046] According to another embodiment, this method may be carried out in vitro or ex vivo. As used herein, ex vivo means that this method is carried out outside of an organism. For example, ex vivo includes ex vivo pulmonary perfusion (EVLP) and treatment of donated blood. As used herein, ex vivo means an experiment, measurement, or treatment carried out in or on tissues or cells (e.g., red blood cells or donor organs) from an organism in an external environment with minimal or some change from the state in which the tissues or cells were placed when they were in vivo. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1 shows schematic diagrams of the cell surface antigen glycan structures terminated with α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal) for types A, H, and B, where triangles indicate the cleavage points of α-acetyl-galactosaminidase EmGH109 and α-galactosidase BfGal110. [Figure 2] Figure 2 shows the deacetylase pathway for A antigen cleavage, along with the corresponding mass spectrometry (MS) results, in which flavonifractor plauti (Fp)GalNAc deacetylase cleaves the acetyl group from the terminal α-N-acetyl-galactosamine of the A antigen (-42 m / z), and the galactosaminide intermediate is then cleaved by flavonifractor plauti (Fp) galactosaminidase (-161 m / z). [Figure 3] Figure 3 shows FACS analysis of A+RBCs treated with different concentrations of EmGH109 or flavonifractor plautiGalNAc deacetylase (FpGalNAc deacetylase) + flavonifractor plautigalactosaminidase (FpGalNAc deacetylase), or treated at 37°C for 1 hour. Anti-H antibody (with secondary FITC labeling added) and APC-labeled anti-A antibody were used for visualization, and the region of H antigen appearance is in the upper left box. Columns A-D compare EmGH109 and FpGalNAcDeAc+FpGalNase at 5 μg / ml (A), 10 μg / ml (B), 50 μg / ml (C), and 50 μg / ml + dextran 40k (D). [Figure 4] Figure 4 compares EmGH109 and FpGalNAcDeAc+FpGalNase at various enzyme concentrations and temperatures (i.e., 4°C, room temperature (RT), and 37°C) in the presence (■) and absence (◆) of dextran. [Figure 5]Figure 5 shows HPAE-PAD analysis of A+B+ and O+ erythrocyte cleavage products, and a comparison of full-length flavonifractor plautigalnac deacetylase (FpGalNAcDeAc) + flavonifractor plautigalactosaminidase (FpGalNase) enzymes with cleaved FpGalNAcDeAc + FpGalNase enzymes in A+ erythrocytes. [Figure 6] Figure 6 shows the pH profiles for (A) FpGalNAc deacetylase and (B) Fp galactosaminidase, respectively. [Figure 7] Figure 7 shows the conversion of A antigen to H antigen on A erythrocytes, analyzed via FACS, for (A) A+RBC control, (B) Flavonifractor plauti GalNAc deacetylase (FpGalNAcDeAc) + Flavonifractor plauti galactosaminidase (FpGalNase) (10 ug / mL), (C) FpGalNAcDeAc + Clostridium tertium (Ct)Ct5757_GalNase (10 ug / mL), and (D) FpGalNAcDeAc + Robinsoniella peoriensis (Rp) galactosaminidase (Rp1021)GalNase (10 ug / mL). [Modes for carrying out the invention]

[0048] The following detailed description will be better understood when read in conjunction with the attached figures. To illustrate the present invention, the drawings show embodiments of the invention. However, the present invention is not limited to the exact configurations, examples, and means shown.

[0049] Terms not directly defined herein shall be understood to have the meanings generally associated therewith, as understood in the technical field of the present invention.

[0050] As used herein, "immobilized enzyme" refers to an enzyme attached to a surface, which may be an inactive, insoluble substance. Enzyme immobilization increases resistance to changes in conditions such as pH and temperature, and can facilitate their removal after use and for enzyme reuse.

[0051] Enzyme immobilization may be achieved by various methods (e.g., affinity tag binding, surface adsorption on glass, capture of resins, alginate beads or matrices, beads, fibers or microspheres, crosslinking to a surface or other enzymes, and covalent bonding to a surface).

[0052] As used herein, “affinity tag binding” refers to the immobilization of an enzyme on a surface (e.g., a porous material using non-covalent or covalent protein tags). Affinity tag binding has been used in protein purification and, more recently, in biocatalytic applications by EziG® (ENGINZYME AB® of Sweden (e.g., PCT / US 1992 / 010113) and PCT / SE 2015 / 050108)). Alternative systems for attaching active oxygen to a surface are known in the art (see, for example, US4088538, US4141857, US4206259, US4218363, US4229536, US4239854, US4619897, US 4748121, US4749653, US4897352, US4954444, US4978619, US5154808, US5914367, US5962279, US6030933, US6291582, US6254645, US10,016,490, and US10,041,055).

[0053] Protein tags are peptide sequences genetically grafted onto recombinant proteins, often removable by chemical or enzymatic means, and are attached to proteins for a variety of purposes. The protein tags listed in Table A are intended as examples and not as an extension thereof. One type of protein tag is affinity tags, which are attached to protein or peptide sequences so that they can be purified from crude biological sources (e.g., from expression systems) using affinity techniques, or to facilitate the immobilization of "tagged" proteins onto surfaces. Examples of affinity tags include chitin-binding domains (CBDs), maltose-binding proteins (MBPs), strep-tags, glutathione-S-transferase (GST), and polyhistidines that bind to metal matrices (His-tags). Another type of protein tag is epitope tags (e.g., V5-tags, Myc-tags, HA-tags, spot-tags, NE-tags), which are short peptide sequences selected to facilitate the production of high-affinity antibodies and are often derived from viral gene sequences to improve immunoreactivity. Epitope tags are used for protein purification and surface immobilization, but are particularly useful in Western blotting, immunofluorescence, and immunoprecipitation experiments. Another type of protein tag is chromatographic tagging (e.g., polyanionic amino acids such as FLAG tags), which can be used to alter the chromatographic properties of a protein to aid in separation and purification or immobilization. Yet another type of protein tagging is solubilization tagging (e.g., maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), and poly(NANP)) and fluorescent tagging (e.g., green fluorescent protein (GFP)). Protein tags enable specific enzymatic modifications, chemical modifications, or the binding of a protein to other components. However, depending on the type or number of tags attached to a protein sequence, the protein's intrinsic function, in this case, its enzymatic function, may be impaired by the tags.Therefore, it is necessary to select a protein tag to ensure that the enzyme activity is not impaired, or the protein tag may be cleaved from the protein before use.

[0054] Table A: Representative Protein Tags JPEG0007862477000001.jpg150165JPEG0007862477000002.jpg180144JPEG00078624770 00003.jpg186138JPEG0007862477000004.jpg176142JPEG0007862477000005.jpg154148

[0055] The use of protein tags is exemplified in this application through the use of polyhistidine protein tags (His-tags) as shown in Sequence IDs 5, 10, 15, 17, 19, 21, 23, 25, 27, 29, and 31. However, those skilled in the art will readily understand that, depending on the purification method used and / or the surface to which the enzyme is bound, enzymes can be purified and / or bound to a surface using any number of other protein tags as described herein. Such protein tags may be selected from one or more of the protein tags listed in Table A, but other such protein tags are known in the art.

[0056] Furthermore, one or more cleavage sites (e.g., the thrombin cleavage sites used in SEQ ID NOs. 15, 17, 19, 21, 23, 25, 27, 29, and 31) may be used to separate protein tags from the enzyme, or otherwise to cleave the enzyme. The cleavage sites may be used for the removal of N-terminal methionine, signal peptides, and / or the conversion of inactive or non-functional proteins into active proteins (i.e., zymogens or proenzymes). Alternatively, cleavage sites may be used to separate two or more enzymes expressed in the same reading frame. Examples of enzymes that can cleave proteins or peptides and will have sequence-specific cleavage sites may be selected from one or more of the following: Arg-C proteinase, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin high specificity (C-terminus of [FYW], not before P), chymotrypsin low specificity (C-terminus of [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, X Factor a, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodobenzoic acid, LvsC, LvsN, NTCB (2-nitro-5-thiocyanbenzoic acid), neutrophil elastase, pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, trypsin.

[0057] Those skilled in the art will understand that the combination of the active galactosaminidase enzyme and the active GalNAc deacetylase enzyme described herein is important for efficiently cleaving the A antigen, and they will also understand that the addition of one or more cleavage sites and / or one or more protein tags is optional, and that such modifications may be selected based on a particular expression system, purification system, and possible surface attachment strategy. Furthermore, other modifications to the galactosaminidase and GalNAc deacetylase sequences are also possible as long as the A antigen cleavage activity is not significantly impaired. Modifications to the galactosaminidase and GalNAc deacetylase sequences may be deletions, insertions, and / or substitutions. Substitutions may be conservative or neutral substitutions. For example, the galactosaminidase and GalNAc deacetylase sequences may share more than 90% sequence identity with the mature enzyme. For example, galactosaminidase sequences and GalNAc deacetylase sequences may share more than 85% sequence identity with the mature enzyme. For example, galactosaminidase sequences and GalNAc deacetylase sequences may share more than 75% sequence identity with the mature enzyme. Alternatively, galactosaminidase and GalNAc deacetylase sequences may have up to 5, 10, 13, 15, 20, or 25% of amino acid modifications.

[0058] As used herein, “adsorption to glass, alginate beads, or matrix” refers to the attachment of an enzyme to the outside of an inert material. Generally, this type of immobilization does not occur by chemical reaction, and the active site of the immobilized enzyme may be blocked by the surface to which it is adsorbed, which may reduce the activity of the adsorbed enzyme.

