JP2008512085A - Cleaved GalNAcT2 polypeptides and nucleic acids - Google Patents

Abstract

The invention features compositions and methods related to truncated variants of GalNAcT2. In particular, the invention features a truncated human GalNAcT2 polypeptide. In addition to nucleic acids encoding such truncated polypeptides, the present invention also features vectors, host cells, expression systems, systems, and methods for expressing and using such polypeptides.

Description

TECHNICAL FIELD OF THE INVENTION The present invention features compositions and methods related to truncated variants of GalNAcT2. In particular, the invention features a truncated human GalNAcT2 polypeptide. In addition to nucleic acids encoding such truncated polypeptides, the invention also features vectors, host cells, expression systems, and methods for expressing and using such polypeptides.

Cross-reference to related applications.This specification is related to US Provisional Application No. 60 / 576,530, filed June 3, 2004, and US Provisional Application No. 60 / 598,584, filed August 3, 2004. , Both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION It has been found in practice that oligosaccharide structures and many types of glycopeptides have a great variety, which are partly synthesized by a number of glycosyltransferases. Glycosyltransferases catalyze the synthesis of glycolipids, glycopeptides, and polysaccharides by transferring mono- or oligosaccharide residues activated to initiate or extend sugar chains to existing receptor molecules. To do. Catalytic reactions are thought to be related to the recognition of the catalytic site of the enzyme in addition to both the donor and acceptor by the appropriate domain.

  Many peptide therapeutics, and many potential peptide therapeutics, are glycosylated peptides. The production of recombinant glycopeptides, in contrast to recombinant non-glycosylated peptides, adds additional processing to the recombinantly produced peptide either in the cell or after the peptide is produced by the cell. A process needs to be provided, where the processing step is performed in vitro. This peptide can be treated enzymatically to introduce one or more glycosyl groups onto the peptide using glycosyltransferases. In particular, glycosyltransferases covalently link one or more glycosyl groups to a peptide.

  The extra in vitro process of peptide processing to make glycopeptides is time consuming and expensive. This is because, in part, the production of recombinant glycosyltransferases for in vitro glycosylation of peptides and glycopeptides to produce glycopeptide therapeutics is burdensome and expensive. As the demand and utility of recombinant glycotherapeutics is increasing, new methods are needed to prepare glycopeptides more efficiently. Furthermore, as increasingly glycopeptides have been found useful in the treatment of various diseases, there is a need for methods that further reduce their production costs. Furthermore, there is a need in the art to develop methods for efficiently producing recombinant glycopeptides for use in developing and improving glycopeptide therapeutics.

  Glycosyltransferases are generally validated in WO 03/031464 (PCT / US02 / 32263) (Patent Document 1), which is incorporated herein by reference in its entirety. One such specific glycosyltransferase useful for the development and production of therapeutic glycopeptides is GalNAcT2. GalNAcT2 or N-acetyl-D-galactosamine transferase catalyzes the transfer of GalNAc from a GalNAc donor to a GalNAc acceptor. The full-length human GalNAcT2 enzyme has been revealed by Bennett et al. (1996, J Biol Chem. 271: 17006-17012 (Non-patent Document 1)). However, the identification of useful variants of this enzyme with enhanced biological activity, such as enhanced catalytic activity or enhanced stability, has not been reported so far.

  In the past, efforts have been made to increase the availability of recombinant glycosyltransferases for in vitro production of glycopeptides. Limited amounts of research have been conducted on recombinant glycosyltransferases that are sometimes suitable for small scale production of oligosaccharides or glycopeptides. For example, White et al. Have revealed a soluble form of human GalNAcT2 (1995, J. Biol. Chem., 270: 24156-24165 (Non-patent Document 2)). In addition, Kurosawa et al. (1994, J Biol Chem. 269: 1402-1409) describe a chicken GalNAcα2,6-sialyltransferase (ST6GalNAcI) that lacks amino acid residues 1-232 from the full-length enzyme. A truncated variant of is described. However, the truncated enzyme described by Kurosawa et al. Lacks the substrate specificity of other ST6GalNAcI enzymes. Accordingly, there remains a need for recombinant glycosyltransferases with activities suitable for “pharmaceutical scale” processes and reactions, including the manufacture of glycopeptide therapeutics. In particular, there is a need for recombinant glycosyltransferases with favorable functional and structural characteristics. In addition to efficient methods for identification and characterization of recombinant glycosyltransferases, there is a need for the production of such glycosyltransferases. The present invention addresses and meets these needs.

WO 03/031464 (PCT / US02 / 32263) Bennett et al., J. Biol Chem. 271: 17006-17012 (1996) White et al., J. Biol. Chem., 270: 24156-24165 (1995) Kurosawa et al., J. Biol Chem. 269: 1402-1409 (1994)

BRIEF SUMMARY OF THE INVENTION In one aspect, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding a truncated human GalNAcT2 polypeptide. A truncated human GalNAcT2 polypeptide lacks all or part of the GalNAcT2 signal domain, provided that the encoded polypeptide is not a human GalNAcT2 truncated mutant polypeptide lacking amino acid residues 1-51. Or additionally lacks all or part of the GalNAcT2 transmembrane domain, or additionally lacks all or part of the GalNAcT2 stem domain.

  In one embodiment, 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.

  In some embodiments, the isolated nucleic acid is an isolated chimeric nucleic acid encoding a fusion polypeptide. The fusion polypeptide can comprise a tag polypeptide covalently linked to a truncated human GalNAcT2 polypeptide, as described herein. Examples of tag polypeptides include maltose binding protein, histidine tag, factor IX tag, glutathione-S-transferase tag, FLAG-tag, and starch-binding domain tag.

  In another aspect, the present invention lacks all or part of the GalNAcT2 signal domain, provided that the encoded polypeptide is not a human GalNAcT2 truncated mutant polypeptide lacking amino acid residues 1-51. An isolated truncated human GalNAcT2 polypeptide is provided that lacks, or additionally lacks, all or part of the GalNAcT2 transmembrane domain, or additionally lacks all or part of the GalNAcT2 backbone domain. In one embodiment, 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. Have In a further aspect, the 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.

  In some embodiments, the isolated truncated GalNAcT2 polypeptide was isolated from a chimeric polypeptide comprising a tag polypeptide covalently linked to the isolated truncated GalNAcT2. Examples of tag polypeptides include maltose binding protein, histidine tag, factor IX tag, glutathione-S-transferase tag, FLAG-tag, and starch-binding domain tag.

  An isolated nucleic acid encoding a truncated GalNAcT2 polypeptide can also be operably linked to a promoter / regulatory sequence, eg, in an expression vector. The invention also includes host cells containing such expression vectors. Host cells are eukaryotic cells or prokaryotic cells, for example. Eukaryotic cells include, for example, mammalian cells, insect cells, and fungal cells. Some preferred mammalian host cells are SF9 cells, SF9 + cells, Sf21 cells, HIGH FIVE cells, or Drosophila Schneider S2 cells. Prokaryotic host cells include, for example, E. coli cells and B. subtilis cells.

  A truncated human GalNAcT2 polypeptide can be produced by using a host cell and growing the recombinant host cell under conditions suitable for expression of the truncated human GalNAcT2 polypeptide. In a preferred embodiment, a fully truncated human GalNAcT2 polypeptide allows the production of glycoproteins or glycopeptides on a commercial scale.

  In a further aspect, the invention includes a method of catalyzing the transfer of a GalNAc moiety to a receptor moiety comprising incubating a truncated human GalNAcT2 polypeptide with the GalNAc moiety and the receptor moiety, wherein the polypeptide Mediates the covalent linkage of the GalNAc moiety to the receptor moiety, thereby catalyzing the transfer of the GalNAc moiety to the receptor moiety, producing a product saccharide, or product glycoprotein, or product glycopeptide . In one embodiment, the receptor moiety is a granulocyte colony stimulating factor (G-CSF) protein. In another embodiment, the receptor moiety is erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferon α, -β and -γ, factor IX, follicle stimulating hormone, interleukin-2, erythropoietin, anti-TNF-α And lysosomal hydrolase. In further embodiments, the polypeptide receptor is a glycopeptide. In some embodiments, the GalNAc moiety comprises a polyethylene glycol moiety. In another embodiment, the product saccharide, product glycoprotein, or product glycopeptide is produced on a commercial scale.

Detailed Description of the Invention Compositions and methods of the invention include truncated variants of human GalNAcT2 polypeptides, isolated nucleic acids encoding these proteins, and methods of use thereof. GalNAcT2 polypeptide catalyzes the transfer of GalNAc from a GalNAc donor to a GalNAc acceptor.

  The glycosyltransferase GalNAcT2 is an essential reagent for glycosylation of therapeutic glycopeptides. In addition, GalNAcT2 is an important reagent for the research and development of therapeutically important glycopeptide and oligosaccharide therapeutics. GalNAcT2 enzymes are typically isolated and purified from natural sources or from tedious and expensive in vitro and recombinant sources. The present invention provides compositions and methods relating to simplified and more cost effective methods for the production of GalNAcT2 enzymes. In particular, the present invention provides compositions and methods for truncated GalNAcT2 enzymes that have improved and useful properties compared to their full-length enzyme counterparts.

  The truncated glycosyltransferases of the present invention are useful for the in vivo and in vitro preparation of glycosylated peptides, as well as the generation of oligosaccharides containing specific glycosyl residues that can be transferred by the truncated glycosyltransferases of the present invention. It is. This is because the truncated form of GalNAcT2 polypeptides was first shown herein to have biological activity equivalent to, and possibly beyond, their full-length polypeptide counterparts. . The present application shows that such truncation mutants not only have biological activity, but truncation mutants can also have enhanced properties of solubility, stability and resistance to proteolytic degradation. Disclosure.

Unless defined otherwise by definition, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

  Certain abbreviations commonly used in the art are used herein, for example, “Ac” is acetyl; “Glc” is glucose; “Glc” is glucosamine; “GlcA” is glucuronic acid. "IdoA" is iduronic acid; "GlcNAc" is N-acetylglucosamine; "NAN" or "sialic acid" or "SA" is N-acetylneuraminic acid; "UDP" is uridine diphosphate; "CMP" is Cytidine monophosphate.

  Each of the following terms used herein has the meaning associated with it in this section.

  As used herein, the article “a, an” means 1 or more (ie, at least 1) for the grammatical object of the article. By way of example, “an element” means one element or more than one element.

  “Encoding” refers to other polymers in biological processes having either a defined nucleotide sequence (ie, rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. And the intrinsic properties of a particular sequence of nucleotides in a nucleic acid, such as a gene, cDNA, or mRNA, for use as a template for the synthesis of macromolecules. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system. The coding strand, whose nucleotide sequence is the same as the mRNA sequence and usually provided in the sequence listing, as well as the non-coding strand used as a template for transcription of a gene or cDNA, is a protein or other product of a gene or cDNA Can mean that you are coding.

  A “coding region” of a gene consists of nucleotide residues in the coding strand of the gene and nucleotides in the non-coding strand of the gene, each homologous or complementary to the coding region of the mRNA molecule produced by transcription of the gene. Become.

  The “coding region” of an mRNA molecule consists of the nucleotide residues of an mRNA molecule that either match the anticodon region of the transfer RNA molecule during translation of the mRNA molecule or encode a stop codon. Thus, the coding region includes nucleotide residues that correspond to amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in a protein export signal sequence).

  An “affinity tag” is a peptide or polypeptide that can be genetically or chemically fused to a second polypeptide for purposes of purification, isolation, targeting, transport, or identification of the second polypeptide. It is a peptide. “Genetic” attachment of an affinity tag to a second protein can be affected by cloning of the nucleic acid encoding the affinity tag adjacent to the nucleic acid encoding the second protein in the nucleic acid vector.

  The term “glycosyltransferase” as used herein means 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 generate sugar nucleotides. Sugar nucleotides are known in the art and include moieties such as, but not limited to, UDP-Gal, UDP-GalNAc, and CMP-NAN.

  An “isolated nucleic acid” is a sequence ordinarily adjacent to a fragment thereof, such as a nucleic acid segment or fragment that is naturally separated from the sequence adjacent to it, eg, a sequence adjacent to that fragment in a naturally occurring genome. Means a DNA fragment removed from The term also applies to nucleic acids that are substantially associated with nucleic acids, such as RNA or DNA or proteins, that naturally accompany it in a cell. The term thus includes, for example, a vector, an autonomously replicating plasmid or virus, or recombinant DNA integrated into prokaryotic or eukaryotic genomic DNA, or other molecules independent of other sequences (e.g., including recombinant DNA present as cDNA or genomic or cDNA fragments produced by PCR or restriction enzyme digestion. This also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

  In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used. “A” means adenosine, “C” means cytidine, “G” means guanosine, “T” means thymidine, and “U” means uridine.

  "Polynucleotide" means a single strand of nucleic acid or parallel and antiparallel strands. Thus, a polynucleotide can be either single-stranded or double-stranded nucleic acid.

  The term “nucleic acid” typically refers to large polynucleotides. However, the terms “nucleic acid” and “polynucleotide” are used interchangeably herein.

  The term “oligonucleotide” typically means a short polynucleotide, generally no more than about 50 nucleotides. If the nucleotide sequence is represented by a DNA sequence (i.e. A, T, G, C), this also includes RNA sequences (i.e. A, U, G, C) where `` U '' is replaced with `` T '' It seems to be understood.

  To describe the nucleic acid sequence, the usual notation is used herein: the left-hand end of the single-stranded nucleic acid sequence is the 5 ′ end; the left-hand direction of the double-stranded nucleic acid sequence is the 5 ′ direction It is called.

  A first defined nucleic acid sequence is, for example, when the last nucleotide of a first nucleic acid sequence is chemically linked via a phosphodiester bond to the first nucleotide of a second nucleic acid sequence. It is referred to as “directly adjacent” to the second defined nucleic acid sequence. Conversely, a first defined nucleic acid sequence is such that, for example, the first nucleotide of a first nucleic acid sequence is chemically linked via a phosphodiester bond to the last nucleotide of a second nucleic acid sequence. Also referred to as “directly adjacent” to the second defined nucleic acid sequence.

