Enzymes for Sialylation of Glycans

Abstract

Described herein are fusion proteins, e.g., fusion proteins comprising enzymatically active portion(s) of ST6Gall or B4GalT1 as well as methods for producing them, nucleic acid molecule(s) encoding the fusion protein(s), vectors comprising the nucleic acid molecule(s), and host cell(s) comprising the vector(s). Also described herein are methods of sialyating immunoglobulin G (IgG) antibodies.

Claims

1. A fusion protein comprising: an N-terminal signal sequence; and an enzymatically active portion of human Alpha-2,6-sialyltransferase 1 (ST6Gal1) comprising SEQ ID NO:4.

2. (canceled)

3. (canceled)

4. (canceled)

5. The fusion protein of claim 1, wherein the signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO:30).

6. (canceled)

7. (canceled)

8. The fusion protein of claim 1, further comprising an affinity tag selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose-binding protein (MBP), chitin binding protein, a streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:31)), FLAG-tag (e.g., DYKDDDDK (SEQ ID NO:32)), a biotin tag, and combinations thereof.

9-21. (canceled)

22. A method for producing a polypeptide comprising: culturing a host cell harboring a nucleotide sequence encoding a fusion protein comprising SEQ ID NO: 6 in a culture medium under conditions permissive for expression of the fusion protein; and isolating the fusion protein from the culture medium.

23. A fusion protein comprising: an N-terminal signal sequence; and an enzymatically active portion of human beta-1,4-galactosyltransferase (B4GalT1) consisting of SEQ ID NO: 43.

24-35. (canceled)

36. The fusion protein of claim 22, consisting of SEQ ID NO:45.

37-43. (canceled)

44. A method for producing a polypeptide comprising: culturing a host cell harboring a nucleotide sequence encoding a fusion protein comprising SEQ ID NO: 45 in a culture medium under conditions permissive for expression of the fusion protein; and isolating the fusion protein from the culture medium.

45. A method for sialyating immunoglobulin G (IgG) antibodies, the method comprising: a) providing a composition comprising IgG antibodies; b) exposing the composition to a β1,4-galactosyltransferase 1 and an enzymatically active portion of ST6Gal1 comprising SEQ ID NO: 45 in the presence of UDP-Gal and CMP-NANA, thereby producing a composition comprising sialyated IgG (sIgG).

46. A method for sialyating immunoglobulin G (IgG) antibodies, the method comprising: a) providing a composition comprising IgG antibodies; b) exposing the IgG antibodies to a β1,4-galactosyltransferase 1 in the presence of UDP-Gal, thereby producing a composition comprising galactosylated IgG antibodies; and c) exposing the composition comprising galactosylated IgG antibodies to an enzymatically active portion of ST6Gal1 comprising SEQ ID NO: 45 in the presence of CMP-NANA, thereby producing a composition comprising sialyated IgG (sIgG).

47. (canceled)

48. The method of claim 46, further comprising supplementing one or more of the compositions with CMP-NANA.

49. (canceled)

50. The method of claim 46, wherein at least 60% of branched glycans on the Fc region of the antibodies in the composition comprising sIgG are disialylated.

51. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0071] FIG. 1 shows a short, branched core oligosaccharide comprising two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the “α 1,3 arm,” and the second branch is referred to as the “α 1,6 arm,”. Squares: N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose.

[0072] FIG. 2 shows common Fc glycans present in IVIg. Squares: N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose. FIG. discloses SEQ ID NO: 7.

[0073] FIG. 3 shows how immunoglobulins, e.g., IgG antibodies, can be sialylated by carrying out a galactosylation step followed by a sialylation step. Squares: N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose.

[0074] FIG. 4 shows the reaction product of a representative example of the IgG-Fc glycan profile for a reaction starting with IVIg. The the left panel is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; the right panel is the IgG Fc glycan profile for the starting IVIg and hsIgG. Bars, from left to right, correspond to IgG1, IgG⅔, and IgG¾, respectively.

DETAILED DESCRIPTION

[0075] Antibodies are glycosylated at conserved positions in the constant regions of their heavy chain and within the Fab. For example, within the Fc domain, human IgG antibodies have a single N-linked glycosylation site at Asn297 of the CH2 domain. Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For human IgG, the core oligosaccharide normally consists of GlcNAc.sub.2Man.sub.3GlcNAc, with differing numbers of outer residues. Variation among individual IgG’s can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).