[0059] As used herein, “capture” refers to the capture of an enzyme within insoluble beads or microspheres. However, capture may prevent substrate arrival and product efflux. One example is the use of calcium alginate beads, which can be produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride.

[0060] As used herein, "crosslinking" refers to the covalent bonding of enzymes to each other in order to create a matrix consisting almost entirely of enzymes. When designing a crosslinking enzyme reaction, ideally, the binding site should not cover the enzyme's active site, so that the enzyme's activity is affected only by immobilization and not by occlusion of the enzyme's active site. Nevertheless, spacer molecules such as poly(ethylene glycol) may be used to reduce steric hindrance by the substrate.

[0061] As used herein, “covalent bond” refers to the covalent bond of an enzyme to an insoluble support or surface (e.g., silica gel). Due to the strength of the covalent bond between the enzyme and the support or surface, the likelihood of the enzyme detaching from the support or surface is very low.

[0062] As used herein, “crowding agent” refers to any polymer or protein that promotes the aggregation of macromolecules by accumulating enzymes on the cell surface in order to improve enzyme activity. Crowding agents may include, for example, dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll®, hyperbranched glycerol, and inactive proteins (Kuznetsova, IM et al. Int J Mol Sci. (2014) “What Macromolecular Crowding Can Do to a Protein” 15(12):23090-23140).

[0063] As used herein, "dextran" refers to polysaccharides having a molecular weight of 1,000 daltons or more and possessing a linear skeleton of α-linked d-glucopyranosyl repeat units. Dextrans are classified into three structural classes (i.e., classes 1-3) based on a pyranose ring structure containing five carbon atoms and one oxygen atom. Class 1 dextrans contain an α(1→6)-linked d-glucopyranosyl skeleton modified with small side chains of d-glucose branches with α(1→2), α(1→3), and α(1→4) links. The molecular weight, spatial arrangement, type and degree of branching, and branched chain length of Class 1 dextrans vary by 3-5 depending on the microbial strain and culture conditions. Isomaltose and isomalttriose are oligosaccharides with a Class 1 dextran skeleton structure. Class 2 dextrans (alternans) contain an alternating skeletal structure of α(1→3) and α(1→6) linked d-glucopyranosyl units and α(1→3) linked branches. Class 3 dextrans (mutans) have a skeletal structure of continuous α(1→3) linked d-glucopyranosyl units with α(1→6) linked branches.

[0064] As used herein, "pullulans" is a structural polysaccharide primarily produced from starch by the fungus Aureobasidium pullulans, consisting of repeating α(1→6) linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units, occasionally containing maltotetraose units.

[0065] As used herein, "dextrin" refers to a D-glucopyranosyl unit having a shorter chain length than dextran, beginning with a single α(1→6) linkage and linearly followed by α(1→4) linked D-glucopyranosyl units.

[0066] As used herein, “dextran sulfate” is derived from dextran via sulfation.

[0067] As used herein, "Ficoll®" is a neutral, highly branched, high-mass hydrophilic polysaccharide that is readily soluble in aqueous solution.

[0068] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

[0069] (material and method) The chemicals and commercially available enzymes used in this study were purchased from Sigma-Aldrich™ unless otherwise noted. The monosaccharide methylumbelliferyl glycoside was a generous gift from Dr. Hongming Chen, and was used for A antigen subtype 1. penta-MU It was a generous gift from Dr. David Kwan (Kwan et al. 2015).

[0070] Human Fecal Metagenome Library

[0071] For the generation of a human metagenomics fosmid library, blood type AB + Fresh human fecal samples were collected from healthy Asian male volunteers. Direct DNA extraction and fosmid library preparation were performed according to the procedure described in the MoE protocol (Armstrong et al. 2017).

[0072] Fosmid library screening

[0073] 51 x 384 well AB +The blood fosmid library plate was thawed at room temperature and replicated in a 384-well plate containing 50 μl of screening LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO4). The plate was incubated at 37°C for 18 hours in a sealed container with a water reservoir to prevent excessive evaporation. 45 μl of the reaction mixture (100 mM NaH2PO4, pH 7.4, 2% (v / v) Triton-X100, 100 μM GalNAc-α-MU, 100 μM Gal-α-MU) was added to the grown screening plate using a QFill® instrument [Genetix®]. Next, the plates were incubated in a sealed container at 37°C for 24 hours, and the fluorescence of each plate (e.g., 365nm Em:435nm, sweep mode, gain 80) was measured at 1, 2, 4, 8, and 24 hours using a SynergyH1 plate reader [BioTek®]. For all wells, the Z-score was calculated, which is given by the formula: Z-score = (median fluorescence) / standard deviation.

[0074] All positive hits exceeding a certain threshold were rearranged into a new 384-well plate, named the "Simple Substrate Hits" plate, and stored at -70°C. Two screening plates were replicated from the "Simple Substrate Hits" plate and re-screened for either GalNAc-α-MU or Gal-α-MU activity to confirm previously detected activity, and then deconvoluted.

[0075] To determine which hits can cleave the A antigen or B antigen structure, a binding enzyme assay is used to test for 50 μM A antigen subtype 1 tetra-MU or 50 μM B antigen subtype 1tetra-Their activity against MU was measured. This binding assay version was previously described by Kwan (Kwan et al. 2015). The assay was modified to also detect cleavage of subtype 1A antigen by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the binding enzyme. α-N-acetylgalactosaminidase and α-galactosidase cleave the terminal sugar, and H antigen subtype I tri- MU is released. Subsequently, α-fucosidase (AfcA (Katayarna 2004)), β-galactosidase (BgaC (Jeong 2009)), and β-hexosaminidase (SpHex (Williams 2002)) cleave the remaining sugar in the exo form until 4-methylumbelliferyl alcohol is released. This can be detected as an increase in fluorescence. To achieve this, 50 μg / mL of each enzyme was added to the reaction mixture. All positive hits exceeding a certain threshold were rescreened three times, and host cell lines containing vectors lacking the inserts were used as negative controls. All confirmed hits were stored separately in LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 15% (v / v) glycerol, 0.2% (v / v) maltose, 10 mM MgSO4) at -70°C.

[0076] Fosmid hit sequencing

[0077] To isolate fosmid DNA for sequencing, a positive hit fosmid glycerol stock was inoculated into 5 mL of TB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO4) and incubated overnight at 37°C and 220 rpm. Fosmid isolation was performed using the GeneJet® Plasmid Miniprep Kit (Thermo Fisher®). The isolated plasmids were purified from contaminated E. coli (E. coli) DNA using Plasmid-Safe® ATP-dependent DNase (Epicentre®), followed by further purification using the GeneJet® PCR Purification Kit (Thermo Fisher®). The concentration was calculated using the Quant-iT® dsDNAHS assay kit (Invitrogen®) on a Qbit® fluorometer (ThermoFisher®). The expected DNA size was confirmed on a 1% agarose gel. For complete fosmid sequencing, 2 ng of each fosmid was sent to the UBC Sequencing Center (Vancouver, BC, Canada). Each fosmid was individually barcoded and sequenced using the Illumina MiSeq® system.

[0078] All raw Illumina MiSeq™ sequencing data was trimmed and assembled using Python scripts available on GitHub at https: / / github.com / hallamlab / FabFos. Briefly, Trimmomatic was used to remove adapters and low-quality sequences from the reads (Bolger 2014). These reads were screened for vector and host sequences using BWA (Li 2013) and then filtered to remove contaminants using Samtools™ and bam2fastq scripts. These high-quality, purified reads were assembled using MEGAHIT with k-mer values ​​ranging between 71 and 241, increasing in increments of 10 (Li 2015). These libraries often have coverage exceeding 20,000x, and to prevent the accumulation of sequencing errors that would interfere with proper sequence assembly, the minimum k-mer multiplicity was calculated as 1% of the estimated fosmid coverage. Scaffolding was performed using minimus2 outside of a Python script assembly that generates multiple contigs (Treangen 2011). Parameterized commands can be found both in the documentation on the GitHub® page and in the Python script itself.

[0079] Fosmid ORF prediction and hit verification

[0080] Fosmid ORFs were identified using the metagenomic version of Prodigal® (Hyatt 2010) and compared with the CAZy® database using BLASTP® as part of the MetaPathways® v2.5 software package (Konwar 2015). MetaPathways® parameters were length > 60, BLAST score > 20, BLAST score ratio > 0.4, E Value <1×10 -6 That is the case.

[0081] All predicted ORFs with annotations for members of the GH or CBM family (along with known or suspected α-galactosidase and / or α-N-acetylgalactosaminidase activity) were cloned into the pET16b plasmid using the Golden Gate® cloning strategy (Engler 2008), and primer sequences were set in Table B. Proteins were expressed at BL21 (DE3) and cultured in 10 mL of ZY5052 auto-induction medium (Studier 2005) at 37°C and 220 rpm for 20 hours. Cells were harvested by centrifugation (4000 × g, 4°C, 10 min) and resuspended in 1 mL of lysis buffer (100 mM NaH2PO4, pH 7.4, 2% (v / v) Triton-X® 100, 1x Protease Inhibitor EDTA-free [Pierce®]). A ligation assay (Kwan 2015) was performed using 50 μl of crude cell lysate from the candidate cells, and 50 μl of assay buffer (100 mM NaH2PO4, pH 7.4, 50 μg / mL SpHex, 50 μg / mL AfcA, 50 μg / mL BgaC, 100 μM A antigen subtype) was used. 1tetra- The reaction was mixed with MU or 100 μM B antigen subtype 1 tetra-MU) and incubated at 37°C. All reactions were performed in three different ways in a black 96-well plate. Fluorescence (365 / 435 nm) was continuously monitored for 4 hours using a Synergy® H1 plate reader [BioTek®]. Assays from crude extracts showing cleavage activity against A or B antigen were repeated, this time without conjugating enzymes, and the reaction products were isolated via an HF Bond Elut C18 column and analyzed by LC-MS and / or TLC. TLC was performed using a TLC silica gel 60 F254 TLC plate [EMD Millipore Corp.®, Billerica, MA, USA].

