ES2365393T3 - SYNTHESIS OF OLIGOSACÁRIDOS, GLICOLÍPIDOS Y GLICOPROTEÍNAS USING BACTERIAL GLICOSILTRANSPHERASES. - Google Patents

Abstract

Method of creating a substantially uniform glycosphere of a fucosylated oligosaccharide, the method comprising: contacting a recombinant α1,3-fucosyltransferase of Helicobacter pylori strain 1182B with a mixture comprising a donor substrate comprising a fucose residue, and an acceptor substrate comprising a sugar or oligosaccharide comprising at least one Nacethylglucosamine residue, under conditions in which α1,3-fucosyltransferase catalyzes the transfer of fucose preferably to the N-acetylglucosamine residue, thereby producing a glycoform. substantially uniform of a fucosylated oligosaccharide, wherein α1,3-fucosyltransferase is encoded by a nucleic acid sequence having the sequence of Figure 1 or having the amino acid sequence of Figure 1 and wherein the acceptor substrate is Lacto- N-neo-Tetraose (LNnt).

Description

Field of the Invention

[0001] The present invention provides nucleic acid and amino acid sequences of Helicobacter pylori fucosyltransferases. The invention also provides methods for using fucosyltransferases in order to synthesize oligosaccharides, glycoproteins, and glycolipids.

Background of the invention

[0002] Although significant advances in carbohydrate chemistry have been made in recent years, there are still substantial difficulties associated with the chemical synthesis of glycoconjugates, particularly with the formation of the ubiquitous β-1,2-cis-mannoside bond found in mammalian oligosaccharides. On the other hand, at each stage of de novo synthesis of a carbohydrate, regional and stereo-chemical obstacles must be resolved.

[0003] In view of the difficulties associated with the chemical synthesis of glycoconjugates, the use of glycosyltransferases to enzymatically synthesize glycoproteins and glycolipids, having desired oligosaccharide fractions, is a promising approach for preparing said glycoconjugates. Enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity and can be carried out using unprotected substrates. On the other hand, glycosyltransferases have been used to enzymatically modify oligosaccharide fractions and have proven to be very effective in producing specific products with good stereochemical and regiochemical control. The glycosyltransferases of interest include fucosyltransferases, sialyltransferases, galactosyltransferases, and Nacethylglucosaminyltransferases. For a general review, see Crout et al., Curr. Opin. Chem. Biol. 2: 98111 (1998) and Arsequell, et al., Tetrahedon: Assymetry 10: 2839 (1997).

[0004] Many glycoproteins and glycolipids require the presence of a particular glycoform, or the absence of a particular glycoform, in order to present a particular biological activity. For example, many glycoproteins and glycolipids require the presence of particular fucosylated structures to exhibit biological activity. Intercellular recognition mechanisms frequently require a fucosylated oligosaccharide. For example, several glycoproteins that function as cell adhesion molecules, including P-selectin, L-selectin, and E-selectin, bind to specific, surface-surface fucosylated carbohydrate structures, such as Lewis-sialyl structures. x and sialil Lewis-a. Additionally, the specific carbohydrate structures that form the ABO blood group system are fucosylated. The carbohydrate structures in each of the three groups share a Fucα1,2Galβ1-disaccharide unit. In blood group O structures, this disaccharide is the terminal structure; while the structure of blood group A is formed by an α1,3Ga1NAc transferase that adds a terminal GalNAc residue to the disaccharide; and the structure of blood group B is formed by an α1,3 galactosyltransferase that adds a terminal galactose residue.

[0005] Lewis blood group structures can also be subjected to fucosylation. For example, the Lewis-x and Lewis-a structures are respectively Galβ1.4 (Fucα1.3) GlcNac and Galβ1.3 (Fucα1.4) GlcNac. Both mentioned structures can also be sialylated (NeuAcα2,3-) to form the corresponding sialylated structures. Other Lewis blood group structures of interest are the Lewis-y and Lewis-b structures that are respectively Fucα1,2Galβ1,4 (Fucα1,3) GlcNAcβ-OR and Fucα1,2Galβ1,3 (Fucα1,4) Glc-NAc -OR. For a description of the ABO structures and Lewis blood group structures and enzymes involved in their synthesis, see, Essentials of Glycobiology, Varki et al. eds., Chapter 16 (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999).

[0006] Specifically, fucosyltransferases have been used in synthetic routes to transfer a guanosine-5'-diphosphofucose fucose residue to a specific hydroxyl of a saccharide acceptor. A variety of donor substrates and acceptor substrates are known (see Guo et al., Applied Biochem. And Biotech. 68: 1-20 (1997)). For example, Ichikawa prepared sialyl Lewis-x by a method that involves the fucosylation of sialylated lactosamine, with a cloned fucosyltransferase (Ichikawa et al., J. Am. Chem. Soc. 114: 9283-9298 (1992)). Lowe has described a method for expressing non-native fucosylation activity in cells, thereby producing fucosylated glycoproteins on cell surfaces, etc. (U.S. Patent No. 5,955,347).

[0007] Thus, since the biological activity of many commercially important recombinantly produced and transgenic glycoproteins and glycolipids depends on the presence of a particular glycoform, or the absence of a particular glycoform, there is a need for a method effective for enzymatically synthesizing glycoconjugates having the desired fucosylated oligosaccharide fractions. Additionally, there is a need for efficient production of fucosylated oligosaccharides. The present invention satisfies this and other needs.

Brief Summary of the Invention

[0008] The present invention provides proteins and nucleic acids of α-1,3 / 4-fucosyltransferase from H. pylori. The α-1,3 / 4-fucosyltransferase proteins catalyze the transfer of a fucose residue from a donor substrate to an acceptor substrate. In one embodiment, the invention provides α-1,3 / 4-fucosyltransferase nucleic acids with a nucleotide sequence according to SEQ ID NO: 1 encoding α-1,3 / 4-fucosyltransferase proteins that transfer fucose to GIcNAc residues.

[0009] In another embodiment, the α-1,3 / 4-fucosyltransferase nucleic acid is presented according to SEQ ID NO: 1. The invention also provides nucleic acid sequences encoding α-1,3 / 4-fucosyltransferase proteins, which include SEQ ID NO: 2, and which catalyze the transfer of fucose to an N-acetylglucosamine residue or to a residue. of glucose In one aspect, the encoded α-1,3 / 4-fucosyltransferase also includes an amino acid label.

[0010] In another embodiment, the invention provides expression vectors that include the α-1,3 / 4-fucosyltransferase nucleic acids described above, host cells that include expression vectors, and methods for producing α-1,3 proteins / 4-fucosyltransferase using host cells cultured under conditions suitable for the expression of the α-1,3 / 4-fucosyltransferase protein.

[0011] In another embodiment, the invention provides recombinant fucosyltransferase proteins that include an amino acid sequence according to SEQ ID NO: 2 wherein the fucosyltransferase catalyzes the transfer of a fucose residue from a donor substrate to N-acetylglucosamine. In one aspect, fucosyltransferase proteins comprise SEQ ID NO: 2. In another aspect, fucosyltransferase proteins also include an amino acid tag.

[0012] The present invention also provides methods for using the above α-1,3 / 4-fucosyltransferase protein in order to produce fucosylated oligosaccharides. Fucosylated oligosaccharides can be further purified. The acceptor substrate may be N-acetylglucosamine. In one embodiment, the acceptor substrate is Lacto-N-neo-Tetraose (LNnT) and the fucosylated product is Lacto-N-Fucopentaose III (LNFP III). Α-1,3 / 4-fucosyltransferase can be used in combination with other glycosyltransferases to produce a fucosylated oligosaccharide. For example, using lactose as a starting material, LNFP can be produced through the action of an α-1,3 / 4-fucosyltransferase that transfers fucose to N-acetylglucosamine, a β-1,3-N-acetylglucosaminyltransferase, and a β-1,4-galactosyltransferase. Β-1,3acetylglucosaminyltransferase and β-1,4-galactosyltransferase can be bacterial enzymes and, in a preferred embodiment, are from Neisseria gonococcus.

[0013] In another embodiment, the α-1,3 / 4-fucosyltransferase protein of the present invention is used to produce fucosylated glycolipids. The acceptor substrate may be N-acetylglucosamine.

[0014] In another embodiment, the present invention provides a method of producing a fucosylated glycoprotein, combining an α-1,3 / 4-fucosyltransferase described herein with a glycoprotein that includes an appropriate acceptor substrate, under conditions suitable for producing a fucosylated glycoprotein. The acceptor substrate can be selected from Galβ1-OR, Gal, 3 / 4GlcNAc-OR, NeuAcα2,3Galβ1,3 / 4GlcNAc-Or, where R is an amino acid, a saccharide, an oligosaccharide, or an aglycone group having At least one carbon atom. The acceptor substrate may be an N-acetylglucosamine residue. Α-1,3 / 4-fucosyltransferase can also include an amino acid label.

Brief description of the drawings

[0015] Figure 1 provides the nucleic acid sequences (SEQ ID NO: 1) and amino acids (SEQ ID NO: 2) of fucosyltransferase from the strain of H. pylori 1182B.

[0016] Figure 2 provides the nucleic acid sequences (SEQ ID NO: 3) and amino acids (SEQ ID NO: 4) of fucosyltransferase from the strain of H. pylori 1111A.

[0017] Figure 3 provides the nucleic acid sequences (SEQ ID NO: 5) and amino acids (SEQ ID NO: 6) of fucosyltransferase from the strain of H. pylori 1218B.

[0018] Figure 4 provides the nucleic acid sequences (SEQ ID NO: 7) and amino acids (SEQ ID NO: 8) of fucosyltransferase from the strain of H. pylori 19C2B.

[0019] Figure 5 provides the nucleic acid sequences (SEQ ID NO: 9) and amino acids (SEQ ID NO: 10) of fucosyltransferase from the strain of H. pylori 915A.

[0020] Figure 6 provides the nucleic acid sequences (SEQ ID NO: 11) and amino acids (SEQ ID NO: 12) of fucosyltransferase from the strain of H. pylori 26695A.

[0021] Figure 7 provides the nucleic acid sequences (SEQ ID NO: 13) and amino acids (SEQ ID NO: 14) of fucosyltransferase from the strain of H. pylori, 19C2A.

[0022] Figure 8 provides an alignment between amino acid sequence 1182 futB (SEQ ID NO: 15) and a consensus sequence of glycosyltransferase family 10 (SEQ ID NO: 16), that is, the family of fucosyltransferases. Amino acids 23 to 305 of 1182 futB are shown in the upper line and represent the most conserved region of the protein, that is, the catalytic domain of fucosyltransferase.

[0023] Figure 9 provides an alignment between amino acid sequence 1111 futA (SEQ ID NO: 17) and a consensus sequence of glycosyltransferase family 10 (SEQ ID NO: 18), that is, the family of fucosyltransferases. Amino acids 27 to 417 of 1182 futB are shown in the upper line and represent the most conserved region of the protein, that is, the catalytic domain of fucosyltransferase.

[0024] Figure 10 provides an alignment between amino acid sequence 1218 futB (SEQ ID NO: 19) and a consensus sequence of glycosyltransferase family 10 (SEQ ID NO: 20), that is, the family of fucosyltransferases. Amino acids 23 to 399 of 1182 futB are shown in the upper line and represent the most conserved region of the protein, that is, the catalytic domain of fucosyltransferase.

[0025] Figure 11 provides an alignment between amino acid sequence 19C2 futB (SEQ ID NO: 21) and a consensus sequence of glycosyltransferase family 10 (SEQ ID NO: 22), that is, the family of fucosyltransferases. Amino acids 23 to 377 of 1182 futB are shown in the upper line and represent the most conserved region of the disclosed protein, that is, the catalytic domain of fucosyltransferase.

[0026] Figure 12 provides an alignment between the amino acid sequence of strains of H. pylori 1182 FutB (SEQ ID NO: 25), 1111 FutA (SEQ ID NO: 23), 1218 FutB (SEQ ID N .º: 26), 19C2 FutB (SEQ ID NO: 27), 915FutA (SEQ ID NO: 10), 19C2 FutA (SEQ ID NO: 14), and 26695 FutA (SEQ ID NO. : 24). The lower sequence is a consensus sequence (SEQ ID NO: 28 to 37).

[0027] Figure 13 provides an alignment between the nucleic acid sequence of strains of H. pylori 1182 FutB (SEQ ID NO: 1), 1111 FutA (SEQ ID NO: 3), 1218 FutB (SEQ ID #: 5), 19C2 FutB (SEQ ID NO: 7), 915FutA (SEQ ID NO: 38), 19C2 FutA (SEQ ID NO: 13), and 26695 FutA (SEQ ID N. º: 11). The lower sequence is a consensus sequence (SEQ ID NO: 39 to 74).

[0028] Figure 14 provides oligosaccharide structures of Lacto-N-neo-Tetraose (LNnT), a substrate of the fucosyltransferases of H. pylori and Lacto-N-Fucopentaose III (LNFPIII or LNFIII), a product of the fucosyltransferases of

H. pylori.

[0029] Figure 15 provides the results of the analysis of the specificity of the acceptor for fucosyltransferases of

H. pylori.

[0030] Figure 16 provides the synthesis performance of LNFIII using H. pylori fucosyltransferases. Two ion exchange resins were tested: MR3 NH4HCO3 and Dowex1 / Dowex50 resin.

[0031] Figure 17 tests the use of FutB α-1,3 / 4-fucosyltransferase from the strain of H. pylori 1182 to transfer fucose to the asialyltransferrin glycoprotein. The upper panel shows the GC / MS analysis of sialylated transferrin. The bottom panel shows the GC / MS analysis of sialylated transferrin that has been enzymatically asylated and then fucosylated using the FutB α-1,3 / 4-fucosyltransferase from the strain of H. pylori 1182. Explanation for sugar structures: filled squares -GIcNAc, white-mannose circles; stuffed diamonds-galactose; triangles-fucose; sialic acid stars.

Definitions

[0032] Unless otherwise defined, all technical and scientific terms used herein have in general the same meaning as commonly understood by those with usual knowledge in the field to which this invention pertains. In general, the nomenclature used herein and laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid hybridization, which are described below, are those well known and commonly used. in the matter. Conventional techniques are used for the synthesis of nucleic acids and peptides. In general, enzymatic reactions and purification steps are carried out according to the manufacturer's specifications. The techniques and procedures are generally carried out according to conventional methods in the field and several general references (see in general, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.

(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which appear throughout the present document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthesis, which are described below, are those well known and commonly used in the art. Conventional techniques, or modifications thereof, are used for chemical syntheses and chemical analyzes.

[0033] The terms "α-1,3 / 4-fucosyltransferase or fucosyltransferase" or a nucleic acid encoding an "α-1,3 / 4 fucosyltransferase or fucosyltransferase" refers to nucleic acids and polypeptides having an amino acid sequence that is encoded by a nucleic acid according to SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 2 or have a nucleic acid sequence having SEQ ID NO: 1. The nucleic acids and proteins of the invention include both naturally occurring and recombinant molecules.

[0034] The α-1,3 / 4-fucosyltransferase enzymes of the invention can also be recognized by the presence of highly conserved catalytic domains found in a family of fucosyltransferase, glycosyltransferase family 10 proteins, see, for example, gnl | CDD | 16836 pfam00852, Glico_transf_10. Alignments between conserved catalytic domains of 1182 futB, 1111 futA, 1218 futB, and 19C2 futB are shown in Figures 8 to 11 and a consensus sequence of the catalytic domain of members of the family 10 glycosyltransferase.