[0076] The present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having an Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with a NeuAc-α 2,6-Gal terminal linkage). The levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region in a preparation of polypeptides).

[0077] Naturally derived polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (particular human serum pooled from more than 1,000 donors), intravenous immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).

[0078] N-linked oligosaccharide chains are added to a protein in the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14-sugar) is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The structure of this initial oligosaccharide is common to most eukaryotes, and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the “α 1,3 arm,” and the second branch is referred to as the “α 1,6 arm,” as shown in FIG. 1.

[0079] N-glycans can be subdivided into three distinct groups called “high mannose type,” “hybrid type,” and “complex type,” with a common pentasaccharide core (Man (α 1,6)-(Man(α 1,3))-Man(β 1,4)-GlcpNAc(β 1,4)-GlcpNAc(β 1,N)-Asn) occurring in all three groups.

[0080] The more common Fc glycans present in IVIg are shown in FIG. 2.

[0081] Additionally or alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a “complex glycan.” Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.

[0082] “Hybrid glycans” comprise characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.

[0083] Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations including N-acetyl and N-glycolyl groups. The two main types of sialyl residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both N-and O-linked glycans. The glycosidic linkage configurations for these sialyl groups can be either α 2,3 or α 2,6.

[0084] Fc regions are glycosylated at conserved, N-linked glycosylation sites. For example, each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the C.sub.H2 domain. IgA antibodies have N-linked glycosylation sites within the C.sub.H2 and C.sub.H3 domains, IgE antibodies have N-linked glycosylation sites within the C.sub.H3 domain, and IgM antibodies have N-linked glycosylation sites within the C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4 domains.

[0085] Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For example, IgG has a single N-linked biantennary carbohydrate at Asn297 of the C.sub.H2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for C1q and FcyR. For human IgG, the core oligosaccharide normally consists of GlcNAc.sub.2Man.sub.3GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).

[0086] Immunoglobulins, e.g., IgG antibodies, can be sialylated by carrying out a galactosylation step followed by a sialylation step. Beta-1,4-galactosyltransferase 1 (B4GalT) is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5′-diphosphosegalactose ([[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy,hydroxyphosphoryl] [(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] hydrogen phosphate; UDP-Gal) to GlcNAc as a β-1,4 linkage. Alpha-2,6-sialyltransferase 1 (ST6) is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5′-monophospho-Nacetylneuraminicacid ((2R,4S,5R,6R)-5-acetamido-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4-hydroxy-6-(1,2,3-trihydroxypropyl)oxane-2-carboxylic acid; CMP-NANA or CMP-Sialic Acid) to Gal as an α-2,6 linkage. Schematically, the reactions proceed shown in FIG. 3.

[0087] Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an α1,3 branch and/or an α1,6 branch) can be characterized using methods described in WO2014/179601.

[0088] In some embodiments of the hsIgG compositions prepared by the methods described herein, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans on the Fc domain have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. In addition, in some embodiments, at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. Overall, in some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.

Enzymes

[0089] Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, and fusion proteins comprising the same are suitable for use in the methods described herein. B4Galt1 is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes that each encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl. B4Galt1 adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. B4GalT1 is also called GGTB2. Four alternative transcripts encoding four isoforms of B4GALT1 (NCBI Gene ID 2683) are described in Table 1.

TABLE-US-00001 Human B4GALT1 isoforms Transcript Length (nt) Protein SEQ ID NO: Length (aa) Isoform NM_001497.4 4176 NP_001488.2 37 398 1 NM_001378495.1 3999 NP_001365424.1 38 385 2 NM_001378496.1 4053 NP_001365425.1 39 357 3 NM_001378497.1 1520 NP_001365426.1 40 225 4

>NP_001488.2 B4GALT1 [organism=Homo sapiens] [GeneID=2683] [isoform=1] (SEQ ID NO:37)

TABLE-US-00002 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRL PQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS SPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVD LELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVD LIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTI NGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNE PNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

>NP_001365424.1 B4GALT1 [organism=Homo sapiens] [GeneID=2683] [isoform=2] (SEQ ID NO:38)

TABLE-US-00003 MPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGG SNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASN LTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKM GGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYV INQAGDTIFNPAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCF SQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGE DDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTK ETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

>NP_001365425.1 B4GALT1 [organism=Homo sapiens] [GeneID=2683] [isoform=3] (SEQ ID NO:39)