[0082] Table B: Primer sequences JPEG0007862477000006.jpg211139

[0083] HPAE-PAD assay

[0084] The analysis of the enzymatic release of galactosamine was performed on a HPAE-PAD (Dionex™) HPLC system. For the following substrates, the cleavage activities of various proteins were tested: mucin from porcine stomach type II dissolved in 100 mM NaH₂PO₄ pH 7.4 at 7.5 μg / μL, 5 mM A antigen subtype 1 penta- MU present in 100 mM NaH₂PO₄ pH 7.4, and RBCs (hematocrit 50%) from A+, B+ and O- donors present in 1xPBS pH 7.4. Samples containing 10 μg / mL of the enzyme were incubated at 37 °C for two hours and then stored at -80 °C for further analysis. A small amount of the reaction (10 μl) was diluted in H₂O (100 μl) and analyzed on a HPAE-PAD instrument. Separation was performed on a CarboPAC PA200™ (150 mm) column with a guard column, and detection was performed using disposable gold on a polytetrafluoroethylene (PTFE) electrode and a four-potential waveform. The separation conditions were as follows: 100 mM sodium hydroxide and a sodium acetate gradient from 70 to 300 mM were applied over the first 10 minutes of the separation. The eluent was held at the final gradient conditions for 1 minute and then returned to the initial conditions over the next 1 minute. The flow rate was 1.0 ml / min and injections were made every 27 minutes. Standards of free sugars GalNAc, Gal and GalN (10 μM) were applied to the HPAE-PAD to determine the peak elution times for reference.

[0085] Kinetic assays

[0086] All kinetic assays using 4-methylumbelliferone as the leaving group were performed by fluorescence measurement. To avoid measurement errors based on the inner filter effect (Palmier 2007), the linear range of the fluorescent dye was verified using a standard curve.

[0087] Fp galactosaminidase

[0088] Michaelis-Menten parameters for GalN antigen subtype 1 penta-MU and A antigen subtype 1 penta -MU was measured at 100 mM NaH2PO4, pH 7.4, 37°C. It was reacted with 3.4 nM Fp galactosaminidase (5.31 nM FpGalNase_truncA), 0.1 mg / mL SpH-ex, AfcA, 0.2 mg / mL BgaC, and substrates of various concentrations (5 μM to 2 mM) in 100 μl. A series of four reactions were performed, including a control (without Fp galactosaminidase) as a replica. Fluorescence signals (365 / 435 nm) resulting from MU release by hydrolysis were monitored using a Synergy H1® plate reader [BioTek®] and converted to concentrations using MU standard concentration curves measured under identical reaction conditions. Initial velocity (μM / s) was measured and plotted using Grafit7.0® to determine kinetic parameters.

[0089] k cat / K M The parameters are GalN antigen subtype 1 / 2 / 4 tetra- MU and B antigen subtype 1 tetra -MU was measured at pH 7.4 and 37°C. The reaction (total volume 100 μL) was carried out in a black 96-plate well, and the binding assay was performed in 100 mM NaH2PO4 (pH 7.4) with 8.63 nM Fp galactosaminidase, 0.1 mg / mL SpHex, BgaC (BgaA for subtype 2), AfcA, and substrates of various concentrations (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). A series of four reactions were performed with a control (without Fp galactosaminidase) as the replicate. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored using a Synergy H1® plate reader [BioTek®] and converted to concentration using MU standard concentration curves measured under the same reaction conditions. Determine the initial velocity (μM / s), plot it using Grafit7.0 (trademark), and k cat / K M (s -1 *mM -1 The parameters were determined.

[0090] 863.2 nM Fp galactosaminidase (at 100 mM NaH2PO4, pH 7.4) or 369.9 nM FpGH4 (at 50 mM Tris / HCl, pH 7.4, 100 μM NAD) with various substrate concentrations (10 μM to 5 mM) in 100 μl volume. + The Michaelis-Menten parameters for GalN-α-pNP in a clear 96 plate (with 1 mM MnCl2) were measured at 37°C. The reaction was carried out as a series of three reactions with two controls (enzyme-free). Absorption (at 405 nm) resulting from pNP release by hydrolysis was monitored using a Synergy H1® plate reader [BioTek®] and converted to concentration using a p-nitrophenol standard concentration curve measured under identical reaction conditions. The initial rate (μM / s) was measured and plotted using Grafit7.0® to determine the kinetic parameters.

[0091] FpGalNac Deacetylase

[0092] A antigen subtype 1 in 100 mM NaH2PO4, pH 7.4, 37°C penta- For MU, the Michaelis-Menten parameters were measured using the aforementioned binding assay (Kwan 2015). To detect cleavage of subtype 1 (later subtype 4), BgaC (Jeong 2009) was used as the β-galactosidase instead of BgaA (Singh 2014). In addition, A antigen subtype 1 penta-Since MU further contains galactose, the concentration of BgaC was increased to 0.2 mg / mL to compensate for the need to cleave both Gal-β-1,3-β-GlcNAc-β-1,3-Gal-β-MU and Gal-β-MU. Furthermore, Fp-galactosaminidase was included to enable cleavage of the galactosamine-containing intermediate. The 100 μl reaction setup consisted of 3 nM FpGalNAc deacetylase (4.52 nM FpGalNacDeAc_D1ext, 3.55 nM FpGalNacDeAc_D1+2), 0.01 mg / mL Fp-galactosaminidase, 0.1 mg / mL SpH-ex, AfcA, 0.2 mg / mL BgaC, and substrates of varying concentrations (5 μM to 2.5 mM). A series of four reactions were performed, with a control (without FpGalNac deacetylase) as a replica. The fluorescence signals (365 / 435 nm) resulting from MU release due to hydrolysis were monitored using a Synergy H1® plate reader (BioTek®), and the concentrations were converted using MU standard concentration curves measured under the same reaction conditions. The initial velocity (μM / s) was determined and plotted using Grafit7.0 to determine the dynamic parameters.

[0093] k cat / K M The parameters are A antigen subtype 1 / 2 / 4 tetra-The MU concentration was determined at pH 7.4 and 37°C. The reaction (total volume 100 μL) was carried out in a black 96-plate well. The binding assay was performed in 100 mM NaH2PO4 (pH 7.4) with various concentrations of substrates (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM) of 12 nM FpGalNAc deacetylase 0.1 mg / mL SpHex, BgaC (BgaA for subtype II), and AfcA. A series of four reactions were performed with a control (without FpGalNAc deacetylase) as the replicate. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored with a Synergy H1™ plate reader (BioTek™) and converted to concentration using the MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was determined, and then plotted using Grafit™ 7.0 to determine the kcat / KM(s-1*mM-1) parameter.

[0094] GH109 Subtype Dynamics

[0095] A antigen subtype 1 / 2 / 4 tetra-For MU, the kcat / KM parameters were measured at pH 7.4 and 37°C. The reaction (total volume 100 μL) was carried out in a black 96 plate well, and the binding assay was performed in 100 mM NaH2PO4, pH 7.4 with 0.1 mg / mL of substrates (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM NAD+, each SpHex, BgaC (BgaA for subtype 2), and AfcA, at various concentrations. A series of four reactions were performed with a control (without α-N-acetylgalactosaminidase) as the replicate. The fluorescence signals (365 / 435 nm) resulting from MU release by hydrolysis were monitored using a Synergy H1® plate reader [BioTek®] and converted to concentrations using MU standard concentration curves measured under identical reaction conditions. The initial rate (μM / s) was determined and plotted using Grafit7.0® to determine the kcat / KM(s⁻¹*mM⁻¹) parameter.

[0096] Crystal analysis

[0097] Prior to crystallization, FpGalNAcDeAc_D1ext was digested overnight with thrombin (Novagen®) at a concentration of 1 mg / mL using the manufacturer's suggested protocol. The protein was then purified using a HisTrap FF column, the effluent was collected, and the solution was buffered with 10 mM Tris pH 8.0 + 75 mM NaCl and concentrated to 12 mg / mL.

[0098] crystallization

[0099] FpGalNAcDeAc_D1ext (12 mg / mL) was crystallized at a 1:1 protein:reservoir ratio using a suspension diffusion deposition method with a reservoir solution consisting of 0.2 M CaCl2, 0.1 M MES pH6, 18% PEG4000, and 20 mM MnCl2. Rapid bromide immersion was used to derivatize the crystals for phasing, and the crystals were prepared by transferring them to a solution of 1 M NaBr, 25% glycerol, 18% PEG4000, 20 mM CaCl2, and 0.1 M Mes pH for 30 seconds, followed by flash freezing in liquid nitrogen. Crystal complexes with blood group B antigen trisaccharide (B_tri) were prepared by pre-incubating the protein (12 mg / mL) with 10 mM B_tri for 2 hours, then setting up drops under the same conditions as above, but without MnCl2. The crystals were cryoprotected in a reservoir solution supplemented with 25% glycerol.