[0035] A biologically active fucosyltransferase as described herein is a fucosyltransferase that catalyzes the transfer of fucose from a donor substrate, for example, GDP-fucose, to an acceptor molecule on an α-1,3 / 4 bond. The acceptor molecule can be either N-acetylglucosylamine (GlucNAc) or glucose. For example, fucosyltransferases from the following strains of H. pylori transfer fucose to Glc-NAc: Strain 915 FutA, Strain 1111 FutA, Strain 19C2 FutB, and Strain 1182 FutB. The product of the FutA gene of H. pylori strain 19C2 FutA transfers fucose to the reducing glucose of the LNnT acceptor, as did the product of the FutB gene of strain H. pylori 1218, and a novel 26695 FutA protein. In preferred embodiments, fucosyltransferase transfers fucose exclusively to GlcNAc or exclusively to glucose. The acceptor molecule can be a carbohydrate, an oligosaccharide, a glycolipid, or a glycoprotein.

[0036] The H. pylori fucosyltransferase proteins of the invention are useful for transferring a saccharide from a donor substrate to an acceptor substrate. The addition generally takes place at the non-reducing end of an oligosaccharide or a carbohydrate fraction in a molecule. However, in some embodiments, the fucose residue is added to a reducing glucose residue. Biomolecules, as defined herein, include among others, biologically significant molecules such as carbohydrates, oligosaccharides, proteins (for example, glycoproteins), and lipids (for example, glycolipids, phospholipids, sphingolipids and gangliosides).

[0037] The following abbreviations are used herein:

Ara = arabinosyl;

Fru = fructosyl;

Fuc = fucosyl;

Gal = galactosyl;

GalNAc = N-acetylgalactosylamino;

Glc = glucosyl;

GlcNAc = N-acetylglucosylamino;

Man = mannosyl; Y

NeuAc = sialyl (N-acetylneuraminyl)

FT or Fut = fucosyltransferase *

ST = sialyltransferase *

GalT = galactosyltransferase *

[0038] Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not a reducing sugar is actually the saccharide of the reducing end. In accordance with the accepted nomenclature, oligosaccharides are represented herein with the non-reducing end on the left and the reducing end on the right.

[0039] All oligosaccharides described herein are described by the name or abbreviation corresponding to the non-reducing saccharide (eg, Gal), followed by the configuration of the glycosidic bond (α or β), the annular bond, the position annul of the saccharide reducer involved in the bond, and then the name or abbreviation of the saccharide reducer (eg, GlcNAc). The link between two sugars can be expressed, for example, as 2,3, 2 → 3, or (2,3). Each saccharide is a pyranose or furanose.

[0040] The term "sialic acid" refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5acetamido-3,5-dideoxy-D-glyceric-D-galactononulopyranos-1-ionic acid (often abbreviated as Neu5Ac, NeuAc, or NANA) A second member of the family is N-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the Nacetyl group of NeuAc is hydroxylated.A third member of the sialic acid family is acid 2- keto-3-deoxynonulosonic (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265: 2181121819 (1990)). Also included 9-substituted sialic acids, such as 9-O-C1-C6 acyl-Neu5Ac such as 9-O-lactylNeu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-acid-9-deoxy -Neu5Ac. For a review of the sialic acid family, see, for example, Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer- Verlag, New York (1 992)) The synthesis and use of sialic acid compounds in a sialylation process is disclosed in the international application WO 92/16640, published on October 1, 1992.

[0041] An "acceptor substrate" for a glycosyltransferase is an oligosaccharide fraction that can act as an acceptor for a particular glycosyltransferase. When the acceptor substrate is brought into contact with the corresponding glycosyltransferase and sugar donor substrate, and other necessary components of the reaction mixture, and the reaction mixture is incubated for a sufficient period of time, the glycosyltransferase transfers residues of sugar from the sugar donor substrate to the acceptor substrate. Frequently, the acceptor substrate will vary for different types of a particular glycosyltransferase.

[0042] An "acceptor substrate" for an H. pylori fucosyltransferase is an oligosaccharide fraction that can act as an acceptor for H. pylori fucosyltransferase. When the acceptor substrate is brought into contact with the H. pylori fucosyltransferase and the sugar donor substrate (e.g. GDP-fucose), and other necessary components of the reaction mixture, and the reaction mixture is incubated for a sufficient period of time, H. pylori fucosyltransferase transfers fucose residues from the GDP-fucose to the acceptor substrate. Frequently, the acceptor substrate will vary for different types of a particular fucosyltransferase. For example, the acceptor substrate for a mammalian galactoside 2-L-fucosyltransferase (α1,2-fucosyltransferase) will include a Galβ1,4-GlcNAc-R at a non-reducing end of an oligosaccharide; this fucosyltransferase fixes a fucose residue to the Gal through an α1,2 bond. The terminal Galβ1,4-GlcNAc-R and Galβ1,3-GlcNAc-R and the sialylated analogs thereof are acceptor substrates, respectively, for α1,3 and α1,4-fucosyltransferases. However, these enzymes fix the fucose residue to the GlcNAc residue of the acceptor substrate. Therefore, the term "acceptor substrate" is considered contextualized with the particular glycosyltransferase of interest for a particular application. The H. pylori fucosyltransferase described herein will transfer fucose to sialylated or non-sialylated acceptor substrates. Some H. pylori fucosyltransferase described herein will transfer fucose to glucose residues.

[0043] A "donor substrate" for glycosyltransferases is an activated nucleotide sugar. Such activated sugars generally consist of uridine monophosphate, guanosine, and citidine derivatives of sugars (respectively UMP, GMP and CMP) or diphosphate derivatives of sugars (UDP, GDP and CDP, respectively) in which the nucleoside monophosphate or diphosphate serves As a leaving group. For example, a donor substrate for fucosyltransferases is GDP-fucose. The sialyltransferase donor substrates, for example, are activated sugar nucleotides comprising the desired sialic acid. For example, in the case of NeuAc, the activated sugar is CMP-NeuAc.

[0044] A "substantially uniform glycoform" or a "substantially uniform glycosylation pattern" when in relation to a species of glycoprotein, refers to the percentage of acceptor substrates that are glycosylated by the glycosyltransferase of interest (eg, fucosyltransferase). For example, in the case of the α1,3 or α1,4 fucosyltransferase indicated above, a substantially uniform fucosylation pattern exists if substantially all (as defined below) of Galβ1,4-GlcNAc-R and sialylated or non-sialylated analogs thereof are fucosylated in a composition comprising the glycoprotein of interest. Those skilled in the art will understand that the starting material may contain glycosylated acceptor substrates (eg, fucosylated Galβ1,4-GlcNAc-R substrates). Thus, the calculated amount of glycosylation will include acceptor substrates that are glycosylated by the methods of the invention, as well as those acceptor substrates already glycosylated in the starting material.

[0045] The term "substantially" in the above definitions of "substantially uniform" generally means that at least about 60%, at least about 70%, at least about 80%, or more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the substrates Acceptors for a particular glycosyltransferase are glycosylated (for example, fucosylated Galβ1,4-GlcNAc-R substrates).

[0046] The term "substantially identical fucosylation pattern" refers to a glycosylation pattern of a glycoprotein produced by a method of the invention, which is at least or about 80%, more preferably at least about 90% , still more preferably at least about 91%, 92%, 93%, 94%, or 95% and still more preferably at least about 96%, 97%, 98% or 99 % identical to fucosylation of a known glycoprotein. "Known fucosylation pattern" refers to a fucosylation pattern of a known glycoprotein from any source that has any known level of fucosylation.

[0047] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, an α carbon that is attached to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or modified peptide skeletons, although they retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that works in a manner similar to a naturally occurring amino acid.

[0048] "Protein," "polypeptide," or "peptide" refers to a polymer in which the monomers are amino acids and are linked together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are α-amino acids, either the optical isomer L or the optical isomer D can be used. Additionally, unnatural amino acids are also included, for example, β-alanine, phenylglycine and homoarginine. In the present invention, amino acids that are not genetically encoded can also be used. In addition, amino acids that have been modified to include reactive groups can also be used in the invention. All amino acids used in the present invention may be either the D or L isomer. In general, the L isomers are preferred. Additionally, other peptidomimetics are also useful in the present invention. For a general review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

[0049] The term "recombinant" when used in reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells may contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes that are in the native form of the cell where genes are modified and reintroduced into the cell by artificial means. The term also encompasses cells that contain an endogenous nucleic acid from the cell that has been modified without extracting the nucleic acid from the cell; Such modifications include those obtained by gene replacement, mutation at specific points, and related techniques. A "recombinant protein" is one that has been produced by a recombinant cell.

[0050] A "fusion protein" refers to an H. pylori fucosyltransferase protein comprising amino acid sequences that occur in addition to, rather than, that are less than, and / or different from the amino acid sequences. that encode the original or native full-length protein or sub-sequences thereof.

[0051] Fusion protein components include "accessory enzymes" and / or "purification or amino acid labels". An "accessory enzyme" as referenced herein is an enzyme that is involved in the catalysis of a reaction that, for example, forms a substrate for a fucosyltransferase. An accessory enzyme, for example, can catalyze the formation of a nucleotide sugar that is used as a donor fraction by a fucosyltransferase, for example, GDP-fucose. An accessory enzyme may also be one that is used in the generation of a nucleotide triphosphate required for the formation of a nucleotide sugar, in the generation of sugar that is incorporated into nucleotide sugar, for example, fucose. The recombinant fusion protein of the invention can be constructed and expressed as a fusion protein with a molecular "purification tag" at one end, which facilitates the purification of the protein. Such tags can also be used for immobilization of a protein of interest during the glycosylation reaction. Suitable tags include "epitopic tags," which are a protein sequence that is specifically recognized by an antibody. Epitopic tags are generally incorporated into fusion proteins to allow the use of an easily available antibody in order to unequivocally detect or isolate the fusion protein. A "FLAG tag" is a commonly used epitopic tag, specifically recognized by a monoclonal anti-FLAG antibody, consisting of the sequence AspTyrLysAspAspAspAspLys (SEQ ID NO: 75) or a substantially identical variant thereof. Those skilled in the art know other suitable labels, and they include, for example, an affinity tag such as a hexahistidine peptide (SEQ ID NO: 76), which will bind to metal ions such as nickel ions or cobalt. Purification tags also include maltose binding domains and starch binding domains. Those skilled in the art know the protein purification of maltose binding domains. Starch binding domains are described in WO 99/15636. In USSN 60 / 468,374, filed May 5, 2003, affinity purification of a fusion protein comprising a starch binding domain using a beta-cyclodextrin-derived resin (BCD) is described.

[0052] The term "functional domain" in reference to glycosyltransferase refers to a glycosyltransferase domain that confers or modulates an enzyme activity, for example, acceptor substrate specificity, catalytic activity, binding affinity, or other biological activity. or biochemistry. Examples of glycosyltransferase functional domains include, among others, the catalytic domain.

[0053] The expression "expression level" in reference to a protein refers to the amount of a protein produced by a cell. The amount of protein produced by a cell can be measured by the assays and units of activity described herein or that are known to those skilled in the art. Those skilled in the art will know how to measure and describe the amount of protein produced by a cell using respectively a variety of assays and units. Thus, quantification and quantitative description of the level of expression of a protein, for example, an H. pylori fucosyltransferase, can be tested by measuring the enzymatic activity or the units used to describe the activity, or the amount of protein. The amount of protein produced by a cell can be determined by conventional known assays, for example, the Bradford Protein Assay (1976), the Pierce Bicinconinic Protein Assay Kit (Rockford, Illinois),

or as described in U.S. Pat. . 5,641,668.

[0054] The term "enzymatic activity" refers to an activity of an enzyme and can be measured through the assays and units described herein or known to those skilled in the art.

[0055] The term "specific activity" as used herein refers to the catalytic activity of an enzyme, for example, an H. pylori fucosyltransferase protein of the present invention, and can be expressed in units of activity . As used herein, an activity unit catalyzes the formation of 1 µmol of product per minute at a temperature (for example, at 37 ° C) and a pH value (for example, with a pH 7.5) determined . Thus, 10 units of an enzyme is a catalytic amount of that enzyme in which 10 µmol of substrate is converted into 10 µmol of product in a minute at a temperature of, for example, 37 ° C and a pH value of, per example, 7.5.

[0056] A "catalytic domain" refers to a protein domain, or a sub-sequence thereof, that catalyzes an enzymatic reaction performed by the enzyme. For example, a catalytic domain of a fucosyltransferase will include a sufficient sequence of fucosyltransferase to transfer a fucose residue from a donor to an acceptor saccharide. A catalytic domain may include a complete enzyme, a sub-sequence thereof, or it may include additional amino acid sequences that are not bound to the enzyme, or a sub-sequence thereof as found in nature. The α-1,3 / 4-fucosyltransferase enzymes of the invention can also be recognized by the presence of highly conserved catalytic domains found in a family of fucosyltransferase, glycosyltransferase family 10 proteins, see, for example, gnl | CDD | 16836 pfam00852, Glico_transf_10. Alignments between conserved catalytic domains of 1182 futB, 1111 futA, 1218 futB, and 19C2 futB are shown in Figures 8 to 11 and a consensus sequence of the catalytic domain of members of the family glycosyltransferase 10. In each of the α-1 enzymes , 3/4-H. pylori fucosyltransferase listed above, for example, 1182 futB, amino acids 23 to 305; 1111 futA, amino acids 27 to 304; 1218 futB, amino acids 23 to 305; and 19C2 futB amino acids 22 to 277 are highly conserved regions, similar to a region of the consensus sequence of the catalytic domain of glycosyltransferase family 10 that begins approximately at amino acid 11 and ends at amino acid 301, and it is believed that they are the catalytic domains of the enzyme. Thus, in the methods of the invention, for example, glycosprotein fucosylation, polypeptides comprising the fucosyltransferase catalytic domains identified above can be used. In the methods of the invention, for example, production of fucosyltransferase proteins for glycosprotein fucosylation, nucleic acids encoding the fucosyltransferase catalytic domains identified above can also be used.

[0057] A "sub-sequence" refers to a sequence of nucleic acids or amino acids that comprise a part of a larger sequence of nucleic acids or amino acids (eg, protein) respectively.

[0058] The term "nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize with nucleic acids of a similar way to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.

[0059] A "recombinant expression cassette" or simply "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of influencing the expression of a structural gene in hosts compatible with these sequences. Expression cassettes include at least promoters and, optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (eg, a nucleic acid encoding a desired polypeptide), and a promoter. Also, as described herein, additional factors necessary or useful in achieving the expression can be used. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs the secretion of a protein expressed from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression can also be included in an expression cassette.

[0060] A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a foreign source for the particular host cell, or, if it comes from the same source, is modified with respect to its original form. Thus, a heterologous glycoprotein gene in a eukaryotic host cell includes a glycoprotein coding gene that is endogenous with respect to the particular host cell that has been modified. Modification of the heterologous sequence can occur, for example, by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of operably binding to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence.

[0061] The term "isolated" refers to material that is substantially or essentially free of components that interfere with the activity of an enzyme. For a saccharide, protein, or nucleic acid of the invention, the term "isolated" refers to material that is substantially or essentially free of components that normally accompany the material as it is in its native state. Typically, an isolated saccharide, protein, or nucleic acid of the invention is at least about 80% pure, usually at least about 90%, and preferably at least about 95% pure as measured by the intensity of the bands in a gel stained with silver color or another method to determine purity. Purity or homogeneity can be indicated by several means well known in the art. For example, a protein or nucleic acid in a sample can be resolved by polyacrylamide gel electrophoresis, and then the protein or nucleic acid can be visualized by staining. For certain purposes, high resolution of the protein or nucleic acid may be desirable, and an HPLC or similar means of purification may be used, for example.

