U.S. patent application number 12/299194 was filed with the patent office on 2009-11-19 for alpha-1, 4-galactosyltransferase (cgtd) from campylobacter jejuni.
This patent application is currently assigned to National Research Council of Canada. Invention is credited to Michel Gilbert, Scott Houliston, Warren Wakarchuk.
Application Number | 20090286288 12/299194 |
Document ID | / |
Family ID | 38655020 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286288 |
Kind Code |
A1 |
Gilbert; Michel ; et
al. |
November 19, 2009 |
ALPHA-1, 4-GALACTOSYLTRANSFERASE (CgtD) FROM CAMPYLOBACTER
JEJUNI
Abstract
.alpha.-1,4-galactosyltransferase (CgtD) polypeptides, nucleic
acids that encode the polypeptides, including a polypeptide from
Campylobacter jejuni strain LIO87 have been isolated and
characterized A method of producing a galactosylated saccharide
comprising contacting an acceptor saccharide containing a terminal
galactose, a donor substrate comprising a galactose moiety and one
of the CgtD polypeptides is described.
Inventors: |
Gilbert; Michel; (Gatineau,
CA) ; Wakarchuk; Warren; (Ottawa, CA) ;
Houliston; Scott; (Ottawa, CA) |
Correspondence
Address: |
NADA JAIN, P.C.
560 White Plains Road, Suite 460
Tarrytown
NY
10591
US
|
Assignee: |
National Research Council of
Canada
Ottawa
ON
|
Family ID: |
38655020 |
Appl. No.: |
12/299194 |
Filed: |
May 1, 2007 |
PCT Filed: |
May 1, 2007 |
PCT NO: |
PCT/CA2007/000745 |
371 Date: |
July 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60797132 |
May 2, 2006 |
|
|
|
Current U.S.
Class: |
435/97 ; 435/193;
435/254.2; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12Y 204/01038 20130101;
C12P 19/18 20130101; C12N 9/107 20130101; C12N 9/1051 20130101 |
Class at
Publication: |
435/97 ; 435/193;
435/254.2; 435/320.1; 536/23.2 |
International
Class: |
C12P 19/18 20060101
C12P019/18; C12N 9/10 20060101 C12N009/10; C12N 1/19 20060101
C12N001/19; C12N 15/63 20060101 C12N015/63; C07H 21/04 20060101
C07H021/04 |
Claims
1. A method of producing a galactosylated product saccharide, the
method comprising the step of: a) contacting an acceptor substrate
with a donor substrate comprising a galactose moiety and an
isolated or recombinant .alpha.-1,4-galactosyltransferase
polypeptide with at least 80% identity to SEQ ID NO:2; and b)
allowing transfer of a galactose moiety to the acceptor saccharide
to occur, thereby producing the galactosylated product
saccharide.
2. The method of claim 1, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 90% identity to SEQ ID NO:2.
3. The method of claim 1, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 95% identity to SEQ ID NO:2.
4. The method of claim 1, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence of SEQ ID NO:2.
5. The method of claim 1, wherein the method is performed at a
commercial scale of production.
6. A reaction mixture comprising an isolated or recombinant
.alpha.-1,4-galactosyltransferase polypeptide, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 80% identity to SEQ ID NO:2, and
wherein the .alpha.-1,4-galactosyltransferase polypeptide transfers
a galactose moiety from a donor substrate to an acceptor
substrate.
7. The reaction mixture of claim 6, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 90% identity to SEQ ID NO:2.
8. The reaction mixture of claim 6, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 95% identity to SEQ ID NO:2.
9. The reaction mixture of claim 6, wherein the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence of SEQ ID NO:2.
10. An isolated nucleic acid the encodes an
.alpha.-1,4-galactosyltransferase polypeptide of claim 6, wherein
the isolated nucleic acid comprises a nucleic acid sequence with at
least 80% identity to SEQ ID NO: 1.
11. The isolated nucleic acid of claim 10, wherein the isolated
nucleic acid comprises a nucleic acid sequence with at least 90%
identity to SEQ ID NO: 1.
12. The isolated nucleic acid of claim 10, wherein the isolated
nucleic acid comprises a nucleic acid sequence with at least 95%
identity to SEQ ID NO: 1.
13. The isolated nucleic acid of claim 10, wherein the isolated
nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 1.
14. An expression vector comprising a nucleic acid sequence of
claim 10.
15. A host cell comprising the expression vector of claim 14.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/797,132, filed May 2, 2006, which is herein
incorporated by reference for all purposes.
FIELD OF INVENTION
[0002] The invention relates to .alpha.-1,4-galactosyltransferase
(CgtD) polypeptides, nucleic acids that encode the polypeptides,
and methods of using the polypeptides.
BACKGROUND OF THE INVENTION
[0003] Carbohydrates are now recognized as being of major
importance in many cell-cell recognition events, notably the
adhesion of bacteria and viruses to mammalian cells in pathogenesis
and leukocyte-endothelial cell interaction through selectins in
inflammation (Varki (1993) Glycobiology 3: 97-130). Moreover,
sialylated glycoconjugates that are found in bacteria (Preston et
al. (1996) Crit. Rev. Microbiol. 22:139-180; Reuter et al. (1996)
Biol. Chem. Hoppe-Seyler 377:325-342) are thought to mimic
oligosaccharides found in mammalian glycolipids to evade the host
immune response (Moran et al. (1996) FEMS Immunol. Med. Microbiol.
16:105-115). Molecular mimicry of host structures by the saccharide
portion of lipopolysaccharide (LPS) is considered to be a virulence
factor of various mucosal pathogens, which use this strategy to
evade a host immune response (Moran et al. (1996) FEMS Immunol.
Med. Microbiol. 16: 105-115; Moran et al. (1996) J. Endotoxin Res.
3: 521-531).
[0004] The oligosaccharide structures involved in these and other
processes are potential therapeutic agents, but they are time
consuming and expensive to make by traditional chemical means. A
very promising route to production of specific oligosaccharide
structures is through the use of the enzymes which make them in
vivo, the glycosyltransferases. Such enzymes can be used as regio-
and stereoselective catalysts for the in vitro synthesis of
oligosaccharides (Ichikawa et al. (1992) Anal. Biochem. 202:
215-238).
[0005] Large scale enzymatic synthesis of oligosaccharides depends
on the availability of sufficient quantities of the required
glycosyltransferases. However, production of glycosyltransferases
in sufficient quantities for use in preparing oligosaccharide
structures has been problematic. Expression of many mammalian
glycosyltransferases has been achieved involving expression in
eukaryotic hosts which can involve expensive tissue culture media
and only moderate yields of protein (Kleene et al. (1994) Biochem.
Biophys. Res. Commun. 201: 160-167; Williams et al. (1995)
Glycoconjugate J. 12: 755-761). Expression in E. coli has been
achieved for mammalian glycosyltransferases, but these attempts
have produced mainly insoluble forms of the enzyme from which it
has been difficult to recover active enzyme in large amounts (Aoki
et al. (1990) EMBO. J. 9:3171-3178; Nishiu et al. (1995) Biosci.
Biotech. Biochem. 59 (9): 1750-1752). Furthermore, because of the
biological activity of their products, mammalian sialyltransferases
generally act in specific tissues, cell compartments and/or
developmental stages to create precise glycan structures.
Identification of glycosyltransferases that can be used in
enzymatic synthesis of commercially valuable oligosaccharides and
that can be produced in large quantities would thus be useful in
the development of these technologies. The present invention
fulfills this and other needs.
BRIEF SUMMARY OF THE INVENTION
[0006] In one aspect the present invention provides a method of
producing a galactosylated product saccharide by contacting an
acceptor substrate with a donor substrate comprising a galactose
moiety and an isolated or recombinant
.alpha.-1,4-galactosyltransferase polypeptide with at least 80%
identity to SEQ ID NO:2; and allowing transfer of a galactose
moiety to the acceptor saccharide to occur, thereby producing the
galactosylated product saccharide. In one embodiment the
.alpha.-1,4-galactosyltransferase polypeptide comprises an amino
acid sequence with at least 90% or 95% identity to SEQ ID NO:2. In
another embodiment the .alpha.-1,4-galactosyltransferase
polypeptide comprises an amino acid sequence of SEQ ID NO:2. In a
further embodiment the method is performed at a commercial scale of
production. In another embodiment the method includes a step of
isolating the galactosylated product saccharide.
[0007] In another aspect the present invention provides a reaction
mixture comprising an isolated or recombinant
.alpha.-1,4-galactosyltransferase polypeptide that transfers a
galactose moiety from a donor substrate to an acceptor substrate
and that has an amino acid sequence with at least 80%, 90%, 95%, or
100% identity to SEQ ID NO:2.
[0008] In another aspect the invention provides an isolated nucleic
acid that encodes an .alpha.-1,4-galactosyltransferase polypeptide.
In one embodiment the isolated nucleic acid comprises a nucleic
acid sequence with at least 90% or 95% identity to SEQ ID NO: 1. In
another embodiment the isolated nucleic acid comprises a nucleic
acid sequence of SEQ ID NO:1.
[0009] In another aspect the invention provides an expression
vector that includes a nucleic acid sequence with at least 80%, 90%
or 95% identity with SEQ ID NO: 1. The invention also includes host
cells that contain the expression vector and methods to make an
.alpha.-1,4-galactosyltransferase polypeptide, by growing the host
cells under conditions suitable for expression of the
.alpha.-1,4-galactosyltransferase polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 provides .sup.1H-.sup.13C HSQC spectrum of
.alpha.Gal-1,4-.beta.Gal-1,4-.beta.GlcNAc-p-nitrophenyl.
Cross-peaks are labeled according to the lettering and numbering in
FIG. 2 (i.e. a for .beta.GlcNAc, b for .beta.Gal, and c for
.alpha.Gal). The carbon resonances of a4 and b4 exhibit shifts that
are downfield in comparison with their value in monosaccharides,
consistent with their participation in a glycosidic bond.
[0011] FIG. 2 provides .sup.1H and .sup.13C chemical shifts.sup.a
of .alpha.Gal-1,4-.beta.Gal-1,4-.beta.GlcNAc-p-nitrophenyl.
[0012] FIG. 3 provides the alignment of the three full-length
versions of CgtD (SEQ ID NOS:3, 4 and 2, respectively) using the
ClustalW program. The bottom sequence is a consensus sequence (SEQ
ID NO:5). The following symbols are used: "*" means that the
residues or nucleotides in that column are identical in all
sequences in the alignment; ":" means that conserved substitutions
have been observed, as defined on the Clustal website; and "."
means that semi-conserved substitutions are observed. See, e.g.,
www.ebi.ac.uk/clustalw/#.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0013] The lipooligosaccharide (LOS) biosynthesis locus has been
sequenced in various strains of Campylobacter jejuni as part of a
project on the comparative genomics of this locus. See, e.g.,
Gilbert, et al., J. Biol. Chem. 275:3896-3906 (2000); Gilbert, et
al., J. Biol. Chem. 277:327-337 (2002); and Gilbert, et al., in
Campylobacter: Molecular and Cellular Biology. (Horizon Bioscience,
Editors: J. M. Ketley and M. E. Konkel), Chapter 11 (2005).
[0014] The function of some of the encoded protein has been
experimentally determined. C. jejuni LIO87 is a serotype strain of
the LIOR (heat labile) serotyping system. The organization of the
LI087 LOS locus (Class "D", GenBank accession number AF400669) is
distinct from the majority of the C. jejuni LOS loci characterized
so far (Classes "A", "B" and "C", Gilbert et al. 2002). For
example, the LOS locus from C. jejuni LIO87 lacks the cluster of
genes involved in sialic acid biosynthesis and in the expression of
LOS outer cores mimicking gangliosides. The C. jejuni LIO87 LOS
locus includes 10 open reading frames (ORFs). Sequence homology
searches indicated that four of these ORFs (ORFs #1, #2, #3 and
#10) are involved in the biosynthesis of the inner core or the
lipid A. It was not possible to infer the function of the proteins
encoded by the other six open reading frames based on sequence
information.