[0100] Data collection, phasing, structure determination

[0101] The dataset was collected using Canadian Light Source™. The data was integrated using XDS (Kabsch 2010) and scaled with Aimless™ (Evans 2013). Fading and automated structural solutions were performed using CRANK2™ (Skubak 2013) in the CCP4I2™ program suite (Potterton 2018). Structures were checked and refined using alternating cycles of Coot™ (Emsley 2004) and Refmac™ (Vagin 2004). B_tri structural complexes were solved by differential Fourier, and ligands were manually constructed in Coot™ as well as water and metal ions. Differential density maps were used to identify Mn in the apo structure. 2+ Ca in the coordination structure 2+The existence of [the compound] was confirmed. The model was validated by Coot® and Molprobity® (Chen 2010). The atomic coordinates and structure factors of the apo and B_tri complexes are deposited in the Protein Data Bank (PDB) with the following accession numbers: Flavonifractor plautiGalNAc deacetylase protein SEQ ID NO: WP_009260926.1, and Flavonifractor plautigalactosaminidase protein SEQ ID NO: WP_044942952.1.

[0102] Active site mutagenesis

[0103] Based on structural information (not shown) and sequence alignment (not shown), FpGalNAcDeAc_D1min and FpGalNase_truncA were mutated using the QuickChange® protocol (Zhang 2004) with the primers listed in Table B. The mutants were purified via NiNTA and HIC columns as described above. The structural integrity of all mutants was confirmed by CD spectroscopy. This confirmed that all test enzymes were structurally similar to the wild type. For mutants with relatively low activity, the reaction was carried out under the same conditions used for complete kinetic determination. However, k cat / K M As mentioned above, the substrate consumption method was used to measure the values ​​(Vocadlo 2002). In short, [substrate] <K M (K m In low concentrations of substrate (corresponding to ~1 / 5-1 / 10 of the above), k cat / K M The value can be approximated by fitting the reaction time course to a linear curve non-linearly and dividing by the enzyme concentration.

[0104] GH36 System Occurrence Mapping

[0105] The GH36 reference sequence was downloaded from the CAZy® database using the SACCHARIS® cazy_extract.pl script (Jones 2018). A reference tree was constructed using phylogenetic-based protein profiling software, TreeSAPP® (available at https: / / github.com / hallamlab / TreeSAPP), and the sequences were mapped to these trees. Briefly, protein family domains were extracted from all full-length sequences downloaded from CAZy® (Yin 2012) using HMMs from dbCAN. These sequences were then clustered with 70% sequence similarity using UCLUST® to remove redundant sequence spaces and reduce tree size (Edgar 2010). We used RAxML® version 8.2.0 to build the reference tree, determined when to terminate the bootstrap before 1000 replication iterations using "--autoMRE", and selected the optimal protein model using PROTGAMMAAUTO® (Stamatakis2006 and Stamatakis2008).

[0106] Next, the query sequences were mapped to these reference trees using TreeSAPP(trademark). Briefly, the protein sequences were aligned to HMMs using hmmsearch(trademark), and the aligned regions were extracted (Eddy 1998). New query sequences were included in the reference multiple alignment using hmmalign(trademark), and TrimAl(trademark) removed unsave positions from the alignment file (Capella-Gutierrez 2009). Query sequences in the reference trees were classified by insertion using RAxML(trademark). The placement of each query sequence was filtered and concatenated into a single file. Jplace(trademark) file before display in iTOL(trademark) (Matsen 2012 and Letunic 2016).

[0107] RBC assay

[0108] Whole blood from healthy, consenting donors was collected in a citrate vacutainer using a protocol approved by the Clinical Ethics Committee of the University of British Columbia. The tube was rotated at 1000 × g for 4 minutes at room temperature to separate RBCs, which were then washed three times with 1x PBS pH 7.4. For assays in the presence of dextran 40k, the washed RBCs (200 μL, 10% hematocrit) were placed in a tube, the supernatant was partially removed, and the mixture was replaced with 1x PBS pH 7.4 in and without dextran 40k (final concentration 300 mg / mL). In addition, several assays were performed in 1x PBS pH 7.4 + 25% plasma or 100% plasma. The RBCs were carefully mixed and placed on an orbital shaker for 30 seconds. The diluted enzyme solution was then added to a final volume of 200 μL. The tube was very gently vortexed and placed on an orbital shaker at the set temperature for the specified time.

[0109] MTS Card

[0110] After the reaction, RBCs were washed three times with an excess of 1x PBS pH 7.4 and analyzed using a Micro Typing System (MTS) card [MTS (MTS), Florida, USA]. RBCs (12 μl, 5% hematocrit) suspended in diluent [MTS, Florida, USA] were carefully added to the minigel column, leaving a space between the blood and the minigel contents. The MTS card was centrifuged at 156xg for 6 minutes at room temperature using a Beckman Coulter Allegra X-22R (MTS) centrifuge with a sample holder modified as recommended. The degree of antigen removal from the RBC surface was assessed by the position of the RBCs in the minigel after rotation, according to the manufacturer's instructions. RBCs with high surface antigen concentrations agglutinated in interaction with the monoclonal antibody present in the gel column and could not penetrate (MTS (MTS) score 4). RBCs without surface antigen did not agglutinate and migrated to the bottom of the minigel (MTS score 0). RBCs that underwent partial removal of surface antigens moved to positions between these locations and were assigned scores between 0 (absent) and 4 (present) according to the manufacturer's instructions.

[0111] H antigen aggregation assay

[0112] To analyze the conversion of A antigen to H antigen after enzymatic treatment, washed A-ECO-RBCs were mixed in an equal volume with 2 μg / mL of anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213 (Abcam®)), and the appearance of agglutination was monitored within a 30-minute time frame. RBCs that agglutinated with anti-H antibody were assigned a score between 0 (no agglutination within 1800 seconds) and 5 (agglutination within 120 seconds).

[0113] FACS

[0114] Enzyme-treated RBCs were washed twice with 1x PBS pH 7.4, and 1% hematocrit ECO-RBCs were treated with 1 / 100 APC-anti-A antibody (Alexa Fluor® 647 mouse anti-human blood group A: cat no. 565384 (BD Pharmingen®)) and / or anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213 (Abcam®)) at room temperature for 30 minutes, followed by two washes with 1x PBS pH 7.4. For detection of anti-H antibody, a secondary FITC-labeled antibody (goat F(ab′)2 anti-mouse IgM mu chain (FITC): cat no. ab5926 (Abcam®)) was used at a concentration of 1 / 500. The data were reconstituted into 1x PBS pH 7.4 (hematocrit 1%) using a flow cytometer (CytoFLEX® (Beckman Coulter®)) and then evaluated.

[0115] Enzyme adsorption and antigenicity

[0116] To test whether the enzyme could be easily removed from RBCs after treatment, adsorption potential was evaluated. Pacific blue-labeled FpGalNAc deacetylase and FpGalNase (F / P=1) were incubated with RBCs at 37°C for 1 hour, and after several washes, residual fluorescence was measured using a flow cytometer (CytoFLEX® (Beckman Coulter®)).

[0117] Antigenicity was tested by incubating RBCs with 50 μg / mL of each enzyme, mixing the enzyme-treated RBCs with allogeneic or autologous serum, and observing the possibility of agglutination. In addition, to assess potential anti-IgG,-C3d exposure, treated RBCs were tested with anti-IgG,-C3d MTS® cards [MTS®, Florida, USA]. The incubation time was 30 minutes at 37°C.

[0118] Synthesis of antigen subtypes

[0119] The synthesis of A and B antigen subtypes 1 / 2 / 4tetra-MU was performed using the modified protocol described in Kwan (Kwan et al. 2015).

[0120] Two-step H antigen subtype 1 / 2 / 4 tri-MU synthesis

[0121] All three syntheses were performed in 10 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl2, 50 U alkaline phosphorylase, 1.5 equivalents UDP-Gal, and 1.2 equivalents GDP-Fuc (scaled with the LacNAc-MU product) using a 20 mg GalNAc-α-MU scale. Depending on the desired product, various glycosyltransferases were added at a concentration of 100 μg / mL. Specifically, CgtBS42 and Te2FT were used for subtype I, HP0826 and WbgL for subtype II, and LgtD and Te2FT for subtype IV. The reaction was carried out at 37°C, and its progress was controlled by TLC (mobile phase ratio EtAc:MeOH:H2O, 6:2:1). 4-methylumbelliferone was hydrolyzed from the compound via 10% H2SO4 and detected by UV (360 nm). After no further increase in product was observed, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure.

[0122] A antigen subtype 1 / 2 / 4 tetra- MU synthesis

[0123] The final synthesis step is 10 mg H antigen subtype 1 / 2 / 4 tri- The reaction was carried out at 37°C in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl2, 25 U alkaline phosphorylase, 1.5 equivalents UDP-GalNAc, and 100 μg / mL BgtA on a MU scale. After no further increase in product was observed, the reaction was applied to an HF Bond Elut C18 column via TLC, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure. The final product was further purified on a 1.5 × 46 cm HW-40F size efflux column and then lyophilized.

[0124] B antigen subtype 1 / 2 / 4 tetra- MU synthesis

[0125] The final synthesis step is 10 mgH antigen subtype 1 / 2 / 4 tri- The reaction was carried out at 37°C in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 25 U alkaline phosphorylase, 1.5 equivalents of UDP-Gal, and 100 μg / mL BoGT6a on a MU scale. After no further increase in product was observed, the progress was tracked via TLC, and the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure. The final product was further purified on a 1.5 × 46 cm HW-40F size exclusion column and then lyophilized.