[0062] The term "operably linked" refers to a functional link between a nucleic acid expression control sequence (such as a promoter, a signal sequence, or a cluster of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects the transcription and / or production of the nucleic acid corresponding to the second sequence.

[0063] The terms "identical" or percentage of "identity", in the context of two or more nucleic acid sequences

or proteins, refer to two or more sequences or sub-sequences that are equal or have a specified percentage of amino acid or nucleotide residues that are equal, when compared and aligned to obtain maximum correspondence, as measured using one of the following algorithms Sequence comparison or visual inspection.

[0064] The term "substantially identical", in the context of two nucleic acids or proteins, refers to two or more sequences or sub-sequences having more than about 60% identity of nucleic acid or amino acid sequences, 65% , 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of amino acid or nucleotide residues, when compared and aligned to obtain maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that has a length of at least about 50 residues, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over At least about 150 waste. In a more preferred embodiment, the sequences are substantially identical over the full length of the coding regions.

[0065] For sequence comparison, typically a sequence acts as a reference sequence, with which test sequences are compared. When a sequence comparison algorithm is used, test and reference sequences are entered into a computer, sub-sequence coordinates are designated, if necessary, and program parameters of the sequence algorithm are designated. Next, the sequence comparison algorithm calculates the percentage of sequence identity for the test sequence (s) with respect to the reference sequence, based on the designated program parameters.

[0066] Optimal sequence alignment can be performed for comparison, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat’l. Acad Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubell et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) ( Ausubel)).

[0067] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990)

J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software to run BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm first involves identifying pairs of high-scoring sequences (HSPs) by identifying short words of length W in the problem sequence, which either coincide with or meet some positive threshold score of value T when aligned with a word of the same length in a sequence of the database. T is referred to as the threshold word punctuation (neighborhood word) (Altschul et al, supra). These successes of initial next words act as seed values to initiate searches in order to find longer HSPs that contain them. Next, the successes of the words extend in both directions along each sequence to the extent that 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; provided that> 0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the successes of the words in each direction stops when: the cumulative alignment score falls by the amount X with respect to its maximum value achieved; the cumulative score reaches zero or below, due to the accumulation of one or more negative score residue alignments; or the end of any of the sequences is reached. The BLAST W, T, and X algorithm parameters determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as a default values a word length (W) of 11, a hope (E) of 10, M = 5, N = -4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as a default a word length (W) of 3, a hope (E) of 10, and the BLOSUM62 score matrix (see Henikoff & Henikoff, Proc, Natl. Acad Sci USA 89: 10915 (1989)).

[0068] In addition to calculating the percent identity of the sequences, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad Sci. USA 90: 5873-5787 (1993)). A measure of the similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a coincidence between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01 , and most preferably less than about 0.001.

[0069] Another indication that two nucleic acid or protein sequences are substantially identical is that the protein encoded by the first nucleic acid has immunological cross-reactivity with the protein encoded by the second nucleic acid, as described below. Thus, a protein is typically substantially identical to a second protein, for example, when the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under strict conditions, as described below.

[0070] The term "which specifically hybridizes with" refers to the binding, duplex, or hybridization of a molecule only with a particular nucleotide sequence under strict conditions when that sequence is present in a complex mixture (eg, total cell) DNA or RNA

[0071] The term "strict conditions" refers to conditions under which a probe will hybridize with its target sub-sequence, but not with other sequences. The strict conditions depend on the sequences and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. In general, strict conditions are selected so that they are approximately 15 ° C lower than the thermal melting point (Tm) for the specific sequence with a defined ionic strength and pH. The Tm is the temperature (under an ionic strength, pH, and concentration of defined nucleic acids) at which 50% of the probes complementary to the target sequence hybridize with the target sequence in equilibrium. (Since the target sequences are generally present in excess, in Tm, 50% of the probes are occupied in equilibrium). Typically, the strict conditions will be those in which the concentration of salts is less than about 1.0 M of Na ions, typically about 0.01 to 1.0 M of concentration of Na ions (or other salts) at a pH between 7.0 and 8.3, and the temperature is at least about 30 ° C for short probes (for example, between 10 and 50 nucleotides) and at least about 60 ° C for long probes (for example, greater than 50 nucleotides). Strict conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is typically at least twice the background hybridization, preferably 10 times the background hybridization. The following may be exemplary strict hybridization conditions: 50% formamide, 5x SSC, and 1% SDS, incubation at 42 ° C, or, 5x SSC, 1% SDS, incubation at 65 ° C, with 0.2 x SSC washing and 0.1% SDS at 65 ° C. For PCR, a temperature of approximately 36 ° C is typical for low-rigor amplification, although the mating temperatures can vary between approximately 32 and 48 ° C depending on the length of the primers. For a high-precision PCR amplification, a temperature of about 62 ° C is typical, although the high rigor mating temperatures may be between about 50 ° C and about 65 ° C, depending on the length of the primers and the specificity. Typical conditions of cycles for both high and low rigor amplifications include a denaturation phase of between 90 and 95 ° C for between 30 and 120 seconds, a mating phase that lasts between 30 and 120 seconds, and an extension phase of approximately 72 ° C for between 1 and 2 minutes. Protocols and guidelines for low and high rigor amplification reactions are available, for example, in Innis, et al. (1990) PCR Protocols: A Guide to Methods and Application Academic Press, N.Y.

[0072] The terms "specifically binds a protein" or "specifically immunoreactive with", when referring to an antibody refer to a binding reaction that is determinant of the presence of the protein in the presence of a heterogeneous population of proteins and other biological products. Thus, under designated immunoassay conditions, the specified antibodies preferably bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies immunoreactive specifically with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0073] "Conservatively modified variations" of a particular polynucleotide sequence refer to those polynucleotides encoding identical or essentially identical amino acid sequences, or when the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encodes any given protein. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at each position in which an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. These variations of nucleic acids are "silent variations", which constitute a kind of "conservatively modified variations". Each polynucleotide sequence described herein that encodes a protein also describes each possible silent variation, except where otherwise indicated. Experts will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and UGG that is ordinarily the only codon for tryptophan) can be modified to produce a functionally identical molecule by conventional techniques. Therefore, each "silent variation" of a nucleic acid encoding a protein is implicit in each sequence described.

[0074] In addition, experts will recognize that individual substitutions, deletions or additions that alter, add or suppress a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in a sequence encoded are "conservatively modified variations" when the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art.

[0075] Experts will appreciate that many conservative variations of fusion proteins and nucleic acids encoding fusion proteins generate essentially identical products. For example, due to the degeneracy of the genetic code, "silent substitutions" (ie, substitutions of a nucleic acid sequence that do not result in an alteration of an encoded protein) are an implicit characteristic of each nucleic acid sequence that encodes an amino acid. As described herein, the sequences are preferably optimized for expression in a particular host cell used to produce the chimeric glycosyltransferases (eg, yeast, human, and the like). Similarly, "conservative amino acid substitutions", in one or a few amino acids of an amino acid sequence are substituted with different amino acids with highly similar properties (see definition section, supra), are also easily identified so that they are highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence that encodes an amino acid. Said conservatively substituted variations, of any particular sequence, are a feature of the present invention. See also Creighton (1984) Proteins, W.H. Freeman and Company. Additionally, individual substitutions, deletions or additions that alter, add or suppress a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations."

[0076] The implementation of this invention may involve the construction of recombinant nucleic acids and the expression of genes in transfected host cells. Molecular cloning techniques to achieve these ends with those known in the art. Experts are well aware of a wide variety of in vitro cloning and amplification methods suitable for the construction of recombinant nucleic acids, such as expression vectors. Examples of these techniques are found in instructions sufficient for the guidance of experts through many cloning exercises, in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger ); and 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., (1999 Supplement) (Ausubel). Suitable host cells, for the expression of recombinant H. pylori fucosyltransferases are known to those skilled in the art, and include, for example, bacterial cells, including E. coli. In the present invention, eukaryotic cells can also be used, for example, insect cells such as the Sf 9 cell and yeast or fungal cells (for example, Aspergillus niger or yeast).

[0077] Examples of sufficient protocols for expert guidance are found through in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (PCR), amplification of Qβ- replicase and other RNA polymerase mediated techniques in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. Eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al. (1989) Proc. Natl. Acad Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. ( 1990) Gene

89: 117. In Wallace et al., Pat. U.S. No. 5,426,039 improved methods of cloning amplified nucleic acids in vitro are described.

Detailed description of the invention

[0078] The present invention provides for the first time bacterial α-1,3 / 4-fucosyltransferases, that is, H. pylori fucosyltransferases, which transfer fucose from a donor substrate to an acceptor sugar in a glycoprotein. Additionally, fucosyltransferases are useful for producing glycosipids and fucosylated oligosaccharides.

[0079] Specifically, α-1,3 / 4-fucosyltransferases from the following strains of H. pylori were cloned and analyzed: 915A2, 1111A2, 19C2B1, 1182B3, 19C2A5, 26695, and 1218. Fucosyltransferases of the following strains from

H. Pylori transferred fucose to Glc-NAc: 915A2, 1111A2, 19C2FutB, and 1182B3. The FutA gene product of the strain of

H. pylori 19C2A5 transferred fucose to the reducing glucose of the LNnT acceptor, as did the product of the FutB gene of the H. pylori strain 1218. The ability of the FutA gene product of the H. pylori strain 26695 was confirmed. of transferring fucose to glucose.

[0080] A major advantage of the H. pylori α-1,3 / 4 fucosyltransfersas with respect to mammalian α-1,3 / 4-fucosyltransfersas is that the H. pylori enzyme appears to be unaffected by the state of sialylation of the acceptor. Additionally, some of the H. pylori fucosyltransfers add fucose exclusively to the Nacethylglucosamine (glcNAc) residue in acceptor sugars that contain both glucose and glcNAc residues. In contrast, mammalian α-1,3 / 4-fucosyltransferases are sensitive to the degree of sialylation of the acceptor and some mammalian enzymes add residues of both glucose and glcNAc in the same acceptor. Additionally, bacterially expressed enzymes offer great cost savings with respect to the expression of mammalian gene products in systems with Sf9 or CHO.

A. Cloning of H. pylori α-1,3 / 4-fucosyltransferases proteins

[0081] Nucleic acids encoding glycosyltransferases, for example, α-1,3 / 4-fucosyltransferases from H. pylori, and methods of obtaining said nucleic acids, are known to those skilled in the art. Suitable nucleic acids (e.g., cDNA, genomic, or subsequences (probes)) can be cloned, or amplified by in vitro methods such as polymerase chain reaction (PCR), ligase chain reaction (CSF) , the transcription based amplification system (TAS), or the self-sustained sequence replication system (SSR). Experts are well aware of a wide variety of in vitro cloning and amplification methodologies. In Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); 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., (1994 Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864, are examples of these techniques and instructions sufficient for expert guidance through many cloning exercises.

[0082] A DNA encoding an H. pylori α-1,3 / 4-fucosyltransferase, or sub-sequences thereof, can be prepared by any suitable method described above, including, for example, cloning and restriction of appropriate sequences. with restriction enzymes. In a preferred embodiment, nucleic acids encoding H. pylori α-1,3 / 4-fucosyltransferases are isolated by routine cloning methods. A nucleotide sequence of an H. pylori α-1,3 / 4-fucosyltransferase can be used as provided, for example, in GenBank or another sequence database (see above), to provide probes that specifically hybridize with an H. pylori α-1,3 / 4-fucosyltransferase gene in a genomic DNA sample, or with an mRNA, encoding an H. pylori α-1,3 / 4-fucosyltransferase, in a sample of Total RNA (for example, in a Southern or Northern transfer). Once the target nucleic acid encoding an H. pylori α-1,3 / 4-fucosyltransferase has been identified, it can be isolated according to conventional methods known to those skilled in the art (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Volumes 1 to 3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc .; or Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York). In addition, isolated nucleic acids can be cleaved with restriction enzymes to create nucleic acids encoding full-length H. pylori α-1,3 / 4-fucosyltransferase, or sub-sequences thereof, for example, which contain sub-sequences that encode at least one subsequence of a catalytic domain of an α-1,3 / 4-fucosyltransferase of H. pylori. These restriction enzyme fragments, which encode an H. pylori α-1,3 / 4-fucosyltransferase or sub-sequences thereof, can then be ligated, for example, to produce a nucleic acid encoding an α-1 protein. , 3/4-fucosyltransferase from H. pylori.

[0083] A nucleic acid encoding an α-1,3 / 4-fucosyltransferase of H. pylori, or a sub-sequence thereof, can be characterized by performing tests in relation to the expressed product. Assays based on the detection of the physical, chemical, or immunological properties of the expressed protein can be used. For example, a cloned H. pylori α-1,3 / 4-fucosyltransferase can be identified, by the ability of a protein encoded by the nucleic acid to catalyze the transfer of a fucose residue from a donor substrate to an acceptor substrate . In one method, capillary electrophoresis is used to detect reaction products. This highly sensitive assay involves the use of derivatives of either saccharide or aminophenyl disaccharide that are labeled with fluorescein as described in Wakarchuk et al. (1996) J. Biol. Chem. 271 (45): 28271-276. For example, for tests in relation to a Neisseria IgtC enzyme, either FCHASE-AP-Lac or FCHASE-AP-Gal can be used, while for the Neisseria IgtB enzyme FCHASE-AP-GlcNAc is an appropriate reagent (Id.) Other methods for the detection of a fucosylated reaction product include thin layer chromatography and GC / MS.

[0084] In addition, a nucleic acid encoding an α-1,3 / 4-fucosyltransferase from H. pylori, or a sub-sequence thereof, can be chemically synthesized. Suitable methods include the phosphotriester method of Narang et al. (1979) Meth. Enzymol 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted to double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single chain as a template. Experts recognize that, although chemical DNA synthesis is frequently limited to sequences of approximately 100 bases, longer sequences can be obtained by ligation of shorter sequences.

[0085] Nucleic acids encoding H. pylori α-1,3 / 4-fucosyltransferases, or sub-sequences thereof, can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid sequence or sub-sequence is amplified by PCR, using a sense primer that contains a restriction enzyme site (eg, Ndel) and an antisense primer that contains another enzyme restriction site (e.g. , Hindlll). This will produce a nucleic acid encoding the H. pylori α-1,3 / 4-fucosyltransferases or desired sub-sequences and having terminal restriction enzyme sites. This nucleic acid can then be readily ligated into a vector that contains a nucleic acid that encodes the second molecule and that has the appropriate corresponding restriction enzyme sites. Suitable PCR primers can be determined by those skilled in the art using sequence information provided in GenBank or other sources. Appropriate restriction enzyme sites can also be added to the nucleic acid encoding the protein or protein sequence of α-1,3 / 4-fucosyltransferase of H. pylori by site-directed mutagenesis. The plasmid containing the nucleotide sequence or sub-sequence encoding the H. pylori α-1,3 / 4-fucosyltransferase is cleaved with the appropriate restriction endonuclease and then ligated into an appropriate vector, for amplification and / or expression according to conventional methods. . In Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., Eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al. (1989) Proc. Natl. Acad Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. ( 1990) Gene 89: 117, examples of sufficient techniques are found for expert guidance through in vitro amplification methods.