[0015] The function of the CgtD protein was determined
experimentally and the present invention demonstrates for the first
time that the CgtD gene product has
.alpha.-1,4-galactosyltransferase activity. In addition, the enzyme
is also able to transfer galactose from a donor to either LacNAc
(Gal-.beta.-1,4-GlcNAc) or the Lac (Gal-.beta.-1,4-Glc) derivatives
as an acceptor molecule.
II. Definitions
[0016] The following abbreviations are used herein: [0017]
Ara=arabinosyl; [0018] Fru=fructosyl; [0019] Fuc=fucosyl; [0020]
Gal=galactosyl; [0021] GalNAc=N-acetylgalactosaminyl; [0022]
Glc=glucosyl; [0023] GlcNAc=N-acetylglucosaminyl; [0024]
Man=mannosyl; and [0025] NeuAc=sialyl (N-acetylneuraminyl).
[0026] An "acceptor substrate" or an "acceptor saccharide" for a
glycosyltransferase, e.g., a CgtD polypeptide, is an
oligosaccharide moiety that can act as an acceptor for a particular
glycosyltransferase. When the acceptor substrate is contacted with
the corresponding glycosyltransferase and sugar donor substrate,
and other necessary reaction mixture components, and the reaction
mixture is incubated for a sufficient period of time, the
glycosyltransferase transfers sugar residues from the sugar donor
substrate to the acceptor substrate. The acceptor substrate can
vary for different types of a particular glycosyltransferase.
Accordingly, the term "acceptor substrate" is taken in context with
the particular glycosyltransferase of interest for a particular
application. Acceptor substrates for
.alpha.-1,4-glycosyltransferases, e.g., CgtD from C. jejuni LIO87,
and additional glycosyltransferases, are described herein. In
preferred embodiments, a CgtD acceptor substrate has a terminal
galactose residue. CgtD acceptor substrates include, e.g., lactose
or lacNAc or oligosaccharides, glycoproteins, glycolipids or
glycopeptides that comprise a lactose or lacNAc moiety.
[0027] A "donor substrate" for glycosyltransferases is an activated
nucleotide sugar. Such activated sugars generally consist of
uridine, guanosine, and cytidine monophosphate derivatives of the
sugars (UMP, GMP and CMP, respectively) or diphosphate derivatives
of the sugars (UDP, GDP and CDP, respectively) in which the
nucleoside monophosphate or diphosphate serves as a leaving group.
For example, a donor substrate for fucosyltransferases is
GDP-fucose. Donor substrates for CgtE proteins include, e.g.,
UDP-GalNAc or UDP-Gal. Donor substrates for sialyltransferases, for
example, are activated sugar nucleotides comprising the desired
sialic acid. The donor substrate for CgtD is UDP-Gal. For instance,
in the case of NeuAc, the activated sugar is CMP-NeuAc. Bacterial,
plant, and fungal systems can sometimes use other activated
nucleotide sugars.
[0028] Oligosaccharides are considered to have a reducing end and a
non-reducing end, whether or not the saccharide at the reducing end
is in fact a reducing sugar. In accordance with accepted
nomenclature, oligosaccharides are depicted herein with the
non-reducing end on the left and the reducing end on the right. All
oligosaccharides described herein are described with the name or
abbreviation for the non-reducing saccharide (e.g., Gal), followed
by the configuration of the glycosidic bond (.alpha. or .beta.),
the ring bond, the ring position of the reducing saccharide
involved in the bond, and then the name or abbreviation of the
reducing saccharide (e.g., GlcNAc). The linkage between two sugars
may be expressed, for example, as 2,3, 2.fwdarw.3, or (2,3). Each
saccharide is a pyranose or furanose.
[0029] As used herein, a "galactose moiety" refers to a molecule
that includes galactose or that can be derived from galactose.
Galactose moieties are usually monosaccharides, e.g.,
galactose.
[0030] As used herein, a "galactosylated product saccharide" refers
an oligosaccharide, a polysaccharide, or a carbohydrate moiety,
either unconjugated or conjugated to a glycolipid or a
glycoprotein, e.g., a biomolecule, that includes a galactose
moiety. Any of the above galactose moieties can be used, e.g.,
galactose. In preferred embodiments the galactose moiety
transferred by CgtD is galactose.
[0031] In some embodiments other sugar moieties, e.g., fucose,
sialic acid, glucose, GalNAc or GlcNAc, are also added to the
acceptor substrate through the action of additional
glycosyltransferases to produce the galactosylated product
saccharide. In some embodiments, the acceptor substrate comprises a
galactose moiety and the CgtD protein is used to add an additional
galactose moiety, making the galactosylated product saccharide.
[0032] The term "sialic acid" or "sialic acid moiety" refers to any
member of a family of nine-carbon carboxylated sugars. The most
common member of the sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. A third sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J.
Biol. Chem. 265: 21811-21819 (1990)). Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40
(1992); Sialic Acids: Chemistry, Metabolism and Function, R.
Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and
use of sialic acid compounds in a sialylation procedure is
disclosed in international application WO 92/16640, published Oct.
1, 1992.
[0033] Much of the nomenclature and general laboratory procedures
required in this application can be found in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. The
manual is hereinafter referred to as "Sambrook et al."
[0034] The terms "CgtD from C. jejuni LIO87," "CgtD," or a nucleic
acid encoding "CgtD from C. jejuni LIO87" or "CgtD" refer to
nucleic acids and polypeptide polymorphic variants, alleles,
mutants, and interspecies homologs that: (1) have an amino acid
sequence that has at least 60% amino acid sequence identity, 65%,
70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or greater amino acid sequence identity, preferably
over a region of at least about 25, 50, 100, 200, 500, 1000, or
more amino acids, to an amino acid sequence encoded by a CgtD from
C. jejuni LIO87 nucleic acid (for a CgtD from C. jejuni LIO87
nucleic acid sequence, see, e.g., SEQ ID NO:1) or to an amino acid
sequence of a CgtD from C. jejuni LIO87 protein (for a CgtD from C.
jejuni LIO87 protein sequence, see, e.g., SEQ ID NO:2); (2) bind to
antibodies, e.g., polyclonal antibodies, raised against an
immunogen comprising an amino acid sequence of a CgtD from C.
jejuni LIO87 protein, and conservatively modified variants thereof;
(3) specifically hybridize under stringent hybridization conditions
to an anti-sense strand corresponding to a nucleic acid sequence
encoding a CgtD from C. jejuni LIO87 protein, and conservatively
modified variants thereof; (4) have a nucleic acid sequence that
has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or higher nucleotide sequence identity, preferably
over a region of at least about 25, 50, 100, 200, 500, 1000, or
more nucleotides, to a CgtD from C. jejuni LIO87 nucleic acid,
e.g., SEQ ID NO:1, or a nucleic acid encoding the catalytic domain.
Preferably the catalytic domain has at least 90%, preferably at
least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid
identity to the CgtD from C. jejuni LIO87 catalytic domain of SEQ
ID NO:2. A polynucleotide or polypeptide sequence is typically from
a bacteria including, but not limited to, Campylobacter,
Haemophilus, and Pasteurella. The nucleic acids and proteins of the
invention include both naturally occurring or recombinant
molecules. A CgtD from C. jejuni LIO87 protein typically has
.alpha.-1,4-galactosyltransferase activity that can be assayed
according to methods known to those of skill in the art, using
appropriate donor substrates and acceptor substrates, as described
herein. Preferred embodiments include a full length CgtD protein
of, e.g., SEQ ID NO:2 or a nucleic acid that encodes a full length
CgtD protein of, e.g., SEQ ID NO:2.
[0035] "Commercial scale" refers to gram scale production of a
galactosylated product in a single reaction. In preferred
embodiments, commercial scale refers to production of greater than
about 50, 75, 80, 90, 100, 125, 150, 175, or 200 grams of
galactosylated product.
[0036] As used herein, a "truncated CgtD polypeptide" or
grammatical variants, refers to a CgtD polypeptide that has been
manipulated to remove at least one amino acid residue, relative to
a wild type CgtD polypeptide that occurs in nature, so long as the
truncated CgtD polypeptide retains enzymatic activity.
[0037] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0038] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0039] Those of skill recognize that many amino acids can be
substituted for one another in a protein without affecting the
function of the protein, i.e., a conservative substitution can be
the basis of a conservatively modified variant of a protein such as
the disclosed CgtD proteins. An incomplete list of conservative
amino acid substitutions follows. The following eight groups each
contain amino acids that are conservative substitutions for one
another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V), Alanine (A); 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); 7) Serine (S), Threonine (T), Cysteine (C); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0040] The cells and methods of the invention are useful for
producing a galactosylated product, generally by transferring a
galactose moiety from a donor substrate to an acceptor molecule.
The cells and methods of the invention are also useful for
producing a galactosylated product sugar comprising additional
sugar residues, generally by transferring a additional
monosaccharide or a sulfate groups from a donor substrate to an
acceptor molecule. The addition generally takes place at the
non-reducing end of an oligosaccharide, polysaccharide (e.g.,
heparin, carragenin, and the like) or a carbohydrate moiety on a
glycolipid or glycoprotein, e.g., a biomolecule. Biomolecules as
defined here include but are not limited to biologically
significant molecules such as carbohydrates, oligosaccharides,
peptides (e.g., glycopeptides), proteins (e.g., glycoproteins), and
lipids (e.g., glycolipids, phospholipids, sphingolipids and
gangliosides).
[0041] The recombinant proteins of the invention can be constructed
and expressed as a fusion protein with a molecular "purification
tag" at one end, which facilitates purification or identification
of the protein. Such tags can also be used for immobilization of a
protein of interest during the glycosylation reaction. Suitable
tags include "epitope tags," which are a protein sequence that is
specifically recognized by an antibody. Epitope tags are generally
incorporated into fusion proteins to enable the use of a readily
available antibody to unambiguously detect or isolate the fusion
protein. A "FLAG tag" is a commonly used epitope tag, specifically
recognized by a monoclonal anti-FLAG antibody, consisting of the
sequence AspTyrLysAspAspAspAspLys (SEQ ID NO:6) or a substantially
identical variant thereof. Other suitable tags are known to those
of skill in the art, and include, for example, an affinity tag such
as a hexahistidine (SEQ ID NO:7) peptide, which will bind to metal
ions such as nickel or cobalt ions or a myc tag. Proteins
comprising purification tags can be purified using a binding
partner that binds the purification tag, e.g., antibodies to the
purification tag, nickel or cobalt ions or resins, and amylose,
maltose, or a cyclodextrin. Purification tags also include maltose
binding domains and starch binding domains. Purification of maltose
binding domain proteins is known to those of skill in the art.
Starch binding domains are described in WO 99/15636, herein
incorporated by reference. Affinity purification of a fusion
protein comprising a starch binding domain using a betacylodextrin
(BCD)-derivatized resin is described in WO 2005/014779, published
Feb. 17, 2005, herein incorporated by reference in its
entirety.
[0042] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogues of
natural nucleotides that hybridize to nucleic acids in manner
similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence includes the
complementary sequence thereof. The terms "nucleic acid", "nucleic
acid sequence", and "polynucleotide" are used interchangeably
herein.
[0043] The term "operably linked" refers to functional linkage
between a nucleic acid expression control sequence (such as a
promoter, signal sequence, or array of transcription factor binding
sites) and a second nucleic acid sequence, wherein the expression
control sequence affects transcription and/or translation of the
nucleic acid corresponding to the second sequence.
[0044] The term "recombinant" when used with reference to a cell
indicates that the cell replicates a heterologous nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic
acid. Recombinant cells can contain genes that are not found within
the native (non-recombinant) form of the cell. Recombinant cells
can also contain genes found in the native form of the cell wherein
the genes are modified and re-introduced into the cell by
artificial means. The term also encompasses cells that contain a
nucleic acid endogenous to the cell that has been modified without
removing the nucleic acid from the cell; such modifications include
those obtained by gene replacement, site-specific mutation, and
related techniques.