[0126] GalN antigen subtype 1 penta- MU synthesis

[0127] 10 mg of A antigen subtype 1 penta-MU was incubated with 1 μg / mL FpGalNAc deacetylase in 5 mL of 100 mM NaH2PO4 at 37°C for 30 minutes, then 1 mM EDTA was added to stop the reaction. Complete substrate conversion was confirmed by TLC, the reaction product was applied to an HF Bond Elut C18 column, washed with several column volumes of 2% methanol, and the product was eluted with 10% methanol. The solvent was then removed under reduced pressure.

[0128] Protein purification

[0129] All proteins and their cleavage sequences were cloned into pET16b or pET28a via Golden Gate® cloning (Engler 2008) or PIPE cloning (Klock 2008). Primer sequences are listed in Table B.

[0130] Protein production for extended characterization was performed in BL21(DE3) cells, cultured for 20 hours at 37°C and 220 rpm in 200 mL of ZY5052 automated induction medium (Studier 2005), and inoculated into 100 μl of overnight LB cultures. Cells were collected by centrifugation (4000xg, 40°C, 10 min) and resuspended in 10 mL of lysis buffer (50 mM Tris / HCl, 150 mM NaCl, 1% (v / v) glycerol, 40 mM imidazole, pH 7.4, 2 mM DTT, 1x protease inhibitor EDTA-free (Pierce®), 2U benzonase (Novagen®), 0.3 mg / mL lysozyme, 10 mM MgCl2), and then subjected to ultrasonic treatment on ice (3-minute pulse duration; 5-second pulse, 10-second pause, 35% amplitude). After removing cell residue by centrifugation (14000xg, 4°C, 30 min), the supernatant was collected and loaded onto a nickel affinity chromatography column (5 mL HisTrapHP® column (GE®)) using a peristaltic pump. Elution was performed on an AEKTApurifier™ system (GE™), and the protein-containing fractions were identified and pooled by monitoring via SDS-PAGE with a 10–75% gradient of 50 mM Tris / HCl, 400 mM imidazole, pH 7.4, and 2 mM DTT. Buffer exchange to 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, and 2 mM DTT, followed by concentration, was performed in Amicon Ultra-15 Centrifugal Filter Units™ MWCO 10 kDa (Millipore™).

[0131] FpGalNAc deacetylase, Fp galactosaminidase, and their cleavage required a second round of purification. Using Amicon Ultra-15 Centrifugal Filter Units MWCO 10kDa (Millipore®), buffer was replaced before loading the proteins onto a hydrophobic interaction chromatography column (Phenyl Sepharose High Performance Column 10 mL (Pharmacia Biotech®)). Column loading, washing, and elution (gradient 0–100%) were performed through an AEKTApurifier (GE®) using the following buffer conditions: FpGalNAc deacetylase bound to 1x PBS, 800 mM NH2PO4, pH 7.4 and eluted to 1x PBS, pH 7.4; and Fp galactosaminidase bound to 25 mM Tris / HCl, 1 M NaCl, pH 7.4 and eluted to 25 mM Tris / HCl, pH 7.4. Protein-containing fractions were identified and then pooled by SDS-PAGE. Buffer exchange to 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, and concentration were performed in Amicon Ultra-15 Centrifugal Filter Units (trademark) MWCO 10 kDa (Millipore (trademark)).

[0132] Protein characterization

[0133] Optimal pH value

[0134] A antigen subtype 1 penta- MU and GalN antigen subtype 1 penta- The general pH ranges for the activity of FpGalNAc deacetylase and Fp galactosaminidase against MU were determined by the appearance of products on TLC plates at different pH values. Reactions were carried out on a 100 μl scale at 37°C with 50 μM substrate and 1 μg / mL enzyme in appropriate buffer systems. Buffers for pH 4–6 were based on 50 mM citrate / sodium citrate buffer, for pH 6–8 on 50 mM sodium phosphate buffer, and for pH 8–10 on 50 mM glycine / sodium hydroxide buffer.

[0135] To determine the optimal pH value, 5 μg / mL Fp galactosaminidase was incubated in 100 μl of 50 mM sodium phosphate buffer with 200 μM GalN-α-pNP within a pH range (5.8–8.0), and the absorption (at 405 nm) resulting from pNP release was monitored for 1 hour at 37°C using a Synergy H1® plate reader (BioTek®).

[0136] 5 μg / mL FpGalNAc deacetylase and 50 μM A antigen subtype Ipenta-MU were pre-incubated for 10 minutes at 37°C in 25 mM sodium phosphate buffer at various pH ranges (5.8–10.0). The reaction was quenched with 100 mM sodium phosphate buffer pH 7.5, 100 μM EDTA, 5 μg / mL Fp galactosaminidase, 50 μg / mL SpHex, 50 μg / mL AfcA, and 50 μg / mL BgaC, with a final volume of 100 μl. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored for 30 minutes at 37°C using a Synergy H1® plate reader (BioTek®).

[0137] Protein stability

[0138] FpGalNAc deacetylase and FpGalNase were stored in 1x PBS buffer, pH 7.4, at 4°C. After 2 weeks and 12 weeks, A antigen subtype 1 was analyzed. penta- As described regarding the optimal pH for MU, enzyme activity was tested by the binding enzyme reaction between FpGalNAc deacetylase and GalN-α-pNP to FpGalNase.

[0139] FpGalNAc deacetylase inhibitor

[0140] FpGalNAc deacetylase was tested against various potential inhibitors in a binding assay in a 96-well plate format. In 100 μL scales at 37°C, 50 μM A antigen subtype 1penta-MU and 5 μg / mL FpGalNAc deacetylase were reacted with 10 μg / mL Fp galactosaminidase, 50 μg / mL SpHex, 50 μg / mL AfcA, and 50 μg / mL BgaC in 100 mM NaH2PO4 pH 7.4. As inhibitors, EDTA (1, 10, 100 μM), Marimast (1, 10, 100, 1000 μM), DMSO (2%, 4%), and Protease Inhibitor Cocktail EDTA-free (Pierce®) (1x, 2x, and 4x) were tested. Fluorescence (365 / 435 nm) was continuously monitored for 1 hour using a Synergy H1® plate reader (BioTek®). Additives showing a strong effect were retested without the conjugating enzyme, and product formation was analyzed by TLC.

[0141] Limited disassembly

[0142] Limited proteolysis was performed to investigate whether smaller, more stable subdomains of Fp-galactosaminidase exist. Fp-galactosaminidase was treated with thermolysin (protein:protease mass ratio 10:1) at various temperatures (20°C, 37°C, 42°C, 50°C, and 65°C) for 1.5 hours. The samples were then electrophoresed on an SDS-PAGE gel, and stable fragments were identified by electrophoresis at approximately 70 kDa (from the initial 118 kDa). Nearly complete degradation was achieved at an incubation temperature of 50°C. These fragments were sent to the UBC Proteomics Core Facility for peptide identification and were determined to be a C-terminal cleavage of the full-length protein with a cleavage site between amino acids 690–700.

[0143] Glycan array screening

[0144] For glycan array screening, 500 μg of FpGalNAcDeAc_D 2ext was labeled with fluorescein isothiocyanate (FITC) at an F / P ratio of 1 using the Fluorotag® FITC binding kit (Sigma®). Screening was performed on a printed array of Protein-Glycan Interaction Core Facility® version 5.3 of CFG, consisting of 600 glycans in 6 replicates at protein concentrations of 5 and 50 μg / mL. Binding motif analysis was performed using web tools from Emory University (https: / / glycopattern.emory.edu / ).

[0145] (Examples) Example 1: Construction and screening of a metagenomic library

[0146] AB +We constructed a metagenomic library containing large (35–65 kb) fragments of DNA extracted from fecal samples provided by male blood type donors. Such libraries contain multiple genes per bacterium, increasing the probability of expression of at least some of these genes and enabling the expression of small "pathways" of multiple genes. Our library contained approximately 19,500 clones in a 51 × 384 well plate and potentially contained about 800,000 genes; therefore, initial screening of such a library using expensive A antigen substrates was not practical. Rather, we screened using methylumbelliferyl α-glycosides of galactose and N-acetyl-galactosamine (Gal-α-MU and GalNAc-α-MU), which are simple and highly sensitive fluorescence-generating substrates. An initial screening with a mixture of these two substrates yielded a subset of 226 hits. These were re-screened for each individual substrate, and 44 were identified by GalNAcase and 166 by galactosidase activity. The second round of screening was performed using a conjugation enzyme assay (Kwan 2015) with the A and B antigen tetrasaccharide glycoside substrates shown in Figure 1, along with a control group without substrate. The conjugation enzyme was only able to act and release MU if the first Gal or GalNAc was cleaved. Of these hits, 11 contained A antigen cleavage activity, one of which also cleaved the B antigen, while 6 produced fluorescence in the absence of substrate, thus encoding a pathway that produces unrelated fluorescent products.