TABLE-US-00004 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRL PQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS SPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVD LELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVD LIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGMSISRPNAVVGRCRMI RHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQIT VDIGTPS

>NP_001365426.1 B4GALT1 [organism=Homo sapiens] [GeneID=2683] [isoform=4] (SEQ ID NO:40)

TABLE-US-00005 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRL PQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS SPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVD LELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP VLQRQQLDYGIYVINQYEKIRRLLW

TABLE-US-00006 Topology of B4GALT1 isoform 1 (SEQ ID NO:37) Feature AAs Description Lengt h Sequence SE Q ID NO: Topological domain 1 -24 Cytoplasmic 9 MRLREPLLSGSAAMPGASLQRACR 41 Transmembran e 25 -44 Helical; Signal-anchor for type II membrane protein 17 LLVAVCALHLGVTLVYYLAG 42 Topological domain 45 -398 Lumenal 380 RDLSRLPQLVGVSTPLQGGSNSAAAIGQ SSGELRTGGARPPPPLGASSQPRPGGDS SPVVDSGPGPASNLTSVPVPHTTALSLP ACPEESPLLVGPMLIEFNMPVDLELVAK QNPNVKMGGRYAPRDCVSPHKVAIIIPF RNRQEHLKYWLYYLHPVLQRQQLDYGIY VINQAGDTIFNRAKLLNVGFQEALKDYD YTCFVFSDVDLIPMNDHNAYRCFSQPRH ISVAMDKFGFSLPYVQYFGGVSALSKQQ FLTINGFPNNYWGWGGEDDDIFNRLVFR GMSISRPNAVVGRCRMIRHS RDKKNEPNPQRFDRIAHTKETMLSDGLN SLTYQVLDVQRYPLYTQITVDIGTPS 63

TABLE-US-00007 Binding sites of B4GALT1 isoform 1 (SEQ ID NO:37) Position(s) Description Reference(s) 250 Metal binding; Manganese 310 Binding site; UDP-alpha-D-galactose “Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway.” Ramakrishnan B., Ramasamy V., Qasba P.K. J. Mol. Biol. 357:1619-1633(2006) 343 Metal binding; Manganese; via tele nitrogen 355 Binding site; N-acetyl-D-glucosamine “Oligosaccharide preferences of betal,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D.M., Seeberger P.H., Qasba P.K. J. Mol. Biol. 353:53-67(2005) “Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis.” Brown J.R., Yang F., Sinha A., Ramakrishnan B., Tor Y., Qasba P.K., Esko J.D. J. Biol. Chem. 284:4952-4959(2009)

TABLE-US-00008 Post Translational Amino Acid Modifications of B4GALT1 isoform 1 (SEQ ID NO:37) Feature key Position(s) Description Reference(s) Glycosylation 113 N-linked (GlcNAc...) asparagine Disulfide bond 130 .Math. 172 “Oligosaccharide preferences of beta1,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D.M., Seeberger P.H., Qasba P.K. J. Mol. Biol. 353:53-67(2005) “Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway.” Ramakrishnan B., Ramasamy V., Qasba P.K. J. Mol. Biol. 357:1619-1633(2006) Disulfide bond 243 .Math. 262

[0090] The soluble form of B4GalT1 derives from the membrane form by proteolytic processing. The cleavage site is at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO:37).

[0091] In some embodiments, one or more of the amino acids of the B4GalT1 corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO:37) is conserved as compared to (SEQ ID NO:37).

[0092] Provided herein are enzymatically active portions of, e.g., B4GalT1. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO:37), or an ortholog, mutant, or variant of SEQ ID NO:37. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO:38), or an ortholog, mutant, or variant of SEQ ID NO:38. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO:39), or an ortholog, mutant, or variant of SEQ ID NO:39. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO:40), or an ortholog, mutant, or variant of SEQ ID NO:40.

[0093] In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:41. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a transmembrane domain, e.g., SEQ ID NO:42. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:41 or a transmembrane domain, e.g., SEQ ID NO:42.

[0094] In some embodiments, the enzymatically active portion of B4GalT1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO:63, or an ortholog, mutants, or variants thereof.

[0095] In some embodiments, the enzymatically active portion of B4GalT1 comprises amino acids 109-398 of SEQ ID NO:37, or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of B4GalT1 consists of SEQ ID NO:37, or an ortholog, mutant, or variant of SEQ ID NO:37.

