US20030207406A1

US20030207406A1 - Method of transferring at least two saccharide units with a polyglycosyltransferase

Info

Publication number: US20030207406A1
Application number: US10/096,129
Authority: US
Inventors: Karl Johnson; Stephen Roth; Stephanie Buczala
Original assignee: Neose Technologies Inc
Current assignee: Neose Technologies Inc
Priority date: 1995-06-07
Filing date: 2002-03-07
Publication date: 2003-11-06
Also published as: CA2224144A1; US6127153A; EP0832277A4; EP0832277A1; US6379933B1; WO1996040971A1; AU709942B2; AU5965596A; JPH11506927A

Abstract

The present invention relates to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and a gene encoding such a polyglycosyltransferase.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

2. Discussion of the Background:

Biosynthesis of Oligosaccharides

Oligosaccharides are polymers of varying number of residues, linkages, and subunits. The basic subunit is a carbohydrate monosaccharide or sugar, such as mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and the like. The number of different possible stereoisomeric oligosaccharide chains is enormous.

Oligosaccharides and polysaccharides play an important role in protein function and activity, by serving as half-life modulators, and, in some instances, by providing structure. As pointed out above, oligosaccharides are critical to the antigenic variability, and hence immune evasion, of Neisseria, especially gonococcus.

Numerous classical techniques for the synthesis of carbohydrates have been developed, but these techniques suffer the difficulty of requiring selective protection and deprotection. Organic synthesis of oligosaccharides is further hampered by the liability of many glycosidic bonds, difficulties in achieving regioselective sugar coupling, and generally low synthetic yields. In short, unlike the experience with peptide synthesis, traditional synthetic organic chemistry cannot provide for quantitative, reliable synthesis of even fairly simple oligosaccharides.

Recent advances in oligosaccharide synthesis have occurred with the isolation of glycosyltransferases from natural sources. These enzymes can be used in vitro to prepare oligosaccharides and polysaccharides (see, e.g., Roth, U.S. Pat. No. 5,180,674). The advantage of biosynthesis with glycosyltransferases is that the glycosidic linkages formed by enzymes are highly stereo and regiospecific. However, each enzyme catalyzes linkage of specific sugar donor residues to other specific acceptor molecules, e.g., an oligosaccharide or lipid. Thus, synthesis of a desired oligosaccharide has required the use of a different glycosyltransferase for each different saccharide unit being transferred.

More specifically, such glycosyltransferases have only provided for the transfer of a single saccharide unit, specific for the glycosyltransferases. For example a galactosyltransferase would transfer only galactose, a glucosyltransferase would transfer only glucose, an N-acetylglucosamine and a sialyl transferase would transfer only sialic acid.

However, the lack of generality of glycosyltransferase, makes it necessary to use a different glycosyltransferase for every different sugar donor being transferred. As the usefulness of oligosaccharide compounds expands, the ability to transfer more than one sugar donor would provide a tremendous advantage, by decreasing the number of glycosyltransferases necessary to form necessary glycosidic bonds.

In addition, a glycosyltransferase which transferred at least two different sugar donors would be advantageous in synthesizing two glycosidic bonds of at least a trisaccharide, using the same glycosyltransferase.

A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been reported as being cloned from the gonococcal strain F62 (Gotschlich J. Exp. Med (1994) 180, 2181-2190). Five genes lgtA, lgtB, lgtC, lgtD and lgtE are reported, and based on deletion experiments, activities are postulated, as encoding for glycosyltransferases. Due to the uncertainty caused by the nature of the deletion experiments, the exact activity of the proteins encoded by each of the genes was not ascertained and some of the genes are only suggested as being responsible for one or another activity, in the alternative. The gene lgtA is suggested as most likely to code for a GlcNAc transferase.

The transfer of more than one different saccharide moietie, by a polyglycosyltransferase has heretofore been unreported.

SUMMARY OF THE INVENTION

The present invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and nucleic acids encoding a polyglycosyltransferase.

Accordingly, in one aspect, the invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase.

Accordingly, another aspect of the invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase, which transfers both GlcNAc and GalNAc, from the corresponding sugar nucleotides, to a sugar acceptor.

According to another aspect of the invention, a polyglycosyltransferase is obtained from a bacteria of the genus Neisseria, Escherichia or Pseudomonas.

According to another aspect of the invention, is directed to a method of making at least two oligosaccharide compounds, from the same acceptor, with a polyglycosyltransferase.

Accordingly, another aspect of the invention is directed to a method of making at least two oligosaccharide compounds, from the same acceptor, with a polyglycosyltransferase, which transfers both GlcNAc and Ga1NAc, from the corresponding sugar nucleotides, to the sugar acceptor.

According to another embodiment of the present invention is a method of transferring an N-acetylgalactosamine using a glycosyltransferase of SEQ ID NO:3

In specific embodiments, the invention relates to a nucleic acid that has a nucleotide sequence which encodes for polypeptide sequence shown in SEQ ID NO: 3.

The functionally active polyglycosyltransferase of the invention is characterized by catalyzing both the addition of GalNAc β1-3 to Gal and the addition of G1cNAc β1-3 to Gal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: provides the amino acid sequence of a polyglycosyltransferase of SEQ ID NO. 3. [0023]
FIG. 2: provides the polynucleotide sequence of a LOS encoding gene isolated from [0024] N. gonorrhoeae, of which nucleotides 445-1488 encode for a polyglycosyltransferase

