U.S. patent application number 12/955611 was filed with the patent office on 2011-06-16 for compositions and methods for humanization and optimization of n-glycans in plants.
This patent application is currently assigned to BIOLEX THERAPEUTICS, INC.. Invention is credited to KEVIN M. COX, LYNN F. DICKEY, CHARLES G. PEELE.
Application Number | 20110144308 12/955611 |
Document ID | / |
Family ID | 38288381 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110144308 |
Kind Code |
A1 |
DICKEY; LYNN F. ; et
al. |
June 16, 2011 |
COMPOSITIONS AND METHODS FOR HUMANIZATION AND OPTIMIZATION OF
N-GLYCANS IN PLANTS
Abstract
Methods for altering the N-glycosylation pattern of proteins in
higher plants are provided. The methods comprise introducing into
the plant a recombinant construct that provides for the inhibition
of expression of .alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT) in a plant. Use of these
constructs to inhibit or suppress expression of both of these
enzymes, and isoforms thereof, advantageously provides for the
production of endogenous and heterologous proteins having a
"humanized" N-glycosylation pattern without impacting plant growth
and development. Stably transformed higher plants having this
protein N-glycosylation pattern are provided. Glycoprotein
compositions, including monoclonal antibody compositions, having
substantially homogeneous glycosylation profiles, and which are
substantially homogeneous for the G0 glycoform, are also
provided.
Inventors: |
DICKEY; LYNN F.; (CARY,
NC) ; COX; KEVIN M.; (RALEIGH, NC) ; PEELE;
CHARLES G.; (APEX, NC) |
Assignee: |
BIOLEX THERAPEUTICS, INC.
PITTSBORO
NC
|
Family ID: |
38288381 |
Appl. No.: |
12/955611 |
Filed: |
November 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11624164 |
Jan 17, 2007 |
7884264 |
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12955611 |
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60759298 |
Jan 17, 2006 |
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60790373 |
Apr 7, 2006 |
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60791178 |
Apr 11, 2006 |
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60812702 |
Jun 9, 2006 |
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60836998 |
Aug 11, 2006 |
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60860358 |
Nov 21, 2006 |
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Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
C07K 2317/13 20130101;
C07K 16/00 20130101; C07K 2317/732 20130101; C12N 15/8258 20130101;
A61P 35/00 20180101; C07K 2317/41 20130101; C07K 2317/734 20130101;
C07K 14/47 20130101; A61P 37/00 20180101; C07K 2317/72 20130101;
C07K 2317/71 20130101; C12N 15/8257 20130101; A61P 29/00
20180101 |
Class at
Publication: |
530/387.3 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. A composition comprising a glycoprotein produced by a duckweed
plant or duckweed plant cell or nodule that expresses glycoproteins
having an altered N-glycosylation pattern, wherein said altered
N-glycosylation pattern is characterized by a reduction in the
attachment of .alpha.1,3-fucose residues, .beta.1,2-xylose
residues, or both .alpha.1,3-fucose residues and .beta.1,2-xylose
residues to N-glycans of said glycoproteins.
2. The composition of claim 1, wherein said N-glycans are devoid of
said fucose and xylose residues.
3. The composition of claim 2, wherein said glycoprotein has an
N-glycosylation profile characterized by the presence of a single
predominant peak corresponding to the G0 glycan species.
4. The composition of claim 3, wherein said glycoprotein is a
monoclonal antibody of interest.
5. A composition comprising a heterologous glycoprotein that has
been produced in a higher plant, wherein said higher plant
comprises a nucleotide construct comprising a fusion polynucleotide
that is capable of inhibiting expression or function of an
.alpha.1,3-fucosyltransferase (FucT) and a
.beta.1,2-xylosyltransferase (XylT) in said plant, wherein said
fusion polynucleotide is operably linked to a promoter that is
functional in a plant cell.
6. The composition of claim 5, wherein said higher plant is a
member of the Lemnaceae.
7. The composition of claim 5, wherein said fusion polynucleotide
comprises in the 5'-to-3' orientation and operably linked: (a) a
chimeric forward fragment, said chimeric forward fragment
comprising: (i) a first fragment comprising about 500 to about 650
contiguous nucleotides having at least 90% sequence identity to a
nucleotide sequence of about 500 to about 650 contiguous
nucleotides of a polynucleotide encoding said FucT; and (ii) a
second fragment comprising about 500 to about 650 contiguous
nucleotides having at least 90% sequence identity to a nucleotide
sequence of about 500 to about 650 contiguous nucleotides of a
polynucleotide encoding said XylT; (b) a spacer sequence comprising
about 200 to about 700 nucleotides; (c) and a reverse fragment,
said reverse fragment having sufficient length and sufficient
complementarity to said chimeric forward fragment such that said
fusion polynucleotide is transcribed as an RNA molecule capable of
forming a hairpin RNA structure.
8. The composition of claim 5, wherein said glycoprotein has an
N-glycosylation profile characterized by the presence of a single
predominant peak corresponding to the G0 glycan species.
9. The composition of claim 8, wherein said glycoprotein is a
monoclonal antibody of interest.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/624,164, filed Jan. 17, 2007, which claims the benefit of
U.S. Application Ser. No. 60/759,298, filed Jan. 17, 2006, U.S.
Application Ser. No. 60/790,373, filed Apr. 7, 2006, U.S.
Application Ser. No. 60/791,178, filed Apr. 11, 2006, U.S.
Application Ser. No. 60/812,702, filed Jun. 9, 2006, U.S.
Application Ser. No. 60/836,998, filed Aug. 11, 2006, and U.S.
Application Ser. No. 60/860,358, filed Nov. 21, 2006, all of which
are herein incorporated by reference in their entirety.
THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
[0002] A joint research agreement was executed on Mar. 4, 2005, and
Mar. 10, 2005, for ddRNAi technology in cells, tissues, or whole
plants of the family Lemnaceae. The names of the parties executing
the joint research agreement are Biolex, Inc., now Biolex
Therapeutics, Inc., having its registered address at 158 Credle
Street, Pittsboro, N.C. 27312 USA, and Commonwealth Scientific and
Industrial Research Organisation ABN 41 687 119 230, a body
corporate established under the Science and Industry Research Act
1949, of Limestone Avenue, Campbell in the Australian Capital
Territory, Australia.
FIELD OF THE INVENTION
[0003] The present invention is related to the field of plant
molecular biology, more particularly to the field of recombinant
mammalian protein production in plants.
BACKGROUND OF THE INVENTION
[0004] A number of plant species have been targeted for use in
"molecular farming" of mammalian proteins of pharmaceutical
interest. These plant expression systems provide for low cost
production of biologically active mammalian proteins and are
readily amenable to rapid and economical scale-up (Ma et al. (2003)
Nat. Rev. Genet. 4:794-805; Raskin et al. (2002) Trends Biotechnol.
20:522-531). Large numbers of mammalian and plant proteins require
post-translational processing for proper folding, assembly, and
function. Of these modifications, the differences in glycosylation
patterns between plants and mammals offer a challenge to the
feasibility of plant expression systems to produce high quality
recombinant mammalian proteins for pharmaceutical use.
[0005] As peptides move through the endoplasmic reticulum (ER) and
Golgi subcellular compartments, sugar residue chains, or glycans,
are attached, ultimately leading to the formation of glycoproteins.
The linkage between the sugar chains and the peptide occurs by
formation of a chemical bond to only one of four protein amino
acids: asparagine, serine, threonine, and hydroxlysine. Based on
this linkage pattern, two basic types of sugar residue chains in
glycoproteins have been recognized: the N-glycoside-linked sugar
chain (also referred to as N-linked glycan or N-glycan), which
binds to asparagine residues on the peptide; and the
O-glycoside-linked sugar chain, which binds to serine, threonine,
and hydroxylysine residues on the peptide.
[0006] The N-glycoside-linked sugar chains, or N-glycans, have
various structures (see, for example, Takahashi, ed. (1989)
Biochemical Experimentation Method 23--Method for Studying
Glycoprotein Sugar Chain (Gakujutsu Shuppan Center), but share a
common oligomannosidic core (see FIG. 29A). The initial steps in
the glycosylation pathway leading to the formation of N-glycans are
conserved in plants and animals. However, the final steps involved
in complex N-glycan formation differ (Lerouge et al. (1998) Plant
Mo. Biol. 38:31-48; Steinkellner and Strasser (2003) Ann. Plant
Rev. 9:181-192). Plants produce glycoproteins with complex
N-glycans having a core bearing two N-acetylglucosamine (GlcNAc)
residues that is similar to that observed in mammals. However, in
plant glycoproteins this core is substituted by a .beta.1,2-linked
xylose residue (core xylose), which residue does not occur in
humans, Lewis.sup.a epitopes, and an .alpha.1,3-linked fucose (core
.alpha.[1,3]-fucose) instead of an .alpha.1,6-linked core fucose as
in mammals (see, for example, Lerouge et al. (1998) Plant Mol.
Biol. 38:31-48 for a review) (see also FIG. 29B). Both the
.alpha.(1,3)-fucose and .beta.(1,2)-xylose residues reportedly are,
at least partly, responsible for the immunogenicity of plant
glycoproteins in mammals (see, for example, Ree et al. (2000) J.
Biol. Chem. 15:11451-11458; Bardor et al. (2003) Glycobiol.
13:427-434; Garcia-Casado et al. (1996) Glycobiol. 6:471-477).
Therefore removal of these potentially allergenic sugar residues
from mammalian glycoproteins recombinantly produced in plants would
overcome concerns about the use of these proteins as
pharmaceuticals for treatment of humans.
[0007] A number of recombinantly produced glycoproteins currently
serve as therapeutics or are under clinical investigation. Examples
include the interferons (IFNs), erythropoietin (EPO), tissue
plasminogen activator (tPA), antithrombin, granulocyte-macrophage
colony stimulating factor (GM-CSF), and therapeutic monoclonal
antibodies (mABs). The oligosaccharide component of the N-glycan
structures of glycoproteins can influence their therapeutic
efficacy, as well as their physical stability, resistance to
protease attack, pharmacokinetics, interaction with the immune
system, and specific biological activity. See, for example, Jenkins
et al. (1996) Nature Biotechnol. 14:975-981.
[0008] Methods are needed to alter the glycosylation pattern in
plant expression systems, specifically to inhibit plant-specific
glycosylation of the eukaryotic core structure, to advantageously
produce recombinant mammalian proteins with a humanized
glycosylation pattern.
BRIEF SUMMARY OF THE INVENTION
[0009] Methods for altering the N-glycosylation pattern of proteins
in higher plants are provided. The methods comprise stably
transforming the plant with at least one recombinant nucleotide
construct that provides for the inhibition of expression of
.alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT) in a plant. Use of these
constructs to inhibit or suppress expression of one or both of
these enzymes, and isoforms thereof, advantageously provides for
the production of endogenous and heterologous proteins having a
"humanized" N-glycosylation pattern without impacting plant growth
and development. Stably transformed higher plants having this
protein N-glycosylation pattern are provided. In some embodiments,
the plant is a crop plant that is a member of the dicots, such as
pea, alfalfa, and tobacco; in other embodiments, the plant is a
crop plant that is a monocot, such as rice or maize. In yet other
embodiments, the plant is a member of the Lemnaceae family, for
example, a Lemna sp.
[0010] The transgenic plants of the invention have the ability to
produce recombinant mammalian proteins having an N-glycosylation
pattern that is more similar to that of the mammalian host. Thus,
in some embodiments, the recombinantly produced mammalian proteins
are glycoproteins comprising complex N-glycans having a reduction
in the attachment of the plant-specific .alpha.(1,3)-fucose and
.beta.(1,2)-xylose residues. In other embodiments, these
recombinantly produced glycoproteins comprise complex N-glycans
that are devoid of these plant-specific residues. In yet other
embodiments, these recombinantly produced glycoproteins have
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 as the single glycan species
attached to the asparagine glycosylation site(s) within the
glycoprotein.
[0011] In some embodiments, the recombinantly produced glycoprotein
is a monoclonal antibody. In this manner, the present invention
provides transgenic plants that are capable of producing
glycan-optimized monoclonal antibodies that specifically bind a
target protein of interest, including monoclonal antibodies having
increased effector function. Thus, in some embodiments, the
recombinantly produced glycan-optimized monoclonal antibodies
comprise complex N-glycans that have a reduction in the attachment
of .alpha.(1,3)-linked fucose residues, thereby increasing ADCC
activity of these antibodies. In other embodiments, the
recombinantly produced monoclonal antibodies comprise complex
N-linked glycans that are devoid of these plant-specific fucose
residues. In this manner, the present invention provides for the
production of a monoclonal antibody composition, wherein at least
90% or more of the intact antibody is represented by a single
glycoform, more particularly, the G0 glycoform. Thus, in some
embodiments of the invention, the recombinantly produced monoclonal
antibodies have increased effector function, wherein the ADCC
activity is increased and/or the ratio of ADCC/CDC activity is
increased. In some of these embodiments, the recombinantly produced
monoclonal antibodies have decreased CDC activity, which can
advantageously reduce the potential for adverse side effects
related to CDC activation upon administration. These
glycan-optimized monoclonal antibodies advantageously can be used
to alter current routes of administration and current therapeutic
regimens, as their increased effector function means they can be
dosed at lower concentrations and with less frequency, thereby
reducing the potential for antibody toxicity and/or development of
antibody tolerance. Furthermore, their improved effector function
yields new approaches to treating clinical indications that have
been previously been resistant or refractory to treatment with the
corresponding monoclonal antibody produced in other recombinant
host systems.