[0096] A suitable functional portion of an B4GalT1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:43. SEQ ID NO:43

TABLE-US-00009 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQN PNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLD YGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHN AYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYW GWGGEDDDIFNRLVFRGMS ISRPNAVVGRCRMIRHSRDKKNEPNPQRFD RIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

[0097] ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, and fusion proteins comprising the same, are suitable for use in the methods described herein. ST6GAL1, β-galactoside α-2,6-sialyltransferase 1, transfers sialic acid from CMP-sialic acid to the Galβ1.fwdarw.4GlcNAc structure on glycoproteins, such as asialofetuin and asialo-a1-acid glycoprotein. ST6Gal1 is also called as ST6N or SIAT1. Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI Gene ID 6480) are described in Table 1.

TABLE-US-00010 Human ST6GAL1 isoforms Transcript Length (nt) Protein SEQ ID NO: Length (aa) Isoform NM_173216.2 4604 NP_775323.1 28 406 a NM_173217.2 3947 NP_775324.1 29 175 b NM_003032.3 4303 NP_003023.1 28 406 a NM_001353916.2 4177 NP_001340845.1 28 406 a

>NP_001340845.1 (NP_003023.1, NP_775323.1) ST6GAL1 [organism=Homo sapiens] [GeneID=6480] [isoform=a] (SEQ ID NO:28)

TABLE-US-00011 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKS LGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDS SSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNV SMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGR EIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYN EGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQM PWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKT DVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPG FRTIHC

>NP_775324.1 ST6GAL1 [organism=Homo sapiens] [GeneID=6480] [isoform=b] (SEQ ID NO:29)

TABLE-US-00012 MNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNY KTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIM MTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKH LNQGTDEDIYLLGKATLPGFRTIHC

TABLE-US-00013 Topology of ST6Gal1 isoform a (SEQ ID NO:28) Feature AAs Description Lengt h Sequence SEQ ID NO: Topological domain 1 -9 Cytoplasmic 9 MIHTNLKKK 34 Transmembran e 10 -26 Helical; Signal-anchor for type II membrane protein 17 FSCCVLVFLLFAVICVW 35 Topological domain 27 -406 Lumenal 380 KEKKKGSYYDSFKLQTKEFQVLKSLGK LAMGSDSQSVSSSSTQDPHRGRQTLGS LRGLAKAKPEASFQVWNKDSSSKNLIP RLQKIWKNYLSMNKYKVSYKGPGPGIK FSAEALRCHLRDHVNVSMVEVTDFPFN TSEWEGYLPKESIRTKAGPWGRCAVVS SAGSLKSSQLGREIDDHDAVLRFNGAP TANFQQDVGTKTTIRLMNSQLVTTEKR FLKDSLYNEGILIVWDPSVYHSDIPKW YQNPDYNFFNNYKTYRKLHPNQPFYIL KPQMPWELWDILQEISPEEIQPNPPSS GMLGIIIMMTLCDQVDIYEFLPSKRKT DVCYYYQKFFDSACTMGAYHPLLYEKN LVKHLNQGTDEDIYLLGKATLPGFRTI HC 36

TABLE-US-00014 Binding sites of ST6Gal1 isoform a (SEQ ID NO:28) Position(s) Description Reference(s) 189 Substrate; via amide nitrogen “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) 212 Substrate 233 Substrate 353 Substrate; via carbonyl oxygen 354 Substrate 365 Substrate 369 Substrate 370 Substrate “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) 376 Substrate

TABLE-US-00015 Post Translational Amino Acid Modifications of ST6Gal1 isoform a (SEQ ID NO:28) Feature key Position(s) Description Reference(s) Disulfide bond 142 .Math. 406 “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) Glycosylation 149 N-linked (GlcNAc...) asparagine “Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8:651-661(2009); and “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) Glycosylation 161 N-linked (GlcNAc...) asparagine “Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8:651-661(2009) Disulfide bond 184 .Math. 335 “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) Disulfide bond 353 .Math. 364 “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M.G. Acta Crystallogr. D 69:1826-1838(2013) Modified residue 369 Phosphotyrosine “Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.” Mayya V., Lundgren D.H., Hwang S.-I., Rezaul K., Wu L., Eng J.K., Rodionov V., Han D.K. Sci. Signal. 2:RA46-RA46(2009)

[0098] The soluble form of ST6Gal1 derives from the membrane form by proteolytic processing.

