WO2005121331A9 - Polypeptides galnact2 tronques et acides nucleiques - Google Patents

Polypeptides galnact2 tronques et acides nucleiques

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Publication number
WO2005121331A9
WO2005121331A9 PCT/US2005/019442 US2005019442W WO2005121331A9 WO 2005121331 A9 WO2005121331 A9 WO 2005121331A9 US 2005019442 W US2005019442 W US 2005019442W WO 2005121331 A9 WO2005121331 A9 WO 2005121331A9
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WO
WIPO (PCT)
Prior art keywords
polypeptide
galnact2
nucleic acid
vai
leu
Prior art date
Application number
PCT/US2005/019442
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English (en)
Other versions
WO2005121331A2 (fr
WO2005121331A8 (fr
WO2005121331A3 (fr
Inventor
Karl F Johnson
Xi Chen
Susann Taudte
Sami Saribas
Original Assignee
Neose Technologies Inc
Karl F Johnson
Xi Chen
Susann Taudte
Sami Saribas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neose Technologies Inc, Karl F Johnson, Xi Chen, Susann Taudte, Sami Saribas filed Critical Neose Technologies Inc
Priority to JP2007515582A priority Critical patent/JP2008512085A/ja
Priority to EP05758682A priority patent/EP1765992A4/fr
Publication of WO2005121331A2 publication Critical patent/WO2005121331A2/fr
Publication of WO2005121331A8 publication Critical patent/WO2005121331A8/fr
Publication of WO2005121331A9 publication Critical patent/WO2005121331A9/fr
Publication of WO2005121331A3 publication Critical patent/WO2005121331A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention features compositions and methods related to truncated mutants of GalNAcT2.
  • the invention features truncated human GalNAcT2 polypeptides.
  • the invention also features nucleic acids encoding such truncated polypeptides, as well as vectors, host cells, expression systems, and methods of expressing and using such polypeptides.
  • glycosyltransferases catalyze the synthesis of glycolipids, glycopeptides, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain.
  • a catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme.
  • peptide therapeutics are glycosylated peptides.
  • the production of a recombinant glycopeptide as opposed to a recombinant non-glycosylated peptide, requires that a recombinantly-produced peptide is subjected to additional processing steps, either within the cell or after the peptide is produced by the cell, where the processing steps are performed in vitro.
  • the peptide can be treated enzymatically to introduce one or more glycosyl groups onto the peptide, using a glycosyltransferase. Specifically, the glycosyltransferase covalently attaches the glycosyl group or groups to the peptide.
  • Glycosyltransferases are reviewed in general in International (PCT) Patent Application No. WO03/031464 (PCT/US02/32263), which is incorporated herein by reference in its entirety.
  • One such particular glycosyltransferase that has utility in the development and production of therapeutic glycopeptides is GalNAcT2.
  • GalNAcT2 or N- acetyl-D-galactosamine transferase, catalyzes the transfer of GaINAc from a GaINAc donor to a GaINAc acceptor.
  • Full length human GalNAcT2 enzyme is disclosed by Bennett et al. (1996, J Biol Chem. 271:17006-17012). However, the identification of useful mutants of this enzyme, having enhanced biological activity such as enhanced catalytic activity or enhanced stability, has not heretofore been reported.
  • the present invention provides an isolated nucleic acid comprising a nucleic acid sequence that encodes a truncated human GalNAcT2 polypeptide.
  • the truncated human GalNAcT2 polypeptide lacks all or a portion of the GalNAcT2 signal domain, or in addition lacks all or a portion the GalNAcT2 transmembrane domain, or in addition lacks all or a portion the GalNAcT2 stem domain; with the proviso that the encoded polypeptide is not a human GalNAcT2 truncation mutant polypeptide lacking amino acid residues 1-51.
  • the isolated nucleic acid comprises a nucleic acid sequence having at least 90% identity with a nucleic acid selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9. In another embodiment, the isolated nucleic acid comprises a nucleic acid sequence having at least 95% identity with a nucleic acid selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9. In a further embodiment, the isolated nucleic acid comprises a nucleic acid sequence selected from SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9.
  • the isolated nucleic acid is an isolated chimeric nucleic acid encoding a fusion polypeptide.
  • the fusion polypeptide can include a tag polypeptide covalently linked to a truncated human GalNAcT2 polypeptide, as described herein.
  • tag polypeptides include a maltose binding protein, a histidine tag, a Factor DC tag, a glutathione-S-transferase tag, a FLAG-tag, and a starch binding domain tag.
  • the invention provides an isolated truncated human GalNAcT2 polypeptide, that lacks all or a portion of the GalNAcT2 signal domain, or in addition lacks all or a portion the GalNAcT2 transmembrane domain, or in addition lacks all or a portion the GalNAcT2 stem domain; with the proviso that the encoded polypeptide is not a human GalNAcT2 truncation mutant polypeptide lacking amino acid residues 1-51.
  • the isolated truncated human GalNAcT2 polypeptide has at least 90% or 95% identity with a polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO: 10.
  • isolated truncated human GalNAcT2 polypeptide comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO: 10.
  • the isolated truncated GalNAcT2 polypeptide isolated chimeric polypeptide comprising a tag polypeptide covalently linked to the isolated truncated GalNAcT2.
  • tag polypeptides include a maltose binding protein, a histidine tag, a Factor IX tag, a glutathione-S-transferase tag, a FLAG-tag, and a starch binding domain tag.
  • the isolated nucleic acid encoding a truncated GalNAcT2 polypeptide can also be operably linked to a promoter/regulatory sequence, within e.g., an expression vector.
  • the invention also includes host cells that comprise such expression vectors.
  • Host cells can be e.g., eukaryotic or a prokaryotic cells.
  • Eukaryotic cells include, e.g., mammalian cells, an insect cells, and a fungal cells. Some preferred mammalian host cells are SF9 cells, an SF9+ cells, an Sf21 cells, a HIGH FIVE cells or Drosophila Schneider S2 cells.
  • Prokaryotic host cells include, e.g., E. coli cells and B. subtilis cells.
  • the host cells can be used to producing a truncated human GalNAcT2 polypeptide, by growing the recombinant host cells of under conditions suitable for expression of the truncated human GalNAcT2 polypeptide.
  • sufficient truncated human GalNAcT2 polypeptide is made to allow commercial scale production of a glycoprotein or glycopeptide.
  • the invention includes a method of catalyzing the transfer of a
  • GaINAc moiety to an acceptor moiety comprising incubating the truncated human GalNAcT2 polypeptide with a GaINAc moiety and an acceptor moiety, wherein said polypeptide mediates the covalent linkage of said GaINAc moiety to said acceptor moiety, thereby catalyzing the transfer of a GaINAc moiety to an acceptor moiety to produce a product saccharide, or a product glycoprotein, or a product glycopeptide.
  • the acceptor moiety is a granulocyte colony stimulating factor (G-CSF) protein.
  • G-CSF granulocyte colony stimulating factor
  • the acceptor moiety is selected from erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferons alpha, -beta, and -gamma, Factor IX, follicle stimulating hormone, interleukin-2, erythropoietin, anti-TNF-alpha, and a lysosomal hydrolase.
  • the polypeptide acceptor is a glycopeptide.
  • the GaINAc moiety comprises a polyethylene glycol moiety.
  • the product saccharide, product glycoprotein, or product glycopeptide is produced on a commercial scale.
  • FIG. 1 is an image of an electrophoretic gel illustrating the PCR amplification of ppGalNAcT2 genes.
  • PCRl PCR product for ppGalNAcT2-N41R (1596 bp); PCR2, PCR product for ppGalNAcT2-N52K (1563 bp); PCR3, PCR product for ppGalNAcT2-N74G (1497 bp); PCR4, PCR product for ppGalNAcT2-N95G (1434 bp).
  • Figure 2A is a plasmid restriction map for the pCWin2MBP vector.
  • Figure 2B is an image of an electrophoretic gel illustrating the fragments resulting from multiple samples of the pCWin2MBP vector digested by both BamHI and Xhol restriction enzymes.
  • Figure 3 is an image of an electrophoretic gel illustrating the screening of DH5 ⁇ (pCWin2MBP-ppGalNAcT2) colonies by restriction mapping (BamHI and Xhol digestion) for plasmid purified from twelve colonies.
  • Lane M bp ladder. Lanes 1-3, N41R; lanes 4-6, N52K; lanes 8-10, N74G; lanes 11-13, N95G.
  • FIG. 4 is an image of an electrophoretic protein gel illustrating SDS-PAGE for JM 109 (pCWin2MBP-ppGalNAcT2) whole cell lysates after IPTG induction as described elsewhere herein.
  • M Pre-Stained MW Standard; Lane 13, IPTG-induced JMl 09
  • FIG. 5 is an image of an electrophoretic protein gel illustrating SDS-PAGE for JM109 (pCWin2MBP-p ⁇ GalNAcT2) cell lysates.
  • Figure 6 is an image of an electrophoretic protein gel illustrating SDS-PAGE for inclusion bodies isolated from JMl 09 ( ⁇ CWin2MBP- ⁇ pGalNAcT2) cells.
  • M Pre-Stained MW Standard; Lane 13, inclusion bodies from JM109 (pCWin2MBP); Lanes 1-12, inclusion bodies from colonies 1-12; Lanes 1-3, JM109 (pCWin2MBP-p ⁇ GalNAcT2N41R); Lanes 4-6, JMl 09 (pCWin2MBP-ppGalNAcT2N52K); Lanes 7-9, JMl 09 (pCWin2MBP- ppGalNAcT2N74G); Lanes 10-12, JM109 (pCWin2MBP-ppGalNAcT2N95G).
  • Figure 7 is an image of an electrophoretic gel illustrating the protein expression pattern in lysates of cells containing human GalNAcT2 constructs.