[0012] Compositions for practicing the methods of the invention are
provided. The compositions comprise novel isolated polynucleotides
and polypeptides encoding a Lemna minor
.alpha.1,3-fucosyltransferase and .beta.1,2-xylosyltransferase, and
variants and fragments thereof. Recombinant nucleotide constructs
that target expression of these two proteins, or expression of
variants thereof, are also provided, as are plant cells, plant
tissues, plants, and seeds comprising these recombinant
constructs.
BRIEF DESCRIPTION OF THE FORMAL DRAWINGS
[0013] FIG. 1 sets forth the DNA (SEQ ID NO:1; coding sequence set
forth in SEQ ID NO:2) and amino acid (SEQ ID NO:3) sequences for
the Lemna minor .alpha.1,3-fucosyltransferase (FucT). The coding
sequence is shown in bold. Nucleotides denoted by the single
underline (-) correspond to the FucT forward fragment within the
RNAi expression cassette designed to inhibit expression of FucT
(see FIG. 5); nucleotides denoted by the double underline (=)
correspond to the spacer sequence within this RNAi expression
cassette. The FucT reverse fragment of the RNAi expression cassette
is the antisense of the FucT forward fragment shown here.
[0014] FIG. 2 sets forth an alignment of the Lemna minor FucT of
SEQ ID NO:3 with .alpha.1,3-fucosyltransferases from Triticum
aestivum (set forth in SEQ ID NO: 30), Hordeum vulgare (set forth
in SEQ ID NO: 31), Oryza sativa (set forth in SEQ ID NO: 32),
Medicago sativa (set forth in SEQ ID NO: 33), and Arabidopsis
thaliana (set forth in SEQ ID NO: 34). The consensus sequence of
the six sequences is set forth in SEQ ID NO: 35.
[0015] FIG. 3 sets forth the DNA (SEQ ID NO:4; coding sequence set
forth in SEQ ID NO:5) sequence for the Lemna minor
.beta.1,2-xylosyltransferase (XylT) isoform #1 and the encoded
amino acid (SEQ ID NO:6) sequence. Nucleotides denoted by the
single underline (-) correspond to the XylT forward fragment within
the RNAi expression cassette designed to inhibit expression of XylT
(see FIG. 6); nucleotides denoted by the double underline (=)
correspond to the spacer sequence within this RNAi expression
cassette. The XylT reverse fragment of the RNAi expression cassette
is the antisense of the XylT forward fragment shown here.
[0016] FIG. 4 sets forth an alignment of the Lemna minor XylT of
SEQ ID NO:6 with .beta.1,2-xylosyltransferases from from Medicago
sativa (set forth in SEQ ID NO: 36), Oryza sativa (set forth in SEQ
ID NO: 37), Arabidopsis thaliana (set forth in SEQ ID NO: 38),
Saccharum officinarum (set forth in SEQ ID NO: 39), and Nicotiana
tabacum (set forth in SEQ ID NO: 40). The consensus sequence of the
six sequences is set forth in SEQ ID NO: 41.
[0017] FIG. 5 sets forth one strategy for designing a single-gene
RNAi knockout of Lemna minor FucT.
[0018] FIG. 6 sets forth one strategy for designing a single-gene
RNAi knockout of Lemna minor XylT based on the DNA sequence for
XylT isoform #1.
[0019] FIG. 7 sets forth one strategy for designing a double-gene
RNAi knockout of Lemna minor FucT and XylT where the XylT portion
of the RNAi knockout is based on the DNA sequence for XylT isoform
#1.
[0020] FIG. 8 shows the Fuc02 construct comprising an RNAi
expression cassette designed for single-gene RNAi knockout of Lemna
minor FucT. Expression of the FucT inhibitory sequence (denoted by
FucT forward and FucT reverse arrows; see FIG. 5) is driven by an
operably linked expression control element (denoted as
AocsAocsAocsAmasPmas) comprising three upstream activating
sequences (Aocs) derived from the Agrobacterium tumefaciens
octopine synthase gene operably linked to a promoter derived from
an Agrobacterium tumefaciens mannopine synthase gene (AmasPmas).
RbcS leader, rubisco small subunit leader sequence; ADH1, intron of
maize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium
tumefacians nopaline synthetase (nos) terminator sequence.
[0021] FIG. 9 shows the Xyl02 construct comprising an RNAi
expression cassette designed for single-gene RNAi knockout of Lemna
minor XylT. Expression of the XylT inhibitory sequence (denoted by
XylT forward and XylT reverse arrows; see FIG. 6) is driven by the
operably linked AocsAocsAocsAmasPmas expression control element.
RbcS leader, rubisco small subunit leader sequence; ADH1, intron of
maize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium
tumefacians nopaline synthetase (nos) terminator sequence.
[0022] FIG. 10 shows the XF02 construct comprising a chimeric RNAi
expression cassette designed for double-gene RNAi knockout of Lemna
minor FucT/XylT. The hairpin RNA is expressed as a chimeric
sequence (a chimeric hairpin RNA), where fragments of the two genes
are fused together and expressed as one transcript. Expression of
the FucT/XylT inhibitory sequence (denoted by FucT and XylT forward
arrows and XylT and FucT reverse arrows; see FIG. 7) is driven by
the operably linked AocsAocsAocsAmasPmas expression control
element. RbcS leader, rubisco small subunit leader sequence; ADH1,
intron of maize alcohol dehydrogenase 1 gene; nos-ter,
Agrobacterium tumefacians nopaline synthetase (nos) terminator
sequence.
[0023] FIG. 11 shows the XF03 construct comprising an RNAi
expression cassette designed for double-gene RNAi knockout of Lemna
minor FucT/XylT. The cassette expresses two RNAi hairpins, one
targeting expression of FucT, the other targeting expression of
XylT. Expression of the FucT inhibitory sequence (denoted by FucT
forward and FucT reverse arrows; see FIG. 5) is driven by an
operably linked expression control element comprising the Lemna
minor ubiquitin promoter plus 5' UTR (LmUbq promoter) and intron
(LmUbq intron) (see SEQ ID NO:7). Expression of the XylT inhibitory
sequence (denoted by XylT forward and XylT reverse arrows; see FIG.
6) is driven by the operably linked AocsAocsAocsAmasPmas expression
control element. RbcS leader, rubisco small subunit leader
sequence; ADH1, intron of maize alcohol dehydrogenase 1 gene;
nos-ter, Agrobacterium tumefacians nopaline synthetase (nos)
terminator sequence.
[0024] FIG. 12 shows the mAbI04 construct that provides for
co-expression of an IgG1 monoclonal antibody (referred to herein as
mAbI) and the double-gene knockout of Lemna minor FucT and XylT,
wherein a chimeric hairpin RNA targeting expression of the FucT and
XylT is expressed. Expression of the FucT/XylT inhibitory sequence
(denoted by FucT and XylT forward arrows and XylT and FucT reverse
arrows; see FIG. 7) is driven by an operably linked expression
control element comprising the Spirodella polyrrhiza ubiquitin
promoter plus 5' UTR (SpUbq promoter) and intron (SpUbq intron)
(see SEQ ID NO: 8). Expression of the IgG1 light chain is driven by
an operably linked expression control element comprising the L.
minor ubiquitin promoter plus 5' UTR (LmUbq promoter) and intron
(LmUbq intron). Expression of the IgG1 heavy chain is driven by the
operably linked AocsAocsAocsAmasPmas expression control element.
RbcS leader, rubisco small subunit leader sequence; ADH1, intron of
maize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium
tumefacians nopaline synthetase (nos) terminator sequence.
[0025] FIG. 13 shows the mAbI05 construct that provides for
co-expression of mAbI and the double-knockout of Lemna minor FucT
and XylT, wherein two hairpin RNAs are expressed, one targeting
expression of FucT, the other targeting expression of the XylT.
Expression of the FucT inhibitory sequence (denoted by FucT forward
and FucT reverse arrows; see FIG. 5) is driven by an operably
linked expression control element comprising the S. polyrrhiza
ubiquitin promoter plus 5' UTR (SpUbq promoter) and intron (SpUbq
intron). Expression of the XylT inhibitory sequence (denoted by
XylT forward and XylT reverse arrows; see FIG. 6) is driven by an
operably linked expression control element comprising the Lemna
aequinoctialis ubiquitin promoter plus 5' UTR (LaUbq promoter) and
intron (LaUbq intron) (see SEQ ID NO:9). Expression of the IgG1
light chain is driven by an operably linked expression control
element comprising the L. minor ubiquitin promoter plus 5' UTR
(LmUbq promoter) and intron (LmUbq intron). Expression of the IgG1
heavy chain is driven by the operably linked AocsAocsAocsAmasPmas
expression control element. RbcS leader, rubisco small subunit
leader sequence; ADH1, intron of maize alcohol dehydrogenase 1
gene; nos-ter, Agrobacterium tumefacians nopaline synthetase (nos)
terminator sequence.
[0026] FIG. 14 shows the mAbI01 construct that provides for
expression of mAbI, where FucT and XylT expression are not
suppressed. Expression of the IgG1 light chain and IgG1 heavy chain
are independently driven by the operably linked
AocsAocsAocsAmasPmas expression control element. RbcS leader,
rubisco small subunit leader sequence; ADH1, intron of maize
alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium tumefacians
nopaline synthetase (nos) terminator sequence. mAbI01 is referred
to as the "wild-type" mAbI01 construct as the expressed mAbI
exhibits the glycosylation profile of wild-type L. minor.
[0027] FIGS. 15 and 16 show the primary screening data for
transgenic RNAi L. minor plant lines comprising the XF02 construct
of FIG. 10.
[0028] FIG. 17 shows primary screening data for transgenic RNAi L.
minor plant lines comprising the mAbI04 construct of FIG. 12 and
mAbI05 construct of FIG. 13.
[0029] FIG. 18 shows the structure and molecular weight of
derivatized wild-type L. minor mAb N-glycans. "GnGn" represents the
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan species, also referred
to as a G0 N-glycan species. "GnGnX" represents the
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan species with the
plant-specific .beta.(1,2)-xylose residue attached. "GnGnXF"
represents the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan species
with the plant-specific .beta.(1,2)-xylose residue and
plant-specific .alpha.(1,3)-fucose residue attached.
[0030] FIG. 19 shows that the wild-type mAbI01 construct (shown in
FIG. 15) providing for expression of the mAbI monoclonal IgG1
antibody in L. minor, without RNAi suppression of L. minor FucT and
XylT, produces an N-glycosylation profile with three major N-glycan
species, including one species having the .beta.1,2-linked xylose
and one species having both the .beta.1,2-linked xylose and core
.alpha.1,3-linked fucose residues; this profile is confirmed with
liquid chromatography mass spectrometry (LC-MS) (FIG. 20) and MALDI
(FIG. 21) analysis.
[0031] FIG. 22 shows an overlay of the relative amounts of the
various N-glycan species of mAbI produced in the wild-type L. minor
line comprising the mAbI01 construct (no suppression of FucT or
XylT) and in the two transgenic L. minor lines comprising the
mAbI04 construct of FIG. 12 (providing for coexpression of mAbI and
the chimeric RNAi construct targeting both L. minor FucT and XlT).
Note the enrichment of the GnGn (i.e., G0) glycan species, with no
.beta.1,2-linked xylose or core .alpha.1,3-linked fucose residues
attached, and the absence of the species having the
.beta.1,2-linked xylose or both the .beta.1,2-linked xylose and
core .alpha.1,3-linked fucose residues. "MGn" represents an
N-glycan precursor, wherein the trimannose core structure,
Man.sub.3GlcNAc.sub.2, has one N-acetylglucosamine attached to the
1,3 mannose arm. "GnM" represents an N-glycan precursor, wherein
the trimannose core structure, Man.sub.3GlcNAc.sub.2, has one
N-acetylglucosamine attached to the 1,6 mannose arm. These N-glycan
precursors represent a trace amount of the total N-glycans present
in the sample. This profile is confirmed with mass spec (LC-MS)
(FIG. 23) and MALDI (FIG. 24) analysis.
[0032] FIG. 25 shows intact mass analysis of mAbI compositions
produced in wild-type L. minor (line 20) comprising the mAbI01
construct. When XylT and FucT expression are not suppressed in L.
minor, the recombinantly produced mAbI composition is
heterogeneous, comprising at least 9 different glycoforms, with the
G0XF.sup.3 glycoform being the predominate species present. Note
the very minor peak representing the G0 glycoform.