DETAILED DESCRIPTION OF THE INVENTION

As disclosed above, the present invention provides for a method of transferring at least two saccharide units with a polyglycosyltransferase, a gene encoding for a polyglycosyltransferase, and a polyglycosyltransferase. The polyglycosyltransferases of the invention can be used for in vitro biosynthesis of various oligosaccharides, such as the core oligosaccharide of the human blood group antigens, i.e., lacto-N-neotetraose. [0025]
Cloning and expression of a polyglycosyltransferase of the invention can be accomplished using standard techniques, as disclosed herein. Such a polyglycosyltransferase is useful for biosynthesis of oligosaccharides in vitro, or alternatively genes encoding such a polyglycosyltransferase can be transfected into cells, e.g., yeast cells or eukaryotic cells, to provide for alternative glycosylation of proteins and lipids. [0026]
The instant invention is based, in part, on the discovery that a polyglycosyltransferase isolated from [0027] Neisseria gonorrhoeae is capable of transferring both GlcNAc β1-3 to Gal and GalNAc β1-3 to Gal, from the corresponding sugar nucleotides.
A protein, glycosyltransferase activity, is reported by Gotschlich et al U.S. Ser. No. 08/312,387 filed on Sep. 26, 1994, by cloning of a locus involved in the biosynthesis of gonococcal LOS, strain F62. The protein sequence identified as SEQ ID NO: 3, a 348 amino acid protein, has now been discovered to have a polyglycosyltransferase activity. More specifically, the protein sequence identified as SEQ ID NO: 3 has been discovered to transfer both GlcNAc β1-3 to Gal and Ga1NAc β1-3 to Gal, from the corresponding sugar nucleotides. [0028]
In addition to the protein sequence SEQ ID NO: 3 and nucleic acid sequences for encoding them reported in U.S. Ser. No. 08/312,387, new polyglycosyltransferases have been discovered which transfer two different sugar units. This protein is similar to the protein of SEQ ID: 3 with the deletion of one or two of the five glycine units occurring between amino acid nos 86-90 of lgtA. In addition, an additional amino acid sequence -Tyr-Ser-Arg-Asp-Ser-Ser can be appended to the carboxy terminus of Ile (amino acid no 348) of SEQ ID NO: 3, while retaining the polyglycosyltransferase activity. [0029]
A polynucleotide sequence encoding for a polyglycosyltransferase is similar to the sequence of nucleic acids no 445 to 1488 of an LOS isolated from [0030] N. gonorrhoeae (see FIG. 2) in which three or six of the guanine units occurring between nucleic acids no 700 to 715 have been deleted.
Another polynucleotide sequence is similar to the sequence of nucleic acids no 445 to 1488 of an LOS isolated from [0031] N. gonorrhoeae (see FIG. 2), in which nucleic acids sufficient to encode the amino acid sequence -Tyr-Ser-Arg-Asp-Ser-Ser, can be appended to nucleic acid 1488.
Abbreviations used throughout this specification include: Lipopolysaccharide, LPS; Lipooligosaccharide, LOS; N-Acetyl-neuraminic acid cytidine mono phosphate, CMP-NANA; wild type, wt; Gal, galactose; Glc, glucose; NAc, N-acetyl (e.g., GalNAc or GlcNAc). [0032]

Isolation of Genes for Poly Glycosyltransferases

Any Neisseria bacterial cell can potentially serve as the nucleic acid source for the molecular cloning of a polyglycosyltransferase gene. In a specific embodiment, infra, the genes are isolated from [0033] Neisseria gonorrhoeae. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA “library'), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. 1, II). For example, a N. gonorrhoeae genomic DNA can be digested with a restriction endonuclease or endonucleases, e.g., Sau3A, into a phage vector digested with a restriction endonuclease or endonucleases, e.g., BamHI/EcoRI, for creation of a phage genomic library. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using; various restriction enzymes. Alternatively, one may use DNAse the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. [0034]
Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired polyglycosyltransferase gene may be accomplished in a number of ways. For example, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe synthesized with a sequence as disclosed herein (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. [0035]
Suitable probes can be generated by PCR using random primers. In particular a probe which will hybridize to the polynucleotide sequence encoding for a four or five glycine residue (i.e. a twelve or fifteen guanine residue) would be a suitable probe for a polyglycosyltransferase. [0036]
As described above, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example DNA clones that produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, proteolytic activity, or functional properties, in particular polyglycosyltransferase activity, the ability of a polyglycosyltransferase protein to mediate transfer of two different saccharide units to an acceptor molecule. [0037]
