Compositions and methods for protein glycosylation

Abstract

This article describes oligosaccharide transferases for N-glycosylation of target proteins in vitro and in host cells. It also provides methods for using such oligosaccharide transferases, nucleic acids encoding such oligosaccharide transferases, and host cells containing such oligosaccharide transferases. Furthermore, it provides glycoconjugates produced using such oligosaccharide transferases.

Description

[0001] 1. Introduction

[0002] This article describes oligosaccharide transferases for N-glycosylation of target proteins in vitro and in host cells. It also provides methods for using such oligosaccharide transferases, nucleic acids encoding such oligosaccharide transferases, and host cells containing such oligosaccharide transferases. Furthermore, it provides glycoconjugates produced using such oligosaccharide transferases.

[0003] 2. Background

[0004] Glycoconjugate vaccines are widely recognized for their ability to prevent many life-threatening bacterial infections. They are generally considered effective and safe and have been used in humans for over 30 years. Conventional glycoconjugate vaccine production often involves the chemical modification of immunogenic carrier proteins with polysaccharide antigens from pathogenic bacteria. However, in recent years, biotechnological methods for producing glycoconjugate vaccines have emerged, which are expected to reduce production costs, further increase homogeneity, and potentially enhance the efficacy and safety of glycoconjugate vaccine products.

[0005] In eukaryotic cells, N-linked glycosylation is a key post-translational protein modification mechanism involving several enzymes. In prokaryotic cells, N-linked glycosylation is catalyzed by certain bacterial N-oligosaccharide transferases (N-OSTs). *Campylobacter jejuni* (… Campylobacter jejuni, C. jejuni The protein glycosylation gene cluster includes pglB The gene, which encodes membrane-bound N-OST (PglB), Cj PglB Cj It can be found in standard bacterial hosts such as Escherichia coli ( Escherichia coli, E. coli PglB is expressed in [a specific protein / organism] and can be co-expressed via glycosylation of a periplasmic protein carrying at least one surface-exposed D / EYNXS / T (Y, X≠P) glycosylation motif. Cj This technology can transfer bacterial polysaccharide antigens to Campylobacter jejuni proteins and to immunogenic carrier proteins containing engineered glycosylation sites from other organisms. PglB Cj It can transfer Campylobacter jejuni oligosaccharides and, to some extent, transfer the O-antigen lipopolysaccharide structure of Gram-negative bacteria and the capsular antigen polysaccharide of Gram-positive bacteria.

[0006] This invention provides recombinant N-OST with modified substrate specificity and a method for using said recombinant N-OST in the production of glycoconjugate vaccines. Such recombinant N-OST can be advantageously used in the N-glycosylation of proteins.

[0007] 3. Overview

[0008] In one aspect, this article provides a recombinant N-oligosaccharide transferase, wherein the recombinant N-oligosaccharide transferase can detectably link an oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end to a carrier protein at an N-glycosylation concordance sequence.

[0009] In some embodiments, the N-OST activity of the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end, which is ligated to the carrier protein at the N-glycosylation sequence, is detected by ELISA.

[0010] In some implementations, the ELISA signal indicating N-OST activity is detectable if it is >2σ or >3σ above the ELISA background signal.

[0011] In some embodiments, the carrier protein is a natural carrier protein from the same organism as N-OST. In some embodiments, the carrier protein is a heterologous carrier protein from an organism different from N-OST.

[0012] In some embodiments, the carrier protein is selected from: Pseudomonas aeruginosa ( P. aeruginosa Exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus (S. aureus) S. aureus The detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA and Campylobacter jejuni native glycoprotein.

[0013] In some embodiments, the carrier protein has at least one glycosylation motif. In some embodiments, the at least one glycosylation motif comprises D / EYNXS / T (X, Y≠P). In some embodiments, the at least one glycosylation motif comprises Asn-X-Ser(Thr), where X can be any amino acid other than Pro. In some embodiments, the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end comprises an antigen.

[0014] In some embodiments, the antigen includes Escherichia coli (E. coli) E. coli Antigens, Salmonella species ( Salmonella sp) antigen, Pseudomonas species ( Pseudomonas sp.) antigen, Klebsiella genus ( Klebsiellasp.) antigen, Acinetobacter O antigen, Chlamydia trachomatis (sp ... Chlamydia trachomatis Antigen, Vibrio cholerae ( Vibrio cholera Antigens, Listeria species ( Listeria sp.) antigen, Legionella pneumophila (sp.) antigen, Legionella pneumophila ( Legionella pneumophila ) serotype 1-15 antigen, Bordetella parapertussis ( Bordetella parapertussis Antigen, Burkholderia melioides ( Burkholderia mallei ) or Burkholderia melioides ( Burkholderia Burkholderia melioides antigen, Francisella tularensis ( Francisella tularensis Antigens, Campylobacter species ( Campylobacter sp.) antigen; Clostridium difficile (sp.) Clostridium difficile Antigen, Streptococcus pyogenes ( Streptococcus pyrogenes Antigen, agalactiae (Streptococcus agalactiae) Streptococcus agalacticae Antigen, Neisseria meningitidis ( Neisseria meningitidis Antigen, Candida albicans ( Candida albicans Antigen, Haemophilus influenzae ( Haemophilus influenza ) antigen, Enterococcus faecalis ( Enterococcus faecalis Antigen, Brewera brucellosis ( Borrelia burgdorferi Antigen, Neisseria meningitidis ( Neisseria meningitidis Antigen, Haemophilus influenzae ( Haemophilus influenza Antigens, large Leishmania ( Leishmania major Antigen or Shigella sonnei ( Shigella sonnei ) or Streptococcus pneumoniae ( Streptococcus pneumoniae Antigens (e.g., CP1, CP4, etc.).

[0015] In some embodiments, the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end is a Staphylococcus aureus or Salmonella enterica serotype ( Salmonella enterica sv. Polysaccharide. In some embodiments, the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end is Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2 polysaccharide.

[0016] In some embodiments, the recombinant N-oligosaccharide transferase can increase the yield of in vivo or in vitro glycosylation of the carrier protein by the polysaccharide lacking N-acetyl sugar at the reducing end, to produce the glycosylated carrier protein at levels exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0017] In some embodiments, compared to the wild-type form of the recombinant N-oligosaccharide transferase, the recombinant N-oligosaccharide transferase can increase the rate of in vivo or in vitro glycosylation of the carrier protein by more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 17, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 times.

[0018] In some embodiments, the recombinant N-oligosaccharide transferase may be glycosylated in vivo or in vitro with the polysaccharide lacking N-acetyl sugar at the reducing end by at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein.

[0019] In some embodiments, the recombinant N-oligosaccharide transferase includes modifications in one or more amino acids, wherein, in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0020] In some embodiments, the 2.5-4.0 Å distance is the distance to the first terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to the second terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to the third terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to a conserved amino acid (e.g., PglB) in the catalytic center of the recombinant N-oligosaccharide transferase in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein. Cj K522, N311, H 479, G476, Y462, G477, Y77, S80 or S199, see, for example, Figure 2).

[0021] In some embodiments, the modification in one or more amino acids is an amino acid substitution.

[0022] In some embodiments, the one or more amino acids include those that are non-conserved amino acids in the phylogenetic family of N-oligosaccharide transferases. In some embodiments, the non-conserved amino acid is conserved in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the members of the phylogenetic family of N-oligosaccharide transferases.

[0023] In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in two or more amino acids. In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in three or more amino acids. In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in four or more amino acids.

[0024] In some embodiments, at least one of the one or more amino acids is located in the pericytoplasmic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase. In some embodiments, the pericytoplasmic loop of the transmembrane domain is a large outer loop 5 (EL5). In some embodiments, the recombinant N-oligosaccharide transferase is PglB. Cj And the EL5 is PglB Cj EL5.

[0025] In some embodiments, the recombinant N-oligosaccharide transferase further includes a mutation in one or more amino acids of the QLKFYxxR motif. In some embodiments, the Q287LKFYxxR294 motif is the Q287LKFYxxR294 motif. In some embodiments, the Q287LKFYxxR294 motif is PglB. Cj The Q287LKFYxxR294 motif.

[0026] In some embodiments, the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0027] In some embodiments, the bound N-glycosylated carrier protein is a native Campylobacter jejuni glycosylated carrier protein. In some embodiments, the bound N-glycosylated carrier protein is a heterologous Campylobacter jejuni glycosylated carrier protein.

[0028] In some embodiments, the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0029] In some implementations, one or more of the PglB selections are modified. Cj The amino acids Y77, S80, S196, N311, Y462, H479, K522, G476, and G477. In some embodiments, PglB is modified. Cj N311. In some embodiments, the recombinant PglB Cj It includes substitution of N311V or substitution of N311I. In some embodiments, the recombinant PglB Cj It includes the substitution of N311V. In some embodiments, the recombinant PglB Cj It also includes modifications in one or more amino acids selected from Y77 and S80. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions selected from Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, Y77G, S80R, and S80H. In some embodiments, the recombinant PglB... Cj It includes amino acid substitutions selected from Y77H and S80R.

[0030] In some implementations, the recombinant PglB Cj Also included in PglB Cj Amino acid modifications in one or more amino acids of the Q287LKFYxxR294 motif. In some embodiments, the recombinant PglB CjAmino acid modifications comprising one or more amino acids selected from Q287, L288, and K289. In some embodiments, the recombinant PglB Cj It includes one or more amino acid substitutions selected from Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q and R294K.

[0031] In some implementations, the recombinant PglB Cj It includes the amino acid substitution N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions Y77H and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions S80R and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions of Q287P and Y77H or Q287P and S80R. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions S80R, Q287P, and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions of Y77H, Q287P, and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions Y77H, S80R, Q287P, and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions Y77H, S80R, Q287P, K289R, and N311V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions N311V and A699V. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions K482R and D483H.

[0032] In another aspect, this document provides a recombinant N-oligosaccharide transferase (N-OST) comprising a modification in one or more amino acids, wherein, in a structural model of a complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, the amino acid side chain is located within a 2.5-4.0 Å distance of one of three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein. In some embodiments, the modification is an amino acid substitution.

[0033] In some embodiments, the carrier protein is selected from: Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholerae toxin B subunit (CTB), cholerae toxin, detoxified variants of cholerae toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.

[0034] In some embodiments, the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end comprises an antigen. In some embodiments, the antigen includes Escherichia coli antigen, Salmonella spp. antigen, Pseudomonas spp. antigen, Klebsiella spp. antigen, Acinetobacter spp. O antigen, Chlamydia trachomatis antigen, Vibrio cholerae antigen, Listeria spp. antigen, Legionella pneumophila serotypes 1-15 antigen, Bordetella parapertussis antigen, Burkholderia melioides or Burkholderia melioides antigen, Francisella tularensis antigen, Campylobacter spp. antigen; Clostridium difficile antigen, Streptococcus agalactiae antigen, Neisseria meningitidis antigen, Candida albicans antigen, Haemophilus influenzae antigen, Enterococcus faecalis antigen, Borrelia brevicornu antigen, Neisseria meningitidis antigen, Haemophilus influenzae antigen, Leishmania giantiformis antigen, Shigella sonnei antigen, or Streptococcus pneumoniae antigen (e.g., CP1, CP4, etc.).

[0035] In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in two or more amino acids. In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in three or more amino acids. In some embodiments, the recombinant N-oligosaccharide transferase comprises modifications in four or more amino acids.

[0036] In some embodiments, at least one of the one or more amino acids is located in the pericytoplasmic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase. In some embodiments, the pericytoplasmic loop of the transmembrane domain is a large outer loop 5 (EL5). In some embodiments, the recombinant N-oligosaccharide transferase is PglB (PglB) of Campylobacter jejuni. Cj ), and EL5 is PglB Cj EL5.

[0037] In some embodiments, the recombinant N-oligosaccharide transferase further comprises a modification of one or more amino acids in the QLKFYxxR motif. In some embodiments, the recombinant N-oligosaccharide transferase further comprises a modification of one or more amino acids in the Q287LKFYxxR294 motif. In some embodiments, the QLKFYxxR motif is PglB. Cj The Q287LKFYxxR294 motif.

[0038] In some embodiments, the amino acid substitution is a substitution of a non-conserved amino acid in the phylogenetic family of N-oligosaccharide transferases.

[0039] In some embodiments, the bound N-glycosylated polypeptide product is a naturally occurring N-glycosylated carrier protein from the same organism as the recombinant N-oligosaccharide transferase. In some embodiments, the N-glycosylated carrier protein is a heterologous N-glycosylated carrier protein, wherein the oligosaccharide or polysaccharide component of the N-glycosylated carrier protein is from an organism different from the recombinant N-oligosaccharide transferase, and / or the carrier protein component of the N-glycosylated carrier protein is from an organism different from the recombinant N-oligosaccharide transferase.

[0040] In some embodiments, the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0041] In some embodiments, the bound N-glycosylated polypeptide product is a native Campylobacter jejuni glycosylated carrier protein. In some embodiments, the bound N-glycosylated polypeptide product is a heterologous Campylobacter jejuni glycosylated carrier protein. In some embodiments, the heterologous Campylobacter jejuni glycosylated carrier protein is Pseudomonas aeruginosa (…). Pseudomonas aeruginosa )Exotoxin (EPA) - Shigella dysenteriae ( S. dysenteriae ) O1 (EPA-O1), EPA-Staphylococcus aureus capsular polysaccharide type 5 (EPA-CP5) or EPA - Salmonella enterica ( Salmonella enterica, S. enterica LT2 (EPA-LT2).

[0042] In some embodiments, the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein does not have an N-acetyl monosaccharide at its reducing end. In some embodiments, the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0043] In some implementations, one or more of the PglB selections are modified. CjThe amino acids Y77, S80, S196, N311, Y462, H479, K522, G476, and G477. In some embodiments, PglB is modified. Cj N311. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions selected from N311V and N311I. In some embodiments, the recombinant PglB Cj It includes the amino acid substitution N311V. In some embodiments, it is modified with one or more amino acids selected from PglB. Cj The amino acids of Y77 and S80. In some embodiments, the recombinant PglB Cj It includes amino acid substitutions selected from Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, Y77G, S80R, and S80H. In some embodiments, the recombinant PglB... Cj It includes amino acid substitutions selected from Y77H and S80R.

[0044] In some implementations, the recombinant PglB Cj Also includes PglB Cj The modification of one or more amino acids of the Q287LKFYxxR294 motif. In some embodiments, the recombinant PglB Cj It contains modifications comprising one or more amino acids selected from Q287, L288, and K289. In some embodiments, the recombinant PglB Cj It includes substitutions selected from Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q and R294K.

[0045] In some implementations, the recombinant PglB Cj It includes the substitution of N311V. In some embodiments, the recombinant PglB Cj It includes the substitution of Y77H and N311V. In some embodiments, the recombinant PglB Cj It includes substitution S80R and substitution N311V. In some embodiments, the recombinant PglB Cj It includes substitution Q287P and substitution Y77H, or substitution Q287P mutation and substitution S80R. In some embodiments, the recombinant PglB Cj It includes substitution S80R, substitution Q287P, and substitution N311V. In some embodiments, the recombinant PglB Cj It includes substitution of Y77H, substitution of Q287P, and substitution of N311V. In some embodiments, the recombinant PglB CjIt includes substitutions of Y77H, S80R, Q287P, and N311V. In some embodiments, the recombinant PglB Cj This includes substitutions of Y77H, S80R, Q287P, K289R, and N311V. In some embodiments, the recombinant PglB... Cj It includes substitution of N311V and substitution of A699V. In some embodiments, the recombinant PglB Cj Includes replacement of K482R and replacement of D483H.

[0046] In some embodiments, the recombinant N-oligosaccharide transferase can detectably link oligosaccharides or polysaccharides lacking N-acetyl sugar at the reducing end to a carrier protein.

[0047] In some embodiments, the recombinant N-oligosaccharide transferase can detectably link oligosaccharides or polysaccharides having a galactosyl monosaccharide at the reducing end to a carrier protein.

[0048] In some embodiments, the oligosaccharide or polysaccharide is a Staphylococcus aureus or Salmonella enterica serotype oligosaccharide or polysaccharide. In some embodiments, the oligosaccharide or polysaccharide is a Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2 oligosaccharide or polysaccharide.

[0049] In some embodiments, the recombinant N-oligosaccharide transferase can increase the yield of in vivo or in vitro glycosylation of the carrier protein by the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end, to produce the glycosylated carrier protein at levels exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0050] In some embodiments, compared to the wild-type form of the recombinant N-oligosaccharide transferase, the recombinant N-oligosaccharide transferase can increase the in vivo or in vitro glycosylation rate of the carrier protein by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, more than 11-fold, more than 12-fold, more than 13-fold, more than 14-fold, more than 15-fold, more than 17-fold, more than 20-fold, more than 25-fold, more than 30-fold, more than 35-fold, more than 40-fold, more than 45-fold, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold, or more than 100-fold.

[0051] In some embodiments, the recombinant N-oligosaccharide transferase can produce at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 70% of the carrier protein in vivo or in vitro.

[0052] In another aspect, this article provides a recombinant N-oligosaccharide transferase PglB containing N311V substitution. Cj .

[0053] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N311V mutation and the Y77H substitution. Cj .

[0054] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N311V mutation and S80R substitution. Cj .

[0055] In another aspect, this article presents recombinant N-oligosaccharide transferase PglB containing N311V mutations and Y77H mutations and S80R substitutions. Cj .

[0056] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N311V mutation and Q287P substitution. Cj .

[0057] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N311V mutation, Y77H substitution, and Q287P substitution. Cj .

[0058] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N311V mutation, S80R substitution, and Q287P substitution. Cj .

[0059] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB comprising N311V, Y77H, S80R, and Q287P substitutions. Cj .

[0060] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing N311V and A669V substitutions. Cj .

[0061] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB comprising N311V, Y77H, S80R, Q287P, and K289R substitutions. Cj .

[0062] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing K482R and D483H substitutions. Cj .

[0063] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing N311V and A669V substitutions. Cj .

[0064] In another aspect, this article provides a recombinant N-oligosaccharide transferase PglB containing N314V substitution. Cl (PglB Campylobacter gullus ( C. lari )).

[0065] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N314V mutation and the Y79H substitution. Cl .

[0066] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N314V mutation and the S82R substitution. Cl .

[0067] In another aspect, this article presents recombinant N-oligosaccharide transferase PglB containing N314V mutations and Y79H mutations and S82R substitutions. Cl .

[0068] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N314V mutation and Q289P substitution. Cl .

[0069] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N314V mutation, Y79H substitution, and Q289P substitution. Cl .

[0070] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing the N314V mutation, S82R substitution, and Q289P substitution. Cl .

[0071] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB comprising N314V, Y79H, S82R, and Q289P substitutions. Cl .

[0072] In another aspect, this article presents a recombinant N-oligosaccharide transferase PglB containing K488R and D489H substitutions. Cl .

[0073] In another respect, this article provides a nucleic acid that encodes the recombinant N-oligosaccharide transferase described herein.

[0074] In another respect, this article provides a host cell containing the recombinant N-oligosaccharide transferase described herein.

[0075] In some embodiments, the host cell also contains recombinant glycosyltransferase.

[0076] In another aspect, this article provides a host cell containing the nucleic acids described herein.

[0077] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is an Escherichia coli cell.

[0078] In another aspect, this article provides a method for producing biological conjugates, the method comprising culturing host cells as described herein.

[0079] In some embodiments, the host cell comprises a carrier protein and a recombinant N-oligosaccharide transferase. In some embodiments, the host cell also comprises a recombinant glycosyltransferase. In some embodiments, the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0080] In some embodiments, the carrier protein is selected from: Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholerae toxin B subunit (CTB), cholerae toxin, detoxified variants of cholerae toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.

[0081] In some embodiments, the bioconjugate is an N-glycosylated carrier protein. In some embodiments, the bioconjugate is a natural Campylobacter jejuni. N- Glycosylated carrier protein. In some embodiments, the bioconjugate is a heterologous Campylobacter jejuni. N- Glycosylated carrier protein. In some embodiments, the N-glycosylated carrier protein does not have N-acetyl sugar at the reducing end of its oligosaccharide or polysaccharide component. In some embodiments, the N-glycosylated carrier protein has galactose at the reducing end of its oligosaccharide or polysaccharide component.

