KARPIAK JOEL (US)
KEMMLER STEFAN JOCHEN (CH)
KOWARIK MICHAEL THOMAS (CH)
MELBY JOEL (US)
OLLIS ANNE (US)
QUEBATTE JULIEN LAURENT (CH)
WO2016107818A1 | 2016-07-07 | |||
WO2016023018A2 | 2016-02-11 |
Claims 1. A PglB oligosaccharyltransferase (OST) polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set out in SEQ ID NO:1 or 2 or a functional fragment thereof, wherein the PglB oligosaccharyltransferase polypeptide amino acid sequence includes the feature that: at least one residue selected from the group consisting of amino acid X57, X63, X94, X101, X172, X176, X191, X193, X233, X234, X255, X286, X295, X301, X319, X397, X402, X425, X435, X446, X462, X479, X523, X532, X601, X605, X606, X610, X645, X676 and X695 is substituted to a different amino acid to that found at that position in SEQ ID NO:1. 2. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of claim 1, wherein the residue corresponding to amino acid 57 of SEQ ID NO:1 is mutated to R or K or T, preferably to R or T, more preferably to R. 3. The PglB oligosaccharyltransferase polypeptide of functional fragment thereof of any one of claims 1-2 wherein the PglB oligosaccharyltransferase polypeptide amino acid sequence includes the feature that: at least one residue selected from the group consisting of amino acid X78, X84, A155, X293, X300, X301, X306, X308, X462, X464, X479, X523 and X570 is substituted to a different amino acid to that found at that position in SEQ ID NO:1. 4. The PglB OST polypeptide or functional fragment thereof of claim 3 wherein the amino acid sequence includes at least one feature selected from the group consisting of: : X78 is mutated to T; X84 is mutated to W; X155 is mutated to Q; X293 is mutated to C; X300 is mutated to L; X301 is mutated to P or G; X306 is mutated to H; X308 is mutated to W; X462 is mutated to W, N or T; X464 is mutated to L; X479 is mutated to M; X523 is mutated to R; X570 is mutated to R or V. 5. The PglB OST of any one of claims 3-4 wherein the amino acid sequence includes at least one feature selected from the list consisting of: the residue corresponding to amino acid X301 of SEQ ID NO:1 is mutated to P; the residue corresponding to amino acid X462 of SEQ ID NO:1 is mutated to N or W and the residue corresponding to amino acid X479 of SEQ ID NO:1 is mutated to M. 6. The PglB OST of any one of claims 3-5 containing at least, 2, 3, 4, 5 or 6 of the features of the amino acid corresponding to X300 of SEQ ID NO:1 is mutated to L; the amino acid corresponding to X301 of SEQ ID NO:1 is mutated to P, the amino acid corresponding to X308 of SEQ ID NO:1 is mutated to W, the amino acid corresponding to X462 of SEQ ID NO:1 is mutated to W, the amino acid corresponding to X479 of SEQ ID NO:1 is mutated to M and the amino acid corresponding to X570 of SEQ ID NO:1 is mutated to R. 7. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of claims 1-6, wherein the residue corresponding to amino acid X77 of SEQ ID NO:1 is R and the residue corresponding to amino acid X311 of SEQ ID NO: 1 is V. 8. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of claims 1-7, wherein the residues corresponding to amino acids X57, X462 and X479 of SEQ ID NO:1 are substituted with a different amino acid to that found at that position in SEQ ID NO:1; optionally to A57R, Y462W and H479M. 9. The PglB oligosaccharyltransferase polypeptide of any one of claims 1-8 wherein the PglB oligosaccharyltransferase polypeptide is full length, optionally with a length of 712, 713 or 714 amino acids. 10. A PglB from Campylobacter coli (PglBC. coli) wherein the residue corresponding to amino acid X57 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to A57R. 11. A polynucleotide encoding a mutated PglB oligosaccharyltransferase polypeptide as claimed in any one of the preceding claims. 12. A composition or host cell (for example a prokaryotic host cell or an E. coli host cell) comprising at least one PglB oligosaccharyltransferase of any one of claims 1-10 or the polynucleotide of claim 11. 13. A process for preparing a glycosylated protein, comprising the steps of: (a) culturing the host cell of claim 12 under conditions suitable for the production of proteins; and (b) isolating the glycosylated protein from the host cell. 14. A use of the PglB oligosaccharyltransferase or functional fragment thereof of any one of claims 1-57 in the production of a glycosylated protein in which a saccharide is attached to an N residue of a glycosylation consensus sequence, comprising the amino acid sequence Asp/Glu-Z1-Asn-Z2-Ser/Thr wherein Z1 and Z2 may be any natural amino acid except Pro, of a protein. 15. The use of claim 14 wherein the PglB oligosaccharyltransferase or functional fragment thereof is capable of increasing the yield of glycosylation of the protein with the saccharide to produce a glycosylated protein by at least 1.5 fold, 2-fold, 3-fold, 5-fold, 10-fold, 20- fold, 50-fold, 100-fold, 200-fold, 500-fold, 700-fold, 1000-fold compared to a corresponding PglB oligosaccharyltransferase which has the sequence of SEQ ID NO:1. |
Abbreviations and Definitions For the purposes of the descriptions herein, the abbreviations used for the genetically encoded amino acids are conventional and are as follows in Table 1: Table 1 When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about ^-carbon (C ^). For example, whereas “Ala” designates alanine without specifying the configuration about the ^ carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When peptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the N®C direction in accordance with convention. The technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings. All U.S patents and published U.S. patent applications, including all sequences disclosed within such patents and patent applications, referred to herein are expressly incorporated by reference. “Acidic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically, encoded acidic amino acids include L-Glu (E) and L-Asp (D). “Amino acid” or “residue” as used in context of the polypeptides disclosed herein refers to the specific monomer at a sequence position (e.g.., P5 indicates that the “amino acid” or “residue” at position 5 of SEQ ID NO: 2 is a proline.) “Amino acid difference” or “residue difference” refers to a change in the residue at a specified position of a polypeptide sequence when compared to a reference sequence. The polypeptide sequence position at which a particular amino acid or amino acid change (“residue difference”) is present is sometimes described herein as “Xn”, or “position n”, where n refers to the residue position with respect to the reference sequence. For example, a residue difference at position X8, where the reference sequence has a serine, refers to a change of the residue at position X8 to any residue other than serine. As disclosed herein, an enzyme can include one or more residue differences relative to a reference sequence, where multiple residue differences typically are indicated by a list of the specified positions where changes are made relative to the reference sequence (e.g., “one or more residue differences as compared to SEQ ID NO: 1 at the following residue positions: X27, X30, X35, X37, X57, X75, X103, X185, X207, X208, X271, X286, or X296.”). A specific substitution mutation, which is a replacement of the specific residue in a reference sequence with a different specified residue may be denoted by the conventional notation “X(number)X’, where X is the single letter identifier of the residue in the reference sequence, “number” is the residue position in the reference sequence, and X’is the single letter identifier of the residue substitution in the engineered sequence. “Aliphatic amino acid or residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I). “Aromatic amino acid or residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H) it is sometimes classified as a basic residue, or as an aromatic residue as its side chain includes a heteroaromatic ring, herein histidine is classified as a hydrophilic residue or as a “constrained residue” (see below). “Basic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value of greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K). “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the PglB oligosaccharyltransferase enzymes may be codon-optimized for optimal production from the host organism selected for expression. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows. “Conservative” amino acid substitutions or mutations refer to the interchangeability of residues having similar side chains, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. However, as used herein, in some embodiments, conservative mutations do not include substitutions from a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small to small residue, if the conservative mutation can instead be a substitution from an aliphatic to an aliphatic, non-polar to non- polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or constrained to constrained residue. Further, as used herein, A, V, L, or I can be conservatively mutated to either another aliphatic residue or to another non-polar residue. Table 2 below shows exemplary conservative substitutions. Table 2 “Constrained amino acid or residue” refers to an amino acid or residue that has a constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry, because it also has a five-membered ring. “Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure. Each control sequence may be native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. “Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence of the non-reference sequence. For example, a given amino acid sequence, such as that of an engineered PglB oligosaccharyltransferase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. “Cysteine” or L-Cys (C) is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids. The “cysteine- like residues” include cysteine and other amino acids that contain sulfhydryl moieties that are available for formation of disulfide bridges. The ability of L-Cys (C) (and other amino acids with -SH containing side chains) to exist in a peptide in either the reduced free -SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes net hydrophobic or hydrophilic character to a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al., 1984, supra), it is to be understood that for purposes of the present disclosure L-Cys (C) is categorized into its own unique group. “Deletion” refers to modification of the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids making up the polypeptide while retaining enzymatic activity and/or retaining the improved properties of an engineered PglB oligosaccharyltransferase enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. “Derived from” as used herein in the context of engineered enzymes identifies the originating enzyme, and/or the gene encoding such enzyme, upon which the engineering was based. For example, the engineered oligosaccharyltransferase enzyme of SEQ ID NO: 4 was obtained by mutating the PglB oligosaccharyltransferase of SEQ ID NO: 2. Thus, this engineered PglB oligosaccharyltransferase enzyme of SEQ ID NO: 4 is “derived from” the polypeptide of SEQ ID NO: 2 An “engineered PglB oligosaccharyltransferase”, as used herein, refers to a PglB oligosaccharyltransferase-type protein which has been systematically modified, through the insertion of new amino acids into its reference sequence, the deletion of amino acids present in its reference sequence, or the mutation of amino acids in its reference sequence into alternate amino acids, either through a process of random mutagenesis followed by selection of mutants having a particular property or through the intentional introduction of particular amino acid changes into the protein sequence. “Fragment”, as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99%, or more, of the full-length PglB oligosaccharyltransferase polypeptide, for example, the polypeptide of SEQ ID NO: 4. A "functional fragment" or a "biologically active fragment", used interchangeably, herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared and that retains substantially all of the activity of the full-length polypeptide. A functional fragment contains at least 100, 200, 300, 400 or 500 contiguous amino acids “Heterologous” polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell. “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably, about 75% identity, about 85% identity to the target DNA, or with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5× Denhart's solution, 5× saline-sodium phosphate-EDTA (SSPE), 0.2% sodium dodecyl sulfate (SDS) at 42°C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42°C. “High stringency hybridization” refers generally to conditions that are about 10°C or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42°C, followed by washing in 0.1×SSPE, and 0.1% SDS at 65°C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above. “Hydrophilic amino acid or residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).“Hydrophobic amino acid or residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically, encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y). “Hydroxyl-containing amino acid or residue” refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically-encoded hydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr (Y). “Improved enzyme property” refers to any enzyme property made better or more desirable for a particular purpose as compared to that property found in a reference enzyme. For the engineered PglB oligosaccharyltransferase polypeptides described herein, the comparison is generally made to a reference PglB oligosaccharyltransferase enzyme which does not contain the particular mutation which improves enzyme efficiency, although in some embodiments, the reference PglB oligosaccharyltransferase can be another improved engineered PglB oligosaccharyltransferase. Enzyme properties for which improvement can be made include, but are not limited to, enzymatic activity (which can be expressed in terms of yield of N-glycosylated protein), thermal stability, solvent stability, pH activity profile, coenzyme requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, and suppression of acid side-product production. “Insertion” refers to modification of the polypeptide by addition of one or more amino acids to the reference polypeptide. In some embodiments, the improved engineered PglB oligosaccharyltransferase enzymes comprise insertions of one or more amino acids to the naturally occurring PglB oligosaccharyltransferase polypeptide as well as insertions of one or more amino acids to other improved PglB oligosaccharyltransferase polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide. “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally- occurring environment or expression system (e.g., host cell or in vitro synthesis). The improved PglB oligosaccharyltransferase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the improved PglB oligosaccharyltransferase enzyme can be an isolated polypeptide. “Non-conservative substitution” refers to substitution or mutation of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non- conservative substitutions may use amino acids between, rather than within, the defined groups listed above. In one embodiment, a non-conservative mutation affects: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; or (c) the bulk of the side chain. “Non-polar amino acid” or “Non-polar residue” refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A). “Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul, et al., 1990, J. Mol. Biol.215: 403-410 and Altschul, et al., 1977, Nucleic Acids Res.3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul, et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Numerous other algorithms are available that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Additionally, determination of sequence alignment and percent sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided. The ClustalW program is also suitable for determining identity. “Polar amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T). “Preferred, optimal, high codon usage bias codons” refers, interchangeably, to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico, et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for, example, Wada, et al., 1992, Nucleic Acids Res.20:2111-2118; Nakamura, et al., 2000, Nucl. Acids Res.28:292; Duret, et al., supra; Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al., Eds., ASM Press, Washington D.C., p.2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g.., complete protein coding sequences-CDS), expressed sequence tags (EST), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol.266:259-281; Tiwari, et al.., 1997, Comput. Appl. Biosci.13:263-270). “Protein”, “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids. “Reference sequence” refers to a defined sequence to which another (e.g., altered) sequence is compared. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a comparison window to identify and compare local regions of sequence similarity. The term, “reference sequence”, is not intended to be limited to wild-type sequences, and can include engineered or altered sequences. For example, in some embodiments, a “reference sequence” can be a previously engineered or altered amino acid sequence. For instance, a “reference sequence based on SEQ ID NO: 2 having a glycine residue at position X12” refers to a reference sequence corresponding to SEQ ID NO: 2 with a glycine residue at X12 (the un-altered version of SEQ ID NO: 2 has an aspartate at X12). “Small amino acid” or “small residue” refers to an amino acid or residue having a side chain that is composed of a total of three or fewer carbon and/or heteroatoms (excluding the ^-carbon and hydrogens). The small amino acids or residues may be further categorized as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with the above definitions. Genetically-encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D). “Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent sequence identity, as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions. “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure PglB oligosaccharyltransferase composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more or about 99% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species and elemental ion species are not considered to be macromolecular species. In some embodiments, the isolated improved PglB oligosaccharyltransferase polypeptide is a substantially pure polypeptide composition. The invention is further disclosed in the following paragraphs: 1. A PglB oligosaccharyltransferase (OST) polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set out in SEQ ID NO:1 or 2 or a functional fragment thereof, wherein the PglB oligosaccharyltransferase polypeptide amino acid sequence includes the feature that: at least one residue selected from the group consisting of amino acid X57, X63, X94, X101, X172, X176, X191, X193, X233, X234, X255, X286, X295, X301, X319, X397, X402, X425, X435, X446, X462, X479, X523, X532, X601, X605, X606, X610, X645, X676 and X695 is substituted to a different amino acid to that found at that position in SEQ ID NO:1. 2. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 1, wherein the amino acid sequence comprises at least one feature selected from the list consisting of: the residue corresponding to X57 is a hydrophilic residue; the residue corresponding to X63 is chosen from a polar residue and a hydrophobic residue; the residue corresponding to X94 is a polar residue; the residue corresponding to X101 is chosen from an acidic residue, a non-polar residue, a hydrophilic residue, constrained residue, and an aromatic residue; the residue corresponding to X172 is an acidic residue; the residue corresponding to X176 is chosen from a non-polar residue and an acidic residue; the residue corresponding to X191 is chosen from a hydrophilic residue and an aromatic residue; the residue corresponding to X193 is chosen from a non-polar residue, hydrophilic residue, and aromatic residue; the residue corresponding to X233 is an aliphatic residue: the residue corresponding to X234 is chosen from a small residue, hydrophilic residue, and aromatic residue; the residue corresponding to X255 is a hydrophilic residue; the residue corresponding to X286 is chosen from a hydrophobic residue and a polar residue; the residue corresponding to X295 is acidic or aliphatic; the residue corresponding to X301 is chosen from a constrained residue, a non- polar residue and an aromatic residue; the residue corresponding to X319 is chosen from a hydrophilic residue and an aliphatic residue; the residue corresponding to X397 is chosen from a hydrophilic residue and a hydrophobic residue; the residue corresponding to X402 is a hydrophilic residue; the residue corresponding to X425 is a polar residue; the residue corresponding to X435 is chosen from hydrophilic residue and a hydrophobic residue; the residue corresponding to X446 is a non-polar residue; the residue corresponding to X462 is chosen from an aromatic residue, a constrained residue and a small residue; the residue corresponding to X479 is a hydrophobic residue; the residue corresponding to X523 is a basic residue; the residue corresponding to X532 is a hydrophilic residue; the residue corresponding to X601 is a hydrophobic residue; the residue corresponding to X605 is an acidic residue; the residue corresponding to X606 is a constrained residue; the residue corresponding to X610 is a chosen from a hydrophilic residue and a hydrophobic residue; the residue corresponding to X645 is chosen from an aliphatic residue and a hydrophilic residue; the residue corresponding to X676 is chosen from an aromatic residue, a hydrophilic residue, and a non-polar residue; the residue corresponding to X695 is chosen from a polar residue and an aliphatic residue. 3. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 1 or 2, wherein the amino acid sequence comprises at least one feature selected from the group consisting of: residue X57 is chosen from T and R; residue X63 is chosen from L and Q; residue X94 is N; residue X101 is chosen from W, E, H, P, R, M, and G; residue X172 is E; residue X176 is chosen from E and G; residue X191 is chosen from H, D, R, and Y; residue X193 is chosen from T, H, G, and F; X233 is V; residue X234 is chosen from H, C, and W; residue X255 is H; residue X286 is chosen from A, Q, and L; residue X295 is E or L; residue X301 is chosen from P, G, and F; residue X319 is chosen from A, Q, L, and T; residue X397 is chosen from N, L, and Q; residue X402 is chosen from R and H; residue X425 is S; residue X435 is chosen from A, L, F, H, and R; residue X446 is G; residue X462 is chosen from P, C, W, T, and N; residue X479 is M; residue X523 is R; residue X532 is H; residue X601 is G; residue X605 is D; residue X606 is P; residue X610 is chosen from P, L, R, D, and A; residue X645 is chosen from L, S, and H; residue X676 is chosen from Q, W, and G; and residue X695 is chosen from I and Q. 4. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-3, wherein the amino acid sequence comprises at least one feature selected from the list consisting of: the residue corresponding to amino acid 57 of SEQ ID NO: 1 is mutated to R; the residue corresponding to amino acid 101 of SEQ ID NO:1 is mutated to M; the residue corresponding to amino acid 191 of SEQ ID NO:1 is mutated to H, D, R or Y (preferably to Y); the residue corresponding to amino acid 462 of SEQ ID NO:1 is mutated to P or W ; the residue corresponding to amino acid 479 of SEQ ID NO:1 is mutated to M and the residue corresponding to amino acid 676 of SEQ ID NO:1 is mutated to W. 5. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-4, wherein the residue corresponding to amino acid 57 of SEQ ID NO:1 is mutated to R or K or T, preferably to R or T, more preferably to R. 6. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 5, wherein the residue corresponding to amino acid 57 of SEQ ID NO:1 is mutated to R. 7. The PglB oligosaccharyltransferase polypeptide of functional fragment thereof of any one of paragraphs 1-6 wherein the PglB oligosaccharyltransferase polypeptide amino acid sequence includes the feature that: at least one residue selected from the group consisting of amino acid X78, X84, A155, X293, X300, X301, X306, X308, X462, X464, X479, X523 and X570 is substituted to a different amino acid to that found at that position in SEQ ID NO:1. 8. The PglB OST polypeptide of functional fragment thereof of paragraph 7 wherein the amino acid sequence includes at least one feature chosen from the group consisting of: the residue corresponding to X78 is a hydroxyl-containing residue; the residue corresponding to X84 is an aromatic residue; the residue corresponding to X155 is a polar residue; the residue corresponding to X293 is a small residue; the residue corresponding to X300 is an aliphatic residue; the residue corresponding to X301 is chosen from a constrained residue and a non- polar residue; the residue corresponding to X306 is a hydrophilic residue; the residue corresponding to X308 is an aromatic residue; the residue corresponding to X462 is chosen from an aromatic residue and a polar residue; the residue corresponding to X464 is an aliphatic residue; the residue corresponding to X479 is a hydrophobic residue; the residue corresponding to X523 is a basic residue; and the residue corresponding to X570 is chosen from a basic residue and a aliphatic residue. 9. The PglB OST polypeptide of functional fragment thereof of any one of paragraphs 7-8 wherein the amino acid sequence includes at least one feature selected from the group consisting of: : X78 is mutated to T; X84 is mutated to W; X155 is mutated to Q; X293 is mutated to C; X300 is mutated to L; X301 is mutated to P or G; X306 is mutated to H; X308 is mutated to W; X462 is mutated to W, N or T; X464 is mutated to L; X479 is mutated to M; X523 is mutated to R; X570 is mutated to R or V. 10. The PglB OST of any one of paragraphs 7-9 wherein the amino acid sequence includes at least one feature selected from the list consisting of: amino acid X300 is mutated to L; amino acid X301 is mutated to P, amino acid X308 is mutated to W, amino acid X462 is mutated to W, amino acid X479 is mutated to M and amino acid X570 is mutated to R. 11. The PglB OST of any one of paragraphs 7-10 wherein the amino acid sequence includes at least one feature selected from the list consisting of: the residue corresponding to amino acid X301 of SEQ ID NO:1 is mutated to P; the residue corresponding to amino acid X462 of SEQ ID NO:1 is mutated to N or W and the residue corresponding to amino acid X479 of SEQ ID NO:1 is mutated to M. 12. The PglB OST of any one of paragraphs 7-11 wherein the amino acid sequence contains a mutation at the residue corresponding to amino acid X479 of SEQ ID NO:1 to M. 13. The PglB of any one of paragraphs 7-12 wherein the amino acid sequence contains a mutation at the residue corresponding to amino acid X462 of SEQ ID NO:1. 14. The PglB of any one of paragraph 7-13 wherein the amino acid sequence contains a mutation at the residue corresponding to amino acid X462 of SEQ ID NO:1 to W and a mutation at the residue corresponding to amino acid X479 of SEQ ID NO:1 to M. 15. The PglB OST of any one of paragraphs 7-14 containing at least, 2, 3, 4, 5 or 6 of the features of the amino acid corresponding to X300 of SEQ ID NO:1 is mutated to L; the amino acid corresponding to X301 of SEQ ID NO:1 is mutated to P, the amino acid corresponding to X308 of SEQ ID NO:1 is mutated to W, the amino acid corresponding to X462 of SEQ ID NO:1 is mutated to W, the amino acid corresponding to X479 of SEQ ID NO:1 is mutated to M and the amino acid corresponding to X570 of SEQ ID NO:1 is mutated to R. 16. The PglB OST of paragraph 15 wherein amino acid X300 is mutated to L; amino acid X301 is mutated to P, amino acid X308 is mutated to W, amino acid X462 is mutated to W, amino acid X479 is mutated to M and amino acid X570 is mutated to R. 17. The PglB OST of any one of paragraphs 1-16 wherein the amino acid sequence comprises at least one residue difference as compared to the amino acid sequence set forth in SEQ ID NO: 1 in at least one residue position selected from the group consisting of: amino acids X12, X51, X104, X130, X176, X177, X186, X191, X218, X234, X286, X295, X306, X308, X319, X382, X482, and X523. 18. The PglB OST of paragraph 17 wherein the amino acid sequence includes at least one feature chosen from: the residue corresponding to amino acid X12 is a hydroxyl-containing residue; the residue corresponding to amino acid X51 is an aliphatic residue; the residue corresponding to amino acid X104 is an aliphatic residue; the residue corresponding to amino acid X130 is an aliphatic residue; the residue corresponding to amino acid X176 is an acidic residue; the residue corresponding to amino acid X177 is an aliphatic residue; the residue corresponding to amino acid X186 is an aliphatic residue; the residue corresponding to amino acid X191 is an aromatic residue; the residue corresponding to amino acid X218 is a small residue; the residue corresponding to amino acid X234 is chosen from an aromatic residue and a small residue; the residue corresponding to amino acid X286 is chosen from a polar residue and an aliphatic residue; the residue corresponding to amino acid X295 is an aliphatic residue; the residue corresponding to amino acid X306 is a hydrophilic residue; the residue corresponding to amino acid X308 is an aromatic residue; the residue corresponding to amino acid X319 is chosen from an aliphatic residue and a polar residue; the residue corresponding to amino acid X382 is a small residue; the residue corresponding to amino acid X482 is a basic residue; the residue corresponding to amino acid X523 is a basic residue. 19. The PglB OST or functional fragment thereof of paragraph 17 or 18 wherein the amino acid sequence includes at least one feature chosen from the group consisting of: residue X12 is S; residue X51 is L; residue X104 is L; residue X130 is L; residue X176 is E; residue X177 is V; residue X186 is L; residue X191 is Y; residue X218 is A; residue X234 is chosen from C and W; residue X286 is chosen from Q and L; residue X295 is L or E; residue X306 is H; residue X308 is F; residue X319 is chosen from L and Q; residue X382 is S; residue X482 is R; and residue X523 is R. 20. The PglB OST or functional fragment thereof of paragraph any one of paragraphs 17- 19 wherein the amino acid sequence includes at least one feature selected from the group consisting of: the residue corresponding to amino acid X218 of SEQ ID NO:1 is A and the residue corresponding to amino acid X382 of SEQ ID NO:1 is S. 21. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraph 17-20, wherein the wherein the amino acid sequence includes the following features: amino acid X191 is Y; amino acid X286 is Q; amino acid X295 is L or E; amino acid X382 is S; amino acid X482 is R; amino acid X523 is R. 22. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-21, wherein the amino acid sequence comprises a residue difference as compared to the amino acid sequence set forth in SEQ ID NO: 1 in at least one residue position selected from: amino acid X21, amino acid X27, amino acid X42, amino acid X44, amino acid X53, amino acid X80, amino acid X97, amino acid X297, amino acid X317, amino acid X341, amino acid X383, amino acid X388, amino acid X410, amino acid X421, amino acid X480, and amino acid X486. 23. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of Paragraph 22, wherein the amino acid sequence includes at least one feature chosen from the group consisting of: the residue corresponding to amino acid X21 is chosen from a hydroxyl- containing residue and an aliphatic residue; the residue corresponding to amino acid X27 is a chosen from a hydroxyl- containing residue and a hydrophobic residue; the residue corresponding to amino acid X42 is chosen from an aromatic residue and a small residue; the residue corresponding to amino acid X44 is chosen from a hydrophobic residue and a hydrophilic residue; the residue corresponding to amino acid X52 is chosen from a hydrophilic residue and an aliphatic residue; the residue corresponding to amino acid X80 is a small residue; the residue corresponding to amino acid X97 is an aliphatic residue; the residue corresponding to amino acid X297 is chosen from a basic residue and a hydroxyl-containing residue; the residue corresponding to amino acid X317 is a small residue; the residue corresponding to amino acid X341 is an aliphatic residue; the residue corresponding to amino acid X421 is a non-polar residue; the residue corresponding to amino acid X480 is chosen from a hydrophilic residue and a hydrophobic residue; and the residue corresponding to amino acid X486 is a small residue, a hydrophilic residue, and an aliphatic residue. 24. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 22 or 23 , wherein the amino acid sequence includes at least one feature chosen from the group consisting of: amino acid X21 is chosen from S and L; amino acid X27 is chosen from M, S, A and W; amino acid X42 is chosen from W and C; amino acid X44 is chosen from M and H; amino acid X53 is chosen from S, I and H; amino acid X80 is chosen from A and D; amino acid X97 is I; amino acid X297 is chosen from K, R S and Y; amino acid X317 is chosen from S and A; amino acid X341 is L; amino acid X383 is M; amino acid X388 is chosen from M and I; amino acid X410 is I; amino acid X421 is G; amino acid X480 is chosen from W, N, Q, I, T, and M; and amino acid X486 is chosen from C, N, L, H, and V. 25. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 22-24, wherein the wherein the amino acid sequence includes the following features: X297 is R. 26. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-25, wherein the amino acid sequence contains at least one further point mutation at a residue corresponding to amino acid X300, X301, X308, X462, X479 or X570 of SEQ ID NO:1. 27. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 26, wherein the residue corresponding to amino acid X462 of SEQ ID NO:1 is mutated from Y to W. 28. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 26-27, wherein the residue corresponding to amino acid X479 of SEQ ID NO:1 is mutated from H to M. 29. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-28, wherein the residue corresponding to amino acid X300 of SEQ ID NO:1 is mutated from N to L. 30. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-29, wherein residue corresponding to amino acid X301 of SEQ ID NO:1 is mutated from L to P. 31. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-30 wherein residue corresponding to amino acid X308 of SEQ ID NO:1 is mutated from F to W. 32. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-31 wherein residue corresponding to amino acid X570 of SEQ ID NO:1 is mutated from L to R. 33. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-32, wherein the residue corresponding to amino acid X191 of SEQ ID NO:1 is mutated from L to Y. 34. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-33, wherein the residue corresponding to amino acid X286 of SEQ ID NO:1 is mutated from Y to Q. 35. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-34, wherein the residue corresponding to amino acid X295 of SEQ ID NO:1 is mutated from S to L or E. 36. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-35, wherein the residue corresponding to amino acid X382 of SEQ ID NO:1 is mutated from A to S. 37. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-36, wherein the residue corresponding to amino acid X482 of SEQ ID NO:1 is mutated from K to R. 38. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-37, wherein the residue corresponding to amino acid X523 of SEQ ID NO:1 is mutated from T to R. 39. The PglB oligosaccharyltransferase or functional fragment thereof of any one of paragraphs 26-38, wherein the residue corresponding to amino acid X297 of SEQ ID NO:1 is mutated from E to R. 40. The PglB oligosaccharyltransferase or functional fragment thereof of any one of paragraphs 26-39, wherein the residue corresponding to amino acid X80 of SEQ ID NO:1 is mutated from S to D. 41. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-40, wherein the residue corresponding to amino acid X187 of SEQ ID NO:1 is mutated from I to V. 42. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-41, wherein the residue corresponding to amino acid X359 of SEQ ID NO:1 is mutated from I to Q. 43. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 26-42, wherein the residue corresponding to amino acid X406 of SEQ ID NO:1 is mutated from N to I. 44. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-43, wherein the residue corresponding to amino acid X77 of SEQ ID NO:1 is R and the residue corresponding to amino acid X311 of SEQ ID NO: 1 is V. 45. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of paragraph 1, having the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 8. 46. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-44, wherein the residues corresponding to amino acids X57, X462 and X479 of SEQ ID NO:1 are substituted with a different amino acid to that found at that position in SEQ ID NO:1; optionally to A57R, Y462W and H479M. 47. The PglB oligosaccharyltransferase polypeptide or functional fragment thereof of any one of paragraphs 1-46, wherein the residues corresponding to amino acids X57, X300, X301, X308, X462, X479 and X570 of SEQ ID NO:1 are substituted with a different amino acid to that found at that position in SEQ ID NO:1; optionally to A57R, N300L, L301P, F308W, Y462W, H479M, and L570R. 48. The PglB oligosaccharyltransferase polypeptide of any one of paragraphs 1-47 wherein the PglB oligosaccharyltransferase polypeptide is full length, optionally with a length of 712, 713 or 714 amino acids 49. The PglB oligosaccharyltransferase polypeptide of any one of paragraphs 1-48 wherein the PglB oligosaccharyltransferase is from C. jejuni. 50. The PglB oligosaccharyltransferase polypeptide of any one of paragraphs 1-48 wherein the PglB oligosaccharyltransferase is from C. lari. 51. The PglB oligosaccharyltransferase polypeptide of any one of paragraphs 1-48 wherein the PglB oligosaccharyltransferase is from C. coli. 52. The PglB oligosaccharyl transferase polypeptide of any one of paragraphs 1-49 composition wherein the PglB oligosaccharyltransferase is engineered. 53 A PglB from Campylobacter coli (PglBC. coli) wherein the residue corresponding to amino acid X57 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to A57R. 54. The PglB from Campylobacter coli (PglB C. coli ) of paragraph 53 wherein the residue corresponding to amino acid X463 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to Y463W. 55. The PglB from Campylobacter coli (PglB C. coli ) of paragraph 53 or 54 wherein the residue corresponding to amino acid X480 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to H480M. 56. The PglB from Campylobacter coli (PglB C. coli ) of any one of paragraphs 53- 55 wherein the residue corresponding to amino acid X311 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to N311V 57. The PblB from Campylobacter coli (PglB C. coli ) of any one of paragraphs 53-56 wherein the residue corresponding to amino acid X77 of SEQ ID NO:12 is substituted with a different amino acid to that found at that position in SEQ ID NO:12; optionally to Y77R. 58. A polynucleotide encoding a mutated PglB oligosaccharyltransferase polypeptide as found in any one of the preceding paragraphs. 59. A composition or host cell (for example a prokaryotic host cell or an E. coli host cell) comprising at least one PglB oligosaccharyltransferase of any one of paragraphs 1-57 or the polynucleotide of paragraph 58. 60. The composition or host cell of paragraph 59 wherein the at least one PglB oligosaccharyltransferase is engineered. 61. A host cell comprising the polynucleotide encoding a mutated PglB oligosaccharyltransferase of paragraph 58 or a PglB from C.coli having an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, 13 or 14. 62. The host cell of paragraph 61 which is a prokaryotic host cell, optionally an E. coli host cell. 63. The host cell of paragraph 61 or 62 wherein the polynucleotide encoding PglB is integrated into the host cell genome or is expressed from a plasmid. 