Alternatives to isolating a polyglycosyltransferase genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence that encodes a polyglycosyltransferase. In another embodiment, DNA for a polyglycosyltransferase gene can be isolated by PCR using oligonucleotide primers designed from the nucleotide sequences disclosed herein. Other methods are possible and within the scope of the invention. [0038]
The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. In a specific aspect of the invention, the polyglycosyltransferase coding sequence is inserted in an [0039] E. coli cloning vector. Other examples of vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini, these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In specific embodiment, PCR primers containing such linker sites can be used to amplify the DNA for cloning. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
Transformation of host cells with recombinant DNA molecules that incorporate the isolated polyglycosyltransferase gene or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. [0040]
The present invention also relates to vectors containing genes encoding truncated forms of the enzyme (fragments) and derivatives of polyglycosyltransferases that have the same functional activity as a polyglycosyltransferase. The production and use of fragments and derivatives related to polyglycosyltransferases are within the scope of the present invention. In a specific embodiment, the fragment or derivative is functionally active, i.e., capable of mediating transfer of two sugar donors to a acceptor moieties. [0041]
Truncated fragments of the polyglycosyltransferases can be prepared by eliminating N-terminal, C-terminal, or internal regions of the protein that are not required for functional activity. Usually, such portions that are eliminated will include only a few, e.g., between 1 and 5, amino acid residues, but larger segments may be removed. [0042]
Chimeric molecules, e.g., fusion proteins, containing all or a functionally active portion of a polyglycosyltransferase of the invention joined to another protein are also envisioned. A polyglycosyltransferase fusion protein comprises at least a functionally active portion of a non-glycosyltransferase protein joined via a peptide bond to at least a functionally active portion of a polyglycosyltransferase polypeptide. The non-glycosyltransferase sequences can be amino- or carboxy-terminal to the polyglycosyltransferase sequences. Expression of a fusion protein can result in an enzymatically inactive polyglycosyltransferase fusion protein. A recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a functionally active portion of a non-glycosyltransferase protein joined in-frame to the polyglycosyltransferase coding sequence, and preferably encodes a cleavage site for a specific protease, e.g., thrombin or Factor Xa, preferably at the polyglycosyltransferase-non-glycosyltransferase juncture. In a specific embodiment, the fusion protein may be expressed in [0043] Escherichia coli.
In particular, polyglycosyltransferase derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as an polyglycosyltransferase gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of polyglycosyltransferase genes that are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Likewise, the polyglycosyltransferase derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a polyglycosyltransferase including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. [0044]
The genes encoding polyglycosyltransferase derivatives and analogs of the invention can be produced by various methods known in the art (e.g., Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of polyglycosyltransferase, care should be taken to ensure that the modified gene remains within the same translational reading frame as the polyglycosyltransferase gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded. [0045]
Additionally, the polyglycosyltransferase nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TABO linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, in [0046] PCR Technology: Principles and Applications for “DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
While a polyglycosyltransferase has been isolated from a bacteria of [0047] Neisseria gonorrhoeae, polyglycosyltransferases can also be isolated from other bacterial species of Neisseria. Exemplary Neisseria bacterial sources include N. animalis (ATCC 19573), N. canis (ATCC 14687), N. cinerea (ATCC 14685), N. cuniculi (ATCC 14688), N. denitrificans (ATCC 14686), N. elongata (ATCC 25295), N. elongata subsp glycolytica (ATCC 29315), N. elongata subsp. nitroreducens (ATCC 49377), N. flavescens (ATCC 13115), N. gonorrhoeae (ATCC 33084), N. lactamica (ATCC 23970), N. macaca (ATCC 33926), N. meningitidis, N. mucosa (ATCC 19695), N. mucosa subsp. eidelbergensis (ATCC 25998), N. polysaccharea (ATCC 43768), N. sicca (ATCC 29256) and N. subflava (ATCC 49275). In addition polyglycosyltransferases can be isolated from Branhamella catarrhalis, Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa and Pseudomonas cepacia.