  A first defined polypeptide sequence is, for example, when the last amino acid of a first polypeptide sequence is chemically linked via a peptide bond to the first amino acid of a second polypeptide sequence. , Referred to as “directly adjacent” to the second defined polypeptide sequence. Conversely, a first defined polypeptide sequence is such that, for example, the first amino acid of a first polypeptide sequence is chemically linked via a peptide bond to the last amino acid of a second polypeptide sequence. Also referred to as “directly adjacent” to the second defined polypeptide sequence.

  The direction of 5 ′ to 3 ′ nucleotide addition to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the “coding strand”; the sequence on the DNA strand located 5 ′ to the reference point on the DNA is referred to as the “upstream sequence”; the reference on the DNA The sequence on the DNA strand located 3 ′ to the point is called the “downstream sequence”.

  Unless otherwise stated, a “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” as used herein refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If a nucleotide residue position in both regions is occupied by the same nucleotide residue, then these regions are homologous at that position. A first region is homologous to a second region if the position of at least one nucleotide residue in each region is occupied by the same residue. Homology between two regions is expressed as a percentage of the nucleotide residue positions of the two regions occupied by the same nucleotide residues. As an example, a region having the nucleotide sequence 5′-ATTGCC-3 ′ and a region having the nucleotide sequence 5′-TATGGC-3 ′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second region, whereby at least about 50%, preferably at least about 75% of each nucleotide position of those portions, At least about 90%, or at least about 95% is occupied by the same nucleotide residues. More preferably, all nucleotide residue positions in each part are occupied by the same nucleotide residue.

  As used herein, “% match” is used interchangeably with “homology”. The determination of percent identity between two nucleotide or amino acid sequences can be performed using a mathematical algorithm. For example, mathematical algorithms useful for comparing two sequences are the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268), the variant of Karlin and Altschul (1993, Proc. Natl Acad. Sci. USA 90: 5873-5877). This algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J. Mol. Biol. 215: 403-410), for example, the National Medical Library (NLM) Life of the National Institutes of Health (NIH). It can be accessed at the BLAST site on the World Wide Web of the Engineering Information Center (NCBI). BLAST nucleotide searches can be performed by the NBLAST program (referred to as “blastn” on the NCBI website) using the following parameters: To obtain nucleotide sequences homologous to the nucleic acids described herein, Gap penalty = 5; Gap extension penalty = 2; Mismatch penalty = 3; Match reward = 1; Expected value 10.0; and Word size = 11. BLAST protein searches are performed by the XBLAST program (referred to as “blastn” on the NCBI website) or the NCBI “blastp” program with the following parameters: amino acid sequence homologous to the protein molecule described herein To obtain the expected value 10.0, BLOSUM62 scoring matrix.

  To obtain a gapped alignment for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects distant relationships between molecules (same as above) and relationships between molecules that share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters for each program (eg, XBLAST and NBLAST) can be obtained and used on the NCH website of NIH's NLM.

  The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. In calculating percent match, typically exact matches are counted.

  A “polypeptide” is a polymer composed of amino acid residues, related natural structural variants, and their synthetic non-natural analogs linked by peptide bonds, related natural synthetic variants, and their synthetic non- Means a natural analog. A synthetic polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. Thus, as used herein, the term “polypeptide” means a polymer of any size of amino acid residues, provided that the polymer comprises at least two amino acid residues.

  The term “protein” typically means a large peptide, also referred to herein as a “polypeptide”. The term “peptide” typically means a short polypeptide. However, the terms “peptide”, “protein” and “polypeptide” are used interchangeably herein. For example, the term “peptide” can mean an amino acid polymer of several hundred amino acids in addition to an amino acid polymer of three amino acids.

  Amino acids as used herein are represented by their full name, the corresponding three letter code, or the corresponding one letter code, as listed in the table below:

Full name 3 letter code 1 letter code <br/> Aspartic acid Asp D
Glutamate Glu E
Lysine Lys K
Argin R
Histidine His H
Tyrosine Tyr Y
Cysteine Cys C
Asparagine Asn N
Glutamine Gln Q
Serine Ser S
Threonine Thr T
Glycine Gly G
Alanine Ala A
Valin
Leu L
Isoleucine Ile I
Methionine Met M
Proline Pro P
Phenylalanine Phe F
Tryptophan Trp W

  To describe the polypeptide sequence, the usual notation is used herein: the left end of the polypeptide sequence is the amino terminus; the right end of the polypeptide sequence is the carboxy terminus.

  As used herein, the term “therapeutic peptide” means any peptide useful for the treatment of an organism's condition or for improving general health. A therapeutic peptide can perform such changes in an 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”.

  The term “reagent peptide” as used herein means any peptide useful in food biochemistry, bioremediation, the manufacture of small molecule therapeutics, and even the manufacture of therapeutic peptides. Typically, a reagent peptide is an enzyme that can catalyze a reaction that produces a product useful in any of the foregoing fields. The term “reagent peptide” is used interchangeably herein with the terms “reagent polypeptide” and “reagent protein”.

  The term “glycopeptide” as used herein means a peptide having at least one carbohydrate moiety covalently linked thereto. It will be understood that the glycopeptide may be a “therapeutic glycopeptide” as described above. The term “glycopeptide” is used interchangeably herein with the terms “glycopolypeptide” and “glycoprotein”.

  A “vector” is a composition of matter that contains and can be used to deliver an isolated nucleic acid into the interior of a cell. Many vectors are known in the art and include, but are not limited to, linear nucleic acids, nucleic acids associated with ionic or amphoteric compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. The term must also be constructed to include non-plasmid and non-viral compounds, such as polylysine compounds, liposomes, etc., that facilitate the transport of nucleic acids into cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors and the like.

  “Expression vector” means a vector comprising a recombinant nucleic acid comprising an expression control sequence operably linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting factors for expression; other elements for expression are 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 (eg, naked or contained in liposomes) and viruses that incorporate recombinant nucleic acids.

  The term “multicloning site” as used herein is a region of a nucleic acid vector that contains more than one nucleotide sequence that is recognized by at least one restriction enzyme.

  The term “antibiotic resistance marker” as used herein is a sequence of nucleotides that encodes a protein that, when expressed in a living cell, confers the ability of the cell to survive and proliferate in the presence of the antibiotic. Means.

  The term “GalNAcT2” as used herein refers to N-acetyl-D-galactosamine transferase 2.

As used herein, the term “truncated” form of a peptide means a peptide that lacks one or more amino acid residues as compared to the full-length amino acid sequence of the peptide. For example, the peptide “NH ₂ -Ala-Glu-Lys-Leu-COOH” means the N-terminal truncated form of the full-length peptide “NH ₂ -Gly-Ala-Glu-Lys-Leu-COOH”. The terms “truncated” and “truncated variant” are used interchangeably herein. As a non-limiting example, a truncated peptide is a GalNAcT2 polypeptide comprising an active domain, a backbone domain, a transmembrane domain, and a signal domain, wherein the signal domain lacks one N-terminal amino acid residue compared to full-length GalNAcT2. It is.

The term “saccharide” is generally any of the carbohydrates that are chemical entities having the most basic structure (CH ₂ O) _n . Sugars vary in complexity and can include nucleic acids, amino acids, or virtually any other chemical moiety present in a biological system.

  “Monosaccharide” means a single unit of carbohydrate of specified identity.

  “Oligosaccharide” means a molecule composed of several units of carbohydrates of a specified identity. A sugar sequence of typically 2-20 units is referred to as an oligosaccharide.

  “Polysaccharide” means a molecule consisting of many units of carbohydrates of a specified identity. However, two or more units of saccharide are considered to be precisely polysaccharides.

  As used herein, a sugar “donor” is a moiety that can provide a saccharide to a glycosyltransferase such that the glycosyltransferase can transfer the saccharide to a sugar acceptor. In a non-limiting example, the GalNAc donor can be UDP-GalNAc.

  As used herein, a sugar “acceptor” is a moiety that can receive a sugar from a sugar donor. Glycosyltransferases can covalently bind saccharides to sugar receptors. In a non-limiting example, G-CSF can be a GalNAc receptor and the GalNAc moiety can be covalently bound to the GalNAc receptor by GalNAc-transferase. In some embodiments, the sugar receptor is a protein or peptide comprising an O-glycosylation site. In a further embodiment, the sugar receptor is, for example, erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferon α, β and γ, factor IX, follicle stimulating hormone, interleukin-2, erythropoietin, anti-TNF-α, As well as lysosomal hydrolases.

  An “specified size” oligosaccharide consists of an identifiable number of monosaccharide units. For example, an oligosaccharide consisting of 10 monosaccharide units can consist of 10 identical monosaccharide units or 5 monosaccharide units of the first identity and 5 monosaccharide units of the second identity. Is. Furthermore, an oligosaccharide of a specified size consisting of monosaccharide units of non-uniform identity can have monosaccharide units in any order from the beginning to the end of the oligosaccharide.

  “Random size” oligosaccharides can be synthesized using methods that do not provide oligosaccharide products of a specified size. For example, the oligosaccharide synthesis method can provide oligosaccharides ranging from 2 monosaccharide units to 22 saccharide units, including any or all lengths therebetween.

  “Commercial scale” means gram scale production of the product saccharide, or glycoprotein, or glycopeptide in a single reaction. In preferred embodiments, commercial scale means production of greater than about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 g.

The term “sialic acid” refers to any member of the 9 carbon carboxylated sugar family. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonuropyranos-1-one acid (Neu5Ac, Often abbreviated as NeuAc or NANA))). The second member of this family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), where the N-acetyl group of NeuAc is hydroxylated. A third sialic acid family member is 2-keto-3-deoxy-nonurosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al, J. Biol. Chem. 265: 21811-21819 (1990)). 9-O-Lactyl-Neu5Ac or 9-O-Acetyl-Neu5Ac, 9-Deoxy-9-fluoro-Neu5Ac and 9-substituted sialic acids also, for example 9-OC ₁ -C ₆ acyl-Neu5Ac -Azido-9-deoxy-Neu5Ac is also included. For verification of the sialic acid family, see, for example, Varki's “Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function”, R. Schauer, Ed. (Springer-Verlag, New York (1992)). See The synthesis and use of sialic acid compounds in sialylation procedures is disclosed in WO 92/16640 published October 1, 1992.

  As used herein, a “protein, peptide, glycoprotein, or glycopeptide remodeling method” refers to the addition of a sugar residue to a protein, peptide, glycoprotein, or glycopeptide using a glycosyltransferase. means. In a preferred embodiment, the sugar residue is covalently attached to the PEG molecule.

  As used herein, an “unpaired cysteine residue” is a correctly folded protein (ie, a protein with biological activity) that does not form a disulfide bond with another cysteine residue. Means cysteine residue.

  “Insoluble glycosyltransferase” means a glycosyltransferase expressed in bacterial inclusion bodies. Insoluble glycosyltransferases are typically solubilized or denatured using, for example, surfactants or chaotropic reagents or some combination. “Refolding” means the process of restoring the structure of a biologically active glycosyltransferase to a solubilized or denatured glycosyltransferase. Thus, refolding buffer means a buffer that enhances or promotes glycosyltransferase refolding.

  "Redox couple" means a mixture of reduced and oxidized thiol reagents and includes reduced and oxidized glutathione (GSH / GSSG), cysteine / cystine, cysteamine / cystamine, DTT / GSSG, and DTE / GSSG (See, eg, Clark, Cur. Op. Biotech. 12: 202-207 (2001)).

  The term “contact” is used interchangeably herein as follows: together, add, mix, pass over, incubate together, flow over, etc.

  The term “PEG” means poly (ethylene glycol). PEG is an example of a polymer conjugated to a peptide. The use of PEG to derivatize peptide therapeutics has been shown to reduce the immunogenicity of those peptides and prolong the clearance time from the circulation. For example, US Pat. No. 4,179,337 (Davis et al.) Relates to non-immunogenic peptides, such as enzymes and peptide hormones conjugated to polyethylene glycol (PEG) or polypropylene glycol. 10-100 moles of polymer per mole peptide are used 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, such as the recombinant glycosyltransferase fusion protein of the present invention, which can be expressed in units of activity. One unit of activity used herein catalyzes the formation of 1 μmol of product per minute at a given temperature (eg, 37 ° C.) and pH value (eg, pH 7.5). Thus, 10 units of enzyme is the catalytic amount of enzyme that converts 10 μmol of substrate to 10 μmol of product per minute at a temperature such as 37 ° C. and a pH value such as 7.5.

An “N-linked” oligosaccharide is such an oligosaccharide linked to the peptide backbone via asparagine by an asparagine-N-acetylglucosamine linkage. N-linked oligosaccharides are also referred to as “N-glycans”. All N-linked oligosaccharides have a common pentasaccharide core of Man ₃ GlcNAc ₂ . These depend on the presence and number of peripheral sugar side branches (also called antennas) such as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. Optionally, the structure may include a core fucose molecule and / or a xylose molecule.

  “O-linked” oligosaccharides are those oligosaccharides that are linked to the peptide backbone through threonine, serine, hydroxyproline, tyrosine, or other hydroxy-containing amino acids.

  The term “substantially” in the definition of “substantially uniform” generally is at least about 60%, at least about 70%, at least about 80%, or more preferably at least about 90%, and even more preferably It means that at least about 95% of the acceptor substrate for a particular glycosyltransferase is glycosylated.

  "Fusion protein" means a protein that contains additional, alternative, fewer and / or different amino acid sequences in the amino acid sequence encoding the original or native full-length protein or subsequences thereof. .

  The `` stem region '' for glycosyltransferases is located close to the transmembrane domain in native glycosyltransferases, and as a residual signal to maintain glycosyltransferases in the Golgi apparatus, And protein domains, or subsequences thereof, that have been reported to function as protein cleavage sites. The backbone region generally begins with the first hydrophilic amino acid, continues to the hydrophobic transmembrane domain, and ends with the catalytic domain, or possibly the first cysteine residue, followed by the transmembrane domain. Examples of backbone regions include the backbone region of eukaryotic ST6GalNAcI, about 30 to about 207 amino acid residues (see eg, mouse enzyme), amino acids 35 to 278 of a human enzyme or amino acids 37 to 253 of a chicken enzyme; the backbone region of a mammalian GalNAcT2 , Including, but not limited to, about 71 to about 129 amino acid residues (see, eg, rat enzyme).