64. The host cell of paragraph 61, 62 or 63 comprising a further polynucleotide encoding a protein containing at least one glycosylation site comprising the amino acid sequence Asp/Glu-Z1-Asn-Z2-Ser/Thr wherein Z1 and Z2 may be any natural amino acid except Pro. 65. The host cell of any one of paragraphs 61-64 wherein the protein containing at least one glycosylation site is selected from the group consisting of exotoxin A of P. aeruginosa (EPA), CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli sat protein, the passenger domain of E. coli sat protein, C. jejuni AcrA, and C. jejuni natural glycoproteins. 66. The host cell of paragraph 65 wherein the protein containing at least one glycosylation site is exoprotein A of P. aeruginosa (EPA). 67. The host cell of any one of paragraphs 61-66 wherein the protein containing at least one glycosylation site contains 2, 3 or 4 glycosylation sites. 68. The host cell of any one of paragraphs 61-67 comprising at least one polynucleotide encoding glycosyltransferase(s) required for the assembly of a specific oligosaccharide on an undecaprenyl lipid carrier. 69. The host cell of any one of paragraphs 61-68 wherein the specific oligosaccharide is an antigen, for example a bacterial O antigen or a bacterial capsular saccharide antigen. 70. The host cell of paragraph 69 wherein the specific oligosaccharide comprises an E. coli O-antigen, a Salmonella sp O-antigen, a Pseudomonas sp. O-antigen, a Klebsiella sp. O-antigen, an acinetobacter O antigen, a Chlamydia trachomatis antigen, a Vibrio cholera antigen, a Listeria sp. antigen, a Legionella pneumophila serotypes 1 to 15 antigen, a Bordetella parapertussis antigen, a Burkholderia mallei or pseudomallei antigen, a Francisella tularensis antigen, a Campylobacter sp. antigen; a Clostridium difficile antigen, a Streptococcus agalacticae antigen, a a Neisseria meningitidis antigen, a Candida albicans antigen, a Haemophilus influenza antigen, a Enterococcus faecalis antigen, a Borrelia burgdorferi antigen, a Staphylococcus aureus capsular saccharide antigen, a, Haemophilus influenza antigen, a Leishmania major antigen, a Shigella sp., or a Streptococcus pneumoniae capsular saccharide antigen (e.g.serotypes CP1, CP2, CP3, CP4, CP5, CP6 (A and B), CP7 (A,B, C), CP8, CP9 (A, L,N, V), CP10 (A,B,C,F), CP11 (A, B,C,D,F), CP12(A,B,F), CP13, CP14 CP15(A,B,C,F), CP16(A,F), CP17(A,F), CP18(A,B,C,F), CP19(A,B,C,F), CP20,CP21, CP22(A,F), CP23(A,B,F), CP24(A,B,F), CP25(A,F), CP26, CP27,CP28(A,F), CP29, CP31, CP32(A,F), CP33(A,B,C,D,F), CPS34, CP35(A,B,C,D,F), CP36, CP37, CP38, CP39, CP40, CP41(A,F), CP42, CP43, CP44, CP45, CP46, CP47(A,F), CPS48 ). 71. The host cell of any one of paragraphs 61-70 wherein the specific oligosaccharide comprises a residue that is not GlcNAc at the reducing end of the oligosaccharide. 72. The host cell of any one of paragraphs 61-71 wherein the specific oligosaccharide comprises a glucose residue at the reducing terminus. 73. A process for preparing a glycosylated protein, comprising the steps of: (a) culturing the host cell of any one of paragraphs 61-72 under conditions suitable for the production of proteins; and (b) isolating the glycosylated protein from the host cell. 74. An in vitro process for preparing a glycosylated protein, comprising the steps of; i) mixing together: a) the PglB oligosaccharyltransferase of any one of paragraphs 1-57; b) a protein comprising at least one glycosylation consensus sequence comprising the amino acid sequence Asp/Glu-Z1-Asn-Z2-Ser/Thr wherein Z1 and Z2 may be any natural amino acid except Pro; and c) a saccharide chain on a lipid carrier recognised by the PglB; ii) incubating under conditions suitable for the enzymatic activity of PglB to transfer the saccharide chain to the at least one glycosylation consensus sequence of the protein to achieve a glycosylated protein; and iii) isolating the glycosylated protein. 75. A glycosylated protein that is made by the process of paragraph 73 or 74. 76. A use of the PglB oligosaccharyltransferase or functional fragment thereof of any one of paragraphs 1-57 in the production of a glycosylated protein in which a saccharide is attached to an N residue of a glycosylation consensus sequence, comprising the amino acid sequence Asp/Glu-Z 1 -Asn-Z 2 -Ser/Thr wherein Z 1 and Z 2 may be any natural amino acid except Pro, of a protein. 77. The use of paragraph 76 wherein the sugar residue of the saccharide which is covalently attached to the N residue of the glycosylation sequence is not an N-acetyl sugar, for example N-acetyl glucosamine. 78. The use of the PglB oligosaccharyltransferase of paragraph 76 or 77 wherein a glucose residue in the saccharide is covalently attached to the N residue of the glycosylation sequence. 79. The use of any one of paragraphs 76-78 wherein the saccharide is a bacterial antigen, optionally a bacterial capsular polysaccharide antigen or a bacterial O-antigen. 80. The use of any one of paragraphs 76-79 wherein the saccharide is a Gram positive bacterial capsular polysaccharide antigen. 81. The use of any one of paragraphs 76-80 wherein the saccharide is an E. coli O antigen, a Salmonella sp antigen, a Pseudomonas sp. antigen, a Klebsiella sp antigen, a Acinetobacter O antigen, a Chlamydia trachomatis antigen, a Vibrio cholera antigen, a Listeria sp. Antigen, a Legionella pneumonia serotypes 1 to 15 antigen, a Bordetella pertussis antigen, a Bordetalla parapertussis antigen, a Burkholderia mallei or pseudomallei antigen, a Francisella tularensis antigen, a Campylobacter sp antigen, a Clostridium difficile antigen, a Streptococcus pyogenes antigen, a Streptococcus agalactiae antigen, a Enterococcus faecalis antigen, a Borrelia burgdorfei antigen, a Neisseria meningitidis antigen, a Haemophilus influenza antigen, a Leishmania major antigen, a Shigella sp. antigen, a Staphylococcus aureus antigen, a Salmonella enterica antigen or a Streptococcus pneumoniae antigen. 82. The use of paragraph 81 wherein the saccharide is a Streptococcus pneumoniae capsular saccharide antigen. 83. The use of paragraph 82 wherein the Streptococcus pneumoniae capsular saccharide antigen is from serotype 1, 4, 19A, 22F, 23A, 35B or 8, preferably from serotype 8. 84. The use of any one of paragraphs 76-83 wherein the protein is a carrier protein comprising at least 1, 2 or 3 of the glycosylation consensus sequence(s). 85. The use of paragraph 84 wherein the carrier protein is Pseudomonas aeruginosa exoprotein A (EPA), diphtheria toxoid, CRM197, tetanus toxoid, pneumolysin. 86. The use of any one of paragraphs 76-85 wherein the PglB oligosaccharyltransferase or functional fragment thereof is capable of increasing the yield of glycosylation of the protein with the saccharide to produce a glycosylated protein by at least 1.5 fold, 2-fold, 3-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 700-fold, 1000-fold compared to a corresponding PglB oligosaccharyltransferase which has the sequence of SEQ ID NO:1. 87. The use of any one of paragraphs 76-86 wherein the PglB oligosaccharyltransferase or functional fragment thereof is capable of glycosylating at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the protein with a saccharide, wherein a glucose residue of the saccharide is covalently bound to the N residue of the glycosylation consensus sequence. 88. A use of a PglB oligosaccharyltransferase (OST) or functional fragment thereof of from Campylobacter coli (PglB C.coli )in the production of a glycosylated protein in which a saccharide is attached to an N residue of a glycosylation consensus sequence, comprising the amino acid sequence Asp/Glu-Z1-Asn-Z2-Ser/Thr wherein Z1 and Z2 may be any natural amino acid except Pro, of a protein. 89. The use of paragraph 88 wherein the PglB C. coli has an amino acid sequence having at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:12, 13 or 14, optionally to SEQ ID NO:12. 90. The use of paragraph 88 or 89 wherein the saccharide is selected from the group consisting of S. flexneri 2a, 3a and 6, and E. coli O18.