[0045] A "recombinant nucleic acid" refers to a nucleic acid that
was artificially constructed (e.g., formed by linking two
naturally-occurring or synthetic nucleic acid fragments). This term
also applies to nucleic acids that are produced by replication or
transcription of a nucleic acid that was artificially constructed.
A "recombinant polypeptide" is expressed by transcription of a
recombinant nucleic acid (i.e., a nucleic acid that is not native
to the cell or that has been modified from its naturally occurring
form), followed by translation of the resulting transcript.
[0046] A "heterologous polynucleotide" or a "heterologous nucleic
acid", as used herein, is one that originates from a source foreign
to the particular host cell, or, if from the same source, is
modified from its original form. Thus, a heterologous
glycosyltransferase gene in a prokaryotic host cell includes a
glycosyltransferase gene that is endogenous to the particular host
cell but has been modified. Modification of the heterologous
sequence may occur, e.g., by treating the DNA with a restriction
enzyme to generate a DNA fragment that is capable of being operably
linked to a promoter. Techniques such as site-directed mutagenesis
are also useful for modifying a heterologous sequence.
[0047] A "subsequence" refers to a sequence of nucleic acids or
amino acids that comprise a part of a longer sequence of nucleic
acids or amino acids (e.g., polypeptide) respectively.
[0048] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements that are capable of
affecting expression of a structural gene in hosts compatible with
such sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0049] A "fusion CgtD polypeptide" or a "fusion
galactosyltransferase polypeptide" of the invention is a
polypeptide that contains a CgtD or an
.alpha.-1,4-galactosyltransferase catalytic domain. The fusion
polypeptide is capable of catalyzing the synthesis of a sugar
nucleotide (e.g., UDP-Galactose) as well as the transfer of the
sugar residue from the sugar nucleotide to an acceptor molecule.
Typically, the catalytic domains of the fusion polypeptides will be
at least substantially identical to those of glycosyltransferases
and fusion proteins from which the catalytic domains are derived.
In some embodiments, a CgtD polypeptide and an epimerase, e.g.,
UDP-glucose 4' epimerase, polypeptide are fused to form a single
polypeptide. For examples of a galactosyltransferase/UDP-glucose 4'
epimerase see e.g. WO1999/031224, which is herein incorporated by
reference for all purposes.
[0050] An "accessory enzyme," as referred to herein, is an enzyme
that is involved in catalyzing a reaction that, for example, forms
a substrate or other reactant for a glycosyltransferase reaction.
An accessory enzyme can, for example, catalyze the formation of a
nucleotide sugar that is used as a sugar donor moiety by a
glycosyltransferase. An accessory enzyme can also be one that is
used in the generation of a nucleotide triphosphate that is
required for formation of a nucleotide sugar, or in the generation
of the sugar which is incorporated into the nucleotide sugar. One
example of an accessory enzyme is UDP-glucose 4' epimerase, e.g.
GalE from S. thermophilus (accession umber M30175)
[0051] A "catalytic domain" refers to a portion of an enzyme that
is sufficient to catalyze an enzymatic reaction that is normally
carried out by the enzyme. For example, a catalytic domain of a
CgtD polypeptide will include a sufficient portion of the CgtD to
transfer a galactose moiety from a sugar donor to an acceptor
saccharide. A catalytic domain can include an entire enzyme, a
subsequence thereof, or can include additional amino acid sequences
that are not attached to the enzyme or subsequence as found in
nature.
[0052] The term "isolated" refers to material that is substantially
or essentially free from components which interfere with the
activity of an enzyme. For cells, saccharides, nucleic acids, and
polypeptides of the invention, the term "isolated" refers to
material that is substantially or essentially free from components
which normally accompany the material as found in its native state.
Typically, isolated saccharides, proteins or nucleic acids of the
invention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or
85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% pure as measured by band intensity on a silver
stained gel or other method for determining purity. Purity or
homogeneity can be indicated by a number of means well known in the
art, such as polyacrylamide gel electrophoresis of a protein or
nucleic acid sample, followed by visualization upon staining. For
certain purposes high resolution will be needed and HPLC or a
similar means for purification utilized. For oligonucleotides, or
other galactosylated products, purity can be determined using,
e.g., thin layer chromatography, HPLC, or mass spectroscopy.
[0053] The terms "identical" or percent "identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0054] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 60%, preferably 80% or 85%, most
preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% nucleotide or amino acid residue identity, when compared and
aligned for maximum correspondence, as measured using one of the
following sequence comparison algorithms or by visual inspection.
Preferably, the substantial identity exists over a region of the
sequences that is at least about 50 residues in length, more
preferably over a region of at least about 100 residues, and most
preferably the sequences are substantially identical over at least
about 150 residues. In a most preferred embodiment, the sequences
are substantially identical over the entire length of the coding
regions.
[0055] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0056] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally, Current Protocols in Molecular Biology,
F. M. Ausubel et al., eds., Current Protocols, a joint venture
between Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc., (1995 Supplement) (Ausubel)).
[0057] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1990)
J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic
Acids Res. 25: 3389-3402, respectively. Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information (www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al, supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix
(see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)).
[0058] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0059] A further indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below.
[0060] The phrase "hybridizing specifically to", refers to the
binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA.
[0061] The term "stringent conditions" refers to conditions under
which a probe will hybridize to its target subsequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH. The Tm is the temperature (under defined
ionic strength, pH, and nucleic acid concentration) at which 50% of
the probes complementary to the target sequence hybridize to the
target sequence at equilibrium. (As the target sequences are
generally present in excess, at Tm, 50% of the probes are occupied
at equilibrium). Typically, stringent conditions will be those in
which the salt concentration is less than about 1.0 M Na.sup.+ ion,
typically about 0.01 to 1.0 M Na.sup.+ ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. For high
stringency PCR amplification, a temperature of about 62.degree. C.
is typical, although high stringency annealing temperatures can
range from about 50.degree. C. to about 65.degree. C., depending on
the primer length and specificity. Typical cycle conditions for
both high and low stringency amplifications include a denaturation
phase of 90-95.degree. C. for 30-120 sec, an annealing phase
lasting 30-120 sec, and an extension phase of about 72.degree. C.
for 1-2 min. Protocols and guidelines for low and high stringency
amplification reactions are available, e.g., in Innis, et al.
(1990) PCR Protocols: A Guide to Methods and Applications Academic
Press, N.Y.
[0062] The phrases "specifically binds to" or "specifically
immunoreactive with", when referring to an antibody refers to a
binding reaction which is determinative of the presence of the
protein or other antigen in the presence of a heterogeneous
population of proteins, saccharides, and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind preferentially to a particular antigen and do not bind in a
significant amount to other molecules present in the sample.
Specific binding to an antigen under such conditions requires an
antibody that is selected for its specificity for a particular
antigen. A variety of immunoassay formats can be used to select
antibodies specifically immunoreactive with a particular antigen.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with an
antigen. See Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0063] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. In a preferred embodiment,
antibodies that specifically bind to a CgtD protein are produced.
Light chains are classified as either kappa or lambda. Heavy chains
are classified as gamma, mu, alpha, delta, or epsilon, which in
turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. Typically, the antigen-binding region of an antibody
will be most critical in specificity and affinity of binding.
[0064] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0065] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce F
(ab)'.sub.2, a dimer of Fab which itself is a light chain joined to
V.sub.H--C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0066] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Techniques for the production of
single chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g. U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al,
Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994);
Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger,
Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also
be made bispecific, i.e., able to recognize two different antigens
(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659
(1991); and Suresh et al, Methods in Enzymology 121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently
joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No.
4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0067] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels for use in diagnostic assays.
[0068] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to
CgtD protein, polymorphic variants, alleles, orthologs, and
conservatively modified variants, or splice variants, or portions
thereof, can be selected to obtain only those polyclonal antibodies
that are specifically immunoreactive with CgtD proteins and not
with other proteins. This selection may be achieved by subtracting
out antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity).
[0069] An "antigen" is a molecule that is recognized and bound by
an antibody, e.g., peptides, carbohydrates, organic molecules, or
more complex molecules such as glycolipids and glycoproteins. The
part of the antigen that is the target of antibody binding is an
antigenic determinant and a small functional group that corresponds
to a single antigenic determinant is called a hapten.
[0070] A "label" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include .sup.32P, .sup.125I, fluorescent
dyes, electron-dense reagents, enzymes (e.g. as commonly used in an
ELISA), biotin, digoxigenin, or haptens and proteins for which
antisera or monoclonal antibodies are available (e.g., the
polypeptide of SEQ ID NO:2 can be made detectable, e.g., by
incorporating a radiolabel into the peptide, and used to detect
antibodies specifically reactive with the peptide).
[0071] The term "immunoassay" is an assay that uses an antibody to
specifically bind an antigen. The immunoassay is characterized by
the use of specific binding properties of a particular antibody to
isolate, target, and/or quantify the antigen.
[0072] The term "carrier molecule" means an immunogenic molecule
containing antigenic determinants recognized by T cells. A carrier
molecule can be a protein or can be a lipid. A carrier protein is
conjugated to a polypeptide to render the polypeptide immunogenic.
Carrier proteins include keyhole limpet hemocyanin, horseshoe crab
hemocyanin, and bovine serum albumin.
[0073] The term "adjuvant" means a substance that nonspecifically
enhances the immune response to an antigen. Adjuvants include
Freund's adjuvant, either complete or incomplete; Titermax gold
adjuvant; alum; and bacterial LPS.
[0074] The term "contacting" is used herein interchangeably with
the following: combined with, added to, mixed with, passed over,
incubated with, flowed over, etc.
III. CgtD Polypeptides
[0075] The CgtD polypeptides of the inventions comprise an amino
acid sequence that is identical to or shares a specified percent
identity with SEQ ID NO:2. The CgtD polypeptide is an
.alpha.-1,4-galactosyltransferase enzyme and has the functional
activity of transferring galactose from UDP-galactose to an
oligosaccharide comprising a terminal galactose.
[0076] Nucleic acids encoding proteins that are related to the CgtD
protein were also identified in other C. jejuni strains, e.g., ATCC
43429 and ATC43430. The amino acid sequences of these proteins are
found at SEQ ID NO:3 and SEQ ID NO:4. The amino acid sequences of
the ATCC 43429 and ATC43430 are identical and share 58% identity
with the LIO87 CgtD protein. An alignment of the related proteins
is provided in FIG. 2. To date, no activity has been identified for
the CgtD polypeptides from ATCC 43429 and ATC43430.
[0077] The Clustal W program was used to produce the alignments in
FIG. 2. A consensus sequence is included in the figure. Identical,
conserved, and semi-conserved amino acid residues are indicated on
the figure. Unmarked residues denote regions or residues without
apparent conservation.
[0078] Using the alignment generated by Clustal W or similar
programs known to those of skill, identical, conserved, or
semi-conserved residues can be identified and used to predict and
avoid changes in amino acid residues that would be detrimental to
CgtD activity. Such alignments can also be used to identify amino
acid residues that can most likely be changed without affecting
protein activity. Amino acid changes, if desired, can be made by
selecting a conserved residue as identified herein or on the
Clustal W website, or by selecting a modification to one of the
corresponding amino acids in a figure such as FIG. 2.
IV. Isolation of Nucleic Acids Encoding CgtD Polypeptides
[0079] Nucleic acids that encode CgtD polypeptides include nucleic
acids that encode the CgtD polypeptides described above, e.g., SEQ
ID NO:2, and conservatively modified variants of that sequence. The
CgtD polypeptides of the invention catalyze the transfer of a
galactose moiety from a donor substrate to an acceptor
substrate.