  “Catalytic domain” means a protein domain that catalyzes an enzymatic reaction performed by an enzyme, or a partial sequence thereof. For example, the catalytic domain of a sialyltransferase appears to contain sufficient sialyltransferase partial sequence to transfer a sialic acid residue from a donor to an acceptor saccharide. The catalytic domain can include the entire enzyme, their partial sequences, or can include additional amino acid sequences that are not attached to the enzyme, or their partial sequences, as is found in nature.

  The term “isolated” refers to material that is substantially or essentially free from components that interfere with the activity of the enzyme. With respect to sugars, proteins, or nucleic acids of the invention, the term “isolated” refers to a substance that is substantially or essentially free from components that normally accompany the substance, as found in its native state. Means. Typically, an isolated sugar, protein, or nucleic acid of the present invention is at least about 80% pure as measured by other methods of determining band intensity or purity on silver stained gels, Usually at least about 90%, preferably at least about 95% pure. Purity or homology can be shown by a number of means well known in the art. For example, proteins or nucleic acids in a sample can be resolved by polyacrylamide gel electrophoresis, and then proteins or nucleic acids can be visualized by staining. For some purposes, high resolution of proteins or nucleic acids is desirable, and for purification, for example, HPLC or similar means can be utilized.

Explanation
I. Isolated nucleic acids
A. Overview Various truncated variants of human GalNAcT2 are illustrated herein. However, the present invention is not constructed to target a human GalNAcT2 truncated variant polypeptide lacking amino acid residues 1-51.

  A full-length GalNAcT2 nucleic acid encodes a polypeptide having a domain structure similar to other glycosyltransferases, including an N-terminal signal domain, a transmembrane domain, a backbone domain, and an active domain, where the active domain is The majority of the amino acid sequence of such polypeptides can be included. As will be appreciated by those skilled in the art, the presence of a foreign domain structure relative to the active domain of a recombinant GalNAcT2 polypeptide has a negative effect on the solubility, stability and activity of the polypeptide in an aqueous or in vitro environment. Sometimes. For example, although not intending to be bound by a particular theory, the presence of a hydrophobic transmembrane domain on a recombinant GalNAcT2 polypeptide used in an in vitro reaction mixture indicates that the recombinant GalNAcT2 without a hydrophobic transmembrane domain A polypeptide can be less soluble than a polypeptide, and even by acting or destroying the folded structure, it can even reduce the enzymatic activity of the polypeptide.

  Thus, for the purpose of enhancing the activity, stability and / or utility of GalNAcT2 polypeptides, it is desirable to produce recombinant GalNAcT2 nucleic acids that encode GalNAcT2 shorter than full-length GalNAcT2. The present invention provides such modified forms of GalNAcT2. More particularly, the present invention provides an isolated nucleic acid encoding such a truncated polypeptide.

  The nucleic acids of the invention encode a truncated form of the GalNAcT2 polypeptide, which is described in more detail elsewhere herein. Cleaved GalNAcT2 polypeptides encoded by the nucleic acids of the invention are also referred to herein as “truncated variants” and may be cleaved in a variety of ways, as will be appreciated by those skilled in the art. Examples of truncated polypeptides encoded by the nucleic acids of the present invention include polypeptides lacking a single N-terminal residue, polypeptides lacking a single C-terminal residue, single N-terminal residues and single A polypeptide lacking both the C-terminal residues, a polypeptide lacking a continuous sequence of residues from the N-terminus, a polypeptide lacking a continuous sequence of residues from the C-terminus, and combinations thereof However, it is not limited to these.

  Thus, based on the disclosure provided herein, it will be understood that cleavage of a nucleic acid encoding a GalNAcT2 polypeptide can be made for a number of reasons. In one embodiment of the invention, a cleavage is made to remove part or all of the nucleic acid sequence encoding the signal peptide domain of GalNAcT2.

  In another embodiment of the invention, a cut can be made to remove some or all of the nucleic acid sequence encoding the transmembrane domain of GalNAcT2. As a non-limiting example, removal of some or all of the nucleic acid sequence encoding the transmembrane domain can increase the solubility or stability of the encoded GalNAcT2 polypeptide and / or The expression level of the peptide can be increased.

  In yet another embodiment of the invention, a cut is made to remove part or all of the nucleic acid sequence encoding the basic domain of GalNAcT2. As a non-limiting example, removal of some or all of the nucleic acid sequence encoding the backbone domain can increase the solubility or stability of the encoded GalNAcT2 polypeptide and / or the encoded polypeptide The expression level of can be increased.

  Those skilled in the art will understand, based on the description disclosed herein, how to design and create truncated variants of GalNAcT2 described in detail elsewhere in the present invention. In one aspect of the invention, the nucleic acid residue that is cleaved may be a highly conserved residue. In another aspect of the invention, the nucleic acid residue to be cleaved is selected such that the encoded polypeptide has a novel N-terminal amino acid residue that will aid in purification of the expressed polypeptide. Also good. In yet another aspect, the nucleic acid residue to be cleaved may be selected such that the encoded truncated polypeptide does not contain specific secondary and / or tertiary structure.

B. GalNAcT2 Isolated Nucleic Acids The present invention features encoding nucleic acids that are shorter than full-length GalNAcT2. That is, the present invention features a nucleic acid encoding a truncated GalNAcT2 polypeptide, provided that the polypeptide expressed by the nucleic acid retains the biological activity of the full-length protein. In one aspect of the invention, the truncated polypeptide is a human truncated GalNAcT2 polypeptide.

  As will be appreciated by those skilled in the art, a nucleic acid encoding full-length human GalNAcT2 is one or more identifiable polypeptides in addition to an “active domain”, which is a domain that primarily contributes to the catalytic activity of GalNAcT2. Nucleic acid sequences encoding the domain can be included. This is because it is known in the art that full-length GalNAcT2 polypeptides, particularly full-length human GalNAcT2 polypeptides, contain a signal domain, a transmembrane domain, and a backbone domain in addition to the active domain. Thus, a nucleic acid encoding full-length human GalNAcT2 has a signal domain at the amino terminus of the polypeptide followed by a transmembrane domain immediately adjacent to the signal domain, followed immediately by the transmembrane domain. A polypeptide having a backbone domain followed by an active domain that extends to the carboxy terminus of the polypeptide and is immediately adjacent to the backbone domain may be encoded.

  Accordingly, in one embodiment, the isolated nucleic acid of the invention encodes a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide lacks all or part of the GalNAcT2 signal domain. In another embodiment, the isolated nucleic acid of the invention may encode a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide comprises all or part of a GalNAcT2 signal domain and a GalNAcT2 transmembrane domain. Lacks. In yet another embodiment, the nucleic acid of the invention may encode a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide is a GalNAcT2 signal domain, a GalNAcT2 transmembrane domain and a GalNAcT2 backbone domain or Missing some.

  Based on the present disclosure, those skilled in the art will know how to make and use these and other such truncated variants of human GalNAcT2.

  “Biological activity of GalNAcT2” is the ability to transfer a GalNAc moiety from a GalNAc donor to an acceptor molecule. Full-length human GalNAcT2, the sequence of which is shown in SEQ ID NO: 1, exhibits such activity. “Biological activity of a GalNAcT2 truncated polypeptide” is a similar ability to transfer a GalNAc moiety from a GalNAc donor to an acceptor molecule. That is, the truncated GalNAcT2 polypeptide of the present invention can catalyze the same glycosyl transfer reaction as full-length GalNAcT2. In a non-limiting example, a truncated human GalNAcT2 polypeptide encoded by a GalNAcT2 nucleic acid of the invention has the ability to transfer the GalNAc moiety from a UDP-GalNAc donor to a granulocyte colony stimulating factor (G-CSF) receptor. However, such transfer here results in an O-linked covalent bond of the GalNAc moiety to the threonine residue of G-CSF.

  Thus, nucleic acids encoding less than full-length or “truncated” GalNAcT2 are included in the present invention, provided that the truncated GalNAcT2 has GalNAcT2 biological activity.

  The methods and compositions of the present invention are not constructed to be limited only to nucleic acids containing GalNAcT2 truncation mutants as disclosed herein, but rather to transfer of GalNAc to GalNAc receptors. Constructed to encompass any nucleic acid encoding a GalNAcT2 truncated variant prepared according to the disclosure herein, either known or not known to be capable of catalyzing The A modified nucleic acid sequence, i.e. a nucleic acid sequence having a sequence different from that encoding a natural protein, also has a biological activity in which the modified nucleic acid catalyzes the transfer of GalNAc to a GalNAc receptor, for example. As long as it still encodes a truncated protein having it, it is encompassed by the methods and compositions of the invention. These modified nucleic acid sequences include modifications caused by point mutations, modifications due to the degeneracy of the genetic code or natural allelic variants, as well as modifications that are genetically manipulated, ie artificially introduced . Thus, the term nucleic acid specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

  The present invention is at least about 90%, 95%, 97%, 98% or 99% identical to the nucleic acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9 Characterized by an isolated nucleic acid containing nucleic acid sequence. The 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 is cleaved Encodes a type GalNAcT2 polypeptide.

  The invention also encompasses isolated nucleic acid molecules encoding truncated GalNAcT2 polypeptides that contain changes in amino acid residues that are not essential for activity. Such a polypeptide encoded by an isolated nucleic acid of the invention differs in amino acid sequence from any one of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10, It still retains the biological activity of GalNAcT2. By way of non-limiting example, an isolated nucleic acid of the invention encodes 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. Nucleotide sequences can be included. As yet another non-limiting example, an isolated nucleic acid of the invention comprises at least about 90%, 95%, 97% of the amino acid sequence set forth in any one of SEQ ID NO: 8 or SEQ ID NO: 10, Nucleotide sequences that encode polypeptides having amino acid sequences that are 98% or 99% identical can be included.

  The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, mathematical algorithms useful for comparing two sequences are the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268), the variant of 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. Mol. Biol. 215: 403-410) and accessed, for example, on the World Wide Web of Information Center for Biotechnology (NCBI) Can do. BLAST nucleotide searches can be performed by the NBLAST program (referred to as “blastn” on the NCBI website) using the following parameters: To obtain nucleotide sequences homologous to the nucleic acids described herein, Gap penalty = 5; Gap extension penalty = 2; Mismatch penalty = 3; Match reward = 1; Expected value 10.0; and Word size = 11. BLAST protein searches are performed by the XBLAST program (referred to as “blastn” on the NCBI website) or the NCBI “blastp” program with the following parameters: amino acid sequence homologous to the protein molecule described herein To obtain the expected value 10.0, BLOSUM62 scoring matrix. To obtain a gapped alignment for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects distant relationships between molecules and relationships between molecules that share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters for each program (eg, XBLAST and NBLAST) can be obtained and used from the NCH website of NIH's NLM. In general, please refer to the website of Biotechnology Information Center (NCBI) operated by the National Library of Medicine (NLM) and the National Institutes of Health (NIH).

  In another aspect, a nucleic acid useful in the methods and compositions of the invention and encoding a truncated GalNAcT2 polypeptide comprises at least one nucleotide inserted into the nucleic acid sequence of such truncated variant. Can have. Alternatively, the additional nucleic acid encoding the truncated GalNAcT2 polypeptide can have at least one nucleotide deleted from the nucleic acid sequence. Furthermore, a GalNAcT2 nucleic acid encoding a truncated variant and useful in the present invention can have both nucleotide insertions and nucleotide deletions present in a single nucleic acid sequence encoding the truncated polypeptide.

  Techniques for introducing changes into nucleotide sequences that are designed to alter the functional properties of the encoded protein or polypeptide are well known in the art. Such modifications include base deletions, insertions or substitutions and the resulting changes in amino acid sequence. Modification of nucleic acid stability, modification of nucleic acid expression level, modification of expressed polypeptide stability or half-life, modification of expressed polypeptide activity, expressed polypeptide properties, as known to those skilled in the art Nucleic acid insertions and / or deletions can be designed into genes for a number of reasons, including, but not limited to, modification of features and alteration of glycosylation patterns. All such modifications to the nucleotide sequence encoding such a protein are encompassed by the present invention.

  The methods and compositions of the present invention are not intended to be limited by the nature of the nucleic acid utilized. The target nucleic acid included in the methods and compositions of the invention may be a native nucleic acid or a synthesized nucleic acid. The nucleic acid can be DNA or RNA and can be double-stranded, single-stranded, or partially double-stranded. Furthermore, the nucleic acid can be recognized as part of a virus or other macromolecule. See, for example, Fasbender et al, 1996, J. Biol. Chem. 272: 6479-89.

II. In vectors and expression systems and other related aspects, the present invention provides a nucleic acid comprising a promoter / regulatory sequence so that the nucleic acid is preferably capable of directing the expression of a polypeptide encoded by the nucleic acid An isolated nucleic acid encoding a truncated GalNAcT2 polypeptide operably linked to the. Thus, the present invention is described in, for example, Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons). , New York), including expression vectors and methods for introducing foreign DNA into cells by co-expression of foreign DNA in those cells.

  Expression of the truncated GalNAcT2 polypeptide in the cell may be accomplished by creating a plasmid, virus, or other type of vector containing a nucleic acid encoding the appropriate nucleic acid, where the nucleic acid is introduced into the vector. Is operably linked to promoter / regulatory sequences utilized to drive expression of the encoded polypeptide with or without a tag in the cells. In addition, promoters well known in the art that are induced in response to induction of substances such as metals, glucocorticoids are also contemplated in the present invention. Thus, it is understood that the present invention includes the use of promoter / regulatory sequences that are either known or unknown, as well as being capable of driving expression of truncated GalNAcT2 polypeptides operably linked thereto. Will be done.