[0147] Example 2: Hit sequencing and initial analysis

[0148] Eleven plasmids were sequenced on Illumina MiSeq®, and ORFs present in the CAZy® database (http: / / www.cazy.org / ) (Lombard 2014) were identified using Metapathways® software (Konwar 2015). Due to the considerable depth of human microbiome sequencing currently available, it was possible to identify the organisms from which all phosmids originated. From eight of the eleven species derived from duplicate genome fragments of two Bacteroides species, their sequences could be classified into five clusters. The only gene common to all phosmids in cluster B is the GH109 enzyme (B. vulgatus). Cluster A also includes GH109 (B. stercoris), but GH109 is the only CAZy gene found in other Bacteroides-derived phosmids (B. vulgatus). Fosmid N08, derived from the obligate anaerobic bacterium Flavonifracter plautii (Li 2015), contains three ORFs found within CAZy: the apparent glycosylation module CBM32 and two latent glycoside hydrolases GH36 and GH4. Finally, fosmid K05, derived from Collinsella sp., possibly Collinsella tanakaei, does not contain CAZy-related ORFs. Here, the generation of a sublibrary of fosmid K05 allowed for the identification of ORFs with A-cleavage activity, which were later identified as GH36 (not shown).

[0149] Example 3: Analysis of GH109 enzyme

[0150] The GH109 family was discovered based on the A antigen cleavage activity of some of its members. These enzymes have unusual NADs. +This method utilizes a dependent mechanism, which was first discovered in the enzyme described in GH4 Add Yip Ref (2004) J.Amer.Chem.Soc. 126, 8354-8355 (Varrot 2005 and Liu 2007). The three GH109 genes identified here were cloned after removing the signal peptide and tagging with His, and expressed in Escherichia coli BL21 (DE3). These three proteins, BsGH109, ​​BvGH109_1, and BvGH109_2 (not shown), were purified together with standard GH109 from Elizabethkingia meningosepticum (EmGH109) (Liu 2007), and their kinetic parameters were determined. The three novel enzymes showed similar catalytic efficiency to each of the three A subtype substrates tested, mainly reflecting the kinetic parameters of standard EmGH109. In contrast, using an approved MTS card, A + When their A antigen cleavage activity was tested in RBCs, unfortunately, only EmGH109 showed significant activity. The tests were conducted in the presence of dextran 40K as a crowding agent, which has been shown to increase activity by accumulating enzymes on the cell surface (Chapanian 2014). In its absence, even 150 ug / mL of EmGH109 showed no effect, but in the presence of 300 mg / mL of dextran 40K, 15 μg / mL of the enzyme was sufficient (see Figures 3 and 4). Previous studies have also shown that low ionic strength also increases the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is not effective in the overall bloodstream.

[0151] Example 4: Analysis of GH36 derived from fosmid K05 from Collinsella sp.

[0152] The GH36 protein identified in fosmid K05 (named K05GH36) showed activity against GalNAc-α-MU and the A antigen tetrasaccharide. This is consistent with its membership in the GH36 family, which primarily includes α-galactosidase and α-N-acetylgalactosaminidase and performs hydrolysis via a double substitution mechanism involving a covalently bound β-glycosyl enzyme intermediate (Comfort 2007). Phylogenetic analysis placed its sequence within cluster 4 of the GH36 subfamily (Fredslund 2011). Interestingly, this cluster also contains a characterized GH36 derived from Clostridium perfringens, which is known to cleave the A antigen structure (Calcutt 2002). However, when the ability of K05GH36 to remove the A antigen from erythrocytes was tested, its activity was disappointing, scoring only 3 even when used in combination with a crowding agent.

[0153] Example 5: Analysis of fosmid N08 derived from Flavonifractor plautii

[0154] Since these novel enzymes did not offer any advantages, we focused on N08 fosmid from F. plauti, particularly because its gene product cleaves both A and B antigens. Three CAZ-related genes were replicated, their signal peptide sequences removed, and expressed in E. coli BL21(DE3). The resulting enzymes were purified to yields up to 140 mg / L. Surprisingly, when the individual purified proteins were tested against A and B tetrasaccharide substrates, the only cleavage observed was cleavage of the B antigen by N08GH36, with no cleavage of the A antigen by either of them. Therefore, we tested pair combinations of these enzymes and were surprised to find that a mixture of N08CBM32 and N08GH36 rapidly cleaved the A antigen tetrasaccharide. TLC analysis of the reaction mixture with individual enzymes revealed that N08CBM32 catalyzes the conversion of the A antigen to a more polar but still UV-active product, while subsequent addition of N08GH36 released a sugar product that co-migrated with galactosamine along with the H antigen trisaccharide. MS analysis of the reaction mixture showed that N08CBM32 is an A antigen deacetylase, hence the 42 m / z and the decrease in the more polar product, while N08GH36 is a galactosaminidase, exhibiting novel activity within this family (Figure 2). This was further confirmed by high-performance anion exchange chromatography (HPAE-PAD) analysis of the reaction (Figure 5), which showed that treatment of the A antigen by both enzymes releases galactosamine, but not by the individual enzymes. Similar results were obtained with gastric viscous substrates, suggesting that this enzyme may have evolved specifically for this substrate. Therefore, these two enzymes will henceforth be called FpGalNAc deacetylase (FpGalNAcDeAc) and Fp galactosaminidase (FpGalNase).

[0155] This pathway of A antigen degradation had not been characterized until recently, but interestingly, it was suggested more than 50 years ago as an explanation for the so-called "acquired" B phenomenon. In this phenomenon, type A patients infected with Clostridium tertium showed a clear change in blood type to B, similar to forensic samples of human tissue submerged in the Thames River (Ref Judd and Annesley https: / / doi.org / 10.1016 / S0887-7963(96)80087-3,Transfusion medicine reviews(1996)10,111-117) (Gerbal 1975). This was likely because the anti-B antibodies used for typing could not distinguish between terminal Gal and GalN.

[0156] When we examined GH4, the third enzyme in fosmid, we found that it hydrolyzes Gal-α-pNP, GalN-α-pNP, and GlcN-α-pNP, but does not cleave substrates based on the A antigen. Therefore, it does not appear to be directly involved in the conversion of the A antigen. However, these glycosaminidases exhibit novel activity within the GH4 family.

[0157] Example 6: Characterization of FpGalNAc deacetylase

[0158] A more detailed bioinformatics analysis of this gene by Phyre2(trademark) (Kelley 2015) revealed the presence of a previously unknown 308-amino acid domain at the N-terminus, a 145-amino acid CBM32 near the C-terminus, and a linker region between them. Since all constructs, including the unprocessed deacetylase domain, exhibited catalytic activity (Table 2), this basic structure was confirmed by cleavage analysis. Therefore, this protein is classified as a founding member of a new glycan esterase family, CExx.

[0159] It has been proven that all acetamide sugar deacetylases are metalloenzymes that require divalent metal ions (Blair 2005). Consistent with this, when treated with 100 μM EDTA, the enzyme activity was almost completely lost, but Mn 2+ Co 2+ Ni 2+ or Zn 2+ The addition of increased activity. Other inhibitors of (nonmetallic) amidase were ineffective. The optimal pH for this enzyme was around 8 (Figure 6), and its substrate specificity was narrow, limited to different A subtypes and shorter versions. However, among these subtypes, it was not very distinguishable, with only about a twofold difference in specific activity between all of them (Table 2). Such pH dependence and specificity profile are ideal for RBC conversion, as all subtypes of A are deacetylated, but none are.

[0160] The specificity of the CBM portion of the protein was investigated using the Consortium for Functional Glycomics (CFG) glycan array. Preferred targets were glycans with repeating N-acetyllactosamine (LacNAc) structures, as seen in the founding member of the CBM32 family, namely N-acetylglucosaminidase from Clostridium perfringens (Ficko-Blean 2006). However, unlike CBMs, our composition does not exhibit high affinity binding to blood antigen structures. Repeating LacNAc structures, as well as some O-glycans and glycolipids, are common components of the cell surface as common components of complexes and hybrid N-glycans (Cohen 2009). In our case, these likely act as anchor points for the deacetylase domain to bind, allowing its catalytic domain to approach the A antigen without competing with its own substrate. Supporting this model, removal of the domain reduced RBC activity without affecting the cleavage rate of the soluble substrate (Table 2).

[0161] Example 7: Crystallographic analysis of FpGalNAc deacetylase

[0162] To provide structural insight into this novel enzyme activity, the cleaved protein was subjected to crystallization tests, and it was found that FpGalNAcDeAc_D1ext produced crystals that diffracted at the best resolution. A solution of this structure revealed a catalytic domain employing a five-fold β-propeller structure with an active site containing a divalent metal ion coordinated by D100 and H252. Co-crystallization of the enzyme with the B antigen trisaccharide, an analog of the reaction product, revealed its binding mode. At the base of the active site pocket, the non-reducing galactosyl moiety, the distinguishing group between A and B antigens, interacts with H97, E64, and two metal-coordinated water molecules via hydrogen bonding. The remaining ligands are surface-exposed, and polar interactions between the fucosyl group and the S61 and D121 side chains are identified. Since the C1-OH group of the reducing galactosyl moiety is exposed to the solvent, its extension to the substrate (i.e., in GlcNAc) is readily regulated by the enzyme. By modeling the N-acetyl group of A-trisaccharides onto this structure, we were able to create rational mutations in nearby amino acids that may be involved in substrate deacetylation. Since residue E64 was inactive in both mutants, it was found to be important for activity, suggesting it likely plays a direct role in the activation of nucleophilic water molecules (Table 1). Residues coordinating the divalent metals D100, Y315, and H252 were also found to be important, and each mutation resulting in approximately a 5000-fold decrease in rate is consistent with their apparent roles in the binding of divalent metal ions. By analogy with other acetamide sugar deacetylases, we propose that FpGalNAc deacetylase hydrolyzes by polarizing the carbonyl molecule and activating a water molecule for nucleophilic attack on the carbonyl molecule, forming a tetrahedral intermediate. The degradation of this intermediate is facilitated by proton donation to the sugar nitrogen atom by His100.