[0080] Nucleic acids that encode additional CgtD polypeptides based
on the information disclosed herein, and methods of obtaining such
nucleic acids, are known to those of skill in the art. Suitable
nucleic acids (e.g., cDNA, genomic, or subsequences (probes)) can
be cloned, or amplified by in vitro methods such as the polymerase
chain reaction (PCR), the ligase chain reaction (LCR), the
transcription-based amplification system (TAS), or the
self-sustained sequence replication system (SSR). A wide variety of
cloning and in vitro amplification methodologies are well-known to
persons of skill. Examples of these techniques and instructions
sufficient to direct persons of skill through many cloning
exercises are found in Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc.,
San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular
Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)
(Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; and Carr,
European Patent No. 0,246,864.
[0081] A DNA that encodes a CgtD polypeptide, or a subsequences
thereof, can be prepared by any suitable method described above,
including, for example, cloning and restriction of appropriate
sequences with restriction enzymes. In one preferred embodiment,
nucleic acids encoding CgtD polypeptides are isolated by routine
cloning methods. A nucleotide sequence of a CgtD polypeptide as
provided in, for example, SEQ ID NO: 1, can be used to provide
probes that specifically hybridize to a gene encoding a CgtD
polypeptide in a genomic DNA sample; or to an mRNA, encoding a CgtD
polypeptide comprising, in a total RNA sample (e.g., in a Southern
or Northern blot). Once the target nucleic acid encoding a CgtD
polypeptide is identified, it can be isolated according to standard
methods known to those of skill in the art (see, e.g., Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols.
1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987)
Methods in Enzymology, Vol. 152: Guide to Molecular Cloning
Techniques, San Diego: Academic Press, Inc.; or Ausubel et al.
(1987) Current Protocols in Molecular Biology, Greene Publishing
and Wiley-Interscience, New York). Further, the isolated nucleic
acids can be cleaved with restriction enzymes to create nucleic
acids encoding the full-length CgtD polypeptide, or subsequences
thereof, e.g., containing subsequences encoding at least a
subsequence of a catalytic domain of the CgtD polypeptide. These
restriction enzyme fragments, encoding a CgtD polypeptide or
subsequences thereof, may then be ligated, for example, to produce
a nucleic acid encoding a CgtD protein.
[0082] A nucleic acid encoding a CgtD polypeptide, or a subsequence
thereof, can be characterized by assaying for the expressed
product. Assays based on the detection of the physical, chemical,
or immunological properties of the expressed protein can be used.
For example, one can identify a cloned CgtD nucleic acid, by the
ability of a protein encoded by the nucleic acid to catalyze the
transfer of a galactose moiety from a donor substrate to an
acceptor substrate. In one method, capillary electrophoresis is
employed to detect the reaction products. This highly sensitive
assay involves using either saccharide or disaccharide aminophenyl
derivatives which are labeled with fluorescein as described in
Wakarchuk et al. (1996) J. Biol. Chem. 271 (45): 28271-276. To
assay for CgtD activity, Lac-FCHASE can be used as a substrate. The
reaction products of other glycosyltransferases can be detected
using capillary electrophoresis, e.g., to assay for a Neisseria
lgtC enzyme, either FCHASE-AP-Lac or FCHASE-AP-Gal can be used,
whereas for the Neisseria lgtB enzyme an appropriate reagent is
FCHASE-AP-GlcNAc (Wakarchuk, supra). To assay for
.alpha.2,8-sialyltransferase, GM3-FCHASE is used as a substrate.
See, e.g., U.S. Pat. No. 6,503,744, which is herein incorporated by
reference. Other methods for detection of oligosaccharide reaction
products include thin layer chromatography and GC/MS and are
disclosed in U.S. Pat. No. 6,503,744, which is herein incorporated
by reference.
[0083] Also, a nucleic acid encoding a CgtD polypeptide, or a
subsequence thereof, can be chemically synthesized. Suitable
methods include the phosphotriester method of Narang et al. (1979)
Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al.
(1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite
method of Beaucage et al (1981) Tetra. Lett., 22: 1859-1862; and
the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis produces a single stranded oligonucleotide. This can be
converted into double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill recognizes that
while chemical synthesis of DNA is often limited to sequences of
about 100 bases, longer sequences may be obtained by the ligation
of shorter sequences.
[0084] Nucleic acids encoding CgtD polypeptides, or subsequences
thereof, can be cloned using DNA amplification methods such as
polymerase chain reaction (PCR). Thus, for example, the nucleic
acid sequence or subsequence is PCR amplified, using a sense primer
containing one restriction enzyme site (e.g., NdeI) and an
antisense primer containing another restriction enzyme site (e.g.,
HindIII). This will produce a nucleic acid encoding the desired
CgtD polypeptide or a subsequence and having terminal restriction
enzyme sites. This nucleic acid can then be easily ligated into a
vector containing a nucleic acid encoding the second molecule and
having the appropriate corresponding restriction enzyme sites.
Suitable PCR primers can be determined by one of skill in the art
using the sequence information provided in GenBank or other
sources. Appropriate restriction enzyme sites can also be added to
the nucleic acid encoding the CgtD protein or a protein subsequence
thereof by site-directed mutagenesis. The plasmid containing the
CgtD protein-encoding nucleotide sequence or subsequence is cleaved
with the appropriate restriction endonuclease and then ligated into
an appropriate vector for amplification and/or expression according
to standard methods. Examples of techniques sufficient to direct
persons of skill through in vitro amplification methods are found
in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987)
U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and
Applications (Innis et al., eds) Academic Press Inc. San Diego,
Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)
C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh
et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al.
(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.
Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241:
1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and
Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89:
117.
[0085] Some nucleic acids encoding bacterial CgtD proteins can be
amplified using PCR primers based on the sequence of CgtD nucleic
acids disclosed herein. Examples of PCR primers that can be used to
amplify nucleic acid that encode CgtD proteins include the
following primer pairs:
TABLE-US-00001 CJ-636 Primer (SEQ ID NO:8)
TAAAAGGCTACATATGACTGAAATTTCAAGTTTTTGG (NdeI site is underlined)
CJ-639 Primer (SEQ ID NO:9) GGCAAGATGATTGTCGACTTAGGCATTGTTTTTC
(SalI site is underlined)
[0086] In some bacteria, nucleic acids encoding CgtD protein can be
isolated by amplifying a specific chromosomal locus, e.g., the LOS
locus of C. jejuni, and then identifying a CgtD nucleic acid
typically found at that locus (see, e.g., U.S. Pat. No. 6,503,744).
Examples of PCR primers that can be used to amplify an LOS locus
comprising nucleic acids encoding a CgtD protein include the
following primer pairs:
TABLE-US-00002 CJ42: Primer in heptosylTase-II (SEQ ID NO: 10) 5'
GC CAT TAC CGT ATC GCC TAA CCA GG 3' 25 mer CJ43: Primer in
heptosylTase-I (SEQ ID NO: 11) 5' AAA GAA TAC GAA TTT GCT AAA GAG G
3' 25 mer
[0087] Other physical properties of a recombinant CgtD polypeptide
expressed from a particular nucleic acid, can be compared to
properties of known CgtD polypeptides to provide another method of
identifying suitable sequences or domains of the CgtD polypeptide
that are determinants of acceptor substrate specificity and/or
catalytic activity. Alternatively, a putative CgtD polypeptide or
recombinant CgtD polypeptide can be mutated, and its role as a
glycosyltransferase, or the role of particular sequences or domains
established by detecting a variation in the structure of a
carbohydrate normally produced by the non-mutated,
naturally-occurring, or control CgtD polypeptide. Those of skill
will recognize that mutation or modification of CgtD polypeptides
of the invention can be facilitated by molecular biology techniques
to manipulate the nucleic acids encoding the CgtD polypeptides,
e.g., PCR.
[0088] Functional domains of newly identified CgtD polypeptides can
be identified by using standard methods for mutating or modifying
the polypeptides and testing them for activities such as acceptor
substrate activity and/or catalytic activity, as described herein.
The functional domains of the various CgtD polypeptides can be used
to construct nucleic acids encoding CgtD polypeptides and the
functional domains of one or more CgtD polypeptides. These
multi-CgtD fusion proteins can then be tested for the desired
acceptor substrate or catalytic activity.
[0089] In an exemplary approach to cloning nucleic acids encoding
CgtD proteins, the known nucleic acid or amino acid sequences of
cloned CgtD polypeptides are aligned and compared to determine the
amount of sequence identity between various CgtD polypeptides. This
information can be used to identify and select protein domains that
confer or modulate CgtD activities, e.g., acceptor substrate
activity and/or catalytic activity based on the amount of sequence
identity between the CgtD proteins of interest. For example,
domains having sequence identity between the CgtD proteins of
interest, and that are associated with a known activity, can be
used to construct CgtD proteins containing that domain, and having
the activity associated with that domain (e.g., acceptor substrate
specificity and/or catalytic activity).
V. Expression of CgtD Polypeptides in Host Cells
[0090] CgtD proteins of the invention can be expressed in a variety
of host cells, including E. coli, other bacterial hosts, and yeast.
The host cells are preferably microorganisms, such as, for example,
yeast cells, bacterial cells, or filamentous fungal cells. Examples
of suitable host cells include, for example, Azotobacter sp. (e.g.,
A. vinelandii), Pseudomonas sp., Rhizobium sp., Erwinia sp.,
Escherichia sp. (e.g., E. coli), Bacillus, Pseudomonas, Proteus,
Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus
and Klebsiella sp., among many others. The cells can be of any of
several genera, including Saccharomyces (e.g., S. cerevisiae),
Candida (e.g., C. utilis, C. parapsilosis, C. krusei, C.
versatilis, C. lipolytica, C. zeylanoides, C. guilliermondii, C.
albicans, and C. humicola), Pichia (e.g., P. farinosa and P.
ohmeri), Torulopsis (e.g., T. candida, T. sphaerica, T. xylinus, T.
famata, and T. versatilis), Debaryomyces (e.g., D. subglobosus, D.
cantarellii, D. globosus, D. hansenii, and D. japonicus),
Zygosaccharomyces (e.g., Z. rouxii and Z. bailii), Kluyveromyces
(e.g., K. marxianus), Hansenula (e.g., H. anomala and H. jadinii),
and Brettanomyces (e.g., B. lambicus and B. anomalus). Examples of
useful bacteria include, but are not limited to, Escherichia,
Enterobacter, Azotobacter, Erwinia, Klebsielia, Bacillus,
Pseudomonas, Proteus, and Salmonella.
[0091] Once expressed in a host cell, the CgtD polypeptides can be
used to produced galactosylated products. For example, the CgtD
polypeptides can be isolated using standard protein purification
techniques and used in in vitro reactions described herein to make
galactosylated products. Partially purified CgtD polypeptides can
also be used in in vitro reactions to make galactosylated products
as can the permeabilized host cells. The host cells can also be
used in an in vivo system (e.g., fermentative production) to
produce galactosylated products.
[0092] Typically, the polynucleotide that encodes the CgtD
polypeptides is placed under the control of a promoter that is
functional in the desired host cell. An extremely wide variety of
promoters are well known, and can be used in the expression vectors
of the invention, depending on the particular application.
Ordinarily, the promoter selected depends upon the cell in which
the promoter is to be active. Other expression control sequences
such as ribosome binding sites, transcription termination sites and
the like are also optionally included. Constructs that include one
or more of these control sequences are termed "expression
cassettes." Accordingly, the invention provides expression
cassettes into which the nucleic acids that encode fusion proteins
are incorporated for high level expression in a desired host
cell.
[0093] Expression control sequences that are suitable for use in a
particular host cell are often obtained by cloning a gene that is
expressed in that cell. Commonly used prokaryotic control
sequences, which are defined herein to include promoters for
transcription initiation, optionally with an operator, along with
ribosome binding site sequences, include such commonly used
promoters as the beta-lactamase (penicillinase) and lactose (lac)
promoter systems (Change et al., Nature (1977) 198: 1056), the
tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids
Res. (1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl.
Acad. Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P.sub.L
promoter and N-gene ribosome binding site (Shimatake et al., Nature
(1981) 292: 128). The particular promoter system is not critical to
the invention, any available promoter that functions in prokaryotes
can be used.