  In expression systems useful in the present invention, a nucleic acid encoding a truncated GalNAcT2 polypeptide may be fused with one or more additional nucleic acids encoding a functional polypeptide. As a non-limiting example, an affinity tag coding sequence may be inserted into a nucleic acid vector adjacent to, upstream, or downstream of a truncated GalNAcT2 polypeptide coding sequence. As will be appreciated by those skilled in the art, affinity tags are typically inserted into the multiple cloning site in-frame with the truncated GalNAcT2 polypeptide. One of skill in the art will also appreciate that an affinity tag coding sequence can be used to produce a recombinant fusion protein by simultaneously expressing an affinity tag and a truncated GalNAcT2 polypeptide. The expressed fusion protein can then be isolated, purified, or identified by affinity tag.

  Affinity tags useful in the present invention include, but are not limited to, maltose binding protein, histidine tag, factor IX tag, glutathione-S-transferase tag, FLAG-tag, and starch-binding domain tag. Absent. Other tags are well known in the art and the use of such tags in the present invention will be readily understood by those skilled in the art.

  As will be appreciated by those skilled in the art, vectors containing a truncated GalNAcT2 polypeptide of the invention can be used to express a truncated polypeptide as either a non-fusion or fusion protein. The selection of any particular plasmid vector or other DNA vector is not a limiting factor of the present invention, and a wide range of vectors are well known in the art. Furthermore, the selection of specific promoter / regulatory sequences and the functional linkage of those promoter / regulatory sequences to a DNA sequence encoding a truncated GalNAcT2 polypeptide is well within the skill of the artisan. Such techniques are well known in the art, such as Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). As a non-limiting example, a vector useful in one embodiment of the invention is based on the pcWori + vector (Muchmore et al., 1987, Meth. Enzymol. 177: 44-73).

  Accordingly, the present invention includes a vector comprising an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide. Nucleic acid integration into vectors and vector selection is described in Sambrook et al. (Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as is well known in the art.

  In aspects of the invention, an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide is integrated into the genome of the host cell together with a nucleic acid encoding the truncated GalNAcT2 polypeptide. In another aspect of the invention, the cells are transiently transfected with an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide. In yet another aspect of the invention, the cells are stably transfected with an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide.

  For purposes of inserting an isolated nucleic acid into a cell, one of skill in the art will vary the available and necessary methods for introducing the isolated nucleic acid of the invention into the host cell, and selection of the host cell. You will also understand that it depends on. Suitable methods for introducing isolated nucleic acids into host cells are well known in the art. Other suitable methods for transforming or transfecting host cells are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. ), And others such as those found in such laboratory manuals.

  Nucleic acid encoding a truncated GalNAcT2 polypeptide can be purified by any suitable means, as is well known in the art. For example, nucleic acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis. Of course, those skilled in the art will recognize that the purification method depends in part on the size of the DNA to be purified.

  The invention also features a recombinant bacterial host cell comprising a nucleic acid vector, particularly as described elsewhere herein. In one aspect, the recombinant cell is transformed with the vector of the present invention. The transformed vector need not be integrated into the cell genome, nor need it be expressed in the cell. However, this transformed vector could be expressed in cells. In one aspect of the invention, E. coli is used for transformation of the vectors of the invention and expression of proteins therefrom. In another aspect of the invention, the E. coli K-12 strain is useful for the expression of proteins from the vectors of the invention. E. coli strains useful in the present invention include, but are not limited to, JM83, JM101, JM103, JM109, W3110, chi1776, and JA221.

  It will be appreciated that host cells useful in the present invention can be grown and cultured on a small, medium or large scale. For example, the host cells of the present invention are useful for testing the expression of proteins from large quantities of the vectors of the present invention as they are useful for large-scale production of reagents or therapeutic protein products. Techniques useful for culturing host cells and expressing proteins from the vectors contained therein are well known in the art and are therefore not described herein.

  Host cells useful for the methods of the invention, as described above, can be prepared according to various methods, as will be appreciated by those of skill in the art based on the disclosure provided herein. In one aspect, the host cells of the invention can be transformed with the vectors of the invention to produce the transformed host cells of the invention. Transformation, as is known to those skilled in the art, involves the process of inserting a nucleic acid vector into a host cell so that the host cell containing the nucleic acid vector remains alive. Transformation of such nucleic acids into bacterial cells involves the production of stably-transformed host cells, performing biological deposits, propagation of host cells containing the vector, production and isolation of additional vectors. Including, but not limited to, the growth of host cells containing vectors for expression, expression of target proteins encoded by the vectors, and the like.

  Many methods for transforming cells with vectors are well known in the art and are therefore not described herein. As a non-limiting example, competent bacterial cells of the present invention can be transformed with the vectors of the present invention using electroporation. Methods for making “competent” bacterial cells are well known in the art and typically involve the preparation of bacterial cells such that the cells take up foreign DNA. Similarly, methods of electroporation are known in the art and a detailed description of such methods can be found, for example, in Sambrook et al. (1989, supra). Transformation of competent cells with vector DNA can also be achieved using chemical-based methods. An example of a well-known chemical-based method of bacterial transformation is described by Inoue et al. (1990, Gene 96: 23-28). Other transformation methods will be known by those skilled in the art.

  The transformed host cell of the present invention can be used to express a truncated GalNAcT2 polypeptide of the present invention. In an embodiment of the invention, a transformed host cell contains a vector of the invention, which now contains a nucleic acid sequence encoding a truncated polypeptide of the invention. The truncated polypeptide is expressed using expression methods known in the art (eg, 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.

  Methods for isolating expressed polypeptides are well known in the art, and those skilled in the art will know how to best isolate an expressed polypeptide based on the characteristics of a given host cell expression system. Will know what to decide. As a non-limiting example, the expressed polypeptide secreted from the host cell can be isolated from the growth medium. Isolation of the polypeptide from the growth medium can include removal of bacterial cells and cell debris. As another non-limiting example, an expressed polypeptide contained in a host cell can be isolated from the host cell. Isolation of such “intracellular” expressed polypeptides involves the destruction of host cells and removal of cellular debris from the resulting mixture. These methods are not intended to be exclusive representations of the invention, but rather are merely intended to illustrate various adaptations of the invention.

  Purification of the truncated polypeptide expressed in accordance with the present invention can be performed by means known in the art. Those skilled in the art will know how to determine the best method of purification of a polypeptide expressed according to the present invention. Purification methods include the expression host, polypeptide crude extract content, polypeptide size, polypeptide characteristics, desired final product of the polypeptide purification process, and subsequent use of the final product of the polypeptide purification process. There will be selected by one of ordinary skill in the art based on, but not limited to, factors.

  In aspects of the invention, isolation or purification of a truncated polypeptide expressed in accordance with the present invention may not be desirable. In certain aspects of the invention, the expressed polypeptide can be stored or transported within the bacterial host cell in which the polypeptide is expressed. In another aspect of the invention, the expressed polypeptide is used in a crude lysate form produced by lysis of host cells in which the polypeptide is expressed. In yet another aspect of the invention, the expressed polypeptide can be partially isolated or partially purified according to any of the methods shown or described herein. Those skilled in the art will know to isolate or purify the polypeptides of the invention if that is not desired, and will be familiar with the techniques available for the use and preparation of such polypeptides.

  Based on the disclosure provided herein, those skilled in the art will also know how to prepare eukaryotic host cells of the present invention. As described elsewhere herein and known to those of skill in the art based on the disclosure provided herein, an isolated nucleic acid encoding a truncated GalNAcT2 polypeptide can be, for example, a lentivirus-based Genomic integration or plasmid-based transfection can be used and introduced into eukaryotic host cells (Sambrook et al., Third Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In one embodiment of the invention, the eukaryotic host cell is a fungal cell. In another embodiment, a nucleic acid encoding a truncated polypeptide of the invention is cloned into a lentiviral vector that contains a specific promoter sequence for expression of the truncated polypeptide. The lentiviral vector containing the truncated polypeptide is then used to transfect host cells for expression of the truncated polypeptide. Methods of making and using lentiviral vectors, such as those useful in the present invention, are well known in the art and are not further described herein.

  In yet another embodiment, the nucleic acid encoding the truncated polypeptide of the present invention is introduced into a host cell using a viral expression system. Viral expression systems are well known in the art and are not described in detail herein. In one aspect of the invention, the viral expression system is a mammalian viral expression system. In another aspect of the invention, the viral expression system is a baculovirus expression system. Such viral expression systems are typically commercially available from a number of suppliers.

  Those skilled in the art will know how to use host cell-vector expression systems for expression of the truncated polypeptides of the invention. Appropriate cloning and expression vectors for use with eukaryotic hosts are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, NY (2001)). The entirety of which is hereby incorporated by reference.

Insect cells can also be used to express a truncated polypeptide of the invention. In one aspect of the invention, Sf9, SF9 ⁺ , Sf21, High Five ™ or Drosophila Schneider S2 cells can be used. In yet another aspect of the invention, a baculovirus, or baculovirus / insect cell expression system is used, the pAcGP67, pFastBac, pMelBac, or pIZ vector and the polyhedrin, p10, or OpIE3 actin promoter are used to cleave the invention. Type polypeptides can be expressed. In another aspect of the invention, the Drosophila expression system can be used with a pMT or pAC5 vector and an MT or Ac5 promoter.

  The truncated GalNAcT2 polypeptide of the present invention can also be expressed in mammalian cells. In one aspect of the invention, 294, HeLa, HEK, NSO, Chinese hamster ovary (CHO), Jurkat, or COS cells can be used to express a truncated polypeptide of the invention. For mammalian cell expression of a truncated polypeptide, a suitable vector such as pT-Rex, pSecTag2, pBudCE4.1, or pCDNA / His Max vector can be used, for example, with a CMV promoter. As will be appreciated by those skilled in the art, in addition to promoter selection, methods and strategies for introducing one or more promoters used to express a truncated GalNAcT2 polypeptide of the invention into a host cell include: It is well known in the art and will vary depending on the host cell and expression system used.

  A variety of mammalian cell culture systems can be used to express the recombinant protein. Non-limiting examples of mammalian expression systems include monkey kidney fibroblast COS-7 strain described by Gluzman (Cell 23: 175 (1981)) and other cells capable of expressing equivalent vectors Strains such as C127, 3T3, CHO, HeLa and BHK cell lines (tines). Mammalian expression vectors contain an origin of replication, an appropriate promoter and essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, transcription termination sequences, and 5 ′ flanking non-transcribed sequences. For example, DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splicing site, and polyadenylation site may be used to provide the necessary non-transcribed genetic elements.

  The methods available and necessary for the introduction of the isolated nucleic acids of the invention into host cells will vary as will be understood by those skilled in the art and will depend on the choice of the host cell. Suitable methods for introducing an isolated nucleic acid into a host cell are well known in the art. As a non-limiting example, vector DNA is introduced into eukaryotic cells using conventional transfection techniques. As used herein, the term “transfection” is used in various arts with respect to the introduction of foreign nucleic acid (eg, DNA) into a host cell, including DEAE-dextran-mediated transfection, lipofection, or electroporation. Means recognized technology. Suitable methods for transformation or transfection of host cells are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 3nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001) and It can be found in other experimental manuals.

  For example, for stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. In order to identify and select these transformants, a gene that encodes a selectable marker (eg, antibiotic resistance) is generally introduced into the host cells along with the gene of interest. Various 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 present invention or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene survive while other cells die).

III. Polypeptides The truncated GalNAcT2 polypeptides of the present invention can be cleaved by a variety of methods, as known and understood by those of skill in the art, based on the present disclosure. Examples of truncated polypeptides of the present invention are: a polypeptide lacking a single N-terminal residue, a polypeptide lacking a single C-terminal residue, a single N-terminal residue and a single C-terminal residue Including a polypeptide lacking both, 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 combination thereof. However, it is not limited to these.

  As will be appreciated by those skilled in the art, a full-length human GalNAcT2 polypeptide includes one or more identifiable polypeptide domains in addition to an “active domain”, which is a domain that primarily contributes to the catalytic activity of GalNAcT2. be able to. This is because it is known in the art that full-length GalNAcT2 polypeptides, particularly full-length human GalNAcT2 polypeptides, contain a signal domain, a transmembrane domain, and a backbone domain in addition to the active domain. Thus, full-length human GalNAcT2 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 backbone domain immediately adjacent to the transmembrane domain, followed by a poly A polypeptide having an active domain extending to the carboxy terminus of the peptide and immediately adjacent to the backbone domain may be encoded.

  Thus, in one embodiment, a GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide that lacks all or part of a GalNAcT2 signal domain. In another embodiment, a GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide lacking all or part of the GalNAcT2 signaling domain and GalNAcT2 transmembrane domain. In yet another embodiment, the GalNAcT2 polypeptide of the invention is a truncated human GalNAcT2 polypeptide lacking all or part of the GalNAcT2 signal domain, GalNAcT2 transmembrane domain, and GalNAcT2 backbone domain. Based on the present disclosure, those skilled in the art will know how to make and use these and other such truncated variants of human GalNAcT2.

  The size and identity of the truncated GalNAcT2 variants of the present invention is based on the point at which the full-length polypeptide is cleaved. As a non-limiting example, a “Δ40 human truncated GalNAcT2” variant of the invention is a truncated GalNAcT2 of the invention in which amino acids 1 to 40 are deleted from the polypeptide, counting from the N-terminus of the full-length polypeptide. Means polypeptide. Thus, the N-terminus of the Δ40 human truncated GalNAcT2 variant begins with an amino acid residue referred to as “amino acid 41” of the full-length polypeptide. This nomenclature can be applied to all truncated GalNAcT2 polypeptides of the invention, including human GalNAcT2.

  Accordingly, the present invention includes an isolated polypeptide comprising a truncated GalNAcT2 polypeptide. Preferably, the isolated truncated GalNAcT2 polypeptide of the present invention is a polypeptide having the amino acid sequence of any one of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10. Have a degree of agreement of at least about 90%. More preferably, the isolated polypeptide is about 95% identical to a polypeptide of any one of the sequences set forth in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10, and even more preferably Is about 98% identical, even more preferably about 99% identical, and most preferably the isolated polypeptide comprising the truncated GalNAcT2 polypeptide is the same.