  • Lane 1 molecular weight marker
  • lane 2 construct 1 culture before induction
  • lane 3 construct 1 culture after induction
  • lane 4 construct 2 culture before induction
  • lane 5 construct 2 culture after induction
  • lane 9, construct 4 culture after induction lane 10, empty.
  • Figure 8 is an image of an electrophoretic protein gel illustrating the protein content of inclusion bodies from JM109 pCWin2 MBP-GalNAcT2 constructs. Lane 1, MW marker; lane 2, JM109 pCWin2 MBP-GalNAcT2 construct 1 inclusion bodies; lane 3, JM109 ⁇ CWin2 MBP-GalNAcT2 construct 2 inclusion bodies.
  • Figure 9 is an image of an electrophoretic protein gel illustrating the glycoPEGylation of G-CSF by ⁇ 51 GalNAcT2-MBP.
  • Lane 1 glycoPEGylation in the presence of 1 mg/ml G-CSF;
  • lane 2 glycoPEGylation in the presence of 0.7 mg/ml G-CSF;
  • lane 3 glycoPEGylation in the presence of 0.4 mg/ml G-CSF;
  • lane 4 glycoPEGylation in the presence of 0.2 mg/ml G-CSF.
  • the glycoPEGylated G-CSF is visible around 60 kDa.
  • Figures 1OA and 1OB depict a nucleic acid sequence encoding a ⁇ 40 GaIN AcT2 polypeptide.
  • Figures 1 IA and 1 IB depict a nucleic acid sequence encoding a ⁇ 51 GalNAcT2 polypeptide.
  • Figures 12A and 12B depict a nucleic acid sequence encoding a ⁇ 73 GalNAcT2 polypeptide.
  • Figures 13 A and 13B depict a nucleic acid sequence encoding a ⁇ 94 GalNAcT2 polypeptide.
  • Figure 14A is an image of a chromatogram illustrating the elution of ⁇ 51
  • GalNAcT2-MBP that was refolded at pH 5.5 and subsequently eluted from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 14B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 14A. The contents of each lane on the gel are described in the figure.
  • Figure 14C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 14 A.
  • Figure 15 A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP that was refolded at pH 6.5 and subsequently eluted from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 15B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 15 A. The contents of each lane on the gel are described in the figure.
  • Figure 15C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 15 A.
  • Figure 16A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP that was refolded at pH 8.0 and subsequently eluted from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 16B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 16 A. The contents of each lane on the gel are described in the figure.
  • Figure 16C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 16 A.
  • Figure 17A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP that was refolded at pH 8.5 and subsequently eluted from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 17B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 17 A. The contents of each lane on the gel are described in the figure.
  • Figure 17C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 17 A.
  • Figure 18 A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP that was refolded at pH 8.0 and subsequently eluted from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 18B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 18 A. The contents of each lane on the gel are described in the figure.
  • Figure 18C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 18 A.
  • Figure 19 A is an image of a chromatogram illustrating the elution of ⁇ 51
  • GalNAcT2-MBP from a Q-sepharose fast flow column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 19B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 19 A. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 19 A.
  • Figure 19C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 19 A.
  • Figure 20 A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP from a Q-sepharose XL column, using 5 mM NaCl. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y- axis.
  • Figure 2OB is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 2OA. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 2OA.
  • Figure 2OC is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 2OA.
  • Figure 21 A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP from a Q-sepharose XL column, using 50 mM NaCl. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y- axis.
  • Figure 2 IB is an image of two electroplioretic gels used to visualize the eluted fractions set forth in Figure 2 IA. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 21 A.
  • Figure 21C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 21 A.
  • Figure 22A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP from a Q-sepharose XL column, using 100 niM NaCl. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y- axis.
  • Figure 22B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 22A. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 22 A.
  • Figure 22C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 22A.
  • Figure 23 A is an image of a chromatogram illustrating the elution of ⁇ 51
  • GalNAcT2-MBP from a Q-sepharose XL column, using 200 mM NaCl. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y- axis.
  • Figure 23B is an image of two electrophoretic gels used to visualize the eluted fractions set forth in Figure 23 A. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 23 A.
  • Figure 23 C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 23 A.
  • Figure 24A is an image of a chromatogram illustrating the elution of ⁇ 51 GalNAcT2-MBP from a Hydroxyapatite Type I column. Fraction numbers are indicated on the X-axis and the relative absorbance of each fraction is indicated on the Y-axis.
  • Figure 24B is an image of an electrophoretic gel used to visualize the eluted fractions set forth in Figure 24A. The contents of each lane on the gel are described in the figure and correspond to the chromatogram of Figure 24A.
  • Figure 24C is a table illustrating the relative GaINAc transferase activity of the fractions set forth in Figure 24A.
  • Figure 25 is a graph illustrating the relative GaINAc transferase activity of various preparations of refolded ⁇ 51 GalNAcT2-MBP. The refolding conditions of each preparation is indicated on the x-axis, and the relative GaINAc transferase activity is illustrated on the Y- axis.
  • Figure 26 is a graph illustrating the relative GaINAc transferase activity of various preparations of refolded ⁇ 51 GalNAcT2-MBP. The refolding conditions of each preparation is indicated on the x-axis, and the relative GaINAc transferase activity is illustrated on the Y- axis.
  • Figure 27 is an image of three MALDI-TOF spectra demonstrating GaINAc transfer to GCSF mediated by ⁇ 51 GalNAcT2-MBP that has been refolded and purified according to the present invention.
  • Figure 28 is an image of three MALDI-TOF spectra demonstrating GaINAc transfer to GCSF mediated by ⁇ 51 GalNAcT2-MBP that has been refolded and purified according to the present invention.
  • compositions and methods of the present invention encompass truncation mutants of human GalNAcT2 polypeptides, isolated nucleic acids encoding these proteins, and methods of their use.
  • GalNAcT2 polypeptides catalyze the transfer of a GaINAc from a GaINAc donor to a GaINAc acceptor.
  • the glycosyltransferase GalNAcT2 is an essential reagent for glycosylation of therapeutic glycopeptides. Additionally, GalNAcT2 is an important reagent for research and development of therapeutically important glycopeptides and oligosaccharide therapeutics. GalNAcT2 enzymes are typically isolated and purified from natural sources, or from tedious and costly in vitro and recombinant sources.
  • the present invention provides compositions and methods relating to simplified and more cost-effective methods of production of GalNAcT2 enzymes. In particular, the present invention provides compositions and methods relating to truncated GalNAcT2 enzymes that have improved and useful properties in comparison to their full-length enzyme counterparts.
  • Truncated glycosyltransferase enzymes of the present invention are useful for in vivo and in vitro preparation of glycosylated peptides, as well as for the production of oligosaccharides containing the specific glycosyl residues that can be transferred by the truncated glycosyltransferase enzymes of the present invention. This is because it is shown for the first time herein that truncated forms of GalNAcT2 polypeptides possess biological activities comparable to, and in some instances, in excess of their full-length polypeptide counterparts. The present application also discloses that such truncation mutants not only possess biological activity, but also that the truncation mutants may have enhanced properties of solubility, stability and resistance to proteolytic degradation.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an rnRNA molecule which is produced by transcription of the gene.
  • a "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
  • the coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule ⁇ e.g., amino acid residues in a protein export signal sequence).
  • An "affinity tag” is a peptide or polypeptide that may be genetically or chemically fused to a second polypeptide for the purposes of purification, isolation, targeting, trafficking, or identification of the second polypeptide.
  • the "genetic" attachment of an affinity tag to a second protein may be effected by cloning a nucleic acid encoding the affinity tag adjacent to a nucleic acid encoding a second protein in a nucleic acid vector.
  • glycosyltransferase refers to any enzyme/protein that has the ability to transfer a donor sugar to an acceptor moiety.
  • a "sugar nucleotide-generating enzyme” is an enzyme that has the ability to produce a sugar nucleotide.
  • Sugar nucleotides are known in the art, and include, but are not limited to, such moieties as UDP-GaI, UDP-GaINAc, and CMP-NAN.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides. However, the terms “nucleic acid” and “polynucleotide” are used interchangeably herein.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • nucleic acid sequences the left- hand end of a single-stranded nucleic acid sequence is the 5' end; the left-hand direction of a double-stranded nucleic acid sequence is referred to as the 5'-direction.
  • a first defined nucleic acid sequence is said to be "immediately adjacent to" a second defined nucleic acid sequence when, for example, the last nucleotide of the first nucleic acid sequence is chemically bonded to the first nucleotide of the second nucleic acid sequence through a phosphodiester bond.
  • a first defined nucleic acid sequence is also said to be "immediately adjacent to" a second defined nucleic acid sequence when, for example, the first nucleotide of the first nucleic acid sequence is chemically bonded to the last nucleotide of the second nucleic acid sequence through a phosphodiester bond.
  • a first defined polypeptide sequence is said to be "immediately adjacent to" a second defined polypeptide sequence when, for example, the last amino acid of the first polypeptide sequence is chemically bonded to the first amino acid of the second polypeptide sequence through a peptide bond.
  • a first defined polypeptide sequence is said to be "immediately adjacent to" a second defined polypeptide sequence when, for example, the first amino acid of the first polypeptide sequence is chemically bonded to the last amino acid of the second polypeptide sequence through a peptide bond.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • Homology refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
  • a region having the nucleotide sequence 5'- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positionss of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • percent identity is used synonymously with “homology.”
  • the determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J. MoI. Biol.
  • NCBI National Center for Biotechnology Information
  • NLM National Library of Medicine
  • NIH National Institutes of Health
  • BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).