[0082] In some embodiments, the recombinant N-oligosaccharide transferase mutant can increase the rate of bioconjugate production by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times compared to the rate achieved by the wild-type form of the recombinant N-oligosaccharide transferase.

[0083] In some embodiments, the recombinant N-oligosaccharide transferase mutant can increase the bioconjugate production yield to a level exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0084] In some embodiments, at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein in the host cell is glycosylated to form the bioconjugate.

[0085] In some embodiments, the method further includes purifying the bioconjugate from the host cell culture.

[0086] In another aspect, this article provides a method for screening a library of recombinant N-oligosaccharide transferases, wherein each recombinant N-oligosaccharide transferase contains a modification in one or more amino acids, the method comprising contacting each member of the library of recombinant N-oligosaccharide transferases with a carrier protein and an oligosaccharide or polysaccharide lacking N-acetyl sugar at its reducing end to produce a bioconjugate.

[0087] In some embodiments, the bioconjugate is an N-glycosylated carrier protein.

[0088] In some embodiments, the contact occurs in vitro. In some embodiments, the contact occurs in vivo. In some embodiments, the contact occurs within a host cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is an *E. coli* cell.

[0089] In some embodiments, the library of recombinant N-oligosaccharide transferases contains at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 750, or at least 1,000 recombinant N-oligosaccharide transferases.

[0090] In some embodiments, the library of recombinant N-oligosaccharide transferases comprises one or more recombinant N-oligosaccharide transferases described herein.

[0091] In some embodiments, the method further includes analyzing the rate or yield of production of the bioconjugate.

[0092] In some embodiments, the method further includes selecting one or more recombinant N-oligosaccharide transferases from a library of the recombinant N-oligosaccharide transferases.

[0093] In some embodiments, if the recombinant N-oligosaccharide transferase produces the bioconjugate at a rate that is more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times faster than the wild-type form of the recombinant N-oligosaccharide transferase, then one or more of the recombinant N-oligosaccharide transferases are selected.

[0094] In some embodiments, if the N-oligosaccharide transferase mutant produces the bioconjugate in a detectable yield at a level more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times above the background level, then one or more N-oligosaccharide transferase mutants are selected.

[0095] In some embodiments, the recombinant N-oligosyltransferase is selected if it glycosylates at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein in the host cell.

[0096] In another aspect, this paper provides a method for identifying recombinant N-oligosaccharide transferases with altered substrate selectivity compared to wild-type forms of N-oligosaccharide transferases, the method comprising modifying one or more amino acids, in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, wherein the side chain of the amino acid is located within a 2.5–4.0 Å distance of one of three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0097] In some embodiments, the method includes modifying two or more amino acids of the recombinant N-oligosaccharide transferase. In some embodiments, the method includes modifying three or more amino acids of the recombinant N-oligosaccharide transferase. In some embodiments, the method includes modifying four or more amino acids of the recombinant N-oligosaccharide transferase.

[0098] In some embodiments, at least one of the one or more amino acids is located in the pericytoplasmic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase. In some embodiments, the pericytoplasmic loop of the transmembrane domain is a large outer loop 5 (EL5).

[0099] In some embodiments, the method further includes mutating one or more amino acids of the QLKFYxxR motif of the recombinant N-oligosaccharide transferase. In some embodiments, the QLKFYxxR motif is the Q287LKFYxxR294 motif. In some embodiments, the bound N-glycosylated carrier protein is a naturally occurring N-glycosylated carrier protein.

[0100] In some embodiments, the bound N-glycosylated carrier protein is a heterologous N-glycosylated carrier protein. In some embodiments, the recombinant N-oligosaccharide transferase is recombinant PglB. Cj In some embodiments, the bound N-glycosylated carrier protein is a native Campylobacter jejuni N-glycosylated carrier protein. In some embodiments, the bound N-glycosylated carrier protein is a heterologous Campylobacter jejuni N-glycosylated carrier protein.

[0101] In some embodiments, the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein does not have an N-acetyl monosaccharide at its reducing end. In some embodiments, the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0102] In some embodiments, the recombinant N-oligosaccharide transferase has altered in vitro substrate selectivity. In some embodiments, the recombinant N-oligosaccharide transferase has altered in vivo substrate selectivity. 4. Description of the attached drawings

[0104] Figure 1 PglB was described Cj The structures of the natural Campylobacter jejuni heptaose substrate and two non-natural polysaccharide substrates show decreasing glycosylation efficiency from top to bottom. GalNAc: 2-N-acetylgalactosamine, Glc: glucose, DATDH: 2,4-diacetamido-2,4,6-trideoxyhexose, PP-und: undecylenyl-pyrophosphate carrier, Rha: rhamnose, Gal: galactose, GlcNAc: N-acetylglucosamine, ManNAc: N-acetylmannosamine, OAc: O-acetyl modified, FucNAc: N-acetylfucosamine, Man: mannose, Abe: abecosose (3,6-deoxy-D-galactose).

[0105] Figure 2 depicts the situation when PglB is involved. Cj An exemplary modeling of oligosaccharide structures during interactions. Figure 2AExemplary conformations of Campylobacter jejuni OS (light gray sphere- and bar-represented) and Salmonella enterica LT2 repeat units (dark gray bar-represented) in the active site are depicted, with the connection site with the receptor peptide selected as a fixed point in the dynamic modeling process. Figure 2B An exemplary conformation of Campylobacter jejuni OS (light gray sphere- and rod representation) is depicted. Figure 2C An exemplary conformation of the LT2 repeat unit (represented by dark gray bars) of *Salmonella enterica* is depicted. PglB Cj The main chain structure is shown in gray (bands), and the phosphate groups of the membrane are shown as light gray spheres. Residues closely adjacent to the native OS are depicted as light gray sphere-bar representations. Dashed lines illustrate the connectivity of the unstructured outer ring EL5.

[0106] Figure 3 explains the PglB Cj The results of exemplary DWP-ELISA screening of saturated mutagenic libraries with N311 residue randomization. Figure 3A depicts the screening results using the host strain and a detection antibody against Staphylococcus aureus CP5 polysaccharide. Figure 3B depicts the screening results using the host strain and a detection antibody against Salmonella enterica LT2 polysaccharide. Hollow circles indicate library clones; solid triangles indicate positive control clones expressing wild-type PglB (pGVXN1413), and shaded triangles indicate clones expressing inactive PglB. mut The negative control clone (pGVXN408) was used. The sequenced clones were marked with elliptical tags.

[0107] Figure 4 depicts the alignment of bacterial PglB homologues at the following locations: (A) in the EL5 region, including the Campylobacter jejuni 287QLKFYxxR294 motif and Campylobacter jejuni N311, and (B) near the residues of Campylobacter jejuni Y77 / S80 and Campylobacter jejuni K482 / D843. Campylobacter jejuni PglB was used as a search template for protein BLAST, and the ClustalW algorithm (DNASTAR, Madison, WI, USA) was used with MegAlign. TM The program compares non-redundant sequences. PglB is conserved in sequences from other species. Cj The residues are shaded. The relevant Campylobacter jejuni residues are indicated at the top, and the corresponding amino acids in the homologous N-OST sequence are enclosed in boxes.

[0108] Figure 5 The diagram illustrates the potential interactions of PglB with sugars. Cj Amino acid substitutions within residues Y462, G476, G477, and H479 affect the performance of overnight-induced DWP cultures. in vivoThe impact of CP5-EPA production. Reference well (100% value, corrected background): pGVXN1050 (wild-type template plasmid). Mean number and standard deviation of triplicate clones for each variant are depicted.

[0109] Figure 6 illustrates the effect of the PglB variant N311V on glycoprotein formation in shake flasks by Western blot analysis. Figure 6A illustrates the results obtained using LT2-EPA in the host strain *Salmonella enterica* SGSC228 (pGVXN150). Figure 6B illustrates the results obtained using CP5-EPA in the host strain *Escherichia coli* St1717 (pGVXN150, pGVXN393). Figure 6C illustrates the results obtained using O1-EPA in the host strain *Escherichia coli* CLM24 (pGVXN64, pGVXN150). Figure 6D illustrates the results obtained using O1-EPA in the host strain *Escherichia coli* CLM24 (pACYC( pgl mut The results were obtained using EPA-Campylobacter jejuni OS in pGVXN150. Compared with... Figure 5 The same experiments were performed, with biomass-normalized periplasmic extracts, similar loading volumes, and one shake flask culture sample per variant. Wild-type PglB: pGVXN970, PglB N311V: pGVXN1217. Theoretical molecular weight of unglycosylated EPA-6H: 69.4 kDa.

[0110] Figure 7 illustrates the amino acid substitution PglB. Cj The effect of N311V on EPA glycosylation of three heteropolysaccharides and natural oligosaccharides. Hollow symbol: wild-type PglB (pGVXN970), solid symbol: PglB N311V (pGVXN1217). Figure 7A Exemplary results obtained using the host strain and a detection antibody against Staphylococcus aureus CP5 polysaccharide are explained. Figure 7B Exemplary results obtained using the host strain and an antibody targeting the LT2 polysaccharide of Salmonella typhimurium serotype are explained. Figure 7C The results obtained using the host strain and the detection antibody against Shigella dysenteriae O1 polysaccharide were explained. Figure 7D Results obtained using the host strain and detection antibody against Campylobacter jejuni oligosaccharides are explained. Background-corrected ELISA signals from shake-flask cultures, normalized to biomass periplasmic extracts, are plotted as mean and standard deviation for n = 3 biological replicates.

[0111] Figure 8 illustrates the effect of N311V on the expression of HA-labeled PglB and the formation of CP5-EPA in the shake-flask experiment. Figure 8AThe results of anti-HA protein blot analysis of PglB-HA in the host strain of Escherichia coli St1717 (pGVXN150, pGVXN393) were explained. Figure 8B Results of CP5-EPA formation timelines obtained by sandwich ELISA analysis of biomass-normalized periplasmic extracts are explained. Hollow symbols depict results for wild-type PglB-HA. Solid symbols depict results for PglB-HA N311V. Means and standard deviations for n = 3 replicate cultures are shown.

[0112] Figure 9 depicts PglB Cj An exemplary result of the third round of directed evolution, which employs rearrangements of neutral and slightly beneficial mutations. Figure 10 A represents the screening results of a representative 96-well library. Hollow circles represent library clones; solid triangles represent PglB N311V (template plasmid pGVXN1418); shaded triangles represent inactive PglB. mut (pGVXN408). Figure 10 B explains the validation of the improvement in DWP after retransformation. The mean and standard deviation of each variant plasmid n = 3 replicate clones / well are depicted, wt: pGVXN1413, and N311V: pGVXN1418. Figure 10 C explains exemplary results of SDS-PAGE and Western blot analysis of proteins purified by Ni-NTA affinity, which were produced in shake flasks using wild-type PglB (pGVXN970), PglB N311V (pGVXN1217), or PglB S80R-Q287P-N311V (library clone 2B2) (similar loading volumes, with total protein concentration (A)). 280 Adjust to 2 mg / mL -1 The theoretical molecular weight of unglycosylated EPA-6H is 69.4 kDa.

[0113] Figure 10 The residues PglB with randomization were depicted. Cj PglB for K482 and D483 Cj Exemplary screening results for the library. Hollow circles: library clones; solid triangles: wild-type PglB-HA (pGVXN407); hollow triangles: inactive PglB (pGVXN408). The clone Fa8_G10 carrying the double mutation K482R-D483H is marked with a circle.

[0114] Figure 11 The displayed bar chart explains PglB CjK482-D483H improved CP5-EPA production in shake-flask cultures. Biomass-normalized periplasmic protein extracts were prepared 4 h after induction and after overnight (o / n) incubation and analyzed by sandwich ELISA. Solid bars: wild-type PglBCj-HA (pGVXN114), hollow bars: PglBCj-HA K482R-D483H (pGVXN635). n = mean and standard deviation of 3 replicate cultures. Subtract background ELISA absorbance (inactive PglB, pGVXN115).

[0115] Figure 12 The displayed Western blot analysis explained PglB Cj K482-D483H can improve the glycosylation of Staphylococcus aureus CP5 polysaccharide on Staphylococcus aureus Hla. SDS-PAGE and Western blot analysis of periplasmic proteins after HisTrap purification are shown. A similar volume of elution fraction with the highest protein concentration (A280) was loaded.

[0116] 5. Abbreviations

[0117] The abbreviation "CP" used in this article refers to "capsular polysaccharide".

[0118] The abbreviation "EL" used in this article refers to "outer ring".

[0119] The abbreviation "N-OST" used in this article refers to N-oligosaccharide transferase.

[0120] The abbreviation "PglB" used in this article Cj "Indicates Campylobacter jejuni ( ​ ​ ) of N-OST PglB.

[0121] The abbreviation "PglB" used in this article Cl "This indicates Campylobacter rubrum ( ​ ) of N-OST PglB. 6. Detailed Implementation

[0123] This document provides modified N-oligosaccharide transferases (N-OSTs) with altered substrate specificity. Specifically, this document provides modified N-OSTs capable of using oligosaccharides or polysaccharides as substrates for N-glycosylation of proteins at a concordant N-glycosylation sequence that is not usable by the wild-type form of the N-OST (or not usable at detectable levels). In some embodiments, the modified N-OST can use such oligosaccharides or polysaccharides to generate detectable levels of N-glycosylated carrier proteins, for example, in vivo or in vitro. The level of glycosylated carrier proteins can be determined by methods known in the art, including, but not limited to, ELISA, HPLC, LC-MS, etc. (see, for example, sections 6.10, 6.12, and Examples 2-3). In some embodiments, the generation of N-glycosylated carrier proteins is detected by ELISA.

[0124] In some embodiments, if the glycosylated carrier protein can be detected in the assay, then the level of the glycosylated carrier protein is detectable, and the assay signal indicates a level of glycosylated carrier protein that is more than 2 or 3 standard deviations (>2σ or >3σ) above the mean or median background signal, or more than 2, 3, 4, 5, 7, 8, 9, or 10 times above the background signal.

[0125] In some embodiments, the background measurement signal is the mean or median measurement signal from a negative control experiment performed in the absence of N-OST. In some embodiments, the background measurement signal is the mean or median measurement signal from a negative control experiment performed in the presence of wild-type N-OST.

[0126] In some embodiments, in assays for detecting the bioconjugate (e.g., in ELISA assays, by HPLC, LC-MS; see also sections 6.10 and 6.12), the glycosylated carrier protein can be detected at levels more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times above the background level.

[0127] Modifications in the N-OSTs provided herein can be located within a specified distance of monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the glycosylated carrier protein bound by the N-OST. To confirm that such modifications result in altered substrate specificity, any conventional assay for protein glycosylation can be used. Such modified N-OSTs can be used to generate bioconjugates in the prokaryotic host cells described herein. Compositions comprising the resulting bioconjugates are also disclosed herein. In one specific embodiment, such modified N-OSTs are capable of generating detectable levels of glycosylated carrier proteins using oligosaccharides or polysaccharides lacking N-acetyl substitution at the reducing end as substrates.

[0128] In some embodiments, in the assay for detecting the bioconjugate, the recombinant N-oligosaccharide transferase mutant can increase the bioconjugate production yield to a level exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0129] The background level in the determination of bioconjugates can be, for example, the average or median signal obtained in a control experiment performed in the absence of N-OST or using wild-type N-OST.

[0130] 6.1 N-Oligosaccharide transferases

[0131] In one aspect, this document provides a recombinant N-oligosaccharide transferase (N-OST) wherein the recombinant N-OST can detectably link an oligosaccharide or polysaccharide lacking an N-acetyl sugar at its reducing end to a carrier protein. In some embodiments, the recombinant N-OST comprises a modification of one or more amino acids, wherein, in a structural model of the complex of the recombinant N-OST and the glycosylated carrier protein product, the side chain of the amino acid is located within a 0.5-10.0 Å distance of the monosaccharide unit at the reducing end of the polysaccharide component of the bound glycosylated carrier protein product. In some embodiments, the modification is an amino acid substitution. In some embodiments, the recombinant N-OST comprises a modification of one or more amino acids, wherein, in a structural model of the complex of the recombinant N-OST and the glycosylated carrier protein product, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the polysaccharide component of the bound glycosylated carrier protein product. In some embodiments, the modification is an amino acid substitution. See, for example, Figure 2 and Section 6.3.

[0132] In another aspect, this document provides recombinant N-OSTs modified with one or more amino acids, wherein, in a structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 1.0-10.0 Å distance of a monosaccharide unit at the reduced end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein. In some embodiments, the recombinant N-OST is a recombinant N-OST modified with one or more amino acids, wherein, in a structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of three terminal monosaccharide units at the reduced end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein. In some embodiments, the recombinant N-OST may detectably link an oligosaccharide or polysaccharide lacking an N-acetyl sugar at the reduced end to the carrier protein. In some embodiments, the modification is an amino acid substitution. See, for example, Figure 2 and Section 6.3.

[0133] In some embodiments, the 2.5-4.0 Å distance is the distance to the first terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to the second terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to the third terminal monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component. In some embodiments, the 2.5-4.0 Å distance is the distance to a conserved amino acid (e.g., PglB) at the catalytic center of the recombinant N-oligosaccharide transferase in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein. Cj K522, N311, H 479, G476, Y462, G477, Y77, S80 or S199, see, for example, Figure 2).

[0134] In some embodiments, in the structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein, the 0.5-10.0 Å distance to the monosaccharide unit at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein is a distance between about 1.0 and 9.0 Å, about 1.5 and about 8.0 Å, about 2.0 Å and about 6.0 Å, or about 2.5 Å and 4.0 Å. In some embodiments, the 1.0–10.0 Å distance to the monosaccharide unit at the reduced end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein in the structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein is about 1.0 Å, about 1.5 Å, about 2.0 Å, about 2.5 Å, about 3.0 Å, about 3.5 Å, about 4.0 Å, about 4.5 Å, about 5.0 Å, about 5.5 Å, about 6.0 Å, about 6.5 Å, about 7.0 Å, about 7.5 Å, about 8.0 Å, about 8.5 Å, about 9.0 Å, or about 10.0 Å. See, for example, Figure 2 and Section 6.3.

[0135] Assays confirming the activity of the N-OST described herein are well known to those skilled in the art (e.g., ELISA, Western blotting) and include those described in sections 6.10 and 6.12. In some embodiments, the recombinant N-OST comprises a modification of one or more amino acids in a structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein, wherein the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reduced end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein, the recombinant N-OST detectably linking the oligosaccharide or polysaccharide lacking the N-acetyl sugar at the reduced end to the carrier protein, and the activity of the N-OST can be confirmed by the assays described in sections 6.10 or 6.12. In some embodiments, the modification is an amino acid substitution.

[0136] The oligosaccharides and polysaccharides may include any oligosaccharides or polysaccharides described herein. See, for example, Section 6.4.

[0137] The carrier protein may include any carrier protein described herein. See, for example, section 6.5.

[0138] In some embodiments, the recombinant N-OST includes modifications in, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more amino acids, wherein, in a structural model of the complex of the N-OST and the glycosylated carrier protein product, the side chains of the amino acids are located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0139] In some embodiments, at least one or more modifications of one or more amino acids are located in a pericytoplasmic loop within the transmembrane domain of the N-OST, and in a structural model of the complex of the N-OST and the glycosylated carrier protein product, the side chain of said amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reduced end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein. In some embodiments, the pericytoplasmic loop is the large outer loop 5 (EL5) of the N-OST. In some embodiments, the pericytoplasmic loop is PglB. Cj ,PglB Cj EL5 of homologues or naturally occurring variants (regarding PglB) Cj For a list of homologues, see, for example, Figures 4 and 9).

[0140] N-OST may include conserved sequence motifs, such as the QLKFYxxR motif. See, for example, Figure 9. In some embodiments, the recombinant N-OST includes a modification in at least one amino acid of the QLKFYxxR motif. In some embodiments, the QLKFYxxR motif is the Q287LKFYxxR294 motif (see, for example, PglB according to SEQ ID NO:1). Cj In some embodiments, the recombinant N-OST comprises, for example, modifications in at least two, at least three, at least four, or at least five amino acids of the QLKFYxxR motif. In some embodiments, the QLKFYxxR motif is PglB. Cj ,PglB Cj QLKFYxxR motifs of homologues or their naturally occurring variants.