[0033] FIG. 26 shows intact mass analysis of mAbI compositions
produced in transgenic L. minor (line 15) comprising the mAbI04
construct of FIG. 12. When XylT and FucT expression are suppressed
in L. minor using this chimeric RNAi construct, the intact mAbI
composition is substantially homogeneous for G0 N-glycans, with
only trace amounts of precursor N-glycans present (represented by
the GnM and MGn precursor glycan species). In addition, the mAbI
composition is substantially homogeneous for the G0 glycoform,
wherein both glycosylation sites are occupied by the G0 N-glycan
species, with three minor peaks reflecting trace amounts of
precursor glycoforms (one peak showing mAbI having an Fc region
wherein the C.sub.H2 domain of one heavy chain has a G0 glycan
species attached to Asn 297, and the C.sub.H2 domain of the other
heavy chain is unglycosylated; another peak showing mAbI having an
Fc region wherein the C.sub.H2 domain of one heavy chain has a G0
glycan species attached to Asn 297, and the C.sub.H2 domain of the
other heavy chain has the GnM or MGn precursor glycan attached to
Asn 297; and another peak showing mAbI having an Fc region wherein
the Asn 297 glycosylation site on each of the C.sub.H2 domains has
a G0 glycan species attached, with a third G0 glycan species
attached to an additional glycosylation site within the mAbI
structure).
[0034] FIG. 27 shows intact mass analysis of the mAbI compositions
produced in transgenic L. minor (line 72) comprising the mAbI05
construct of FIG. 13. When XylT and FucT expression are suppressed
in L. minor using this construct, the intact mAbI composition is
substantially homogeneous for G0 N-glycans, with only trace amounts
of precursor N-glycan species present (represented by the GnM and
MGn precursor glycan species). In addition, the mAbI composition is
substantially homogeneous (at least 90%) for the G0 glycoform, with
the same three minor peaks reflecting precursor glycoforms as
obtained with the mAbI04 construct.
[0035] FIG. 28 summarizes two possible designs for targeting
expression of individual FucT and XylT genes.
[0036] FIG. 29A shows the common oligomannosidic core structure of
complex N-glycans of glycoproteins produced in plants and animals.
In mammals, the core structure can include a fucose residue in
which 1-position of the fucose is bound to 6-position of the
N-acetylgucosamine in the reducing end through an .alpha. bond
(i.e., .alpha.(1,6)-linked fucose). FIG. 29B shows the
plant-specific modifications to these N-glycans. The mammalian R
groups can be one of the following: (a) R=GlcNAc.beta.(1,2); (b)
R=Gal.beta.(1,4)-GlcNAc.beta.(1,2); (c)
R=NeuAc.alpha.(2,3)-Gal.beta.(1,4)-GlcNAc.beta.(1,2); (d)
R=NeuGc.alpha.(2,3)-Gal.beta.(1,4)-GlcNAc.beta.(1,2); and (e)
R=Gal.alpha.(1,3)-Gal.beta.(1,4)-GlcNAc.beta.(1,2). The plant R
groups can be one of the following: (a) R=null; (b)
R=GlcNAc.beta.(1,2);
##STR00001##
Abbreviations: Man, mannose; GlcNAc, N-acetylglucosamine; Xyl,
xylose; Fuc, fucose; Gal, galactose; NeuAc (neuraminic acid (sialic
acid); *, reducing end of sugar chain that binds to asparagine.
[0037] FIG. 30 shows the G0, G0X, and G0XF.sup.3 species of
N-linked glycans of glycoproteins referred to in the description
and claims of the present invention, along with the alternate
nomenclature used herein.
[0038] FIG. 31 sets forth the partial cDNA (SEQ ID NO:19; coding
sequence set forth in SEQ ID NO:20) sequence for the Lemna minor
.beta.1,2-xylosyltransferase (XylT) isoform #2, and partial amino
acid (SEQ ID NO:21) sequence encoded thereby. Nucleotides denoted
by the single underline (-) correspond to the XylT forward fragment
within the RNAi expression cassette designed to inhibit expression
of XylT (see FIG. 33); nucleotides denoted by the double underline
(=) correspond to the spacer sequence within this RNAi expression
cassette. The XylT reverse fragment of the RNAi expression cassette
is the antisense of the XylT forward fragment shown here.
[0039] FIG. 32 sets forth an alignment of the Lemna minor XylT
isoform #1 of SEQ ID NO:6 with the Lemna minor partial-length XylT
isoform #2 of SEQ ID NO:21.
[0040] FIG. 33 sets forth one strategy for designing a single-gene
RNAi knockout of Lemna minor XylT based on the partial DNA sequence
for XylT isoform #2.
[0041] FIG. 34 sets forth one strategy for designing a double-gene
RNAi knockout of Lemna minor FucT and XylT, where the XylT portion
of the RNAi knockout is based on the partial DNA sequence for XylT
isoform #2.
[0042] FIG. 35 shows receptor binding activity of the CHO-derived
and SP2/0-derived mAbI product for the Fc.gamma.RIIIa on freshly
isolated human NK cells.
[0043] FIG. 36 shows receptor binding activity of the wild-type
Lemna-derived mAbI product and the transgenic Lemna-derived mAbI
product for the Fc.gamma.RIIIa on freshly isolated human NK cells
collected from Donor 1.
[0044] FIG. 37 shows receptor binding activity of the wild-type
Lemna-derived mAbI product and the transgenic Lemna-derived mAbI
product for the Fc.gamma.RIIIa on freshly isolated human NK cells
collected from Donors 2 and 3.
[0045] FIG. 38 shows receptor binding activity of the Sp2/0-derived
mAbI product, the wild-type Lemna-derived mAbI product, and the
transgenic Lemna-derived mAbI product for the mouse
Fc.gamma.RIV.
[0046] FIG. 39 shows a diagram of the MDXA04 binary expression
vector for RNAi silencing of FucT and XylT activity in Lemna.
Hatched regions show the position of the heavy (H) and light (L)
chain variable region gene sequences of fully human mAb1 kappa
antibody MDX-060 and the chimeric hairpin RNA (RNAi) designed to
target silencing of endogenous Lemna genes encoding FucT and XylT.
Promoters: P1, P2, and P3; terminator: T; selectable marker: SM;
left border: LB; right border, RB. The MDXA01 expression vector
used to express the MDX-060 mAb in wild-type Lemna did not contain
the hairpin RNA region.
[0047] FIG. 40 shows glycosyltransferase activity in Lemna
wild-type and MDX-060 LEX.sup.Opt RNAi lines. Microsomal membranes
from wild-type (WT) and MDX-060 LEX.sup.Opt RNAi (line numbers are
indicated) plants were incubated in the presence of a reaction
buffer containing GDP-Fuc, UDP-Xyl and GnGn-dabsyl-peptide
acceptor. Mass peaks corresponding to fucosylated (white bars) or
xylosylated (black bars) products synthesized by microsomes from
each line were measured by positive reflectron mode MALDI-TOF MS
and normalized, in percent, to the WT positive control. Boiled
wildtype membranes (BWT) indicate background ion counts.
[0048] FIG. 41 shows SDS-PAGE of plant extracts and protein A or
hydroxyapatite purified samples from MDX-060 LEX.sup.Opt under
non-reducing (FIG. 41A) and reducing (FIG. 41B) conditions,
respectively. MAb purified from a CHO cell line (MDX-060 CHO) was
used as a positive control. Mark12 molecular weight markers were
included on the gels. Gels were stained with Colloidal Blue.
[0049] FIG. 42 shows the spectra obtained from negative, reflectron
mode MALDI-TOF mass spectrometric analysis of 2-AA labeled
N-glycans released from MDX-060 mAbs expressed in CHO (MDX-060
CHO), wild-type Lemna (MDX-060 LEX), or Lemna transformed with the
XylT/FucT RNAi construct (MDX-060 LEX.sup.Opt). Significant peaks
are identified by the corresponding mass ([M-H].sup.-). The *
indicates the location of matrix artifacts.
[0050] FIG. 43 shows the spectra obtained from NP-HPLC-QTOF MS
analysis of 2-AA labeled N-glycans released from MDX-060 mAbs
expressed in CHO (MDX-060 CHO), wild-type Lemna (MDX-060 LEX), or
Lemna transformed with the XylT/FucT RNAi construct (MDX-060
LEX.sup.Opt). 2-AA labeled N-glycans were separated by normal phase
chromatography and detected by fluorescence. The most abundant
peaks from each sample (labeled a-i) were characterized by on-line
negative mode QTOF MS and their corresponding QTOF mass spectra
([M-2H].sup.2-) are shown.
[0051] FIG. 44 shows in vitro activity of MDX-060 mAbs as measured
by flow cytometric analysis of MDX-060 CHO, LEX, or glyco-optimized
LEX.sup.Opt mAb binding to CD30 expressed on L540 cells. L540 cells
were incubated with increasing concentrations of the indicated
antibody as outlined in Example 6 herein below. Geo Mean
Fluorescence Intensity (GMFI) is plotted against the various
concentrations of mAb used. .box-solid.: MDX-060 CHO;
.tangle-solidup.: MDX-060 LEX; : MDX-060 LEX.sup.opt.
[0052] FIG. 45 shows equilibrium binding of glyco-optimized and
wild-type mAb to two different human FcR.gamma.IIIa allotypes
(Val.sup.158 or Phe.sup.158). The binding signal as a function of
FcR.gamma.IIIa was fit to a one-site binding model. .box-solid.:
MDX-060 CHO; .tangle-solidup.: MDX-060 LEX; : MDX-060
LEX.sup.opt.
[0053] FIG. 46 shows ADCC activity of MDX-060 mAb derived from CHO,
LEX (wild-type Lemna glycosylation), or LEX.sup.Opt (RNAi
transgenic Lemna). Human effector cells from a
Fc.gamma.RIIIaPhe.sup.158 homozygote donor and a
Fc.gamma.RIIIaPhe/Val.sup.158 heterozygote donor were incubated
with BATDA-labeled L540 cells at an effector:target ratio of 50:1
in the presence of increasing concentrations of the indicated
antibodies. Specific percent lysis at each mAb concentration is
plotted. Human mAb1 not recognizing antigen on L540 cells was used
as an isotype control in all experiments. EC.sub.50 values
(.mu.g/mL), binding constants and maximal percent lysis were
calculated using GraphPad Prism 3.0 software. .box-solid.: MDX-060
CHO; .tangle-solidup.: MDX-060 LEX; : MDX-060 LEX.sup.opt.
[0054] FIG. 47 shows intact mass analysis of the MDX-060 LEX mAb
compositions produced in wild-type L. minor comprising the MDXA01
construct. When XylT and FucT expression are not suppressed in L.
minor, the recombinantly produced MDX-060 LEX mAb composition
comprises at least 7 different glycoforms, with the G0XF.sup.3
glycoform being the predominate species present. Note the absence
of a peak representing the G0 glycoform.
[0055] FIG. 48 shows glycan mass analysis of the heavy chain of the
MDX-060 LEX mAb produced in wild-type L. minor comprising the
MDXA01 construct. When XylT and FucT expression are not suppressed
in L. minor, the predominate N-glycan species present is
G0XF.sup.3, with additional major peaks reflecting the G0X species.
Note the minor presence of the G0 glycan species.
[0056] FIG. 49 shows intact mass analysis of the MDX-060
LEX.sup.Opt mAb compositions produced in transgenic L. minor
comprising the MDXA04 construct. When XylT and FucT expression are
suppressed in L. minor, the intact mAb composition contains only G0
N-glycans. In addition, the composition is substantially
homogeneous for the G0 glycoform (peak 2), wherein both
glycosylation sites are occupied by the G0 N-glycan species, with
two minor peaks reflecting trace amounts of precursor glycoforms
(peak 1, showing mAb having an Fc region wherein the C.sub.H2
domain of one heavy chain has a G0 glycan species attached to Asn
297, and the C.sub.H.sup.2 domain of the other heavy chain is
unglycosylated; and peak 3, showing mAb having an Fc region wherein
the Asn 297 glycosylation site on each of the C.sub.H2 domains has
a G0 glycan species attached, with a third G0 glycan species
attached to an additional glycosylation site within the mAb
structure).
[0057] FIG. 50 shows glycan mass analysis of the heavy chain of the
MDX-060 LEX.sup.Opt mAb produced in transgenic L. minor comprising
the MDXA04 construct. When XylT and FucT expression are suppressed
in L. minor, the only readily detectable N-glycan species attached
to the Asn 297 glycosylation sites of the C.sub.H2 domains of the
heavy chains is G0.
[0058] FIGS. 51A (MALDI analysis) and 51B (HPLC analysis) show that
the homogeneous glycosylation profile exhibited by mAbI produced in
transgenic L. minor (line 24) comprising the mAbI04 RNAi construct
was consistently observed with scaled-up production. This
glycosylation profile was consistent over the 8-month period of
continuous maintenance of the transgenic line via clonal
expansion.