  • PSI-Blast or PHI-Blast can be used to perfonn an iterated search which detects distant relationships between molecules (id.) and relationships between molecules which share a common pattern.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs can be used as available on the website of the National Center for Biotechnology Information of the National Library of Medicine at the National Institutes of Health.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. A "polypeptide,” as the term is used herein, therefore refers to any size polymer of amino acid residues, provided that the polymer contains at least two amino acid residues.
  • protein typically refers to large peptides, also referred to herein as “polypeptides.”
  • peptide typically refers to short polypeptides.
  • peptide may refer to an amino acid polymer of three amino acids, as well as an amino acid polymer of several hundred amino acids.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • a "therapeutic peptide” as the term is used herein refers to any peptide that is useful to treat a disease state or to improve the overall health of a living organism.
  • a therapeutic peptide may effect such changes in a living organism when administered alone, or when used to improve the therapeutic capacity of another substance.
  • the term “therapeutic peptide” is used interchangeably herein with the terms “therapeutic polypeptide” and “therapeutic protein.”
  • a "reagent peptide” as the term is used herein refers to any peptide that is useful in food biochemistry, bioremediation, production of small molecule therapeutics, and even in the production of therapeutic peptides.
  • reagent peptides are enzymes capable of catalyzing a reaction to produce a product useful in any of the aforementioned areas.
  • the term “reagent peptide” is used interchangeably herein with the terms “reagent polypeptide” and "reagent protein.”
  • glycopeptide refers to a peptide having at least one carbohydrate moiety covalently linked thereto. It will be understood that a glycopeptide may be a "therapeutic glycopeptide,” as described above.
  • glycopeptide is used interchangeably herein with the terms “glycopolypeptide” and “glycoprotein.”
  • a "vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear nucleic acids, nucleic acids associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Expression vector refers to a vector comprising a recombinant nucleic acid comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant nucleic acid.
  • a "multiple cloning site" as the term is used herein is a region of a nucleic acid vector that contains more than one sequence of nucleotides that is recognized by at least one restriction enzyme.
  • an "antibiotic resistance marker” as the term is used herein refers to a sequence of nucleotides that encodes a protein which, when expressed in a living cell, confers to that cell the ability to live and grow in the presence of an antibiotic.
  • GalNAcT2 refers to N-acetyl-D-galactosamine transferase 2.
  • a "truncated" form of a peptide refers to a peptide that is lacking one or more amino acid residues as compared to the full-length amino acid sequence of the peptide.
  • the peptide "NH2-Ala-Glu-Lys-Leu-COOH” is an N-terminally truncated form of the full-length peptide "NH2-Gly-Ala-Glu-Lys-Leu-COOH.”
  • the terms "truncated form” and “truncation mutant” are used interchangeably herein.
  • a truncated peptide is a GalNAcT2 polypeptide comprising an active domain, a stem domain, a transmembrane domain, and a signal domain, wherein the signal domain is lacking a single N-terminal amino acid residue as compared to the full length GalNAcT2.
  • saccharides refers in general to any carbohydrate, a chemical entity with the most basic structure Of(CH 2 O) n . Saccharides vary in complexity, and may also include nucleic acid, amino acid, or virtually any other chemical moiety existing in biological systems.
  • Olet al. refers to a molecule consisting of several units of carbohydrates of defined identity. Typically, saccharide sequences between 2-20 units may be referred to as oligosaccharides.
  • Polysaccharide refers to a molecule consisting of many units of carbohydrates of defined identity. However, any saccharide of two or more units may correctly be considered a polysaccharide.
  • a saccharide "donor” is a moiety that can provide a saccharide to a glycosyltransferase so that the glycosyltransferase may transfer the saccharide to a saccharide acceptor.
  • a GaINAc donor may be UDP-GaINAc.
  • a saccharide "acceptor” is a moiety that can accept a saccharide from a saccharide donor.
  • a glycosyltransferase can covalently couple a saccharide to a saccharide acceptor.
  • G-CSF may be a GaINAc acceptor, and a GaINAc moiety may be covalently coupled to a GaINAc acceptor by way of a GaINAc- transferase.
  • a saccharide acceptor is a protein or peptide comprising an O glycosylation site.
  • saccharide acceptors include, e.g., erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferons alpha, -beta, and -gamma, Factor IX, follicle stimulating hormone, interleukin-2, erythropoietin, anti-TNF-alpha, and a lysosomal hydrolase
  • An oligosaccharide with a "defined size” is one which consists of an identifiable number of monosaccharide units.
  • an oligosaccharide consisting of 10 monosaccharide units is one which may consist of 10 identical monosaccharide units or 5 monosaccharide units of a first identity and 5 monosaccharide units of a second identity.
  • an oligosaccharide of defined size that consists of monosaccharide units of heterogeneous identity may have the monosaccharide units in any order from beginning to end of the oligosaccharide.
  • An oligosaccharide of "random size" is one which may be synthesized using methods that do not provide oligosaccharide products of defined size.
  • a method of oligosaccharide synthesis may provide oligosaccharides that range from two monosaccharide units to twenty-two saccharide units, including any or all lengths in between.
  • Communication scale refers to gram scale production of a product saccharide, or glycoprotein, or glycopeptide in a single reaction. In preferred embodiments, commercial scale refers to production of greater than about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 grams.
  • 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-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-l-onic acid (often abbreviated as Neu5Ac, NeuAc, or NANA).
  • a second member of the family is N-glycolyl- neuraminic acid (Neu5Gc or NeuGc), in which the N- acetyl group of NeuAc is hydroxylated.
  • a third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. Q.9S6) J. Biol. Chem. 261: 11550-11557; Kanamori et al, J. Biol. Chem. 265: 21811-21819 (1990)). Also included are 9-substituted sialic acids such as a 9-0-C 1 -C 6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy- Neu5Ac.
  • KDN 2-keto-3-deoxy-nonulosonic acid
  • 9-substituted sialic acids such as a 9-0-C 1 -C 6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-ace
  • sialic acid family see, e.g., Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992)).
  • the synthesis and use of sialic acid compounds in a sialylation procedure is disclosed in international application WO 92/16640, published October 1, 1992.
  • a "method of remodeling a protein, a peptide, a glycoprotein, or a glycopeptide” as used herein, refers to addition of a sugar residue to a protein, a peptide, a glycoprotein, or a glycopeptide using a glycosyltransferase.
  • the sugar residue is covalently attached to a PEG molecule.
  • an "unpaired cysteine residue” as used herein, refers to a cysteine residue, which in a correctly folded protein (i.e., a protein with biological activity), does not form a disulfide bind with another cysteine residue.
  • an "insoluble glycosyltransferase” refers to a glycosyltransferase that is expressed in bacterial inclusion bodies. Insoluble glycosyltransferases are typically solubilized or denatured using e.g., detergents or chaotropic agents or some combination. "Refolding” refers to a process of restoring the structure of a biologically active glycosyltransferase to a glycosyltransferase that has been solubilized or denatured. Thus, a refolding buffer, refers to a buffer that enhances or accelerates refolding of a glycosyltransferase.
  • a "redox couple” refers to mixtures of reduced and oxidized thiol reagents and include reduced and oxidized glutathione (GSH/GSSG), cysteine/cystine, cysteamine/cystamine, DTT/GSSG, and DTE/GSSG. (See, e.g., Clark, Cur. Op. Biotech. 12:202-207 (2001)).
  • contacting is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc.
  • PEG refers to polyethylene glycol
  • PEG is an exemplary polymer that has been conjugated to peptides.
  • the use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides and prolong the clearance time from the circulation.
  • U.S. Pat. No. 4,179,337 (Davis et al.) concerns non- immunogenic peptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of polymer are used per mole peptide and at least 15% of the physiological activity is maintained.
  • the term "specific activity" as used herein refers to the catalytic activity of an enzyme, e.g., a recombinant glycosyltransferase fusion protein of the present invention, and may be expressed in activity units.
  • one activity unit catalyzes the formation of 1 ⁇ mol of product per minute at a given temperature (e.g., at 37°C) and pH value (e.g., at pH 7.5).
  • 10 units of an enzyme is a catalytic amount of that enzyme where 10 ⁇ mol of substrate are converted to 10 ⁇ mol of product in one minute at a temperature of, e.g., 37 °C and a pH value of, e.g., 7.5.
  • N-linked oligosaccharides are those oligosaccharides that are linked to a peptide backbone through asparagine, by way of an asparagine-N-acetylglucosamine linkage. N- linked oligosaccharides are also called “N-glycans.” All N-linked oligosaccharides have a common pentasaccharide core of Man 3 GIcNAc 2 . They differ in the presence of, and in the number of branches (also called antennae) of peripheral sugars such as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. Optionally, this structure may also contain a core fucose molecule and/or a xylose molecule.
  • O-linked oligosaccharides are those oligosaccharides that are linked to a peptide backbone through threonine, serine, hydroxyproline, tyrosine, or other hydroxy-containing amino acids.
  • substantially in the above definitions of "substantially uniform” generally means at least about 60%, at least about 70%, at least about 80%, or more preferably at least about 90%, and still more preferably at least about 95% of the acceptor substrates for a particular glycosyltransferase are glycosylated.
  • a "fusion protein” refers to a protein comprising amino acid sequences that are in addition to, in place of, less than, and/or different from the amino acid sequences encoding the original or native full-length protein or subsequences thereof.
  • a "stem region" with reference to glycosyltransferases refers to a protein domain, or a subsequence thereof, which in the native glycosyltransferases is located adjacent to the trans-membrane domain, and has been reported to function as a retention signal to maintain the glycosyltransferase in the Golgi apparatus and as a site of proteolytic cleavage.
  • Stem regions generally start with the first hydrophilic amino acid following the hydrophobic transmembrane domain and end at the catalytic domain, or in some cases the first cysteine residue following the transmembrane domain.