Expression of a Polyglycosyltransferase

The gene coding for a polyglycosyltransferase, or a functionally active fragment or other derivative thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can also be supplied by the native polyglycosyltransferase gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protein-coding sequence. Preferably, however, a bacterial expression system is used to provide for high level expression of the protein with a higher probability of the native conformation. Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccine virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. [0048]
Preferably, the periplasmic form of the polyglycosyltransferase (containing a signal sequence) is produced for export of the protein to the [0049] Escherichia coli periplasm or in an expression system based on Bacillus subtillis.
Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). [0050]
Expression of nucleic acid sequence encoding a polyglycosyltransferase or peptide fragment may be regulated by a second nucleic acid sequence so that the polyglycosyltransferase or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a polyglycosyltransferase may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. For expression in bacteria, bacterial promoters are required. Eukaryotic viral or eukaryotic promoters, including tissue specific promoters, are preferred when a vector containing a polyglycosyltransferase gene is injected directly into a subject for transient expression, resulting in heterologous protection against bacterial infection, as described in detail below. Promoters which may be used to control polyglycosyltransferase gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 198 1, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the O-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and the like [0051]
Expression vectors containing polyglycosyltransferase gene inserts can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of “marker” gene functions, and (d) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR with incorporation of radionucleotides or stained with ethidiuin bromide to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted polyglycosyltransferase gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., 3-galactosidase activity, PhoA activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. If the polyglycosyltransferase gene is inserted within the marker gene sequence of the vector, recombinants containing the polyglycosyltransferase insert can be identified by the absence of the marker gene function. In the fourth approach, recombinant expression vectors can be identified by assaying for the activity of the polyglycosyltransferase gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the polyglycosyltransferase gene product in in vitro assay systems, e.g., polyglycosyltransferase activity. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. [0052]
Biosynthesis of Oligosaccharides [0053]
The polyglycosyltransferase of the present invention can be used in the biosynthesis of oligosaccharides. The polyglycosyltransferases of the invention are capable of stereospecific conjugation of two specific activated saccharide unit to specific acceptor molecules. Such activated saccharides generally consist of uridine or guanosine diphosphate and cytidine monophosphate derivatives of the saccharides, in which the nucleoside mono- and diphosphate serves as a leaving group. Thus, the activated saccharide may be a saccharide-UDP, a saccharide-GDP, or a saccharide-CMP. In specific embodiments, the activated saccharide is UDP-G1cNAc, UDP-Ga1NAc, or UDP-Gal. [0054]
Within the context of the claimed invention, two different saccharide units means saccharides which differ in structure and/or stereochemistry at a position other than C[0055] ₁and accordingly the pyranose and furanose of the same carbon backbone are considered to be the same saccharide unit, while glucose and galactose (i.e. C₄isomers) are considered different.
A glycosyltransferase typically has a catalytic activity of from about 1 to 250 turnovers/sec in order to be considered to possess a specific glycosyltransferase activity. [0056]
Accordingly each individual glycosyltransferase activity of the polyglycosyltransferase of the present invention, is within the range of from 1 to 250 turnovers/sec, preferably from 5 to 100 turnovers/sec, more preferably from 10 to 30 turnovers/sec. [0057]
In addition to absolute glycosyltransferase activity, the relative rates of each of the identified glycosyltransferase activities, in the polyglycosyltransferase, has relative activities of from 0.1 to 10 times, preferably from 0.2 to 5 times, more preferably from 0.5 to 2 times and most preferably from 0.8 to 1.5, the rate of any one of the other glycosyltransferase activities. [0058]
The term “acceptor moiety” as used herein refers to the molecules to which the polyglycosyltransferase transfers activated sugars. [0059]
For the synthesis of an oligosaccharide, a polyglycosyltransferase is contacted with an appropriate activated saccharide and an appropriate acceptor moiety under conditions effective to transfer and covalently bond the saccharide to the acceptor molecule. Conditions of time, temperature, and pH appropriate and optimal for a particular saccharine unit transfer can be determined through routine testing; generally, physiological conditions will be acceptable. Certain co-reagents may also be desirable; for example, it may be more effective to contact the polyglycosyltransferase with the activated saccharide and the acceptor moiety in the presence of a divalent cation. [0060]
According to the invention, the polyglycosyltransferase enzymes can be covalently or non-covalently immobilized on a solid phase support such as SEPHADEX, SEPHAROSE, or poly(acrylamide-co-N-acryloxysucciimide) (PAN) resin. A specific reaction can be performed in an isolated reaction solution, with facile separation of the solid phase enzyme from the reaction products. Immobilization of the enzyme also allows for a continuous biosynthetic stream, with the specific polyglycosyltransferases attached to a solid support, with the supports arranged randomly or in distinct zones in the specified order in a column, with passage of the reaction solution through the column and elution of the desired oligosaccharide at the end. An efficient method for attaching the polyglycosyltransferase to a solid support and using such immobilized polyglycosyltransferases is described in U.S. Pat. No. 5,180,674, issued Jan. 19, 1993 to Roth, which is specifically incorporated herein by reference in its entirety. [0061]
An oligosaccharide, e.g., a disaccharide, prepared using a polyglycosyltransferase of the present invention can serve as an acceptor moiety for further synthesis, either using other polyglycosyltransferases of the invention, or glycosyltransferases known in the art (see, e.g., Roth, U.S. Pat. No. 5,180,674). [0062]
Alternatively, the polyglycosyltransferases of the present invention can be used to prepare GalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-Glc or GalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-GlcNAc from lactose or lactosamine respectively, in which a polyglycosyltransferase is used to synthesize both the GlcNAc β1-3-Gal and GalNAc β1-3 Gal linkages. [0063]
Accordingly, a method for preparing an oligosaccharide having the structure GalNAcβ1-3-Galβ1-4-G1cNAc β1-3-Galβ1-4-Glc comprises sequentially performing the steps of: [0064]
a) contacting a reaction mixture comprising an activated GlcNAc (such as UDP-GlcNAc) to lactose with a polyglycosyltransferase having an amino acid sequence of SEQ ID NO:3, or a functionally active fragment thereof; [0065]
b) contacting a reaction mixture comprising an activated Gal (i.e. UDP-Gal) to the acceptor moiety comprising a GlcNAcβ1-3-Galβ1-4-Glc residue in the presence of a β1-4-galactosyltransferase; and [0066]
c) contacting a reaction mixture comprising an activated GalNAc (i.e. UDP-GalNAc) to the acceptor moiety comprising a Galβ1-4-GlcNAcβ1-3-Galβ1-4-Glc residue in the presence of the polyglycosyltransferase of step a). [0067]
A suitable β1-4 galactosyltransferase can be isolated from bovine milk. [0068]
Oligosaccharide synthesis, using a polyglycosyltransferase is generally conducted at a temperature of from 15 to 38° C., preferably from 20 to 25° C. While enzymatic activity is comparable at 25° C. and 37° C., the polyglycosyltransferase stability is greater at 25° C. [0069]
In a preferred embodiment polyglycosyltransferase activity is observed in the absence of α-lactalbumin. [0070]
In a preferred embodiment polyglycosyltransferase activity is observed at the same pH, more preferably at pH 6.5 to 7.5. [0071]
In a preferred embodiment polyglycosyltransferase activity is observed at the same temperature. [0072]
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. [0073]