[0141] In some embodiments, the recombinant N-OST comprises a modification of one or more amino acids, wherein, in a structural model of the complex of the N-OST and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein, and also comprises a modification of one or more amino acids in the QLKFYxxR motif.

[0142] In some embodiments, the amino acid modification comprises amino acid substitution. The amino acid can substitute for a natural protein-forming amino acid or an artificial amino acid. In some embodiments, the amino acid modification comprises the substitution of non-conserved amino acids (i.e., modification of non-conserved amino acids between N-OSTs from different organisms). In some embodiments, the non-conserved amino acids are conserved in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of members of the phylogenetic family of N-oligosaccharide transferases. In some embodiments, the non-conserved amino acids are conserved in between about 10% and about 90%, between about 20% and about 80%, between about 30% and about 70%, or between about 40% and about 60% of members of the phylogenetic family of N-oligosaccharide transferases.

[0143] In some embodiments, the recombinant N-OST can increase the rate of in vivo or in vitro glycosylation of the carrier protein by about 2 to about 100 times, about 5 to about 80 times, about 10 to about 60 times, about 10 to about 20 times, or about 20 to about 40 times, compared to the rate of the wild-type form of the recombinant N-OST. In some embodiments, the recombinant N-OST can increase the rate of in vivo or in vitro glycosylation of the carrier protein by more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 17, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 times compared to the rate of the wild-type form of the recombinant N-OST.

[0144] In some embodiments, the glycosylation rate of the recombinant N-OST and the wild-type form of the recombinant N-OST can be compared by comparing the rates at which recombinant N-OST glycosylates carrier proteins with polysaccharides or oligosaccharides lacking N-acetyl sugar at the reducing end.

[0145] In some embodiments, the rate at which the recombinant N-OST is used with a polysaccharide or oligosaccharide glycosylated carrier protein lacking N-acetyl sugar at the reducing end is compared with the rate at which wild-type N-OST is used with a polysaccharide or oligosaccharide glycosylated carrier protein lacking N-acetyl sugar at the reducing end.

[0146] In some embodiments, the rate at which the wild-type N-OST is used to glycosylate a carrier protein of a polysaccharide or oligosaccharide lacking N-acetyl sugar at the reducing end is defined as a relative rate of 100%.

[0147] In some embodiments, the rate at which the recombinant N-OST is used to glycosylate a carrier protein with a polysaccharide or oligosaccharide lacking an N-acetyl sugar at the reducing end is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the relative rate of wild-type N-OST.

[0148] In some embodiments, the recombinant N-OST can increase the in vivo or in vitro glycosylation yield of the polysaccharide to the carrier protein by about 2 to about 100 times, about 5 to about 80 times, about 10 to about 60 times, about 10 to about 20 times, or about 20 to about 40 times, compared to the yield achieved with the wild-type form of recombinant N-OST. In some embodiments, the recombinant N-OST can increase the in vivo or in vitro glycosylation yield of the polysaccharide to the carrier protein by more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 17, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 times compared to the yield achieved using the wild-type form of recombinant N-OST.

[0149] In some embodiments, the recombinant N-OST can generate an in vivo or in vitro glycosylation level of the carrier protein between about 1% and about 70%, about 3% and about 65%, about 5% and about 60%, about 5% and about 55%, about 10% and about 50%, about 15% and about 45%, about 20% and about 40%, or about 25% and about 35%. In some embodiments, the recombinant N-OST can generate an in vivo or in vitro glycosylation level of the carrier protein of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0150] In some embodiments, the carrier protein comprises two or more N-glycosylated consensual sequences. In some embodiments, the recombinant N-OST can glycosylate all N-glycosylated consensual sequences of the carrier protein in vitro or in vivo. In some embodiments, the recombinant N-OST can glycosylate at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of all N-glycosylated consensual sequences of the carrier protein in vitro or in vivo. In some embodiments, the recombinant N-OST can glycosylate between about 10% and about 70%, between 20% and about 60%, or between about 30% and about 50% of all N-glycosylated consensual sequences of the carrier protein in vitro or in vivo.

[0151] In some embodiments, the carrier protein comprises one or more N-glycosylated consensual sequences. In some embodiments, the carrier protein is a population of carrier proteins. In some embodiments, the recombinant N-OST may glycosylate at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of all N-glycosylated consensual sequences in the carrier proteins of the population of carrier proteins, either in vitro or in vivo. In some embodiments, the recombinant N-OST may glycosylate at between about 10% and about 70%, between 20% and about 60%, or between about 30% and about 50% of all N-glycosylated consensual sequences in the carrier proteins of the population of carrier proteins, either in vitro or in vivo.

[0152] The recombinant N-OST can be derived from any organism possessing N-OST. In some embodiments, the recombinant N-OST is derived from eukaryotes. In some embodiments, the recombinant N-OST is derived from prokaryotes. In some embodiments, the recombinant N-OST is derived from Campylobacter jejuni (…). ​ , ​ Campylobacter coli ( ​ , ​ Campylobacter gullii () ​ , ​ ), Uppsala Campylobacter ( ​ , ​ ), Curvularia ( ​ , ​ ), Campylobacter conc. ​ , C. ​ Campylobacter humanis ( ​ , ​ ), Campylobacter filamentosa ( ​ , ​ ), Campylobacter showa ( Campylobacter showae , C.showae ), autotrophic sulfurmonas ( Sulfurimonas autotrophica , S. autotrophica ), denitrifying sulfurmonas ( Sulfurimonas denitrificans , S. denitrificans ), Sulfurospirillum Deleyian ( S. deleyianum ), Sulfuricurvum kujiense ( S. kujiense ), Nautilus deep-sea ( N. profundicola ), Sulfurous sp. NBC37-1, succinate-producing Worlinella ( Wolinella succinogenes , W. succinogenes), Caminibacter mid-Atlanticus ( C. Mid-Atlantic ), Nitratiruptor sp. SB155-2, Helicobacter pylori (Gastroenteritis Helicobacter pylori) Helicobacter pylori , H.pullorum ), Helicobacter pauciflorus ( Helicobacter canadensis , H. Canadian ), Helicobacter Wingham ( Helicobacter winghamensis ), Desulfurobacterium thermolithotr ( D. thermolithotr ), Desulfomicrobium baculatum ( D. baculatum ), common desulfurization vibrio ( Desulfovibrio vulgaris , D. vulgaris ), Desulfovibrio alkaliphilus ( D. alkaliphile ), Lake Retba desulfurization bacteria ( Desulfohalobium retbaense , D. retbaense ), Deferribacter desulfuricans ( D. desulfuricans ), Desulfovibrio salexigenes ( D. of the Salem family ), Desulfovibrio lazy ( D. salexigenes ), Desulfovibrio aespoeensis ( D. Aesop's ), Cand. Puniceispirillum marinum , Calditerribrio nitroreducens ( C. nitroreducens ) or thermophilic bacteria that produce pyrophoric methane ( Methanothermus fervidus , M. fervidus ).

[0153] In some embodiments, the recombinant N-OST is derived from Campylobacter ( ). Campylobacter Prokaryotes. In some embodiments, the recombinant N-OST is derived from Campylobacter jejuni or Campylobacter guillier (e.g., from Campylobacter jejuni). pglB Gene product PglB, PglB Cj Or from Campylobacter gull pglB Gene product PglB, PglB Cl ).

[0154] In some implementations, the recombinant N-OST is recombinant PglB. Cj Recombinant PglB Cj Homologues or naturally occurring PglB Cj Recombination forms of variants. PglB Cj Homologues can include naturally occurring PglB. Cj Homologues (e.g., illustrated in Figures 4 and 6) and non-naturally occurring PglBCj Homologues. ikB Cj Homologues may include PglB as described in SEQ ID NO:1 Cj Proteins having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0155] In some implementations, the recombinant N-OST is recombinant PglB. Cl Recombinant PglB Cl Homologues or naturally occurring PglB Cl Recombination forms of variants. PglB Cl Homologues can include naturally occurring PglB. Cl Homologues (e.g., illustrated in Figure 4) and non-naturally occurring PglB Cl Homologues. ikB Cl Homologues may include PglB of SEQ ID NO:2 Cl Proteins having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0156] Some amino acid positions are conserved in different members of the phylogenetic N-OST family. See, for example, Figure 4. Some amino acid positions are conserved in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of members of the phylogenetic N-OST family. In some embodiments, the amino acid at the conserved amino acid position is modified in the recombinant N-OST provided herein.

[0157] In some embodiments, the recombined modified N-OST comprises a PglB fragment, for example, PglB Cj Fragment or PglB Cl Fragment. In some embodiments, the PglB fragment comprises at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or at least 650 consecutive amino acids of full-length PglB.

[0158] (a) PglB Cj Modification

[0159] In some implementations, the modified N-OST described herein is a modified wild-type N-OST, for example, wild-type PglB. Cj In some embodiments, the wild-type PglB Cj It is the wild-type PglB of SEQ ID NO:1 Cj Or its naturally occurring variants:

[0160]

[0161] In some implementations, PglB is modified Cj One or more of the amino acids Y77, S80, S196, N311, Y462, H479, K522, G476 or G477, or any combination thereof.

[0162] In some implementations, PglB is modified Cj The amino acid N311. In some embodiments, the modification of N311 is an N311V or N311I substitution. In some embodiments, the modification of N311 is an N311V substitution.

[0163] In some implementations, PglB is modified Cj The amino acids N311 and Y77. In some embodiments, the modification of Y77 is a substitution of Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, or Y77G. In some embodiments, the modification of Y77 is a substitution of Y77H.

[0164] In some implementations, PglB is modified Cj The amino acids N311 and S80. In some embodiments, the modification of S80 is an S80R substitution or an S80H substitution. In some embodiments, the modification of S80 is an S80R substitution.

[0165] In some implementations, the recombinant PglB Cj Included in PglB Cj The modification of at least one amino acid in the Q287LKFYxxR294 motif. In some embodiments, the modification of PglB... Cj At least one amino acid of Q287, L288, or K289. In some embodiments, the recombinant PglB Cj Includes Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q, or R294K substitutions.

[0166] In another embodiment, this document provides recombinant PglB containing N311V substitution. Cj .

[0167] In another embodiment, this document provides recombinant PglB comprising N311V substitution and Y77H substitution. Cj .

[0168] In another embodiment, this document provides recombinant PglB comprising N311V substitution and S80R substitution. Cj .

[0169] In another embodiment, this document provides recombinant PglB comprising Y77H substitution and Q287P substitution. Cj .

[0170] In another embodiment, this document provides recombinant PglB comprising S80R replacement and Q287P replacement. Cj .

[0171] In another embodiment, this document provides recombinant PglB comprising N311V substitution, S80R substitution, and Q287P substitution. Cj .

[0172] In another embodiment, this document provides recombinant PglB comprising N311V substitution, Y77H substitution, and Q287P substitution. Cj .

[0173] In another embodiment, this document provides a recombinant PglB comprising the N311V mutation, Y77H substitution, S80R substitution, and Q287P substitution. Cj .

[0174] In another embodiment, this document provides a recombinant PglB comprising N311V substitution, Y77H substitution, S80R substitution, Q287P substitution, and K289R substitution. Cj .

[0175] In another embodiment, this document provides recombinant PglB comprising N311V substitution and A699V substitution. Cj .

[0176] In another embodiment, this document provides recombinant PglB comprising K482R and D483H substitutions. Cj .

[0177] In some embodiments, the substitution may be a conservative amino acid substitution (e.g., from one basic amino acid to another). In some embodiments, the substitution may be a non-conservative amino acid substitution (e.g., from a basic amino acid to an acidic amino acid).

[0178] (b) PglB Cl Modification

[0179] In some implementations, the modified N-OST described herein is a modified wild-type NOST, for example, wild-type PglB. Cl (PglB of Campylobacter rubrum). In some embodiments, the wild-type PglB Cj It is the wild-type PglB of SEQ ID NO:2 Cl Or its naturally occurring variants:

[0180]

[0181] In some implementations, PglB is modified Cl One or more of the amino acids Y79, S82, N314, K488, or D489, or any combination thereof.

[0182] In some implementations, PglB is modified Cl The amino acid N314. In some embodiments, the modification of N314 is an N314V or N314I substitution. In some embodiments, the modification of N314 is an N314V substitution.

[0183] In some implementations, PglB is modified Cl The amino acids N314 and Y797. In some embodiments, the modification of Y79 is a substitution of Y79H, Y79T, Y79W, Y79R, Y79K, Y79A, or Y79G. ​​In some embodiments, the modification of Y79 is a substitution of Y79H.

[0184] In some implementations, PglB is modified Cl The amino acids N314 and S82. In some embodiments, the modification of S82 is an S82R substitution or an S82H substitution. In some embodiments, the modification of S82 is an S82R substitution.

[0185] In some implementations, the recombinant PglB Cl Included in PglB Cl The modification of at least one amino acid in the QLKFYxxR motif. In some embodiments, at least one amino acid, Q289, is modified. In some embodiments, the recombinant PglB Cl Includes Q289P, Q289K, or Q289R replacements.

[0186] In another embodiment, this document provides recombinant PglB containing N314V substitution. Cl .

[0187] In another embodiment, this document provides recombinant PglB comprising N314V substitution and Y79H substitution. Cl .

[0188] In another embodiment, this document provides recombinant PglB comprising N314V substitution and S82R substitution. Cl .

[0189] In another embodiment, this document provides recombinant PglB comprising Y79H replacement and Q289P replacement. Cl .

[0190] In another embodiment, this document provides recombinant PglB comprising S82R substitution and Q289P substitution. Cl .

[0191] In another embodiment, this document provides recombinant PglB comprising N314V substitution, S82R substitution, and Q289P substitution. Cl .

[0192] In another embodiment, this document provides recombinant PglB comprising N314V substitution, Y79H substitution, and Q289P substitution. Cl .

[0193] In another embodiment, this document provides a recombinant PglB comprising the N314V mutation, aY79H substitution, S82R substitution, and Q289P substitution. Cl .

[0194] In some embodiments, the substitution may be a conservative amino acid substitution (e.g., from one basic amino acid to another). In some embodiments, the substitution may be a non-conservative amino acid substitution (e.g., from a basic amino acid to an acidic amino acid).

[0195] 6.2 Screening Methods

[0196] In another aspect, this paper provides a method for screening a library of recombinant N-OST provided herein, the method comprising contacting each member of the recombinant N-OST library with a carrier protein and an oligosaccharide or polysaccharide lacking N-acetyl sugar at its reduced end to produce a bioconjugate.

[0197] In some embodiments, the bioconjugate is an N-glycosylated carrier protein.

[0198] The oligosaccharides and polysaccharides may include any oligosaccharides or polysaccharides described herein. See, for example, Section 6.4.

[0199] The carrier protein may include any carrier protein described herein. See, for example, section 6.5.

[0200] In some embodiments, the contact occurs in vitro. In some embodiments, the contact occurs in vivo. In some embodiments, the contact occurs in the host cells described herein. In some embodiments, the host cells are prokaryotic cells. In some embodiments, the host cells are *E. coli* cells.

[0201] In some embodiments, the recombinant N-OST library contains one or more recombinant N-OSTs provided herein. In some embodiments, the recombinant N-OST library contains at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 750, or at least 1,000 recombinant N-OSTs. In some embodiments, the recombinant N-OST library contains between about 2 and about 1,000, between about 10 and about 800, between about 50 and about 600, between about 100 and about 400, or between about 100 and about 200 recombinant N-OSTs.

[0202] In some embodiments, the method further includes determining the rate or yield of production of the bioconjugate. In some embodiments, the method further includes determining the conjugation level of the bioconjugate (e.g., the glycosylation level calculated as a percentage of the glycosylated carrier protein). Methods for determining the rate or yield of production of the bioconjugate or the level of conjugation of the bioconjugate are known in the art. See, for example, sections 6.9, 6.10, 6.12 and Examples 2-3.

[0203] In some embodiments, the method further includes selecting one or more recombinant N-OSTs from a library of recombinant N-OSTs.

[0204] In some embodiments, one or more recombinant N-OSTs are selected if the recombinant N-OST produces the bioconjugate at a rate that is about 2 to 100 times faster than that of wild-type N-OST, about 5 to 80 times faster, about 10 to 60 times faster, about 10 to 40 times faster, about 10 to 30 times faster, or about 10 to 20 times faster. In some embodiments, if the recombinant N-OST produces the bioconjugate at a rate that is more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times faster than wild-type N-OST, then one or more of the recombinant N-OST are selected.

[0205] In some embodiments, the one or more recombinant N-OSTs are selected if the N-OST mutant produces a bioconjugate in amounts between about 2 to about 100 times, about 5 to about 80 times, about 10 to about 60 times, about 10 to about 40 times, about 10 to about 30 times, or about 10 to about 20 times compared to wild-type N-OST. In some embodiments, if the N-OST mutant produces a bioconjugate in amounts exceeding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times the amount of wild-type N-OST, then one or more N-oligosaccharide transferase mutants are selected.

[0206] In some embodiments, one or more recombinant N-OSTs are selected if the recombinant N-OST is glycosylated to a carrier protein at a concentration of about 1% to about 70%, about 3% to about 65%, about 5% to about 60%, about 10% to about 55%, about 15% to about 50%, about 20% to about 45%, about 20% to about 40%, or about 25% to about 35% in vitro (e.g., in a reaction vessel), or in vivo (e.g., in host cells). In some embodiments, one or more recombinant N-OSTs are selected if the recombinant N-OST is glycosylated to a carrier protein at a concentration of at least 1%, at least 3%, 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% in vitro (e.g., in a reaction vessel), or in vivo (e.g., in host cells).

[0207] In another aspect, this paper provides a method for identifying recombinant N-OSTs provided herein, the recombinant N-OSTs having altered substrate selectivity compared to the wild-type form of recombinant N-OSTs, the method comprising substituting one or more amino acids (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids) in a structural model of the complex of the recombinant N-OST and the N-glycosylated carrier protein, wherein the side chain of the amino acid is located within a 2.5–4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0208] In some embodiments, the N-OST mutant has altered in vitro substrate selectivity. In some embodiments, the N-OST mutant has altered in vivo substrate selectivity.

[0209] 6.3 Structural Model

[0210] The structural models used to describe the recombinant N-OST disclosed herein comprise complexes of recombinant N-OST and bound N-glycosylated carrier proteins, and can be obtained using any method known to those skilled in the art. For example, structural models can be obtained using X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR). Exemplary methods for obtaining structural models of protein complexes are described, for example, in Bernhard Rupp's work. Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology Garland Science, 1st edition (October 20, 2009); Eaton E. Lattman and Patrick J. Loll; Protein Crystallography: AConcise Guide, Johns Hopkins University Press ; First edition (March 26, 2008); Arthur G. Palmer III and Wayne J. Fairbrother; Protein NMR Spectroscopy, 2nd Edition: Principles and Practice Academic Press, 2nd edition (December 28, 2005). The structural model can be, for example, an X-ray or NMR structural model of the complex of N-OST and the bound N-glycosylated carrier. In some embodiments, the structural model can be a homology model of the complex of recombinant N-OST and the bound N-glycosylated carrier protein. See, for example, Example 1 and Figure 2. The oligosaccharide or polysaccharide of the N-glycosylated carrier protein and the carrier protein component can be modeled in the product conformation or in the substrate conformation.

[0211] The structural model used to describe the recombinant N-OST disclosed herein may include any N-OST disclosed herein, any carrier protein, or any oligosaccharide or polysaccharide. See, for example, sections 6.1, 6.4, and 6.5.

[0212] The structural model may include a model of the full-length recombinant N-OST or a fragment thereof. For example, the structural model may be constructed using recombinant N-OST, recombinant wild-type N-OST, or N-OST purified from a organism expressing N-OST. In some embodiments, the structural model includes the catalytic site of the N-OST. The structural model may model N-OST from any organism possessing N-OST. The structural model may model any recombinant N-OST described herein. See, for example, Figure 4. In some embodiments, the structural model is a homology model generated using the experimentally revealed structure of *Campylobacter gullii* PglB (PDBid 3RCE) as a template. See, for example, Example 1. The structural model may be constructed using the oligosaccharides or polysaccharides and / or carrier proteins described herein. The N-OST, carrier protein, and oligosaccharide or polysaccharide components used to construct the structural model may all be from the same organism or from two or three different organisms.