  The present invention also provides analogs of polypeptides, including truncated GalNAcT2 polypeptides disclosed herein. Analogs differ from the native protein or peptide by conservative amino acid sequence differences or by modifications that do not affect the sequence, or both.

For example, conservative amino acid changes can be made that alter the primary sequence of the protein or peptide but do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups:
Glycine, alanine;
Valine, isoleucine, leucine;
Aspartic acid, glutamic acid;
Asparagine, glutamine;
Serine, threonine;
Lysine, arginine;
Phenylalanine, tyrosine.

  Modifications (usually without changing the primary sequence) include in vivo or in vitro chemical derivatization of the polypeptide, eg, acetylation, carboxylation. Also includes glycosylation modifications, such as those made by modifying the glycosylation pattern of a polypeptide during its synthesis and processing, or in further processing steps; for example, for mammalian glycosylation or deglycosylation enzymes By exposure to enzymes that affect glycosylation of the polypeptide. Also encompassed are sequences having phosphorylated amino acid residues, such as, for example, phosphotyrosine, phosphoserine, or phosphothreonine.

  Modified using conventional molecular biology techniques to improve their resistance to proteolysis, or to optimize solubility properties, or to make them more suitable as therapeutic agents Polypeptides are also included. Analogs of such polypeptides include those containing residues other than natural L-amino acids, such as D-amino acids or non-natural synthetic amino acids. The peptides of the present invention are not limited to the products of any of the processes listed herein.

  Fragments of the truncated GalNAcT2 polypeptide of the present invention are included in the present invention, provided that the fragment has the biological activity of the full-length polypeptide. That is, the truncated GalNAcT2 polypeptide of the present invention can catalyze the same glycosyl transfer reaction as full-length GalNAcT2. As a non-limiting example, a truncated human GalNAcT2 polypeptide has the ability to transfer a GalNAc moiety from a UDP-GalNAc donor to a granulocyte colony stimulating factor (G-CSF) receptor, where such transfer is , Resulting in an O-linked covalent bond to the G-CSF threonine residue of the GalNAc moiety. Thus, less than full length, or “truncated” GalNAcT2 is included in the present invention, provided that the truncated GalNAcT2 has GalNAcT2 biological activity.

  In another aspect of the invention, a composition comprising an isolated truncated GalNAcT2 polypeptide described herein can comprise a highly purified truncated GalNAcT2 polypeptide. Alternatively, a composition comprising a truncated GalNAcT2 polypeptide can comprise a cell lysate prepared from the cells used to express a particular truncated GalNAcT2 polypeptide. Furthermore, the truncated GalNAcT2 polypeptide of the present invention can be any one of a number of cells suitable for expression of the polypeptide, such as cells well known to those of skill in the art, as described in detail elsewhere herein. Can be expressed in one.

  The substantially pure protein isolated and obtained as described herein can be purified according to known techniques of protein purification, here to monitor purification at each stage of the technique. In addition, immunological, enzymatic or other assays are used. Protein purification methods are well known in the art and are described, for example, in a paper by Deutscher et al. (1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

IV. Methods The present invention features a method of expressing a truncated polypeptide. Polypeptides that can be expressed according to the methods of the invention include truncated GalNAcT2 polypeptides. More preferably, polypeptides that can be expressed according to the methods of the present invention include, but are not limited to, truncated human GalNAcT2 polypeptides. In a preferred embodiment, the polypeptide that can be expressed according to the method of the invention is a polypeptide comprising any one of the polypeptide sequences shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10. .

  In one embodiment, the invention features a method of expressing a truncated GalNAcT2 polypeptide encoded by an isolated nucleic acid of the invention, as described elsewhere herein. The truncated GalNAcT2 polypeptide has the property of catalyzing the transfer of the GalNAc moiety to the receptor moiety. In one aspect of the invention, a method of expressing a truncated GalNAcT2 polypeptide comprises the steps of cloning the isolated nucleic acid of the invention into an expression vector, inserting the expression vector construct into a host cell, and from there Expressing a truncated GalNAcT2 polypeptide.

  In addition to methods of polypeptide expression, expression systems and the construction of recombinant host cells for polypeptide expression are discussed extensively in detail elsewhere herein. It will be appreciated that methods of expression of truncated polypeptides of the present invention include, but are not limited to, all such methods described herein. In some expression systems, the truncated GalNAcT2 polypeptide of the invention is expressed as an insoluble protein, eg, an encapsulated protein in a bacterial host cell. A method for refolding an insoluble glycosyltransferase comprising a GalNAcT2 polypeptide is described in US Provisional Patent Application No. 60 / 542,210 filed February 4, 2004; US Patent Provisional Application filed August 6, 2004 No. 60 / 599,406; U.S. Provisional Application No. 60 / 627,406 filed on November 12, 2004; and International Patent Application PCT / US05 / 03856 filed on Feb. 4, 2005. Each of which is incorporated herein by reference as a reference for all purposes.

  The invention also features a method of catalyzing the transfer of a GalNAc moiety to a GalNAc acceptor moiety, wherein the GalNAc transfer reaction comprises a GalNAc donor moiety and a GalNAc acceptor moiety of the truncated GalNAcT2 polypeptide of the present invention. Performed by incubation together. In one aspect, a truncated GalNAcT2 polypeptide of the invention mediates covalent linkage of a GalNAc moiety to a GalNAc receptor moiety, thereby catalyzing the transfer of the GalNAc moiety to a receptor moiety.

  In one embodiment of the invention, the truncated GalNAcT2 polypeptide useful in the transglycosylation reaction is a truncated human GalNAcT2 polypeptide. In one aspect, the human GalNAcT2 glycosyltransferase reaction involves the transfer of GalNAc residues from a GalNAc donor to a GalNAc acceptor.

  As a non-limiting example, a method for catalyzing the transfer of a GalNAc moiety to an acceptor moiety involves incubating a truncated GalNAcT2 polypeptide with a UDP-GalNAc GalNAc donor and a granulocyte colony stimulating factor (G-CSF) acceptor moiety. Wherein the truncated GalNAcT2 polypeptide mediates the transfer of GalNAc from a UDP-GalNAc donor to a GCSF receptor.

  Accordingly, in one embodiment, the invention also features a polypeptide receptor moiety. In one embodiment of the invention, the polypeptide receptor moiety is human growth hormone. In another embodiment, the polypeptide receptor moiety is erythropoietin. In yet another embodiment, the polypeptide receptor moiety is interferon-α. In another embodiment, the polypeptide receptor moiety is interferon-beta. In another embodiment of the invention, the polypeptide receptor moiety is interferon-γ. In yet another aspect of the invention, the polypeptide receptor moiety is a liposomal hydrolase. In another embodiment, the polypeptide receptor moiety is a blood factor polypeptide. In yet another embodiment, the polypeptide receptor moiety is anti-tumor necrosis factor-α. In another embodiment of the invention, the polypeptide receptor moiety is a follicle stimulating hormone.

  In one embodiment, the invention also features a method of transferring a GalNAc-polyethylene glycol complex to a receptor molecule. In one aspect, the receptor molecule is a polypeptide. In another aspect, the receptor molecule is a glycopeptide. Compositions and methods useful for the design, generation and transfer of GalNAc-polyethylene glycol conjugates to receptor molecules are described in PCT International Patent Application WO 03/031464 (PCT / US02 / 32263) and US Patent Application No. 2004/0063911. Each of which is incorporated herein by reference in its entirety. Assay methods for glycosyltransferase activity are well known in the art. Various assays have been published for detecting glycosyltransferases that can be used in accordance with the present invention. The following illustrates such an assay useful for detecting glycosyltransferase activity, but should not be considered limiting. Furukawa et al. (1985, Biochem. J, 227: 573-582) discusses borate-impregnated paper electrophoresis assays and fluorescence assays. Roth et al. (1983, Exp'l Cell Research 143: 217-225) describe the application of the borate assay to the previously calorimetric glucuronyltransferase. Benau et al. (1990, J. Histochem. Cytochem., 38: 23-30) describe a histochemical assay based on the reduction of diazonium salts by NADH. See also Roth US Pat. No. 6,284,493, which is incorporated herein by reference.

Experimental Examples The invention will now be described with reference to the following examples. These examples are provided merely for purposes of illustration, and the invention is not to be construed as limited to these examples, but rather becomes apparent as a result of the content provided herein. And is configured to encompass all variations.

Example 1: Cloning, expression, and refolding of human polypeptide N-acetylgalactosaminyltransferase II (GalNAcT2) in E. coli JM109 In order to evaluate the sialyltransferase activity of truncated variants of human GalNAcT2, The construct was designed and produced. The four mutants produced were a truncated mutant Δ40 having a lysine corresponding to R41 of full-length human GalNAcT2 shown in SEQ ID NO: 2 as its new N-terminal residue, and its new N-terminal residue. A truncated mutant Δ51 having a lysine corresponding to K52 of full-length human GalNAcT2 shown in SEQ ID NO: 2 as a group, G74 of full-length human GalNAcT2 shown in SEQ ID NO: 2 as its new N-terminal residue And a truncated mutant Δ94 having a glycine corresponding to G95 of full-length human GalNAcT2 shown in SEQ ID NO: 2 as its new N-terminal residue.

  The truncated human polypeptide N-acetylgalactosaminyltransferase II (GalNAcT2) was expressed as a maltose binding protein (MBP) -fusion protein in inclusion bodies derived from E. coli JM109 cells. The production of active enzyme was tested by refolding and assay for two polypeptide receptors. Thus, the generation of several truncated forms of the human polypeptide GalNAcT2 as a maltose binding protein fusion protein in E. coli JM109 cells has been described herein. Recombinant proteins are refolded from isolated inclusion bodies using the Hampton FoldIt screening kit (Hampton Research, Aliso Vieja, CA). All four constructs were expressed in JM109 E. coli at a level of about 2 g / L culture medium.

  PCR (polymerase chain reaction) amplification consists of 5 μl template DNA (11 μg / ml, 100-fold diluted pBKS-Full ppGalNAcT2), 40 pmol 5′-primer and 3′-primer, 10 nmol dNTP mix, and 5 units Herculase (Trademark) Containing DNA polymerase with a final reaction volume of 50 μl under conditions of 31 cycles of denaturation at 95 ° C. for 45 seconds, annealing at 62 ° C. for 45 seconds, and extension at 74 ° C. for 170 seconds. . The PCR product was subjected to 1% agarose gel electrophoresis. The DNA fragment was cleaved and purified by QIAEX II gel extraction kit (Qiagen, Valencia, CA). Table 1 illustrates the primers used for the PCR reaction.

(Table 1) Primers used for cloning of ppGalNAcT2

  The gel purified PCR product was digested with BamHI and XhoI, gel purified again and ligated into the pCWin2MBP vector previously digested with the same restriction enzymes. The ligated product was transformed into E. coli DH5α electrically competent cells. Transformants were seeded on LB agar plates containing 50 μg / ml kanamycin and incubated overnight at 37 ° C. Three colonies were picked for each construct and cultured in LB medium containing 15 μg / ml kanamycin. Plasmid DNA was purified by QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.) And screened by restriction mapping with BamHI and XhoI. A plasmid with the correct digestion pattern was transformed into JM109 chemically competent cells.

  JM109 cells were cultured overnight in a 15 ml culture tube containing 6 ml LB medium and 15 μg / ml kanamycin at 37 ° C. with high speed shaking (250 rpm). For each culture, 2 mL of the starting culture was transferred to a 50 ml centrifuge tube containing 23 ml LB medium with 15 μg / ml kanamycin and incubated at 37 ° C. for 3 hours with high speed shaking. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added at a final concentration of 0.4 mM to induce protein expression. After shaking for an additional 3 hours at 37 ° C. (250 rpm), the cells were collected by centrifugation at 3,500 × g for 10 minutes. The cell pellet was then resuspended in 0.6 ml of 20 mM Tris-HCl buffer (pH 8.5) containing 1% Triton X-100. Then lysozyme (100 μg) and DNaseI (2 μg) were added. This mixture was shaken for 45 minutes at 37 ° C. in an incubator shaker and then transferred to a 1.5 ml microcentrifuge tube. The lysate was separated from inclusion bodies (IB) by centrifugation at 14,000 rpm for 5 minutes.

  Prepare each sample for SDS-PAGE separation by mixing 5 μl of total cell suspension, lysate or inclusion body suspension with 5 μl of 2xTris-Glycine SDS sample buffer and 1.1 μl of DTT (1M) did. The mixture was heated at 98 ° C. for 5 minutes, cooled to room temperature, and loaded into each well of a 1.0 mm × 15 well 4-20% Tris-glycine gradient gel. Electrophoresis was performed at 120V for 100 minutes. The gel was then stained for 2 hours and decolorized with distilled water (see FIGS. 4-6).

  Inclusion bodies are placed in a lysis buffer containing 4 M guanidine-HCl, 100 mM Tris-HCl (pH 9.0), 5 mM EDTA, and 10 mM DTT at a concentration of 20 mg / ml (high protein concentration) or 2 mg / ml (low protein). Concentration). Inclusion body refolding with the Hampton FoldIt screening kit was performed according to the manufacturer's protocol (Hampton Products, Aliso Viejo, Calif.) Except that 1/10 volume was used (100 μl-scale).

The non-radioactive enzyme activity assay of the lysate was performed using 50 mM MES buffer (pH 6.0), MnCl ₂ (15 mM), MgCl ₂ (15 mM), NaCl (0.15 M), UDP-GalNAc (5 mM), 1.5 μg G- CSF (receptor) and a final volume of 10 μl containing 2.15 μl of lysate sample were run overnight at 37 ° C. in 0.5 μl microcentrifuge tubes. As a negative control, the enzyme was replaced with H ₂ O. Purified recombinant ppGalNAcT2 (0.5 μl) from the Sf9 baculovirus expression system was used as a positive control. An assay for refolded inclusion bodies was described for the lysate except that interferon α2b (4 μg) was used as the enzyme acceptor and the sample volume added to the reaction mixture was 5.65 μl. It was done in the same manner as the one.