  • Exemplary stem regions include, but is not limited to, the stem region of eukaryotic ST ⁇ GalNAcI, amino acid residues from about 30 to about 207 (see e.g., the murine enzyme), amino acids 35-278 for the h uman enzyme or amino acids 37-253 for the chicken enzyme; the stem region of mammalian GalNAcT2, amino acid residues from about 71 to about 129 (see e.g., the rat enzyme).
  • a "catalytic domain” refers to a protein domain, or a subsequence thereof, that catalyzes an enzymatic reaction performed by the enzyme.
  • a catalytic domain of a sialyltransferase will include a subsequence of the sialyltransferase sufficient to transfer a sialic acid residue from a donor to an acceptor saccharide.
  • a catalytic domain can include an entire enzyme, a subsequence thereof, or can include additional amino acid sequences that are not attached to the enzyme, or a subsequence thereof, as found in nature.
  • isolated refers to material that is substantially or essentially free from components which interfere with the activity of an enzyme.
  • a saccharide, protein, or nucleic acid of the invention refers to material that is substantially or essentially free from components which normally accompany the material as found in its native state.
  • 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 band intensity on a silver stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art.
  • 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.
  • high resolution of the protein or nucleic acid may be desirable and HPLC or a similar means for purification, for example, may be utilized.
  • GalNAcT2 nucleic acids encode polypeptides that have a domain structure similar to other glycosyltransferases, including an N-terminal signal domain, a transmembrane domain, a stem domain, and an active domain, wherein the active domain may comprise the majority of the amino acid sequence of such polypeptides.
  • domain structure(s) extraneous to the active domain of recombinant GalNAcT2 polypeptides may have a negative effect on the solubility, stability and activity of the polypeptide in an aqueous or in vitro environment.
  • the presence of a hydrophobic transmembrane domain on a recombinant GaIN AcT2 polypeptide used in an in vitro reaction mixture may render the polypeptide less soluble than a recombinant GalNAcT2 polypeptide without a hydryophobic transmembrane domain, and further, may even decrease the enzymatic activity of the polypeptide by affecting or destabilizing the folded structure.
  • GalNAcT2 nucleic acids that encode GalNAcT2 that is shorter than full-length GalNAcT2, for the purpose of enhancing the activity, stability and/or utility of GalNAcT2 polypeptides.
  • the present invention provides such modified forms of GalNAcT2. More particularly, the present invention provides isolated nucleic acids encoding such truncated polypeptides.
  • Nucleic acids of the present invention encode truncated forms of GalNacT2 polypeptides, as described in greater detail elsewhere herein.
  • a truncated GalNAcT2 polypeptide encoded by a nucleic acid of the present invention also referred to herein as a "truncation mutant,” may be truncated in various ways, as would be understood by the skilled artisan.
  • Examples of truncated polypeptides encoded by a nucleic acid of the present invention include, but are not limited to, a polypeptide lacking a single N-terminal residue, a polypeptide lacking a single C-terminal residue, a polypeptide lacking both an single N- terminal residue and a single C-terminal residue, a polypeptide lacking a contiguous sequence of residues from the N-terminus, a polypeptide lacking a contiguous sequence of residues from the C-terminus, and any combinations thereof.
  • truncations of nucleic acids encoding GalNAcT2 polypeptides may be made for numerous reasons.
  • a truncation may be made in order to remove part or all of the nucleic acid sequence encoding the signal peptide domain of an GalNAcT2.
  • a truncation may be made in order to remove part or all of a nucleic acid sequence encoding a transmembrane domain of an GalNAcT2.
  • removal of a part or all of a nucleic acid sequence encoding a transmembrane domain may increase the solubility or stability of the encoded GalNAcT2 polypeptide and/or may increase the level of expression of the encoded polypeptide.
  • a truncation may be made in order to remove part or all of a nucleic acid sequence encoding a stem domain of an GalNAcT2.
  • removal of a part or all of a nucleic acid sequence encoding a stem domain may increase the solubility or stability of the encoded GalNAcT2 polypeptide and/or may increase the level of expression of the encoded polypeptide.
  • the nucleic acid residue at which a truncation is made may be a highly-conserved residue.
  • the nucleic acid residue at which a truncation is made may be selected such that the encoded polypeptide has a new N-terminal amino acid residue that will aid in the purification of the expressed polypeptide.
  • the nucleic acid residue at which a truncation is made may be selected such that the encoded truncated polypeptide does not contain a specific secondary and/or tertiary structure.
  • the present invention features nucleic acids encoding smaller than full-length GalNAcT2. That is, the present invention features a nucleic acid encoding a truncated GalNAcT2 polypeptide, provided the polypeptide expressed by the nucleic acid retains the biological activity of the full-length protein.
  • a truncated polypeptide is a human truncated GalNAcT2 polypeptide.
  • a nucleic acid encoding a full-length human GalNAcT2 may contain a nucleic acid sequence encoding one or more identifyable polypeptide domains in addition to the "active domain," the domain primarily responsible for the catalytic activity, of GalNAcT2. This is because it is known in that art that a full-length GalNAcT2 polypeptide, and in particular, a full-length human GalNAcT2 polypeptide, contains a signal domain, a transmembrane domain, and a stem domain, in addition to an active domain.
  • a nucleic acid encoding a full-length human GalNAcT2 may encode a polypeptide that has a signal domain at the amino-terminus of the polypeptide, followed by a transmembrane domain immediately adjacent to the signal domain, followed by a stem domain that is immediately adjacent to the transmembrane domain, followed by an active domain that extends to the carboxy-terminus of the polypeptide and is located immediately adjacent to the stem domain.
  • an isolated nucleic acid of the invention may encode a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide is lacking all or a portion of the GalNAcT2 signal domain.
  • an isolated nucleic acid of the invention may encode a truncated human
  • a nucleic acid of the invention may encode a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide is lacking the GalNAcT2 signal domain, the GalNAcT2 transmembrane domain and all or a portion the GalNAcT2 stem domain.
  • the "biological activity of GalNAcT2" is the ability to transfer a GaINAc moiety from a GaINAc donor to an acceptor molecule.
  • Full-length human GalNAcT2 the sequence of which is set forth in SEQ ID NO:1, exhibits such activity.
  • the "biological activity of a GalNAcT2 truncated polypeptide” is similarly the ability to transfer a GaINAc moiety from a GaINAc donor to an acceptor molecule. That is, a truncated GalNAcT2 polypeptide of the present invention can catalyze the same glycosyltransfer reaction as the full-length GalNAcT2.
  • a truncated human GalNAcT2 polypeptide encoded by a GalNAcT2 nucleic acid of the invention has the ability to transfer a GaINAc moiety from a UDP-GaINAc donor to a granulocyte-colony stimulating factor (G-CSF) acceptor, wherein such a transfer results in the O-linked covalent coupling of a GaINAc moiety to a threonine residue of G-CSF.
  • G-CSF granulocyte-colony stimulating factor
  • GalNAcT2 is included in the present invention provided that the truncated GalNAcT2 has GalNAcT2 biological activity.
  • the methods and compositions of the invention should not be construed to be limited solely to a nucleic acid comprising a GalNAcT2 truncation mutant as disclosed herein, but rather, should be construed to encompass any nucleic acid encoding a GalNAcT2 truncated mutant, prepared in accordance with the disclosure herein, either known or unknown, which is capable of catalyzing transfer of a GaINAc to a GaINAc acceptor. Modified nucleic acid sequences, i.e.
  • nucleic acid sequences having sequences that differ from the nucleic acid sequences encoding the naturally-occurring proteins are also encompassed by methods and compositions of the invention, so long as the modified nucleic acid still encodes a truncated protein having the biological activity of catalyzing the transfer of a GaINAc to a GaINAc acceptor, for example.
  • modified nucleic acid sequences include modifications caused by point mutations, modifications due to the degeneracy of the genetic code or naturally occurring allelic variants, and further modifications that have been introduced by genetic engineering, i.e., by the hand of man.
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
  • the present invention features an isolated nucleic acid comprising a nucleic acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to a nucleic acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:9.
  • the present invention also features an isolated nucleic acid sequence comprising any one of the sequences set forth in SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:9, wherein the isolated nucleic acid encodes a truncated GalNAcT2 polypeptide.
  • the present invention also encompasses isolated nucleic acid molecules encoding a truncated GalNAcT2 polypeptide that contains changes in amino acid residues that are not essential for activity.
  • Such polypeptides encoded by an isolated nucleic acid of the invention differ in amino acid sequence from any one of the sequences set forth in SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO: 10, yet retain the biological activity of GalNAcT2.
  • an isolated nucleic acid of the invention may include a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4.
  • an isolated nucleic acid of the invention may include a nucleotide sequence encoding a polypeptide that has an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NO:8 or SEQ ID NO: 10.
  • the determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci.
  • NBLAST and XBLAST programs of Altschul, et al. (1990, J. MoI. Biol. 215:403- 410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site.
  • BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).
  • PSI-Blast or PHI- Blast can be used to perform an iterated search which detects distant relationships between molecules and relationships between molecules which share a common pattern.
  • the default parameters of the respective programs can be used. See, generally, the internet website for the National Center for Biotechnology Information, which is maintained by the National Library of Medicine and the National Institutes of Health.
  • a nucleic acid useful in the methods and compositions of the present invention and encoding a truncated GalNAcT2 polypeptide may have at least one nucleotide inserted into the nucleic acid sequence of such a truncated mutant.
  • an additional nucleic acid encoding a truncated GalNAcT2 polypeptide may have at least one nucleotide deleted from the nucleic acid sequence.
  • a GalNAcT2 nucleic acid encoding a truncated mutant and useful in the invention may have both a nucleotide insertion and a nucleotide deletion present in a single nucleic acid sequence encoding the truncated polypeptide.