[0086] Other physical properties of a cloned H. pylori α-1,3 / 4-fucosyltransferase protein expressed from a particular nucleic acid can be compared with H-α-1,3 / 4-fucosyltransferase properties pylori known to provide another method of identifying suitable sequences or domains of the H. pylori α-1,3 / 4-fucosyltransferases that are determinants of the specificity of the acceptor substrate and / or catalytic activity. Alternatively, a putative H. pylori α-1,3 / 4-fucosyltransferase gene or recombinant H. pylori α-1,3 / 4-fucosyltransferase gene can be mutated, and its role can be established as α-1,3 / 4-fucosyltransferases, or the role of particular sequences or domains by detecting a variation in the structure of a carbohydrate normally produced by non-mutated α-1,3 / 4-fucosyltransferases, of natural origin, or control.

[0087] Functional domains of H. pylori α-1,3 / 4-fucosyltransferases can be identified using conventional methods to mutate or modify H. pylori g α-1,3 / 4-fucosyltransferases by testing the modified proteins or mutated in relation to activities such as acceptor substrate activity and / or catalytic activity, as described herein. The functional domains of the various α-1,3 / 4-fucosyltransferases of H. pylori can be used to construct nucleic acids encoding α-1,3 / 4-fucosyltransferases that comprise the functional domains of one or more α- 1,3 / 4-fucosyltransferase. Then, these fusion proteins can be tested in relation to the desired acceptor substrate or catalytic activity.

[0088] In an exemplary approach to the cloning of nucleic acids encoding α-1,3 / 4-fucosyltransferase proteins, known nucleic acid sequences or amino acids of cloned glycosyltransferases are aligned and compared to determine the amount of sequence identity between various glycosyltransferases. This information can be used to identify and select protein domains that confer or modulate glycosyltransferase activities, for example, acceptor substrate activity and / or catalytic activity based on the amount of sequence identity between the glycosyltransferases of interest. For example, domains that have sequence identity between the fucosyltransferases of interest, and that are associated with a known activity, can be used to construct fucosyltransferase proteins that contain that domain, and that have the activity associated with that domain (e.g., specificity of the acceptor substrate and / or catalytic activity).

B. Fusion protein comprising accessory enzymes involved in the formation of nucleotide sugars

[0089] In some embodiments, the fusion polypeptides of the invention include, in addition to the catalytic domain (s) of α-1,3 / 4-fucosyltransferases and / or other functional domains, at least a catalytic domain of an accessory enzyme. Accessory enzymes include, for example, those enzymes that are involved in the formation of a nucleotide sugar. The accessory enzyme may be involved in the fixation of sugar to a nucleotide, or it may be involved, for example, in the creation of sugar or the nucleotide. In general, nucleotide sugar is one that is used as a saccharide donor by the glycosyltransferase catalytic domain of the particular fusion polypeptide. Α-1,3 / 4-fucosyltransferases use GDP-fucose as a sugar donor. For example, in PCT / CA98 / 01180, USSN 09 / 211,691 presented on December 14, 1998, examples of fusion proteins are found that comprise a functional domain of a glycosyltransferase and an accessory enzyme and methods for performing said fusions.

[0090] Accessory enzymes that are involved in the synthesis of nucleotide sugars are well known to those skilled in the art. For a review of bacterial polysaccharide synthesis and gene nomenclature, see, for example, Reeves et al., Trends Microbiol. 4: 495-503 (1996). The methods described above for obtaining nucleic acids encoding glycosyltransferase are also applicable to obtaining nucleic acids encoding enzymes involved in the formation of nucleotide sugars. For example, one of the nucleic acids known in the art can be used, some of which are listed below, directly or as zonda to isolate a corresponding nucleic acid from other organisms of interest.

[0091] An example of a fusion polypeptide provided by the invention is used to produce a fucosylated soluble oligosaccharide. The donor nucleotide sugar for fucosyltransferases is GDP-fucose, which is relatively expensive to produce. To reduce the cost of production of the fucosylated oligosaccharide, the invention provides fusion polypeptides that can convert the relatively inexpensive GDP-mannose into GDP-fucose, and then catalyze the transfer of the fucose to an acceptor saccharide. These fusion polypeptides include a catalytic domain of at least one of a GDP-mannose dehydratase, a GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase, or a GDP-4-keto-6-deoxy -L-glucose 4-reductase. When each of these enzymatic activities is provided, GDP-mannose can be converted to GDP-fucose.

[0092] Stevenson et al. (1996) J. Bacteriol. 178: 4885-4893 (GenBank Accession No. U38473) describes the nucleotide sequence of a cluster of E. coli genes encoding GDP-fucose synthesizing enzymes. It had been published that this gene cluster included an open reading frame for GDP-mannose dehydratase (nucleotides 8633-9754; Stevenson et al., Supra). It was recently discovered that this gene cluster also contains an open reading frame that encodes an enzyme that has both 3.5 and 4-reductase epimerization activities (see US Joint Assignment Patent No. 6,500,661, published 31 December 2002), and therefore is capable of converting the reaction product of GDP-mannose dehydratase (GDP-4-keto-6-deoxyanose) into GDP-fucose. This ORF, which is designated YEF B, is among nucleotides 9757-10722. Before the discovery that YEF B encodes an enzyme that has two activities, it was not known whether one or two enzymes were necessary for the conversion of GDP-4-keto-6-deoximanose to GDP-fucose. The nucleotide sequence of a gene encoding the human Fx enzyme is found in GenBank Accession number U58766.

[0093] Fusion polypeptides including a catalytic mannosyltransferase domain and a catalytic domain of a GDP-Man pyrophosphorylase (EC 2.7.7.22), which converts Man-1-P into GDP-Man, are also provided. Suitable genes of many organisms are known, including E. coli: GenBank U13629, AB010294, D43637 D13231, Bastin et al., Gene 164: 17-23 (1995), Sugiyama et al., J. Bacteriol. 180: 2775-2778 (1998), Sugiyama et al., Microbiology 140 (Pt 1): 59-71 (1994), Kido et al., J. Bacteriol. 177: 2178-2187 (1995); Klebsiella pneumoniae: GenBank AB010296, AB010295, Sugiyama et al., J. Bacteriol. 180: 2775-2778 (1998); Salmonella enterica: GenBank X56793 M29713, Stevenson et al., J. Bacteriol. 178: 4885-4893 (1996).

[0094] The fusion polypeptides of the invention for the fucosylation of a saccharide acceptor can also use enzymes that provide a minor or "scavenge" pathway for the formation of GDP-fucose. In this pathway, free fucose is phosphorylated by fucokinase to form fucose 1-phosphate, which, together with guanosine 5'-triphosphate (GTP), is used by the GDP-fucose pyrophosphorylase to form GDP-fucose (Ginsburg et al. , JJ Biol. Chem., 236: 2389-2393 (1961) and Reitman, J. Biol. Chem., 255: 9900-9906 (1980)). Accordingly, a fucosyltransferase catalytic domain can be linked to a catalytic domain of a GDP-fucose pyrophosphorylase, for which suitable nucleic acids are described in U.S. patent application. in process with the present, joint assignment, serial number 08 / 826,964, filed on April 9, 1997. Nucleic acids encoding fucokinase are described for, for example, Haemophilus influenzae (Fleischmann et al. (1995) Science 269: 496-512) and E. coli (Lu and Lin (1989) Nucleic Acids Res. 17: 4883-4884).

[0095] Additional accessory enzymes from which a catalytic domain can be obtained are those that are involved in the formation of reagents consumed in a glycosyltransferase cycle. For example, any of the various phosphate kinases is useful as an accessory enzyme. Polyphosphate kinase (EC 2.7.4.1), for example, catalyzes the formation of ATP; nucleoside phosphate kinases (EC 2.7.4.4) can form the respective nucleoside diphosphates; creatine phosphate kinase (ES 2.7.3.2); myokinase (EC 2.7.4.3); N-acetylglucosamine acetyl kinase (EC 2.7.1.59); acetyl phosphate kinase; and pyruvate kinase (EC 2.7.1.40).

C. Expression cassettes and host cells to express recombinant H. pylori α-1,3 / 4-fucosyltransferase proteins

[0096] The fusion proteins of the invention can be expressed in a variety of host cells, including E. coli, other bacterial hosts, and yeast. The host cells are preferably microorganisms, such as, for example, yeast cells, bacterial cells, or filamentous fungal cells. Examples of suitable host cells include, for example, Azotobacter sp. (for example, A. vinelandii), Pseudomonas sp., Rhizobium sp., Erwinia sp., Escherichia sp. (for example, E. coli), Bacillus, Pseudomonas, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus and Klebsiella sp., among many others. The cells can be of any of several genera, including Saccharomyces (for example, S. cerevisiae), Candida (for example, C. utilis, C. parapsilosis, C. krusei, C. versatilis, C. lipolytica, C. zeylanoides , C. guilliermondii, C. albicans, and C. humicola), Pichia (for example, P. farinosa and P. ohmen), Torulopsis (for example, T. candida, T. sphaerica, T. xylinus, T. famata, and T. versatilis), Debaryomyces (for example, D. subglobosus, D. cantarellii,

D. balloonsus, D. hansenii, and D. japonicus), Zygosaccharomyces (for example, Z. rouxii and Z. bailii), Kluyveromyces (for example, K. marxianus), Hansenula (for example, H. anomala and H. jadinii ), and Brettanomyces (for example, B. lambicus and

B. anomalus). Examples of useful bacteria include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Klebsielia.

[0097] Typically, the polynucleotide encoding the α-1,3 / 4-fucosyltransferase protein is placed under the control of a promoter that is functional in the desired host cell. An extremely wide variety of promoters is widely known, and they can be used in the expression vectors of the invention, depending on the particular application. Commonly, the selected promoter depends on the cell in which the promoter will be active. Optionally, other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also included. Constructs that include one or more of these control sequences are called "expression cassettes." Accordingly, the invention provides expression cassettes in which nucleic acids encoding fusion proteins are incorporated, for high level expression in a desired host cell.

[0098] Expression control sequences that are suitable for use in a particular host cell are frequently obtained by cloning a gene that is expressed in that cell. Commonly used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, together with sequences from ribosome binding sites, include commonly used promoters such as systems. β-lactamase (penicillinase) and lactose (lac) promoters (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8 : 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25); and the lambda-derived PL promoter and the ribosome binding site of N genes (Shimatake et al., Nature (1981) 292: 128). The particular promoter system is not critical to the invention, any available promoter that functions in prokaryotes can be used.

[0099] For the expression of α-1,3 / 4-fucosyltransferase proteins in prokaryotic cells other than E. coli, a promoter that functions in the particular prokaryotic species is required. Such promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used. For example, the trp-lac hybrid promoter works in Bacillius in addition to E. coli.

[0100] A ribosome binding site (RBS) is suitably included in the expression cassettes of the invention. An RBS in E. coli, for example, consists of a nucleotide sequence between 3 and 9 nucleotides in length between 3 and 11 nucleotides upstream of the initiation codon (Shine and Dalgamo, Nature (1975) 254: 34; Steitz, In Biological regulation and developtment: Gene expression (ed. RF. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY).

[0101] For the expression of α-1,3 / 4-fucosyltransferase proteins in yeast, suitable promoters include GAL1-10 (Johnson and Davies (1984) Mol. Cell. Biol. 4: 1440-1448) ADH2 ( Russell et al. (1983) J. Biol. Chem. 258: 26742682), PHO5 (EMBO J. (1982) 6: 675-680), and MFα (Herskowitz and Oshima (1982) in The Molecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, NY pp. 181-209). Another suitable promoter for use in yeast is the ADH2 / GAPDH hybrid promoter as described in Cousens et al., Gene 61: 265-275 (1987). For filamentous fungi such as, for example, strains of Aspergillus fungi (McKnight et al., US Patent No. 4,935,349), examples of useful promoters include those derived from glycolytic genes from Aspergillus nidulans, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093 2099 (1985)) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al.).

[0102] In the present invention, either constitutive or regulated promoters can be used. Regulated promoters can be advantageous since host cells can be grown to high densities before inducing the expression of fusion proteins. The high level expression of heterologous proteins slows cell growth in some situations. An inducible promoter is a promoter that directs the expression of a gene where the level of expression is altered by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemical agents. These promoters are referred to herein as "inducible" promoters, which allow the timing of glycosyltransferase or enzyme expression involved in the synthesis of nucleotide sugars to be controlled. For E. coli and other bacterial host cells, those skilled in the art know inducible promoters. They include, for example, the lac promoter, the bacteriophage lambda PL promoter, the trp-lac hybrid promoter (Amann et al. (1983) Gene 25: 167, from Boer et al., (1983) Proc. Nat ' 1. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol .; Tabor et al. (1985) Proc. Nat'l Acad. Sci. USA 82: 1074-8). These promoters and their use are described in Sambrook et al., Supra. A particularly preferred inducible promoter, for prokaryotic expression is a dual promoter that includes a tac promoter component linked to a promoter component obtained from a gene or genes encoding enzymes involved in galactose metabolism (e.g., a promoter of a UDP galactose 4-epimerase (galE) gene. The dual tac-gal promoter is described in PCF Patent Application Publication No. WO 98/20111.

[0103] A construct that includes a polynucleotide of interest operably linked to gene expression control signals that, when placed in an appropriate host cell, drives the expression of the polynucleotide is called an "expression cassette." Expression cassettes encoding the fusion proteins of the invention are frequently placed in expression vectors for introduction into the host cell. Vectors typically include, in addition to an expression cassette, a nucleic acid sequence that allows the vector to replicate independently in one or more selected host cells. In general, this sequence is one that allows the vector to replicate independently of the host's chromosomal DNA, and includes origins of replication or autonomous replication sequences. Such sequences are well known for a variety of bacteria. For example, the origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria. Alternatively, the vector can be replicated upon integration into the genomic complement of the host cell and replicated as the cell undergoes DNA replication. A preferred expression vector, for the expression of enzymes, in bacterial cells is pTGK, which includes a dual tac-gal promoter and is described in PCT Patent Application Publication No. WO98 / 20111.

[0104] The construction of polynucleotide constructs generally requires the use of vectors capable of replicating in bacteria. A plethora of kits for the purification of bacterial plasmids are commercially available (see, for example, EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech; StrataCleanJ, from Statagene; and, QIAexpress Expression System, Qiagen). Then, isolated and purified plasmids can be further manipulated to produce other plasmids, and can be used to transfect cells. Cloning into Streptomyces or Bacillus is also possible.

[0105] Selectable markers are frequently incorporated into the expression vectors used to express the polynucleotides of the invention. These genes can encode a gene product, such as a protein, necessary for the survival or growth of transformed host cells cultured in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline. Alternatively, selectable markers can encode proteins that complement auxotrophic deficiencies or provide critical nutrients not available from complex media, for example, the gene encoding D-alanine racemase for Bacilli. Frequently, the vector will have a selectable marker that is functional in, for example, E. coli, or other cells in which the vector replicates before being introduced into the host cell. Those skilled in the art know several selectable markers and they are described, for example, in Sambrook et al., Supra.

[0106] The construction of suitable vectors containing one or more of the components listed above utilizes conventional ligation techniques as described in the references cited above. Isolated DNA fragments or plasmids are cleaved, customized, and re-linked in the desired manner to generate the required plasmids. To confirm the correct sequences in the constructed plasmids, the plasmids can be analyzed by conventional techniques such as by restriction endonuclease digestion, and / or sequencing according to known methods. Molecular cloning techniques to achieve these ends are known in the art. Experts widely know a wide variety of in vitro cloning and amplification methods suitable for the construction of recombinant nucleic acids. In Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, CA (Berger); and 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., (1998 Supplement) (Ausubel), are examples of these techniques and instructions sufficient for guidance of expert people, through many cloning exercises.