[0213] In some embodiments, the bound N-glycosylated carrier protein comprises a native oligosaccharide or polysaccharide component (from the same organism as N-OST). In some embodiments, the bound N-glycosylated carrier protein comprises a heterologous oligosaccharide or polysaccharide component (from an organism different from N-OST). In some embodiments, the bound N-glycosylated carrier protein comprises a native oligosaccharide or polysaccharide component of Campylobacter jejuni (from Campylobacter jejuni). In some embodiments, the bound N-glycosylated carrier protein comprises a heterologous oligosaccharide or polysaccharide component of Campylobacter jejuni (not from Campylobacter jejuni).

[0214] In the structural model, physical distances, such as those known to those skilled in the art, can be determined using any method or software tool known to them, including the physical distance between certain N-OST amino acid side chains and monosaccharide units at the reducing ends of oligosaccharide or polysaccharide components of the bound N-glycosylated carrier protein. See, for example, Chang, G. et al., An internalcoordinate Monte-Carlo Method for Searching Conformational Space. J. Am. Chem. Soc., 1989, 111, 4379; Saunders, M. et al., Conformations of cycloheptadecane: A Comparison of Methods for Conformational Searching. J. Am. Chem. Soc. 1990, 112, 1419.

[0215] 6.4 Oligosaccharides and Polysaccharides

[0216] Oligosaccharides linked to carrier proteins via recombinant N-OST as provided herein can have 2-100 monosaccharide units, for example, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 monosaccharide units. Polysaccharides linked to carrier proteins via recombinant N-OST as provided herein can have more than 100 monosaccharide units, for example, 101, 110, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more monosaccharide units.

[0217] The carrier protein or N-OST may comprise any N-OST or any carrier protein disclosed herein. See, for example, sections 6.1 and 6.5.

[0218] In some embodiments, the sugar at the reducing end of the oligosaccharide or polysaccharide is a pentose, hexose, or heptose. In some embodiments, the sugar at the reducing end of the oligosaccharide or polysaccharide is a pentose aldose or pentose ketose. In some embodiments, the pentose is D-arabinose, D-lythose, D-ribose, D-xylose, D-ribulose, or D-xylulose. In some embodiments, the sugar at the reducing end of the oligosaccharide or polysaccharide is a hexose aldose or hexose ketose. In some embodiments, the hexose is, for example, D-allose, D-azose, D-glucose, D-mannose, D-gulose, D-iduroose, D-galactose, D-talose, D-allulose, D-fructose, D-sorbose, or D-tagatose. In some embodiments, the sugar at the reducing end of the oligosaccharide or polysaccharide is a deoxy or dideoxy sugar, for example, rhamnose, fucose, or apicoose. In some embodiments, the sugar at the reducing end of the oligosaccharide or polysaccharide is analdecyl heptanose or ketulose heptanose. In some embodiments, the heptanose is mannoheptanose.

[0219] The oligosaccharides and polysaccharides linked to the carrier protein via the recombinant N-OST provided herein can originate from any organism, such as prokaryotes or eukaryotes. In some embodiments, the oligosaccharides or polysaccharides are derived from pathogenic organisms, such as human pathogens or animal pathogens (e.g., livestock or pets). In some embodiments, the oligosaccharides or polysaccharides are derived from bacterial organisms. In some embodiments, the oligosaccharides or polysaccharides can be derived from *Escherichia coli*, *Salmonella* species (e.g., *Salmonella enterica* subsp. *enteroides*). 、 Salmonella enterica subsp. Salmonella enterica subsp. Arizona, Salmonella enterica subsp. Bis-Arizona, Salmonella enterica subsp. Howton, Salmonella enterica subsp. Bongore ( S. bongori ) and enteric Salmonella Indus subsp. Indos, Pseudomonas species (Pseudomonas aeruginosa), Klebsiella species (e.g., Klebsiella pneumoniae) K. pneumonia Acinetobacter spp., Chlamydia trachomatis, Vibrio cholerae, Listeria spp. (e.g., Listeria monocytogenes) L. monocytogenes Legionella pneumophila, Bordetella parapertussis, Burkholderia melioides and Burkholderia melioides, Burkholderia tularensis, Campylobacter species (Campylobacter jejuni); Clostridium difficile, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Streptococcus agalactiae, Neisseria meningitidis, Candida albicans, Haemophilus influenzae, Enterococcus faecalis, Leptospira brevicornu, Neisseria meningitidis, Haemophilus influenzae, Leishmania macrophylla.

[0220] In some embodiments, the oligosaccharide or polysaccharide contains an antigen, such as an epitope that is immunogenic in humans or animals (e.g., livestock or pets). In some embodiments, the oligosaccharide or polysaccharide contains the O antigen of *E. coli* (e.g., *E. coli*).O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, O20, O21, O22, O23, O24, O25, O26, O27, O28, O29, O30, O32, O 33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O46, O48 , O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, O6 3. O64, O65, O66, O68, O69, O70, O71, O73, O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O9 5. O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, O111, O112, O113, O114, O115, O116, O117, O118, O119 , O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O1 32. O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O15 5. O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O1 67, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, O187), Salmonella species (Salmonella enterica subsp. enterica, Salmonella enterica Salam, Salmonella enterica Arizona, Salmonella enterica Bi-Arizona, Salmonella enterica Howton, Salmonella Bongore, or Salmonella enterica Indica) antigens and O types 1-67 (as detailed in

[44] ), Pseudomonas species (Pseudomonas aeruginosa O serotypes 1-20

[45] ), Klebsiella species (e.g.,Klebsiella pneumoniae serotypes O1, O2 (and subserotypes), O3, O4, O5, O6, O7, O8, O9, O10, O11, O12,

[46] ), Acinetobacter spp. O antigen (e.g., Acinetobacter baumannii identified in

[47] , A. baumannii O antigen), Chlamydia trachomatis O antigen (serotypes A, B, C, D, E, F, G, H, I, J, K, L1, L2, L3), Vibrio cholerae O antigen O1 to 155, Listeria species, especially Listeria monocytogenes 1, 2, 3, 4 and their subserotypes, Legionella pneumophila serotypes 1-15 O antigen, Bordetella parapertussis O antigen, Burkholderia melioides and Burkholderia melioides O antigen, Francisella tularensis, Campylobacter species (Campylobacter jejuni); Clostridium difficile (serotypes A, G, H, K, S1, S4, D, Cd-5, K Toma) et al. 1988, and Clostridium perfringens ( C. perfringens Capsular polysaccharides of serotypes A, B, C, D and E, Staphylococcus aureus types 5 and 8, Streptococcus pyogenes (group B streptococcal capsular polysaccharides), Escherichia coli, Streptococcus agalactiae (group A streptococcal capsular polysaccharides), Neisseria meningitidis (serotypes A, B, C, W, Y, X), Candida albicans, Haemophilus influenzae, Enterococcus faecalis capsular polysaccharide type IV; and other surface polysaccharide structures, such as Brønsted brevis glycolipid (

[48] ), Neisseria meningitidis pili protein O-glycan [49, 50] and lipooligosaccharide (LOS), Haemophilus influenzae LOS, Leishmania sigmata lipophosphatidylcholine [51, 52]), tumor-associated carbohydrate antigens (malaria glycosylphosphatidylinositol, Mycobacterium tuberculosis arabinomannan

[53] .

[0221] In some embodiments, the oligosaccharide or polysaccharide is Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus, S. aureus ) or enteric Salmonella serotype ( Salmonella enterica sv., S. enterica sv. Polysaccharide. In some embodiments, the polysaccharide is Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2 polysaccharide.

[0222] In some embodiments, the oligosaccharide or polysaccharide comprises an N-acetyl sugar at the reducing end. In some embodiments, the oligosaccharide or polysaccharide comprising an N-acetyl sugar at the reducing end may include, for example, the O antigen of *E. coli* (e.g., O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, O20, O21, O22, O23, O24, O25, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O46, ...). O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, O63, O64, O65, O66, O68, O69, O70, O71, O73, O74, O75, O76, O77 , O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O 105, O106, O107, O108, O109, O110, O111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O1 28. O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O15 O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, O187), capsular polysaccharides of Staphylococcus aureus (e.g., CP5 or CP8), and Francisella tularensis. Schu4 Capsular polysaccharides, capsular polysaccharides of Streptococcus pneumoniae capsules (e.g., CP1, 4, 5, 12, 25, 38, 44, 45, or 46). 、Neisseria meningitidis piliin O glycan [49, 50], Burkholderia melioides and Burkholderia melioides O antigen, Bordetella parapertussis O antigen, Legionella pneumophila serotypes 1-15 O antigen, Listeria species O antigen, especially Listeria monocytogenes 1, 2, 3, 4 O antigen, Pseudomonas species O antigen (Pseudomonas aeruginosa O serotypes 1-20

[45] ), Klebsiella species O antigen (e.g., Klebsiella pneumoniae serotypes O1, O2 (and subserotypes), O3, O4, O5, O6, O7, O8, O9, O10, O11, O12,

[46] ), Shigella species O antigen (e.g., Shigella dysenteriae ( S. dysenteriae ) 、 Shigella sonnei ( S. sonnei ) 、 Shigella flexneri ( S. flexneri ) 、 Shigella boydii ( S. boydii ), Acinetobacter O antigen (e.g., Acinetobacter baumannii O antigen identified in

[47] ) or Listeria O antigen.

[0223] N-acetyl sugars may contain an amino-acetyl (N-acetyl) substituent at one or more carbon atoms of the sugar. For example, N-acetyl sugars may contain an N-acetyl substituent at the C2 atom of a monosaccharide unit (such as a glucose unit (N-acetylglucosamine)).

[0224] In some embodiments, the oligosaccharide or polysaccharide comprises a sugar at the un-N-acetylated reducing end. In some embodiments, the oligosaccharide or polysaccharide comprising an un-N-acetylated sugar at the reducing end may include, for example, *Escherichia coli* O20, *Salmonella* species (e.g., *Salmonella enterica* subsp. *enteroides*, *Salmonella enterica* subsp. *salami*, *Salmonella enterica* subsp. *argentina ... or *Salmonella enterica* subsp. *indicum* or *Salmonella typhi* (…). S. Typhi antigen, O antigen of types 1-67, group A streptococci (Streptococcus pyogenes) S. pyrogenes )) 、 Group B Streptococcus and Streptococcus pneumoniae CPS Serotypes (encoding wchA, wcjG, or wcjH in their capsular gene clusters, i.e., all serotypes except CP1, 4, 5, 12, 25, 38, 44, 45, and 46) capsular polysaccharides or enteric Salmonella serotypes ( Salmonella enterica sv., S. enterica sv. O antigen.

[0225] In some embodiments, the oligosaccharide or polysaccharide comprises Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2 polysaccharide, Vibrio cholerae O antigen (e.g., O1 to 155) or Listeria species O antigen (e.g., Listeria monocytogenes types 1, 2, 3, and 4).

[0226] In some embodiments, the oligosaccharide or polysaccharide contains DN-acetylfucosaccharide (D-FucNAc) residues at its reducing end, such as capsular polysaccharides of Staphylococcus aureus serotypes 5 and 8 or Pseudomonas aeruginosa O antigen serotypes O2, O5, O11, and O16.

[0227] In some embodiments, the oligosaccharide or polysaccharide contains a 4-amino-dN-acetylfucosamine (D-FucNAc4N) residue at its reducing end, for example, from Streptococcus pneumoniae (e.g., serotype 1), Shigella sonnei O antigen, or Shigella-like seromonas (e.g., *Streptococcus pneumoniae* O antigen, or *Shigella seromonas*). Plesiomonas shigelloides Some oligosaccharides or polysaccharides of O17.

[0228] In some embodiments, the oligosaccharide or polysaccharide contains a DN-acetyl quinosamine (D-QuiNAc) residue at its reducing end, such as Pseudomonas aeruginosa O antigen serotypes O6, O1, or Tularemia flavus serotype Schu4.

[0229] In some embodiments, the oligosaccharide or polysaccharide contains galactose residues at its reducing end, for example, in Salmonella LT2.

[0230] In some embodiments, the oligosaccharide or polysaccharide comprises Streptococcus pneumoniae capsular polysaccharide serotype 5, Escherichia coli O1, O2, and Cronobacter sakazakii O5, i.e., polysaccharides and oligosaccharides having a reducing end D-GlcNAc linked to L-rhamnose 1-4 in the β configuration.

[0231] 6.5 Carrier Proteins

[0232] The carrier protein can be linked to oligosaccharides or polysaccharides via the recombinant N-OST provided herein. See, for example, Section 6.1.

[0233] The carrier protein can be any natural carrier protein (from the same organism as N-OST) or any heterologous carrier protein (from a different organism than N-OST). In some embodiments, the carrier protein is an immunogen. The carrier protein can be a full-length protein or a fragment thereof. Exemplary carrier proteins include, but are not limited to, *Pseudomonas aeruginosa* exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, *Staphylococcus aureus* detoxified hemolysin A, aggregation factor A, aggregation factor B, *Escherichia coli* FimH, *Escherichia coli* FimHC, *Escherichia coli* heat-labile enterotoxin, detoxified variants of *Escherichia coli* heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, *Escherichia coli* sat protein, the passenger domain of *Escherichia coli* sat protein, *Campylobacter jejuni* AcrA, and *Campylobacter jejuni* natural glycoprotein. In some embodiments, the carrier protein is *Pseudomonas aeruginosa* exotoxin A (EPA).

[0234] In some embodiments, the recombinant N-OST N-glycosylated carrier protein described herein is modified, for example, in a manner that makes the protein less toxic and / or more sensitive to glycosylation. In some embodiments, the carrier protein is modified such that the number of glycosylation sites in the carrier protein is maximized in a particular manner that allows for the administration of lower concentrations of the protein, for example, in an immunogenic composition, in its bioconjugate form. Thus, in some embodiments, the carrier protein described herein is modified to contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycosylation sites than are typically associated with a carrier protein (e.g., relative to the number of glycosylation sites associated with a carrier protein in its native / natural (e.g., “wild-type”) state). In some embodiments, the introduction of a glycosylation site is accompanied by the insertion of a glycosylation concordant sequence (e.g., (i) a concordant sequence Asn-X-Ser(Thr), where X is independently selected from any amino acid other than Pro; or (ii) a concordant sequence D / EXNZS / T, where X and Z are independently selected from any amino acid other than Pro) at any location in the primary structure of the protein. Such an introduction of a glycosylation site may be accompanied by, for example, the addition of a new amino acid to the primary structure of the protein (addition of a glycosylation site in whole or in part), or modification of existing amino acids in the protein to generate a glycosylation site (not by adding an amino acid to the protein, but by mutating selected amino acids of the protein to form a glycosylation site). Those skilled in the art will recognize that the amino acid sequence of a protein can be readily modified using methods known in the art, such as recombinant methods including modifying the nucleic acid sequence encoding the protein. In specific embodiments, the glycosylation concordant sequence is introduced into specific regions of the carrier protein, such as the surface structure of the protein, at the N or C terminus of the protein, and / or in a disulfide-bonded loop at the base of the protein. In some implementations, for more efficient glycosylation, the classic 5-amino acid glycosylation concordance sequence can be extended with lysine residues, and thus the inserted concordance sequence can encode 5, 6, or 7 amino acids that will be inserted into or replace the amino acids of the receptor protein.

[0235] The N-OST may include any N-OST disclosed herein. See, for example, Section 6.1.

[0236] In some embodiments, the carrier protein includes a “tag,” which is a sequence of amino acids that allows for the isolation and / or identification of the carrier protein. For example, adding a tag to the carrier protein described herein can be used to purify the protein, and therefore can be used to purify conjugate vaccines containing the tagged carrier protein. Exemplary tags that may be used herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine-tags or 6XHis-tags), FLAG-tags, and HA tags. In some embodiments, the tags used herein are removable, for example, by chemical reagents or enzymatic methods once they are no longer needed, e.g., after the protein has been purified.

[0237] 6.6 Nucleic Acid

[0238] In another respect, this article provides nucleic acids encoding the recombinant N-OST provided herein (e.g., Section 6.1).

[0239] In some implementations, the nucleic acid encodes recombinant PglB Cj The modified amino acids are one or more of Y77, S80, S196, N311, Y462, H479, K522, G476, or G477.

[0240] In some implementations, the nucleic acid encodes PglB Cj PglB is modified Cj The amino acid N311. In some embodiments, the modification of N311 is an N311V or N311I substitution. In some embodiments, the modification of N311 is an N311V substitution.

[0241] In some implementations, the nucleic acid encodes recombinant PglB Cj The amino acids N311 and Y77 are modified. In some embodiments, the modification of Y77 is a substitution of Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, or Y77G. In some embodiments, the modification of Y77 is a substitution of Y77H.

[0242] In some implementations, the nucleic acid encodes recombinant PglB Cj PglB is modified Cj The amino acids N311 and S80. In some embodiments, the modification of S80 is an S80R substitution or an S80H substitution. In some embodiments, the modification of S80 is an S80R substitution.

[0243] In some implementations, the nucleic acid encodes recombinant PglB Cj The recombinant PglB Cj Included in PglBCj The modification of at least one amino acid in the Q287LKFYxxR294 motif. In some embodiments, the nucleic acid encodes recombinant PglB. Cj PglB is modified Cj At least one amino acid selected from Q287, L288, or K289. In some embodiments, the recombinant PglB Cj Includes Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q, or R294K substitutions.

[0244] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing an N311V substitution. Cj .

[0245] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing N311V and Y77H substitutions. Cj .

[0246] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing N311V and S80R substitutions. Cj .

[0247] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing Y77H and Q287P substitutions. Cj .

[0248] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing S80R and Q287P substitutions. Cj .

[0249] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing N311V replacement, S80R replacement, and Q287P replacement. Cj .

[0250] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing N311V, Y77H, and Q287P substitutions. Cj .

[0251] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing the N311V mutation, Y77H substitution, S80R substitution, and Q287P substitution. Cj .

[0252] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB comprising N311V replacement, Y77H replacement, S80R replacement, Q287P replacement, and K289R replacement. Cj .

[0253] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing N311V and A699V substitutions. Cj .

[0254] In another embodiment, this document provides a nucleic acid encoding a recombinant PglB containing K482R and D483H substitutions. Cj .

[0255] 6.7 Host Cell

[0256] In another aspect, this document provides host cells comprising the recombinant N-OSTs provided herein. In some embodiments, the host cells comprise two or more recombinant N-OSTs provided herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more recombinant N-OSTs).

[0257] In another aspect, this document provides a host cell comprising nucleic acids provided herein (e.g., encoding recombinant N-OST provided herein (e.g., Section 6.1)). See, for example, Section 6.6. In some embodiments, the host cell comprises two or more nucleic acids provided herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids).

[0258] In some embodiments, the host cell contains one or more other enzymes (e.g., glycosyltransferases) that can be used for the production of bioconjugates or the N-glycosylation of carrier proteins. In some embodiments, at least one of the other enzymes that can be used for the production of bioconjugates is a recombinant enzyme. In some embodiments, the host cell contains two or more other enzymes that can be used for the production of bioconjugates (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more other enzymes).

[0259] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is an *E. coli* cell. In some embodiments, the host cell comprises the recombinant N-OST provided herein. See, for example, section 6.1. In some embodiments, the host cell comprises a carrier protein and the recombinant N-OST provided herein. See, for example, sections 6.1 and 6.5. In some embodiments, the host cell comprises a carrier protein, the recombinant N-OST provided herein, and a recombinant glycosyltransferase. In some embodiments, the recombinant N-OST is recombinant PglB. Cj See, for example, section 6.1(a).