[0094] For expression of CgtD proteins in prokaryotic cells other
than E. coli, a promoter that functions in the particular
prokaryotic species is required. Such promoters can be obtained
from genes that have been cloned from the species, or heterologous
promoters can be used. For example, the hybrid trp-lac promoter
functions in Bacillus in addition to E. coli.
[0095] A ribosome binding site (RBS) is conveniently included in
the expression cassettes of the invention. An RBS in E. coli, for
example, consists of a nucleotide sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation codon
(Shine and Dalgarno, Nature (1975) 254: 34; Steitz, In Biological
regulation and development: Gene expression (ed. R. F. Goldberger),
vol. 1, p. 349, 1979, Plenum Publishing, NY).
[0096] For expression of the CgtD proteins in yeast, convenient
promoters include GAL1-10 (Johnson and Davies (1984) Mol. Cell.
Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J. Biol. Chem.
258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), and MF.alpha.
(Herskowitz and Oshima (1982) in The Molecular Biology of the Yeast
Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring
Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209). Another
suitable promoter for use in yeast is the ADH2/GAPDH hybrid
promoter as described in Cousens et al., Gene 61:265-275 (1987).
For filamentous fungi such as, for example, strains of the fungi
Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349), examples of
useful promoters include those derived from Aspergillus nidulans
glycolytic genes, such as the ADH3 promoter (McKnight et at., EMBO
J. 4: 2093 2099 (1985)) and the tpiA promoter. An example of a
suitable terminator is the ADH3 terminator (McKnight et al.).
[0097] Either constitutive or regulated promoters can be used in
the present invention. Regulated promoters can be advantageous
because the host cells can be grown to high densities before
expression of the fusion proteins is induced. High level expression
of heterologous proteins slows cell growth in some situations. An
inducible promoter is a promoter that directs expression of a gene
where the level of expression is alterable by environmental or
developmental factors such as, for example, temperature, pH,
anaerobic or aerobic conditions, light, transcription factors and
chemicals. Such promoters are referred to herein as "inducible"
promoters, which allow one to control the timing of expression of
the glycosyltransferase or enzyme involved in nucleotide sugar
synthesis. For E. coli and other bacterial host cells, inducible
promoters are known to those of skill in the art. These include,
for example, the lac promoter, the bacteriophage lambda P.sub.L
promoter, the hybrid trp-lac promoter (Amann et al. (1983) Gene 25:
167; de Boer et al. (1983) Proc. Nat'l. Acad. Sci. USA 80: 21), and
the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol.;
Tabor et al. (1985) Proc. Nat'l Acad. Sci. USA 82: 1074-8). These
promoters and their use are discussed in Sambrook et al., supra. A
particularly preferred inducible promoter for expression in
prokaryotes is a dual promoter that includes a tac promoter
component linked to a promoter component obtained from a gene or
genes that encode enzymes involved in galactose metabolism (e.g., a
promoter from a UDPgalactose 4-epimerase gene (galE)). The dual
tac-gal promote is described in PCT Patent Application Publ. No.
WO98/20111.
[0098] A construct that includes a polynucleotide of interest
operably linked to gene expression control signals that, when
placed in an appropriate host cell, drive expression of the
polynucleotide is termed an "expression cassette." Expression
cassettes that encode the fusion proteins of the invention are
often placed in expression vectors for introduction into the host
cell. The vectors typically include, in addition to an expression
cassette, a nucleic acid sequence that enables the vector to
replicate independently in one or more selected host cells.
Generally, this sequence is one that enables the vector to
replicate independently of the host chromosomal DNA, and includes
origins of replication or autonomously replicating sequences. Such
sequences are well known for a variety of bacteria. For instance,
the origin of replication from the plasmid pBR322 is suitable for
most Gram-negative bacteria. Alternatively, the vector can
replicate by becoming integrated into the host cell genomic
complement and being replicated as the cell undergoes DNA
replication. A preferred expression vector for expression of the
enzymes is in bacterial cells is pTGK, which includes a dual
tac-gal promoter and is described in PCT Patent Application Publ.
NO. WO98/20111.
[0099] The construction of polynucleotide constructs generally
requires the use of vectors able to replicate in bacteria. A
plethora of kits are commercially available for the purification of
plasmids from bacteria (see, for example, EasyPrepJ, FlexiPrepJ,
both from Pharmacia Biotech; StrataCleanJ, from Stratagene; and,
QIAexpress Expression System, Qiagen). The isolated and purified
plasmids can then be further manipulated to produce other plasmids,
and used to transfect cells. Cloning in Streptomyces or Bacillus is
also possible.
[0100] Selectable markers are often incorporated into the
expression vectors used to express the polynucleotides of the
invention. These genes can encode a gene product, such as a
protein, necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that confer resistance to antibiotics or other toxins,
such as ampicillin, neomycin, kanamycin, chloramphenicol, or
tetracycline. Alternatively, selectable markers may encode proteins
that complement auxotrophic deficiencies or supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. Often, the vector will have one
selectable marker that is functional in, e.g., E. coli, or other
cells in which the vector is replicated prior to being introduced
into the host cell. A number of selectable markers are known to
those of skill in the art and are described for instance in
Sambrook et al., supra.
[0101] Construction of suitable vectors containing one or more of
the above listed components employs standard ligation techniques as
described in the references cited above. Isolated plasmids or DNA
fragments are cleaved, tailored, and re-ligated in the form desired
to generate the plasmids required. To confirm correct sequences in
plasmids constructed, the plasmids can be analyzed by standard
techniques such as by restriction endonuclease digestion, and/or
sequencing according to known methods. Molecular cloning techniques
to achieve these ends are known in the art. A wide variety of
cloning and in vitro amplification methods suitable for the
construction of recombinant nucleic acids are well-known to persons
of skill. Examples of these techniques and instructions sufficient
to direct persons of skill through many cloning exercises are found
in Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego,
Calif. (Berger); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1998 Supplement) (Ausubel).
[0102] A variety of common vectors suitable for use as starting
materials for constructing the expression vectors of the invention
are well known in the art. For cloning in bacteria, common vectors
include pBR322 derived vectors such as pBLUESCRIPT.TM., and
.lamda.-phage derived vectors. In yeast, vectors include Yeast
Integrating plasmids (e.g., Ylp5) and Yeast Replicating plasmids
(the YRp series plasmids) and pGPD-2. Expression in mammalian cells
can be achieved using a variety of commonly available plasmids,
including pSV2, pBC12BI, and p91023, as well as lytic virus vectors
(e.g., vaccinia virus, adeno virus, and baculovirus), episomal
virus vectors (e.g., bovine papillomavirus), and retroviral vectors
(e.g., murine retroviruses).
[0103] The methods for introducing the expression vectors into a
chosen host cell are not particularly critical, and such methods
are known to those of skill in the art. For example, the expression
vectors can be introduced into prokaryotic cells, including E.
coli, by calcium chloride transformation, and into eukaryotic cells
by calcium phosphate treatment or electroporation. Other
transformation methods are also suitable.
[0104] Translational coupling may be used to enhance expression.
The strategy uses a short upstream open reading frame derived from
a highly expressed gene native to the translational system, which
is placed downstream of the promoter, and a ribosome binding site
followed after a few amino acid codons by a termination codon. Just
prior to the termination codon is a second ribosome binding site,
and following the termination codon is a start codon for the
initiation of translation. The system dissolves secondary structure
in the RNA, allowing for the efficient initiation of translation.
See Squires, et. al. (1988), J. Biol. Chem. 263: 16297-16302.
[0105] The CgtD polypeptides can be expressed intracellularly, or
can be secreted from the cell. Intracellular expression often
results in high yields. If necessary, the amount of soluble, active
fusion protein may be increased by performing refolding procedures
(see, e.g., Sambrook et al., supra.; Marston et al., Bio/Technology
(1984) 2: 800; Schoner et al., Bio/Technology (1985)3:151). In
embodiments in which the CgtD polypeptides are secreted from the
cell, either into the periplasm or into the extracellular medium,
the DNA sequence is linked to a cleavable signal peptide sequence.
The signal sequence directs translocation of the fusion protein
through the cell membrane. An example of a suitable vector for use
in E. coli that contains a promoter-signal sequence unit is
pTA1529, which has the E. coli phoA promoter and signal sequence
(see, e.g., Sambrook et at., supra.; Oka et al., Proc. Natl. Acad.
Sci. USA (1985) 82: 7212; Talmadge et al., Proc. Natl. Acad. Sci.
USA (1980) 77: 3988; Takahara et al., J. Biol. Chem. (1985) 260:
2670). In another embodiment, the CgtD proteins are fused to a
subsequence of protein A or bovine serum albumin (BSA), for
example, to facilitate purification, secretion, or stability.
[0106] The CgtD polypeptides of the invention can also be further
linked to other bacterial proteins. This approach often results in
high yields, because normal prokaryotic control sequences direct
transcription and translation. In E. coli, lacZ fusions are often
used to express heterologous proteins. Suitable vectors are readily
available, such as the pUR, pEX, and pMR100 series (see, e.g.,
Sambrook et al., supra.). For certain applications, it may be
desirable to cleave the non-glycosyltransferase and/or accessory
enzyme amino acids from the fusion protein after purification. This
can be accomplished by any of several methods known in the art,
including cleavage by cyanogen bromide, a protease, or by Factor
X.sub.a (see, e.g., Sambrook et al., supra.; Itakura et al.,
Science (1977) 198: 1056; Goeddel et al., Proc. Natl. Acad. Sci.
USA (1979) 76: 106; Nagai et al., Nature (1984) 309: 810; Sung et
al., Proc. Natl. Acad. Sci. USA (1986) 83: 561). Cleavage sites can
be engineered into the gene for the fusion protein at the desired
point of cleavage.
[0107] More than one recombinant protein may be expressed in a
single host cell by placing multiple transcriptional cassettes in a
single expression vector, or by utilizing different selectable
markers for each of the expression vectors which are employed in
the cloning strategy.
[0108] A suitable system for obtaining recombinant proteins from E.
coli which maintains the integrity of their N-termini has been
described by Miller et al. Biotechnology 7:698-704 (1989). In this
system, the gene of interest is produced as a C-terminal fusion to
the first 76 residues of the yeast ubiquitin gene containing a
peptidase cleavage site. Cleavage at the junction of the two
moieties results in production of a protein having an intact
authentic N-terminal reside.
VI. Purification of CgtD Polypeptides
[0109] The CgtD proteins of the present invention can be expressed,
e.g., as intracellular proteins or as proteins that are secreted
from the cell, and can be used in this form, in the methods of the
present invention. For example, a crude cellular extract containing
the expressed intracellular or secreted CgtD polypeptide can used
in the methods of the present invention.
[0110] Alternatively, the CgtD polypeptide can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, column chromatography, gel
electrophoresis and the like (see, generally, R. Scopes, Protein
Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in
Enzymology Vol. 182. Guide to Protein Purification, Academic Press,
Inc. N.Y. (1990)). Substantially pure compositions of at least
about 70, 75, 80, 85, 90% homogeneity are preferred, and 92, 95, 98
to 99% or more homogeneity are most preferred. The purified
proteins may also be used, e.g., as immunogens for antibody
production.
[0111] To facilitate purification of the CgtD polypeptides of the
invention, the nucleic acids that encode the proteins can also
include a coding sequence for an epitope or "tag" for which an
affinity binding reagent is available, i.e. a purification tag.
Examples of suitable epitopes include the myc and V-5 reporter
genes; expression vectors useful for recombinant production of
fusion proteins having these epitopes are commercially available
(e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His and
pcDNA3.1/V5-His are suitable for expression in mammalian cells).
Additional expression vectors suitable for attaching a tag to the
CgtD polypeptide of the invention, and corresponding detection
systems are known to those of skill in the art, and several are
commercially available (e.g., FLAG'' (Kodak, Rochester N.Y.).