Examples Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Example 1 Plasmids and strain used to assess the activity of PglB variants The activity of PglB variants was tested in a derivative of E. coli W3110 with deletions of genes as described in WO 14/057109A1. Capsular polysaccharide-encoding loci were stably integrated into the E. coli chromosome based on the methods detailed in WO2014057109A1. Other required elements, including PglB, carrier proteins, supplementary biosynthetic enzymes and regulatory proteins were variously expressed from plasmids pEXT21 (Spec resistant, IPTG-inducible), pEXT22 (Kan resistant, IPTG- inducible), pEC415 (Kan resistant, arabinose-inducible) or pLAFR (tet resistant, constitutive) or derivatives thereof. Plasmids: Example 2 Detection of glycosylation by ELISA from small scale cultures Variants of PglB were tested for their ability to catalyse the glycosylation of Exoprotein A from Pseudomonas aeruginosa (EPA) containing D/E-Z1-N-Z2-S/T glycosylation sites (where Z 1 and Z 2 are not P) using a polysaccharide corresponding to that of S. pneumoniae serotype 8. Therefore a E. coli host cell was transformed with plasmids encoding glycosyltransferase genes required for the construction of a S. pneumoniae serotype 8 capsular polysaccharide, a variant PglB gene and EPA containing glycosylation sites. Expression of the genes was induced using IPTG and arabinose and the E. coli host cells were grown overnight to allow expression of glycosyltransferases, PglB and EPA and glycosylation of EPA as follows. The wells of a 96 deep well plate were filled with 1ml of TB media and each well was inoculated with a single colony of host cell E. coli and incubated at 37 degrees C overnight. Samples of each well were used to inoculate main cultures in a 96 deep well plate containing of 1ml of TB supplemented with 10mM MgCl 2 and appropriate antibiotics and were grown until an OD600 of 1.3-1.5 was reached. Cells were incubated with 1mM IPTG and 0.1% arabinose overnight at 37 degrees C. Periplasmic extracts were made by centrifuging the plates, removing supernatant and adding 0.2ml of 50mM Tris-HCl pH 7.5, 175mM NaCl, 5mM EDTA followed by shaking at 4 degrees C to suspend the cells.10 ^l of 10mg/ml polymyxin B was added to each well and the cells were incubated for 1 hour at 4 degrees C. The plate was centrifuged and the supernatant removed. In order to isolate the glycosylated protein from the periplasmic extract, 120 ^l of a 25% slurry of IMAC resin in 30mM Tris pH 8.0, 10mM imidazole, 500mM NaCl was added to each well of a 96 well filter plate (Acroprep Advance) and placed on top of a Nunc ELISA plate. The plate was centrifuged and the flow through discarded.150 ^l of periplasmic extract and 37.5ml of 5x binding buffer (150mM Tris pH 8.0, 50mM imidazole, 2.5M NaCl was added to each well. The samples were incubated for 30 minutes at room temperature. The plate was centrifuged and the flow through discarded and three more washing steps were carried out. Finally the glycosylated protein was eluted with 30mM Tris pH 8.0, 500mM imidazole, 200mM NaCl, ready for use in an ELISA assay. A sandwich ELISA was performed by coating the wells of a 96-well plate with an antibody that recognizes the saccharide part of the glycosylated protein (for example, a monoclonal antibody against S. pneumoniae serotype 8) diluted in PBS. The plate was incubated overnight at 4 degrees C to allow coating. The plate was then washed with PBS containing 0.1% Tween. The plate was then blocked for 2 hours at room temperature using 5% bovine serum albumin in PBST. The plate was washed in PBST. The sample was diluted in PBST containing 1% BSA and incubated in the coated wells for one hour at room temperature. After washing a detection antibody, for example anti-Histag – horseradish peroxidase diluted in PBST containing 1% BSA was added to each well and incubated for one hour at room temperature. The plate was then washed before adding 3,3’,5,5’-Tetramethylbenzidine liquid substrate, Supersensitive, for ELISA (Sigma- Aldrich). After a few minutes, the reaction was stopped by addition of 2M sulfuric acid. The results were obtained by reading the OD at 450nm. Results As a starting point, mutations were generated in a PglB which already contained a mutation at N311V. A first round of variant generation identified Y77R as a mutation which further increased the OST activity of PglB (see Figure 1). A PglB containing mutations at N311V and Y77R was subjected to mutation and promising variants were selected, sequenced and analysed for OST activity as described above. The fold increase in oligosaccharyltransferase activity of each variant was calculated and the results are shown in Table 3. Table 3 Improvement in engineered PglB OST activity in transferring S. pneumoniae 8 saccharide to a protein as determined by ELISA ROUND 2
The mutations 57T, 57R, 63L, 63Q, 94N, 101W, 101E, 101H, 101P, 101R, 101M, 101G, 172E, 176E, 176G, 191H, 191D, 101R, 101Y, 193T, 193H, 193G, 193F, 233V, 234H, 234C, 234W, 255H, 286A, 286Q, 286L, 301P, 301G, 301F, 319A, 319Q, 319L, 319T, 397N, 397L, 397Q, 402R, 402H, 425S, 435A, 435L, 435F, 435H, 435R, 446G, 462,P, 462C, 462W, 462T, 462N 479M, 523R, 532H, 601G, 605D, 606P, 610P, 610L, 610R, 610D, 610A, 645L, 645S, 645H, 676Q, 676W, 676G, 695I and 695Q were noted as mutations which appeared in several PglB variants which were capable of enhanced catalysis of the addition of a S. pneumoniae serotype 8 saccharide to a carrier protein. Out of these, the A57R mutation was selected as a promising mutation to take forward into further rounds due to its high increase in OST activity, its frequency of appearance in promising variants and its position in the PglB structure. The ability of residues 462 and 479 to synergise in order to produce higher fold increases in OST activity when both residues are mutated is demonstrated in the following table showing results for individual : In a further round of experiments, the favourable A57R mutation was added to the Y77R and N311V mutations. Further mutations were added to PglB with A57R, Y77R and N311V mutations. The new mutations were tested for increased PglB activity by ELISA using S. pneumoniae PS8 as the saccharide added to EPA. The results are shown in Table 4 below. Table 4 – Improvement in engineered PglB OST activity in transferring S. pneumoniae 8 saccharide to a protein as determined by ELISA ROUND 3 From this round, the combination of 462W and 479M was found in 52 separate PglB variants, producing fold increases in OST activity of up to 15 fold, or 8-12 fold where these mutations were the only new mutations present. These mutations are considered as important for improving the efficiency of PglB for glycosylation of proteins with saccharides containing a glucose residue at the reducing end of the saccharide. The mutations N300L, L301P, F308W and L570R were also noted as mutations which appeared in several PglB variants which were capable of enhanced catalysis of the addition of a S. pneumoniae serotype 8 saccharide to a carrier protein. N300L, L301P, F308W, Y462W, H479M and L570R were selected as promising mutations to take forward into further rounds of mutation/selection