  • nucleic acid insertions and/or deletions may be designed into the gene for numerous reasons, including, but not limited to modification of nucleic acid stability, modification of nucleic acid expression levels, modification of expressed polypeptide stability or half-life, modification of expressed polypeptide activity, modification of expressed polypeptide properties and characteristics, and changes in glycosylation pattern. All such modifications to the nucleotide sequences encoding such proteins are encompassed by the present invention.
  • nucleic acid encompassed by methods and compositions of the invention may be native or synthesized nucleic acid.
  • the nucleic acid may be DNA or RNA and may exist in a double-stranded, single-stranded or partially double-stranded form. Furthermore, the nucleic acid may be found as part of a virus or other macromolecule. See, e.g., Fasbender et al, 1996, J. Biol. Chem. 272:6479-89.
  • the invention includes an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the polypeptide encoded by the nucleic acid.
  • the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in those cells, as described, for example, in Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • Expression of a truncated GalNAcT2 polypeptide in a cell may be accomplished by generating a plasmid, viral, or other type of vector comprising a nucleic acid encoding the appropriate nucleic acid, wherein the nucleic acid is operably linked to a promoter/regulatory sequence which serves to drive expression of the encoded polypeptide, with or without tag, in cells in which the vector is introduced, hi addition, promoters which are well known in the art which are induced in response to inducing agents such as metals, glucocorticoids, and the like, are also contemplated in the invention.
  • promoters which are well known in the art which are induced in response to inducing agents such as metals, glucocorticoids, and the like, are also contemplated in the invention.
  • the invention includes the use of any promoter/regulatory sequence, which is either known or unknown, and which is capable of driving expression of the truncated GalNAcT2 polypeptide operably linked there
  • a nucleic acid encoding a truncated GalNAcT2 polypeptide may be fused to one or more additional nucleic acids encoding a functional polypeptide.
  • an affinity tag coding sequence may be inserted into a nucleic acid vector adjacent to, upstream from, or downstream from a truncated GalNAcT2 polypeptide coding sequence.
  • an affinity tag will typically be inserted into a multiple cloning site in frame with the truncated GalNAcT2 polypeptide.
  • an affinity tag coding sequence can be used to produce a recombinant fusion protein by concomitantly expressing the affinity tag and truncated GalNAcT2 polypeptide. The expressed fusion protein can then be isolated, purified, or identified by means of the affinity tag.
  • Affinity tags useful in the present invention include, but are not limited to, a maltose binding protein, a histidine tag, a Factor IX tag, a glutathione-S-transferase tag, a FLAG-tag, and a starch binding domain tag.
  • Other tags are well known in the art, and the use of such tags in the present invention would be readily understood by the skilled artisan.
  • a vector comprising a truncated GalNAcT2 polypeptide of the present invention may be used to express the truncated polypeptide as either a non-fusion or as a fusion protein.
  • Selection of any particular plasmid vector or other DNA vector is not a limiting factor in this invention and a wide plethora of vectors are well-known in the art. Further, it is well within the skill of the artisan to choose particular promoter/regulatory sequences and operably link those promoter/regulatory sequences to a DNA sequence encoding a truncated GalNAcT2 polypeptide.
  • a vector useful in one embodiment of the present invention is based on the pcWori+ vector (Muchmore et al., 1987, Meth. Enzymol. 177:44-73).
  • the invention thus includes a vector comprising an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide.
  • a nucleic acid encoding a truncated GalNAcT2 polypeptide.
  • the incorporation of a nucleic acid into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • GalNAcT2 polypeptide is integrated into the genome of a host cell in conjunction with a nucleic acid encoding a truncated GalNAcT2 polypeptide.
  • a cell is transiently transfected with an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide.
  • a cell is stably transfected with an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide.
  • a nucleic acid encoding a truncated GalNAcT2 polypeptide may be purified by any suitable means, as are well known in the art.
  • the nucleic acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis.
  • the method of purification will depend in part on the size of the DNA to be purified.
  • the present invention also features a recombinant bacterial host cell comprising , inter alia, a nucleic acid vector as described elsewhere herein, hi one aspect, the recombinant cell is transformed with a vector of the present invention.
  • the transformed vector need not be integrated into the cell genome nor does it need to be expressed in the cell. However, the transformed vector will be capable of being expressed in the cell, hi one aspect of the invention, E. coli is used for transformation of a vector of the present invention and expression of protein therefrom, hi another aspect of the invention, a K- 12 strain of E. coli is useful for expression of protein from a vector of the present invention.
  • Strains of E. coli useful in the present invention include, but are not limited to, JM83, JMlOl, JM103, JM109, W3110, chil776, and JA221.
  • a host cell useful in the present invention will be capable of growth and culture on a small scale, medium scale, or a large scale.
  • a host cell of the invention is useful for testing the expression of a protein from a vector of the invention equally as much as it is useful for large scale production of a reagent or therapeutic protein product.
  • Techniques useful in culturing host cells and expressing protein from a vector contained therein are well known in the art and will therefore not be listed herein.
  • a host cell useful in methods of the present invention may be prepared according to various methods, as would be understood by the skilled artisan when arniend with the disclosure set forth herein, hi one aspect, a host cell of the present invention may be transformed with a vector of the present invention to produce a transformed host cell of the invention. Transformation, as known to the skilled artisan, includes the process of inserting a nucleic acid vector into a host cell, such that the host cell containing the nucleic acid vector remains viable.
  • Such transformation of nucleic acid into a bacterial cell is useful for purposes including, but not limited to, creation of a stably-transformed host cell, making a biological deposit, propagating the vector-containing host cell, propagating the vector- containing host cell for the production and isolation of additional vector, expression of target protein encoded by vector, and the like.
  • a competent bacterial cell of the invention may be transformed by a vector of the invention using electroporation.
  • Methods of making bacterial cells "competent" are well-known in the art, and typically involve preparation of the bacterial cells so that the cells take up exogenous DNA.
  • methods of electroporation are known in the art, and detailed descriptions of such methods may be found, for example, in Sambrook et al. (1989, supra).
  • the transformation of a competent cell with vector DNA may be also accomplished using chemical-based methods.
  • One example of a well-known chemical-based method of bacterial transformation is described by Inoue, et al. (1990, Gene 96:23-28). Other methods of transformation will be known to the skilled artisan.
  • a transformed host cell of the present invention may be used to express a truncated GalNAcT2 polypeptide of the present invention.
  • a transformed host cell contains a vector of the invention, which contains therein a nucleic acid sequence encoding an truncated polypeptide of the invention.
  • the truncated polypeptide is expressed using any expression method known in the art (for example, IPTG).
  • IPTG IPTG
  • the expressed truncated polypeptide may be contained within the host cell, or it may be secreted from the host cell into the growth medium.
  • an expressed polypeptide that is secreted from a host cell may be isolated from the growth medium. Isolation of a polypeptide from a growth medium may include removal of bacterial cells and cellular debris. By way of another non-limiting example, an expressed polypeptide that is contained within a host cell may be isolated from the host cell. Isolation of such an "intracellular" expressed polypeptide may include disruption of the host cell and removal of cellular debris from the resultant mixture.
  • Purification of a truncated polypeptide expressed in accordance with the present invention may be effected by any means known in the art. The skilled artisan will know how to determine the best method for the purification of a polypeptide expressed in accordance with the present invention. A purification method will be chosen by the skilled artisan based on factors such as, but not limited to, the expression host, the contents of the crude extract of the polypeptide, the size of the polypeptide, the properties of the polypeptide, the desired end product of the polypeptide purification process, and the subsequent use of the end product of the polypeptide purification process.
  • isolation or purification of a truncated polypeptide expressed in accordance with the present invention may not be desired.
  • an expressed polypeptide may be stored or transported inside the bacterial host cell in which the polypeptide was expressed.
  • an expressed polypeptide may be used in a crude lysate form, which is produced by lysis of a host cell in which the polypeptide was expressed, hi yet another embodiment of the invention, an expressed polypeptide may be partially isolated or partially purified according to any of the methods set forth or described herein. The skilled artisan will know when it is not desirable to isolate or purify a polypeptide of the invention, and will be familiar with the techniques available for the use and preparation of such polypeptides.
  • a eukaryotic host cell of the invention When armed with the disclosure set forth herein, the skilled artisan would also know how to prepare a eukaryotic host cell of the invention.
  • an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide may be introduced into a eukaryotic host cell, for example, using a lentivirus-based genomic integration or plasmid- based transfection (Sambrook et al., Third Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)).
  • a eukaryotic host cell is a fungal cell.
  • a nucleic acid encoding a truncated polypeptide of the invention is cloned into a lentiviral vector containing a specific promoter sequence for expression of the truncated polypeptide.
  • the truncated polypeptide-containing lentiviral vector is then used to transfect a host cell for expression of the truncated polypeptide.
  • a nucleic acid encoding a truncated polypeptide of the invention is introduced into a host cell using a viral expression system.
  • Viral expression systems are well-known in the art, and will not be described in detail herein.
  • a viral expression system is a mammalian viral expression system.
  • a viral expression system is a baculo virus expression system. Such viral expression systems are typically commercially available from numerous vendors.
  • Insect cells can also be used for expression of a truncated polypeptide of the present invention.
  • Sf9, SF9 + , Sf21, High FiveTM or Drosophila Schneider S2 cells can be used.
  • a baculovirus, or a baculo virus/insect cell expression system can be used to express a truncated polypeptide of the invention using a pAcGP67, pFastBac, pMelBac, or pIZ vector and a polyhedrin, plO, or OpIE3 actin promoter.
  • a Drosophila expression system can be used with a pMT or pAC5 vector and an MT or Ac5 promoter.
  • a truncated GalNAcT2 polypeptide of the invention can also be expressed in mammalian cells.
  • 294, HeLa, HEK, NSO, Chinese hamster ovary (CHO), Jurkat, or COS cells can be used to express a truncated polypeptide of the invention.