Another example of a suitable tag is a polyhistidine sequence,
which is capable of binding to metal chelate affinity ligands.
Typically, six adjacent histidines (SEQ ID NO:7) are used, although
one can use more or less than six. Suitable metal chelate affinity
ligands that can serve as the binding moiety for a polyhistidine
tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E. (1990)
"Purification of recombinant proteins with metal chelating
adsorbents" In Genetic Engineering: Principles and Methods, J. K.
Setlow, Ed., Plenum Press, NY; commercially available from Qiagen
(Santa Clarita, Calif.)). Other purification or epitope tags
include, e.g., AU1, AU5, DDDDK (SEQ ID NO:12) (EC5), E tag, E2 tag,
Glu-Glu, a 6 residue peptide, EYMPME (SEQ ID NO: 13), derived from
the Polyoma middle T protein, HA, HSV, IRS, KT3, S tage, S1 tag, T7
tag, V5 tag, VSV-G, .beta.-galactosidase, Gal4, green fluorescent
protein (GFP), luciferase, protein C, protein A, cellulose binding
protein, GST (glutathione S-transferase), a step-tag, Nus-S,
PPI-ases, Pfg 27, calmodulin binding protein, dsb A and fragments
thereof, and granzyme B. Epitope peptides and antibodies that bind
specifically to epitope sequences are commercially available from,
e.g., Covance Research Products, Inc.; Bethyl Laboratories, Inc.;
Abcam Ltd.; and Novus Biologicals, Inc.
[0112] Purification tags also include maltose binding domains and
starch binding domains. Proteins comprising purification tags can
be purified using a binding partner that binds the purification
tag, e.g., antibodies to the purification tag, nickel or cobalt
ions or resins, and amylose, maltose, or a cyclodextrin.
Purification tags also include starch binding domains, E. coli
thioredoxin domains (vectors and antibodies commercially available
from e.g. Santa Cruz Biotechnology, Inc. and Alpha Diagnostic
International, Inc.), and the carboxy-terminal half of the SUMO
protein (vectors and antibodies commercially available from e.g.,
Life Sensors Inc.). Starch binding domains, such as a maltose
binding domain from E. coli and SBD (starch binding domain) from an
amylase of A. niger, are described in WO 99/15636, herein
incorporated by reference. Affinity purification of a fusion
protein comprising a starch binding domain using a betacyclodextrin
(BCD)-derivatized resin is described in WO 2005/014779, published
Feb. 17, 2005, herein incorporated by reference in its entirety. In
some embodiments, a CgtD polypeptide comprises more than one
purification or epitope tag.
[0113] Other haptens that are suitable for use as tags are known to
those of skill in the art and are described, for example, in the
Handbook of Fluorescent Probes and Research Chemicals (6th Ed.,
Molecular Probes, Inc., Eugene Oreg.). For example, dinitrophenol
(DNP), digoxigenin, barbiturates (see, e.g., U.S. Pat. No.
5,414,085), and several types of fluorophores are useful as
haptens, as are derivatives of these compounds. Kits are
commercially available for linking haptens and other moieties to
proteins and other molecules. For example, where the hapten
includes a thiol, a heterobifunctional linker such as SMCC can be
used to attach the tag to lysine residues present on the capture
reagent.
[0114] One of skill would recognize that modifications can be made
to the catalytic or functional domains of the CgtD polypeptide
without diminishing their biological activity. Some modifications
may be made to facilitate the cloning, expression, or incorporation
of the catalytic domain into a fusion protein. Such modifications
are well known to those of skill in the art and include, for
example, the addition of codons at either terminus of the
polynucleotide that encodes the catalytic domain to provide, for
example, a methionine added at the amino terminus to provide an
initiation site, or additional amino acids (e.g., poly H is) placed
on either terminus to create conveniently located restriction
enzyme sites or termination codons or purification sequences.
VII. Fusion CgtD Proteins
[0115] In some embodiments, the recombinant cells of the invention
express fusion proteins that have more than one enzymatic activity
that is involved in synthesis of a desired galactosylated
oligosaccharide. The fusion polypeptides can be composed of, for
example, a CgtD polypeptide that is joined to a an accessory
enzyme, e.g., [UDP-GalNAc 4' epimerase or a UDP-glucose
4'epimerase. Fusion proteins can also be made using catalytic
domains or other truncations of the enzymes. For example, a
polynucleotide that encodes a CgtD polypeptide can be joined,
in-frame, to a polynucleotide that encodes, e.g., a UDP-GalNAc 4'
epimerase or a UDP-glucose 4'epimerase. The resulting fusion
protein can then catalyze not only the synthesis of the activated
galactose molecule, but also the transfer of the galactose moiety
to the acceptor molecule. The fusion protein can be two or more
galactose cycle enzymes linked into one expressible nucleotide
sequence. The fusion CgtD polypeptides of the present invention can
be readily designed and manufactured utilizing various recombinant
DNA techniques well known to those skilled in the art. Exemplary
fusion proteins are described in PCT Patent Application
PCT/CA98/01180, which was published as WO99/31224 on Jun. 24, 1999
and which discloses CMP-sialic acid synthase from Neisseria fused
with an .alpha.2,3-sialyltransferase from Neisseria. In some
embodiments, more that one fusion CgtD polypeptide is expressed in
the cell. Fusion protein can also comprise purification or epitope
tags as described herein.
VIII. Donor Substrates and Acceptor Substrates
[0116] Suitable donor substrates used by the CgtD polypeptides
include e.g., UDP-Gal. Guo et al., Applied Biochem. and Biotech.
68: 1-20 (1997).
[0117] Typically, acceptor substrates include a terminal lactose or
LacNAc derivatives for addition of a galactose residue by an a 1,4
linkage. Examples of suitable acceptors include a terminal Gal that
is linked to GlcNAc or Glc by a .beta.1,4 linkage, and a terminal
Gal that is .beta.1,3-linked to either GlcNAc or GalNAc. Suitable
acceptors, include, for example, lactose and LacNAc, and other
acceptors that can be determined by those of skill in the art. The
terminal residue to which the galactose moiety is attached can
itself be attached to, for example, H, a saccharide,
oligosaccharide, or an aglycone group having at least one
carbohydrate atom. In some embodiments, the acceptor residue is a
portion of an oligosaccharide that is attached to a peptide, a
protein, a lipid, or a proteoglycan, for example.
[0118] Suitable acceptor substrates used by the CgtD polypeptides
and methods of the invention include, but are not limited to,
polysaccharides and oligosaccharides. The CgtD polypeptides
described herein can also be used in multienzyme systems to produce
a desired product from a convenient starting material.
[0119] Suitable acceptor substrates used by the CgtD polypeptides
and methods of the invention include, but are not limited to,
proteins, lipids, peptides, glycoproteins, glycolipids,
glycopeptides, gangliosides and other biological structures (e.g.,
whole cells) that can be modified by the methods of the invention.
These acceptor substrates will typically comprise the
polysaccharide or oligosaccharide molecules described above.
Exemplary structures, which can be modified by the methods of the
invention include any a of a number glycolipids, glycoproteins and
carbohydrate structures on cells known to those skilled in the
art.
[0120] The present invention provides CgtD polypeptides that are
selected for their ability to produce oligosaccharides,
glycoproteins and glycolipids having desired oligosaccharide
moieties. Similarly, if present, accessory enzymes are chosen based
on an desired activated sugar substrate or on a sugar found on the
product oligosaccharide.
[0121] For synthesis of glycoproteins, one can readily identify
suitable CgtD polypeptides by reacting various amounts of a CgtD
polypeptide of interest (e.g., 0.01-100 mU/mg protein) with a
glycoprotein (e.g., at 1-10 mg/ml) to which is linked an
oligosaccharide that has a potential acceptor site for
glycosylation by the CgtD protein of interest. The abilities of the
recombinant CgtD proteins of the present invention to add a sugar
residue at the desired acceptor site are compared, and a CgtD
polypeptide having the desired property (e.g., acceptor substrate
specificity or catalytic activity) is selected.
[0122] In general, the efficacy of the enzymatic synthesis of
oligosaccharides, glycoproteins, and glycolipids, having desired
galactosylated oligosaccharide moieties, can be enhanced through
use of recombinantly produced CgtD polypeptides of the present
invention. Recombinant techniques enable production of the
recombinant CgtD polypeptides in the large amounts that are
required for large-scale in vitro oligosaccharide, glycoprotein and
glycolipid modification.
[0123] In some embodiments, suitable oligosaccharides,
glycoproteins, and glycolipids for use by the CgtD polypeptides and
methods of the invention can be glycoproteins and glycolipids
immobilized on a solid support during the glycosylation reaction.
The term "solid support" also encompasses semi-solid supports.
Preferably, the target glycoprotein or glycolipid is reversibly
immobilized so that the respective glycoprotein or glycolipid can
be released after the glycosylation reaction is completed. Many
suitable matrices are known to those of skill in the art. Ion
exchange, for example, can be employed to temporarily immobilize a
glycoprotein or glycolipid on an appropriate resin while the
glycosylation reaction proceeds. A ligand that specifically binds
to the glycoprotein or glycolipid of interest can also be used for
affinity-based immobilization. For example, antibodies that
specifically bind to a glycoprotein are suitable. Also, where the
glycoprotein of interest is itself an antibody or contains a
fragment thereof, one can use protein A or G as the affinity resin.
Dyes and other molecules that specifically bind to a glycoprotein
or glycolipid of interest are also suitable.
[0124] Preferably, when the acceptor saccharide is a truncated
version of the full-length glycoprotein, it preferably includes the
biologically active subsequence of the full-length glycoprotein.
Exemplary biologically active subsequences include, but are not
limited to, enzyme active sites, receptor binding sites, ligand
binding sites, complementarity determining regions of antibodies,
and antigenic regions of antigens.
IX. Production of Galactosylated Products
[0125] CgtD polypeptides can be used to make galactosylated
products in in vitro reactions mixes or by in vivo reactions, e.g.,
by fermentative growth of recombinant microorganisms that comprise
nucleotides that encode CgtD polypeptides.
[0126] A. In Vitro Reactions
[0127] The CgtD polypeptides can be used to make galactosylated
products in in vitro reactions mixes. The in vitro reaction
mixtures can include permeabilized microorganisms comprising the
CgtD polypeptides, partially purified CgtD polypeptides, or
purified CgtD polypeptides; as well as donor substrates acceptor
substrates, and appropriate reaction buffers. For in vitro
reactions, the recombinant glycosyltransferase proteins, such as
CgtD polypeptides, acceptor substrates, donor substrates and other
reaction mixture ingredients are combined by admixture in an
aqueous reaction medium. Additional glycosyltransferases can be
used in combination with the CgtD polypeptides, depending on the
desired galactosylated product. The medium generally has a pH value
of about 5 to about 8.5. The selection of a medium is based on the
ability of the medium to maintain pH value at the desired level.
Thus, in some embodiments, the medium is buffered to a pH value of
about 7.5. If a buffer is not used, the pH of the medium should be
maintained at about 5 to 8.5, depending upon the particular
galactosyltransferase used. For CgtD polypeptides, the pH range is
preferably maintained from about 7.0 to 8.0. For
sialyltransferases, the range is preferably from about 5.5 to about
8.0.
[0128] Enzyme amounts or concentrations are expressed in activity
units, which is a measure of the initial rate of catalysis. One
activity unit catalyzes the formation of 1 .mu.mol of product per
minute at a given temperature (typically 37.degree. C.) and pH
value (typically 7.5). Thus, 10 units of an enzyme is a catalytic
amount of that enzyme where 10 .mu.mol of substrate are converted
to 10 .mu.mol of product in one minute at a temperature of
37.degree. C. and a pH value of 7.5.
[0129] The reaction mixture may include divalent metal cations
(Mg.sup.2+, Mn.sup.2+). The reaction medium may also comprise
solubilizing detergents (e.g., Triton or SDS) and organic solvents
such as methanol or ethanol, if necessary. The enzymes can be
utilized free in solution or can be bound to a support such as a
polymer. The reaction mixture is thus substantially homogeneous at
the beginning, although some precipitate can form during the
reaction.