EXAMPLE 1

Synthesis of GalNAcβ1-3-Galβ1-4-G1cNAcβ1-3-Ga1β1-4-Glc: [0074]
Lactose was contacted with UDP-N-acetylglucosamine and a β-galactoside β1-3 N-acetylglucosaminyl transferase of SEQ ID NO: 3, in a 0.5 M HEPES buffered aqueous solution at 25° C. The product trisaccharide was then contacted with UDP-Gal and a β-N-acetylglucosaminoside β1-4 Galactosyltransferase isolated from bovine milk, in a 0.05 M HEPES buffered aqueous solution at 37° C. The product tetrasaccharide was then contacted with UDP-N-acetylgalactosamine and a β-galactoside β1-3 N-acetylgalactosaminyl transferase of SEQ ID NO: 3, in a 0.05 M HEPES buffered aqueous solution at 25° C. The title pentasaccharide was isolated by conventional methods. [0075]
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described therein. [0076]
1 8 1 5859 DNA Neisseria gonorrhoeae 1 ctgcaggccg tcgccgtatt caaacaactg cccgaagccg ccgcgctcgc cgccgccaac 60 aaacgcgtgc aaaacctgct gaaaaaagcc gatgccgcgt tgggcgaagt caatgaaagc 120 ctgctgcaac aggacgaaga aaaagccctg tacgctgccg cgcaaggttt gcagccgaaa 180 attgccgccg ccgtcgccga aggcaatttc cgaaccgcct tgtccgaact ggcttccgtc 240 aagccgcagg ttgatgcctt cttcgacggc gtgatggtga tggcggaaga tgccgccgta 300 aaacaaaacc gcctgaacct gctgaaccgc ttggcagagc agatgaacgc ggtggccgac 360 atcgcgcttt tgggcgagta accgttgtac agtccaaatg ccgtctgaag ccttcaggcg 420 gcatcaaatt atcgggagag taaattgcag cctttagtca gcgtattgat ttgcgcctac 480 aacgtagaaa aatattttgc ccaatcatta gccgccgtcg tgaatcagac ttggcgcaac 540 ttggatattt tgattgtcga tgacggctcg acagacggca cacttgccat tgccaaggat 600 tttcaaaagc gggacagccg tatcaaaatc cttgcacaag ctcaaaattc cggcctgatt 660 ccctctttaa acatcgggct ggacgaattg gcaaagtcgg gggggggggg gggggaatat 720 attgcgcgca ccgatgccga cgatattgcc tcccccggct ggattgagaa aatcgtgggc 780 gagatggaaa aagaccgcag catcattgcg atgggcgcgt ggctggaagt tttgtcggaa 840 gaaaaggacg gcaaccggct ggcgcggcac cacaaacacg gcaaaatttg gaaaaagccg 900 acccggcacg aagacatcgc cgcctttttc cctttcggca accccataca caacaacacg 960 atgattatgc ggcgcagcgt cattgacggc ggtttgcgtt acgacaccga gcgggattgg 1020 gcggaagatt accaattttg gtacgatgtc agcaaattgg gcaggctggc ttattatccc 1080 gaagccttgg tcaaataccg ccttcacgcc aatcaggttt catccaaaca cagcgtccgc 1140 caacacgaaa tcgcgcaagg catccaaaaa accgccagaa acgatttttt gcagtctatg 1200 ggttttaaaa cccggttcga cagcctagaa taccgccaaa caaaagcagc ggcgtatgaa 1260 ctgccggaga aggatttgcc ggaagaagat tttgaacgcg cccgccggtt tttgtaccaa 1320 tgcttcaaac ggacggacac gccgccctcc ggcgcgtggc tggatttcgc ggcagacggc 1380 aggatgaggc ggctgtttac cttgaggcaa tacttcggca ttttgtaccg gctgattaaa 1440 aaccgccggc aggcgcggtc ggattcggca gggaaagaac aggagattta atgcaaaacc 1500 acgttatcag cttggcttcc gccgcagaac gcagggcgca cattgccgca accttcggca 1560 gtcgcggcat cccgttccag tttttcgacg cactgatgcc gtctgaaagg ctggaacggg 1620 caatggcgga actcgtcccc ggcttgtcgg cgcaccccta tttgagcgga gtggaaaaag 1680 cctgctttat gagccacgcc gtattgtggg aacaggcatt ggacgaaggc gtaccgtata 1740 tcgccgtatt tgaagatgat gtcttactcg gcgaaggcgc ggagcagttc cttgccgaag 1800 atacttggct gcaagaacgc tttgaccccg attccgcctt tgtcgtccgc ttggaaacga 1860 tgtttatgca cgtcctgacc tcgccctccg gcgtggcgga ctacggcggg cgcgcctttc 1920 cgcttttgga aagcgaacac tgcgggacgg cgggctatat tatttcccga aaggcgatgc 1980 gttttttctt ggacaggttt gccgttttgc cgcccgaacg cctgcaccct gtcgatttga 2040 tgatgttcgg caaccctgac gacagggaag gaatgccggt ttgccagctc aatcccgcct 2100 tgtgcgccca agagctgcat tatgccaagt ttcacgacca aaacagcgca ttgggcagcc 2160 tgatcgaaca tgaccgccgc ctgaaccgca aacagcaatg gcgcgattcc cccgccaaca 2220 cattcaaaca ccgcctgatc cgcgccttga ccaaaatcgg cagggaaagg gaaaaacgcc 2280 ggcaaaggcg cgaacagtta atcggcaaga ttattgtgcc tttccaataa aaggagaaaa 2340 gatggacatc gtatttgcgg cagacgacaa ctatgccgcc tacctttgcg ttgcggcaaa 2400 aagcgtggaa gcggcccatc ccgatacgga aatcaggttc cacgtcctcg atgccggcat 2460 cagtgaggaa aaccgggcgg cggttgccgc caatttgcgg ggggggggta atatccgctt 2520 tatagacgta aaccccgaag atttcgccgg cttcccctta aacatcaggc acatttccat 2580 tacgacttat gcccgcctga aattgggcga atacattgcc gattgcgaca aagtcctgta 2640 tctggatacg gacgtattgg tcagggacgg cctgaagccc ttatgggata ccgatttggg 2700 cggtaactgg gtcggcgcgt gcatcgattt gtttgtcgaa aggcaggaag gatacaaaca 2760 aaaaatcggt atggcggacg gagaatatta tttcaatgcc ggcgtattgc tgatcaacct 2820 gaaaaagtgg cggcggcacg atattttcaa aatgtcctgc gaatgggtgg aacaatacaa 2880 ggacgtgatg caatatcagg atcaggacat tttgaacggg