[0260] In some embodiments, the host cell used to produce the bioconjugates described herein is engineered to contain heterologous nucleic acids, such as heterologous nucleic acids encoding one or more carrier proteins and / or heterologous nucleic acids encoding one or more proteins, such as genes encoding one or more proteins. In some embodiments, heterologous nucleic acids encoding proteins involved in glycosylation pathways (e.g., prokaryotic and / or eukaryotic glycosylation pathways) are introduced into the host cell described herein. Such nucleic acids may encode proteins, including, but not limited to, oligosaccharide transferases and / or glycosaccharide transferases. Heterologous nucleic acids (e.g., nucleic acids encoding carrier proteins and / or nucleic acids encoding other proteins (e.g., proteins involved in glycosylation)) can be introduced into the host cell described herein using any method known to those skilled in the art, such as electroporation, thermally stimulated chemical transformation, natural transformation, phage transduction, and conjugation. In some embodiments, heterologous nucleic acids are introduced into the host cell described herein using plasmids, for example, plasmids (e.g., expression vectors) expressing the heterologous nucleic acids in the host cell. In some embodiments, the heterologous nucleic acid is introduced into the host cell described herein using the insertion method described in International Patent Application Publication No. WO 2014 / 057109.

[0261] In some embodiments, additional modifications may be introduced (e.g., using recombinant techniques) into the host cell described herein. For example, host cell nucleic acids (e.g., genes) encoding proteins may be deleted or modified in a manner that renders them inactive / dysfunctional (i.e., the deleted or modified host cell nucleic acids no longer encode functional proteins or no longer encode any proteins) in the host cell background (genome), the proteins forming components that may compete with or interfere with glycosylation pathways (e.g., compete with or interfere with one or more heterologous genes recombinantly introduced into the host cell involved in glycosylation). In some embodiments, when nucleic acids are deleted from the genome of the host cell provided herein, they are replaced with desired sequences, such as sequences that can be used for glycoprotein production.

[0262] Exemplary genes that can be deleted (and, in some cases, replaced with other desired nucleic acid sequences) in host cells include: host cell genes involved in glycolipid biosynthesis, such as... waaL (See, for example, Feldman et al., 2005, PNAS USA 102:3016-3021), lipid A core biosynthetic cluster ( waa ), galactose clusters ( gal ), arabinose clusters ( ara ), colonic acid clusters ( wc ), capsular polysaccharide clusters, undecylenoyl-p biosynthetic genes (e.g. uppS, uppP ), und-P reusable genes, metabolic enzymes involved in nucleotide activation and sugar biosynthesis, common antigenic clusters of intestinal bacteria, and prophage O antigen modification clusters such as gtrABS cluster.

[0263] The host cells described herein can produce the N-glycosylated carrier proteins described herein. In some embodiments, the N-glycosylated carrier proteins produced by the host cells described herein are antigens, such as viral or bacterial antigens that can be used in vaccines. In some embodiments, the N-glycosylated carrier proteins produced by the host cells described herein can be any carrier protein described herein, wherein the carrier protein is modified by the host cells described herein to have one or more beneficial characteristics, such as N-glycosylation.

[0264] Some of the examples below describe the application of the methods described herein in Gram-negative Escherichia coli host cells; however, any host cell known to those skilled in the art can be used to produce N-glycosylated carrier proteins, including archaea, prokaryotic host cells other than Escherichia coli, and eukaryotic host cells.

[0265] Exemplary prokaryotic host cells that can be used according to the methods described herein include, but are not limited to, species of Escherichia coli (Escherichia coli). Escherichia species), species of the genus Shigella ( Shigella species), species of the genus Klebsiella ( Klebsiella species) Xhantomonas Species, Salmonella species ( Salmonella species), Yersinia species ( Yersinia species), species of the genus Lactococcus ( Lactococcus species), species of the genus Lactobacillus ( Lactobacillus species), species of the genus *Pseudomonas* ( Pseudomonasspecies), species of the genus Corynebacterium ( Corynebacterium species), species of the genus Streptomyces ( Streptomyces species), species of the genus Streptococcus ( Streptococcus species), species of the genus Staphylococcus ( Staphylococcus species), species of the genus Bacillus ( Bacillus species) and Clostridium species ( Clostridium species).

[0266] In some embodiments, the host cell described herein comprises a genome in which one or more DNA sequences have been incorporated, wherein said DNA sequences encode proteins or contain operons / gene clusters involved in protein N-glycosylation. For example, in some embodiments, the host cell described herein comprises a genome in which one or more of the following have been inserted: DNA encoding N-OST, DNA encoding glycosyltransferases, DNA encoding carrier proteins, and containing rfb DNA of gene clusters, including DNA of capsular polysaccharide gene clusters, and / or DNA encoding epimerases.

[0267] The host cell may include the recombinant N-OST provided herein or a nucleic acid encoding the recombinant N-OST provided herein, wherein the recombinant N-OST may be derived from any organism possessing N-OST, including eukaryotes or prokaryotes. In some embodiments, the N-OST protein or the nucleic acid encoding N-OST is derived from the genus Campylobacter (e.g., from Campylobacter jejuni). pglB Gene).

[0268] The host cell described herein may contain glycosyltransferases known in the art or nucleic acid sequences encoding glycosyltransferases known in the art. In some embodiments, the glycosyltransferase is the glycosyltransferase described in International Patent Application Publication No. WO2011 / 138361, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the glycosyltransferase is derived from Gram-positive bacteria, for example, from Staphylococcus aureus. In some embodiments, the glycosyltransferase is capsular polysaccharide 5 from Staphylococcus aureus. 。 In some embodiments, the glycosyltransferase is capsular polysaccharide 8 from Staphylococcus aureus. In some embodiments, the glycosyltransferase is derived from Gram-negative bacteria, such as Escherichia coli. In some embodiments, the glycosyltransferase is derived from eukaryotes.

[0269] The host cell described herein may contain or produce carrier proteins known in the art, or contain nucleic acid sequences encoding carrier proteins known in the art. The carrier proteins produced by the host cell described herein contain at least one N-glycosylated concordant sequence, for example, the concordant sequence (i) Asn-X-Ser(Thr), wherein X is independently selected from any amino acid other than Pro; or (ii) D / EXNZS / T, wherein X and Z are independently selected from any amino acid other than Pro. Therefore, the host cell may contain a DNA sequence encoding an N-glycosylated concordant sequence. The host cell may include any carrier protein known in the art, including the carrier proteins described in Section 5.5. In some embodiments, the carrier protein is Pseudomonas aeruginosa exotoxin A (EPA), including EPA that has been modified to contain at least one N-glycosylated concordant sequence. In some embodiments, the carrier protein is cholera toxin B. In some embodiments, the carrier protein is AcrA. In some embodiments, the carrier protein is HLA. In some embodiments, the carrier protein is ClfA.

[0270] 6.8 Bioconjugates

[0271] The bioconjugates described herein are conjugates between a protein prepared in a host cell (e.g., any carrier protein described herein (e.g., section 6.5)) and an oligosaccharide or polysaccharide (e.g., any oligosaccharide or polysaccharide described herein; see, e.g., section

[00201] ), wherein the oligosaccharide or polysaccharide is linked to the protein by a host cell mechanism (e.g., N-linking). In some embodiments, the oligosaccharide or polysaccharide is an antigen (e.g., any antigen described herein; see, e.g., section 6.4). Glycoconjugates may include bioconjugates, as well as glycoantigen (e.g., oligosaccharide and polysaccharide)-protein conjugates prepared by other means (e.g., by chemical linking of the protein and the glycoantigen).

[0272] The recombinant N-OST described herein (see, for example, Section 6.1) can be used to produce host cells that produce bioconjugates comprising N-glycosylated carrier proteins. In some embodiments, bioconjugates are provided herein comprising carrier proteins N-glycosylated with antigens (e.g., oligosaccharides or polysaccharides) described herein. In some embodiments, the carrier protein is EPA. The bioconjugates described herein may, for example, but not limited to, comprise any carrier protein described herein. The bioconjugates described herein may, for example, but not limited to, comprise any oligosaccharides or polysaccharides described herein.

[0273] In some embodiments, the heterologous Campylobacter jejuni glycosylated carrier protein is Pseudomonas aeruginosa exotoxin (EPA)-Shigella dysenteriae O1 (EPA-O1), EPA-Staphylococcus aureus capsular polysaccharide type 5 (EPA-CP5), or EPA - Enteric Salmonella LT2 (EPA-LT2).

[0274] In some embodiments, this document provides a bioconjugate comprising EPA and one or more of the different oligosaccharides or polysaccharides described herein.

[0275] In some embodiments, this document provides a bioconjugate comprising a carrier protein conjugated to one or more of the following Escherichia coli O1, O2, O4, O6, O7, O8, O11, O15, O16, O17, O18, O20, O22, O25, O73, O75, and / or O83. In some embodiments, the carrier protein is EPA.

[0276] In some embodiments, this document provides a bioconjugate comprising a carrier protein conjugated to one or more different Pseudomonas aeruginosa polysaccharides. In some embodiments, the carrier protein is EPA.

[0277] In some embodiments, this document provides a bioconjugate comprising a carrier protein conjugated to one or more different Klebsiella pneumoniae polysaccharides. In one specific embodiment, the carrier protein is EPA.

[0278] 6.9 Methods for producing bioconjugates

[0279] In some embodiments, the recombinant N-OST provided herein (see, for example, section 6.1) can be used to produce the biological conjugates provided herein (see, for example, section 6.8), such as glycoconjugates. In some embodiments, the recombinant N-OST provided herein can be used to produce conjugate vaccines, i.e., vaccines containing oligosaccharides or polysaccharides (see, for example, section 5.4) and protein antigens of the pathogen against which the vaccine is designed.

[0280] In another aspect, this document provides a method for producing biological conjugates, the method comprising culturing host cells provided herein in a cell culture medium (see, for example, section 6.7). In some embodiments, the host cells contain nucleic acids encoding recombinant modified N-OSTs provided herein (see, for example, sections 6.1 and 6.6). In some embodiments, the host cells contain nucleic acids encoding carrier proteins described herein (see, for example, sections 6.5 and 6.6). In some embodiments, the carrier protein has one or more N-glycosylated concordant sequences. In some embodiments, the host cells contain nucleic acids encoding glycosyltransferases (see, for example, sections 6.6 and 6.7).

[0281] In some embodiments, the bioconjugate is an N-glycosylated carrier protein. The N-glycosylated carrier protein may comprise an oligosaccharide or polysaccharide component, including any oligosaccharide or polysaccharide described herein. See, for example, section

[00201] . The N-glycosylated carrier protein may comprise any carrier protein described herein. See, for example, section 6.5. In some embodiments, the bioconjugate is a native Campylobacter jejuni N-glycosylated polypeptide (comprising Campylobacter jejuni oligosaccharide or polysaccharide components and a Campylobacter jejuni carrier protein). In some embodiments, the bioconjugate is a heterologous Campylobacter jejuni glycosylated polypeptide (comprising polysaccharide components and / or carrier proteins not derived from Campylobacter jejuni). In some embodiments, the glycosylated polypeptide does not have an N-acetyl sugar at its reducing end. In some embodiments, the glycosylated polypeptide has galactose at its reducing end.

[0282] In some embodiments, when using host cells comprising the recombinant N-OST of the present invention, the bioconjugate is produced at a rate between about 2 to 100 times faster, about 5 to 80 times faster, about 10 to 60 times faster, about 10 to 20 times faster, or about 20 to 40 times faster than when using host cells comprising a wild-type form of recombinant N-OST. In some embodiments, when using host cells comprising the recombinant N-OST of the present invention, the bioconjugate is produced at a rate more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times faster than when using host cells comprising the wild-type form of recombinant N-OST.

[0283] In some embodiments, when using host cells comprising the recombinant N-OST of the present invention, the bioconjugate is produced at a yield between about 2 to about 100 times, about 5 to about 80 times, about 10 to about 60 times, about 10 to about 20 times, or about 20 to about 40 times, compared to when using host cells comprising a wild-type form of recombinant N-OST. In some embodiments, when using host cells comprising the recombinant N-OST of the present invention, the bioconjugate is produced in yields greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times greater than when using host cells comprising the wild-type form of recombinant N-OST.

[0284] In some embodiments, about 1% to about 70%, about 3% to about 65%, about 5% to about 60%, about 10% to about 55%, about 15% to about 50%, about 20% to about 45%, about 20% to about 45%, about 25% to about 40%, or about 30% to about 35% of the carrier protein in the host cell are glycosylated to form the bioconjugate.

[0285] In some embodiments, at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein in the host cell are glycosylated to form the bioconjugate.

[0286] In some embodiments, the method further includes purifying the bioconjugate from the host cell culture. Methods for purifying bioconjugates (such as N-glycosylated carrier proteins) from host cell cultures are known in the art. See, for example, Jan-Christer Janson. Protein Purification: Principles, High Resolution Methods, and Applications. Wiley; 3rd edition (March 22, 2011).

[0287] 6.10 Analytical Methods

[0288] Multiple methods can be used to analyze the structural composition and glycan length of the bioconjugates or N-glycosylated carrier proteins described in this paper.

[0289] In one implementation, hydrazine hydrolysis can be used to analyze glycans. First, the polysaccharides are released from their protein carriers by incubation with hydrazine according to the manufacturer's instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK). The nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, allowing the attached glycan to be released. The N-acetyl group is lost during this process and must be reconstituted by re-N-acetylation. The free glycan is purified on a carbon column and subsequently labeled at the reduced end with the fluorescent 2-aminobenzamide (Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB. Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid). Anal Biochem. September 20, 1995; 230(2):229-38). Based on the HPLC protocol of Royle et al. (Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM. An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins.) Anal Biochem. On May 1, 2002 (304(1):70-90), labeled polysaccharides were separated on a GlycoSep-N column (GL Sciences). The resulting fluorescence chromatograms indicated the length and number of polysaccharide repeating units. Structural information could be collected by collecting individual peaks and then performing MS / MS analysis. This confirmed the monosaccharide composition and sequence of the repeating units and also identified the homogeneity of the polysaccharide composition. Specific peaks at low molecular weights could be analyzed by MALDI-MS / MS, and the results could be used to confirm the polysaccharide sequence. Each peak corresponds to a polymer composed of a certain number of repeating units and their fragments. The chromatograms thus allowed for the measurement of polymer length distribution. Elution time indicated polymer length, and fluorescence intensity correlated with the molar abundance of various polymers.

[0290] In another embodiment, SDS-PAGE or capillary gel electrophoresis can be used to evaluate glycans and glycoconjugates. The polymer length of the O-antigen glycan synthesized here is defined by the number of repeating units assembled from linear chains. This means that the typical ladder-like pattern is a consequence of the different numbers of repeating units that make up the glycan. Thus, two adjacent bands separated by size in SDS-PAGE or other techniques differ by only a single repeating unit. When analyzing glycoproteins for glycan size, these differences in dispersion are utilized: unglycosylated carrier proteins and glycoconjugates with different polymer chain lengths are separated according to their electrophoretic mobilities. The number of the first detectable repeating units (n1) and the average number of repeating units (n) present on the glycoconjugate are measured. average These parameters can be used to verify batch-to-batch consistency or polysaccharide stability.

[0291] In another implementation, high-quality MS and size exclusion HPLC can be used to measure the size of the intact glycoconjugate.

[0292] In another embodiment, the anthrone-sulfuric acid assay can be used to measure the polysaccharide yield (Leyva A, Quintana A, Sánchez M, Rodríguez EN, Cremata J, Sánchez JC. Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation). Biologicals March 2008; 36(2):134-41. (Electronic publication on November 26, 2007).

[0293] (a) Changes in the Use of Glycosylation Sites

[0294] To confirm that the use of a specific site in a protein has been altered, the use of glycosylation sites can be quantified. Methods for doing so are listed below.

[0295] Glycopeptides LC-MS / MS: Glycoconjugates are digested with a protease, and the peptides are separated by a suitable chromatographic method (C18, hydrophilic interaction HPLC HILIC, GlycoSepN column, SE HPLC, AE HPLC), and the different peptides are identified using MS / MS. This method can be used with or without prior glycan shortening achieved by chemical (Smith degradation) or enzymatic methods. Quantification of glycopeptide peaks using UV detection in the 215–280 nm range allows for relative determination of glycosylation sites.

[0296] Size exclusion HPLC: Earlier elution times from SE HPLC columns reflect higher glycosylation site usage. See also (a).

[0297] (b) Homogeneity

[0298] The homogeneity (homogeneity of linked sugar residues) of glycoconjugates can be assessed by measuring glycan length and hydrodynamic radius.

[0299] 6.11 Benefits

[0300] The recombinant N-OST provided herein (see, for example, Section 5.1) and the methods provided herein using the recombinant N-OST provided herein (see, for example, Section 6.1) (see, for example, Sections 6.2 and 6.9) have particular commercial importance and relevance because they allow for the rapid, high-yield, large-scale, and low-cost fermentation of highly homogeneous bioconjugate products (e.g., glycoconjugate products or conjugate vaccine products). The recombinant N-OST provided herein enables the economically viable production of commercially and therapeutically valuable bioconjugates, such as conjugate vaccines. It is anticipated that enzymatic production methods using the recombinant N-OST provided herein will yield more homogeneous and reproducible bioconjugate products than commonly used chemical synthesis methods. It is anticipated that the reproducibility and robustness of bioconjugate production methods using the recombinant N-OST provided herein will contribute to reduced production costs. It is generally believed that the homogeneity of particular biotherapeutic conjugate vaccines can affect the clinical safety of pharmaceutical products.

[0301] 6.12 Analytical methods for testing benefits

[0302] Yield.Yield is measured as the amount of carbohydrates derived from one liter of bacterial production culture grown in a bioreactor under controlled and optimized conditions. After purification of the glycoconjugate, carbohydrate yield can be measured directly by anthrone assay or by an ELISA using carbohydrate-specific antiserum. Indirect measurement is possible by calculating the theoretical amount of carbohydrates per gram of protein using well-known BCA, Lowry, or Bardford assays and glycan length and structure. Alternatively, yield can be measured by drying the glycoprotein product from a volatile buffer and weighing it using a balance.

[0303] Homogeneity. Homogeneity refers to the variability in glycan length and the number of possible glycosylation sites. The methods listed above can be used for this purpose. SE-HPLC allows for the measurement of hydrodynamic radii. A higher number of glycosylation sites in a support results in a greater variation in the hydrodynamic radius compared to a support with fewer glycosylation sites. However, when analyzing individual glycan chains, they are more homogeneous due to their more controlled length. Glycan length is measured by hydrazine hydrolysis, SDS-PAGE, and CGE. Additionally, homogeneity can also refer to changes in the usage pattern of certain glycosylation sites, ranging to a wider / narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0304] The invention is further described in the following paragraphs:

[0305] 1. A recombinant N-oligosaccharide transferase (N-OST) that can detectably link an oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end to a carrier protein at an N-glycosylation concordance sequence.

[0306] 2. The method of paragraph 1, wherein the N-OST activity of the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end, ligated to the carrier protein at the N-glycosylation sequence, is detected by ELISA.

[0307] 3. The method of paragraph 1 or 2, wherein the ELISA signal indicating the N-OST activity is detectable if it is >2σ or >3σ above the ELISA background signal.

[0308] 4. A recombinant N-oligosaccharide transferase of any of paragraphs 1-3, wherein the carrier protein is a natural carrier protein from the same organism as N-OST.

[0309] 5. A recombinant N-oligosaccharide transferase of any of paragraphs 1-3, wherein the carrier protein is a heterologous carrier protein from an organism different from N-OST.

[0310] 6. The recombinant N-oligosaccharide transferase in paragraph 5, wherein the carrier protein is selected from: Pseudomonas aeruginosa ( P. aeruginosa Exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus (S. aureus) S. aureus The detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA and Campylobacter jejuni native glycoprotein.

[0311] 7. A recombinant N-oligosaccharide transferase of any one of paragraphs 1-6, wherein the carrier protein has at least one glycosylation motif.

[0312] 8. The recombinant N-oligosaccharide transferase of paragraph 7, wherein the at least one glycosylation motif comprises D / EYNXS / T (X, Y≠P).

[0313] 9. The recombinant N-oligosaccharide transferase of paragraph 7, wherein the at least one glycosylation motif comprises Asn-X-Ser(Thr), wherein X can be any amino acid other than Pro.

[0314] 10. A recombinant N-oligosaccharide transferase of any of paragraphs 1-9, wherein the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end contains an antigen.