[0059] FIG. 52 shows that suppression of FucT and XylT expression
using the chimeric RNAi mAbI04 construct of FIG. 12 results in
endogenous glycoproteins having a homogeneous glycosylation pattern
consistent with that observed for recombinant glycoproteins. For
this figure, the .beta.1,2-linked xylose residue attached to the
trimannose core structure is designated by the star symbol.
[0060] FIG. 53 shows the structure of complex N-glycans described
in Example 6 below. M=mannose; Gn=N-acetylglucosamine; A=galactose;
X=xylose; F=fucose.
DETAILED DESCRIPTION OF THE INVENTION
[0061] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the inventions are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0062] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0063] The present invention provides compositions and methods for
altering the N-glycosylation pattern of homologous and heterologous
polypeptides produced in a plant, particularly a plant that serves
as an expression system for recombinant proteins of interest. The
methods comprise the use of nucleotide constructs comprising one or
more sequences that are capable of inhibiting expression of
.alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT) in a plant. Compositions for
use in practicing the methods of the invention, and compositions
comprising glycoproteins having a substantially homologous
glycosylation profile, including glycan-optimized monoclonal
antibodies with improved effector function, are also provided.
DEFINITIONS
[0064] "Polypeptide" refers to any monomeric or multimeric protein
or peptide.
[0065] "Biologically active polypeptide" refers to a polypeptide
that has the capability of performing one or more biological
functions or a set of activities normally attributed to the
polypeptide in a biological context. Those skilled in the art will
appreciate that the term "biologically active" includes
polypeptides in which the biological activity is altered as
compared with the native protein (e.g., suppressed or enhanced), as
long as the protein has sufficient activity to be of interest for
use in industrial or chemical processes or as a therapeutic,
vaccine, or diagnostics reagent. Biological activity can be
determined by any method available in the art. For example, the
biological activity of members of the interferon family of proteins
can be determined by any of a number of methods including their
interaction with interferon-specific antibodies, their ability to
increase resistance to viral infection, or their ability to
modulate the transcription of interferon-regulated gene targets. In
like manner, biological activity of monoclonal antibodies can be
determined by any of a number of methods including, but not limited
to, assays for measuring binding specificity and effector function,
for example, using assays for antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
activity.
[0066] By "host cell" is intended a cell that comprises a
heterologous nucleic acid sequence of the invention. Though the
nucleic acid sequences of the invention, and fragments and variants
thereof, can be introduced into any cell of interest, of particular
interest are plant host cells. In some embodiments, the plant host
cells are cells of a plant that serves as a host for expression of
recombinant proteins, for example, a plant expression system for
production of recombinant mammalian proteins of interest as noted
herein below.
[0067] By "heterologous polypeptide of interest" is intended a
polypeptide that is not expressed by the host cells in nature.
Conversely, a "homologous polypeptide" is intended a polypeptide
that is naturally produced within the cells of the host.
Heterologous and homologous polypeptides that undergo
post-translational N-glycosylation are referred to herein as
heterologous or homologous glycoproteins. In accordance with the
methods of the present invention, the N-glycosylation pattern of
both heterologous and homologous glycoproteins is altered within
the cells of a plant host so that these glycoproteins have an
N-glycosylation pattern that is more similar to that observed with
mammalian hosts.
[0068] Exemplary heterologous polypeptide and heterologous
nucleotide sequences of interest include, but are not limited to,
sequences that encode mammalian polypeptides, such as insulin,
growth hormone, .alpha.-interferon, .beta.-interferon,
.beta.-glucocerebrosidase, .beta.-glucoronidase, retinoblastoma
protein, p53 protein, angiostatin, leptin, erythropoietin (EPO),
granulocyte macrophage colony stimulating factor, plasminogen,
tissue plasminogen activator, blood coagulation factors, for
example, Factor VII, Factor VIII, Factor IX, and activated protein
C, alpha 1-antitrypsin, monoclonal antibodies (mAbs), Fab
fragments, single-chain antibodies, cytokines, receptors, hormones,
human vaccines, animal vaccines, peptides, and serum albumin.
[0069] For purposes of the present invention, the terms "N-glycan,"
"N-linked glycan," and "glycan" are used interchangeably and refer
to an N-linked oligosaccharide, e.g., one that is or was attached
by an N-acetylglucosamine (GlcNAc) residue linked to the amide
nitrogen of an asparagine residue in a protein. The predominant
sugars found on glycoproteins are glucose, galactose, mannose,
fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine
(GlcNAc), and sialic acid (e.g., N-acetyl-neuraminic acid (NeuAc)).
The processing of the sugar groups occurs cotranslationally in the
lumen of the ER and continues in the Golgi apparatus for N-linked
glycoproteins.
[0070] By "oligomannosidic core structure" or "trimannose core
structure" of a complex N-glycan is intended the core structure
shown in FIG. 29A, wherein the core comprises three mannose (Man)
and two N-acetylglucosamine (GlcNAc) monosaccharide residues that
are attached to the asparagine residue of the glycoprotein. The
asparagine residue is generally within the conserved peptide
sequence Asn-Xxx-Thr or Asn-Xxx-Ser, where Xxx is any residue
except proline, aspartate, or glutamate. Subsequent glycosylation
steps yield the final complex N-glycan structure. The trimannose
core structure is denoted herein as "Man.sub.3GlcNAc.sub.2."
[0071] The N-glycans attached to glycoproteins differ with respect
to the number of branches (antennae) comprising peripheral sugars
(e.g., GlcNAc, galactose, fucose, and sialic acid) that are added
to the trimannose core structure. N-glycans are commonly classified
according to their branched constituents (e.g., complex, high
mannose, or hybrid). A "complex" type N-glycan typically has at
least one GlcNAc attached to the 1,3 mannose arm and at least one
GlcNAc attached to the 1,6 mannose arm of a "trimannose" core.
Where one GlcNAc is attached to each mannose arm, the species of
N-linked glycan is denoted herein as
"GlcNAc.sub.2Man.sub.3GlcNAc.sub.2" or "GnGn." Where only one
GlcNac is attached, the N-glycan species is denoted herein as
"GlcNAc.sub.1Man.sub.3GlcNAc.sub.2", wherein the GlcNac is attached
to either the 1,3 mannose arm (denoted "MGn" herein) or the 1,6
mannose arm (denoted "GnM" herein) (see FIG. 30). Complex N-glycans
may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc")
sugar residues that are optionally modified with sialic acid or
derivatives (e.g., "NeuAc," where "Neu" refers to neuraminic acid
and "Ac" refers to acetyl). Where a galactose sugar residue is
attached to each GlcNAc on each mannose arm, the species of
N-linked glycan is denoted herein as
"Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2." Complex N-glycans may
also have intrachain substitutions comprising "bisecting" GlcNAc
and core fucose ("Fuc"). Complex N-glycans may also have multiple
antennae on the "trimannose core," often referred to as "multiple
antennary glycans." A "high mannose" type N-glycan has five or more
mannose residues. A "hybrid" N-glycan has at least one GlcNAc on
the terminal of the 1,3 mannose arm of the trimannose core and zero
or more mannoses on the 1,6 mannose arm of the trimannose core.
[0072] The terms "G0 glycan" and "G0 glycan structure" and "G0
glycan species" are used interchangeably and are intended to mean
the complex N-linked glycan having the
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 structure, wherein no terminal
sialic acids (NeuAcs) or terminal galactose (Gal) sugar residues
are present. If a G0 glycan comprises a fucose ("Fuc") residue
attached to the trimannose core structure, it is referred to herein
as a "G0F.sup.3 glycan" (having the plant-specific
.alpha.1,3-linked fucose residue) or "G0F.sup.6 glycan" (having the
mammalian .alpha.1,6-linked fucose residue). In plants, a G0 glycan
comprising the plant-specific .beta.1,2-linked xylose residue
attached to the trimannose core structure is referred to herein as
a "G0X glycan," and a G0 glycan comprising both the plant-specific
.beta.1,2-linked xylose residue and plant-specific
.alpha.1,3-linked fucose residue attached to the trimannose core
structure is referred to herein as a "G0XF.sup.3 glycan."
[0073] The terms "G1 glycan" and "G1 glycan structure" and "G1
glycan species" are used interchangeably and are intended to mean
the complex N-linked glycan having the
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 structure, wherein one terminal
galactose (Gal) residue is attached to either the 1,3 mannose or
1,6 mannose arm, and no terminal sialic acids are present. The
terms "G2 glycan" and "G2 glycan structure" and "G2 glycan species"
are used interchangeably and are intended to mean the complex
N-linked glycan having the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
structure, wherein a terminal galactose (Gal) residue is attached
to the 1,3 mannose arm and the 1,6 mannose arm, and no terminal
sialic acids are present.
[0074] The term "glycoform" as used herein refers to a glycoprotein
containing a particular carbohydrate structure or structures. Thus,
for example, a "G0 glycoform" refers to a glycoprotein that
comprises only G0 glycan species attached to its glycosylation
sites. It is recognized that a glycoprotein having more than one
glycosylation site can have the same glycan species attached to
each glycosylation site, or can have different glycan species
attached to different glycosylation sites. In this manner,
different patterns of glycan attachment yield different glycoforms
of a glycoprotein.
[0075] The term "glycosylation profile" is intended to mean the
characteristic "fingerprint" of the representative N-glycan species
that have been released from a glycoprotein composition or
glycoprotein product, either enzymatically or chemically, and then
analyzed for their carbohydrate structure, for example, using
LC-HPLC, or MALDI-TOF MS, and the like. See, for example, the
review in Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp.
28-57; herein incorporated by reference in its entirety.
[0076] The terms "substantially homogeneous," "substantially
uniform," and "substantial homogeneity" in the context of a
glycosylation profile for a glycoprotein composition or
glycoprotein product are used interchangeably and are intended to
mean a glycosylation profile wherein at least 80%, at least 85%, at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
of the total N-glycan species within the profile are represented by
one desired N-glycan species, with a trace amount of precursor
N-glycan species appearing in the profile. By "trace amount" is
intended that any given precursor N-glycan species that is present
in the glycosylation profile is present at less than 5%, preferably
less than 4%, less than 3%, less than 2%, less than 1%, and even
less than 0.5% or even less than 0.1% of the total amount of
N-glycan species appearing in the profile. By "precursor" N-glycan
species is intended an N-glycan species that is incompletely
processed. Examples of precursor N-glycan species present in trace
amounts in the glycoprotein compositions or glycoprotein products
of the invention, and thus appearing in the glycosylation profiles
thereof, are the Man3GlcNAc2, MGn (GlcNac1Man3GlcNAc2 wherein
GlcNac1 is attached to the 1,3 mannose arm), and GnM
(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,6 mannose
arm) precursor N-glycan species described above.
[0077] Thus, for example, where the desired N-glycan species within
a glycoprotein product or composition is G0, a substantially
homogeneous glycosylation profile for that product or composition
would be one wherein at least 80%, at least 85%, at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the total
amount of N-glycan species appearing in the glycosylation profile
for the product or composition is represented by the G0 glycan
species, with a trace amount of precursor N-glycan species
appearing in the glycosylation profile. For such a composition, a
representative precursor N-glycan species appearing in its
glycosylation profile would be the Man3GlcNAc2, MGn
(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose
arm), and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to
the 1,6 mannose arm) precursor N-glycan species described
above.
[0078] The terms "substantially homogeneous," "substantially
uniform," and "substantial homogeneity" in the context of a
glycoprotein composition or glycoprotein product are used
interchangeably and are intended to mean the glycoprotein product
or glycoprotein composition wherein at least 80%, at least 85%, at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
of the glycoprotein present in the product or composition is
represented by one desired glycoform, with a trace amount of
precursor or undesired glycoforms being present in the composition.
By "trace amount" is intended that any given precursor or undesired
glycoform that is present in the glycoprotein product or
composition is present at less than 5%, preferably less than 4%,
less than 3%, less than 2%, less than 1%, and even less than 0.5%
or even less than 0.1% of the total glycoprotein. By "precursor"
glycoform is intended a glycoform wherein at least one
glycosylation site is either unglycosylated, or is occupied by an
N-glycan species that represents a precursor of the desired
N-glycan species, or a glycoform wherein one or more additional
glycosylation sites is present, relative to the desired glycoform,
and is occupied by (i.e., has attached thereto) the desired
N-glycan species or an undesired N-glycan species.
[0079] Thus, for example, a substantially homogeneous glycoprotein
composition or product comprising the G0 glycoform is a composition
or product wherein at least 80%, 80%, at least 85%, at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the
glycoprotein present in the product or composition is represented
by the G0 glycoform, wherein all anticipated glycosylation sites
are occupied by the G0 glycan species, with a trace amount of
precursor or undesired glycoforms being present in the composition.
In such a composition, a representative precursor glycoform would
be one in which glycosylation sites are unoccupied, and an
exemplary undesired glycoform would be a glycoform having a mixture
of G0 glycan and G0X or G0XF3 glycan species attached to its
glycosylation sites.