ctgtttaaag gcggggtgtg 2940 ttatgcgaac agccgtttca actttatgcc gaccaattat gcctttatgg cgaacgggtt 3000 tgcgtcccgc cataccgacc cgctttacct cgaccgtacc aatacggcga tgcccgtcgc 3060 cgtcagccat tattgcggct cggcaaagcc gtggcacagg gactgcaccg tttggggtgc 3120 ggaacgtttc acagagttgg ccggcagcct gacgaccgtt cccgaagaat ggcgcggcaa 3180 acttgccgtc ccgccgacaa agtgtatgct tcaaagatgg cgcaaaaagc tgtctgccag 3240 attcttacgc aagatttatt gacggggcag gccgtctgaa gccttcagac ggcatcggac 3300 gtatcggaaa ggagaaacgg attgcagcct ttagtcagcg tattgatttg cgcctacaac 3360 gcagaaaaat attttgccca atcattggcc gccgtagtgg ggcagacttg gcgcaacttg 3420 gatattttga ttgtcgatga cggctcgacg gacggcacgc ccgccattgc ccggcatttc 3480 caagaacagg acggcaggat caggataatt tccaatcccc gcaatttggg ctttatcgcc 3540 tctttaaaca tcgggctgga cgaattggca aagtcggggg ggggggaata tattgcgcgc 3600 accgatgccg acgatattgc ctcccccggc tggattgaga aaatcgtggg cgagatggaa 3660 aaagaccgca gcatcattgc gatgggcgcg tggttggaag ttttgtcgga agaaaacaat 3720 aaaagcgtgc ttgccgccat tgcccgaaac ggcgcaattt gggacaaacc gacccggcat 3780 gaagacattg tcgccgtttt ccctttcggc aaccccatac acaacaacac gatgattatg 3840 aggcgcagcg tcattgacgg cggtttgcgg ttcgatccag cctatatcca cgccgaagac 3900 tataagtttt ggtacgaagc cggcaaactg ggcaggctgg cttattatcc cgaagccttg 3960 gtcaaatacc gcttccatca agaccagact tcttccaaat acaacctgca acagcgcagg 4020 acggcgtgga aaatcaaaga agaaatcagg gcggggtatt ggaaggcggc aggcatagcc 4080 gtcggggcgg actgcctgaa ttacgggctt ttgaaatcaa cggcatatgc gttgtacgaa 4140 aaagccttgt ccggacagga tatcggatgc ctccgcctgt tcctgtacga atatttcttg 4200 tcgttggaaa agtattcttt gaccgatttg ctggatttct tgacagaccg cgtgatgagg 4260 aagctgtttg ccgcaccgca atataggaaa atcctgaaaa aaatgttacg cccttggaaa 4320 taccgcagct attgaaaccg aacaggataa atcatgcaaa accacgttat cagcttggct 4380 tccgccgcag agcgcagggc gcacattgcc gataccttcg gcagtcgcgg catcccgttc 4440 cagtttttcg acgcactgat gccgtctgaa aggctggaac aggcgatggc ggaactcgtc 4500 cccggcttgt cggcgcaccc ctatttgagc ggagtggaaa aagcctgctt tatgagccac 4560 gccgtattgt gggaacaggc gttggatgaa ggtctgccgt atatcgccgt atttgaggac 4620 gacgttttac tcggcgaagg cgcggagcag ttccttgccg aagatacttg gttggaagag 4680 cgttttgaca aggattccgc ctttatcgtc cgtttggaaa cgatgtttgc gaaagttatt 4740 gtcagaccgg ataaagtcct gaattatgaa aaccggtcat ttcctttgct ggagagcgaa 4800 cattgtggga cggctggcta tatcatttcg cgtgaggcga tgcggttttt cttggacagg 4860 tttgccgttt tgccgccaga gcggattaaa gcggtagatt tgatgatgtt tacttatttc 4920 tttgataagg aggggatgcc tgtttatcag gttagtcccg ccttatgtac ccaagaattg 4980 cattatgcca agtttctcag tcaaaacagt atgttgggta gcgatttgga aaaagatagg 5040 gaacaaggaa gaagacaccg ccgttcgttg aaggtgatgt ttgacttgaa gcgtgctttg 5100 ggtaaattcg gtagggaaaa gaagaaaaga atggagcgtc aaaggcaggc ggagcttgag 5160 aaagtttacg gcaggcgggt catattgttc aaatagtttg tgtaaaatat aggggattaa 5220 aatcagaaat ggacacactg tcattcccgc gcaggcggga atctaggtct ttaaacttcg 5280 gttttttccg ataaattctt gccgcattaa aattccagat tcccgctttc gcggggatga 5340 cggcgggggg attgttgctt tttcggataa aatcccgtgt tttttcatct gctaggtaaa 5400 atcgccccaa agcgtctgca tcgcggcgat ggcggcgagt ggggcggttt ctgtgcgtaa 5460 aatccgtttt ccgagtgtaa ccgcctgaaa gccggcttca aatgcctgtt gttcttcctg 5520 ttctgtccag ccgccttcgg gcccgaccat aaagacgatt gcgccggacg ggtggcggat 5580 gtcgccgagt ttgcaggcgc ggttgatgct cataatcagc ttggtgtttt cagacggcat 5640 tttgtcgagt gcttcacggt agccgatgat gggcagtacg gggggaacgg tgttcctgcc 5700 gctttgttcg cacgcggaga tgacgatttc ctgccagcgt gcgaggcgtt tggcggcgcg 5760 ttctccgtcg aggcggacga tgcagcgttc gctgatgacg ggctgtatgg cggttacgcc 5820 gagttcgacg cttttttgca gggtgaaatc catgcgatc 5859 2 126 PRT Neisseria gonorrhoeae 2 Leu Gln Ala Val Ala Val Phe Lys Gln Leu Pro Glu Ala Ala Ala Leu 1 5 10 15 Ala Ala Ala Asn Lys Arg Val Gln Asn Leu Leu Lys Lys Ala Asp Ala 20 25 30 Ala Leu Gly Glu Val Asn Glu Ser Leu Leu Gln Gln Asp Glu Glu Lys 35 40 45 Ala Leu Tyr Ala Ala Ala Gln Gly Leu Gln Pro Lys Ile Ala Ala Ala 50 55 60 Val Ala Glu Gly Asn Phe Arg Thr Ala Leu Ser Glu Leu Ala Ser Val 65 70 75 80 Lys Pro Gln Val Asp Ala Phe Phe Asp Gly Val Met Val Met Ala Glu 85 90 95 Asp Ala Ala Val Lys Gln Asn Arg Leu Asn Leu Leu Asn Arg Leu Ala 100 105 110 Glu Gln Met Asn Ala Val Ala Asp Ile Ala Leu Leu Gly Glu 115 120 125 3 348 PRT Neisseria gonorrhoeae 3 Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Asn Gln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser Arg Ile Lys Ile Leu Ala 50 55 60 Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Gly Gly Glu Tyr Ile Ala Arg Thr 85 90 95 Asp Ala Asp Asp Ile Ala Ser Pro Gly Trp Ile Glu Lys Ile Val Gly 100 105 110 Glu Met Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu 115 120 125 Val Leu Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Lys 130 135 140 His Gly Lys Ile Trp Lys Lys Pro Thr Arg His Glu Asp Ile Ala Ala 145 150 155 160 Phe Phe Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg 165 170 175 Arg Ser Val Ile Asp Gly Gly Leu Arg Tyr Asp Thr Glu Arg Asp Trp 180 185 190 Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Val Ser Lys Leu Gly Arg Leu 195 200 205 Ala Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln 210 215 220 Val Ser Ser Lys His Ser Val Arg Gln His Glu Ile Ala Gln Gly Ile 225 230 235 240 Gln Lys Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr 245 250 255 Arg Phe Asp Ser Leu Glu Tyr Arg Gln Thr Lys Ala Ala Ala Tyr Glu 260 265 270 Leu Pro Glu Lys Asp Leu Pro Glu Glu Asp Phe Glu Arg Ala Arg Arg 275 280 285 Phe Leu Tyr Gln Cys Phe Lys Arg Thr Asp Thr Pro Pro Ser Gly Ala 290 295 300 Trp Leu Asp Phe Ala Ala Asp Gly Arg Met Arg Arg Leu Phe Thr Leu 305 310 315 320 Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Leu Ile Lys Asn Arg Arg Gln 325 330 335 Ala Arg Ser Asp Ser Ala Gly Lys Glu Gln Glu Ile 340 345 4 306 PRT Neisseria gonorrhoeae 4 Met Asp Ile Val Phe Ala Ala Asp Asp Asn Tyr Ala Ala Tyr Leu Cys 1 5 10 15 Val Ala Ala Lys Ser Val Glu Ala Ala His Pro Asp Thr Glu Ile Arg 20 25 30 Phe His Val Leu Asp Ala Gly Ile Ser Glu Glu Asn Arg Ala Ala Val 35 40 45 Ala Ala Asn Leu Arg Gly Gly Gly Asn Ile Arg Phe Ile Asp Val Asn 50 55 60 Pro Glu Asp Phe Ala Gly Phe Pro Leu Asn Ile Arg His Ile Ser Ile 65 70 75 80 Thr Thr Tyr Ala Arg Leu Lys Leu Gly Glu Tyr Ile Ala Asp Cys Asp 85 90 95 Lys Val Leu Tyr Leu Asp Thr Asp Val Leu Val Arg Asp Gly Leu Lys 100 105 110 Pro Leu Trp Asp Thr Asp Leu Gly Gly Asn Trp Val Gly Ala Cys Ile 115 120 125 Asp Leu Phe Val Glu Arg Gln Glu Gly Tyr Lys Gln Lys Ile Gly Met 130 135 140 Ala Asp Gly Glu Tyr Tyr Phe Asn Ala Gly Val Leu Leu Ile Asn Leu 145 150 155 160 Lys Lys Trp Arg Arg His Asp Ile Phe Lys Met Ser Cys Glu Trp Val 165 170 175 Glu Gln Tyr Lys Asp Val Met Gln Tyr Gln Asp Gln Asp Ile Leu Asn 180 185 190 Gly Leu Phe Lys Gly Gly Val Cys Tyr Ala Asn Ser Arg Phe Asn Phe 195 200 205 Met Pro Thr Asn Tyr Ala Phe Met Ala Asn Gly Phe Ala Ser Arg His 210 215 220 Thr Asp Pro Leu Tyr Leu Asp Arg Thr Asn Thr Ala Met Pro Val Ala 225 230 235 240 Val Ser His Tyr Cys Gly Ser Ala Lys Pro Trp His Arg Asp Cys Thr 245 250 255 Val Trp Gly Ala Glu Arg Phe Thr Glu Leu Ala Gly Ser Leu Thr Thr 260 265 270 Val Pro Glu Glu Trp Arg Gly Lys Leu Ala Val Pro Pro Thr Lys Cys 275 280 285 Met Leu Gln Arg Trp Arg Lys Lys Leu Ser Ala Arg Phe Leu Arg Lys 290 295 300 Ile Tyr 305 5 337 PRT Neisseria gonorrhoeae 5 Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Ala Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Gly Gln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Pro Ala 35 40 45 Ile Ala Arg His Phe Gln Glu Gln Asp Gly Arg Ile Arg Ile Ile Ser 50 55 60 Asn Pro Arg Asn Leu Gly Phe Ile Ala Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Glu Tyr Ile Ala Arg Thr Asp Ala 85 90 95 Asp Asp Ile Ala Ser Pro Gly Trp Ile Glu Lys Ile Val Gly Glu Met 100 105 110 Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu Val Leu 115 120 125 Ser Glu Glu Asn Asn Lys Ser Val Leu Ala Ala Ile Ala Arg Asn Gly 130 135 140 Ala Ile Trp Asp Lys Pro Thr Arg His Glu Asp Ile Val Ala Val Phe 145 150 155 160 Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg Arg Ser 165 170 175 Val Ile Asp Gly Gly Leu Arg Phe Asp Pro Ala Tyr Ile His Ala Glu 180 185 190 Asp Tyr Lys Phe Trp Tyr Glu Ala Gly Lys Leu Gly Arg Leu Ala Tyr 195 200 205 Tyr Pro Glu Ala Leu Val Lys Tyr Arg Phe His Gln Asp Gln Thr Ser 210 215 220 Ser Lys Tyr Asn Leu Gln Gln Arg Arg Thr Ala Trp Lys Ile Lys Glu 225 230 235 240 Glu Ile Arg Ala Gly Tyr Trp Lys Ala Ala Gly Ile Ala Val Gly Ala 245 250 255 Asp Cys Leu Asn Tyr Gly Leu Leu Lys Ser Thr Ala Tyr Ala Leu Tyr 260 265 270 Glu Lys Ala Leu Ser Gly Gln Asp Ile Gly Cys Leu Arg Leu Phe Leu 275 280 285 Tyr Glu Tyr Phe Leu Ser Leu Glu Lys Tyr Ser Leu Thr Asp Leu Leu 290 295 300 Asp Phe Leu Thr Asp Arg Val Met Arg Lys Leu Phe Ala Ala Pro Gln 305 