[0163] Table 1|A antigen Type2 tetra -Specific activity of its variant FpGalNAcDeAc_D1min against MU cleavage JPEG0007862477000007.jpg77153

[0164] Example 8: Characterization of FpGalNAcDeAc and FpGalNase

[0165] In phylogenetic sequencing, FpGalNase was placed in a new subgroup of the GH36 family (5) (Fredslund 2011). The 390-amino acid catalytic domain is located at the center of this large (1079 amino acid) protein and has a potential glycosylation domain at its C-terminus. Removal of this C-terminal domain did not affect the enzyme's dynamic parameters with soluble substrates (Table 2), but deacetylation A + It reduced the cleavage efficiency of RBCs. This enzyme is specific to galactosamine-containing sugars and does not cleave GalNAc residues under any of the conditions tested. However, it has fairly broad specificity for cleaving de-N-acetylated galactosaminides ranging from simple aryl glycosides GalN-α-pNP to higher-order ones. In fact, (Table 2) the k of the three A subtypes tested cat / K M The values ​​were all similar to each other and also similar to the deacetylase values. k for cleavage of B antigen cat / K M The value was more than 2000 times lower than the value for the corresponding GalN antigen, but it was still sufficient to produce a positive hit on the original screen. This specificity for the deacetylated α-galactose constituent substrate, combined with its optimal pH of approximately 6.5–7.0, makes it suitable for use in blood type conversion with deacetylase (Figure 6).

[0166] Table 2 | Dynamic parameters of FpGalNAcDeAc and FpGalNase constructs against different antigen substrates JPEG0007862477000008.jpg149160

[0167] Example 9: Cleavage of A antigen from RBCs

[0168] A + B + and O + Type A RBCs were incubated with FpGalNAcDeAc and FpGalNase individually, as well as in a mixture, and the released sugars were analyzed by HPAE-PAD ion chromatography. Neither enzyme released any sugar products individually. However, when a mixture of the two was used, galactosamine was released. + It was clearly released from RBC, but B + or O + It was not released from the RBC surface and showed high specificity only for the A antigen. This is very important because it indicates that GalNAc is not released from the RBC surface under other conditions. The cleaved form of FpGalNase was also effective, but its activity was somewhat lower.

[0169] Next, we performed tests to remove the antigen from RBCs using industry-standard MTS® cards. RBCs were loaded onto these antibody-conjugated columns and centrifuged. RBCs without antigen moved to the bottom of the column and were scored as 0, while untreated RBCs containing the corresponding antigen adhered to the top and were scored as 4, with the degree of antigen removal being ranked by the intermediate score. FpGalNase treatment alone did not remove A or B antigenicity at the concentrations shown in Table 3, which is consistent with its inactivity to the GalNAc substrate and low activity to Gal. Incubation with FpGalNAcDeAc removed antigenicity by converting acetamide to an amine, weakening the binding of the anti-A antibody used. The minimum amount of enzyme required for complete antigen deacetylation was evaluated using FpGalNAcDeAc alone and in combination with FpGalNase, both with and without dextran 300 mg / ml as a crowding agent. FpGalNase concentrations up to 3 μg / ml were sufficient without dextran, but the inclusion of 300 mg / ml of dextran reduced the required load to 0.5 μg / ml (Table 3). The conventional best enzyme, EmGH109, ​​was ineffective in the absence of dextran unless a low-salt buffer was used, but in the presence of dextran, the minimum effective concentration was 15 μg / ml, which was a 30-fold higher load. Versions of FpGalNAcDeAc lacking CBM were far less effective.

[0170] Table 3|A + B + and AB + Results of MTS cards when RBCs are processed with EmGH109, ​​FpGalNAcDeAc, and FpGalNase. JPEG0007862477000009.jpg140156

[0171] Since the MTS® card test does not assess the complete conversion of the A antigen, and antibodies to detect the GalN antigen were unavailable, we focused on detecting the newly formed H antigen on treated RBCs. FpGalNase was functional at a concentration of only 5 μg / ml and resulted in an increase in H antigen levels simultaneously with the loss of the A antigen, as confirmed by FACS analysis shown in Figure 3. By measuring the agglutination time in the presence of anti-H antibody, we were able to identify several individuals with A + The functionality of both enzymes for RBC donors, and their ability to perform whole blood reaction under conditions unattainable by other blood-converting enzymes, were demonstrated. Thus, this enzyme pair can perform A at a much lower enzyme load than the best conventional enzymes require. + RBCs are converted to type O "general donor" RBCs. However, before transfusing these RBCs to a patient, it is recommended to remove all trace amounts of enzymes used in the conversion, most likely by washing the cells after centrifugation, to avoid harmful immune reactions. To confirm that this can be achieved, fluorescently labeled samples of FpGalNAcDeAc and FpGalNase are used. + The RBCs were treated, and then FACS analysis was used to confirm that simple washing was indeed effective (Figure 3).

[0172] Further characterization of the produced A-ECO RBCs may be useful to assess their full viability for use in transfusion medicine, but the possibility of directly incorporating the enzyme into plasma, as is possible during blood collection, may allow for easy and cost-effective implementation into existing automated routines of blood collection and storage, away from the process. In particular, the stability of the enzyme was tested, as shown in Table 4. Table 4: Storage stability of galactosaminidase and GalNAc deacetylase JPEG0007862477000010.jpg92157

[0173] Example 10: GalNAc deacetylase and galactosaminidase fusion derived from Clostridium tertium

[0174] In the search for similar enzymes, we identified a novel Clostridium tertium spontaneous fusion of galactosaminidase and GalNAc deacetylase linked by a CBM (GH36 domain-CBM-deacetylation domain). Initial tests showed that this enzyme cleaved the A antigen on erythrocytes (using the same mechanism: first deacetylation, then galactosamine cleavage), but not very efficiently (i.e., similar to EmGH109). While the Clostridium tertium deacetylation domain is less efficient than F. plauti GalNAc deacetylase, when assisted by F. plauti GalNAc deacetylase, the Clostridium tertium galactosaminidase domain exhibits similar activity to F. plauti galactosaminidase on erythrocytes.

[0175] Example 11: Alternative GalNAc deacetylase and galactosaminidase enzymes

[0176] The data show that Clostridium tertium, galactosaminidase (Ct5757_GalNAse), and Rp1021 have comparable enzymatic activity for the conversion of GalN antigen to H antigen (second reaction step).

[0177] Furthermore, data on alternative GalNAc deacetylase and galactosaminidase enzymes were collected and compared with the GalNAc deacetylase and galactosaminidase of Flavonifractor plauti. As shown in Table 5, the MTS scores for anti-A antibodies on treated A RBCs were shown for the Clostridium tertium native fusion of galactosaminidase and GalNAc deacetylase, which requires the presence of dextran to effectively cleave the A antigen and also shows good activity (Ct5757_DeAcase) when combined with Flavonifractor plauti galactosaminidase (FpGalNase). Furthermore, Table 6 shows that while Robinsoniera peoliensis (Rp) Rp3672 and Rp3671 can deacetylate the A antigen on RBCs, their efficiency is lower than that of FpGalNAcDeAcase, and their activity is only achieved in the presence of a crowding agent (i.e., dextran 40k).

[0178] Table 5: MTS scores of anti-A antibodies on treated A RBCs JPEG0007862477000011.jpg53153

[0179] Table 6: MTS scores of Robinsonia peoliensis (Rp) 3671 and 3672 JPEG0007862477000012.jpg36148

[0180] Figure 7 shows the conversion of A antigen to H antigen on A RBCs, analyzed by FACS screening, for (A) A+RBC control, (B) Flavonifractor plauti GalNAc deacetylase (FpGalNAcDeAc) + Flavonifractor plauti galactosaminidase (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertium (Ct)Ct5757_GalNase (10 μg / mL), and (D) FpGalNAcDeAc + Robinsoniera peoliensis (Rp) galactosaminidase (Rp1021)GalNase (10 μg / mL). The data shows that Clostridium tertium (Ct)Ct5757_GalNase and Robinsonia peoliensis (Rp) galactosaminidase (Rp1021)GalNase exhibit similar enzymatic activity to Flavonifractor plautigalactosaminidase in the conversion of GalN antigen to H antigen (second reaction step).

[0181] While various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the invention according to the general knowledge of those skilled in the art, and such modifications include the substitution of known equivalents to any aspect of the invention in substantially the same way and to achieve the same results. Numerical ranges encompass the numerical values ​​that define the range. In this specification, the term “comprising” is used as a non-restrictive term substantially equivalent to the phrase “including, but not limited to,” and the term “comprises” has the corresponding meaning. Where used herein, the singular forms “a,” “an,” and “the” include multiple referents unless explicitly stated in the context. Thus, for example, a reference to “a thing” includes multiple such things. Citations of references herein do not constitute an admission that such references are prior art of embodiments of the present invention. The present invention includes all embodiments and variations substantially as described above with reference to the examples and drawings.

[0182] (array) The Flavonifractor plauti DNA sequence was modified from a naturally occurring DNA sequence (GalNAc deacetylase 2311 / 2319nt / galactosaminidase 3228 / 3237nt). In particular, there was a difference in the length of the sequence used for protein purification, which resulted in the removal of the signal peptide and the addition of an N-terminal His tag via the vector backbone.