[0107] A variety of common vectors suitable for use with starting materials for the construction of expression vectors of the invention are widely known in the art. For cloning in bacteria, common vectors include vectors derived from pBR322 such as pBLUESCMPT ™, and vectors derived from phage . In yeast, the vectors include yeast integration plasmids (eg, YIp5) and Yeast Replication plasmids (YRp series plasmids) and pGPD-2. Mammalian cell expression can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI and p91023, as well as lytic virus vectors (eg, vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (by for example, bovine papillomavirus), and retroviral vectors (for example, murine retroviruses).

[0108] The methods for introducing expression vectors into a chosen host cell are not particularly critical, and such methods are known to those skilled in the art. For example, expression vectors can be introduced into prokaryotic cells, including E. coli, by transformation with calcium chloride, and into eukaryotic cells by treatment with calcium phosphate or electroporation. Other transformation methods are also suitable.

[0109] To enhance the expression, translational coupling can be used. The strategy uses an open, short, upstream reading frame, derived from a highly expressed native gene with respect to the translational system, which is located downstream of the promoter, and a ribosome binding site followed, after a few codons of amino acids, for a termination codon. Just before the termination codon is a second ribosome binding site, and after the termination codon a start codon is found for translation initiation. The system dissolves the secondary structure in the RNA, allowing effective translation initiation. See Squires, et. to the. (1988), J. Biol. Chem. 263: 16297-16302.

[0110] The α-1,3 / 4-fucosyltransferase proteins can be expressed intracellularly, or they can be secreted from the cell. Intracellular expression frequently results in high yields. If necessary, the amount of active, soluble fusion protein can be increased to perform refolding procedures (see, for example, Sambrook et al., Supra .; Marston et al., Bio / Technology (1984) 2: 800 ; Schoner et al., Bio / Technology (1985) 3: 151). In embodiments in which the α-1,3 / 4-fucosyltransferase proteins are secreted from the cell, either to the periplasm or to the extracellular medium, the DNA sequence is linked to a cleavable signal peptide sequence. The signal sequence directs the translocation of the fusion protein through the cell membrane. An example of a vector suitable for use in E. coli containing a signal promoter-sequence unit is pTA1529, which has the E. coli phoA promoter and a signal sequence (see, for example, Sambrook et al., Supra .; Oka et al., Proc. Natl. Acad Sci USA (1985) 82: 7212; Talmadge et al. Proc. Natl. Acad. Sci. USA (1980) 77: 3988; Takahara et al., J. Biol. Chem. (1985) 260: 2670). In another embodiment, the fusion proteins are fused to a bovine serum albumin protein (BSA) sub-sequence, for example, to facilitate purification, secretion, or stability.

[0111] The α-1,3 / 4-fucosyltransferase proteins of the invention can also be additionally linked to other bacterial proteins. This approach often results in high yields, because normal prokaryotic control sequences direct transcription and translation. In E. coli, lacZ fusions are frequently used to express heterologous proteins. Suitable vectors are readily available, such as the pUR, pEX, and pMR100 series (see, for example, Sambrook et al., Supra.). For certain applications, it may be desirable to cleave the amino acids from accessory and / or non-glycosyltransferase enzymes with respect to the fusion protein after purification. This can be achieved by any of several methods known in the art, including cleavage with cyanogen bromide, a protease, or with Factor Xa (see, for example, Sambrook et al., Supra .; Itakura et al., Science (1977) 198: 1056; Goeddel et al., Proc. Natl. Acad. Sci USA (1979) 76: 106; Nagai et al., Nature (1984) 309: 810; Sung et al., Proc. Natl. Acad. Sci USA (1986) 83: 561). Excision sites in the gene for the fusion protein at the desired cleavage point can be obtained by genetic engineering.

[0112] In a single host cell, more than one recombinant protein can be expressed by placing multiple transcription cassettes in a single expression vector, or using different selectable markers for each of the expression vectors that are used in the cloning strategy .

[0113] A suitable system for obtaining recombinant E. coli proteins that maintains the integrity of its N-terminal ends has been described by Miller et al. Biotechnology 7: 698-704 (1989). In this system, the gene of interest is produced as a C-terminal fusion with the first 76 residues of the yeast ubiquitin gene that contains a peptidase cleavage site. The cleavage at the junction of the two fractions results in the production of a protein that has an intact authentic N-terminal residue.

D. Purification of α-1,3 / 4-fucosyltransferase proteins

[0114] The H. pylori fucosyltransferase proteins of the present invention can be expressed as intracellular proteins or as proteins that are secreted from the cell, and can be used in this way, in the methods of the present invention. For example, in the methods of the present invention a crude cell extract containing the secreted or intracellular H. pylori fucosyltransferase protein expressed can be used.

[0115] Alternatively, H. pylori fucosyltransferase proteins can be purified according to conventional procedures of the art, including precipitation with ammonium sulfate, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R Scopes, Protein Purification, Springer-Verlag, NY (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. NY (1990)). Substantially pure compositions with a homogeneity of at least about 70 to 90% are preferred, and most preferred are with a homogeneity of between 98 and 99% or greater. Purified proteins can also be used, for example, as immunogens for antibody production.

[0116] To facilitate the purification of the H. pylori α-1,3 / 4-fucosyltransferase proteins of the invention, the nucleic acids encoding the fusion proteins may also include a coding sequence for an epitope or "tag" for which an affinity binding reagent is available, that is, a purification tag. Examples of suitable epitopes include the myc and V-5 reporter genes; useful expression vectors commercially available for recombinant production of fusion proteins having these epitopes (for example, the pcDNA3.1 / Myc-His and pcDNA3.1 / V5-His vectors of Invitrogen (Carlsbad CA) are suitable for the expression in mammalian cells). Those skilled in the art know additional expression vectors suitable for labeling the H. pylori α-1,3 / 4-fucosyltransferase proteins of the invention, and corresponding detection systems, and several of them are commercially available ( for example, FLAG "(Kodak, Rochester NY). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to affinity ligands of metal chelates. Typically, six adjacent histidines are used (SEQ ID NO. : 76), although a number greater than or less than six may be used Suitable suitable affinity ligands of metal chelates that can serve as a binding fraction for a polyhistidine tag include nitrile-tri-acetic acid (NTA) (Hochuli, E. (1990) “Purification of recombinant proteins with metal chelating adsorbents” in Genetic Engineering: Principles and Methods, JK-Setlow, Ed., Plenum Press, NY; commercially available from Qiagen (Santa Clari ta, CA)).

[0117] Purification tags also include maltose binding domains and starch binding domains. Those skilled in the art know the purification of proteins with maltose binding domains. Starch binding domains are described in WO 99/15636. Affinity purification of a fusion protein comprising a starch binding domain using a beta-cyclodextrin-derived resin (BCD) is described in USSN 60 / 468,374, filed May 5, 2003.

[0118] Those skilled in the art know other haptens that are suitable for use as labels, and they are described, for example, in the Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene OR). For example, dinitrophenol (DNP), digoxigenin, barbiturates (see, for example, US Patent No. 5,414,085), and various types of fluorophores are useful as haptens, as are derivatives of these compounds. . There are commercially available kits to bind haptens and other fractions to proteins and other molecules. For example, when the hapten includes a thiol, a heterobifunctional linker such as SMCC can be used to fix the tag to lysine residues present in the capture reagent.

[0119] Experts will recognize that modifications can be made to the catalytic or functional domains of α1,3 / 4-fucosyltransferase without decreasing their biological activity. Some modifications can be made to facilitate cloning, expression, or incorporation of the catalytic domain into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, the addition of codons at any of the terminal ends of the polynucleotide encoding the catalytic domain to provide, for example, a methionine added at the amino terminal end with the in order to provide an initiation site, or additional amino acids (eg, poly His) located at any of the terminal ends to create properly located restriction enzyme sites or termination codons or purification sequences.

E. Uses of H. pylori fucosyltransferase proteins

[0120] The invention provides H. pylori α-1,3 / 4-fucosyltransferase proteins and methods of using H. pylori α-1,3 / 4-fucosyltransferase proteins to enzymatically synthesize glycoproteins, glycolipids, and oligosaccharide fractions. The glycosyltransferase reactions of the invention take place in a reaction medium comprising at least one H. pylori α-1,3 / 4-fucosyltransferase, an acceptor substrate, and a donor substrate, and typically a soluble divalent metal cation . In some embodiments, accessory enzymes and substrates for the catalytic fraction of the accessory enzyme are also present, so that the accessory enzymes can synthesize the donor substrate for H. pylori α-1,3 / 4-fucosyltransferase.

[0121] Several methods of using glycosyltransferases to synthesize glycoproteins and glycolipids having desired oligosaccharide fractions are known. Exemplary methods are described, for example, in WO 96/32491, Ito et al. (1993) Pure Appl. Chem. 65: 753, and US patents 5,352,670, 5,374,541, and 5,545,553.

[0122] H. pylori fucosyltransferase proteins prepared as described herein can be used in combination with additional glycosyltransferases. For example, a combination of recombinant sialyltransferase fusion protein and a recombinant H. pylori α-1,3 / 4-fucosyltransferase fusion can be used. By performing two glycosyltransferase reactions sequentially in a single vessel, the overall yields are improved with respect to procedures in which an intermediate species is isolated. On the other hand, the cleaning and elimination of solvents and additional by-products is reduced. Similarly, recombinant glycosyltransferases can be used with a recombinant accessory enzyme, which may or may not be present as a fusion protein. In other embodiments, H. pylori α-1,3 / 4-fucosyltransferase and additional glycosyltransferases are produced in the same cell

or accessory enzymes, and they are used to synthesize a desired final product.

[0123] Products generated by the above processes can be used without purification. However, well known, conventional techniques, for example, thin or thick layer chromatography, ion exchange chromatography, or membrane filtration, can be used for the recovery of glycosylated saccharides. In addition, for example, membrane filtration can be used, using a nanofiltration or reverse osmosis membrane as described in common assignment patent No. 735695. As a further example, filtration can be used to extract proteins. with membrane in which the membranes have a molecular weight cut-off value of between about 1,000 and about 10,000 Daltons. As an alternative example, nanofiltration or reverse osmosis can then be used to extract salts. Nanofilter membranes are a class of reverse osmosis membranes that pass monovalent salts but retain non-charged polyvalent and solute salts greater than between about 200 and about 1,000 Daltons, depending on the membrane used. Thus, for example, the oligosaccharides produced by the compositions and methods of the present invention can be retained in the membrane and the contaminating salts will pass through it.

F. Donor Substrates and Accepting Substrates

[0124] Suitable donor substrates used by the H. pylori fucosyltransferase proteins and other glycosyltransferases in the methods of the invention include, among others, UDP-Glc, UDP-GlcNAc, UDP-Gal, UDP-GalNAc, GDP-Man , GDP-Fuc, UDP-GlcUA, and CMP-sialic acid. Guo et al., Applied Biochem. and Biotech. 68: 1-20 (1997)

[0125] Suitable acceptor substrates used by H. pylori fucosyltransferase proteins and methods of the invention include, among others, polysaccharides, oligosaccharides, lipids and glycolipids. For example, the LNnT oligosaccharide can be fucosylated to form LNFIII. The fucosyltransferases described herein can also be used in multi-enzyme systems to produce a desired product from a suitable starting material. For example, LNFIII was prepared on a multigram scale from lactose using H. pylori α-1,3 / 4-fucosyltransferases from strain 1182 described herein, in combination with β-1,3-N-acetylglucosaminyltransferase (IgtA ) of Neisseria gonococcus and β-1,4-galactosyltransferase (IgtB) of Neisseria gonococcus.

[0126] Suitable acceptor substrates used by H. pylori fucosyltransferase proteins and the methods of the invention include, among others, proteins, lipids, gangliosides and other biological structures (eg, whole cells) that can be modified by the methods of the invention Exemplary structures, which can be modified by the methods of the invention, include any of a series of glycolipids, glycoproteins and carbohydrate structures in cells known to those skilled in the art as set forth in Table 1.

Table 1

Growth Hormones and Growth Factors Receptors and Chimeric Receptors

•

G-CSF • CD4

•

GM-CSF • Tumor Necrosis Factor (TNF) receptor

•

 TPO • Alfa-CD20

•

 EPO • MAb-CD20

•

EPO variants • MAb-alpha-CD3

•

α-TNF • TNF MAb-receptor

•

Leptin • MAb-CD4 • PSGL-1 Enzymes and Inhibitors • MAb-PSGL-1

•

 t-PA • Complement

•

variants of t-PA • GlyCAM or its chimera

•

Urokinase • N-CAM or its chimera

•

 Factors VII, VIII, IX, X • LFA-3

•

 DNase • CTLA-IV

•

Glucocerebrosidase

•

Hirudin Monoclonal Antibodies (Immunoglobulins)

•

 Α1 antitrypsin • MAb-anti-RSV

•

Antithrombin III • MAb-anti-receptor IL-2 • MAb-anti-CEA Cytokines and Cytokines • MAb-anti-receptor IIb / IIIa Chimeric platelet • MAb-anti-EGF

•

Interleukin-1 (IL-1), 1B, • MAb-anti-receptor Her-2 2, 3, 4

•

Interferon-α (IFN-α) Cells

•

 IFN-α-2b • Red blood cells

•

 IFN-β • White blood cells (for example, T cells,

•

 IFN-γ B cells, dendritic cells, macrophages

•

Diphtheria toxin NK cells, neutrophils, monocytes and the like) chimeric-IL-2 • Stem cells

[0127] Examples of suitable acceptor substrates, used in reactions catalyzed with fucosyltransferase, and examples of suitable acceptor substrates used in reactions catalyzed with sialyltransferase 5 are described in Guo et al., Applied Biochem. and Biotech. 68: 1-20 (1997), although not limited to the latter.

[0128] The present invention provides fucosyltransferase proteins (eg, fucosyltransferases) of H. pylori that are selected for their ability to produce glycoproteins and glycolipids having desired oligosaccharide moieties. Similarly, accessory enzymes are chosen, if they were present, based on a

10 desired activated sugar substrate or in a sugar found in the product oligosaccharide.

[0129] Suitable H. pylori fucosyltransferase proteins can be readily identified by reacting various amounts of an H. pylori α-1,3 / 4-fucosyltransferase protein of interest (for example, between 0.01 and 100 mU / mg of protein) with a glycoprotein (for example, between 1 and 10 mg / ml) to which an oligosaccharide is bound

15 having a potential acceptor site for glycosylation by the fusion protein of interest. The capabilities of the recombinant glycosyltransferase fusion proteins of the present invention to add a sugar residue at the desired acceptor site are compared, and an H. pylori fucosyltransferase protein having the desired property is selected (e.g., specificity of the acceptor substrate or catalytic activity).

[0130] In general, the efficacy of enzymatic synthesis of glycoproteins and glycolipids, having desired oligosaccharide fractions, can be enhanced through the use of H. pylori α-1,3 / 4-fucosyltransferase proteins produced from recombinantly, of the present invention. Recombinant techniques enable the production of recombinant H. pylori α-1,3 / 4-fucosyltransferase proteins in the high amounts needed for large-scale modification of glycoproteins and glycolipids.

[0131] The glycoproteins and glycolipids suitable for use by the H. pylori fucosyltransferase proteins and the methods of the invention may be glycoproteins and glycolipids immobilized on a solid support during the glycosylation reaction. The term "solid support" also includes semi-solid supports. Preferably, the target glycoprotein or glycolipid is reversibly immobilized so that the respective glycoprotein or glycolipid can be released after the glycosylation reaction has been completed. Those skilled in the art know many suitable matrices. For example, ion exchange can be used to temporarily immobilize a glycoprotein or glycolipid in an appropriate resin while the glycosylation reaction proceeds. A ligand that specifically binds to the glycoprotein or glycolipid of interest can also be used for affinity-based immobilization. For example, antibodies that specifically bind to a glycoprotein are suitable. In addition, when the glycoprotein of interest itself is an antibody or contains a fragment thereof, protein A or G can be used as an affinity resin. Dyes and other molecules that specifically bind to a glycoprotein or glycolipid of interest are also suitable.