  • a suitable vector such as pT-Rex, pSecTag2, pBudCE4.1, or pCDNA/His Max vector can be used, along with, for example, a CMV promoter.
  • mammalian cell culture systems can be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.
  • Mammalian expression vectors may comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • vector DNA can be introduced into a eukaryotic cell using conventional transfection techniques.
  • transfection refers to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 3nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001), and other such laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a truncated polypeptide of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can ' be identified by drug selection ⁇ e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a truncated GalNAcT2 polypeptide of the present invention may be truncated in various ways, as would be known and understood by the skilled artisan, when armed with the present disclosure.
  • Examples of truncated polypeptides of the present invention include, but are not limited to, a polypeptide lacking a single N-terminal residue, a polypeptide lacking a single C-terminal residue, a polypeptide lacking both an single N-terminal residue and a single C-terminal residue, a polypeptide lacking a contiguous sequence of residues from the N-terminus, a polypeptide lacking a contiguous sequence of residues from the C-terminus, and any such combinations thereof.
  • a full-length human GalNAcT2 polypeptide may contain one or more identifyable polypeptide domains in addition to the "active domain," the domain primarily responsible for the catalytic activity, of GalNAcT2. This is because it is known in that art that a full-length GalNAcT2 polypeptide, and in particular, a full-length human GalNAcT2 polypeptide, contains a signal domain, a transmembrane domain, and a stem domain, in addition to an active domain.
  • a full-length human GalNAcT2 may have a signal domain at the amino-terminus of the polypeptide, followed by a transmembrane domain immediately adjacent to the signal domain, followed by a stem domain that is immediately adjacent to the transmembrane domain, followed by an active domain that extends to the carboxy-terminus of the polypeptide and is located immediately adjacent to the stem domain.
  • a GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide lacking all or a portion of the GalNAcT2 signal domain
  • a GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide lacking the GalNAcT2 signal domain and all or a portion of the GalNAcT2 transmembrane domain
  • a GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide lacking the GalNAcT2 signal domain, the GalNAcT2 transmembrane domain and all or a portion the GalNAcT2 stem domain.
  • a truncated GalNAcT2 mutant of the present invention is based on the point at which the full-length polypeptide is truncated.
  • a " ⁇ 40 human truncated GalNAcT2" mutant of the invention refers to a truncated GalNAcT2 polypeptide of the invention in which amino acids 1 through 40, counting from the N-terminus of the full-length polypeptide, are deleted from the polypeptide. Therefore, the N-terminus of the ⁇ 40 human truncated GalNAcT2 mutant begins with the amino acid residue that would be referred to as "amino acid 41" of the full- length polypeptide.
  • amino acid 41 amino acid residue that would be referred to as "amino acid 41" of the full- length polypeptide.
  • the present invention therefore also includes an isolated polypeptide comprising a truncated GalNAcT2 polypeptide.
  • an isolated truncated GalNAcT2 polypeptide of the present invention has at least about 90% identity to a polypeptide having the amino acid sequence of any one of the sequences set forth in SEQ ED NO:4, SEQ ID NO: 8 or SEQ DD NO:10.
  • the isolated polypeptide is about 95% identical, and even more preferably, about 98% identical, still more preferably, about 99% identical, and most preferably, the isolated polypeptide comprising a truncated GalNAcT2 polypeptide is identical to the polypeptide set forth in one of SEQ DD NO:4, SEQ DD NO: 8 or SEQ DD NO:10.
  • the present invention also provides for analogs of polypeptides which comprise a truncated GalNAcT2 polypeptide as disclosed herein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.
  • conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.
  • Conservative amino acid substitutions typically include substitutions within the following groups:
  • Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phospliotyrosine, phosphoserine, or phosphothreonine.
  • polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L- amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
  • the peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
  • Fragments of a truncated GalNAcT2 polypeptide of the invention are included in the present invention, provided the fragment possesses the biological activity of the full- length polypeptide. That is, a truncated GalNAcT2 polypeptide of the present invention can catalyze the same glycosyltransfer reaction as the full-length GalNAcT2.
  • a truncated human GalNAcT2 polypeptide has the ability to transfer a GaINAc moiety from a UDP-GaINAc donor to a granulocyte-colony stimulating factor (G- CSF) acceptor, wherein such a transfer results in the O-linked covalent coupling of a GaINAc moiety to a threonine residue of G-CSF. Therefore, a smaller than full-length, or "truncated,” GalNAcT2 is included in the present invention provided that the truncated GalNAcT2 has GalNAcT2 biological activity.
  • G- CSF granulocyte-colony stimulating factor
  • compositions comprising an isolated truncated GalNAcT2 polypeptide as described herein may include highly purified truncated GalNAcT2 polypeptides.
  • compositions comprising truncated GalNAcT2 polypeptides may include cell lysates prepared from the cells used to express the particular truncated GalNAcT2 polypeptides.
  • truncated GalNAcT2 polypeptides of the present invention may be expressed in one of any number of cells suitable for expression of polypeptides, such cells being well-known to one of skill in the art, as described in detail elsewhere herein.
  • Substantially pure protein isolated and obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure.
  • Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).
  • the present invention features a method of expressing a truncated polypeptide.
  • Polypeptides which can be expressed according to the methods of the present invention include a truncated GalNAcT2 polypeptide. More preferably, polypeptides which can be expressed according to the methods of the present invention include, but are not limited to, a truncated human GalNAcT2 polypeptide. In a preferred embodiment, a polypeptide which can be expressed according to the methods of the present invention is a polypeptide comprising any one of the polypeptide sequences set forth in SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO: 10.
  • the present invention features a method of expressing a truncated GalNAcT2 polypeptide encoded by an isolated nucleic acid of the invention, as described elsewhere herein, wherein the expressed truncated GalNAcT2 polypeptide has the property of catalyzing the transfer of a GaINAc moiety to an acceptor moiety.
  • a method of expressing a truncated GalNAcT2 polypeptide includes the steps of cloning an isolated nucleic acid of the invention into an expression vector, inserting the expression vector construct into a host cell, and expressing a truncated GalNAcT2 polypeptide therefrom.
  • Methods of expression of polypeptides are discussed in extensive detail elsewhere herein. Methods of expression of a truncated polypeptide of the present invention will be understood to include, but not to be limited to, all such methods as described herein.
  • the truncated GalNAcT2 polypeptides of the invention are expressed as insoluble proteins, e.g., in an inclusion protein in a bacterial host cell. Methods of refolding insoluble glycosyltransferases, including GalNAcT2 polypeptides, are disclosed in U.S. Provisional Patent Application Serial No. 60/542,210, filed February 4, 2004; U.S.
  • the present invention also features a method of catalyzing the transfer of a GaINAc moiety to a GaINAc acceptor moiety, wherein the GalNAc-transfer reaction is carried out by incubating a truncated GalNAcT2 polypeptide of the invention with a GaINAc donor moiety and a GaINAc acceptor moiety.
  • a truncated GalNAcT2 polypeptide of the invention mediates the covalent linkage of a GaINAc moiety to a GaINAc acceptor moiety, thereby catalyzing the transfer of a GaINAc moiety to an acceptor moiety.
  • a truncated GalNAcT2 polypeptide useful in a glycosyltransfer reaction is a truncated human GalNAcT2 polypeptide, hi one aspect, the human GalNAcT2 glycosyltransfer reaction involves the transfer of a GaINAc residue from a GaINAc donor to a GaINAc acceptor.
  • a method of catalyzing the transfer of a GaINAc moiety to an acceptor moiety includes the steps of incubating a truncated GalNAcT2 polypeptide with UDP-GaINAc GaINAc donor and a granulocyte colony stimulating factor (G-CSF) acceptor moiety, wherein the truncated GalNAcT2 polypeptide mediates the transfer of GaINAc from the UDP-GaINAc donor to the GCSF acceptor.
  • G-CSF granulocyte colony stimulating factor
  • the present invention also features a polypeptide acceptor moiety.
  • a polypeptide acceptor moiety is a human growth hormone.
  • a polypeptide acceptor moiety is an erythropoietin.
  • a polypeptide acceptor moiety is an interferon- alpha.
  • a polypeptide acceptor moiety is an interferon-beta.
  • a polypeptide acceptor moiety is an interferon- gamma.
  • a polypeptide acceptor moiety is a lysosomal hydrolase.
  • a polypeptide acceptor moiety is a blood factor polypeptide.
  • a polypeptide acceptor moiety is an anti-tumor necrosis factor- alpha.
  • a polypeptide acceptor moiety is follicle stimulating hormone.
  • the present invention also features a method of transferring a GalNAc-polyethyleneglycol conjugate to an acceptor molecule.
  • an acceptor molecule is a polypeptide.
  • an acceptor molecule is a glycopeptide.
  • Compositions and methods useful for designing, producing and transferring a GalNAc- polyethyleneglycol conjugate to an acceptor molecule are discussed at length in International (PCT) Patent Application No. WO03/031464 (PCT/US02/32263) and U.S. Patent Application No. 2004/0063911 , each of which is incorporated herein by reference in its entirety. Methods of assaying for glycosyltransferase activity are well-known in the art.
  • Example 1 Cloning, Expression, and Refolding of Human Polypeptide N- acetylgalactosaminyltransferase II (GalNAcT2) in E. co/z JM109
  • Truncated human polypeptide N-acetylgalactosaminyltransferase II was expressed as maltose binding protein (MBP)-fusion proteins in inclusion bodies from E. coli JM 109 cells.
  • MBP maltose binding protein
  • the production of active enzyme was examined by refolding and assaying against two polypeptide acceptors. Therefore, described herein is the generation of several truncated forms of human polypeptide GalNAcT2 as maltose binding protein fusion proteins in E. coli JM109 cells.