[0130] The temperature at which an above process is carried out can
range from just above freezing to the temperature at which the most
sensitive enzyme denatures. That temperature range is preferably
about 0.degree. C. to about 45.degree. C., and more preferably at
about 20.degree. C. to about 37.degree. C.
[0131] The reaction mixture so formed is maintained for a period of
time sufficient to obtain the desired high yield of desired
oligosaccharide determinants present on oligosaccharide groups
attached to the biomolecule to be glycosylated. For large-scale
preparations, the reaction will often be allowed to proceed for
between about 0.5-240 hours, and more typically between about 1-36
hours.
[0132] One or more of the glycosyltransferase reactions can be
carried out as part of a glycosyltransferase cycle. Preferred
conditions and descriptions of glycosyltransferase cycles have been
described. A number of glycosyltransferase cycles (for example,
sialyltransferase cycles, galactosyltransferase cycles, and
fucosyltransferase cycles) are described in U.S. Pat. No. 5,374,541
and WO 9425615 A. Other glycosyltransferase cycles are described in
Ichikawa et al. J. Am. Chem. Soc. 114:9283 (1992), Wong et al. J.
Org. Chem. 57: 4343 (1992), DeLuca, et al., J. Am. Chem. Soc.
117:5869-5870 (1995), and Ichikawa et al. In Carbohydrates and
Carbohydrate Polymers. Yaltami, ed. (ATL Press, 1993).
[0133] For the above glycosyltransferase cycles, the concentrations
or amounts of the various reactants used in the processes depend
upon numerous factors including reaction conditions such as
temperature and pH value, and the choice and amount of acceptor
saccharides to be glycosylated. Because the glycosylation process
permits regeneration of activating nucleotides, activated donor
sugars and scavenging of produced PPi in the presence of catalytic
amounts of the enzymes, the process is limited by the
concentrations or amounts of the stoichiometric substrates
discussed before. The upper limit for the concentrations of
reactants that can be used in accordance with the method of the
present invention is determined by the solubility of such
reactants.
[0134] Preferably, the concentrations of activating nucleotides,
phosphate donor, the donor sugar and enzymes are selected such that
glycosylation proceeds until the acceptor is consumed. The
considerations discussed below, while in the context of a
sialyltransferase, are generally applicable to other
glycosyltransferase cycles.
[0135] Each of the enzymes is present in a catalytic amount. The
catalytic amount of a particular enzyme varies according to the
concentration of that enzyme's substrate as well as to reaction
conditions such as temperature, time and pH value. Means for
determining the catalytic amount for a given enzyme under
preselected substrate concentrations and reaction conditions are
well known to those of skill in the art.
[0136] B. In Vivo Reactions
[0137] The CgtD polypeptides can be used to make galactosylated
products by in vivo reactions, e.g., fermentative growth of
recombinant microorganisms comprising the CgtD polypeptides.
Fermentative growth of recombinant microorganisms can occur in the
presence of medium that includes an acceptor substrate, e.g.
lactose or LacNAc and a donor substrate or a precursor to a donor
substrate, e.g., galactose. See, e.g., Priem et al., Glycobiology
12:235-240 (2002). The microorganism takes up the acceptor
substrate and the donor substrate or the precursor to a donor
substrate and the addition of the donor substrate to the acceptor
substrate takes place in the living cell. The microorganism can be
altered to facilitate uptake of the acceptor substrate, e.g., by
expressing a sugar transport protein. For example, where lactose is
the acceptor saccharide, E. coli cells that express the LacY
permease can be used. Other methods can be used to decrease
breakdown of an acceptor saccharide or to increase production of a
donor saccharide or a precursor of the donor saccharide. In some
embodiments, production of galactosylated products is enhanced by
manipulation of the host microorganism. For example, in E. coli,
break down of sialic acid can be minimized by using a host strain
that is lacking, e.g., CMP-sialate synthase (NanA-). (In some
strains of E. coli, CMP-sialate synthase appears to be a catabolic
enzyme.) Also in E. coli, when lactose is, for example, the
acceptor saccharide or an intermediate in synthesizing the
galactosylated product, lactose breakdown can be minimized by using
host cells that are LacZ-.
[0138] C. Characterization of and Isolation of Galactosylated
Products
[0139] The production of galactosylated products can be monitored
by e.g., determining that production of the desired product has
occurred or by determining that a substrate such as the acceptor
substrate has been depleted. Those of skill will recognize that
galactosylated products such as oligosaccharide, can be identified
using techniques such as chromatography, e.g., using paper or TLC
plates, or by mass spectrometry, e.g., MALDI-TOF spectrometry, or
by NMR spectroscopy. Methods of identification of galactosylated
products are known to those of skill in the art and are found,
e.g., in U.S. Pat. No. 6,699,705, which is herein incorporated by
reference for all purposes and in Varki et al., Preparation and
Analysis of Glycoconjugates, in Current Protocols in Molecular
Biology, Chapter 17 (Ausubel et al. eds, 1993).
[0140] The products produced using CgtD polypeptides can be used
without purification. However, standard, well known techniques, for
example, thin or thick layer chromatography, ion exchange
chromatography, or membrane filtration can be used for recovery of
galactosylated saccharides. Also, for example, membrane filtration,
utilizing a nanofiltration or reverse osmotic membrane as described
in commonly assigned AU Patent No. 735695 may be used. As a further
example, membrane filtration wherein the membranes have a molecular
weight cutoff of about 1000 to about 10,000 Daltons can be used to
remove proteins. As another example, nanofiltration or reverse
osmosis can then be used to remove salts. Nanofilter membranes are
a class of reverse osmosis membranes which pass monovalent salts
but retain polyvalent salts and uncharged solutes larger than about
200 to about 1000 Daltons, depending upon the membrane used. Thus,
for example, the oligosaccharides produced by the compositions and
methods of the present invention can be retained in the membrane
and contaminating salts will pass through. Glycoprotein
galactosylated products can be isolated or purified using standard
protein purification techniques, including those described
herein.
[0141] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a nucleic acid" includes a plurality of such
nucleic acids and reference to "the polypeptide" includes reference
to one or more polypeptides and equivalents thereof known to those
skilled in the art, and so forth.
[0142] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed. All citations are
incorporated herein by reference.
EXAMPLES
Example 1
Cloning Expression and Characterization of CgtD from C. jejuni
LIO87
[0143] ORF #8 from C. jejuni LIO87 (SEQ ID NO:1) was cloned and
expressed in Escherichia coli. The extracts from Escherichia coli
that overexpress the protein of SEQ ID NO:2 were assayed with
UDP-Glc, UDP-GlcNAc, UDP-Gal and UDP-GalNAc as donors and the
fluorescent derivatives of Glc, Gal, Lac, LacNAc and
Gal-.alpha.-1,4-Lac as acceptors. The protein product of ORF #8
(SEQ ID NO:2) was active with UDP-Gal as donor and either the
LacNAc (Gal-.beta.-1,4-GlcNAc) or the Lac (Gal-.beta.-1,4-Glc)
derivatives as acceptor. SEQ ID NO:2 was designated CgtD
(Campylobacter glycosyltransferase; D) and further
characterized.
Example 2
Further Characterization of CgtD Activity
[0144] To further characterize the enzymatic activity of CgtD, the
gene encoding CgtD from C. jejuni LIO87 was cloned in pCWori+ as a
C-terminal fusion with the E. coli maltose-binding (MalE) protein
(construct CJL-99). CJL-99 was electroporated in E. coli AD202.
CgtD expressed well as a MalE fusion (about 150 units per liter).
The activity was assessed on the donors and acceptors determined
optimal in Example 1. Activity was optimal at pH 7 to 8. Inclusion
of divalent cations increased activity. Some diva lent cations were
more beneficial than others e.g., MnCl.sub.2 yielded about 15%
better activity than MgCl.sub.2.
Example 3
Determining the Regio- and Stereo-Specificity of CgtD
[0145] The regio- and stereo-specificity of CgtD were determined
using purified MalE-CgtD (CJL-99). 19 mg of
.alpha.Gal-1,4-.beta.Gal-1,4-.beta.GlcNAc-p-nitrophenyl were
synthesized. The .sup.1H NMR resonances were assigned to the
trisaccharide compound through the use of 2D homonuclear COSY and
TOCSY spectra as shown in FIG. 1. These assignments were used to
identify cross-peaks in an .sup.1H-.sup.13C HSQC spectrum, which
correlates the chemical shift of a proton atom with its directly
bonded carbon neighbor (FIG. 2). Because inter-residue
connectivities can be established across glycosidic bonds using a
.sup.1H-.sup.13C HMBC pulse sequence, HMBC spectra of the
trisaccharide compound were acquired to establish the covalent
linkages between sugar residues. These data confirmed that CgtD
transferred a Gal residue to a Gal on the disaccharide precursor
through an .alpha.1.fwdarw.4 linkage. Consistent with the HMBC
spectra, the carbon resonance at position C4 of .beta.Gal displayed
a downfiled shift in comparison with monosaccharide values. This
shift is a qualitative indicator of a glycosidic linkage with the
adjacent .beta.Gal residue.
Example 4
BLAST Sequence Searches for CgtD Homologues
[0146] A BLASTP search using CgtD from C. jejuni LIO87 (SEQ ID
NO:2) did not reveal any significant homologue from organisms other
than C. jejuni. Two C. jejuni strains (ATCC 43429 and ATC43430)
have homologues of CgtD (SEQ ID NO:3 and SEQ ID NO:4) that are
identical between themselves and share 58% identity with CgtD from
C. jejuni LIO87 (FIG. 3). To date, no activity has been identified
for the CgtD polypeptides from ATCC 43429 and ATC43430.
[0147] The Clustal W program was used to produce the alignments in
FIG. 3. The following symbols are used: "*" all sequences in the
alignment have identical e residues; ":" conserved substitutions
are present; and "." semi-conserved substitutions are present. See,
e.g., www.ebi.ac.uk/clustalw/#. The website defines conserved
substitutions by designating amino acids in the following groups as
interchangeable: A, V, F, P, M, I, L and W; D and E; R, H and K;
and S, T, Y, H, C, N, G and Q. The bottom sequence is a consensus
sequence generated by the program.