[0132] The recombinant fusion protein of the invention can be constructed and expressed as a fusion protein with a molecular "tag" at one end, which facilitates the purification of the protein, that is, a purification tag. Such tags can also be used for immobilization of a protein of interest during the glycosylation reaction. Suitable tags include "epitopic tags," which are a protein sequence that is specifically recognized by an antibody. Epitopic tags are generally incorporated into fusion proteins to allow the use of an readily available antibody in order to unequivocally detect or isolate the fusion protein. A "FLAG tag" is a commonly used epitopic tag, specifically recognized by a monoclonal anti-FLAG antibody, consisting of the sequence AspTyrLysAspAspAspAspLys (SEQ ID NO: 75) or a substantially identical variant thereof. An mcy tag is another commonly used epitopic tag. Those skilled in the art know other suitable labels, and they include, for example, an affinity tag such as a hexahistidine peptide (SEQ ID NO: 76), which will bind to metal ions such as nickel ions or cobalt. Purification tags also include maltose binding domains and starch binding domains. The purification of proteins with maltose binding domains is known to those skilled in the art. Starch binding domains are described in WO 99/15636. Affinity purification of a fusion protein comprising a starch binding domain using a beta-cyclodextrin-derived resin (BCD) is described in USSN 60 / 468,374, filed May 5, 2003.

[0133] Preferably, when the glycoprotein is a truncated version of the full length glycoprotein, it preferably includes the biologically active sub sequence of the full length glycoprotein. Exemplary biologically active sub-sequences include, among others, enzyme active sites, receptor binding sites, ligand binding sites, antibody complementarity determining regions, and antigenic antigen regions.

[0134] In some embodiments, the H. pylori fucosyltransferase proteins and methods of the present invention are used to enzymatically synthesize a glycoprotein or glycolipid having a substantially uniform glycosylation pattern. Glycoproteins and glycolipids include a saccharide or oligosaccharide that is bound to a protein, glycoprotein, lipid, or glycolipid for which an alteration of the glycoform is desired. The saccharide or oligosaccharide includes a structure that can function as an acceptor substrate for a glycosyltransferase. When the acceptor substrate is glycosylated, the desired oligosaccharide fraction is formed. The desired oligosaccharide fraction is that which communicates the desired biological activity to the glycoprotein or glycolipid to which it is attached. In the compositions of the invention, the preselected saccharide residue is linked to at least about 30% of the potential acceptor sites of interest. More preferably, the preselected saccharide residue is linked with at least about 50% of the potential acceptor substrates of interest, and even more preferably with at least 70% of the potential acceptor substrates of interest. In situations where the starting glycoprotein or glycolipid exhibits heterogeneity in the oligosaccharide fraction of interest (for example, some of the oligosaccharides in the starting glycoprotein or glycolipid already have the preselected saccharide residue attached to the acceptor substrate of interest), the percentages mentioned include said previously established saccharide residues.

[0135] The term "altered" refers to the glycoprotein or glycolipid of interest having a glycosylation pattern that, after application of H. pylori fucosyltransferase proteins and the methods of the invention, is different with respect to the observed in the glycoprotein as it was originally produced. An example of such glycoconjugates are glycoproteins in which glycoproteins of glycoproteins are different from those found in glycoprotein when it is produced by cells of the organism from which the glycoprotein is native. Also provided are H. pylori fucosyltransferase proteins and methods of using said fusion proteins to enzymatically synthesize glycoproteins and glycolipids in which the glycosylation pattern of these glycoconjugates is modified compared to the glycosylation pattern of glycoconjugates as produced. Originally by means of a host cell, which can be of the same species or a different species from the cells from which the native glycoconjugates are produced.

[0136] Differences in glycosylation patterns can be assessed not only by structural analysis of glycoproteins and glycolipids, but also by comparison of one or more biological activities of glycoconjugates. For example, a glycoprotein having an "altered glycoform" includes one that exhibits an improvement in one or more biological activities of the glycoprotein after the glycosylation reaction compared to the unmodified glycoprotein. For example, an altered glycoconjugate includes one that, after the application of H. pylori fucosyltransferase proteins and the methods of the invention, exhibits a higher binding affinity for a ligand or receptor of interest, a greater therapeutic half-life, a reduced antigenicity, and a focus on specific tissues. The amount of improvement observed is preferably statistically significant, and more preferably it is at least about 25% improvement, and even more preferably it is at least about 50%, 60%, 70%, and Even more preferably it is at least 80%.

G. Fucosyltransferase reactions

[0137] H. pylori fucosyltransferase proteins, acceptor substrates, donor substrates and other ingredients of the reaction mixture, including other glycosyltransferases and accessory enzymes, are combined by mixing in an aqueous reaction medium. The medium in general has a pH value between about 5 and about 8.5. The selection of a medium is based on the ability of the medium to maintain the pH value at the desired level. Thus, in some embodiments, the medium is buffered at a pH value of approximately

7.5. If a buffer is not used, the pH of the medium should be maintained at about 5 to 8.5, depending on the particular glycosyltransferase used. For fucosyltransferases, the pH range is preferably maintained between about 6.0 and 8.0. For sialyltransferases, the range is preferably between about 5.5 and about 7.5.

[0138] Enzyme amounts or concentrations are expressed in units of activity, which are a measure of the initial rate of catalysis. A unit of activity catalyzes the formation of 1 μmol of product per minute at a temperature (typically 37 ° C) and a determined pH value (typically 7.5). Thus, 10 units of an enzyme is a catalytic amount of that enzyme in which 10 μmol of substrate is converted into 10 μmol of product in one minute at a temperature of 37 ° C and a pH value of 7.5.

[0139] The reaction mixture may include divalent metal cations (Mg2 +, Mn2 +). The reaction medium may also comprise solubilizing detergents (eg, Triton or SDS) and organic solvents such as methanol

or ethanol, if necessary. Enzymes can be used free in solution or they can be attached to a support such as a polymer. In this way, the reaction mixture is substantially homogeneous in the beginning, although, during the reaction, it may form some precipitate.

[0140] The temperature at which an earlier process is carried out may be between just above freezing to the temperature at which the most sensitive enzyme is denatured. The temperature range is preferably between about 0 ° C and about 45 ° C, and more preferably between about 20 ° C and about 37 ° C.

[0141] The reaction mixture thus formed is maintained for a period of time sufficient to obtain the desired high yield of the desired oligosaccharide products, including determinants present in oligosaccharide groups attached to the glycoprotein to glycosylate. For large scale preparations, the reaction will often be allowed to proceed for between about 0.5 and 240 hours, and more typically between about 1 and 18 hours.

[0142] In embodiments in which more than one glycosyltransferase is used to obtain the oligosaccharide products, enzymes and reagents for a second glycosyltransferase reaction can be added to the reaction medium once the first glycosyltransferase reaction is almost complete . For some enzyme combinations, glycosyltransferases and corresponding substrates can be combined in a single initial reaction mixture; preferably, the enzymes in said simultaneous reactions do not form a product that cannot serve as an acceptor for the other enzyme. By sequentially carrying out two glycosyltransferase reactions in a single vessel, the overall yields are improved with respect to procedures in which an intermediate species is isolated. On the other hand, the cleaning and elimination of solvents and additional by-products is reduced. Additionally, in some embodiments, fucosyltransferase and additionally glycosyltransferases or accessory enzymes are expressed in the same host cell and the desired product is synthesized within the host cell.

[0143] One or more of the glycosyltransferase reactions can be carried out as part of a glycosyltransferase cycle. Preferred conditions and descriptions of glycosyltransferase cycles have been described. In the patent

U.S. No. 5,374,541 and WO 9425615 A describe a series of glycosyltransferase cycles (eg, sialyltransferase cycles, galactosyltransferase cycles, and fucosyltransferase cycles). Other glycosyltransferase cycles are described in Ichikawa et al. J. Am. Chem. Soc. 114: 9283 (1992), Wong et al. J. Org. Chem. 57: 4343 (1992), DeLuca et al., J. Am. Chem. Soc. 117: 5869-5870 (1995), and Ichikawa et al. In Carbohydrates and Carbohydrate Polymers. Yaltami, ed. (ATL Press, 1993).

[0144] Other glycosyltransferases can be substituted in similar transferase cycles as described in detail for fucosyltransferases and sialyltransferases. In particular, glycosyltransferase can also be, for example, glycosyltransferases, for example, Alg8 (Stagljov et al., Proc. Natl. Acad. Sci USA 91: 5977 (1994)) or Alg5 (Heesen et al. Eur. J. Biochem. 224: 71 (1994)), N-acetylgalactosaminyltransferases such as, for example, α (1,3) Nacethylgalactosaminyltransferase, β (1,4) N-acetylgalactosaminyltransferases (Nagata et al. J. Biol. Chem. 267: 12082 -12089 (1992) and Smith et al. J. Biol. Chem. 269: 15162 (1994)) and N-acetylgalactosaminyltransferase polypeptide (Homa et al. J. Biol. Chem. 268: 12609 (1993)). Suitable N-acetylglucosaminyltransferases include GnTI (2.4.1.141, Hull et al., BBRC 176: 608 (1991)), GnTII, and GnTIII (Ihara et al. J. Biochem. 113: 692 (1993)), GnTV (Shoreiban et al. J. Biol. Chem.

268: 15381 (1993)), N-acetylglucosaminyltransferase with O bridges (Bierhuizen et al. Proc. Natl. Acad. SCI USA 89: 9326 (1992)), N-acetylglucosamine-1-phosphate transferase (Rajput et al. Biochem J. 285: 985 (1992), and hyaluronan synthase The suitable mannosyltransferases include α (1,2) mannosyltransferase, α (1,3) mannosyltransferase, β (1,4) mannosyltransferase, Dol-P-Man synthase, OCh1, and Pmt1.

[0145] For the previous glycosyltransferase cycles, the concentrations or amounts of the various reagents used in the processes depend on numerous factors that include reaction conditions such as temperature and pH value, and the choice and amount of acceptor saccharides to glycosylate. Because the glycosylation process allows regeneration of activation nucleotides, activated donor sugars and scavenging of PPi produced, in the presence of catalytic amounts of enzymes, the process is limited by the concentrations or amounts of the stoichiometric substrates described. previously. The upper limit for reagent concentrations that can be used according to the method of the present invention is determined by the solubility of said reagents.

[0146] Preferably, the concentrations of activation nucleotides, phosphate donor, donor sugar and enzymes are selected such that glycosylation proceeds until the acceptor is consumed.

[0147] Each of the enzymes is present in a catalytic amount. The catalytic amount of a particular enzyme varies according to the concentration of the substrate of that enzyme as well as the reaction conditions such as temperature, time and pH value. Those skilled in the art are well aware of means to determine the catalytic amount for a given enzyme under pre-selected substrate concentrations and reaction conditions.

[0148] The fucosyltransferase reaction can be carried out using an oligosaccharide or polysaccharide as an acceptor molecule. Suitable acceptor substrates used by H. pylori fucosyltransferase proteins and the methods of the invention include, among others, polysaccharides, oligosaccharides, lipids, and glycolipids. For example, the LNnT oligosaccharide can be fucosylated to form LNFIII. The fucosyltransferases described herein can also be used in multiple enzyme systems to produce a desired product from a suitable starting material. For example, LNFIII was prepared on a multigram scale from lactose using the H. pylori α-1,3 / 4-fucosyltransferases of strain 1182 described herein, in combination with Neisseria β-1,3-acetylglucosaminyltransferase (1gtA) gonococcus and β-1,4-galactosyltransferase (1gtB) from Neisseria gonococcus.

[0149] The recombinant fucosyltransferase fusion protein used in the methods of the invention is chosen based on its ability to fucosylate the fucosyltransferase acceptor substrates of interest. Preferably, the fucosyltransferase is tested for suitability using a fucosyltransferase acceptor substrate that is fixed to a soluble saccharide or oligosaccharide. The use of a soluble saccharide or oligosaccharide acceptor substrate in the assay to determine fucosyltransferase activity allows to select a fucosyltransferase that produces the desired oligosaccharide product.

[0150] The fucosyltransferase reaction can be carried out using a lipid or glycolipid as an acceptor molecule. Many saccharides require the presence of particular fucosylated structures to present biological activity. Frequently, intercellular recognition mechanisms require a fucosylated oligosaccharide. For example, several proteins that function as cell adhesion molecules, including P-selectin, E-selectin, bind to cell-surface fucosylated carbohydrate structures, for example, the sialyl Lewis x and sialyl Lewis a structures. Additionally, the specific carbohydrate structures that form the ABO blood group system are fucosylated. The carbohydrate structures in each of the three groups share a Fucα1,2Galβ1-disaccharide unit. In structures of blood group O, this disaccharide is the terminal structure, the structure of group A is formed by an α1,3 GalNAc transferase that adds a terminal GalNAc residue to the disaccharide. The structure of group B is formed by an α1,3 galactosyltransferase that adds a terminal galactose residue. Lewis's blood group structures are also fucosylated. For example, the structures of Lewis x and Lewis a are respectively Galβ1.4 (Fucα1.3) GlcNac and Galβ1.4 (Fucα1.4) GlcNac. Both mentioned structures can also be sialylated (NeuAcα2,3-) to form the corresponding sialylated structures. Other Lewis blood group structures of interest are the Lewis and and b structures that are respectively Fucαl, 2Gaβ1.4 (Fucα1,3) GlcNAcβ-OR and Fucαl, 2Galβ1,3 (Fucα1,4) GlcNAc-OR. For a description of the structures of the ABO and Lewis blood groups and the enzymes involved in their synthesis, see, Essentials of Glycobiology, Varki et al. eds., Chapter 16 (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999).

[0151] The recombinant fucosyltransferase fusion protein used in the methods of the invention is chosen based on its ability to fucosylate the fucosyltransferase acceptor substrates of interest. Preferably, the fucosyltransferase is tested for suitability using a fucosyltransferase acceptor substrate that is fixed to a lipid or glycolipid. The use of an acceptor substrate linked to glycolipid, instead of an acceptor substrate that is part of a soluble oligosaccharide, in the assay to determine fucosyltransferase activity allows to select a fucosyltransferase that produces the fucosylation pattern selected in the glycolipid.

[0152] Fucosyltransferases have been used in synthetic routes to transfer a unit of fucose from guanosine5'-diphosphofucose to a specific hydroxyl of a saccharide acceptor. For example, Ichikawa prepared sialyl Lewis-X by a method that involves the fucosylation of sialylated lactosamine with a cloned fucosyltransferase (Ichikawa et al., J. Am. Chem. Soc. 114: 9283-9298 (1992)). Lowe has described a method for expressing non-native fucosylation activity in cells, thereby producing fucosylated glycoproteins, cell surfaces, etc. (U.S. Patent No. 5,955,347).

[0153] In one embodiment, the methods of the invention are practiced by contacting a substrate, which has an acceptor fraction for a fucosyltransferase, with a reaction mixture that includes a fucose donor fraction, a fucosyltransferase, and other reagents required for fucosyltransferase activity. The substrate is incubated in the reaction mixture for a sufficient time and under appropriate conditions, to transfer fucose from the fucose donor fraction to the fucosyltransferase acceptor fraction. In preferred embodiments, fucosyltransferase catalyzes the fucosylation of at least 60% of the respective acceptor fractions of fucosyltransferase in the composition.