  The DNA fragment for the ppGalNAcT2 gene (about 1.5 kb) was successfully amplified by PCR as shown in FIG. Vector plasmid DNA pCWin2MBP was digested with BamHI and XhoI and purified on a 1% agarose gel. The gel-purified DNA fragment was digested with the same two enzymes and purified. After digestion, the DNA fragment was visually confirmed on an agarose gel (FIG. 2B).

  BamHI and XhoI digestion of plasmids purified from 12 selected colonies showed the expected correct pattern on a 1% agarose gel. The size of this vector was approximately 6.2 kb and the insert was approximately 1.5 kb. The maltose-binding protein (MBP) expressed in JM109 transformed with the pCWin2MBP vector plasmid showed a band at approximately 43 kDa. Over 90% of the total cellular protein was MBP. The # 2 colony of construct N41R expressed a shorter protein than expected, indicating the appearance of a mutation. All other 11 colonies showed a band at approximately 100 kDa of the MBP-ppGalNAcT2 fusion protein, with more than 80% of the total protein being the target fusion protein.

  Gel electrophoresis showed that most of MBP was expressed as a soluble form in the cell lysate (FIGS. 1 and 2). Proteins overexpressed in the wrong construct (colony # 2) for N41R were also found in the cell lysate. However, most of the MBP-ppGalNAcT2 fusion protein was in inclusion bodies (lanes 1 and 3-12 in FIGS. 1 and 2). More than 90% of the proteins in inclusion bodies were MBP fusion proteins of interest.

  In summary, the four truncated forms of the human polypeptide GalNAcT2 were successfully cloned into the pCWin2MBP vector and expressed as an MBP fusion protein in inclusion bodies in E. coli JM109. The expression level of the enzyme in the inclusion bodies was about 2 g / L. As estimated from SDS-PAGE, more than 80% of the inclusion bodies were target MBP-ppGalNAcT2 fusion proteins.

Example 2: Development of protein refolding conditions for MBP-human GalNAcT2 expressed in E. coli
Refolding experiments for MBP-GalNAcT2 were performed on a 1 ml scale under 4 different MBP-GalNAcT2 DNA constructs and 16 different possible refolding conditions. Refolding was performed using the Hampton Research Foldit kit (Hampton Research, Aliso Viejo, Calif.) And the assay was performed by detecting the radioactivity of [ ³ H] UDP-GalNAc added to the MuC-2 peptide and using GalNAc interferon. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) using addition to α-2b and G-CSF was performed. This data illustrates that MBP-GalNAcT2 expressed in E. coli can be refolded into an active enzyme. It is clear that the active configuration of MBP-GalNAcT2 constructs 1 and 2 was identified under refolding conditions 8 and 15, as observed by the Hampton Research's Foldit kit (Hampton Research, Aliso Viejo, CA) . [ ³ H] UDP-GalNAc assay showed success and was later confirmed by interferon α-2b (IFα-2b) and granulocyte colony stimulating factor (G-CSF) -based glycosyltransferase assay It was. Specific methods and data for this study are presented herein.

As described elsewhere herein, the GalNAcT2 construct used in the present invention comprises DNA encoding various amino-terminally truncated amino acid variants of the original human GalNAcT2 protein, which is described below. Including the following constructs beginning with the N-terminal amino acid:
Construct 1-pCWin2 MBP-GalNAcT2-R41 Arginine (924aa, 103682.5MW)
Construct 2-pCWin2 MBP-GalNAcT2-K52 lysine (913aa, 102286.0MW)
Construct 3-pCWin2 MBP-GalNAcT2-G74 glycine (891aa, 99799.3MW) and Construct 4-pCWin2 MBP-GalNAcT2-G95 glycine (870aa, 97419.8MW).

  The construct was first expanded to 2 ml of starting culture by inoculation with 2 ml of Martone L-broth containing 10 μg / ml kanamycin sulfate, stripped from a specific glycerol stock culture with a pipette tip. This procedure was performed on all four starting cultures on all four constructs. The starting culture was incubated overnight at 37 ° C. and tumbled at 250 rpm. From the overnight culture, 275 ml of Martone L-broth-culture containing 4 types of 10 μg / ml kanamycin sulfate was prepared. Each of these cultures was inoculated with 275 μL of one of the 2 mL starting cultures of constructs 1 through 4. These 275 ml cultures were incubated overnight at 37 ° C. with shaking at 250 rpm.

  Finally, 1 L of Martone L-broth culture containing 4 types of 10 μg / ml kanamycin sulfate was prepared. Each of these cultures was inoculated with 40 ml of one of the 275 mL cultures of constructs 1 to 4. These 1 L cultures were incubated at 37 ° C. with shaking at 250 rpm until the measured OD600 reached approximately 1.0. When this point was reached, IPTG was added to each of the four 1 L cultures to a final concentration of 0.4 mM. The culture was then incubated overnight at 37 ° C. with shaking at 250 rpm.

  A 1 L culture containing the JM109 pCWin2 MBP-GalNAcT2 construct numbered 1-4 was transferred to a 1 L centrifuge bottle. The culture was then centrifuged at 5000 rpm and 4 ° C. for 30 minutes. The supernatant was removed and the pellet was weighed. The pellet from each sample was then washed and inclusion bodies (IB) were isolated. The pellet of each construct was first resuspended in 15 ml of 20 mM Tris-HCl (pH 8.5), 5 mM EDTA and then lysed by passing twice through a French press at 12,000 psi.

  Next, the lysate of each construct was centrifuged in a 50 ml disposable tube at 5000 rpm and 25 ° C. for 5 minutes. The supernatant was removed and the pellet was resuspended in 25 ml of 20 mM Tris-HCl (pH 8.5), 1% Triton X-100. The suspension was incubated for 10 minutes at room temperature. The suspension was then centrifuged at 5000 rpm and 25 ° C. for 5 minutes. The supernatant was then removed and the sample was resuspended a second time in 25 ml of 20 mM Tris-HCl (pH 8.5), 1% Triton X-100 and incubated for 10 minutes at room temperature. The suspension was centrifuged again at 5000 rpm and 25 ° C. for 5 minutes. The supernatant was removed and a third wash was performed by resuspending the pellet in 25 ml of 20 mM Tris-HCl (pH 8.5), 1% Triton X-100. This suspension was allowed to stand at room temperature for 10 minutes, and then centrifuged at 5000 rpm and 25 ° C. for 5 minutes. The supernatant from each sample was removed and the pellet was weighed. The pellet was then diluted to 20 mg / ml by resuspending in an appropriate amount of 20 mM Tris-HCl (pH 8.5), 5 mM EDTA. A 1 ml aliquot was made from these suspensions for each of the four constructs and stored at -20 ° C. These aliquots were marked IB or “TWIB” washed 3 times.

  Solubilization buffer was prepared with the following components: 6 M guanidine hydrochloride, 5 mM EDTA, 50 mM Tris-HCl (pH 8) and 10 mM DTT. 1 ml of this solution was added to a 20 mg aliquot of TWIB to give a 20 mg / ml solution. This solution was incubated overnight on the bench to solubilize IB. This procedure was performed on TWIB aliquots of each MBP-GalNAcT2 construct to provide protein refolding experiments.

  The Hampton Foldit Screening kit was used (Hampton Products, Aliso Viejo, Calif.) To screen for refolding conditions that could produce an activated form of MBP-GalNAcT2 expressed in E. coli. The composition of each refolding buffer is found in Table 2.

(Table 2) Refolding conditions from Hampton Research Foldit kit (Hampton Research, Aliso Viejo, CA)

  For a given refolding condition, 950 μL of refolding buffer is combined with 50 μL of solubilized protein (for high protein concentration conditions) or 995 μL of refolding buffer is combined with 5 μL of solubilized protein ( For low protein concentration conditions). The refolding reaction was placed overnight on a rotary shaker in a cold room (4 ° C.).

  From the results obtained in this screening, it was determined that refolding conditions 3, 8, 11, 12, 15 and 16 produced the most promising results for constructs 1 and 2. Additional refolding reactions were performed under such conditions using G-50 gel filtration instead of dialysis to obtain a more concentrated protein refolded sample (see Refold Purification section for methods). From these experiments, it was further refined and it was found that conditions 8 and 11 were optimal. More specifically, condition 15 was found to be optimal for overnight incubation rotations and condition 8 was still optimal for 5 days of incubation.

The protein refolded sample was first purified by dialysis against 20 mM Tris-HCl (pH 8.5). 100 μL of each refolded sample was dialyzed. Dialysis was performed in a beaker containing 20 mM Tris-HCl (pH 8.5) with slow stirring. Samples were placed at 4 ° C. and dialyzed overnight. The resulting retentate was used in a radioactivity assay as described elsewhere herein. As an alternative to obtaining a more concentrated protein sample, MBP-GalNAcT2 refolded sample was purified using a G-50 Macro Spin column (Harvard Bioscience, Holliston, Mass.). The cap was removed from the G-50 column and the column was placed in a 2 ml microcentrifuge tube. To each column was added H ₂ O (500 μl) and the columns were incubated for 15 minutes and hydrated. The columns were then centrifuged at ˜2000 × g for 4 minutes before they were transferred to a new 2 ml centrifuge tube. Each refolding solution (150 μl) was loaded onto one column. The column was then centrifuged at 2000 xg for ~ 2 minutes. The resulting permeate represented a purified refolded sample.

Radiolabeled [ ³ H] -UDP-GalNAc assay was performed, and the activity of refolded MBP-GalNAcT2 expressed in E. coli was determined by monitoring the addition of radiolabeled GalNAc to the peptide receptor. The receptor was a MuC-2-like peptide having the sequence MVTPTPTPTC (SEQ ID NO: 16). This peptide was dissolved in 1M Tris-HCl (pH 8.0). Initial screening was performed on refolded protein samples purified by dialysis. Subsequently, the refolded sample was freshly refolded and purified by G-50 gel filtration. The assay included a protein refolding sample, a GalNAcT2 from baculovirus as a positive control, a negative control sample with all components except the enzyme, and a maximum input sample containing all components other than the enzyme. A total of 19 samples were tested. Assay solutions consisting of these components are listed in Table 3:

(Table 3) GalNAcT2 assay reaction composition

For each of the refolded samples, 30 μL of the reaction mixture was combined with 20 μL of the refolded sample. For the negative control, 20 μL H ₂ O was combined with 30 μL reaction mixture. For the positive control, 1 μL of GalNAcT2 baculovirus enzyme was added in addition to 19 μL of H ₂ O to form a 30 μL reaction mixture. For the “maximum input” sample, 30 μL of the reaction mixture was combined with 20 μL of dH ₂ O. The reaction was incubated at 37 ° C. for 30 minutes. 100 ml DOWEX AG 1X8 (chlorine form) was washed with 100 ml resin and 100 ml H ₂ O and mixed thoroughly. Water was decanted off from the resin and another 100 ml of H ₂ O was added, mixed and removed. The resin was resuspended one last time in 100 ml dH ₂ O. After incubation of the GalNAcT2 assay reaction for 30 minutes, 1 ml of resin resuspended in H ₂ O was added to each reaction (except for the maximum input sample). The sample was vortexed briefly and then loaded onto the filter column and allowed to flow out into the scintillation vial by gravity. 5 ml of scintillation fluid was added to each sample and standard. Samples were shaken briefly and loaded on a scintillation counter to measure radioactivity.

To determine whether E. coli-expressed refolded MBP-GalNAcT2 transfers GalNAc from a UDP-GalNAc donor to the interferon α-2b receptor, an IFα-2b assay was performed. From data obtained from refold screening (see [ ³ H] UDP-GalNAcT2 assay described elsewhere in this specification), MBP in refolding buffers 8 and 15 as determined by radioactivity assay -GalNAc constructs 1 and 2 were shown to yield the most active enzyme. Thus, in the IFα-2b assay, constructs 1 and 2 in refolding buffers 8 and 15 were assayed for transferase activity. In addition, as a positive control, GalNAcT2 from the baculovirus system was further assayed.

This assay consists of reaction buffer (27 mM MES (pH 7), 200 mM NaCl, 20 mM MgCl ₂ , 20 mM MnCl ₂ , and 0.1% Tween 80), IFα-2b protein (2 mg / ml in 5 OmM MES (pH 6), 150 mM NaCl). 0.05% Tween 80, 0.05% NaN ₃ ), and 100 mM UDP-GalNAc. This assay solution was prepared as shown in Table 4 for each reaction.

(Table 4) IFα-2b receptor GalNAcT2 activity assay parameters

For each refolded sample, 4.4 μL of sample was added to 15 μL of reaction solution. For positive control, 1 μL of standard GalNAcT2 baculovirus was added to one tube along with 3.4 μL of H ₂ O. The reaction was incubated for several days on a rotary shaker at 32 ° C., during which time it was assayed by MALDI at overnight and day 5 time points.

The assay was performed to determine whether MBP-GalNAcT2 expressed and refolded in E. coli transfers GalNAc from a UDP-GalNAc donor to a G-CSF receptor. Construct 2 in refolding buffer 8 was assayed for GalNAcT2 activity as described above. In addition, GalNAcT2 from the positive control baculovirus was assayed. Assay reaction buffer (27mM MES (pH7), 200mM NaCl, 20mM MgCl 2, 20mM MnCl 2, and 0.1% Tween 80), G- CSF protein (H ₂ O in 2 mg / ml), and 100 mM UDP- Made of GalNAc. Assay solutions were prepared for each reaction as shown in Table 5.

(Table 5) Parameters of G-CSF receptor GalNAcT2 activity assay

For the refolded sample, 4.4 μL of sample was added to 15 μL of reaction solution. For positive control, 1 μL of standard GalNAcT2 baculovirus was added to one tube along with 3.4 μL of H ₂ O. The reaction was incubated for 4 days on a rotary shaker at 32 ° C., at the end of which a sample was taken and assayed by MALDI.