[0148] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
131888DNACampylobacter jejuniCampylobacter jejuni LIO87
alpha-1,4-galactosyltransferase (Campylobacter glycosyltransferase
D, CgtD) ORF #8 1atgactgaaa tttcaagttt ttggtatact cctaaaggct
ataagggtat aggtttaatg 60gaaattctta cgattaaatc ttggcttgat catgggtata
aattccatct atatacttat 120aatttagaag ataaaatttt tttaaaattc
caagagctgt ttgataattt tatacttaaa 180gatgcaaatg aaatcatacc
ttttgaagaa tattttagcg atgatagggg agctggagta 240gctgcttttt
cagatttttt taggtttaat ctactttatc tcaggggggg ggtatgggtg
300gatcttgata tggtgtgttt aaaccattat gattatgata aaaaagaata
tattttttct 360aaggaaattg ataatgatct aagcaaagct agaatcacaa
cttcactcct taaatttcca 420aaacaaagtg aatttggaaa attaattata
gatgaagcaa aaaagattgt tgatgataac 480aaaataattc cttggggtat
tataggtcct tggtttttag ctaaatgggt taaagaatat 540gatttagaaa
aacatgctct agactataaa gatacttgtc aaatttcttg tggtaatact
600agagatttta tagataaaaa aattttcgat aaaaacagac tttgtttgca
tttattttct 660gaaatgtgga aaatttataa aatgaataaa aatcattttt
ataaatcatg catttatgga 720tttttacttc aaaagcacaa tatccttgat
ttatgtctta aattaaatta taatcttagt 780ttttgcgata aacattatga
taaattcctt ccttttatta atataaaaaa taaaataaga 840ttttattttc
gccacccaaa aaagattttt aagaaaaaca atgcctaa 8882295PRTCampylobacter
jejuniCampylobacter jejuni LIO87 alpha-1,4-galactosyltransferase
(Campylobacter glycosyltransferase D, CgtD) ORF #8 2Met Thr Glu Ile
Ser Ser Phe Trp Tyr Thr Pro Lys Gly Tyr Lys Gly1 5 10 15Ile Gly Leu
Met Glu Ile Leu Thr Ile Lys Ser Trp Leu Asp His Gly 20 25 30Tyr Lys
Phe His Leu Tyr Thr Tyr Asn Leu Glu Asp Lys Ile Phe Leu 35 40 45Lys
Phe Gln Glu Leu Phe Asp Asn Phe Ile Leu Lys Asp Ala Asn Glu 50 55
60Ile Ile Pro Phe Glu Glu Tyr Phe Ser Asp Asp Arg Gly Ala Gly Val65
70 75 80Ala Ala Phe Ser Asp Phe Phe Arg Phe Asn Leu Leu Tyr Leu Arg
Gly 85 90 95Gly Val Trp Val Asp Leu Asp Met Val Cys Leu Asn His Tyr
Asp Tyr 100 105 110Asp Lys Lys Glu Tyr Ile Phe Ser Lys Glu Ile Asp
Asn Asp Leu Ser 115 120 125Lys Ala Arg Ile Thr Thr Ser Leu Leu Lys
Phe Pro Lys Gln Ser Glu 130 135 140Phe Gly Lys Leu Ile Ile Asp Glu
Ala Lys Lys Ile Val Asp Asp Asn145 150 155 160Lys Ile Ile Pro Trp
Gly Ile Ile Gly Pro Trp Phe Leu Ala Lys Trp 165 170 175Val Lys Glu
Tyr Asp Leu Glu Lys His Ala Leu Asp Tyr Lys Asp Thr 180 185 190Cys
Gln Ile Ser Cys Gly Asn Thr Arg Asp Phe Ile Asp Lys Lys Ile 195 200
205Phe Asp Lys Asn Arg Leu Cys Leu His Leu Phe Ser Glu Met Trp Lys
210 215 220Ile Tyr Lys Met Asn Lys Asn His Phe Tyr Lys Ser Cys Ile
Tyr Gly225 230 235 240Phe Leu Leu Gln Lys His Asn Ile Leu Asp Leu
Cys Leu Lys Leu Asn 245 250 255Tyr Asn Leu Ser Phe Cys Asp Lys His
Tyr Asp Lys Phe Leu Pro Phe 260 265 270Ile Asn Ile Lys Asn Lys Ile
Arg Phe Tyr Phe Arg His Pro Lys Lys 275 280 285Ile Phe Lys Lys Asn
Asn Ala 290 2953283PRTCampylobacter jejuniCampylobacter jejuni ATCC
43429 alpha-1,4-galactosyltransferase (Campylobacter
glycosyltransferase D, CgtD) 3Met Lys Gln Glu Ile Ser Ser Phe Trp
Tyr Thr Pro Arg Gly Tyr Lys1 5 10 15Gly Ile Gly Leu Met Glu Leu Leu
Ser Ile Lys Ser Phe Ile Asp Asn 20 25 30Gly Tyr Lys Phe Ile Leu Tyr
Thr Tyr Asn Leu Asp Asp Lys Ile Phe 35 40 45Lys Lys Leu Asp Glu Leu
Phe Asp Asp Phe Glu Leu Lys Asp Ala Asn 50 55 60Glu Ile Val Ser Phe
Lys Asn Tyr Phe Arg Asp Asp Arg Gly Ser Gly65 70 75 80Val Ala Ala
Phe Ser Asp Tyr Phe Arg Tyr Asn Leu Leu Tyr Leu Lys 85 90 95Lys Lys
Lys Arg Gly Gly Val Trp Val Asp Leu Asp Met Ile Cys Leu 100 105
110Asn Tyr Ile Asp Leu Asn Glu Glu Tyr Ile Phe Thr Gln Glu Val Asp
115 120 125Glu Asp Asn Lys Lys Ser Arg Ile Thr Thr Ser Phe Leu Lys
Phe Ser 130 135 140Arg Tyr Ser Asp Phe Gly Lys Asn Leu Ile Gln Glu
Ala Glu Lys Ile145 150 155 160Ile Asn Lys Arg Lys Lys Ile Ser Trp
Gly Val Ile Gly Pro Trp Phe 165 170 175Leu Ala Asp His Val Lys Lys
Cys Gly Leu Glu Asn Phe Val Trp Asp 180 185 190Tyr Lys Arg Thr Cys
Gln Ile Pro Trp Cys Asn Val Lys Ile Phe Leu 195 200 205Asp Asn Thr
Ser Ile Asp Ile Ser Gln Pro Phe Leu His Leu Phe Ser 210 215 220Glu
Met Trp Arg Leu Asn Asn Met Glu Lys Asn Thr Phe His Gln Met225 230
235 240Gly Val Tyr Gly Gln Leu Leu Lys Lys His Glu Ile Glu Lys Leu
Tyr 245 250 255Asn Gln Ile Asn Thr Cys Leu Lys Thr Ser Met Leu Asp
Asn Ile Ala 260 265 270Ser Phe Leu Thr Lys Phe Phe Ile Lys Lys Leu
275 2804283PRTCampylobacter jejuniCampylobacter jejuni ATCC 43430
alpha-1,4-galactosyltransferase (Campylobacter glycosyltransferase
D, CgtD) 4Met Lys Gln Glu Ile Ser Ser Phe Trp Tyr Thr Pro Arg Gly
Tyr Lys1 5 10 15Gly Ile Gly Leu Met Glu Leu Leu Ser Ile Lys Ser Phe
Ile Asp Asn 20 25 30Gly Tyr Lys Phe Ile Leu Tyr Thr Tyr Asn Leu Asp
Asp Lys Ile Phe 35 40 45Lys Lys Leu Asp Glu Leu Phe Asp Asp Phe Glu
Leu Lys Asp Ala Asn 50 55 60Glu Ile Val Ser Phe Lys Asn Tyr Phe Arg
Asp Asp Arg Gly Ser Gly65 70 75 80Val Ala Ala Phe Ser Asp Tyr Phe
Arg Tyr Asn Leu Leu Tyr Leu Lys 85 90 95Lys Lys Lys Arg Gly Gly Val
Trp Val Asp Leu Asp Met Ile Cys Leu 100 105 110Asn Tyr Ile Asp Leu
Asn Glu Glu Tyr Ile Phe Thr Gln Glu Val Asp 115 120 125Glu Asp Asn
Lys Lys Ser Arg Ile Thr Thr Ser Phe Leu Lys Phe Ser 130 135 140Arg
Tyr Ser Asp Phe Gly Lys Asn Leu Ile Gln Glu Ala Glu Lys Ile145 150
155 160Ile Asn Lys Arg Lys Lys Ile Ser Trp Gly Val Ile Gly Pro Trp
Phe 165 170 175Leu Ala Asp His Val Lys Lys Cys Gly Leu Glu Asn Phe
Val Trp Asp 180 185 190Tyr Lys Arg Thr Cys Gln Ile Pro Trp Cys Asn
Val Lys Ile Phe Leu 195 200 205Asp Asn Thr Ser Ile Asp Ile Ser Gln
Pro Phe Leu His Leu Phe Ser 210 215 220Glu Met Trp Arg Leu Asn Asn
Met Glu Lys Asn Thr Phe His Gln Met225 230 235 240Gly Val Tyr Gly
Gln Leu Leu Lys Lys His Glu Ile Glu Lys Leu Tyr 245 250 255Asn Gln
Ile Asn Thr Cys Leu Lys Thr Ser Met Leu Asp Asn Ile Ala 260 265
270Ser Phe Leu Thr Lys Phe Phe Ile Lys Lys Leu 275
2805300PRTArtificial SequenceDescription of Artificial Sequence
Campylobacter jejuni alpha-1,4-galactosyltransferase (Campylobacter
glycosyltransferase D, CgtD) consensus sequence 5Met Lys Gln Glu
Ile Ser Ser Phe Trp Tyr Thr Pro Arg Gly Tyr Lys1 5 10 15Gly Ile Gly
Leu Met Glu Leu Leu Ser Ile Lys Ser Phe Ile Asp Asn 20 25 30Gly Tyr
Lys Phe Ile Leu Tyr Thr Tyr Asn Leu Asp Asp Lys Ile Phe 35 40 45Lys
Lys Leu Asp Glu Leu Phe Asp Asp Phe Glu Leu Lys Asp Ala Asn 50 55
60Glu Ile Val Ser Phe Lys Asn Tyr Phe Arg Asp Asp Arg Gly Ser Gly65
70 75 80Val Ala Ala Phe Ser Asp Tyr Phe Arg Tyr Asn Leu Leu Tyr Leu
Lys 85 90 95Lys Lys Lys Arg Gly Gly Val Trp Val Asp Leu Asp Met Ile
Cys Leu 100 105 110Asn Tyr Ile Asp Leu Asn Lys Glu Glu Tyr Ile Phe
Thr Gln Glu Val 115 120 125Asp Glu Asp Asn Lys Lys Ser Arg Ile Thr
Thr Ser Phe Leu Lys Phe 130 135 140Ser Arg Tyr Ser Asp Phe Gly Lys
Asn Leu Ile Gln Glu Ala Glu Lys145 150 155 160Ile Ile Asn Lys Arg
Lys Lys Ile Ser Trp Gly Val Ile Gly Pro Trp 165 170 175Phe Leu Ala
Asp His Val Lys Lys Cys Gly Leu Glu Asn Phe Val Trp 180 185 190Asp
Tyr Lys Arg Thr Cys Gln Ile Pro Trp Cys Asn Val Lys Ile Phe 195 200
205Leu Asp Asn Thr Ser Ile Asp Ile Ser Gln Pro Phe Leu His Leu Phe
210 215 220Ser Glu Met Trp Arg Leu Asn Asn Met Glu Lys Asn Thr Phe
His Gln225 230 235 240Met Gly Val Tyr Gly Gln Leu Leu Lys Lys His
Glu Ile Glu Lys Leu 245 250 255Tyr Asn Gln Ile Asn Thr Cys Leu Lys
Thr Ser Asp Lys Met Leu Asp 260 265 270Asn Ile Ala Ser Phe Leu Asn
Ile Lys Asn Lys Thr Lys Phe Phe Ile 275 280 285Lys Lys Leu Lys Lys
Ile Phe Lys Lys Asn Asn Ala 290 295 30068PRTArtificial
SequenceDescription of Artificial Sequence"FLAG tag" epitope tag
6Asp Tyr Lys Asp Asp Asp Asp Lys1 576PRTArtificial
SequenceDescription of Artificial Sequence hexahistidine affinity
tag, polyhistidine epitope tag, purification tag, six adjacent
histidines 7His His His His His His1 5837DNAArtificial
SequenceDescription of Artificial SequencePCR amplification primer
CJ-636 8taaaaggcta catatgactg aaatttcaag tttttgg 37934DNAArtificial
SequenceDescription of Artificial SequencePCR amplification primer
CJ-639 9ggcaagatga ttgtcgactt aggcattgtt tttc 341025DNAArtificial
SequenceDescription of Artificial SequencePCR amplification primer
CJ42, primer in heptosylTase-II 10gccattaccg tatcgcctaa ccagg
251125DNAArtificial SequenceDescription of Artificial SequencePCR
amplification primer CJ43, primer in heptosylTase-I 11aaagaatacg
aatttgctaa agagg 25125PRTArtificial SequenceDescription of
Artificial SequenceDDDDK EC5 purification tag, epitope tag 12Asp
Asp Asp Asp Lys1 5136PRTArtificial SequenceDescription of
Artificial Sequencepurification tag, epitope tag peptide derived
from Polyoma middle T protein 13Glu Tyr Met Pro Met Glu1 5
* * * * *
References