due to their ability to increase OST activity, their frequency of appearance in efficient PglB variants and their position in the molecular structure of PglB. These residues were added to the Y77R, N311V and A57R mutations and further point mutations were tested for their ability to improve the activity of PglB to add a S. pneumoniae serotype 8 saccharide to a protein. The results are shown in Table 5 below Table 5 – Improvement in engineered PglB OST activity in transferring S. pneumoniae 8 saccharide to a protein as determined by ELISA ROUND 4 From this round L191Y, Y286Q, S295L, A382S, K482R and T523R mutations were added to the previously tested mutations resulting in a reference PglB containing the following mutations: Y77R, N311V, A57R, N300L, L301P, F308W, Y462W, H479M, L570R, L191Y, Y286Q, S295L, A382S, K482R and T523R. In the next round, further mutations were tested for their ability to further increase the efficiency of a PglB containing Y77R, N311V, A57R, N300L, L301P, F308W, Y462W, H479M, L570R, round L191Y, Y286Q, S295L, A382S, K482R and T523R further and the results are shown in Table 6 below. Table 6 - Improvement in engineered PglB OST activity in transferring S. pneumoniae 8 saccharide to a protein as determined by ELISA ROUND 5 From this round a E297R was selected due to the increase in activity produced where this mutation was present and the frequency of appearance of this mutation. A S80D mutation was also selected as a promising residue due to its frequency of appearance in variants with higher levels of OST activity. Summary of evolution Over the course of this study, the activity of PglB in the context of adding a S. pneumoniae serotype 8 saccharide to a protein containing a glycosylation consensus sequence was increased by over three orders of magnitude (Figure 1). The introduction of A57R into a PglB into a PglB enzyme already containing Y77R and N311V mutations led to a 24 fold increase in PglB activity. The further addition of N300L, L301P, F308W, Y462W, H479M and L750R led to a cumulative increase in activity of 360 fold. The further incorporation of L191Y, Y286Q, S295L, A382S, K482R and T523R led to a cumulative increase of activity of 2520 fold and the further inclusion of a E297R mutation led to a cumulative 5040 fold increase in activity. Further rounds of evolution allowed small increases in PglB activity, however the largest increases in activity were achieved in rounds 1-3 (see Figure 1). Example 3 Measurement of PglB activity at shake flask volume Some of the mutated PglB enzymes were used in larger scale assays in order to confirm increases in activity in glycosylating an EPA protein containing 3 glycosylation consensus sequences with S. pneumoniae serotype 8 saccharide. Electrocompetent E. coli strains were transformed with the required plasmids by electroporation. The cells were allowed to recover for 1 hour and plated onto agar plates containing appropriate antibiotics and 2mM MgCl2. The plates were incubated overnight. A preculture was made by inoculating TB media containing appropriate antibiotics and 10mM MgCl 2 with cells from the plate and incubating overnight. The main culture was started by diluting the preculture in TB medium containing appropriate antibiotics and 10mM MgCl2 to an OD600nm of 0.1. The culture was grown to an OD600nm of 0.8-1.0 and the cells were then induced using an appropriate inducer (e.g. arabinose of IPTG). The cell were then incubated overnight. A periplasmic extract was made by the following process. The culture was centrifuged to pellet the E. coli. The supernatant was discarded and the pellet resuspended in 30mM Tris- HCl pH 8.5, 1mM EDTA, 20% sucrose. Lysozyme was added to a final concentration of 1mg/ml and the cells were incubated with the lysozyme for 25 minutes at 4 degrees C with shaking. After centrifugation, the periplasmic extract was retained. 1ml of periplasmic extract was mixed with 0.25ml of 150mM Tris pH8.0, 50mM imidazole, 2.5M NaCl and 20mM MgCl 2 .0.2ml of a 50% slurry or pre-equilibrated NiNTA agarose (Qiagen) was added and the sample incubated for 20 minutes at room temperature with shaking. The IMAC resin was centrifuged and the supernatant discarded. 0.5ml of 30mM Tris pH 8.0, 10mM imidazole, 500mM NaCl with 0.1% n-Dodecyl-B- maltose was added, the resin centrifuged and the supernatant discarded. The resin was further washed three times with 30mM Tris pH 8.0, 10mM imidazole, 500mM NaCl. 0.2ml of elution buffer (30mM Tris pH 8.0, 10mM imidazole, 50mM NaCl) was added to the resin and incubated for 5 minutes at room temperature. The eluate was recovered and used for further analysis. The amount of glycosylation was assessed by SDS-PAGE and western blotting. After running the samples on an SDS-PAGE, the proteins were transferred to nitrocellulose membrane. The membrane was blocked with 10% milk for at least 10 minutes. After blocking the membrane was incubated with a first antibody (a mouse Mab against S. pneumoniae serotype 8 for example) in PBS-T containing 1% milk for 1 hour. After washing with PBS-T, the membrane was incubated with a second antibody - HRP conjugate (anti-mouse IgG Fc HRP) in PBS-T with 1% milk for an hour. The membrane was washed in PBS-T and developed using BioFX TMB One Component HRP membrane substrate. Results Figure 1 shows a gel in which enhanced levels of EPA glycosylated with S. pneumoniae serotype 8 capsular saccharide were obtained. The most important increases in OST activity were achieved in rounds 1-3, with smaller fold increases in activity being achieved in subsequent rounds. At a shake flask scale, yield increases of well over 1,000 fold were achieved. Example 4 The mutated PglB oligosaccharyltransferases show enhanced efficiency at catalyzing glycosylation with further saccharides Further experiments were carried out to investigate whether the modified PglB OSTs from each round could produce higher yields of further bioconjugates where different saccharides were bonded to the EPA protein. The protocols of example 2 and 3 were used to make bioconjugates of S. pneumoniae serotype 22F covalently bonded to modified EPA. The results of ELISA and western blotting show that good yields of S. pneumoniae serotype 22F-EPA conjugate could be achieved using the modified PglBs generated from rounds 3, 4 and 5 of example 2. The yield using a round 3 PglB with mutations at A57R, Y77R and N311V is good but is improved further by using the PglB from round 4 which contains additional point mutations at N300L, L301P, F308W, Y462W, H479M and L570R. The yield is further improved by using the round 5 PglB containing further mutations at L191Y, Y286Q, S295L and A382S. The mutated PglB OSTs were also efficient at catalysing the addition of S. pneumoniae serotype 23A saccharide to a protein as shown in Figure 3A. The mutated PglB OSTs were also efficient at catalysing the addition of S. pneumoniae serotype 35B to a protein as shown in Figure 3B. The inclusion of substitutions at A57R, Y77R and N311V led to the improved activity shown in Figure 3B. The mutated PglB OSTs were also efficient at catalysing the addition of S. pneumoniae serotype 19A to a protein as shown in Figure 4. Sequence Listing SEQ ID NO:1
M L