310 315 320 Tyr Arg Lys Ile Leu Lys Lys Met Leu Arg Pro Trp Lys Tyr Arg Ser 325 330 335 Tyr 6 280 PRT Neisseria gonorrhoeae 6 Met Gln Asn His Val Ile Ser Leu Ala Ser Ala Ala Glu Arg Arg Ala 1 5 10 15 His Ile Ala Asp Thr Phe Gly Ser Arg Gly Ile Pro Phe Gln Phe Phe 20 25 30 Asp Ala Leu Met Pro Ser Glu Arg Leu Glu Gln Ala Met Ala Glu Leu 35 40 45 Val Pro Gly Leu Ser Ala His Pro Tyr Leu Ser Gly Val Glu Lys Ala 50 55 60 Cys Phe Met Ser His Ala Val Leu Trp Glu Gln Ala Leu Asp Glu Gly 65 70 75 80 Leu Pro Tyr Ile Ala Val Phe Glu Asp Asp Val Leu Leu Gly Glu Gly 85 90 95 Ala Glu Gln Phe Leu Ala Glu Asp Thr Trp Leu Glu Glu Arg Phe Asp 100 105 110 Lys Asp Ser Ala Phe Ile Val Arg Leu Glu Thr Met Phe Ala Lys Val 115 120 125 Ile Val Arg Pro Asp Lys Val Leu Asn Tyr Glu Asn Arg Ser Phe Pro 130 135 140 Leu Leu Glu Ser Glu His Cys Gly Thr Ala Gly Tyr Ile Ile Ser Arg 145 150 155 160 Glu Ala Met Arg Phe Phe Leu Asp Arg Phe Ala Val Leu Pro Pro Glu 165 170 175 Arg Ile Lys Ala Val Asp Leu Met Met Phe Thr Tyr Phe Phe Asp Lys 180 185 190 Glu Gly Met Pro Val Tyr Gln Val Ser Pro Ala Leu Cys Thr Gln Glu 195 200 205 Leu His Tyr Ala Lys Phe Leu Ser Gln Asn Ser Met Leu Gly Ser Asp 210 215 220 Leu Glu Lys Asp Arg Glu Gln Gly Arg Arg His Arg Arg Ser Leu Lys 225 230 235 240 Val Met Phe Asp Leu Lys Arg Ala Leu Gly Lys Phe Gly Arg Glu Lys 245 250 255 Lys Lys Arg Met Glu Arg Gln Arg Gln Ala Glu Leu Glu Lys Val Tyr 260 265 270 Gly Arg Arg Val Ile Leu Phe Lys 275 280 7 6 PRT Neisseria gonorrhoeae 7 Tyr Ser Arg Asp Ser Ser 1 5 8 348 PRT Neisseria gonorrhoeae 8 Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Asn Gln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser Arg Ile Lys Ile Leu Ala 50 55 60 Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Gly Gly Glu Tyr Ile Ala Arg Thr 85 90 95 Asp Ala Asp Asp Ile Ala Ser Pro Gly Trp Ile Glu Lys Ile Val Gly 100 105 110 Glu Met Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu 115 120 125 Val Leu Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Lys 130 135 140 His Gly Lys Ile Trp Lys Lys Pro Thr Arg His Glu Asp Ile Ala Ala 145 150 155 160 Phe Phe Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg 165 170 175 Arg Ser Val Ile Asp Gly Gly Leu Arg Tyr Asp Thr Glu Arg Asp Trp 180 185 190 Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Val Ser Lys Leu Gly Arg Leu 195 200 205 Ala Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln 210 215 220 Val Ser Ser Lys His Ser Val Arg Gln His Glu Ile Ala Gln Gly Ile 225 230 235 240 Gln Lys Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr 245 250 255 Arg Phe Asp Ser Leu Glu Tyr Arg Gln Thr Lys Ala Ala Ala Tyr Glu 260 265 270 Leu Pro Glu Lys Asp Leu Pro Glu Glu Asp Phe Glu Arg Ala Arg Arg 275 280 285 Phe Leu Tyr Gln Cys Phe Lys Arg Thr Asp Thr Pro Pro Ser Gly Ala 290 295 300 Trp Leu Asp Phe Ala Ala Asp Gly Arg Met Arg Arg Leu Phe Thr Leu 305 310 315 320 Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Leu Ile Lys Asn Arg Arg Gln 325 330 335 Ala Arg Ser Asp Ser Ala Gly Lys Glu Gln Glu Ile 340 345

Claims

What is claimed as new and desired to be secured by Letters Patent of the United States is:

1. A method of transferring at least two saccharide units with a single enzyme comprising contacting an acceptor moiety and two different sugar donors with a polyglycosyltransferase.

2. The method of claim 1, wherein said two saccharide units are N-acetylglucosamine and N-acetylgalactosamine.

3. The method of claim 1, wherein said acceptor moiety has a galactose at a non-reducing end.

4. The method of claim 1, wherein said polyglycosyltransferase is isolated from Neisseria.

5. The method of claim 1, wherein said polyglycosyltransferase is isolated from Neisseria gonorrhoeae.

6. A method of transferring N-acetylgalactosamine, to an acceptor moiety, comprising contacting an N-acetylgalactosamine donor and an acceptor moiety with an N-acetylglucosaminyl transferase.

7. The method of claim 6, wherein said N-acetylglucosaminyl transferase is isolated from Neisseria.

8. The method of claim 6, wherein said N-acetylglucosaminyl transferase is isolated from Neisseria gonorrhoeae.

9. The method of claim 6, wherein said acceptor moiety has a galactose at a non-reducing end.

10. The method of claim 8, wherein said Neisseria gonorrhoeae is ATCC 33084.