[0080] The term "antibody" is used in the broadest sense and covers
fully assembled antibodies, antibody fragments that can bind
antigen (e.g., Fab', F'(ab).sub.2, Fv, single chain antibodies,
diabodies), and recombinant peptides comprising the foregoing.
Antibodies represent one of the many glycoproteins contemplated by
the methods and compositions of the present invention. Derivatives
of antibodies are also contemplated by the present invention.
Derivatives include fusion proteins comprising an immunoglobulin or
portion thereof, such as an Fc region having a C.sub.H2 domain.
[0081] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts.
[0082] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light- and heavy-chain variable
domains.
[0083] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR) regions. The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al. (1991) NIH Publ. No. 91-3242, Vol. I,
pages 647-669).
[0084] The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effecter functions,
such as Fc receptor (FcR) binding, participation of the antibody in
antibody-dependent cellular cytotoxicity (ADCC), opsonization,
initiation of complement-dependent cytotoxicity (CDC activity), and
mast cell degranulation.
[0085] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et
al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments. Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab')2 fragment that has two antigen-combining sites and
is still capable of cross-linking antigen.
[0086] "Fv" is the minimum antibody fragment that contains a
complete antigen recognition and binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0087] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (C.sub.H1) of the heavy
chain. Fab fragments differ from Fab' fragments by the addition of
a few residues at the carboxy terminus of the heavy-chain C.sub.H1
domain including one or more cysteines from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs
of Fab' fragments that have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0088] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0089] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of human immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA, and IgA2. The heavy-chain constant domains that correspond to
the different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known. Different isotypes have different
effector functions. For example, human IgG1 and IgG3 isotypes
mediate antibody-dependent cell-mediated cytotoxicity (ADCC)
activity.
[0090] Immunoglobulins have conserved N-linked glycosylation of the
Fc region of each of the two heavy chains. Thus, for example,
immunoglobulins of the IgG type have glycosylated C.sub.H2 domains
bearing N-linked oligosaccharides at asparagine 297 (Asn-297).
Different glycoforms of immunoglobulins exist depending upon the
particular N-glycan species attached to each of these two
glycosylation sites, and depending upon the degree to which both
sites are glycosylated within an immunoglobulin composition.
[0091] "Nucleotide sequence of interest" as used herein with
reference to expression of heterologous polypeptides refers to any
polynucleotide sequence encoding a heterologous polypeptide
intended for expression in a host, particularly a plant host, for
example, in a higher plant, including members of the
dicotyledonaceae and monocotyledonaceae. For example,
polynucleotide sequences encoding therapeutic (e.g., for veterinary
or medical uses) or immunogenic (e.g., for vaccination)
polypeptides can be expressed using transformed plant hosts, for
example, duckweed, according to the present invention.
[0092] The use of the term "polynucleotide" is not intended to
limit the present invention to polynucleotides comprising DNA.
Those of ordinary skill in the art will recognize that
polynucleotides can comprise ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides
and ribonucleotides include both naturally occurring molecules and
synthetic analogues. The polynucleotides of the invention also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0093] The terms "inhibit," "inhibition," and "inhibiting" as used
herein refer to any decrease in the expression or function of a
target gene product, including any relative decrement in expression
or function up to and including complete abrogation of expression
or function of the target gene product. The term "expression" as
used herein in the context of a gene product refers to the
biosynthesis of that gene product, including the transcription
and/or translation and/or assembly of the gene product. Inhibition
of expression or function of a target gene product (i.e., a gene
product of interest) can be in the context of a comparison between
any two plants, for example, expression or function of a target
gene product in a genetically altered plant versus the expression
or function of that target gene product in a corresponding
wild-type plant. Alternatively, inhibition of expression or
function of the target gene product can be in the context of a
comparison between plant cells, organelles, organs, tissues, or
plant parts within the same plant or between plants, and includes
comparisons between developmental or temporal stages within the
same plant or between plants. Any method or composition that
down-regulates expression of a target gene product, either at the
level of transcription or translation, or down-regulates functional
activity of the target gene product can be used to achieve
inhibition of expression or function of the target gene
product.
[0094] The term "inhibitory sequence" encompasses any
polynucleotide or polypeptide sequence that is capable of
inhibiting the expression of a target gene product, for example, at
the level of transcription or translation, or which is capable of
inhibiting the function of a target gene product. Examples of
inhibitory sequences include, but are not limited to, full-length
polynucleotide or polypeptide sequences, truncated polynucleotide
or polypeptide sequences, fragments of polynucleotide or
polypeptide sequences, variants of polynucleotide or polypeptide
sequences, sense-oriented nucleotide sequences, antisense-oriented
nucleotide sequences, the complement of a sense- or
antisense-oriented nucleotide sequence, inverted regions of
nucleotide sequences, hairpins of nucleotide sequences,
double-stranded nucleotide sequences, single-stranded nucleotide
sequences, combinations thereof, and the like. The term
"polynucleotide sequence" includes sequences of RNA, DNA,
chemically modified nucleic acids, nucleic acid analogs,
combinations thereof, and the like.
[0095] It is recognized that inhibitory polynucleotides include
nucleotide sequences that directly (i.e., do not require
transcription) or indirectly (i.e., require transcription or
transcription and translation) inhibit expression of a target gene
product. For example, an inhibitory polynucleotide can comprise a
nucleotide sequence that is a chemically synthesized or in
vitro-produced small interfering RNA (siRNA) or micro RNA (miRNA)
that, when introduced into a plant cell, tissue, or organ, would
directly, though transiently, silence expression of the target gene
product of interest. Alternatively, an inhibitory polynucleotide
can comprise a nucleotide sequence that encodes an inhibitory
nucleotide molecule that is designed to silence expression of the
gene product of interest, such as sense-orientation RNA, antisense
RNA, double-stranded RNA (dsRNA), hairpin RNA (hpRNA),
intron-containing hpRNA, catalytic RNA, miRNA, and the like. In yet
other embodiments, the inhibitory polynucleotide can comprise a
nucleotide sequence that encodes a mRNA, the translation of which
yields a polypeptide that inhibits expression or function of the
target gene product of interest. In this manner, where the
inhibitory polynucleotide comprises a nucleotide sequence that
encodes an inhibitory nucleotide molecule or a mRNA for a
polypeptide, the encoding sequence is operably linked to a promoter
that drives expression in a plant cell so that the encoded
inhibitory nucleotide molecule or mRNA can be expressed.
[0096] Inhibitory sequences are designated herein by the name of
the target gene product. Thus, for example, an
".alpha.1,3-fucosyltransferase (FucT) inhibitory sequence" would
refer to an inhibitory sequence that is capable of inhibiting the
expression of a FucT, for example, at the level of transcription
and/or translation, or which is capable of inhibiting the function
of a FucT. Similarly, a ".beta.1,2-xylosyltransferase (XylT)
inhibitory sequence" would refer to an inhibitory sequence that is
capable of inhibiting the expression of a XylT, at the level of
transcription and/or translation, or which is capable of inhibiting
the function of a XylT. When the phrase "capable of inhibiting" is
used in the context of a polynucleotide inhibitory sequence, it is
intended to mean that the inhibitory sequence itself exerts the
inhibitory effect; or, where the inhibitory sequence encodes an
inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, or
double-stranded RNA polynucleotides), or encodes an inhibitory
polypeptide (i.e., a polypeptide that inhibits expression or
function of the target gene product), following its transcription
(for example, in the case of an inhibitory sequence encoding a
hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its
transcription and translation (in the case of an inhibitory
sequence encoding an inhibitory polypeptide), the transcribed or
translated product, respectively, exerts the inhibitory effect on
the target gene product (i.e., inhibits expression or function of
the target gene product).
[0097] The term "introducing" in the context of a polynucleotide,
for example, a nucleotide construct of interest, is intended to
mean presenting to the plant the polynucleotide in such a manner
that the polynucleotide gains access to the interior of a cell of
the plant. Where more than one polynucleotide is to be introduced,
these polynucleotides can be assembled as part of a single
nucleotide construct, or as separate nucleotide constructs, and can
be located on the same or different transformation vectors.
Accordingly, these polynucleotides can be introduced into the host
cell of interest in a single transformation event, in separate
transformation events, or, for example, in plants, as part of a
breeding protocol. The methods of the invention do not depend on a
particular method for introducing one or more polynucleotides into
a plant, only that the polynucleotide(s) gains access to the
interior of at least one cell of the plant. Methods for introducing
polynucleotides into plants are known in the art including, but not
limited to, transient transformation methods, stable transformation
methods, and virus-mediated methods.
[0098] "Transient transformation" in the context of a
polynucleotide is intended to mean that a polynucleotide is
introduced into the plant and does not integrate into the genome of
the plant.
[0099] By "stably introducing" or "stably introduced" in the
context of a polynucleotide introduced into a plant is intended the
introduced polynucleotide is stably incorporated into the plant
genome, and thus the plant is stably transformed with the
polynucleotide.
[0100] "Stable transformation" or "stably transformed" is intended
to mean that a polynucleotide, for example, a nucleotide construct
described herein, introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof, more particularly, by the progeny of multiple
successive generations. In some embodiments, successive generations
include progeny produced vegetatively (i.e., asexual reproduction),
for example, with clonal propagation. In other embodiments,
successive generations include progeny produced via sexual
reproduction. A higher plant host that is "stably transformed" with
at least one nucleotide construct that is capable of inhibiting
expression of a FucT and/or XylT as described herein refers to a
higher plant host that has the nucleotide construct(s) integrated
into its genome, and is capable producing progeny, either via
asexual or sexual reproduction, that also comprise the inhibitory
nucleotide construct(s) stably integrated into their genome, and
hence the progeny will also exhibit the desired phenotype of having
an altered N-glycosylation pattern characterized by a reduction in
the attachment of .alpha.1,3-fucose and/or .beta.1,2-xylose
residues to the N-glycans of homologous and heterologous
glycoproteins produced therein.
[0101] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds,
plant cells, and progeny of same. Parts of transgenic plants are to
be understood within the scope of the invention to comprise, for
example, plant cells, protoplasts, tissues, callus, embryos as well
as flowers, ovules, stems, fruits, leaves, roots, root tips, and
the like originating in transgenic plants or their progeny
previously transformed with a DNA molecule of the invention and
therefore consisting at least in part of transgenic cells. As used
herein, the term "plant cell" includes, without limitation, cells
of seeds, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen, and
microspores.
[0102] The class of plants that can be used in the methods of the
invention is generally as broad as the class of higher plants
amenable to transformation techniques, including both
monocotyledonous (monocot) and dicotyledonous (dicot) plants.
Examples of dicots include, but are not limited to, legumes
including soybeans and alfalfa, tobacco, potatoes, tomatoes, and
the like. Examples of monocots include, but are not limited to,
maize, rice, oats, barley, wheat, members of the duckweed family,
grasses, and the like. "Lower-order plants" refers to non-flowering
plants including ferns, horsetails, club mosses, mosses,
liverworts, hornworts, algae, for example, red, brown, and green
algae, gametophytes, sporophytes of pteridophytes, and the like. In
some embodiments, the plant of interest is a member of the duckweed
family of plants.
[0103] The term "duckweed" refers to members of the family
Lemnaceae. This family currently is divided into five genera and 38
species of duckweed as follows: genus Lemna (L. aequinoctialis, L.
disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L.
miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L.
turionifera, L. valdiviana); genus Spirodela (S. intermedia, S.
polyrrhiza, S. punctata); genus Wolffia (Wa. angusta, Wa. arrhiza,
Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
elongata, Wa. globosa, Wa. microscopica, Wa. neglecta); genus
Wolfiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina,
Wl. lingulata, Wl. repunda, Wl. rotunda, and Wl. neotropica) and
genus Landoltia (L. punctata). Any other genera or species of
Lemnaceae, if they exist, are also aspects of the present
invention. Lemna species can be classified using the taxonomic
scheme described by Landolt (1986) Biosystematic Investigation on
the Family of Duckweeds: The family of Lemnaceae--A Monograph Study
(Geobatanischen Institut ETH, Stiftung Rubel, Zurich).
[0104] The term "duckweed nodule" as used herein refers to duckweed
tissue comprising duckweed cells where at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells are
differentiated cells. A "differentiated cell," as used herein, is a
cell with at least one phenotypic characteristic (e.g., a
distinctive cell morphology or the expression of a marker nucleic
acid or protein) that distinguishes it from undifferentiated cells
or from cells found in other tissue types. The differentiated cells
of the duckweed nodule culture described herein form a tiled smooth
surface of interconnected cells fused at their adjacent cell walls,
with nodules that have begun to organize into frond primordium
scattered throughout the tissue. The surface of the tissue of the
nodule culture has epidermal cells connected to each other via
plasmadesmata. Members of the duckweed family reproduce by clonal
propagation, and thus are representative of plants that clonally
propagate.
[0105] "Duckweed-preferred codons" as used herein refers to codons
that have a frequency of codon usage in duckweed of greater than
17%.
[0106] "Lemna-preferred codons" as used herein refers to codons
that have a frequency of codon usage in the genus Lemna of greater
than 17%.