[0183] Informal array list

[0184] Sequence ID 2

[0185] Description: Flavonifractor plauti GalNAc deacetylase (protein sequence) MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVAD GEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAK SEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTN GIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0186] Sequence ID 4

[0187] Description: Flavonifractor Plauti GalNAc deacetylase (removal signal peptide protein sequence) ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSP TIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAE NSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEY ELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0188] Sequence ID 5

[0189] explanation: His tag Flavonifractor Plauti GalNAc deacetylase (pET16a-protein sequence) MG THHHHHHHHHHSSGADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDML SPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYA ENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEY ELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0190] Sequence ID 7

[0191] Description: Flavonifractor plautigalactosaminidase

[0192] Sequence ID 9

[0193] Description: Flavonifractor plautigalactosaminidase (removal signal peptide protein sequence)

[0194] Sequence ID 10

[0195] explanation: His tag Flavonifractor plautigalactosaminidase (pET16a-protein sequence) MG THHHHHHHHHH

[0196] Sequence ID 12

[0197] Description: Clostridium tertium isolated protein sequence 099345757.1-Ct5757 (a fusion of galactosaminidase and GalNAc deacetylase linked by CBM (original protein sequence))

[0198] Sequence ID 14

[0199] Description: Isolated protein sequence of Clostridium tertium 5757 (Ct5757) excluding the signal peptide (identification number 099345757.1-Ct5757)

[0200] Sequence ID 15

[0201] explanation: His tag and Clostridium tertium 5757 (Ct5757) fusion protein sequence expression constructs having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0202] Sequence ID 17

[0203] explanation: His tag and a Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH SSGLVPRGSHSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLR GVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWM IMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVEL RESDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0204] Sequence ID 19

[0205] explanation: His tag and a galactosaminidase expression construct of the Clostridium tertium 5757 (Ct5757) protein sequence having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCN CGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0206] Sequence ID 21

[0207] explanation: His tag and a construct expressing Robinsoniera peoliensis Rp1021 galactosaminidase protein having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0208] Sequence ID 23

[0209] explanation: His tag and a Ruthenibacterium lactatiformans R18755GalNAc deacetylase protein sequence expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH SSGLVPRGSHEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTG PNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGT PSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDF VQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0210] Sequence ID 25

[0211] explanation: His tag and a Robinsoniera peoliensis Rp 3671GalNAc deacetylase protein expression construct with a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0212] Sequence ID 27

[0213] explanation: His tag and a Robinsoniera peoliensis Rp 3672GalNAc deacetylase protein expression construct with a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0214] Sequence ID 29

[0215] explanation: His tag and a construct expressing the Robinsoniera peoliensis Rp3671GalNAc deacetylase protein Rp3671 (in the pET28a vector) which has a thrombin cleavage site. MGSS HHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKE VQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDK GIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALP EPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVI YNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVT VFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGIN WGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEE FQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0216] Sequence ID 31

[0217] explanation: His tagand a Robinsoniera peoliensis Rp 3672_GalNAc deacetylase_protein expression construct (in pET28a vector) having a thrombin cleavage site. MGSS HHHHHH

[0218] Sequence ID 32

[0219] Description: Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence HSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQ INYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPN RTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRES SDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0220] Sequence ID 33

[0221] Description: Ruthenibacterium lactatiformans R18755GalNAc deacetylase protein sequence HEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTAT IYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPG HNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQ PGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0222] Sequence ID 34

[0223] Description: Robinsonia peoliensis Rp3671 GalNAc deacetylase protein HSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQT EDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTD STIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENV AFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSD TTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFD EPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWG VEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEF QEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0224] Sequence ID 35

[0225] Description: Robinsonia peoliensis Rp3672_GalNAc deacetylase_protein

[0226] Sequence ID 36

[0227] Description: Clostridium tertium 5757 (Ct5757) galactosaminidase protein sequence HYNLIDNISVEKLDTDISQANENVFLNGNIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQ DFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0228] Sequence ID 37

[0229] Description: Robinsonia peoliensis Rp1021 galactosaminidase protein sequence

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Epub 2011 Jul 30.

Claims

1. An isolated nucleic acid encoding a GalNAc deacetylase selected from one or more of SEQ ID NOs: 1 and SEQ ID NOs:

3.

2. An isolated nucleic acid encoding a galactosaminidase selected from one or more of SEQ ID NOs: 6 and SEQ ID NOs:

8.

3. A vector comprising the nucleic acid described in claim 1 or 2 and a heterogeneous nucleic acid.

4. The vector according to claim 3, wherein the heterogeneous nucleic acid encodes a polypeptide selected from one or more of the protein tag and cleavage sites.

5. The protein tags include albumin-binding protein (ABP), alkaline phosphatase (AP), AU1 epitope, AU5 epitope, AviTag, bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxyl carrier protein (BCCP), blue tongue virus tag (B-tag), single-domain camel antibody (C-tag), calmodulin-binding peptide (CBP or calmodulin-tag), chloramphenicol acetyltransferase (CAT), and cellulose. Binding domain (CBP), chitin-binding domain (CBD), choline-binding domain (CBD), dihydrofolate reductase (DHFR), DogTag, E2 epitope, E-tag, FLAG epitope (FLAG-tag), galactose-binding protein (GBP), green fluorescent protein (GFP), Glu-Glu (EE-tag), glutathione S-transferase (GST), human influenza hemagglutinin (HA), HaloTag (trademark), alternating histidine and glutamine tags (HQ tag), alternating histidine and asparagine tags (HN tag), histidine affinity tag (HAT), horseradish peroxidase (HRP), HSV epitope, isopept tag (Isop-tag), ketosteroid isomerase (KSI), KT3 epitope, LacZ, luciferase, maltose-binding protein (CBP), Myc epitope (Myc-tag), NE-tag, NusA, PDZ domain, PDZ ligand, polyarginine (Arg-tag), polyaspartic acid (Asp-tag), polycysteine ​​(Cys-tag), polyglutamic acid (Glu-tag), Polyhistidine (His-tag), polyphenylalanine (Phe-tag), Profinity eXact, Protein C, Rho1D4 tag, S1-tag, S-tag, Softag 1, Softag 3, SnoopTag Jr, SnoopTag, SpotTag, SpyTag (Spy-tag), streptavidin-binding peptide (SBP), Staphylococcus aureus protein A (Protein A), Staphylococcus aureus protein G (Protein G), StrepTag, streptavidin (SBP-tag), StrepTag II, Sdy-tag, low molecular weight ubiquitin-like modifier (SUMO),The vector according to claim 4, selected from one or more of the following: tandem affinity purification (TAP), T7 epitope, tetracysteine ​​tag (TC tag), thioredoxin (Trx), TrpE, Ty tag, ubiquitin, universal, V5 tag, VSV-G or VSV- tag, and Xpress tag.

6. A vector comprising the nucleic acid according to claim 1 or 2.

7. An isolated nucleic acid, wherein the isolated nucleic acid is (a) Encode a GalNAc deacetylase selected from one or more of SEQ ID NOs: 1 and 3; or (b) Encode a galactosaminidase selected from one or more of SEQ ID NOs: 6 and SEQ ID NOs: 8; Isolated nucleic acids.

8. A vector comprising the nucleic acid described in claim 7 and a heterogeneous nucleic acid.

9. The vector according to claim 8, wherein the heterogeneous nucleic acid encodes a polypeptide selected from one or more of the protein tag and cleavage sites.

10. The protein tags include albumin-binding protein (ABP), alkaline phosphatase (AP), AU1 epitope, AU5 epitope, AviTag, bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxyl carrier protein (BCCP), blue tongue virus tag (B-tag), single-domain camel antibody (C-tag), calmodulin-binding peptide (CBP or calmodulin-tag), chloramphenicol acetyltransferase (CAT), and cellulose. Binding domain (CBP), chitin-binding domain (CBD), choline-binding domain (CBD), dihydrofolate reductase (DHFR), DogTag, E2 epitope, E-tag, FLAG epitope (FLAG-tag), galactose-binding protein (GBP), green fluorescent protein (GFP), Glu-Glu (EE-tag), glutathione S-transferase (GST), human influenza hemagglutinin (HA), HaloTag (trademark), alternating histidine and glutamine tags (HQ tag), alternating histidine and asparagine tags (HN tag), histidine affinity tag (HAT), horseradish peroxidase (HRP), HSV epitope, isopept tag (Isop-tag), ketosteroid isomerase (KSI), KT3 epitope, LacZ, luciferase, maltose-binding protein (CBP), Myc epitope (Myc-tag), NE-tag, NusA, PDZ domain, PDZ ligand, polyarginine (Arg-tag), polyaspartic acid (Asp-tag), polycysteine ​​(Cys-tag), polyglutamic acid (Glu-tag), Polyhistidine (His-tag), polyphenylalanine (Phe-tag), Profinity eXact, Protein C, Rho1D4 tag, S1-tag, S-tag, Softag 1, Softag 3, SnoopTag Jr, SnoopTag, SpotTag, SpyTag (Spy-tag), streptavidin-binding peptide (SBP), Staphylococcus aureus protein A (Protein A), Staphylococcus aureus protein G (Protein G), StrepTag, streptavidin (SBP-tag), StrepTag II, Sdy-tag, low molecular weight ubiquitin-like modifier (SUMO),The vector according to claim 9, selected from one or more of the following: tandem affinity purification (TAP), T7 epitope, tetracysteine ​​tag (TC tag), thioredoxin (Trx), TrpE, Ty tag, ubiquitin, universal, V5 tag, VSV-G or VSV- tag, and Xpress tag.

Images