[0099] In some embodiments, one or more of the amino acids of the ST6Gal1 corresponding to amino acids 142, 149, 161, 184, 189, 212, 233, 335, 353, 354, 364, 365, 369, 370, 376, or 406 of ST6Gal1 isoform a (SEQ ID NO:28) is conserved as compared to SEQ ID NO:28.

[0100] Also provided herein is an enzymatically active portion of, e.g., ST6Gal1. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform a (SEQ ID NO:28), or an ortholog, mutant, or variant of SEQ ID NO:28. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform b (SEQ ID NO:29), or an ortholog, mutant, or variant of SEQ ID NO:29.

[0101] In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:34. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a transmembrane domain, e.g., SEQ ID NO:35. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:34 or a transmembrane domain, e.g., SEQ ID NO:35.

[0102] In some embodiments, the enzymatically active portion of ST6Gal1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO:36, or an ortholog, mutants, or variants thereof.

[0103] In some embodiments, the enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO:28 (SEQ ID NO:4), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO:4, or an ortholog, mutant, or variant of SEQ ID NO:4.

[0104] A suitable functional portion of an ST6Gal1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:3 or SEQ ID NO:4. SEQ ID NO:4

TABLE-US-00016 AKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFS AEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAV VSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQL VTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRK LHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCD QVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGT DEDIYLLGKATLPGFRTIHC

Variants

[0105] In some embodiments, the enzyme(s) described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent, e.g., β-galactoside α-2,6-sialyltransferase activity or β-1,4-galactosyltransferase activity.

[0106] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0107] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence.

[0108] For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0109] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Fusion Proteins

[0110] Also provided herein are fusion protein(s) comprising enzyme(s) or portions thereof as described herein.

[0111] In one embodiment, the fusion protein comprises a signal sequence. In some embodiments, the signal sequence is about 15 to about 20 amino acid, e.g., about 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the signal sequence comprises a hydrophobic core region (h-region) flanked by an n-region and a c-region. In some embodiments, the c-region comprises a signal peptidase consensus cleavage site.

[0112] In some embodiments, signal sequence is an N-terminal signal sequence.

[0113] In some embodiments, the signal sequence is an azurocidin signal sequence. In some embodiments, the azurocidin signal sequence comprises or consists of MTRLTVLALLAGLLASSRA (SEQ ID NO:30). In some embodiments, the signal sequence is a serum albumin signal sequence. In some embodiments, the serum albumin signal sequence comprises or consists of MKWVTFISLLFLFSSAYS (SEQ ID NO:46). In some embodiments, the signal sequence is an immunoglobulin heavy chain signal sequence. In some embodiments, the immunoglobulin heavy chain signal sequence comprises or consists of MDWTWRVFCLLAVTPGAHP (SEQ ID NO:47). In some embodiments, the signal sequence is an immunoglobulin light chain signal sequence. In some embodiments, the immunoglobulin light chain signal sequence comprises or consists of MDWTWRVFCLLAVTPGAHP (SEQ ID NO:48).

[0114] In some embodiments, the signal sequence is a cystatin-S signal sequence. In some embodiments, the cystatin-S signal sequence comprises or consists of MARPLCTLLLLMATLAGALA (SEQ ID NO:49). In some embodiments, the signal sequence is an IgKappa signal sequence. In some embodiments, the IgKappa signal sequence comprises or consists of MDMRAPAGIFGFLLVLFPGYRS (SEQ ID NO:50). In some embodiments, the signal sequence is a trypsonigen 2 signal sequence. In some embodiments, the trysonigen 2 signal sequence comprises or consists of MRSLVFVLLIGAAFA (SEQ ID NO:51). In some embodiments, the signal sequence is potassium channel blocker signal sequence. In some embodiments, the potassium channel blocker sequence comprises or consists of MSRLFVFILIALFLSAIIDVMS (SEQ ID NO:52).