  As shown in Table 22, pellet mass and inclusion body mass were determined for each of the four cultures of 1L JM109 pCWin2 MBP-GalNAcT2 constructs 1-4.

(Table 6) Cell pellet weight relative to inclusion body weight

  The expression of MBP-GalNAcT2 was observed by SDS-Page gel analysis of JM109 pCWin2 MBP-GalNAcT2 whole cell sample before and after induction with IPTG (FIG. 7). Protein gels show a clear increase in protein expression in the induced state compared to the uninduced state. In addition, there is a discernible band at ˜100 kDa, which increases substantially after induction and correlates with the expected size of the MBP-GalNAcT2 band.

The protein sample was diluted by combining 50 μL of the protein sample with 950 μL of H ₂ O. Samples were then analyzed using a UV spectrophotometer. The protein concentration was calculated from the absorbance and molar extinction coefficient: as shown in Table 7, construct 1-0.65 mg / ml / 1 A ₂₈₀ units, construct 2-0.64 mg / ml / 1 A ₂₈₀ units.

Table 7 Protein concentration of 1L JM109 pCWin2 MBP-GalNAcT2 culture after solubilization and G-50 purification

  Inclusion bodies obtained from JM109 pCWin2 MBP-GalNAcT2 constructs 1 and 2 were analyzed using SDS-PAGE to prove the presence of MBP-GalNAcT2. This protein was clearly observed in both lanes of the gel and flowed to approximately 100 kDa (FIG. 8).

All four constructs were tested in the [ ³ H] UDP-GalNAcT2 assay under all 16 refolding conditions available in the Hampton Foldit kit (Hampton Research, Aliso Viejo, Calif.). The refolded cleaved enzyme was purified by dialysis and then tested for activity using a radioactivity assay as shown in Table 8.

Table 8. GalNAcT2 activity assay results for refolded protein

  The results of this assay show that refolding conditions 3, 8, 11, 12, 15 and 16 resulted in the highest CPM and thus the highest possible GalNAcT2 activity. Furthermore, it was clear that construct 2 produced the maximum number of positive hits in this assay and was therefore studied with a focus on this construct.

Table 9 Results of focused overnight refolded truncated enzyme

  In this assay, construct 2 was tested under the refolding conditions 3, 8, 11, 12, 15, and 16 of 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 radioactivity assay. The results show that maximum activity was obtained from refolding condition 15 after overnight incubation on a rotator.

(Table 10) GalNAcT2 activity results of 5-day refolding experiment

  In this assay, construct 2 was rotated overnight at 4 ° C. and allowed to stand at 4 ° C. for 5 days before refolding conditions 3, 8, 11, 12 of the Hampton Foldit kit (Hampton Research, Aliso Viejo, Calif.). , 15 and 16 below. These refolded enzymes were purified by G-50 gel filtration and then tested for activity by radioactivity assay. The results show that construct 8 showed the highest activity after standing in refolding buffer 8 for 5 days. Therefore, it was determined that conditions 8 and 15 had the greatest potential for the production of properly folded and active MBP-GalNAcT2.

  The IFα-2b assay was performed on constructs 1 and 2 refolded overnight in refolding buffer 15 (1-15 and 2-15, respectively) and incubated at 32 ° C. for 5 days. At 16 hours and 5 days, IFα-2b reaction was collected. The results show that the parent peak of IFα-2b is MW-19267. The success of the reaction was indicated by the addition of ˜203 molecular weight to the peak. From the data on day 5 for refolds 1-15 and 2-15, the appearing peaks were found at ˜119478 and ˜19473, respectively, with a difference of about 203 MW. This data illustrated that GalNAc was added to IFα-2b by the refolded GalNAcT2 protein, thereby confirming the activity reported elsewhere in the radioactivity assay.

  In addition, the IFα-2b assay was performed with the refolded enzyme of constructs 1 and 2 in refolding buffer 8 for 5 days (1-8 and 2-8, respectively). The IFα-2b reaction was again incubated at 32 ° C. for 3 days. The reaction was analyzed at the third day. The results show that the parent peak of IFα-2b is MW-19263. The success of the reaction was indicated by the addition of ˜203 molecular weight to the peak. From the third day data for refolds 1-8 and 2-8, the appearing peaks were again observed at ˜19462 and ˜19469, respectively, and the difference was also about 203 MW. This data again confirmed that GalNAc was added to IFα-2b by the refolded GalNAcT2 protein and was reported by a radioactivity assay.

  A G-CSF assay was performed on the refolded enzyme for 5 days in construct 2 in refolding buffer 8. The G-CSF reaction was incubated at 32 ° C. for 4 days. The reaction was analyzed at the fourth day. The parent peak of G-CSF was expected to be MW-18786. The success of the reaction was indicated by the addition of ˜203 molecular weight to the peak. From the third day data on refolded enzyme 2-8, an emerging peak was observed at ˜19001, with a difference of about 203 MW. This data also indicates that GalNAc was added to G-CSF by the refolded GalNAcT2 protein and was reported by the radioactivity and IFα-2b assays reported elsewhere in this specification. It was confirmed.

  In summary, the data presented herein illustrates that E. coli expressed MBP-GalNAcT2 can be refolded into an active enzyme. The active configuration of MBP-GalNAcT2 constructs 1 and 2 was obtained under refolding conditions 8 and 15 found in the Hampton Research's Foldit kit (Hampton Research, Aliso Viejo, CA). The production of functional refolded proteins is demonstrated using radioactivity, IFα-2b and G-CSF assays, which are GalNAc polypeptides by the GalNAcT2 truncated variants of the present invention. Revealed the transition to.

  As discussed elsewhere herein, a GalNAcT2 truncated variant of the present invention is a polyglycosyl-polyethyleneglycol (“glycosyl-PEG”) conjugate, also known as “glycoPEGylation” of a polypeptide. It is also useful for transfer to peptides. Using a purified and refolded Δ51 GalNAcT2-MBP fusion made according to the present invention, Δ51 GalNAcT2-MBP is capable of transferring a GalNAc-sialic acid (SA) -PEG complex to G-CSF. It showed that there is.

In order to glycoPEGylate G-CSF, a glycoPEGylation reaction mixture was prepared. This reaction mixture consists of 5 μl Δ51 GalNAcT2-MBP (20 μU), 2 μl GalNAc-α2,6-sialyltransferase (ST6GalNAcI), 6.25 mM MnCl ₂ , 15 mM UDP-GalNAc, 0.75 mM CMP-SA-PEG (20K), and 2 mg 2 μl to 10 μl of / ml G-CSF was included. Gel electrophoresis of the reaction product revealed that Δ51 GalNAcT2-MBP transferred the GalNAc-sialic acid (SA) -PEG complex to G-CSF (FIG. 9).

Example 3: Purification of Δ51 GalNAcT2-MBP and Optimization of Refolding The development of Δ51 GalNAcT2 refolding and purification presented herein demonstrates the utility of two column purification methods for GalNAcT2 mutants. Yes. The use of binding mode Q Sepharose Fast Flow and binding and flow-through mode Q Sepharose XL as the first purification step was investigated. Q Sepharose XL in flow-through mode using a NaCl concentration of 10 OmM in the load led to the best recovery and purity of the active Δ51 GalNAcT2-MBP. The use of type I hydroxyapatite was considered as the second column step. Initial data indicates that Δ51 GalNAcT2-MBP can bind to this resin and be eluted as an active enzyme by a phosphate gradient.

  Δ51 GalNAcT2-MBP was cloned and expressed as described elsewhere herein. The collected cell pellets were resuspended in 10 mM Tris / 5 mM EDTA (pH 7.5) (5 mL / g cells) to generate double washed inclusion bodies (DWIB) containing expressed Δ51 GalNAcT2-MBP. It became cloudy and dissolved twice using a microfluidizer at 12,000 psi. Inclusion bodies were collected by centrifugation for 20 minutes at 6,000 rpm in Sorvall RC-3B. The pellet was washed twice by resuspending it in the previous buffer with a 5 mL / g pellet followed by centrifugation at 6,000 rpm for 20 minutes. DWIB was aliquoted and stored at -20 ° C.

  Initial studies showed that urea solubilization led to higher Δ51 GalNAcT2-MBP activity of the refolded material than guanidine hydrochloride solubilization. Therefore Δ51 GalNAcT2-MBP was solubilized in 7 M urea / 50 mM Tris / 10 mM DTT / 5 mM EDTA (pH 8.0) for all subsequent experiments.

1. Refolding experiment-pH search (scout)
A pH search was performed to determine the best pH for Δ51 GalNAcT2-MBP refolding.

(Table 11) Reaction parameters for pH search of Δ51 GalNAcT2-MBP refolding conditions

  Δ51 GalNAcT2-MBP refolding was performed by solubilization of 2.5 g of DWIB in 250 mL of 7 M urea / 50 mM Tris / 10 mM DTT / 5 mM EDTA (pH 8.0) at 4 ° C. 50 mL of solubilized Δ51 GalNAcT2-MBP DWIB was added to 1 L of refolding buffer at 4 ° C. with stirring (21-fold dilution—0.5 mg / mL). Refolding proceeded for 20.5 hours at 4 ° C. with stirring.

  The refolds were filtered using Cuno Zeta Plus BioCap (Cuno, Meriden, CT), concentrated 4 times, and 5 dia of 10 mM Tris / 5 mM NaCl (pH 8) on a 1ft2 3OkDa MWCO TFF (regenerated cellulose) filter. Diafiltration was performed at a constant volume of filtration volumes (diavolumes).

  Concentrated and diafiltered refolds were loaded onto a pre-equilibrated 48 mL Q Sepharose fast flow column (Amersham Biosciences, Piscataway, NJ) and low salt buffer (10 mM Tris / 5 mM NaCl (pH 8. 0)) 2 column volumes (CV). The protein was eluted with a 15 CV gradient of 0-50% high salt buffer (10 mM Tris / 1M NaCl, pH 8.0) followed by a 1 CV gradient up to 100% high salt buffer. The column was regenerated with 0.5M NaOH.

  The highest Δ51 GalNAcT2-MBP activity was achieved using Refold 2a conditions (pH 8.0) combined with urea solubilization. Active Δ51 GalNAcT2-MBP eluted early during QSFF elution. 1 L of refold yielded a total of 42 OmU of Δ51 GalNAcT2-MBP.

  Additional refolding conditions for Δ51 GalNAcT2-MBP were screened. 55 mM MES (pH 6.5), 264 mM NaCl, 11 mM KCl, refolding buffer containing 0.055% PEG 3350 and 550 mM L-arginine, and 55 mM Tris-HCl (pH 8.0), 10.56 mM NaCl, 0.44 mM KCl, A refolding buffer containing 0.055% PEG 3350 and 550 mM L-arginine was screened. Four conditions using two buffers: solubilization at pH 6.5, followed by refolding at pH 6.5, solubilization at pH 6.5, followed by refolding at pH 8.0, Solubilization at pH 8.0, followed by refolding at pH 6.5, solubilization at pH 8.0, followed by refolding at pH 8.0 was screened. Assay of Δ51 GalNAcT2-MBP refolded under all four conditions revealed enzymatic activity capable of transferring GalNAc to GCSF.

2. Δ51 GalNAcTI-MBP purification The use of Q Sepharose Fast Flow (QSFF) and Q Sepharose XL (QXL) (Amersham Biosciences, Piscataway, NJ) in Δ51 GalNAcT2-MBP purification was tested. QSFF was used in combined mode. For this purpose, concentrated and diafiltered Δ51 GalNAcT2-MBP refold (in 10 mM Tris / 5 mM NaCl, pH 8.0) -A was loaded onto a pre-equilibrated 50 mL QSFF column and 15 CV A gradient from 10 mM Tris / 5 mM NaCl (pH 8.0) to 50% 10 mM Tris / 1 M NaCl (pH 8.0) (B), followed by a second gradient from 50 to 100% B above 1 CV. Used and eluted.

  QXL was used in combined mode and flow-through mode. The NaCl concentration in the concentrated and diafiltered Δ51 GalNAcT2-MBP refold (each 4OmL = 160mL refold volume) was adjusted to 5, 50, 100, and 20OmM NaCl and then loaded onto a 3.9mL QXL column. . The column was washed with 2 CV and the bound protein was eluted with a 30 CV gradient from A to B.

  Δ51 GalNAcT2-MBP was tightly bound to QSFF resin under the above conditions with 5 mM NaCl in loading buffer and equilibration buffer. The active Δ51 GalNAcT2-MBP eluted at the beginning of the main peak, which appeared as doublet on a non-reducing 4-20% Tris-glycine gel. The main contaminant is a currently unidentified band that flowed at a slightly lower molecular weight adjacent to the 98 kDa marker band. Various other contaminants eluted with inactive Δ51 GalNAcT2-MBP in the remainder of this large peak.

  When the same conditions as QSFF binding (ie 5 mM NaCl) were applied, Δ51 GalNAcT2-MBP bound tightly to the QXL resin. Increased Δ51 GalNAcT2-MBP activity was observed in flow-through and washing at high NaCl concentrations at this load. Interestingly, the large contaminating band observed in the QSFF purification was not observed in this flow-through when the load contained 50 and 100 mM NaCl. At both these NaCl concentrations, most of the active Δ51 GalNAcT2-MBP was observed in flow-through and washing; only some remaining Δ51 GalNAcT2-MBP activity was detected on the left shoulder of the elution peak. As observed in the QSFF resin, a bulk amount of contaminating bands was observed in the main elution peak. Most of the active Δ51 GalNAcT2-MBP was located in the flow-through when the load salt concentration was adjusted to 200 mM, but no significant purification was achieved under these conditions. In conclusion, the optimal NaCl concentration for use of QXL in flow-through mode is higher than 50 mM NaCl but lower than 200 mM NaCl. Based on these data, to use an anion exchange resin in flow-through mode, 100 mM NaCl is the appropriate concentration in loading buffer and equilibration buffer.