[0315] 11. The recombinant N-oligosaccharide transferase of paragraph 10, wherein the antigen includes *Escherichia coli* (…). E. coli Antigens, Salmonella species ( Salmonella sp) antigen, Pseudomonas species ( Pseudomonas sp.) antigen, Klebsiella genus ( Klebsiella sp.) antigen, Acinetobacter O antigen, Chlamydia trachomatis (sp ... Chlamydia trachomatis Antigen, Vibrio cholerae ( Vibrio cholera Antigens, Listeria species ( Listeria sp.) antigen, Legionella pneumophila (sp.) antigen, Legionella pneumophila ( Legionella pneumophila ) serotype 1-15 antigen, Bordetella parapertussis ( Bordetella parapertussis Antigen, Burkholderia melioides ( Burkholderia mallei ) or Burkholderia melioides ( Burkholderia Burkholderia melioides antigen, Francisella tularensis (Francisella tularensis Antigens, Campylobacter species ( Campylobacter sp.) antigen; Clostridium difficile (sp.) Clostridium difficile Antigen, Streptococcus pyogenes ( Streptococcus pyrogenes Antigen, agalactiae (Streptococcus agalactiae) Streptococcus agalacticae Antigen, Neisseria meningitidis ( Neisseria meningitidis Antigen, Candida albicans ( Candida albicans Antigen, Haemophilus influenzae ( Haemophilus influenza ) antigen, Enterococcus faecalis ( Enterococcus faecalis Antigen, Brewera brucellosis ( Borrelia burgdorferi Antigen, Neisseria meningitidis ( Neisseria meningitidis Antigen, Haemophilus influenzae ( Haemophilus influenza Antigens, large Leishmania ( Leishmania major Antigen or Shigella sonnei ( Shigella sonnei ) or Streptococcus pneumoniae ( Streptococcus pneumoniae )antigen.

[0316] 12. A recombinant N-oligosaccharide transferase of any of paragraphs 1-10, wherein the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end is a Staphylococcus aureus or Salmonella enterica serum type polysaccharide.

[0317] 13. The recombinant N-oligosaccharide transferase of paragraph 12, wherein the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end is Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2 polysaccharide.

[0318] 14. A recombinant N-oligosaccharide transferase from any of paragraphs 1-13, wherein the recombinant N-oligosaccharide transferase is PglB (PglB) of Campylobacter jejuni. Cj ) or PglB of Campylobacter gullii (PglB Cl ).

[0319] 15. A recombinant N-oligosaccharide transferase of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase comprises a modification in one or more amino acids, wherein, in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0320] 16. The recombinant N-oligosaccharide transferase of paragraph 13, wherein the modification in one or more amino acids is an amino acid substitution.

[0321] 17. The recombinant N-oligosaccharide transferase of paragraph 13 or 16, wherein one or more amino acids are among the non-conserved amino acids in the phylogenetic family of N-oligosaccharide transferases.

[0322] 18. The recombinant N-oligosaccharide transferase of paragraph 17, wherein the non-conserved amino acid is conserved in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the phylogenetic family of N-oligosaccharide transferases.

[0323] 19. A recombinant N-oligosaccharide transferase of any of paragraphs 14-18, wherein the recombinant N-oligosaccharide transferase comprises modifications in two or more amino acids.

[0324] 20. A recombinant N-oligosaccharide transferase of any of paragraphs 14-18, wherein the recombinant N-oligosaccharide transferase comprises modifications in three or more amino acids.

[0325] 21. A recombinant N-oligosaccharide transferase of any of paragraphs 14-18, wherein the recombinant N-oligosaccharide transferase comprises modifications in four or more amino acids.

[0326] 22. A recombinant N-oligosaccharide transferase of any of paragraphs 14-21, wherein at least one of the one or more amino acids is located in the cytoplasmic pericyclic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase.

[0327] 23. The recombinant N-oligosaccharide transferase of paragraph 22, wherein the cytoplasmic pericytic loop of the transmembrane domain is the large outer loop 5 (EL5).

[0328] 24. The recombinant N-oligosaccharide transferase of paragraph 23, wherein the recombinant N-oligosaccharide transferase is PglB (PglB) of Campylobacter jejuni. Cj ) or PglB of Campylobacter gullii (PglB Cl ), and EL5 is PglB Cj or PglB Cl EL5.

[0329] 25. A recombinant N-oligosaccharide transferase of any of paragraphs 14-24, wherein the recombinant N-oligosaccharide transferase further comprises a mutation in one or more amino acids of the QLKFYxxR motif.

[0330] 26. The recombinant N-oligosaccharide transferase of paragraph 25, wherein the QLKFYxxR motif is the Q287LKFYxxR294 motif.

[0331] 27. The recombinant N-oligosaccharide transferase of paragraph 26, wherein the Q287LKFYxxR294 motif is PglB. Cj The Q287LKFYxxR294 motif.

[0332] 28. A recombinant N-oligosaccharide transferase from any of paragraphs 14-27, wherein the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0333] 29. A recombinant N-oligosaccharidase of any of paragraphs 14-28, wherein the protein component of the N-glycosylated carrier protein bound thereto is Campylobacter jejuni protein.

[0334] 30. A recombinant N-oligosaccharidotransferase of any of paragraphs 14-28, wherein the protein component of the N-glycosylated carrier protein bound thereto is selected from: Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholerae toxin B subunit (CTB), cholerae toxin, detoxified variant of cholerae toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.

[0335] 31. A recombinant N-oligosaccharide transferase of any of paragraphs 28-30, wherein the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0336] 32. A recombinant N-oligosaccharide transferase, wherein one or more of the enzymes modified from PglB are present. Cj The amino acids Y77, S80, S196, N311, Y462, H479, K482, D483, K522, G476 and G477.

[0337] 33. The recombinant N-oligosaccharide transferase in paragraph 32, wherein PglB is modified. Cj N311.

[0338] 34. The recombinant N-oligosaccharide transferase in paragraph 33, including recombinant PglB. Cj Includes replacement of N311V or replacement of N311I.

[0339] 35. The recombinant N-oligosaccharide transferase of paragraph 34, wherein the recombinant PglB Cj Includes replacement of N311V.

[0340] 36. The recombinant N-oligosaccharide transferase mutant of paragraph 32, wherein the recombinant PglB Cj It also includes modifications in one or more amino acids selected from Y77 and S80.

[0341] 37. The recombinant N-oligosaccharide transferase mutant of paragraph 36, wherein the recombinant PglB Cj It includes amino acid substitutions selected from Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, Y77G, S80R, and S80H.

[0342] 38. The recombinant N-oligosaccharide transferase mutant of paragraph 37, wherein the recombinant PglB Cj It includes amino acid substitutions selected from Y77H and S80R.

[0343] 39. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-38, wherein the recombinant PglB Cj Also included in PglB Cj Amino acid modifications in one or more amino acids of the Q287LKFYxxR294 motif.

[0344] 40. The recombinant N-oligosaccharide transferase mutant of paragraph 39, wherein the recombinant PglB Cj Amino acid modifications included in one or more amino acids selected from Q287, L288, K289 and R294.

[0345] 41. The recombinant N-oligosaccharide transferase mutant of paragraph 40, wherein the recombinant PglB Cj It includes one or more amino acid substitutions selected from Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q and R294K.

[0346] 42. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It contains amino acid substitution N311V.

[0347] 43. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid substitutions Y77H and N311V.

[0348] 44. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid replacements S80R and N311V.

[0349] 45. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid substitutions of Q287P and Y77H or Q287P and S80R.

[0350] 46. ​​A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid replacements S80R, Q287P, and N311V.

[0351] 47. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid substitutions Y77H, Q287P, and N311V.

[0352] 48. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid substitutions Y77H, S80R, Q287P, and N311V.

[0353] 49. A recombinant N-oligosaccharide transferase mutant of any of paragraphs 28-31, wherein the recombinant PglB Cj It includes amino acid substitutions Y77H, S80R, Q287P, K289R, and N311V.

[0354] 50. The recombinant N-oligosaccharide transferase mutant of paragraph 28, wherein the recombinant PglB Cj It includes amino acid substitutions N311V and A699V.

[0355] 51. The recombinant N-oligosaccharide transferase mutant of paragraph 28, wherein the recombinant PglB Cj It includes amino acid substitutions K482R and D483H.

[0356] 52. A recombinant N-oligosaccharide transferase of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase can increase the effect of the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end on the carrier protein. in vivo N-glycosylation or in vitroThe yield of N-glycosylation is determined to produce glycosylated carrier proteins at levels exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0357] 53. A recombinant N-oligosaccharide transferase of any of the preceding paragraphs, wherein, compared to the wild-type form of the recombinant N-oligosaccharide transferase, the recombinant N-oligosaccharide transferase enables the oligosaccharide or polysaccharide lacking N-acetyl sugar at the reducing end to affect the carrier protein. in vivo N-glycosylation or in vitro The rate of N-glycosylation increases by more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times.

[0358] 54. A recombinant N-oligosaccharide transferase of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase mutant may be glycosylated in vivo or in vitro with at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein.

[0359] 55. A recombinant N-oligosaccharide transferase (N-OST) comprising a modification in one or more amino acids, wherein, in a structural model of a complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, the amino acid side chain is located within a 2.5-4.0 Å distance of one of three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0360] 56. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the recombinant N-oligosaccharide transferase is PglB (PglB) of Campylobacter jejuni. Cj ) or PglB of Campylobacter gullii (PglBCl )

[0361] 57. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the carrier protein is selected from: Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholerae toxin B subunit (CTB), cholerae toxin, detoxified variant of cholerae toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.

[0362] 58. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the oligosaccharide or polysaccharide component contains an antigen.

[0363] 59. The recombinant N-oligosaccharide transferase of paragraph 58, wherein the antigens include Escherichia coli antigen, Salmonella spp. antigen, Pseudomonas spp. antigen, Klebsiella spp. antigen, Acinetobacter spp. O antigen, Chlamydia trachomatis antigen, Vibrio cholerae antigen, Listeria spp. antigen, Legionella pneumophila serotypes 1-15 antigen, Bordetella parapertussis antigen, Burkholderia melioides or Burkholderia melioides antigen, Francisella tularensis antigen, Campylobacter spp. antigen; Clostridium difficile antigen, Streptococcus pyogenes antigen, Streptococcus agalactiae antigen, Neisseria meningitidis antigen, Candida albicans antigen, Haemophilus influenzae antigen, Enterococcus faecalis antigen, Leptospira brevicornu antigen, Neisseria meningitidis antigen, Haemophilus influenzae antigen, Leishmania giantiformis antigen or Shigella sonnei or Streptococcus pneumoniae antigen.

[0364] 60. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-59, wherein the recombinant N-oligosaccharide transferase comprises modifications in two or more amino acids.

[0365] 61. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-59, wherein the recombinant N-oligosaccharide transferase comprises modifications in three or more amino acids.

[0366] 62. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-59, wherein the recombinant N-oligosaccharide transferase comprises modifications in four or more amino acids.

[0367] 63. A recombinant N-oligosaccharide transferase of any of paragraphs 55-59, wherein at least one of the one or more amino acids is located in the cytoplasmic pericyclic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase.

[0368] 64. The recombinant N-oligosaccharide transferase of paragraph 63, wherein the cytoplasmic pericytic loop of the transmembrane domain is the large outer loop 5 (EL5).

[0369] 65. The recombinant N-oligosaccharide transferase of paragraph 64, wherein the recombinant N-oligosaccharide transferase is PglB (PglB) of Campylobacter jejuni. Cj ), and EL5 is PglB Cj EL5.

[0370] 66. A recombinant N-oligosaccharide transferase of any of paragraphs 55-59, wherein the recombinant N-oligosaccharide transferase further comprises a modification in one or more amino acids of the QLKFYxxR motif.

[0371] 67. The recombinant N-oligosaccharide transferase of paragraph 66, wherein the recombinant N-oligosaccharide transferase further comprises a modification in one or more amino acids of the Q287LKFYxxR294 motif.

[0372] 68. The recombinant N-oligosaccharide transferase of paragraph 66, wherein the QLKFYxxR motif is PglB Cj The Q287LKFYxxR294 motif.

[0373] 69. A recombinant N-oligosaccharide transferase of any of paragraphs 55-68, wherein the modification is an amino acid substitution.

[0374] 70. The recombinant N-oligosaccharide transferase of paragraph 69, wherein the amino acid substitution is a non-conservative amino acid substitution.

[0375] 71. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the bound N-glycosylated polypeptide product is a naturally occurring N-glycosylated carrier protein from the same organism as the recombinant N-oligosaccharide transferase.

[0376] 72. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the N-glycosylated carrier protein is a heterologous N-glycosylated carrier protein, wherein the oligosaccharide or polysaccharide component of the N-glycosylated carrier protein is from an organism different from the recombinant N-oligosaccharide transferase, and / or the carrier protein component of the N-glycosylated carrier protein is from an organism different from the recombinant N-oligosaccharide transferase.

[0377] 73. The recombinant N-oligosaccharide transferase of paragraph 55, wherein the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0378] 74. The recombinant N-oligosaccharide transferase of paragraph 73, wherein the bound N-glycosylated polypeptide product is a natural Campylobacter jejuni glycosylated carrier protein.

[0379] 75. The recombinant N-oligosaccharide transferase of paragraph 73, wherein the bound N-glycosylated polypeptide product is a heterologous Campylobacter jejuni glycosylated carrier protein.

[0380] 76. The recombinant N-oligosaccharide transferase of paragraph 75, wherein the heterologous Campylobacter jejuni glycosylated carrier protein is Pseudomonas aeruginosa exotoxin (EPA)-Shigella dysenteriae O1 (EPA-O1), EPA-Staphylococcus aureus capsular polysaccharide type 5 (EPA-CP5), or EPA - Enteric Salmonella LT2 (EPA-LT2).

[0381] 77. The recombinant N-oligosaccharide transferase of paragraph 73, wherein the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein does not have an N-acetyl monosaccharide at its reducing end.

[0382] 78. The recombinant N-oligosaccharide transferase of paragraph 73, wherein the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0383] 79. A recombinant N-oligosaccharide transferase, wherein one or more of the following are modified: PglB Cj The amino acids Y77, S80, S196, N311, Y462, H479, K482, D483, K522, G476 and G477.

[0384] 80. The recombinant N-oligosaccharide transferase in paragraph 79, which modifies PglB Cj N311.

[0385] 81. Recombinant N-oligosaccharide transferases in paragraph 79, including recombinant PglB. Cj It includes amino acid substitutions selected from N311V and N311I.

[0386] 82. The recombinant N-oligosaccharide transferase in paragraph 81, including recombinant PglB. Cj It contains amino acid substitution N311V.

[0387] 83. The recombinant N-oligosaccharide transferase of paragraph 79, wherein one or more of the enzymes are modified from PglB. Cj The amino acids of Y77 and S80.

[0388] 84. The recombinant N-oligosaccharide transferase of paragraph 83, wherein the recombinant PglBCj It includes amino acid substitutions selected from Y77H, Y77T, Y77W, Y77R, Y77K, Y77A, Y77G, S80R, and S80H.

[0389] 85. The recombinant N-oligosaccharide transferase in paragraph 84, including recombinant PglB. Cj It includes amino acid substitutions selected from Y77H and S80R.

[0390] 86. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-86, wherein the recombinant PglB Cj Also includes PglB Cj The modification of one or more amino acids of the Q287LKFYxxR294 motif.

[0391] 87. The recombinant N-oligosaccharide transferase of paragraph 86, wherein the recombinant PglB Cj It contains one or more amino acids selected from Q287, L288 and K289.

[0392] 88. The recombinant N-oligosaccharide transferase of paragraph 87, wherein the recombinant PglB Cj It includes substitutions selected from Q287P, Q287K, Q287R, L288M, L288F, L288I, L288C, K289R, K289N, K289Q and R294K.

[0393] 89. A recombinant N-oligosaccharide transferase of any of paragraphs 73-78, wherein the recombinant PglB Cj Includes replacement of N311V.

[0394] 90. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj Includes replacement Y77H and replacement N311V.

[0395] 91. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj Includes replacement S80R and replacement N311V.

[0396] 92. The recombinant N-oligosaccharide transferase in any of paragraphs 73-78, including recombinant PglB. Cj It includes substitutions Q287P and Y77H or substitutions Q287P mutations and substitutions S80R.

[0397] 93. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB CjThis includes replacement S80R, replacement Q287P, and replacement N311V.

[0398] 94. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj This includes replacement Y77H, replacement Q287P, and replacement N311V.

[0399] 95. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj This includes replacement Y77H, replacement S80R, replacement Q287P, and replacement N311V.

[0400] 96. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj This includes replacing Y77H, S80R, Q287P, K289R, and N311V.

[0401] 97. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj Includes replacement N311V and replacement A699V.

[0402] 98. A recombinant N-oligosaccharide transferase of any one of paragraphs 73-78, wherein the recombinant PglB Cj It includes the K482R substitution mutation and the D483H substitution.

[0403] 99. A recombinant N-oligosaccharide transferase, wherein the recombinant N-oligosaccharide transferase can detectably link an oligosaccharide or polysaccharide lacking an N-acetyl sugar at the reducing end to a carrier protein.

[0404] 100. A recombinant N-oligosaccharide transferase of any of paragraphs 99, wherein the recombinant N-oligosaccharide transferase can detectably link an oligosaccharide or polysaccharide having a galactosyl monosaccharide at a reducing end to a carrier protein.

[0405] 101. The recombinant N-oligosaccharide transferase of paragraph 100, wherein the oligosaccharide or polysaccharide is a serotype oligosaccharide or polysaccharide of Staphylococcus aureus or Salmonella enterica.

[0406] 102. The recombinant N-oligosaccharide transferase of paragraph 100, wherein the oligosaccharide or polysaccharide is an oligosaccharide or polysaccharide of Staphylococcus aureus CP5 or Salmonella enterica typhus serotype LT2.

[0407] 103. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-102, wherein the recombinant N-oligosaccharide transferase can increase the in vivo or in vitro N-glycosylation yield of the polysaccharide lacking N-acetyl sugar at the reducing end to produce the glycosylated carrier protein at a level more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times above the background level in an assay for detecting the N-glycosylated carrier protein.

[0408] 104. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-102, wherein, compared with the wild-type form of the recombinant N-oligosaccharide transferase, the recombinant N-oligosaccharide transferase can increase the in vivo or in vitro N-glycosylation yield of the polysaccharide to the carrier protein by more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 17 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, more than 40 times, more than 45 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times.

[0409] 105. A recombinant N-oligosaccharide transferase of any one of paragraphs 55-102, wherein said recombinant N-oligosaccharide transferase can produce at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein. in vivo N-glycosylation level or in vitro Glycosylation level.

[0410] 106. A recombinant N-oligosaccharide transferase PglB Cj It contains N311V substitution.

[0411] 107. A recombinant N-oligosaccharide transferase PglB Cj It contains the N311V mutation and the Y77H substitution.

[0412] 108. A recombinant N-oligosaccharide transferase PglB Cj It contains the N311V mutation and the S80R substitution.

[0413] 109. A recombinant N-oligosaccharide transferase PglB Cj It contains N311V mutation, Y77H mutation and S80R substitution.

[0414] 110. A recombinant N-oligosaccharide transferase PglB Cj It contains the N311V mutation and the Q287P substitution.

[0415] 111. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V mutation, Y77H substitution and Q287P substitution.

[0416] 112. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V mutation, S80R substitution and Q287P substitution.

[0417] 113. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V replacement, Y77H replacement, S80R replacement and Q287P replacement.

[0418] 114. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V substitution and A669V substitution.

[0419] 115. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V replacement, Y77H replacement, S80R replacement, Q287P replacement and K289R replacement.

[0420] 116. A recombinant N-oligosaccharide transferase PglB Cj It includes K482R substitution and D483H substitution.

[0421] 117. A recombinant N-oligosaccharide transferase PglB Cj It includes N311V substitution and A669V substitution.

[0422] 118. A recombinant N-oligosaccharide transferase PglB Cl (PglB Campylobacter rubrum), which contains N314V substitution.

[0423] 119. A recombinant N-oligosaccharide transferase PglB Cl It contains the N314V mutation and the Y79H substitution.

[0424] 120. A recombinant N-oligosaccharide transferase PglB ClIt contains the N314V mutation and the S82R substitution.

[0425] 121. A recombinant N-oligosaccharide transferase PglB Cl It contains N314V mutation, Y79H mutation and S82R substitution.