[0107] "Lemna gibba-preferred codons" as used herein refers to
codons that have a frequency of codon usage in Lemna gibba of
greater than 17% where the frequency of codon usage in Lemna gibba
was obtained from the Codon Usage Database (GenBank Release 113,0;
at
http://www.kazusa.or.jp/codon/cgibin/showcodon.cgi?species=Lemna+gibba+[g-
bpln]).
[0108] "Translation initiation codon" refers to the codon that
initiates the translation of the mRNA transcribed from the
nucleotide sequence of interest.
[0109] "Translation initiation context nucleotide sequence" as used
herein refers to the identity of the three nucleotides directly 5'
of the translation initiation codon.
[0110] "Secretion" as used herein refers to translocation of a
polypeptide across both the plasma membrane and the cell wall of a
host plant cell.
[0111] "Operably linked" as used herein in reference to nucleotide
sequences refers to multiple nucleotide sequences that are placed
in a functional relationship with each other. Generally, operably
linked DNA sequences are contiguous and, where necessary to join
two protein coding regions, in reading frame.
Isolated Polynucleotides and Polypeptides
[0112] The present invention provides isolated polynucleotides and
polypeptides that are involved in further modification of plant
N-linked glycans (also referred to as "N-glycans"), particularly an
.alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT) identified in Lemna minor, a
member of the duckweed family, and variants and fragments of these
polynucleotides and polypeptides. Inhibition of the expression of
one or both of these proteins, or biologically active variants
thereof, in a plant that expresses these proteins beneficially
yields an N-glycosylation pattern that has a reduction in the
attachment of .alpha.1,3-fucose and .beta.1,2-xylose residues to
glycoprotein N-glycans. In some embodiments of the invention, the
methods disclosed herein provide for complete inhibition of
expression of FucT and XylT, yielding an N-glycosylation pattern of
glycoproteins produced within a plant wherein the N-linked glycans
are devoid of .alpha.1,3-fucose and .beta.1,2-xylose residues.
[0113] The full-length cDNA sequence, including 5'- and 3'-UTR, for
L. minor alpha 1-3 fucosyltransferase (FucT) is set forth in FIG.
1; see also SEQ ID NO:1 (open reading frame set forth in SEQ ID
NO:2). The predicted amino acid sequence encoded thereby is set
forth in SEQ ID NO:3. At least two isoforms of the L. minor FucT
gene have been identified; the homology between the isoforms is
about 90%. The encoded protein shares some similarity with other
FucTs from other higher plants. See FIG. 2. For example, the L.
minor FucT sequence shares approximately 50.1% sequence identity
with the Arabidopsis thaliana FucT shown in FIG. 2.
[0114] The full-length cDNA sequence, including 5'- and 3'-UTR, for
L. minor .beta.1-2 xylosyltransferase (XylT) (isoform #1) is set
forth in FIG. 3; see also SEQ ID NO:4 (ORF set forth in SEQ ID
NO:5). The predicted amino acid sequence encoded thereby is set
forth in SEQ ID NO:6. At least two isoforms of the L. minor XylT
gene have been identified; the homology between the isoforms is
about 90%. The encoded protein shares some similarity with other
XylTs from other higher plants. See FIG. 4. For example, the L.
minor XylT shares approximately 56.4% sequence identity with the
Arabidopsis thaliana XylT shown in FIG. 4. A partial-length cDNA
sequence, including 3'-UTR, for L. minor .beta.1-2
xylosyltransferase (XylT) (isoform #2) is set forth in FIG. 31; see
also SEQ ID NO:19 (ORF set forth in SEQ ID NO:20). The predicted
amino acid sequence encoded thereby is set forth in SEQ ID NO:21.
The partial-length XylT isoform #2 shares high sequence identity
with the corresponding region of the full-length XylT isoform #1,
as can be seen from the alignment shown in FIG. 32.
[0115] The invention encompasses isolated or substantially purified
polynucleotide or protein compositions. An "isolated" or "purified"
polynucleotide or protein, or biologically active portion thereof,
is substantially or essentially free from components that normally
accompany or interact with the polynucleotide or protein as found
in its naturally occurring environment. Thus, an isolated or
purified polynucleotide or protein is substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Optimally, an "isolated"
polynucleotide is free of sequences (optimally protein encoding
sequences) that naturally flank the polynucleotide (i.e., sequences
located at the 5' and 3' ends of the polynucleotide) in the genomic
DNA of the organism from which the polynucleotide is derived. For
example, in various embodiments, the isolated polynucleotide can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or
0.1 kb of nucleotide sequence that naturally flank the
polynucleotide in genomic DNA of the cell from which the
polynucleotide is derived. A protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating
protein. When the protein of the invention or biologically active
portion thereof is recombinantly produced, optimally culture medium
represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight)
of chemical precursors or non-protein-of-interest chemicals.
[0116] The coding sequence for the L. minor FucT gene is set forth
as nucleotides (nt) 243-1715 of SEQ ID NO:1 and as SEQ ID NO:2, and
the amino acid sequence for the encoded FucT polypeptide is set
forth in SEQ ID NO:3. The coding sequence for the L. minor XylT
isoform #1 gene is set forth as nucleotides 63-1592 of SEQ ID NO:4
and as SEQ ID NO:5, and the amino acid sequence for the encoded
XylT polypeptide is set forth in SEQ ID NO:6. The coding sequence
for the partial-length L. minor XylT isoform #2 gene is set forth
as nucleotides 1-1276 of SEQ ID NO:19 and as SEQ ID NO:20, and the
amino acid sequence for the encoded partial-length XylT polypeptide
is set forth in SEQ ID NO:21.
[0117] In particular, the present invention provides for isolated
polynucleotides comprising nucleotide sequences encoding the amino
acid sequences shown in SEQ ID NOS:3, 6, and 21. Further provided
are polypeptides having an amino acid sequence encoded by a
polynucleotide described herein, for example those set forth in SEQ
ID NOS:1, 2, 4, 5, 19, and 20, and fragments and variants thereof.
Nucleic acid molecules comprising the complements of these
nucleotide sequences are also provided. It is recognized that the
coding sequence for the FucT and/or XylT gene can be expressed in a
plant for overexpression of the encoded FucT and/or XylT. However,
for purposes of suppressing or inhibiting the expression of these
proteins, the respective nucleotide sequences of SEQ ID NO:1, 2, 4,
5, 19, and 20 will be used to design constructs for suppression of
expression of the respective FucT and/or XylT protein. Thus,
polynucleotides, in the context of suppressing the FucT protein
refers to the FucT coding sequences and to polynucleotides that
when expressed suppress or inhibit expression of the FucT gene, for
example, via direct or indirect suppression as noted herein below.
Similarly, polynucleotides, in the context of suppressing or
inhibiting the XylT protein refers to the XylT coding sequences and
to polynucleotides that when expressed suppress or inhibit
expression of the XylT gene, for example, via direct or indirect
suppression as noted herein below.
[0118] Fragments and variants of the disclosed polynucleotides and
proteins encoded thereby are also encompassed by the present
invention. By "fragment" is intended a portion of the FucT or XylT
polynucleotide or a portion of the FucT or XylT amino acid sequence
encoded thereby. Fragments of a polynucleotide may encode protein
fragments that retain the biological activity of the native protein
and hence have FucT activity or XylT activity as noted elsewhere
herein. Alternatively, fragments of a polynucleotide that are
useful as hybridization probes generally do not encode fragment
proteins retaining biological activity. Fragments of a FucT or XylT
polynucleotide can also be used to design inhibitory sequences for
suppression of expression of the FucT and/or XylT polypeptide.
Thus, for example, fragments of a nucleotide sequence may range
from at least about 15 nucleotides, 20 nucleotides, about 50
nucleotides, about 100 nucleotides, about 150 nucleotides, about
200 nucleotides, about 250 nucleotides, about 300 nucleotides,
about 350 nucleotides, about 400 nucleotides, about 450
nucleotides, about 500 nucleotides, about 550 nucleotides, about
600 nucleotides, about 650 nucleotides, about 700 nucleotides,
about 750 nucleotides, about 800 nucleotides, and up to the
full-length polynucleotide encoding the proteins of the
invention.
[0119] A fragment of a FucT polynucleotide that encodes a
biologically active portion of a FucT protein of the invention will
encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400,
450, 475, 500 contiguous amino acids, or up to the total number of
amino acids present in a full-length FucT protein of the invention
(for example, 509 amino acids for SEQ ID NO:3). A fragment of a
XylT polynucleotide that encodes a biologically active portion of a
full-length XylT protein of the invention will encode at least 15,
25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 475 contiguous
amino acids, or up to the total number of amino acids present in a
full-length XylT protein of the invention (for example, 490 amino
acids for SEQ ID NO:3). A fragment of a XylT polynucleotide that
encodes a biologically active portion of a partial-length XylT
protein of the invention will encode at least 15, 25, 30, 50, 100,
150, 200, 250, 300, 350, 400 contiguous amino acids, or up to the
total number of amino acids present in a partial-length XylT
protein of the invention (for example, 490 amino acids for SEQ ID
NO:21)
[0120] Thus, a fragment of a FucT or XylT polynucleotide may encode
a biologically active portion of a FucT or XylT protein,
respectively, or it may be a fragment that can be used as a
hybridization probe or PCR primer, or used to design inhibitory
sequences for suppression, using methods disclosed below. A
biologically active portion of a FucT or XylT protein can be
prepared by isolating a portion of one of the FucT or XylT
polynucleotides of the invention, respectively, expressing the
encoded portion of the FucT or XylT protein (e.g., by recombinant
expression in vitro), and assessing the activity of the encoded
portion of the FucT or XylT polypeptide. Polynucleotides that are
fragments of an FucT or XylT nucleotide sequence comprise at least
15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, or 1450
contiguous nucleotides, or up to the number of nucleotides present
in a FucT or XylT polynucleotide disclosed herein (for example,
1865, 1473, 1860, 1530, 1282, or 1275 nucleotides for SEQ ID NOS:1,
2, 4, 5, 19, and 20, respectively).
[0121] "Variants" is intended to mean substantially similar
sequences. For polynucleotides, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more sites
within the native polynucleotide and/or a substitution of one or
more nucleotides at one or more sites in the native polynucleotide.
As used herein, a "native" polynucleotide or polypeptide comprises
a naturally occurring nucleotide sequence or amino acid sequence,
respectively. For polynucleotides, conservative variants include
those sequences that, because of the degeneracy of the genetic
code, encode the amino acid sequence of one of the FucT or XylT
polypeptides of the invention. Naturally occurring allelic variants
such as these can be identified with the use of well-known
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant polynucleotides also include synthetically derived
polynucleotides, such as those generated, for example, by using
site-directed mutagenesis but which still encode a FucT or XylT
protein of the invention. Generally, variants of a particular
polynucleotide of the invention (for example, SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20,
fragments thereof, and complements of these sequences) will have at
least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to that particular polynucleotide as determined by
sequence alignment programs and parameters described elsewhere
herein.
[0122] Variants of a particular polynucleotide of the invention
(i.e., the reference polynucleotide) can also be evaluated by
comparison of the percent sequence identity between the polypeptide
encoded by a variant polynucleotide and the polypeptide encoded by
the reference polynucleotide. Thus, for example, an isolated
polynucleotide that encodes a polypeptide with a given percent
sequence identity to the FucT or XylT polypeptide of SEQ ID NO:3,
SEQ ID NO:6, or SEQ ID NO:21, respectively, is disclosed. Percent
sequence identity between any two polypeptides can be calculated
using sequence alignment programs and parameters described
elsewhere herein. Where any given pair of polynucleotides of the
invention is evaluated by comparison of the percent sequence
identity shared by the two polypeptides they encode, the percent
sequence identity between the two encoded polypeptides is at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity.
[0123] "Variant" protein is intended to mean a protein derived from
the native protein by deletion or addition of one or more amino
acids at one or more sites in the native protein and/or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is, the
enzymatic activity of attaching the .alpha.1,3-linked fucose
residue (activity of FucT) or .beta.1,2-linked xylose residue
(activity of XylT) to glycoprotein N-glycans in plants as described
herein. Such variants may result from, for example, genetic
polymorphism or from human manipulation. Biologically active
variants of a native FucT or XylT protein of the invention will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the amino acid sequence for the native protein
as determined by sequence alignment programs and parameters
described elsewhere herein. A biologically active variant of a
protein of the invention may differ from that protein by as few as
1-15 amino acid residues, as few as 1-10, such as 6-10, as few as
5, as few as 4, 3, 2, or even 1 amino acid residue.
[0124] The proteins of the invention may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in
the art. For example, amino acid sequence variants and fragments of
the FucT and XylT proteins can be prepared by mutations in the DNA.
Methods for mutagenesis and polynucleotide alterations are well
known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad.
Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.
154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.
(1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff et al. (1978) Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by reference. Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be
optimal.