Type I hydroxyapatite (80 μm) (BioRad, Hercules, CA) was tested as a second column step. With active Δ51 GalNAcT2-MBP partially purified on QSFF (using binding and elution mode), active Δ51 GalNAcT2-MBP is useful for binding to type I HA resin and for further purification of proteins It was investigated whether it was. For this purpose, the I-type HA column 2.25 mL, was pre-equilibrated in _{5mM NaPO 4 / 5mM NaCl (pH7.0} ) (C). The active Δ51 GalNAcT2-MBP eluted from QSFF was adjusted to pH 7.0 with 1M HCl and loaded onto a Type I HA column. Proteins with 5CV gradient of 20CV gradient subsequently 50 to 100% D in _{0~50% 300mM NaPO 4 / 5mM NaCl} (pH7.0) (D), and eluted. The column was regenerated with 0.5M NaOH. The obtained data indicate that Δ51 GalNAcT2-MBP binds to type I hydroxyapatite resin and is eluted as an active enzyme.

  Each and all disclosures of patents, patent applications, and publications listed herein are hereby incorporated by reference in their entirety.

  While the invention has been described with reference to specific embodiments, it is obvious that other embodiments and modifications of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. I will. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

For the purpose of illustrating the invention, drawings of certain embodiments of the invention are shown. However, the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.
FIG. 1 is an electrophoresis gel image illustrating PCR amplification of the ppGalNAcT2 gene. M, 1kb DNA ladder; PCR1, ppGalNAcT2-N41R PCR product (1596bp); PCR2, ppGalNAcT2-N52K PCR product (1563bp); PCR3, ppGalNAcT2-N74G PCR product (1497bp); PCR4, ppGalNAcT2-N95G PCR product (1434bp). FIG. 2A is a plasmid restriction map of the pCWin2MBP vector. FIG. 2B is an electrophoresis gel image illustrating fragments from multiple samples of the pCWin2MBP vector digested with both BamHI and XhoI restriction enzymes. FIG. 3 is an electrophoresis gel image illustrating the screening of DH5α (pCWin2MBP-ppGalNAcT2) colonies by restriction maps (BamHI and XhoI digestion) of plasmids purified from 12 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 electrophoretic protein gel image illustrating an SDS-PAGE of JM109 (pCWin2MBP-ppGalNAcT2) whole cell lysate after IPTG induction as described elsewhere in this specification. M, pre-stained MW standard; lane 13, IPTG induced JM109 (pCWin2MBP); lanes 1-12, proteins in all cells of colonies 1-12; lanes 1-3, JM109 (pCWin2MBP-ppGalNAcT2N41R); lanes 4-6 JM109 (pCWin2MBP-ppGalNAcT2N52K); Lanes 7-9, JM109 (pCWin2MBP-ppGalNAcT2N74G); Lanes 10-12, JM109 (pCWin2MBP-ppGalNAcT2N95G). FIG. 5 is an electrophoretic protein gel image illustrating SDS-PAGE of JM109 (pCWin2MBP-ppGalNAcT2) cell lysate. M, pre-stained MW standard; lane 13, lysate of JM109 (pCWin2MBP); lane 1-12, lysate of colony 1-12; lanes 1-3, JM109 (pCWin2MBP-ppGalNAcT2N41R); lanes 4-6, JM109 ( pCWin2MBP-ppGalNAcT2N52K); Lanes 7-9, JM109 (pCWin2MBP-ppGalNAcT2N74G); Lanes 10-12, JM109 (pCWin2MBP-ppGalNAcT2N95G). FIG. 6 is an electrophoretic protein gel image illustrating SDS-PAGE of inclusion bodies isolated from JM109 (pCWin2MBP-ppGalNAcT2) cells. M, pre-stained MW standard; lane 13, inclusions from JM109 (pCWin2MBP); lanes 1-12, inclusions from colonies 1-12; lanes 1-3, JM109 (pCWin2MBP-ppGalNAcT2N41R); lanes 4-6, JM109 (pCWin2MBP-ppGalNAcT2N52K); Lanes 7-9, JM109 (pCWin2MBP-ppGalNAcT2N74G); Lanes 10-12, JM109 (pCWin2MBP-ppGalNAcT2N95G). FIG. 7 is an electrophoresis gel image illustrating the protein expression pattern in the lysate of cells containing the human GalNAcT2 construct. 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 after induction 2 cultures; lane 6, construct 3 culture before induction; lane 7, construct 3 culture after induction; lane 8, construct 4 culture before induction; lane 9, construct 4 culture after induction Things; Lane 10, empty. FIG. 8 is an electrophoresis protein gel image illustrating the protein content of inclusion bodies from the JM109 pCWin2 MBP-GalNAcT2 construct. Lane 1, MW marker; Lane 2, JM109 pCWin2 MBP-GalNAcT2 construct 1 inclusion body; Lane 3, JM109 pCWin2 MBP-GalNAcT2 construct 2 inclusion body. FIG. 9 is an image of an electrophoretic protein gel illustrating glycoPEGylation of G-CSF with Δ51 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 PEGylation; Lane 4, glycoPEGylation in the presence of 0.2 mg / ml G-CSF. GlycoPEGylated G-CSF is found around 60 kDa. Figures 10A and 10B show the nucleic acid sequence encoding the Δ40 GalNAcT2 polypeptide. FIGS. 11A and 11B show the nucleic acid sequence encoding the Δ51 GalNAcT2 polypeptide. Figures 12A and 12B show the nucleic acid sequence encoding the Δ73 GalNAcT2 polypeptide. FIGS. 13A and 13B show the nucleic acid sequence encoding the Δ94 GalNAcT2 polypeptide. FIG. 14A is an image of a chromatogram illustrating the elution of Δ51 GalNAcT2-MBP refolded at pH 5.5 and subsequently eluted from a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 14B is an image of two electrophoretic gels used for visualizing the eluted fraction shown in FIG. 14A. The contents of each lane on the gel are shown in the figure. FIG. 14C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 14A. FIG. 15A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP refolded at pH 6.5 and subsequently eluted from a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 15B is an image of two electrophoretic gels used to view the eluted fraction shown in FIG. 15A. The contents of each lane on the gel are shown in the figure. FIG. 15C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 15A. FIG. 16A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP refolded at pH 8.0 and subsequently eluted from a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 16B is an image of the two electrophoretic gels used to view the eluted fraction shown in FIG. 16A. The contents of each lane on the gel are shown in the figure. FIG. 16C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 16A. FIG. 17A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP refolded at pH 8.5 and subsequently eluted from a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 17B is an image of two electrophoretic gels used to view the eluted fraction shown in FIG. 17A. The contents of each lane on the gel are shown in the figure. FIG. 17C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 17A. FIG. 18A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP refolded at pH 8.0 and subsequently eluted from a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 18B is an image of the two electrophoretic gels used to view the eluted fraction shown in FIG. 18A. The contents of each lane on the gel are shown in the figure. FIG. 18C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 18A. FIG. 19A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP on a Q-Sepharose fast flow column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 19B is an image of two electrophoretic gels used for visualizing the eluted fraction shown in FIG. 19A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 19A. FIG. 19C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 19A. FIG. 20A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP from a Q-Sepharose XL column using 5 mM NaCl. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 20B is an image of two electrophoretic gels used for visualizing the eluted fraction shown in FIG. 20A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 20A. FIG. 20C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 20A. FIG. 21A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP from a Q-Sepharose XL column using 50 mM NaCl. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 21B is an image of two electrophoretic gels used to view the eluted fraction shown in FIG. 21A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 21A. FIG. 21C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 21A. FIG. 22A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP from a Q-Sepharose XL column using 100 mM NaCl. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 22B is an image of the two electrophoretic gels used to view the eluted fraction shown in FIG. 22A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 22A. FIG. 22C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 22A. FIG. 23A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP from a Q-Sepharose XL column using 200 mM NaCl. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 23B is an image of two electrophoretic gels used to view the eluted fraction shown in FIG. 23A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 23A. FIG. 23C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 23A. FIG. 24A is a chromatogram image illustrating the elution of Δ51 GalNAcT2-MBP from a hydroxyapatite type I column. Fraction numbers are shown on the X axis, and relative absorbance of each fraction is shown on the Y axis. FIG. 24B is an image of the electrophoresis gel used for visualizing the eluted fraction shown in FIG. 24A. The contents of each lane on the gel are shown in the figure and correspond to the chromatogram of FIG. 24A. FIG. 24C is a table illustrating the relative GalNAc transferase activity of the fractions shown in FIG. 24A. FIG. 25 is a graph illustrating the relative GalNAc transferase activity of various preparations of refolded Δ51 GalNAcT2-MBP. The refolding conditions for each preparation are shown on the x-axis and the relative GalNAc transferase activity is shown on the Y-axis. FIG. 26 is a graph illustrating the relative GalNAc transferase activity of various preparations of refolded Δ51 GalNAcT2-MBP. Refolding conditions for each preparation are shown on the x-axis and relative GalNAc transferase activity is shown on the Y-axis. FIG. 27 is three MALDI-TOF spectra revealing GalNAc transfer to GCSF mediated by Δ51 GalNAcT2-MBP refolded and purified according to the present invention. FIG. 28 is three MALDI-TOF spectra revealing GalNAc transfer to GCSF mediated by Δ51 GalNAcT2-MBP refolded and purified according to the present invention.

Claims (30)

  An isolated nucleic acid comprising a nucleic acid sequence encoding a truncated human GalNAcT2 polypeptide, wherein the truncated human GalNAcT2 polypeptide is a human GalNAcT2 truncated variant wherein the encoded polypeptide lacks amino acid residues 1-51 An isolated nucleic acid that lacks all or part of the GalNAcT2 signal domain, provided that it is not a polypeptide.   The truncated human GalNAcT2 polypeptide further lacks all or part of the GalNAcT2 transmembrane domain, provided that the encoded polypeptide is not a human GalNAcT2 truncation mutant polypeptide lacking amino acid residues 1-51. 2. The isolated nucleic acid of claim 1, wherein   The truncated human GalNAcT2 polypeptide further lacks all or part of the GalNAcT2 backbone domain, provided that the encoded polypeptide is not a human GalNAcT2 truncation mutant polypeptide lacking amino acid residues 1-51. The isolated nucleic acid according to claim 2.   A nucleic acid sequence encoding a truncated human GalNAcT2 polypeptide, wherein the nucleic acid sequence has 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. 2. The isolated nucleic acid according to claim 1, comprising:   5. The isolated nucleic acid of claim 4, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9.   5. The isolated nucleic acid according to claim 4, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9.   An isolated chimeric nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a tag polypeptide covalently linked to a second polypeptide encoded by the isolated nucleic acid of claim 1. Chimeric nucleic acid.   8. The isolated chimera of claim 7, wherein the tag polypeptide is selected from the group consisting of 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. Nucleic acid.   An isolated, truncated human GalNAcT2 polypeptide that lacks all or part of the GalNAcT2 signal domain, provided that the polypeptide is not a human GalNAcT2 polypeptide truncation mutant that lacks amino acid residues 1-51 Truncated human GalNAcT2 polypeptide.   The truncated human GalNAcT2 polypeptide further lacks all or part of the GalNAcT2 transmembrane domain, provided that the polypeptide is not a human GalNAcT2 polypeptide truncation mutant lacking amino acid residues 1-51. 10. The isolated truncated human GalNAcT2 polypeptide of claim 9.   The truncated human GalNAcT2 polypeptide further lacks all or part of the GalNAcT2 backbone domain, provided that the polypeptide is not a human GalNAcT2 polypeptide truncation mutant lacking amino acid residues 1-51. Item 11. An isolated truncated human GalNAcT2 polypeptide according to Item 10.   10. The isolated truncated human GalNAcT2 polypeptide of claim 9, having at least 90% identity with a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10.   10. The isolated truncated human GalNAcT2 polypeptide of claim 9, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10.   10. The isolated truncated human GalNAcT2 polypeptide of claim 9, consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10.   10. An isolated chimeric polypeptide comprising a tag polypeptide covalently linked to the isolated truncated GalNAcT2 polypeptide of claim 9.   16. The isolated chimera of claim 15, wherein the tag polypeptide is selected from the group consisting of 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. Polypeptide.   2. The isolated nucleic acid of any of claims 1 further comprising a promoter / regulatory sequence operably linked thereto.   An expression vector comprising the isolated nucleic acid according to claim 1.   A recombinant cell comprising the isolated expression vector of claim 18.   20. The recombinant cell according to claim 19, which is a eukaryotic cell or a prokaryotic cell.   21. The recombinant cell of claim 20, wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, and a fungal cell.   22. The recombinant cell of claim 21, wherein the insect cell is selected from the group consisting of SF9 cells, SF9 + cells, Sf21 cells, HIGH FIVE cells, or Drosophila Schneider S2 cells.   21. The recombinant cell of claim 20, wherein the prokaryotic cell is selected from the group consisting of an E. coli cell and a B. subtilis cell.   21. A method for producing a truncated human GalNAcT2 polypeptide, comprising the step of growing the recombinant cell of claim 20 under conditions suitable for expression of the truncated human GalNAcT2 polypeptide.   A method of catalyzing the transfer of a GalNAc moiety to a receptor moiety, comprising the step of incubating the polypeptide of claim 9 with a GalNAc moiety and a receptor moiety, wherein the polypeptide comprises the GalNAc moiety. A method of mediating covalent linkage to the receptor moiety, thereby catalyzing the transfer of the GalNAc moiety to the receptor moiety to produce a product saccharide, or product glycoprotein, or product glycopeptide.   26. The method of claim 25, wherein the receptor moiety is granulocyte colony stimulating factor (G-CSF) protein.   The receptor moiety is composed of erythropoietin, human growth hormone, granulocyte colony stimulating factor, interferons α, β and γ, factor IX, follicle stimulating hormone, interleukin-2, anti-TNF-α, and lysosomal hydrolase 26. The method of claim 25, wherein   26. The method of claim 25, wherein the polypeptide receptor is a glycopeptide.   26. The method of claim 25, wherein the GalNAc moiety comprises a polyethylene glycol moiety.   26. The method of claim 25, wherein the product saccharide, product glycoprotein, or product glycopeptide is produced on a commercial scale.

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