  • the recombinant proteins are refolded from isolated inclusion bodies using the Hampton FoIdIt screen kit (Hampton Research, Aliso Vieja, CA). All four constructs were expressed in JMl 09 E. coli at levels of approximately 2g/L culture media.
  • PCR Polymerase Chain Reaction
  • amplifications were performed in a final reaction volume of 50 ⁇ l containing 5 ⁇ l of template DNA (11 ⁇ g/ml, 100-fold diluted pBKS-Full ppGalNAcT2), 40 pmol of 5'- primer and 3'- primer, 10 nmol of dNTP mixture, and 5 units of HerculaseTM Enhanced DNA Polymerase under the conditions of 31 cycles of denaturation at 95 0 C for 45 seconds, annealing at 62°C for 45 seconds, and extension at 74°C for 170 seconds.
  • PCR products were subjected to 1% agarose gel electrophoresis. DNA fragments were excised and purified by QIAEX II gel extraction kit (Qiagen, Valencia, CA). Table 1 illustrates the primers used in the PCR reactions.
  • JMl 09 cells were cultured in a 15 ml culture tube containing 6 ml LB medium and 15 ⁇ g/ml of kanamycin overnight at 37°C with rapid shaking (250 rpm). For each culture, two milliliters of starting culture was transferred to a 50 ml centrifuge tube containing 23 ml LB medium with 15 ⁇ g/ml kanamycin and incubated at 37 0 C with rapid shaking for 3 hours. Isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce the protein expression.
  • IPTG Isopropyl-1-thio- ⁇ -D-galactopyranoside
  • Each sample for SDS-PAGE separation was prepared by mixing 5 ⁇ l of whole cells suspension, lysate, or inclusion bodies suspension with 5 ⁇ l of 2 x Tris-Glycine SDS sample buffer and 1.1 ⁇ l of DTT (1 M). The mixture was heated at 98 0 C for 5 minutes, cooled to room temperature, and loaded to each well of a 1.0 mm x 15 well 4-20% Tris-Glycine gradient gel. The electrophoresis was conducted at 120 V for 100 minutes. The gel was then stained for 2 hours and de-stained with distilled water (see Figures 4-6).
  • Inclusion bodies were dissolved at 20 mg/ml (high protein concentration) or 2 mg/ml concentration (low protein concentration) in solubilizing buffer containing 4 M Guanidine-HCl, 100 mM Tris-HCl, pH 9.0, 5 mM EDTA, and 10 mM DTT. Refolding of inclusion bodies by Hampton FoIdIt Screen Kit was carried out by following the manufacturer's protocol, except that a 10-fold less volume was used (100 ⁇ l -scale) (Hampton Products, Aliso Viejo, CA).
  • Non-radioactive enzyme activity assays for lysates were carried out in a 0.5 ⁇ l microcentrifuge tube at 37°C for overnight in a final volume of 10 ⁇ l containing 50 mM MES buffer, pH 6.0, MnCl 2 (15 mM), MgCl 2 (15 mM), NaCl (0.15 M), UDP-GaINAc (5 mM), 1.5 ⁇ g G-CSF (acceptor), and 2.15 ⁇ l of lysate sample. Enzyme was substituted by H 2 O as a negative control. Purified recombinant ppGalNAcT2 (0.5 ⁇ l) from Sf9 baculovirus expression system was used as the positive control.
  • DNA fragments for ppGalNAcT2 genes were successfully amplified by PCR as shown in Figure 5.
  • Vector plasmid DNA pCWin2MBP was digested by BamHI and Xhol, and purified on a 1% agarose gel. The gel purified DNA fragment was digested by the same two enzymes and purified. After digestion, the DNA fragments were clean as visualized on an agarose gel ( Figure 2B).
  • BamHI and Xhol digestion of the plasmids purified from the selected twelve colonies showed predicted correct pattern on a 1% agarose gel. The size of the vector was around 6.2 kb, and the inserts were approximately 1.5 kb.
  • Maltose-binding protein (MBP) expressed in the JM 109 transformed with pCWin2MBP vector plasmid showed a band at around 43 kDa. Over 90% of the proteins in the whole cells are MBP. The #2 colony of the construct N41R expressed a shorter protein than expected, indicating the occurrence of mutation. All other eleven colonies showed a band at about 100 kDa for MBP-ppGalNAcT2 fusion proteins, with over 80% of the total proteins were the target fusion proteins.
  • Refolding experiments on MBP-GalNAcT2 were carried out on a 1 ml scale, with four different MBP-GaIN AcT2 DNA constructs and under 16 different possible refolding conditions. Refolding was performed using the Hampton Research Foldit kit (Hampton Research, Aliso Viejo, CA) and the assays were performed via radioactive detection of [ 3 H] UDP-GaINAc addition to a MuC-2 peptide and via matrix-assisted laser desorption ionization mass spectrometry (MALDI) analysis utilizing addition of GaINAc to Interferon ⁇ -2b and G- CSF.
  • MALDI matrix-assisted laser desorption ionization mass spectrometry
  • GalNAcT2 constructs used in the present invention comprised DNA encoding various amino terminal amino acid truncation mutants of the original human GalNAcT2 protein, including the following constructs, which begin with the N-terminal amino acid as indicated:
  • IL Martone L-Broth cultures containing lO ⁇ g/ml Kanamycin sulfate were prepared. Each of these cultures was inoculated with 40 ml of one of the 275 ml cultures of constructs 1 though 4. These IL cultures were incubated at 37 0 C, with shaking at 250rpm, until the OD600 measured approximately 1.0. Upon reaching this point, IPTG was added to each of the four IL cultures to a final concentration of 0.4mM. Cultures were then allowed incubate overnight at 37 0 C, with shaking at 250rpm.
  • Caps were removed from the G-50 columns and columns were placed into 2 ml microcentrifuge tubes. H 2 O (500 ⁇ l) was added to each column and the columns were allowed to incubate for 15 minutes to hydrate. The columns were then centrifuged at ⁇ 2000 x g for 4 minutes after which they were transferred to new 2 ml centrifuge tubes. Each refold solution (150 ⁇ l) was applied to one of the columns. Columns were then centrifuged at 2000 x g for ⁇ 2 minutes. Resulting permeates represented the purified refold samples.
  • a radiolabeled [ 3 H]-UDP-GaINAc assay was performed to determine the activity of the E.coli-expressed refolded MBP-GalNAcT2 by monitoring the addition of radiolabeled GaINAc to a peptide acceptor.
  • the acceptor was a MuC-2 - like peptide having the sequence MVTPTPTPTC (SEQ ID NO: 16).
  • the initial screen was performed on refolded protein samples which had been purified by dialysis. Subsequent refold samples were freshly refolded and purified by G-50 gel filtration.
  • the assay included protein refold samples, GalNAcT2 from Baculovirus as a positive control, a negative control sample with all the components except enzyme and a maximum input sample which contained all components except enzyme. A total of 19 samples were tested.
  • the assay solution consisted of the components listed in Table 3:
  • constructs 1 and 2 in refold buffers 8 and 15 were assayed for transferase activity. Additionally, as a positive control, GalNAcT2 from a Baculovirus system was assayed as well.
  • the assay solution was prepared as shown in Table 4 for each reaction.
  • the above assay was performed to determine whether E.coli-expressed refolded MBP-GalNAcT2 could transfer GaINAc to G-CSF acceptor from a UDP-GaINAc donor.
  • construct 2 in refold buffer 8 was assayed for GalNAcT2 activity.
  • GalNAcT2 from Baculovirus was assayed.
  • the assay solution was prepared for each reaction as shown in Table 5.
  • T aao u i ble 5 Parameters for G-CSF acceptor GalNAcT2 activity assay
  • MBP-GalNAcT2 The expression of MBP-GalNAcT2 was observed by way of the SDS-Page gel analysis of JM 109 pCWin2 MBP-GalNAcT2 whole cell samples before and after induction by IPTG (Figure 7).
  • the protein gel shows a clear increase in protein expression in the induced state compared to the uninduced state. Furthermore there is a distinct band at ⁇ 100kDa that substantially increases after induction which correlates to the expected size of the MBP-GalNAcT2 band.
  • Protein samples were diluted by combining 950 ⁇ L of H 2 O with 50 ⁇ L of protein sample. Samples were then analyzed using a UV spectrophotometer. Protein concentration was calculated from absorption values and the molar extinction coefficients: Construct 1 — 0.65mg/ml per 1 A 28 o unit, Construct 2 - 0.64mg/ml per 1 A 280 unit, as shown in Table 7.
  • VlL CMP x (nmoles Donor) x lOO ⁇ l/ml
  • construct 2 was tested under refold conditions 3, 8, 11, 12, 15 and 16 from the Hampton Foldit kit (Hampton Research, Aliso Viejo, CA). These refolded enzymes were purified by G-50 gel filtration and then tested for activity by the radioactive assay. Results indicate that after overnight incubation on a rotator, greatest activity was obtained from refold condition 15.
  • construct 2 was tested under refold conditions 3, 8, 11, 12, 15 and 16 from the Hampton Foldit kit (Hampton Research, Aliso Viejo, CA) after being rotated overnight at 4°C and left resting at 4°C for 5 days. These refolded enzymes were purified by G-50 gel filtration and then tested for activity by the radioactive assay. Results indicated that after 5 days in refold buffer 8, construct 8 displayed the highest activity. Therefore it was determined that conditions 8 and 15 had the greatest potential for producing a properly folded and active MBP-GalNAcT2.
  • An IF ⁇ -2b assay was performed on overnight refolds of constructs 1 and 2 in refold buffer 15 (1-15 and 2-15, respectively) and was incubated at 32°C for 5 days. Time points were taken of the IF ⁇ -2b reaction at 16 hours and 5 days. The results indicate that the parental peak for IF ⁇ -2b is at MW -19267. A successful reaction would be indicated by addition of -203 molecular weight to that peak. From the 5 day data for refolds 1-15 and 2- 15, a developing peak was observed at -119478 and -19473 respectively, a difference of approximately 203 MW.