[0115] In some embodiments, the signal sequence is an alpha conotoxin Ip 1.3 signal sequence. In some embodiments, the alpha conotoxin Ip1.3 signal sequence comprises or consists of MGMRMMFIMFMLVVLATTVVS (SEQ ID NO:53). In some embodiments, the signal sequence is an alfa-galactosidase signal sequence. In some embodiments, the alfa-galactosidase signal sequence comprises or consists of MRAFLFLTACISLPGVFG (SEQ ID NO:54). In some embodiments, the signal sequence is a cellulase signal sequence. In some embodiments, the cellulase signal sequence comprises or consists of MKFQSTLLLAAAAGSALA (SEQ ID NO:55). In some embodiments, signal sequence is an aspartic proteinase nepenthesin-1 signal sequence. In some embodiments, the aspartic proteinase nepenthesin-1 signal sequence comprises or consists of MASSLYSFLLALSIVYIFVAPTHS (SEQ ID NO:56). In some embodiments, the signal sequence is an acid chitinase signal sequence. In some embodiments, the acid chitinase signal sequence comprises or consists of MKTHYSSAILPILTLFVFLSINPSHG (SEQ ID NO:57). In some embodiments, the signal sequence is a K28 prepro-toxin signal sequence. In some embodiments, the K28 prepro-toxin signal sequence comprises or consists of MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYARG (SEQ ID NO:58). In some embodiments, the signal sequence is a killer toxin zygocin precursor signal sequence. In some embodiments, the killer toxin zygocin precursor signal sequence comprises or consists of MKAAQILTASIVSLLPIYTSA (SEQ ID NO:59). In some embodiments, the signal sequence is a cholera toxin signal sequence. In some embodiments, the cholera toxin signal sequence comprises or consists of MIKLKFGVFFTVLLSSAYA (SEQ ID NO:60). In some embodiments, the signal sequence is a human growth hormone signal sequence. In some embodiments, the human growth hormone signal sequence comprises or consists of MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO:61)

[0116] In some embodiments, the fusion protein comprises one or more affinity tag(s). In some embodiments, the purification tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose-binding protein (MBP), chitin binding protein, a streptavidin tag (e.g., Strep-Tag®, e.g., Trp—Ser—His—Pro—Gln—Phe—Glu—Lys (SEQ ID NO—31)), FLAG-tag (e.g., DYKDDDDK (SEQ ID NO:32)), a biotin tag (e.g., AviTag™) and combinations thereof.

[0117] In some embodiments, the affinity tag is situated towards the N-terminal side of the enzyme or portion thereof. In some embodiments, the affinity tag is N-terminal.

[0118] In some embodiments, the affinity tag is situated towards the C-terminal side of the enzyme or portion thereof. In some embodiments, the affinity tag is C-terminal.

[0119] In some embodiments, the affinity tag is a polyhistidine tag. In some embodiments, the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO:11), HHHHH (SEQ ID NO:12), HHHHHH, (SEQ ID NO:13), HHHHHHH (SEQ ID NO:14), HHHHHHHH (SEQ ID NO:15), HHHHHHHHH (SEQ ID NO:16), and HHHHHHHHHH (SEQ ID NO:17). In some embodiments, the polyhistidine tag is a hexahistidine tag (e.g., HHHHHH (SEQ ID NO:13)).

[0120] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45. SEQ ID NO:44

TABLE-US-00017 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQN PNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLD YGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHN AYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYW GWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDR IAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPRD

SEQ ID NO:45

TABLE-US-00018 gssplldmGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVD LELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVD LIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTI NGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNE PNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSpr dhhhhhhh

[0121] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:3 or SEQ ID NO:5. SEQ ID NO:3

TABLE-US-00019 gssplldmlehhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYL SMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEG YLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTA NFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIP KWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQ PNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMG AYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC

SEQ ID NO:5

TABLE-US-00020 hhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYK GPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTK AGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKT TIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNF FNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLG IIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKN LVKHLNQGTDEDIYLLGKATLPGFRTIHC

Expression Systems

[0122] To use the enzyme(s) and/or fusion protein(s) described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the enzyme(s) and/or fusion protein(s) can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the enzyme(s) and/or fusion protein(s). The nucleic acid encoding the enzyme(s) and/or fusion protein(s) can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

[0123] To obtain expression, a sequence encoding the enzyme(s) and/or fusion protein(s) is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0124] The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins.

[0125] In some embodiments, the promoter is selected from the group consisting of human cytomegalovirus (CMV), EF-1 α (EF1A), elongation factor 1α short (EFS), CMV enhancer chicken β-Actin promoter and rabbit β-Globin splice acceptor site (CAG), hybrid CBA (CBh), spleen focus-forming virus (SFFV), murine stem cell virus (MSCV), simian virus 40 (SV40), mouse phosphoglycerate kinase 1 (mPGK), human phosphoglycerate kinase 1 (hPGK), and ubiquitin C (UBC) promoters. In some embodiments, the promoter is a human cytomegalovirus promoter (CMV).