[0154] The specificity for a selected substrate is only the first criterion that a preferred fucosyltransferase should meet. The fucosyltransferase used in the method of the invention may also preferentially subject a variety of substrates to fucosylation, and support the scaling up of the reaction to allow fucosylation of at least about 500 mg of substrate. More preferably, the fucosyltransferase will support the fucosylation reaction scale to allow the synthesis of at least about 1 kg, and more preferably, at least 10 kg of substrate with relatively low cost and infrastructure requirements.

[0155] Acceptable fractions suitable for binding, catalyzed with fucosyltransferase, of a fucose residue include, among others, GlcNAc-OR, Galβ1,3GlcNAc-OR, NeuAcα2,3Galβ1,3GlcNAc-OR, Galβ1,4GlcNAc-OR and NeuAcα2 , 3Galβ1,4GlcNAc-OR, where R is an amino acid, a saccharide, an oligosaccharide or an aglycone group having at least one carbon atom. R is linked to or is part of a substrate. The appropriate fucosyltransferase, for a particular reaction, is chosen based on the type of fucose bond that is desired (for example, α2, α3, or α4), the particular acceptor of interest, and the ability of the fucosyltransferase to achieve high yield desired fucosylation. Previously, suitable fucosyltransferases and their properties have been described.

[0156] If a sufficient proportion of the oligosaccharides bound to the substrate, in a composition, does not include a fucosyltransferase acceptor fraction, a suitable acceptor can be synthesized. For example, a preferred method of synthesizing an acceptor for a fucosyltransferase involves the use of a GlcNAc transferase to fix a GlcNAc residue to an acceptor fraction of GlcNAc transferase, which is present in oligosaccharides bound to the substrate. In preferred embodiments, a transferase having the ability to glycosylate a large portion of the potential acceptor fractions of interest is chosen. Next, the resulting GlcNAcβ-OR can be used as an acceptor for a fucosyltransferase.

[0157] The resulting GlcNAcβ-OR fraction can be subjected to galactosylation before the reaction of the fucosyltransferase, generating, for example, a Galβ1,3GlcNAc-OR or Gal β1,4GlcNAc-OR residue. In some embodiments, the galactosylation and fucosylation steps can be carried out simultaneously. Choosing a fucosyltransferase that requires the galactosylated acceptor, only the desired product is formed. Thus, this method entails:

(to)

 galactosylation of a compound of the formula GlcNAcβ-OR with a galactosyltransferase in the presence of a UDP-galactose under conditions sufficient to form the compounds Galβ1,4GlcNAcβ-OR or Galβ1,3GlcNAcOR; Y

(b)

 fucosylation of the compound formed in (a) using a fucosyltransferase in the presence of GDP-fucose under conditions sufficient to form a compound selected from:

Fucα1,2Galβ1,4GlcNAc1β-O1R; Fucα1,2Galβ1,3GlcNAc-OR; Fucα1,2Galβ1,4GlcNAc1β-O1R; Fucα1,2Galβ1,3GalNAc-OR; Galβ1.4 (Fuc1, α3) GlcNAcβ-OR; or Galβ1.3 (Fucα1.4) GlcNAc-OR.

[0158] Additional fucose residues may be added to the above structures including an additional fucosyltransferase, having the desired activity. For example, the methods may form oligosaccharide determinants such as Fucα1,2Galβ1,4 (Fucα1,3) GlcNAcβ-OR and Fucα1,2Galβ1,3 (Fucα1,4) GlcNAc-OR. Thus, in another preferred embodiment, the method includes the use of at least two fucosyltransferases. Multiple fucosyltransferases are used either simultaneously or sequentially. When fucosyltransferases are used sequentially, it is generally preferred that the glycoprotein is not purified between the multiple stages of fucosylation. When multiple fucosyltransferases are used simultaneously, the enzymatic activity can be obtained from two independent enzymes or, alternatively, from a single enzyme having more than one fucosyltransferase activity.

[0159] The fucosyltransferase reaction can be carried out by contacting recombinant fucosyltransferase protein of the present invention with a mixture that includes, for example, multiple copies of a species of glycoprotein, preferably having a greater portion of the latter one or more linked oligosaccharide groups that include an acceptor substrate for a fucosyltransferase; fucose donor substrate; and other reagents required for fucosyltransferase activity. The glycoprotein is incubated in the reaction mixture for a sufficient time and under appropriate conditions, to transfer fucose from a donor substrate to a fucosyltransferase acceptor substrate.

[0160] The recombinant fucosyltransferase fusion protein used in the methods of the invention is chosen based on its fucosylation capacity of the fucosyltransferase acceptor substrates of interest. Preferably, the fucosyltransferase is tested for suitability using a fucosyltransferase acceptor substrate that is bound to a glycoprotein. The use of an acceptor substrate linked to glycoprotein, instead of an acceptor substrate that is part of a soluble oligosaccharide, in the assay to determine fucosyltransferase activity, allows to select a fucosyltransferase that produces the fucosylation pattern selected in the glycoprotein.

[0161] In a preferred embodiment, the recombinant fucosyltransferase fusion protein of the present invention has a high level of expression in cells and / or high enzymatic activity (eg, high specificity for a selected substrate and / or high catalytic activity ). In another preferred embodiment, fucosyltransferase is useful in a method for fucosylation of a commercially important transgenic or recombinant glycoprotein. The fucosyltransferase used in the method of the invention can also preferably fucosylate a variety of glycoproteins effectively, and support increasing the scale of the reaction to allow fucosylation of at least about 500 mg of the glycoprotein. More preferably, the fucosyltransferase will support the fucosylation reaction scale to allow the synthesis of at least about 1 kg, and more preferably, at least 10 kg of recombinant glycoprotein with relatively low cost and infrastructure requirements.

[0162] In an exemplary embodiment, the method of the invention results in the formation in a glycoprotein of at least one ligand for a selectin. Confirmation of ligand formation is tested in an operative manner by probing the ability of the glycoprotein to interact with a selectin. The interaction between a glycoprotein and a specific selectin is measurable through methods familiar to those familiar with the subject (see, for example, Jutila et al., J. Immunol. 153: 3917-28 (1994); Edwards et al., Cytometry 43 (3): 211-6 (2001); Stahn et al., Glycobiology 8: 311-319 (1998); Luo et al., Cell Biochem 80 (4): 522-31 (2001) ; Dong et al., J. Biomech. 33 (1): 35-43 (2000); Jung et al., J. Immunol. 162 (11): 6755-62 (1999); Keramidaris et al., J. Allergy Clin. Immunol. 107 (4): 734-8 (2001); Fieger et al., Biochim. Biophys. A cta 1524 (1): 75-85 (2001); Bruehl et al., J. Biol. Chem 275 (42): 32642-8 (2000); Tangemann et al., J. Exp. Med. 190 (7): 935-42 (1999); Scalia et al., Circ. Res. 84 (1): 93-102 (1999); Alon et al., J. Cell Biol. 138 (5): 1169-80 (1997); Steegmaier et al., Eur. J; Immunol. 27 (6): 1339-45 (1997 ); Stewart et al., J. Med. Chem. 44 (6): 988-1002 (2001); Schurmann et al, Gut 36 (3): 411-8 (1995); Burrows et al., J. Clin Pathol 47 (10): 939-44 (1994)).

[0163] Acceptable substrates for the fixation, catalyzed with fucosyltransferase, of a fucose residue include, among others, GIcNAc-OR, Galβ1,3GlcNAc-OR, NeuAcα2,3Galβ1,3GlcNAc-OR, Galβ1,4GlcNAc-OR and NeuAcα2 , 3Galβ1,4GlcNAc-OR, where R is an amino acid, a saccharide, an oligosaccharide or an aglycone group having at least one carbon atom. R is linked to or is part of a glycoprotein. The appropriate fucosyltransferase for a particular reaction is chosen based on the type of fucose bond that is desired (eg, α2, α3, or α4), the particular acceptor of interest, and the ability of the fucosyltransferase to achieve the desired high yield of fucosylation Previously, suitable fucosyltransferases and their properties have been described.

[0164] If a sufficient proportion of the oligosaccharides linked to glycoprotein in a composition does not include a fucosyltransferase acceptor substrate, a suitable acceptor can be synthesized. For example, a preferred method of synthesizing an acceptor for a fucosyltransferase involves the use of a GlcNAc transferase to fix a GlcNAc residue to a GlcNAc transferase acceptor substrate, which is present in the protein-linked oligosaccharides. In preferred embodiments, a transferase having the ability to glycosylate a high portion of the potential acceptor substrates of interest is chosen. Next, the resulting GlcNAcβ-OR can be used as an acceptor for a fucosyltransferase.

[0165] The resulting GlcNAcβ-OR fraction can be subjected to galactosylation before the reaction of fucosyltransferase, generating, for example, a Galβ1,3GlcNAc-OR or Gal β1,4GlcNAc-OR residue. In some embodiments, the galactosylation and fucosylation steps can be carried out simultaneously. Thus, this method entails:

(to)

 galactosylation of a compound of the formula GlcNAcβ-OR with a galactosyltransferase in the presence of a UDP-galactose under conditions sufficient to form the compounds Galβ1,4GlcNAcβ-OR or Galβ1,3GlcNAcOR; Y

(b)

 fucosylation of the compound formed in (a) using a fucosyltransferase in the presence of GDP-fucose under conditions sufficient to form a compound selected from:

Fucα1,2Galβ1,4GlcNAc1β-O1R; Fucα1,2Galβ1,3GlcNAc-OR; Fucα1,2Galβ1,4GlcNAc1β-O1R; Fucα1,2Galβ1,3GalNAc-OR; Galβ1.4 (Fuc1, α3) GlcNAcβ-OR; or Galβ1.3 (Fucα1.4) GlcNAc-OR.

[0166] Additional fucose residues can be added to a fucosylated glycoprotein by treating the fucosylated peptide with a fucosyltransferase, having the desired activity. For example, the methods may form oligosaccharide determinants such as Fucα1,2Galβ1,4 (Fucα1,3) GlcNAcβ-OR and Fucα1,2Galβ1,3 (Fucα1,4) GlcNAc-OR. Thus, in another preferred embodiment, the method includes the use of at least two fucosyltransferases. Multiple fucosyltransferases are used either simultaneously or sequentially. When fucosyltransferases are used sequentially, it is generally preferred that the glycoprotein is not purified between the multiple stages of fucosylation. When multiple fucosyltransferases are used simultaneously, the enzymatic activity can be obtained from two independent enzymes or, alternatively, from a single enzyme having more than one fucosyltransferase activity.

H. Synthesis of oligosaccharides with multiple enzymes

[0167] As described above, in some embodiments, two or more enzymes can be used to form a desired oligosaccharide or oligosaccharide determinant in a glycoprotein or glycolipid. For example, a particular oligosaccharide determinant may require the addition of a galactose, a sialic acid, and a fucose to exhibit a desired activity. Accordingly, the invention provides methods in which two or more enzymes, for example, glycosyltransferases, trans-sialidases, or sulfotransferases, are used to obtain a high yield synthesis of a desired oligosaccharide determinant.

[0168] In a preferred embodiment, LNFIII was prepared from lactose using H. pylori α-1,3 / 4-fucosyltransferases from strain 1182 described herein, in combination with β-1,3N-acetylglucosaminyltransferase (1gtA) of Neisseria gonococcus and β-1,4-galactosyltransferase (1gtB) of Neisseria gonococcus. Those experts will recognize that in this embodiment of the invention other enzymes of β-1,3-Acetylglucosaminyltransferase and β-1,4-galactosyltransferase can be used.

[0169] In some cases, an oligosaccharide linked to glycoprotein or glycolipid will include an acceptor substrate for the particular glycosyltransferase of interest when the in vivo biosynthesis of the glycoprotein or glycolipid occurs. Such glycoproteins or glycolipids can be subjected to glycosylation using H. pylori fucosyltransferase proteins and the methods of the invention without a prior modification of the glycosylation pattern of the glycoprotein or glycolipid, respectively. However, in other cases, a glycoprotein or glycolipid of interest will lack a suitable acceptor substrate. In such cases, the methods of the invention can be used to alter the glycosylation pattern of the glycoprotein or glycolipid so that the oligosaccharides linked to the glycoprotein or glycolipid then include an acceptor substrate for fixation, catalyzed with glycosyltransferase, of a unit of preselected saccharide of interest to form a desired oligosaccharide fraction.

[0170] Oligosaccharides linked to glycoprotein or glycolipid first, optionally, can be "trimmed" ("trimmed"), either in whole or in part, to expose either an acceptor substrate for glycosyltransferase or either a fraction to which one or more appropriate residues can be added to obtain a suitable acceptor substrate. Enzymes such as glycosyltransferases and endoglycosidases are useful for fixation and trimming reactions. For example, a glycoprotein having oligosaccharides of the "high mannose" type can be truncated by means of a mannosidase to obtain an acceptor substrate which, upon fixing one or more preselected saccharide units, forms the desired oligosaccharide determinant.

[0171] The methods are also useful for synthesizing a desired oligosaccharide fraction into a protein or lipid that, in its native form, is not glycosylated. An appropriate acceptor substrate for the corresponding glycosyltransferase can be fixed to said proteins or lipids prior to glycosylation using the methods of the present invention. See, for example, US Patent No. 5,272,066 for methods of obtaining polypeptides having suitable acceptors for glycosylation.

[0172] Thus, in some embodiments, the invention provides methods for in vitro sialylation of groups of saccharides present in a glycoconjugate that first involves the modification of the glyconjugate to create a suitable acceptor. The following are examples of preferred methods of synthesis, with multiple enzymes, of desired oligosaccharide fractions.

Fucosylated and sialylated oligosaccharide fractions

[0173] Oligosaccharide determinants that confer a desired biological activity on a glycoprotein are frequently subjected to sialylation in addition to being subjected to fucosylation. Accordingly, the invention provides methods in which an oligosaccharide linked to glycoprotein is subjected to sialylation and fucosylation with high yields.

[0174] Sialylation can be achieved using either a trans-sialidase or a sialyltransferase, except when a particular fraction requires a sialic acid with an α2,6 bond, in which a sialyltransferase is used. Previously, suitable examples of each type of enzyme have been described. These methods involve the sialylation of an acceptor for a sialyltransferase or a trans-sialidase by bringing the acceptor into contact with the appropriate enzyme in the presence of an appropriate donor substrate. For sialyltransferases, CMP-sialic acid is a preferred donor substrate. However, trans-sialidases preferably use a donor substrate that includes a leaving group to which trans-sialidase cannot add sialic acid.

[0175] Acceptor substrates of interest include, for example, Galβ-OR. In some embodiments, the acceptor substrates are contacted with a sialyltransferase in the presence of CMP-sialic acid under conditions under which sialic acid is transferred to the non-reducing end of the acceptor substrate to form the compound NeuAcα2,3GalβOR or NeuAcα2,6Galβ- OR. In this formula, R is an amino acid, a saccharide, an oligosaccharide or an aglycone group having at least one carbon atom. R is linked to or is part of a glycoprotein. An α2,8-sialyltransferase can also be used to fix a second sialic acid residue or multiple sialic acid residues to the above structures.

[0176] To obtain an oligosaccharide fraction that is both sialylated and fucosylated, the sialylated acceptor is contacted with a fucosyltransferase as described above. The sialyltransferase and fucosyltransferase reactions are generally carried out sequentially, since most sialyltransferases are not active in a fucosylated acceptor. However, FT VII acts only on a sialylated acceptor substrate. Therefore, FTVII can be used in a simultaneous reaction with a sialyltransferase.