  • a G-CSF assay was performed on the 5-day refolded enzymes of construct 2 in refold buffer 8. The G-CSF reaction was allowed to incubate at 32°C for 4 days. The reaction was analyzed at the 4 day time point. The parental peak for G-CSF is expected at MW ⁇ 18786. A successful reaction would be indicated by addition of -203 molecular weight to that peak. From the 3 day data for refolded enzymes 2-8, a developing peak was observed at -19001, a difference of approximately 203 MW. This data again indicated that GaINAc was added to G-CSF by the refolded GalNAcT2 protein and confirmed what was reported by the radioactive assay and the IF ⁇ -2b assay as reported elsewhere herein.
  • GaIN AcT2 truncation mutants of the present invention are also useful for the transfer of a glycosyl-polyethyleneglycol ("glycosyl-PEG") conjugate to a polypeptide, also known as "glycoPEGylation" of a polypeptide.
  • glycosyl-PEG glycosyl-polyethyleneglycol
  • glycoPEGylation also known as "glycoPEGylation” of a polypeptide.
  • a glycoPEGylation reaction mixture was prepared in order to glycoPEGylate G- CSF.
  • the reaction mixture contained 5 ⁇ l of ⁇ 51 GalNAcT2-MBP (20 ⁇ U), 2 ⁇ l of GaINAc- ⁇ 2,6-sialyltransferase (ST ⁇ GalNAcI), 6.25 mM MnCl 2 , 15 mM UDP-GaINAc, 0.75 mM CMP-SA-PEG (20K), and between 2 ⁇ l and 10 ⁇ l of 2 mg/ml G-CSF.
  • Gel electrophoresis of the reaction products demonstrated that ⁇ 51 GalNAcT2-MBP transferred a GalNAc-sialic acid (SA)-PEG conjugate to G-CSF (Figure 9).
  • Example 3 Optimization of Purification and Refolding of ⁇ 51 GalNAcT2-MBP
  • ⁇ 51 GalNAcT2 refolding and purification development as set forth herein demonstrates the utility of a two column purification procedure for purification of GaIN AcT2 mutants.
  • Q Sepharose Fast Flow in binding mode and Q Sepharose XL in binding and flow through mode as an initial purification step has been explored.
  • Q Sepharose XL in flow through mode using a NaCl concentration of 10OmM in the load led to best recovery and purity of active ⁇ 51 GalNAcT2-MBP.
  • the use of Hydroxy apatite Type I has been considered as a second column step.
  • Initial data indicate ⁇ 51 GalNAcT2-MBP binds to this resin and can be eluted as an active enzyme with a phosphate gradient.
  • ⁇ 51 GalNAcT2-MBP was cloned and expressed as set forth elsewhere herein.
  • DWIBs double-washed inclusion bodies
  • harvested cell pellet was resuspended in 1OmM Tris/ 5mM EDTA pH 7.5 (5mL/g cells) and lysed in two passes using a micro fluidizer at 12,000psi.
  • Inclusion bodies were harvested by centrifugation at 6,000 rpm for 20 min in a Sorvall RC-3B. The pellet was washed twice by resuspension in above buffer at 5mL/g pellet followed by centrifugation at 6,000 RPM for 20min.
  • DWBs were aliquoted and stored at -2O 0 C.
  • ⁇ 51 GalNAcT2-MBP refolds were performed by solubilizing 2.5g of DWIB ' s in 250 mL of 7M urea/ 5OmM Tris/ 1OmM DTT/ 5mM EDTA pH 8.0 at 4 0 C. 5OmL solubilized ⁇ 51 GalNAcT2-MBP DWIB 's were added to IL of refold buffer at 4 0 C while stirring (21- fold dilution - 0.5mg/mL). Refolding was allowed to proceed for 20.5h at 4 0 C with stirring.
  • Refolds were filtered using a Cuno Zeta Plus BioCap (Cuno, Meriden, CT), concentrated 4-fold and diafiltered on a 1 ft2 3OkDa MWCO TFF (regenerated cellulose) filter at constant volume with 5 diavolumes of 1OmM Tris/ 5mM NaCl pH 8.
  • ⁇ 51 GalNAcT2-MBP bound tightly to QSFF resin under above conditions with 5mM NaCl in load and equilibration buffers. Active ⁇ 51 GalNAcT2-MBP eluted at the beginning of the major peak and appears as a doublet on a nonreduced 4-20% Tris-glycine gel.
  • the major contaminant is a currently unidentified band running at a slightly lower molecular weight close to the 98kDa marker band. A variety of other contaminants elute with inactive ⁇ 51 GalNAcT2-MBP in the remainder of the major peak.
  • ⁇ 51 GalNAcT2-MBP bound tightly to QXL resin if the same conditions as for QSFF binding were applied (i.e. 5mM NaCl). Increasing ⁇ 51 GalNAcT2-MBP activity was observed in flow through and wash at higher NaCl concentrations in the load. Interestingly, the major contaminating band observed in QSFF purification was not visible in the flow through if the load contained 50 and 10OmM NaCl. At both NaCl concentrations the majority of active ⁇ 51 GalNAcT2-MBP could be found in flow through and wash; only some residual ⁇ 51 GalNAcT2-MBP activity was detected in the left shoulder of the elution peak.
  • Hydroxyapatite Type I 80 ⁇ m (BioRad, Hercules, CA) was examined as a second column step. Active ⁇ 51 GalNAcT2-MBP partially purified over QSFF (using bind and elute mode) was used to investigate if active ⁇ 51 GalNAcT2-MBP would bind to an HA Type I resin and would be useful to further purify the protein. For this purpose, a 2.25 mL HA Type I column was pre-equilibrated with 5mM NaPO4/ 5mM NaCl pH 7.0 (C). Active ⁇ 51 GalNAcT2-MBP eluted from QSFF was adjusted to pH 7.0 with IM HCl and applied onto the HA Type I column.
  • the protein was eluted using a 20 CV gradient from 0-50% 30OmM NaPO4/ 5mM NaCl pH 7.0 (D), followed by a 5 CV gradient from 50-100% D.
  • the column was regenerated using 0.5M NaOH.
  • the data obtained indicate that ⁇ 51 GalNAcT2-MBP binds to hydroxyapatite type I resin and can be eluted as an active enzyme.
  • Arg Lys GIu Asp Trp Asn GIu lie Asp Pro lie Lys Lys Lys Asp Leu 1 5 10 15 His His Ser Asn GIy GIu GIu Lys Ala GIn Ser Met GIu Thr Leu Pro 20 25 30
  • Cys GIy GIy Ser Leu GIu lie lie Pro Cys Ser Arg VaI GIy His VaI 305 310 315 320
  • GIy Ser Leu lie Lys Leu GIn GIy Cys Arg GIu Asn Asp Ser Arg GIn 465 470 475 480
  • Lys Trp GIu Gin lie GIu GIy Asn Ser Lys Leu Arg His VaI GIy Ser 485 490 495
  • GIu lie lie Leu VaI Asp Asp Tyr Ser Asn Asp Pro GIu Asp GIy Ala 100 105 110
  • Leu Leu GIy Lys lie GIu Lys VaI Arg VaI Leu Arg Asn Asp Arg Arg 115 120 125 Glu GIy Leu Met Arg Ser Arg VaI Arg GIy Ala Asp Ala Ala GIn Ala 130 135 140

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Abstract

L'invention se rapporte à des compositions et des procédés portant sur des mutants de GalNAcT2. Plus précisément, l'invention concerne des polypeptides GalNAcT2 humains tronqués. Elle porte aussi sur des acides nucléiques codant ces polypeptides tronqués, ainsi que sur des vecteurs, des cellules hôtes, des systèmes d'expression et des procédés d'expression et d'utilisation de ces polypeptides.
PCT/US2005/019442 2004-06-03 2005-06-03 Polypeptides galnact2 tronques et acides nucleiques WO2005121331A2 (fr)

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JP2007515582A JP2008512085A (ja) 2004-06-03 2005-06-03 切断型ＧａｌＮＡｃＴ２ポリペプチドおよび核酸
EP05758682A EP1765992A4 (fr) 2004-06-03 2005-06-03 Polypeptides galnact2 tronques et acides nucleiques

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US57653004P 2004-06-03 2004-06-03
US60/576,530 2004-06-03
US59858404P 2004-08-03 2004-08-03
US60/598,584 2004-08-03

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US7696163B2 (en) 2001-10-10 2010-04-13 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
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US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
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US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US20060040856A1 (en) 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
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EP1799249A2 (fr) 2004-09-10 2007-06-27 Neose Technologies, Inc. Interferon alpha glycopegyle
DK2586456T3 (en) 2004-10-29 2016-03-21 Ratiopharm Gmbh Conversion and glycopegylation of fibroblast growth factor (FGF)
EP1858543B1 (fr) 2005-01-10 2013-11-27 BioGeneriX AG Facteur de stimulation de colonie de granulocytes glycopegylé
EP2386571B1 (fr) 2005-04-08 2016-06-01 ratiopharm GmbH Compositions et méthodes utilisées pour la préparation de mutants par glycosylation de l'hormone de croissance humaine résistant à la protéase
EP1888098A2 (fr) 2005-05-25 2008-02-20 Neose Technologies, Inc. Formulations d'erythropoietine glycopegylees
US20080255026A1 (en) 2005-05-25 2008-10-16 Glycopegylated Factor 1X Glycopegylated Factor Ix
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EP2049144B8 (fr) 2006-07-21 2015-02-18 ratiopharm GmbH Glycosylation de peptides par l'intermédiaire de séquences de glycosylation à liaison o
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