[0126] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the enzyme(s) and/or fusion protein(s) and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

[0127] In some embodiments, the expression vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). See, e.g., Zufferey et al., “Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors,” Journal of Virology 73(4):2886-92 (1999).

[0128] The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the enzyme(s) and/or fusion protein(s), e.g., expression in plants, animals, bacteria, fungus, protozoa, etc.

[0129] Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

[0130] Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the enzyme(s) and/or fusion protein(s).

[0131] In some embodiments, the host cells are stably transformed.

[0132] In some embodiments, the host cells are grow under non-hypoxic conditions.

[0133] The enzyme(s) and/or fusion protein(s) described herein can be produced by any protein production system known in the art, such as host cell based expression systems, synthetic biology platforms, or cell-free protein production platforms. In some embodiments, the protein production system is capable of post-translational modification(s), including, but not limited to one or more of glycosylation, e.g., N-glycosylated proteins, disulfide bond formation, and tyrosine phosphorylation. See, e.g., Boh and Ng, “Impact of Host Cell Line Choice on Glycan Profile,” Critical Reviews in Biotechnology 38(6):851-67 (2018).

[0134] In some embodiments, the host cell is a mammalian host cell. In some embodiments, the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, NS0 myeloma cells, Sp2/0 hybridoma mouse cells, human embryonic kidney cells (HEK), HT-1080 human cells, and derivatives thereof.

[0135] In some embodiments, the host cell is a non-human mammalian host cell. In some embodiments, the non-human mammalian host cell is selected from CHO cells, BHK-21 cells, murine NS0 myeloma cells, Sp2/0 hybridoma cells, and derivatives thereof.

[0136] In some embodiments, the host cell is a human mammalian host cell. In some embodiments, the human cell is selected from the group consisting of HEK, PER.C6, CEVEC’s amniocyte production (CAP), AGE1.HM, HKB-11, HT-1080 cells, and derivatives thereof.

[0137] In some embodiments, the host cell is a human embryonic kidney cell (HEK, ATCC® CRL-1573™) or derivative thereof.

[0138] In some embodiments, the HEK cell expresses a temperature sensitive allele of the SV40 T antigen. In some embodiments, the HEK cell is resistant against the Ricin toxin after ethymethanesulfonate (EMS) mutagenesis and lack N-acetylglucosaminyltransferase I activity, e.g., encoded by the MGAT1 gene. In some embodiments, the HEK cell predominantly modifies glycoproteins with the Man5GlcNAc2 N-glycan. In some embodiments, the HEK cell expresses the tetR repressor, enabling tetracycline-inducible protein expression.

[0139] In some embodiments, the HEK derivative is selected from the group consisting of HEK293, HEK293T (293tsA1609neo, ATCC® CRL-3216™), HEK293T/17 (ATCC® CRL-11268™), HEK293T/17 SF (ATCC® ACS-4500™), HEK293S, HEK293SG, HEK293FTM, HEK293SGGD, HEK293FTM, HEK293E, and HKB-11.

[0140] Synthetic biology platforms, such as those described in Kightlinger et al., “Synthetic Glycobiology: Parts, Systems, and Applications,” ACS Synth. Biol. 9:1534-62 (2020) are also suitable for producing the enzyme(s) and/or fusion protein(s) described herein.

[0141] Also provided herein are vectors and cells comprising the vectors, as well as kits comprising the proteins and nucleic acids described herein, e.g., for use in a method described herein.

EXAMPLES

[0142] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Hypersialylated IgG Preparation

[0143] IgG in which more than 60% of the overall branched glycans are disialylated can be prepared as follows.

[0144] Briefly, a mixture of IgG antibodies is exposed to a sequential enzymatic reaction using β1,4 galactosyltransferase 1 (B4-GalT) and α2,6-sialyltransferase (ST6-Gal1) enzymes. The B4-GalT does not need to be removed from the reaction before addition of ST6-Gal1 and no partial or complete purification of the product is needed between the enzymatic reactions.

[0145] The galactosyltransferase enzyme selectively adds galactose residues to pre-existing asparagine-linked glycans. The resulting galactosylated glycans serve as substrates to the sialic acid transferase enzyme which selectively adds sialic acid residues to cap the asparagine-linked glycan structures attached to. Thus, the overall sialylation reaction employed two sugar nucleotides (uridine 5′-diphosphogalactose (UDPGal) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA)). The latter is replenished periodically to increase disialylated product relative to monosialylated product. The reaction includes the co-factor manganese chloride.