[0426] 122. A recombinant N-oligosaccharide transferase PglB Cl It contains the N314V mutation and the Q289P substitution.

[0427] 123. A recombinant N-oligosaccharide transferase PglB Cl It includes the N314V mutation, Y79H substitution, and Q289P substitution.

[0428] 124. A recombinant N-oligosaccharide transferase PglB Cl It includes the N314V mutation, S82R substitution, and Q289P substitution.

[0429] 125. A recombinant N-oligosaccharide transferase PglB Cl It includes N314V replacement, Y79H replacement, S82R replacement and Q289P replacement.

[0430] 126. A recombinant N-oligosaccharide transferase PglB Cl It includes K488R substitution and D489H substitution.

[0431] 127. A nucleic acid that encodes a recombinant N-oligosaccharide transferase of any one of segments 1-126.

[0432] 128. A host cell comprising any one of paragraphs 1-127 of a recombinant N-oligosaccharide transferase.

[0433] 129. The host cell of paragraph 128, said host cell further comprising recombinant glycosyltransferase.

[0434] 130. A host cell containing the nucleic acid described in paragraph 128.

[0435] 131. The host cell of any of the preceding paragraphs, wherein the host cell is a prokaryotic cell.

[0436] 132. The host cell of paragraph 131, wherein the host cell is an Escherichia coli cell.

[0437] 133. A method for producing a biological conjugate, the method comprising culturing host cells of any of the above paragraphs in a cell culture medium.

[0438] 134. The method of paragraph 133, wherein the host cell comprises a carrier protein and a recombinant N-oligosaccharide transferase.

[0439] 135. The method of paragraph 134, wherein the host cell further comprises a recombinant glycosyltransferase.

[0440] 136. The method in paragraph 134, wherein the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0441] 137. The method in paragraph 134 uses carrier proteins selected from: Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, aggregation factor A, aggregation factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholerae toxin B subunit (CTB), cholerae toxin, detoxified variants of cholerae toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.

[0442] 138. The method of paragraph 134, wherein the bioconjugate is an N-glycosylated carrier protein.

[0443] 139. The method of paragraph 134, wherein the bioconjugate is a natural Campylobacter jejuni. N- Glycosylated carrier proteins.

[0444] 140. The method of paragraph 134, wherein the bioconjugate is a heterologous Campylobacter jejuni. N- Glycosylated carrier proteins.

[0445] 141. The method of paragraph 138, wherein the N-glycosylated carrier protein does not have N-acetyl sugar at the reducing end of its oligosaccharide or polysaccharide component.

[0446] 142. The method of paragraph 141, wherein the N-glycosylated carrier protein has galactose at the reducing end of its oligosaccharide or polysaccharide component.

[0447] 143. The method of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase mutant can increase the rate of bioconjugate production by more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 17, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 times compared to the rate achieved by the wild-type form of the recombinant N-oligosaccharide transferase.

[0448] 144. The method in any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase mutant can increase the yield of bioconjugate production to a level exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times.

[0449] 145. The method of any of the preceding paragraphs, wherein at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein in the host cell is glycosylated to form the bioconjugate.

[0450] 146. The method of any of the preceding paragraphs, the method further comprising purifying the bioconjugate from the host cell culture.

[0451] 147. A method for screening a library of recombinant N-oligosaccharide transferases, wherein each recombinant N-oligosaccharide transferase comprises a modification in one or more amino acids, the method comprising contacting each member of the library of recombinant N-oligosaccharide transferases with a carrier protein and an oligosaccharide or polysaccharide lacking N-acetyl sugar at its reducing end to produce a bioconjugate.

[0452] 148. The method of paragraph 147, wherein the bioconjugate is an N-glycosylated carrier protein.

[0453] 149. The method of paragraphs 147 or 148, wherein the contact occurs in vitro.

[0454] 150. The method of either paragraph 147 or 148, wherein the contact occurs within the body.

[0455] 151. The method of paragraph 150, wherein the contact occurs in a host cell.

[0456] 152. The method of paragraph 151, wherein the host cell is a prokaryotic cell.

[0457] 153. The method of paragraph 151, wherein the host cell is an Escherichia coli cell.

[0458] 154. The method of any one of paragraphs 147-153, wherein the library of said recombinant N-oligosaccharide transferases comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 500, at least 750, or at least 1,000 recombinant N-oligosaccharide transferases.

[0459] 155. The method of paragraph 147, wherein the library of said recombinant N-oligosaccharide transferase comprises one or more recombinant N-oligosaccharide transferases from any of paragraphs 1-97.

[0460] 156. The method of paragraph 147, the method further comprising selecting one or more recombinant N-oligosaccharide transferases from a library of the recombinant N-oligosaccharide transferases.

[0461] 157. The method of paragraph 156, wherein if the recombinant N-oligosyltransferase produces the bioconjugate at a rate more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times faster than the wild-type form of the recombinant N-oligosyltransferase, then the one or more recombinant N-oligosyltransferases are selected.

[0462] 158. The method of paragraph 156, wherein if the N-oligosaccharide transferase mutant produces the bioconjugate at a level exceeding the background level by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times, then the one or more N-oligosaccharide transferase mutants are selected.

[0463] 159. The method of paragraph 156, wherein the recombinant N-oligosyltransferase glycosylates at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the carrier protein in the host cell, then the one or more recombinant N-oligosyltransferases are selected.

[0464] 160. The method of any of paragraphs 133-159, the method further comprising analyzing the rate or yield of production of the bioconjugate.

[0465] 161. A method for identifying a recombinant N-oligosaccharide transferase having altered substrate specificity compared to a wild-type form of an N-oligosaccharide transferase, the method comprising modifying one or more amino acids, in a structural model of a complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, wherein the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

[0466] 162. The method of paragraph 161, the method comprising modifying two or more amino acids of the recombinant N-oligosaccharide transferase.

[0467] 163. The method of paragraph 161, the method comprising modifying three or more amino acids of the recombinant N-oligosaccharide transferase.

[0468] 164. The method of paragraph 161, the method comprising modifying four or more amino acids of the recombinant N-oligosaccharide transferase.

[0469] 165. The method of paragraph 161, wherein at least one of the one or more amino acids is located in the cytoplasmic pericyclic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase.

[0470] 166. The method of paragraph 165, wherein the cytoplasmic ring of the transmembrane domain is the large outer ring 5 (EL5).

[0471] 167. The method of paragraphs 161-166, the method further comprising mutating one or more amino acids of the QLKFYxxR motif of the recombinant N-oligosaccharide transferase.

[0472] 168. The method of paragraph 167, wherein the QLKFYxxR motif is the Q287LKFYxxR294 motif.

[0473] 169. The method of any of paragraphs 161-168, wherein the bound N-glycosylated carrier protein is a naturally occurring N-glycosylated carrier protein.

[0474] 170. The method of any of paragraphs 161-168, wherein the bound N-glycosylated carrier protein is a heterologous N-glycosylated carrier protein.

[0475] 171. The method of paragraph 161, wherein the recombinant N-oligosaccharide transferase is recombinant PglB. Cj .

[0476] 172. The method of paragraph 171, wherein the bound N-glycosylated carrier protein is a naturally occurring Campylobacter jejuni N-glycosylated carrier protein.

[0477] 173. The method of paragraph 171, wherein the bound N-glycosylated carrier protein is a heterologous Campylobacter jejuni N-glycosylated carrier protein.

[0478] 174. The method of paragraph 171, wherein the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein does not have an N-acetyl monosaccharide at its reducing end.

[0479] 175. The method of paragraph 171, wherein the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein has a galactosyl monosaccharide at its reducing end.

[0480] 176. The method of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase has altered in vitro substrate selectivity.

[0481] 177. The method of any of the preceding paragraphs, wherein the recombinant N-oligosaccharide transferase has altered in vivo substrate selectivity.

[0482] 7. Examples

[0483] 7.1 Example 1: PglBCj Modeling and Computer environment Oligosaccharide conjugate

[0484] Using the experimental Campylobacter gull structure as a template (PDBid 3RCE), a set of homology models for Campylobacter jejuni PglB was prepared using different homology modeling methods. The model prepared using the HHpredB method (Söding J, Biegert A, Lupas AN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005:33, W244-8.) was selected because its coordinates were better scored by the QMEAN model property assessment tool (Benkert P, Biasini M, Schwede T. Toward the estimation of the absolute quality of individual protein structuremodels. Bioinformatics. 2011: 27:343-350). Next, Mg was transferred from the Campylobacter gull structure (PDBid 3RCE). 2+Ions and receptor peptides. To visualize the orientation of proteins in the cell membrane, a phospholipid bilayer model was derived from entries in the OPM database of Campylobacter guillier (Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 2012 40:D370-6.). The first repeating unit of the native heptasaccharide ligand of Campylobacter jejuni and the LT2 polysaccharide of Salmonella intestinalis was parameterized to explain charge and tautomerism and the resulting low-energy conformation (Shelley JC, Cholleti A, Frye LL, Greenwood JR, Timlin MR, Uchimaya M: J Comput Aided Mol Des. 2007, (12):681-91). These low-energy conformations were then incorporated into the model, and conformations were sampled (Kolossváry, I.; Guida, WC Low-modeConformational Search Elucidated. Application to C39H80 and Flexible Docking of 9-Deazaguanine Inhibitors to PNP. J. Comput. Chem. 1999, 20, 1671). The sugar was defined as a freely moving substructure, and positional constraints were placed on all atoms in the protein backbone and on all atoms within a 6 Å radius around OS. Distance constraints were applied to the CO-N and N-C1 bonds between the first sugar unit and ASP-N. 1000 steps of conformational sampling were established using OPLS (Kaminski, GA; Friesner, RA; Tirado-Rives, J.; Jorgensen, WJJ Phys. Chem. B 2001, 105, 6474). This included a fully atomic water model and 35 kcal / mol. -1 A window is used to filter possible conformations, which are then processed using 21 kJ / mol. -1 A similar scheme with a narrower energy window is refined.

[0485] PglB CjThe selection of potential oligosaccharide interaction residues as mutagenic targets required the preparation of a homology model, which was prepared using the experimentally revealed structure of Campylobacter guillier PglB (PDBid 3RCE) as a template. The first repeating unit of the native Campylobacter jejuni N-glycan substrate and the heterologous Salmonella enterica LT2 polysaccharide was simulated with PglB. Cj The combination of these components. The monosaccharide subunit composition of the repeating units of the natural oligosaccharide substrate and the two heteropolysaccharides analyzed in this study showed... Figure 1 In this study, the oligosaccharide structure was conformally sampled while simultaneously attaching the amide nitrogen of an asparagine residue within the receptor peptide. It was assumed that the conformation of the enzyme and therefore the ligand was product-like, i.e., just before release from the active site. Therefore, the structural model did not account for any factors related to the initial binding of the undecyprene-pyrophosphate-linked oligosaccharide (OS) substrate. Figure 2A Examples of predicted conformations of the first repeating unit of Campylobacter jejuni OS and Salmonella enterica LT2 are shown in the figure. Substantial differences exist between the two oligosaccharides in the total interaction with protein residues and their relative orientation at the binding site. Snapshots of thermodynamically favorable conformations are shown. Figure 2B and 2C In the middle. Although natural OS substrates give it and wild-type PglB Cj The surrounding residues provide numerous hydrogen-bonded couplers, but the LT2O antigen repeat unit lacks similar interactions, thus supporting a mechanistic model where low transfer efficiency is caused by poor binding between the carbohydrate substrate and the active site. The LT2O antigen repeat unit consists of four hexoses lacking N-acetyl substituents. Figure 1 This limits the possibility of hydrogen bond formation.

[0486] To select in PglB WT The mutagenic sites within the sequence were used to identify amino acid side chains located within a 2.5–4 Å distance of the native Campylobacter jejuni N-glycan. The following residues matched this criterion: Y77, S80, S196, N311, Y462, H479, and K522. Figure 2B In the cases of G476 and G477, the carbonyl oxygen atom of the predicted polypeptide backbone is located close to the innermost sugar. Figure 2B ).

[0487] 7.2 Example 2: Mutagenesis of predicted sugar-interacting residues

[0488] The bacterial strains and plasmids used in this study are described in Table 1. *Escherichia coli* W3110 was used. waaL As a product for the production of EPA-Campylobacter jejuni OS and EPA- in in vivo glycosylation experiments ShigellaO1 glycoconjugate and EPA-CP5 host strain. Enteric Salmonella typhimurium serotype SGSC228 (which produces LT2 polysaccharide but lacks functionality) waaL Genes were used to produce LT2-EPA. Supercompetent *E. coli* cells were used for the initial transformation of constructed plasmid libraries and variants. *E. coli* DH5α was used as the standard host for plasmid production and storage.

[0489] Table 1. Strains and plasmids

[0490]

[0491] Codon-optimized PglB was expressed from the low-copy plasmid pEXT21, obtained from the gene synthesis service Genescript. The following construction was used to build... pglB Template plasmid for the library: using restriction sites Kpn I and BamH I(pGVNX1049) will be codon-optimized pglB-HA pGVXN925 was PCR-subcloned into pACT3Kan, and then a TAA stop codon was inserted before the sequence encoding the HA peptide tag using QuikChange (pGVNX1050). The reduced-size pACT3Kan-derived template plasmid pGVXN1413 was constructed as follows, which lacks... lacI Repressed gene (semi-constitutive expression of PglB): This involves the repression of pGVXN1050 (encoding wild-type PglB). Kpn I- BamH I fragment is linked into pGVXN1415 BamH I- Kpn In the I vector main strand fragment. The following is the construction of a smaller template plasmid pGVXN1418 (PglB N311V) for the second round library: pGVXN1050... AscI - BamH Fragment I (Wild Type) pglB The third and last gene) is linked into pGVXN1415. BamH I- AscI Within the fragment. All plasmids were verified by DNA sequencing.

[0492] Add appropriate antibiotics to all growth media to ensure plasmid maintenance (ampicillin, Amp: 100 mg / L). -1 Chloramphenicol, Cm: 10 mg / L -1 Kanamycin, Kan: 30 mg / L -1 Spectinomycin, Sp: 80 mg / L -1 Tetracycline, Tet: 20 mg / L-1 ).

[0493] The mutagenic primers and sequencing services were obtained from Microsynth (Balgach, Switzerland). QuikChange used pGVXN1050, pGVXN1415, or pGVXN1418 as templates to construct PglB variants and libraries. Sequencing was then used to validate the desired mutation.

[0494] Only the forward primer sequences for each primer pair are given, with the respective reverse complementary sequences used for the reverse primers. Degenerate codons at mutation sites are underlined. Saturation mutagenesis of N311: 5'-GC TTC ATG TAC TTCAAC GTT NNK Saturation mutagenesis of CAG ACG ATC CAA GAA GTG G-3' (SEQ ID NO:3), Y77: 5'-CATCAG CCG AAC GAT CTG AGT NNK TAC GGT AGC TCT CTG TCC G-3'(SEQ ID NO:4), four amino acids (Ala, Ser, Cys, Gly) of G476-G477 randomized 5'-C GAT GTT AAA ACG CTG GTC GAC KSTKST AAA CAC CTG GGC AAG G-3'(SEQ ID NO:5), S80 saturated mutagenesis 5'-CG AAC GAT CTGAGT TAT TAC GGT NNK TCT CTG TCC GCG CTG ACC-3'(SEQ ID NO:6), Q287 saturation mutagenesis 5'-GGT GTT GAT CCG ATT CTG TAC NNK CTG AAA TTT TAT ATC TTC CGC TCA G-3'(SEQID NO:7), 5'-GTT GAT CCG ATT CTG TAC CAG (Saturation-induced mutagenesis of L288) NNK AAA TTT TAT ATCTTC CGC TCA GAT G-3'(SEQ ID NO:8), 5'-GAT CCG ATT CTG TAC CAGCTG, a saturated mutagenesis of K289 NNK TTT TAT ATC TTC CGC TCA GAT GAA TCG-3'(SEQ ID NO:9), Saturated Mutagenesis of F290 5'-CCG ATT CTG TAC CAG CTG AAA NNKTAT ATC TTC CGC TCA GAT GAA TCG-3'(SEQ IDNO:10), Saturated Mutagenesis of Y291 5'-CG ATT CTG TAC CAG CTG AAA TTT NNK ATC TTC CGC TCAGAT GAA TCG-3'(SEQ ID NO:11), 5'-G TAC CAG CTG AAA TTT TAT ATCTTC (Saturated mutagenesis of R294) NNK TCA GAT GAA TCG GCA AAC CTG-3' (SEQ ID NO:12). A first-round library was constructed using wild-type PglB plasmids pGVXN1050 or pGVXN1413 as templates. A second-round library was constructed using pGVXN1418 (PglB N311V) or pGVXN1930 (PglB-HA N311V) as templates. The initial mutant library was based on pGVXN1050 as the template plasmid. However, it was found that such libraries repeatedly generated variant plasmids with a 2.1 kB reduction in the vector backbone, attributed to recombination events at repetitive sequences present in the original pACT3 sequence. This size reduction led to… lacI The loss of the repressor gene, in turn, promoted a 2-fold increase in semiconstitutive expression of PglB and EPA-CP5 levels (disclosed elsewhere). To avoid such undesirable recombination, pGVXN1413, pGVXN1418, and pGVXN1930 were used as templates in subsequent libraries.

[0495] Following the manufacturer's instructions (Stratagene), a shuffled library of neutral and slightly beneficial second-round variants was constructed using the Multi Site-Directed Mutagenesis Kit. A mixture of three oligonucleotides was used, with primers targeting the Y77 region: 5'-CAT CAG CCG AAC GAT CTG AGT. YMT TAC GGT MGT TCT CTG TCC GCG CTG AC-3' (SEQ ID NO:13), and primer 5'-C GGT GTT GATCCG ATT CTG TAC targeting the EL5 region. MVGWTKMAK TTT TAT ATC TTC CGC TCA GAT GAA TCG -3' and 5'- C GGTGTT GAT CCG ATT CTG TAC MVGWTKCGTA 4:1 molar ratio mixture of TTT TAT ATC TTC CGC TCA GAT GAA TCG-3' (SEQ ID NO:14). The modified PglB variant N311V (pGVXN1418) was used as a template.

[0496] Libraries with less than 20% wild-type clones were used for screening. Plasmid libraries were generated as follows: at least 1000 XL10-Gold colonies (5000 colonies for shuffled libraries) were resuspended in phosphate-buffered saline (PBS), followed by plasmid purification using a standard microparticle preparation kit. The plasmid library was then transformed into *E. coli* and *Salmonella enterica* expression strains using a standard electroporation procedure.

[0497] In addition to reducing the concentration of IPTG added during induction to 30 µM to minimize inclusion body formation, mutant libraries and individual variant plasmids were screened in 96-well plates as previously described. The PglB variant plasmid was isolated from the expression strains by re-transformation of the plasmid preparation in chemocompetent *E. coli* DH5α and selection for kanamycin-only resistance. Mutations were characterized by Sanger sequencing of the purified plasmids using two overlapping reads. *Salmonella enterica* SGSG228 (pGVXN150), *E. coli* St1717 (pGVXN150, pGVXN393), and *E. coli* W3110 were used in chemo or electrocompetent *Salmonella enterica* SGSG228 (pGVXN150, pGVXN393), *E. coli* St1717 (pGVXN150, pGVXN393), and *E. coli* W3110. waaL (pGVXN64, pGVXN150) were used as host strains for screening LT2-EPA, CP5-EPA and O1-EPA DWP-ELISA, respectively.