[0125] Thus, the polynucleotides of the invention include both the
naturally occurring FucT and XylT sequences as well as mutant
forms. Likewise, the proteins of the invention encompass both
naturally occurring FucT and XylT proteins as well as variations
and modified forms thereof. Such variants will continue to possess
the desired activity. Thus, where expression of a functional
protein is desired, the expressed protein will possess the desired
FucT or XylT activity, i.e., the enzymatic activity of attaching
the .alpha.1,3-linked fucose residue (activity of FucT) or
.beta.1,2-linked xylose residue (activity of XylT) to glycoprotein
N-glycans in plants as described herein. Where the objective is
inhibition of expression or function of the FucT and/or XylT
polypeptide, the desired activity of the variant polynucleotide or
polypeptide is one of inhibiting expression or function of the
respective FucT and/or XylT polypeptide. Obviously, where
expression of a functional FucT or XylT variant is desired, the
mutations that will be made in the DNA encoding the variant must
not place the sequence out of reading frame and optimally will not
create complementary regions that could produce secondary mRNA
structure. See, EP Patent Application Publication No. 75,444.
[0126] Where a functional protein is desired, the deletions,
insertions, and substitutions of the protein sequences encompassed
herein are not expected to produce radical changes in the
characteristics of the protein. However, when it is difficult to
predict the exact effect of the substitution, deletion, or
insertion in advance of doing so, one skilled in the art will
appreciate that the effect will be evaluated by routine screening
assays, including the assays for monitoring FucT and XylT activity
described herein below in the Experimental section.
[0127] Variant polynucleotides and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different FucT or XylT coding sequences can be manipulated to
create a new FucT or XylT protein possessing the desired
properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. For example, using this approach, sequence motifs encoding a
domain of interest may be shuffled between the FucT or XylT gene of
the invention and other known FucT or XylT genes, respectively, to
obtain a new gene coding for a protein with an improved property of
interest. Strategies for such DNA shuffling are known in the art.
See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.
(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0128] The comparison of sequences and determination of percent
identity and percent similarity between two sequences can be
accomplished using a mathematical algorithm. In a preferred
embodiment, the percent identity between two amino acid sequences
is determined using the Needleman and Wunsch (1970) J. Mol. Biol.
48:444-453 algorithm, which is incorporated into the GAP program in
the GCG software package (available at www.accelrys.com), using
either a BLOSSUM62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet another preferred embodiment, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package, using a BLOSUM62 scoring
matrix (see Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA
89:10915) and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of
parameters (and the one that should be used if the practitioner is
uncertain about what parameters should be applied to determine if a
molecule is within a sequence identity limitation of the invention)
is using a BLOSUM62 scoring matrix with a gap weight of 60 and a
length weight of 3).
[0129] The percent identity between two amino acid or nucleotide
sequences can also be determined using the algorithm of Meyers and
Miller (1989) CABIOS 4:11-17 which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4.
[0130] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found,
for example, in Sambrook et al. (2001) Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.) and Tijssen (1993) Hybridization With Nucleic Acid
Probes, Part I: Theory and Nucleic Acid Preparation (Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Ltd., NY, N.Y.).
[0131] For purposes of the present invention, "stringent
conditions" encompass conditions under which hybridization will
only occur if there is less than 25% mismatch between the
hybridization molecule and the target sequence. "Stringent
conditions" may be broken down into particular levels of stringency
for more precise definition. Thus, as used herein, "moderate
stringency" conditions are those under which molecules with more
than 25% sequence mismatch will not hybridize; conditions of
"medium stringency" are those under which molecules with more than
15% mismatch will not hybridize, and conditions of "high
stringency" are those under which sequences with more than 10%
mismatch will not hybridize. Conditions of "very high stringency"
are those under which sequences with more than 6% mismatch will not
hybridize.
[0132] The FucT and XylT polynucleotides of the invention can be
used as probes for the isolation of corresponding homologous
sequences in other organisms, more particularly in other plant
species. In this manner, methods such as PCR, hybridization, and
the like can be used to identify such sequences based on their
sequence homology to the sequences of the invention. See, for
example, Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview,
N.Y.) and Innis et al. (1990), PCR Protocols: A Guide to Methods
and Applications (Academic Press, New York). Polynucleotide
sequences isolated based on their sequence identity to the entire
FucT or XylT polynucleotides of the invention (i.e., SEQ ID NOS:1
and 2 for FucT; SEQ ID NOS:4 and 5 for XylT isoform #1 of SEQ ID
NO:6; and SEQ ID NOS:19 and 20 for XylT isoform #2 of SEQ ID NO:21)
or to fragments and variants thereof are encompassed by the present
invention.
[0133] In a PCR method, oligonucleotides primers can be designed
for use in PCR reactions for amplification of corresponding DNA
sequences from cDNA or genomic DNA extracted from any organism of
interest. Known methods of PCR include, but are not limited to,
methods using paired primers, nested primers, single specific
primers, degenerate primers, gene-specific primers, vector-specific
primers, partially-mismatched primers, and the like. Methods for
designing PCR primers and PCR cloning are generally known in the
art and are disclosed in Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols:
A Guide to Methods and Applications (Academic Press, New York);
Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New
York); and Innis and Gelfand, eds. (1999) PCR Methods Manual
(Academic Press, New York).
[0134] In a hybridization method, all or part of a known nucleotide
sequence can be used as a probe that selectively hybridizes to
other corresponding polynucleotides present in a population of
cloned genomic DNA fragments or cDNA fragments (i.e., cDNA or
genomic libraries) from another organism of interest. The so-called
hybridization probes may be genomic DNA fragments, cDNA fragments,
RNA fragments, or other oligonucleotides, and may be labeled with a
detectable group such as .sup.32P, or any other detectable marker.
Probes for hybridization can be made by labeling synthetic
oligonucleotides based on the nucleotide sequence of interest, for
example, the FucT or XylT polynucleotides of the invention.
Degenerate primers designed on the basis of conserved nucleotides
or amino acid residues in the known nucleotide or encoded amino
acid sequence can additionally be used. Methods for construction of
cDNA and genomic libraries, and for preparing hybridization probes,
are generally known in the art and are disclosed in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by
reference.
[0135] For example, all or part of the specific known FucT or XylT
polynucleotide sequence may be used as a probe that selectively
hybridizes to other FucT or XylT nucleotide and messenger RNAs. To
achieve specific hybridization under a variety of conditions, such
probes include sequences that are unique and are preferably at
least about 10 nucleotides in length, and more optimally at least
about 20 nucleotides in length. This technique may be used to
isolate other corresponding FucT or XylT nucleotide sequences from
a desired organism or as a diagnostic assay to determine the
presence of a FucT or XylT coding sequences in an organism.
Hybridization techniques include hybridization screening of plated
DNA libraries (either plaques or colonies; see, for example, Innis
et al., eds. (1990) PCR Protocols: A Guide to Methods and
Applications (Academic Press, New York)).
[0136] Thus, in addition to the native FucT and XylT
polynucleotides and fragments and variants thereof, the isolated
polynucleotides of the invention also encompass homologous DNA
sequences identified and isolated from other organisms by
hybridization with entire or partial sequences obtained from the
FucT or XylT polynucleotides of the invention or variants thereof.
Conditions that will permit other DNA sequences to hybridize to the
DNA sequences disclosed herein can be determined in accordance with
techniques generally known in the art. For example, hybridization
of such sequences may be carried out under various conditions of
moderate, medium, high, or very high stringency as noted herein
above.
Methods of the Invention
[0137] The present invention is directed to methods for altering
protein glycosylation patterns in higher plants, particularly in
higher plants that serve as hosts for production of recombinant
proteins, particularly recombinant mammalian proteins of
pharmaceutical interest. The methods find use in producing higher
plants that are capable of producing recombinant proteins having an
N-glycosylation pattern that more closely resembles that found in
mammals. Compositions of the invention include higher plants that
are stably transformed to comprise an altered N-glycosylation
pattern of their endogenous (i.e., homologous) and recombinantly
produced heterologous proteins. In some embodiments, the higher
plants are transgenic plants that produce monoclonal antibodies
(mAbs) to mammalian proteins that have enhanced ADCC activity
relative to mAbs produced in a control plant that has not had the
glycosylation machinery altered to reduce the plant-specific
attachment of .alpha.1,3-fucose residues to the N-glycans of
homologous and heterologous glycoproteins produced therein.
[0138] The methods of the invention target the suppression (i.e.,
inhibition) of the expression of one or both of the enzymes
involved the production of complex glycoproteins in higher plants.
Of particular interest is suppression of a fucosyltransferase or
one or more isoforms thereof, suppression of a xylosyltransferase
or one or more isoforms thereof, or suppression of expression of
both of these proteins and one or more isoforms thereof. It is
recognized that suppression of the fucosyltransferase and/or
xylosyltransferase and one or more isoforms thereof can be
accomplished transiently. Alternatively, by stably suppressing the
expression of the fucosyltransferase and/or xylosyltransferase, it
is possible to produce transgenic higher plants that carry over
from generation to generation, either asexually or sexually, the
ability to produce glycoproteins having an N-glycosylation pattern
that more closely resembles that found in mammals, more
particularly, in humans. This advantageously provides for the
production of recombinant mammalian glycoproteins that have reduced
attachment of the plant .beta.1,2-linked xylose residue and/or
.alpha.1,3-linked fucose residue to glycoprotein N-glycans.
[0139] Inhibition of the expression of one or advantageously both
of these proteins in a plant, for example, a dicotyledonous or
monocotyledonous plant, for example, a duckweed plant, can be
carried out using any method known in the art. In this manner, a
polynucleotide comprising an inhibitory sequence for FucT, XylT, or
a combination thereof is introduced into the host cell of interest.
For transient suppression, the FucT or XylT inhibitory sequence can
be a chemically synthesized or in vitro-produced small interfering
RNA (siRNA) or micro RNA (miRNA) that, when introduced into the
host cell, would directly, though transiently, inhibit FucT, XylT,
or a combination thereof, by silencing expression of these target
gene product(s).
[0140] Alternatively, stable suppression of expression of FucT,
XylT, or a combination thereof is desirable as noted herein above.
Thus, in some embodiments, the activity of the FucT or the XylT
polypeptide of the invention is reduced or eliminated by
transforming a plant cell with an expression cassette that
expresses a polynucleotide that inhibits the expression of the FucT
or XylT, or both. The polynucleotide may inhibit the expression of
the FucT or XylT, or both, directly, by preventing transcription or
translation of the FucT or XylT messenger RNA, or indirectly, by
encoding a polypeptide that inhibits the transcription or
translation of a gene encoding the FucT or XylT, or both. Methods
for inhibiting or eliminating the expression of a gene in a plant
are well known in the art, and any such method may be used in the
present invention to inhibit the expression of FucT or XylT, or
both.
[0141] Thus, in some embodiments, expression of the FucT and/or
XylT protein can be inhibited by introducing into the plant a
nucleotide construct, such as an expression cassette, comprising a
sequence that encodes an inhibitory nucleotide molecule that is
designed to silence expression of the FucT and/or XylT gene product
of interest, such as sense-orientation RNA, antisense RNA,
double-stranded RNA (dsRNA), hairpin RNA (hpRNA), intron-containing
hpRNA, catalytic RNA, miRNA, and the like. In other embodiments,
the nucleotide construct, for example, an expression cassette, can
comprise a sequence that encodes a mRNA, the translation of which
yields a polypeptide of interest that inhibits expression or
function of the FucT and/or XylT gene product of interest. Where
the nucleotide construct comprises a sequence that encodes an
inhibitory nucleotide molecule or a mRNA for a polypeptide of
interest, the sequence is operably linked to a promoter that drives
expression in a plant cell so that the encoded inhibitory
nucleotide molecule or mRNA can be expressed.
[0142] In accordance with the present invention, the expression of
a FucT or XylT gene is inhibited if the protein level of the FucT
or XylT is statistically lower than the protein level of the same
FucT or XylT in a plant that has not been genetically modified or
mutagenized to inhibit the expression of that FucT or XylT. In
particular embodiments of the invention, the protein level of the
FucT or XylT, or both, in a modified plant according to the
invention is less than 95%, less than 90%, less than 80%, less than
70%, less than 60%, less than 50%, less than 40%, less than 30%,
less than 20%, less than 10%, or less than 5% of the protein level
of the same FucT or XylT in a plant that is not a mutant or that
has not been genetically modified to inhibit the expression of that
FucT or XylT, or both the FucT or XylT. The expression level of the
FucT or XylT, or both, may be measured directly, for example, by
assaying for the level of FucT or XylT, or both, expressed in the
plant cell or plant, or indirectly, for example, by observing the
effect in a transgenic plant at the phenotypic level, i.e., by
transgenic plant analysis, observed as a reduction, or even
elimination, of the attachment of .beta.1,2-xylose and/or
.alpha.1,3-fucose residues to the glycoprotein N-glycans in the
plant.