[0146] A representative example of the IgG-Fc glycan profile for such a reaction starting with IVIg and the reaction product is shown in FIG. 4. In FIG. 4, on the left is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; on the right is the IgG Fc glycan profile for the starting IVIg and hsIgG. In this study, glycan profiles for the different IgG subclasses are derived via glycopeptide mass spectrometry analysis. The peptide sequences used to quantify glycopeptides for different IgG subclasses were: IgG1 = EEQYNSTYR (SEQ ID NO:7), IgG⅔ EEQFNSTFR (SEQ ID NO:8), IgG¾ EEQYNSTFR (SEQ ID NO:9) and EEQFNSTYR (SEQ ID NO:10).

[0147] The glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg the sum of all the nonsialylated glycans is more than 80% and the sum of all sialylated glycans is < 20%. For the reaction product, the sum for all nonsialylated glycans is < 20% and the sum for all sialylated glycans is more than 80%. Nomenclature for different glycans listed in the glycoprofile use the Oxford notation for N linked glycans.

Example 2: Alternative Sialylation Condition

[0148] Alternative suitable reaction conditions for galactosylation and sialylation to create hsIgG in, e.g., 50 mM BIS-TRIS pH 6.9 include: galactosylation of IgG antibodies (e.g., pooled IgG antibodies, pooled immunoglobulins or IVIg) as follows: 7.4 mM MnCl.sub.2; 38 .Math.mol UDP-Gal/g IgG antibody; and 7.5 units B4GalT/g IgG antibody with 16-24 hours of incubation at 37° C. followed by sialylation in 7.4 mM MnCl.sub.2; 220 .Math.mol CMP-NANA/g IgG antibody (added twice: once at the start of the reaction and again after 9-10 hrs); and 15 units ST6-Gal1/g IgG antibody with 30-33 hours of incubation at 37°. The reaction can be carried out by adding the ST6-Gal1 and CMP-NANA to the galactosylation reaction. Alternatively, all of the reactants can be combined at the outset and the CMP-NANA supplemented.

Example 3: Production of ST6Gal

[0149] A fusion protein that includes an enzymatically active portion of ST6Gal was designed for high level expression in HEK cells and ease of purification. SEQ ID NO:6 is the immature fusion protein which includes a portion of human ST6Gal (SEQ ID NO:4), a 6 HIS tag (SEQ ID NO: 13), a signal sequence from azurocidin (MTRLTVLALL AGLLASSRAGSSPLLD (SEQ ID NO:62); 19 aa is signal is underlined) and amino acids resulting from the cloning process. SEQ ID NO:3 is the secreted form, and SEQ ID NO:5 includes the 6 HIS tag (SEQ ID NO: 13) and the ST6GalT portion. SEQ ID NO: 6

TABLE-US-00021 MTRLTVLALLAGLLASSRAGSSPLLDMLEHHHHHHHHMAKPEASFQVWNK DSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHV NVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSGSLKSSQLG REIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLY NEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQ MPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRK TDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLP GFRTIHC

SEQ ID NO: 3

TABLE-US-00022 gssplldmlehhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYL SMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEG YLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTA NFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIP KWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQ PNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMG AYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC

SEQ ID NO: 4

TABLE-US-00023 AKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFS AEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAV VSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQL VTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRK LHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCD QVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGT DEDIYLLGKATLPGFRTIHC

SEQ ID NO: 5

TABLE-US-00024 hhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYK GPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTK AGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKT TIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNF FNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLG IIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKN LVKHLNQGTDEDIYLLGKATLPGFRTIHC

[0150] HEK293 cells (Expi293F®cells; Life Technologies) were stably transfected with a vector expressing a polypeptide having SEQ ID NO: 6 under the control of a CMV promotor. To produce ST6GalT fusion protein, the stably transfected, and clonally selected cells were counted and seeded on Day 0 at a cell density of 0.4E6 cells/mL, grown at 37° C., 5% CO2, 130-150 rpm. On Day 4, a 10% glucose/media feed was added to the cells. Growth was monitored by daily. On Day 7 cell supernatants were harvested, sterile filtered through a 0.45 micron filter and then a 0.2 micron filter.

OTHER EMBODIMENTS

[0151] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.