[0498] The host strains used for the production of EPA-CP5, EPA-LT2, and EPA-O1 in the shake-flask experiments were similar to those in the DWP experiments. *E. coli* W3110 was used... waaL (pACYC( pgl mut pGVXN150) is used for EPA- Cj The host strain produced by OS. The kinetics of glycoprotein formation were recorded as follows: Biomass-normalized periplasmic protein extracts were prepared periodically after induction, followed by sandwich ELISA. Three aliquots containing LB medium (5 g / L) were used. -1 Yeast extract, 10 g / L -1 and 5 g L -1 Pre-culture tubes containing NaCl were inoculated with individual colonies from fresh streaked or transformed plates and incubated overnight at 37°C and 160 rpm. The pre-culture was then inoculated 1:50 with triplicate aliquots of LB-M9 medium (5 g / L) containing 50% (v / v). -1Yeast extract, 10 g / L -1 Tryptone, 12.8 g / L -1 Na2HPO4∙7H2O, 3.0 g L -1 KH2PO4, 0.5 g L -1 NaCl, 1.0 g L -1 A conical flask containing NH4Cl, 2 mM MgSO4∙7H2O, and 0.1 mM CaCl2 was prepared and incubated at 37 °C and 160 rpm. The solution was then heated to 0.5 OD. 600 Add 1 mM IPTG and 4 g L -1 L-arabinose was used for induction, and the stirrer speed was reduced to 100 rpm. Salmonella In the case of the host strain, EPA / EPA-LT2 degradation was observed in LB-M9 shake flask cultures after overnight induction. Degradation can be prevented by using a high-intensity composite medium (2 YT, 10 g L). -1 Yeast extract, 14 g / L -1 Tryptone (M9 salt) will induce OD 600 Reduce to 0.3-0.4, and after induction, switch to static incubation (completely anaerobic growth).

[0499] Periplasmic extracts were prepared. The extracts were diluted 1000–20,000 times in PBS containing 1% w / v milk powder and analyzed by sandwich ELISA. Only dilutions producing unsaturated ELISA signals (absorbance below 1.0 at 450 nm) were used for data analysis. To purify the hexahistidine-labeled protein, periplasmic extracts from triplicate overnight shake-flask cultures were combined and Ni-affinity chromatography was performed using a HiTrap FF column (GE Healthcare) according to standard protocols. SDS-PAGE and Coomassie staining were performed using standard methods. The relative combined intensities of the EPA-LT2 glycoforms were quantified using ImageJ software (imagej.nih.gov).

[0500] Periplasmic protein extracts were appropriately diluted and analyzed by sandwich ELISA in 96-well plates. The capture antibody for all ELISA analyses was protein G purified goat-anti-EPA antiserum. Rabbit anti-Staphylococcus aureus CP5 and rabbit anti-... Salmonella O:5 / O:4 (Staten Serum Institute, Denmark), Rabbit Anti- ShigellaO1 and rabbit anti-Campylobacter jejuni were used to detect EPA-linked oligosaccharides and polysaccharides. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Bio-Rad, Reinach, Switzerland) and Ultra-TMB substrate (Thermo-Scientific / Pierce) were used for ELISA development. The HRP reaction was stopped by adding 2M H2SO4, and the absorbance (ELISA signal) at 450 nm relative to air was measured using a plate reader. An appropriate development time was selected to avoid signal saturation (maximum absorbance ≤ 1.0 at 450 nm). The result was obtained by subtracting the value from the expression PglB. mut The average absorbance values ​​of samples derived from the syngeneic control strain were used to correct for background. Western blot analysis of periplasmic and total cellular proteins was performed as previously described. EPA and PglB-HA were detected using rabbit anti-EPA (Sigma-Aldrich, Buchs, Switzerland) and rabbit anti-HA (Sigma-Aldrich) primary antibodies, respectively. Oligosaccharides and polysaccharides conjugated to EPA were detected using the same antibodies used for ELISA.

[0501] Using PglB of Campylobacter jejuni OS Cj In the model, the amide group of N311 forms a direct hydrogen bond with the C6 hydroxyl group of the second monosaccharide, i.e., GalNAc (counting from the reducing end). Figure 2A ).Will pglB The plasmid (in which N311 is randomly replaced with all other 19 amino acids) was transformed into a nontoxic form expressing LT2 polysaccharide and Pseudomonas aeruginosa exotoxin (EPA). salmonella genus In the strain, three clones exhibiting significantly improved glycosylation efficiency compared to wild-type PglB were screened in 96-well plates using a glycoprotein-specific ELISA (Figure 3A). In all of them, residue N311 was mutated to valine (codons: 2xGTT, 1xGTG).

[0502] The same library was then transformed into *E. coli* cells expressing *Staphylococcus aureus* capsular polysaccharide type 5 (CP5) and EPA. Seven clones exhibiting enhanced glycosylation efficiency compared to wild-type PglB were identified by measuring CP5-EPA productivity using ELISA (Figure 3B). Six of these clones carried the amino acid substitution N311V (codons GTG and GTT), and one showed the mutant N311I (codon ATT). The high proportion of viable clones (≈80%) indicates that N311 is highly resistant to mutations, consistent with the considerable variability at this position in the homologous N-OST sequence (Figure 4).

[0503] The above mutagenesis treatment localizes Y77 to another amino acid closely adjacent to the oligosaccharide binding site. Y77 is located in the periplasmic ring of the transmembrane domain and, according to our model, can interact with the oligosaccharide through water-mediated hydrogen bonds and direct hydrogen bonds. Figure 2A The residuals were found to be highly resistant to mutation (80-90% of CP5-EPA clones were generated in saturated mutagenesis libraries); this again corresponds to the high degree of variability in the homologous protein sequence (Figure 4). Only neutral amino acid substitutions (Y77L, Y77F) were found, but no improved variants were identified.

[0504] Still in PglB Cj The model models residues Y462, G476, G477, and H479 as being located close to the bound natural oligosaccharides. Figure 2A However, they are highly conserved in bacterial N-OST. Although variations are limited to naturally occurring amino acid substitutions, they are observed in Y462, G476, G477, and H479 PglB. Cj The CP5-EPA glycoconjugate signal was reduced by 50-90% in the variants, with notable exceptions being G476P and H479N, which were found to be neutral mutations. Figure 5 Random combinations of the small amino acids alanine, serine, cysteine, and glycine at G476–G477 all result in reduced or eliminated CP5-EPA production.

[0505] In summary, this first round of mutagenesis, targeting amino acids closely adjacent to the oligosaccharide binding site of PglB, identified N311 as the site with the highest impact on improving glycosylation yield. Additionally, Y77 was identified as mutation-resistant.

[0506] 7.3 Example 3: Effect of N311V on glycoprotein formation rate

[0507] PglB was analyzed in shake flask cultures. Cj Effects of variant N311V on in vivo glycosylation rates of different oligosaccharide / polysaccharide substrates (Figure 6). Expression of PglB from low copy number vectors Cj N311V and PglBwt. Cells expressing the mutant N-OST produced 8-fold more LT2-EPA after overnight induction (Fig. 6A). The fold increases after 2h and 4h of induction were 22-fold and 11-fold, respectively. The initial rate of CP5-EPA formation increased by 5.1-fold (Fig. 6B). The initial rate of O1-EPA formation was also increased by 2-fold in mutant N311V (Fig. 6C), although this polysaccharide substrate was not used for library screening. Conversely, for the natural Campylobacter jejuni OS substrate with PglB, the EPA production rate was significantly higher. in vivoGlycosylation was not found to have a significant effect. The increase in ELISA signal over time and the beneficial or neutral effect of N311V correspond to the Western blot results of exemplary periplasmic protein samples (Figure 7).

[0508] To analyze the reasons for the beneficial effects of N311V production, we constructed wild-type PglB and PglB N311V variants using a C-terminal hemagglutinin (HA) peptide tag. This allowed us to track PglB expression levels during experiments. The PglB-HA-specific bands in biomass-normalized whole-cell protein samples derived from the mutants exhibited lower intensity and greater variability than those from wild-type PglB. Figure 8A The degradation products, corresponding in size to the C-terminal periplasmic domain, appear after induction, indicating the destabilizing effect of the mutation. Although they have a significant negative effect on PglB stability, they are present in PglB expression... Cj EPA-CP5 production in N311V cells (tracked by ELISA) increased significantly again. Figure 8B ).

[0509] 7.4 Example 4: Mutagenesis and Screening of Other Wheels

[0510] Based on the principle of iterative saturation mutagenesis, PglB is used. Cj N311V was used as a template for randomization of Y77 and S80. The latter residue also varied among PglB homologues (Fig. 4) and faced the modeled oligosaccharide substrate binding site, which was located just above the outer loop EL1 extending from the membrane (Fig. 2). Both Y77 and S80 were found to be highly resistant to mutations, exhibiting 70-80% active clones when screened for LT2-EPA production (Table 1). The 10 clones with the highest ELISA signals were sequenced, and Y77 was modified by changing different amino acids, showing a bias towards residues containing basic side chains (Table 2). In the NNK library of randomized S80, the variant S80R was dominant among the best-performing clones (Table 2).

[0511] Table 2. PglB mutations in the second round of saturated mutagenesis library Cj Mutation tolerance of residues and amino acid substitutions identified in the 10 clones with the highest LT2-EPA ELISA signal. Clones were counted as active when the background-corrected ELISA signal exceeded 10% of the mean of the N311V control wells.

[0512]

[0513] PglB in the N-terminal portion of EL5 Cj Q 287 LKFYxxR294 The motif is highly conserved in the PglB sequence of Campylobacter species, but not in the N-OST of more distantly related species (Fig. 9). Since the innermost two glycosubunits of the N-linked glycans in Campylobacter species are observed to be similar (first, 2,4-diacetamido-2,4,6-trideoxyhexose; second, N-acetyl-hexosamine), it is hypothesized that Campylobacter-specific Q... 287 LKFYxxR 294 The residues of the motif may affect oligosaccharide specificity. Saturated mutagenesis libraries were generated at these positions using the modified variant N311V as a template. Significant differences between the first and second parts of the motif were observed when screened for LT2-EPA production in host strains (Table 2). Although saturated mutagenesis of Q287, L288, and K289 yielded 60–80% viable clones, the neighboring residues F290, Y291, and R294 were highly mutagenic. The 10 best-performing clones from the Q287, L288, and K289 libraries exhibited non-random amino acid substitutions (Table 2). Proline and the positively charged amino acids lysine and arginine were overexpressed at position Q287. Alternating hydrophobic residues (M, I, F, or C) were exclusively found at L288. A bias towards residues with amides (Q, N) or positively charged side chains (R) was observed at position K289 (Table 2).

[0514] In the final step, neutral and slightly beneficial mutations were identified regarding residues Y77, S80, Q287, L288, and K289. When screening 720 clones of this library for LT2-EPA production, numerous positive outliers were identified, one example of which is given in… Figure 10 In section A, clones with at least a 2.5-fold increase in ELISA signal compared to the average signal of template control clones on the same plate were sequenced (n = 14). S80R was detected in 79% (n = 11) of these clones, Q287P in 43% (n = 6) and Y77H in 29% (n = 4). Dual mutants Y77H-N311V and S80R-N311V were found to be 2-fold and 4-fold, respectively. Q287P appeared only in combination with Y77H or S80R. Y77S, L288I, L288F, K289R, and K289Q were found once or twice in combination with the more frequently observed mutations, indicating that these amino acid substitutions were neutral.

[0515] Representative improved variant plasmids were retransformed into EPA-LT2 expression strains and re-screened in triplicate DWP mini-cultures. Figure 10B). Significant additive beneficial effects were confirmed with Y77H (2.1-fold relative to N311V) and S80R (1.8-fold relative to N311V). When combined with S80R-N311V, Q287P resulted in a further 1.7-fold increase in EPA-LT2 ELISA signal, leading to a 15-fold overall improvement relative to wild-type PglB in overnight induced DWP cultures. Figure 10 B). A cumulative improvement in EPA-LT2 formation was also observed when substances purified from shake-flask cultures by Ni-NTA affinity chromatography were analyzed by SDS-PAGE and Western blotting. Figure 10 C). Sugar type and its relationship Salmonella Serum-type O:4 and O:5 specific antibodies were hybridized to detect branched abicosyls within LT2 polysaccharide and its O-acetylated form, respectively. Image quantification software was used for PglB... Cj The combined strength of the glycosidic band (>80 kDa) increased by 9 times and 16 times, respectively, for variants N311V and S80R-Q287P-N311V.

[0516] In summary, it has been confirmed that recombinant N-OSTs with altered substrate specificity have been successfully identified. Specifically, N-OSTs capable of conjugating carrier proteins containing oligosaccharides or polysaccharides lacking an N-acetyl group in the monosaccharide unit at the non-reducing end of the oligosaccharide or polysaccharide have been identified. Advantageously, the identified N-OSTs contain certain amino acid substitutions and allow for the production of medically relevant glycovaccines at increased rates and yields compared to wild-type N-OSTs.

[0517] 7.5 Example 5: Mutagenesis of Campylobacter jejuni residues K482 and D483

[0518] Using forward oligonucleotide primers 5'-GTA GAT GGT GGA AAG CAT TTW GGT NDTNDT AATTTT TTC CCT TCT TTT GCT TTA AGC -3' and reverse primer 5'- GCT TAA AGC AAA AGA AGG GAAAAA ATT AHNAHN ACC WAA ATG CTT TCC ACC ATC TAC -3' (mutated codons with underscores; N = A, T, G, or C; D = A, T, or G; W = A or T), using QuikChange to convert non-conservative PglB CjResidues K482 and D483 were simultaneously randomized to 12 non-redundant, chemically distinct amino acids (S, N, I, V, D, G, F, Y, C, L, H, R). The IPTG-induced plasmid pGVXN407 (encoding the gene sequence of wild-type PglB with a C-terminal HA tag) was used as a template. The library's characteristics were verified by sequencing 20 randomly selected clones. All expected nucleotides were detected at the mutated codons, and the library exhibited wild-type characteristics. pglB The proportion of cloned sequences was less than 15%. The library named Fa was transformed into the expression strain *Escherichia coli* St1717 (pGVXN150, pGVXN393) and screened in 96-well plates as previously described (Ihssen). people, 2012, BMC Biotechnolgy 12:67). Exemplary screening results are shown in the sample of a total of 801 clones screened. Figure 10 Cloning Fa8_G10 (in...) Figure 10 (Marked with circles) When rescreened in 8 replicate wells, it showed a significantly increased glycoprotein-specific ELISA signal and was found to contain double-mutant PglB. Cj K482R-D483H (Table 3). The amino acid variation K482R-D483H was introduced into the wild-type low-copy-number plasmid pGVXN114 via QuikChange. pglB The sequence was used to generate plasmid pGVXN635. Wild-type and variant plasmids were transformed into expression strain *Escherichia coli* St1717 (pGVXN150, pGVXN393), and CP5-EPA production was analyzed in triplicate shake-flask experiments as previously described. people, 2012, BMC Biotechnolgy 12:67). As determined by sandwich ELISA, the double mutant PglB C j K482R-D483H promotes a 1.2-2.0-fold increase in CP5-EPA levels. Figure 11 ).

[0519] Table 2. Residues PglB with randomization Cj Rescreening and sequencing of the best-performing clones from libraries Fa of K482 and D483.

[0520]

[0521] wild-type PglB C jThe K482R-D483H variant plasmids pGVXN114 and pGVXN635 were transformed into *Escherichia coli* St2457 (pGVXN570, pGVXN393), which express *Staphylococcus aureus* α-hemolysin (Hla) with engineered glycosylation sites. Strains from overnight pre-cultures were inoculated into 1-liter flask cultures (SOB medium + chloramphenicol, ampicillin, tetracycline, and spectinomycin) to an OD value of 0.1. 600， It was then incubated at 37°C with shaking. At an OD of approximately 1.0... 600 The results were obtained by adding 1 mM IPTG and 2 g L, respectively. -1 L-arabinose was used to induce the expression of PglB and Hla. IPTG was omitted in the control experiment. The induced cultures were incubated overnight at 37°C with shaking until harvest. The total incubation time was approximately 23 hours.

[0522] In experiments with induction, 1200 OD were harvested for both strains, while in experiments without PglB induction, a total of 1500 OD were harvested from overnight cultures. After washing the precipitated cells once with 0.9% NaCl, the cells were resuspended to 50 OD in resuspension buffer (25% sucrose, 10 mM EDTA, 200 mM Tris∙HCl, pH 8.5). 600 The cell suspension was swirl for 20 min. The cells were then separated by centrifugation and resuspended in osmotic shock buffer (10 mM Tris∙HCl, pH 8.5) to 50 OD. 600 The mixture was incubated for 30 min with gentle stirring. After another centrifugation step, 20 mM MgCl2, 0.5 M NaCl, 10 mM imidazole, and 30 mM Tris∙HCl (pH 8.0) were added to the supernatant. His-labeled Hla and CP5-Hla were purified from the supernatant (= osmotic shock fluid) according to standard procedures. The coverage of all four experiments was analyzed by SDS-PAGE (Coomassie staining), anti-CP5 Western blotting, and anti-His Western blotting. 280 Elution peak fraction (1 mL) ( Figure 12 ). CP5-specific bands (with dashed boxes) for expression of PglB. Cj Compared to strain K482R-D483H (pGVXN635) which expresses wild-type PglB CjThe (pGVXN114) strain showed a stronger effect. Enhancement was observed in both IPTG-induced and non-induced shake-flask cultures. The total intensity of the CP5-specific band in the molecular weight range of 50–110 kDa was quantified using ImageJ software (http: / / ImageJ.nih.gov / ij / ). The gray value of the local background was subtracted. PglB expression... Cj The HisTrap eluent of strain K482R-D483H contained more than wild-type PglB. Cj The HisTrap eluent of the strain contained 2.0 times more CP5-Hla.

[0523] The scope of this invention is not limited to the specific embodiments described herein. In fact, those skilled in the art will understand from the foregoing description and drawings that various modifications are possible to the subject matter provided herein, in addition to those described. Such modifications are intended to fall within the scope of the appended claims.

[0524] This article cites numerous publications, patents, and patent applications, the contents of which are incorporated in their entirety through citation.

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Claims

1. A recombinant N-oligosaccharide transferase, which is PglB (PglB) of Campylobacter jejuni of SEQ ID NO:

1. Cj The modified form of the recombinant N-oligosaccharide transferase (N-OST) can detectably link an oligosaccharide or polysaccharide lacking an N-acetyl sugar at the reducing end to a carrier protein at an N-glycosylation concordance sequence, wherein one or more of PglB are modified as follows. Cj The amino acids N311, Y77, S80, Q287, L288, K289, R294, K482, and D483 are: N311 is replaced with V, Y77 is replaced with H / T / W / R / K / A or G, S80 is replaced with R or H, Q287 is replaced with P / K or R, L288 is replaced with M / F or I, K289 is replaced with R / N or Q, R294 is replaced with K, or K482 is replaced with R and D483 is replaced with H.

2. The recombinant N-oligosaccharide transferase of claim 1, wherein the recombinant N-oligosaccharide transferase comprises a modification in one or more amino acids, and in a structural model of the complex of the recombinant N-oligosaccharide transferase and the N-glycosylated carrier protein, the side chain of the amino acid is located within a 2.5-4.0 Å distance of one of the three terminal monosaccharide units at the reducing end of the oligosaccharide or polysaccharide component of the bound N-glycosylated carrier protein.

3. The recombinant N-oligosaccharide transferase of claim 1, wherein the recombinant N-oligosaccharide transferase comprises modifications in two or more amino acids.

4. The recombinant N-oligosaccharide transferase of claim 3, wherein at least one of the one or more amino acids is located in the cytoplasmic pericyclic loop of the transmembrane domain of the recombinant N-oligosaccharide transferase.

5. The recombinant N-oligosaccharide transferase of claim 1, wherein the recombinant N-oligosaccharide transferase further comprises a mutation in one or more amino acids of the QLKFYxxR motif.

6. The recombinant N-oligosaccharide transferase of claim 5, wherein PglB is modified. Cj N311.

7. The recombinant N-oligosaccharide transferase according to claim 1, wherein the recombinant PglB Cj It also includes modifications in one or more amino acids selected from Y77 and S80.

8. The recombinant N-oligosaccharide transferase according to any one of claims 1-7, wherein the recombinant PglB Cj Also included in PglB Cj Amino acid modifications in one or more amino acids of the Q287LKFYxxR294 motif.

9. The recombinant N-oligosaccharide transferase according to claim 8, wherein the recombinant PglB Cj Amino acid modifications included in one or more amino acids selected from Q287, L288, K289 and R294.

10. A nucleic acid encoding a recombinant N-oligosaccharide transferase according to any one of claims 1-9.

11. A host cell comprising a recombinant N-oligosaccharide transferase according to any one of claims 1-9.

12. A method for producing a bioconjugate, the method comprising culturing the host cell of claim 11 in a cell culture medium.

13. The method of claim 12, further comprising purifying the bioconjugate from the host cell culture.

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