[0177] If trans-sialidase is used to achieve sialylation, fucosylation and sialylation reactions can be carried out either simultaneously or sequentially, in any order. The protein to be modified is incubated with a reaction mixture containing an adequate amount of a trans-sialidase, a sialic acid donor substrate, a fucosyltransferase (capable of creating an α1,3 or α1,4 bond), and a donor substrate of suitable fucosil (for example, GDPfucose).

Determinants of galactosylated, fucosylated and sialylated oligosaccharides

[0178] The invention also provides methods for enzymatically synthesizing oligosaccharide fractions that are galactosylated, fucosylated and sialylated. In these methods, either a sialyltransferase or a trans-sialidase can be used (for sialic acid with α2.3 bond only).

[0179] The trans-sialidase reaction involves incubation of the protein to be modified with a reaction mixture containing an adequate amount of a galactosyltransferase (galβ1.3 or galβ1.4), a suitable galactosyl donor (for example, UDP-galactose), a trans-sialidase, a suitable sialic acid donor substrate, a fucosyltransferase (capable of creating an α1.3 or α1.4 bond), a suitable fucosyl donor substrate (e.g. GDP-fucose), and a divalent metal ion. These reactions can be carried out either sequentially or simultaneously.

[0180] If a sialyltransferase is used, the method involves incubation of the protein to be modified with a reaction mixture containing an adequate amount of a galactosyltransferase (galβ1.3 or galβ1.4), a suitable galactosyl donor (for example , UDP-galactose), a sialyltransferase (α2.3 or α2.6) and a suitable sialic acid donor substrate (eg, sialic CMP acid). The reaction is allowed to proceed substantially until it is completed, and then a fucosyltransferase (capable of creating an α1.3 or α1.4 bond) and a suitable fucosyl donor substrate (eg, GDP-fucose). If a fucosyltransferase that requires a sialylated substrate (for example, FT VII) is used, the reactions can be carried out simultaneously.

Sialyltransferase reactions

[0181] As described above, in some embodiments, the present invention provides an H. pylori fucosyltransferase protein and methods for fucosylation of a glycoprotein after the sialylation of the glycoprotein. In a preferred embodiment, the fusion proteins and methods of the invention synthesize glycoproteins that have a substantially uniform sialylation pattern. The fucosylation of the sialylated glycoprotein is then carried out, thereby producing a population of fucosylated glycoproteins in which the members have a substantially uniform pattern of fucosylation.

[0182] The glycoprotein may be contacted with a sialyltransferase and a sialic acid donor substrate for a sufficient time and under appropriate reaction conditions, to transfer sialic acid from the sialic acid donor substrate to saccharide groups. Sialyltransferases comprise a family of glycosyltransferases that transfer sialic acid from the CMP-sialic acid of the donor substrate to the acceptor oligosaccharide substrates. In preferred embodiments, the sialyltransferases are recombinant sialyltransferase fusion proteins. Provisional sialyltransferase reactions are described in provisional application US No. 60 / 035,710, filed on January 16, 1997, and non-provisional application US No. 09 / 007,741, filed on January 15, 1998.

[0183] In some embodiments, saccharide fractions in a glycoprotein having sialylation patterns altered by the H. pylori fucosyltransferase proteins of the present invention have a higher percentage of sialylated terminal galactose residues than the unaltered glycoprotein. Preferably, more than about 80% of the terminal galactose residues present in the oligosaccharides linked to glycoprotein will be subjected to sialylation following the use of the methods. More preferably, the use of H. pylori fucosyltransferase proteins and the methods of the invention will result in more than about 90% sialylation, and even more preferably more than about 95% sialylation of terminal galactose residues. Most preferably, essentially 100% of the terminal galactose residues present in the glycoproteins in the composition are subjected to sialylation following the modification using the methods of the present invention. The fusion proteins and methods of the invention are typically capable of achieving the desired level of sialylation in about 48 hours or less, and more preferably in about 24 hours or less.

 [0184] At least 15 different sialyltransferases of mammals have been documented, and to date the cDNAs of thirteen of them have been cloned (in relation to the systematic nomenclature used herein, see, Tsuji et al. ( 1996) Glycobiology 6: v-xiv). These cDNAs can be used to produce the recombinant sialyltransferase fusion proteins of the invention.

[0185] Preferably, for glycosylation of glycoprotein carbohydrates with N bonds and / or O bonds, sialyltransferase transfers sialic acid to the terminal sequence Galβ1,4-OR or GalNAc-OR, where R is an amino acid, a saccharide , an oligosaccharide or an aglycone group that has at least one carbon atom and is linked to or forms part of a glycoprotein. Galβ1,4-GlcNAc is the penultimate most common sequence that underlies terminal sialic acid in completely sialylated carbohydrate structures. At least three of the cloned mammalian sialyltransferases meet this specificity requirement of the acceptor, and it has been shown that each of them transfers sialic acid to carbohydrate groups with N bonds and O bonds of glycoproteins.

[0186] In some embodiments, the sialylation methods of the invention exhibit an increase in commercial viability through the use of bacterial sialyltransferases, either recombinantly produced or produced in native bacterial cells. Recently, two bacterial sialyltransferases have been reported; an ST6GaIII from Photobacterium damsela (Yamamoto et al. (1996) J. Biochem. 120: 104-110) and an ST3Gal V from Neisseria meningitidis (Gilbert et al. (1996) J. Biol. Chem. 271: 28271-28276) . The two recently described bacterial enzymes transfer sialic acid to the Galβ1,4GlcNAc sequence in oligosaccharide substrates.

[0187] A viral α2,3-sialyltransferase on which it has been reported recently is also suitable for testing and its possible use in the sialylation methods of the invention (Sujino et al. (2000) Glycobiology B10: 312-320) . This enzyme, v-ST3Gall, was obtained from cells infected with myxoma virus and is apparently related to mammalian ST3GallV as indicated by comparison of the respective amino acid sequences. The v-ST3Gall catalyzes the sialylation of Type 1 (Galβ1,3-GlcNAcβ1-R), Type II (Galβ1,4GlcNAc-β1-R) and III (Galβ1,3GalNAcβ1-R) acceptors. The enzyme can also transfer sialic acid to fucosylated acceptor substrates (for example, Lewis-x and Lewis-a).

[0188] An example of a sialyltransferase that is useful in the claimed methods is ST3Gal lll, which is also referred to as α (2,3) sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal of a glycoside Galβ1,3GlcNAc, Galβ1,3GalNAc or Galβ1,4GlcNAc (see, for example, Wen et al. (1992) J. Biol.

Chem. 267: 21011; Van den Eijnden et al. (1991) J. Biol. Chem. 256: 3159). Sialic acid bonds with a Gal with the formation of an α bond between the two saccharides. The junction (bond) between saccharides is between position 2 of NeuAc and position 3 of Gal. This particular enzyme can be isolated from rat liver (Weinstein et al. (1982) J. Biol. Chem. 257: 13845); the genomic DNA sequences (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) and human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 13941401) are known, which facilitates the production of this enzyme by recombinant expression. In a preferred embodiment, the claimed sialylation methods use a rat ST3Gal lll.

[0189] Other sialyltransferases, including those listed above, are also useful in a large-scale economic and effective process for the sialylation of commercially important glycoproteins. As described above, a simple test to find the usefulness of these other enzymes is to react several amounts of each enzyme (between 1 to 100 mU / mg protein) with an easily available glycoprotein protein such as asialo-α- AGP (at between 1 and 10 mg / ml) to compare the capacity of the sialyltransferase of interest of sialilar glycoproteins. The results can be compared, for example, with one or both of an ST6Gall or an ST3Gallll (for example, a bovine or human enzyme), depending on the particular sialic acid bond desired. Alternatively, for this evaluation, instead of asialo-α1 AGP, other glycoproteins, or oligosaccharides with N or O bonds enzymatically released from the peptide structural backbone, or saccharides that are produced by other methods or purified can be used from natural products such as milk. However, preferably, sialyltransferases are tested using an oligosaccharide that is linked to a glycoprotein. Sialyltransferases that have a capacity of, for example, sialylation of oligosaccharides with N-bonds or with O-bonds, of glycoproteins, more effective than ST6Gall are useful in a large-scale practical process for the sialylation of glycoproteins.

[0190] The invention also provides methods of altering the sialylation pattern of a glycoprotein before fucosylation by adding sialic acid in an α2.6Gal bond as well as the α2.3Gal bond, which are both found in oligosaccharides with N bonds, of human plasma glycoproteins. In this embodiment, ST3Gal lll and ST6Gall sialyltransferases are both present in the reaction and provide proteins that have a reproducible relationship of the bonds formed in the resialylation reaction. Thus, a mixture of the two enzymes can be valuable if both links in the final product are desired.

[0191] In the glycoprotein to be modified by the sialylation methods described herein, an acceptor substrate for sialyltransferase is present. Suitable acceptors include, for example, galactosylated acceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc (lactose), GalNAc-O-Ser, GalNac -O-Thr, and other acceptors known to those skilled in the art (see, for example, Paulson et al. (1978) J. Biol. Chem. 253: 5617-5624). Typically, the acceptors are included in oligosaccharide chains that are attached to asparagine, serine, or threonine residues present in a protein.

Examples

[0192] The following examples are offered to illustrate, but not limit, the claimed invention.

Example 1: Cloning of Helicobacter pylori fucosyltransferases.

[0193] Putative fucosyltransferase genes of the following Helicobacter pylori strains were amplified by PCR, cloned into vectors for expression in E. coli: strain 915 FutA, strain 1111 FutA, strain 19C2 FutB, strain 1182 FutB, strain 19C2 FutA, strain 26695 FutA, and strain 1218 FutB. Figures 1 to 7 provide nucleic acid and amino acid sequences. An alignment of amino acid sequences is provided in Figure 12; an alignment of nucleic acid sequences is provided in Figure 13.

[0194] Putative fucosyltransferase proteins were screened for α1,3 / 4-fucosyltransferase activity using LNnT and GDP-fucose substrates. The oligo structures of LNaT and a product, LNFPIII, are shown in Figure 14.

[0195] Cultures of one hundred milliliters of E. coli transformed with H. pylori fucosyltransferase were grown to OD600 of 0.8 and induced with IPTG, and were harvested. Cell lysates were created using a French press. Fucosyltransferase enzymes were tested for enzyme activity and acceptor specificity using the LNnT substrate. The reactions contained 3 mM GDP-fucose, 3 mM LNnT, 50 mM Tris pH 7.5, 20 mM MnCl2, and 15% bacterial lysate. The reactions were incubated at 37 ° C for twenty four hours.

[0196] The reaction products were separated using the following TLC-buffer system: 7 IPA: 2 H2O: 1 Acetic acid. The samples were methylated, hydrolyzed, reduced with sodium borodeuteride, acetylated and analyzed by GC / MS together with LNnT and LNF3 samples. Results are shown in Figure 15. A value of Glc with respect to Glc-NAc close to 1 favors the fucosylation of Glc-NAc. A value of Glc with respect to Glc-NAc close to 0 favors the fucosylation of Glc. Fucosyltransferases of the following strains of H. pylori transferred fucose to Glc-NAc: strain 915 FutA, strain 1111 FutA, strain 19C2 FutB, and strain 1182 FutB. The FutA gene product of H. pylori strain 19C2A transferred fucose to the reducing glucose of the LNnT acceptor, as did the FutB gene product of H. pylori strain 1218 FutB. A product of the novel FutA gene of strain 26695 of H. pylori also catalyzed the transfer of fucose to glucose.

Example 2: Production of oligosaccharides using Helicobacter pylori fucosyltransferases

[0197] One-liter cultures of E. coli expressing H. pylori fucosyltransferases were grown, induced and harvested. Lysates were used to synthesize LNFIII from LNnT. Two different ion exchange resins were tested for purification of LNFDIII. Reaction mixtures were centrifuged at

5,000 RPM for thirty minutes. The samples were then processed by ultrafiltration using hollow fiber ultrafiltration membranes with a molecular weight cutoff value of 10 kD. An ion exchange chromatography was applied using either 1 ml of resin per 1 ml of synthesis (70%) in NH4HCO3 MR3 column or 2 ml of resin per 1 ml of synthesis (82%) in Dowex1 / Dowex50 column. The samples were then passed on a P2 Size Exclusion column and then lyophilized. The results are shown in Figure 16. The yields using the Dowex resin approached 50%, while the yields of the NH4HCO3 MR3 column approached 70%.

[0198] LNFIII was prepared from lactose using lysates of E. coli cells expressing H. pylori α-1,3 / 4-fucosyltransferases from strain 1182 described herein, in combination with β-1,3-N-acetylglucosaminyltransferase ( 1gtA) of Neisseria gonococcus and β-1,4-galactosyltransferase (1gtB) of Neisseria gonococcus on a multigram scale. Those experts will recognize that, in this embodiment of the invention, other enzymes of β-1,3N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase can be used.

Example 3: Production of glycoproteins using Helicobacter pylori fucosyltransferases.

[0199] Using asialyltransferrin as a substrate, the ability of fucosyltransferase of H. pylori strain 1182B to add fucose to acceptor molecules in glycoprotein was tested. 1182B fucosyltransferase was produced in E. coli cells as described above. The reactions were carried out in a buffer containing 50 mM Tris pH 7.5, 20 mM MnCl 2, 200 μg of asialyltransferrin, and 5 mM GDP-fucose. The reactions were started by adding 15% v / v of the bacterial lysate. The reaction was incubated overnight at 37 ° C. Samples were analyzed using GC / MS. The results are shown in Figure 17.

[0200] It is understood that the examples and embodiments described herein are for illustrative purposes only and that, considering them, those skilled in the art will suggest various modifications or changes.

Claims (12)

one.

Method of creating a substantially uniform glycosphere of a fucosylated oligosaccharide, the method comprising: contacting a recombinant α1,3-fucosyltransferase of Helicobacter pylori strain 1182B with a mixture comprising a donor substrate comprising a fucose residue, and an acceptor substrate comprising a sugar or oligosaccharide comprising at least one Nacethylglucosamine residue, under conditions in which α1,3-fucosyltransferase catalyzes the transfer of fucose preferably to the N-acetylglucosamine residue, thereby producing a glycoform. substantially uniform of a fucosylated oligosaccharide, wherein α1,3-fucosyltransferase is encoded by a nucleic acid sequence having the sequence of Figure 1 or having the amino acid sequence of Figure 1 and wherein the acceptor substrate is Lacto- N-neo-Tetraose (LNnt).

2.

The method of claim 1, wherein the method further comprises a step of purification of the fucosylated oligosaccharide.

3.

Method of claim 1, wherein the donor substrate is GDP fucose.

Four.

Method of claim 1, wherein the fucosyltransferase comprises an amino acid tag.

5.

Method of claim 1, wherein the acceptor substrate is fixed to a glycoprotein.

6.

Method of claim 1, wherein the fucosylated oligosaccharide is Lacto-N-Fucopentaose III (LNFP III).

7.

The method of claim 1, wherein the mixture further comprises lactose, a β-1,3-Nacethylglucosaminyltransferase, and a β-1,4-galactosyltransferase.

8.

Method of claim 7, wherein the β-1,3-N-acetylglucosaminyltransferase is a bacterial enzyme.

9.

Method of claim 8, wherein the β-1,3-N-acetylglucosaminyltransferase is from Neisseria gonococcus.

10.

Method of claim 7, wherein the β-1,4-galactosyltransferase is a bacterial enzyme.

eleven.

Method of claim 10, wherein the β-1,4-galactosyltransferase is from Neisseria gonococcus.

12.

Method of claim 7, wherein the fucosylated oligosaccharide is Lacto-N-Fucopentaose III (LNFP III).