[0143] In other embodiments of the invention, the activity of FucT
or XylT, or both, is reduced or eliminated by transforming a plant
cell with an expression cassette comprising a polynucleotide
encoding a polypeptide that inhibits the activity of FucT or XylT,
or both. The activity of a FucT or XylT is inhibited according to
the present invention if the activity of the FucT or XylT is
statistically lower than the activity of the same FucT or XylT in a
plant that has not been genetically modified to inhibit the
activity of that FucT or XylT. In particular embodiments of the
invention, the activity of the FucT or XylT in a modified plant
according to the invention is less than 95%, less than 90%, less
than 80%, less than 70%, less than 60%, less than 50%, less than
40%, less than 30%, less than 20%, less than 10%, or less than 5%
of the activity of the same FucT or XylT in a plant that has not
been genetically modified to inhibit the expression of that FucT or
XylT. The activity of a FucT or XylT is "eliminated" according to
the invention when it is not detectable by the assay methods
described elsewhere herein.
[0144] In other embodiments, the activity of a FucT or XylT, or
both, may be reduced or eliminated by disrupting the gene encoding
the FucT or XylT, or both of these genes. The invention encompasses
mutagenized plants, particularly plants that are members of the
duckweed family, that carry mutations in a FucT or XylT gene, or
mutations in both genes, where the mutations reduce expression of
the FucT and/or XylT gene or inhibit the activity of the encoded
FucT and/or XylT.
[0145] The methods of the invention can involve any method or
mechanism known in the art for reducing or eliminating the activity
or level of FucT and/or XylT in the cells of a higher plant,
including, but not limited to, antisense suppression, sense
suppression, RNA interference, directed deletion or mutation,
dominant-negative strategies, and the like. Thus, the methods and
compositions disclosed herein are not limited to any mechanism or
theory of action and include any method where expression or
function of FucT and/or XylT is inhibited in the cells of the
higher plant of interest, thereby altering the N-glycosylation
pattern of endogenous and heterologous glycoproteins produced in
the plant.
[0146] For example, in some embodiments, the FucT inhibitory
sequence or the XylT inhibitory sequence (or both) is expressed in
the sense orientation, wherein the sense-oriented transcripts cause
cosuppression of the expression of one or both of these enzymes.
Alternatively, the FucT and/or XylT inhibitory sequence (e.g.,
full-length sequence, truncated sequence, fragments of the
sequence, combinations thereof, and the like) can be expressed in
the antisense orientation and thus inhibit endogenous FucT and/or
XylT expression or function by antisense mechanisms.
[0147] In yet other embodiments, the FucT and/or XylT inhibitory
sequence or sequences are expressed as a hairpin RNA, which
comprises both a sense sequence and an antisense sequence. In
embodiments comprising a hairpin structure, the loop structure may
comprise any suitable nucleotide sequence including for example 5'
untranslated and/or translated regions of the gene to be
suppressed, such as the 5' UTR and/or translated region of the FucT
polynucleotide of SEQ ID NO:1 or 2, or the 5' UTR and/or translated
region of the XylT polynucleotide of SEQ ID NO:4, 5, 19, or 20, and
the like. In some embodiments, the FucT or XylT inhibitory sequence
expressed as a hairpin is encoded by an inverted region of the FucT
or XylT nucleotide sequence. In yet other embodiments, the FucT
and/or XylT inhibitory sequences are expressed as double-stranded
RNA, where one FucT and/or XylT inhibitory sequence is expressed in
the sense orientation and another complementary sequence is
expressed in the antisense orientation. Double-stranded RNA,
hairpin structures, and combinations thereof comprising FucT
nucleotide sequences, XylT nucleotide sequences, or combinations
thereof may operate by RNA interference, cosuppression, antisense
mechanism, any combination thereof, or by means of any other
mechanism that causes inhibition of FucT and/or XylT expression or
function.
[0148] Thus, many methods may be used to reduce or eliminate the
activity of a FucT or XylT, or both of these proteins, and any
isoforms thereof. By "isoform" is intended a naturally occurring
protein variant of the FucT or XylT protein of interest, where the
variant is encoded by a different gene. Generally, isoforms of a
particular FucT or XylT protein of interest are encoded by a
nucleotide sequence having at least 90% sequence identity to the
nucleotide sequence encoding the FucT or XylT protein of interest.
More than one method may be used to reduce or eliminate the
activity of a single plant FucT or XylT, and isoforms thereof.
Non-limiting examples of methods of reducing or eliminating the
activity of a plant FucT or XylT are given below.
Polynucleotide-Based Methods:
[0149] In some embodiments of the present invention, a plant cell
is transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of FucT or
XylT, or both. The term "expression" as used herein refers to the
biosynthesis of a gene product, including the transcription and/or
translation of the gene product. For example, for the purposes of
the present invention, an expression cassette capable of expressing
a polynucleotide that inhibits the expression of at least one FucT
or XylT, or both, is an expression cassette capable of producing an
RNA molecule that inhibits the transcription and/or translation of
at least one FucT or XylT, or both. The "expression" or
"production" of a protein or polypeptide from a DNA molecule refers
to the transcription and translation of the coding sequence to
produce the protein or polypeptide, while the "expression" or
"production" of a protein or polypeptide from an RNA molecule
refers to the translation of the RNA coding sequence to produce the
protein or polypeptide.
[0150] Examples of polynucleotides that inhibit the expression of a
FucT or XylT, or both, are given below.
[0151] Sense Suppression/Cosuppression
[0152] In some embodiments of the invention, inhibition of the
expression of FucT or XylT, or both, may be obtained by sense
suppression or cosuppression. For cosuppression, an expression
cassette is designed to express an RNA molecule corresponding to
all or part of a messenger RNA encoding a FucT or XylT, or both, in
the "sense" orientation. Overexpression of the RNA molecule can
result in reduced expression of the native gene. Accordingly,
multiple plant lines transformed with the cosuppression expression
cassette are screened to identify those that show the greatest
inhibition of FucT or XylT expression.
[0153] The polynucleotide used for cosuppression may correspond to
all or part of the sequence encoding the FucT or XylT, all or part
of the 5' and/or 3' untranslated region of a FucT or XylT
transcript, or all or part of both the coding sequence and the
untranslated regions of a transcript encoding FucT or XylT. In some
embodiments where the polynucleotide comprises all or part of the
coding region for the FucT or XylT protein, the expression cassette
is designed to eliminate the start codon of the polynucleotide so
that no protein product will be transcribed.
[0154] Cosuppression may be used to inhibit the expression of plant
genes to produce plants having undetectable protein levels for the
proteins encoded by these genes. See, for example, Broin et al.
(2002) Plant Cell 14:1417-1432. Cosuppression may also be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Methods for using
cosuppression to inhibit the expression of endogenous genes in
plants are described in Flavell et al. (1994) Proc. Natl. Acad.
Sci. USA 91:3490-3496; Jorgensen et al. (1996) Plant Mol. Biol.
31:957-973; Johansen and Carrington (2001) Plant Physiol.
126:930-938; Broin et al. (2002) Plant Cell 14:1417-1432;
Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; Yu et al.
(2003) Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,034,323,
5,283,184, and 5,942,657; each of which is herein incorporated by
reference. The efficiency of cosuppression may be increased by
including a poly-dT region in the expression cassette at a position
3' to the sense sequence and 5' of the polyadenylation signal. See,
U.S. Patent Publication No. 20020048814, herein incorporated by
reference. Typically, such a nucleotide sequence has substantial
sequence identity to the sequence of the transcript of the
endogenous gene, optimally greater than about 65% sequence
identity, more optimally greater than about 85% sequence identity,
most optimally greater than about 95% sequence identity. See, U.S.
Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by
reference.
[0155] Antisense Suppression
[0156] In some embodiments of the invention, inhibition of the
expression of FucT or XylT, or both, may be obtained by antisense
suppression. For antisense suppression, the expression cassette is
designed to express an RNA molecule complementary to all or part of
a messenger RNA encoding the FucT or XylT. Overexpression of the
antisense RNA molecule can result in reduced expression of the
native gene. Accordingly, multiple plant lines transformed with the
antisense suppression expression cassette are screened to identify
those that show the greatest inhibition of FucT or XylT
expression.
[0157] The polynucleotide for use in antisense suppression may
correspond to all or part of the complement of the sequence
encoding the FucT or XylT, all or part of the complement of the 5'
and/or 3' untranslated region of the FucT or XylT transcript, or
all or part of the complement of both the coding sequence and the
untranslated regions of a transcript encoding the FucT or XylT. In
addition, the antisense polynucleotide may be fully complementary
(i.e., 100% identical to the complement of the target sequence) or
partially complementary (i.e., less than 100% identical to the
complement of the target sequence) to the target sequence.
Antisense suppression may be used to inhibit the expression of
multiple proteins in the same plant. See, for example, U.S. Pat.
No. 5,942,657. Furthermore, portions of the antisense nucleotides
may be used to disrupt the expression of the target gene.
Generally, sequences of at least 50 nucleotides, 100 nucleotides,
200 nucleotides, 300, 400, 450, 500, 550, or greater may be used.
Methods for using antisense suppression to inhibit the expression
of endogenous genes in plants are described, for example, in Liu et
al. (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos.
5,759,829 and 5,942,657, each of which is herein incorporated by
reference. Efficiency of antisense suppression may be increased by
including a poly-dT region in the expression cassette at a position
3' to the antisense sequence and 5' of the polyadenylation signal.
See, U.S. Patent Publication No. 20020048814, herein incorporated
by reference.
[0158] Double-Stranded RNA Interference
[0159] In some embodiments of the invention, inhibition of the
expression of a FucT or XylT, or both, may be obtained by
double-stranded RNA (dsRNA) interference. For dsRNA interference, a
sense RNA molecule like that described above for cosuppression and
an antisense RNA molecule that is fully or partially complementary
to the sense RNA molecule are expressed in the same cell, resulting
in inhibition of the expression of the corresponding endogenous
messenger RNA.
[0160] Expression of the sense and antisense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and an antisense sequence. Alternatively, separate
expression cassettes may be used for the sense and antisense
sequences. Multiple plant lines transformed with the dsRNA
interference expression cassette or expression cassettes are then
screened to identify plant lines that show the greatest inhibition
of FucT or XylT expression. Methods for using dsRNA interference to
inhibit the expression of endogenous plant genes are described in
Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964,
Liu et al. (2002) Plant Physiol. 129:1732-1743, and WO 99/49029, WO
99/53050, WO 99/61631, and WO 00/49035; each of which is herein
incorporated by reference.
[0161] Hairpin RNA Interference and Intron-Containing Hairpin RNA
Interference
[0162] In some embodiments of the invention, inhibition of the
expression of FucT or XylT, or both, may be obtained by hairpin RNA
(hpRNA) interference or intron-containing hairpin RNA (ihpRNA)
interference. These methods are highly efficient at inhibiting the
expression of endogenous genes. See, Waterhouse and Helliwell
(2003) Nat. Rev. Genet. 4:29-38 and the references cited
therein.
[0163] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited, and an
antisense sequence that is fully or partially complementary to the
sense sequence. Thus, the base-paired stem region of the molecule
generally determines the specificity of the RNA interference. hpRNA
molecules are highly efficient at inhibiting the expression of
endogenous genes, and the RNA interference they induce is inherited
by subsequent generations of plants. See, for example, Chuang and
Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;
Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; and
Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38. Methods
for using hpRNA interference to inhibit or silence the expression
of genes are described, for example, in Chuang and Meyerowitz
(2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al.
(2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell
(2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMC
Biotechnology 3:7, and U.S. Patent Publication No. 20030175965;
each of which is herein incorporated by reference. A transient
assay for the efficiency of hpRNA constructs to silence gene
expression in vivo has been described by Panstruga et al. (2003)
Mol. Biol. Rep. 30:135-140, herein incorporated by reference.
[0164] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith
et al. (2000) Nature 407:319-320. In fact, Smith et al. show 100%
suppression of endogenous gene expression using ihpRNA-mediated
interference. Methods for using ihpRNA interference to inhibit the
expression of endogenous plant genes are described, for example, in
Smith et al. (2000) Nature 407:319-320; Wesley et al. (2001) Plant
J. 27:581-590; Wang and Waterhouse (2001) Curr. Opin. Plant Biol.
5:146-150; Waterhouse and Helliwell (2003) Nat. Rev. Genet.
4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295, and
U.S. Patent Publication No. 20030180945, each of which is herein
incorporated by reference.
[0165] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the antisense sequence do
not correspond to an endogenous RNA. In this embodiment, the sense
and antisense sequence flank a loop sequence that comprises a
nucleotide sequence corresponding to all or part of the endogenous
messenger RNA of the target gene. Thus, it is the loop region that
determines the specificity of the RNA interference. See, for
example, WO 02/00904, herein incorporated by reference.
[0166] Transcriptional gene silencing (TGS) may be accomplished
through use of hpRNA constructs wherein the inverted repeat of the
hairpin shares sequence identity with the promoter region of a gene
to be silenced. Processing of the hpRNA into short RNAs which can
interact with the homologous promoter region may trigger
degradation or methylation to result in silencing (Aufsatz et al.
(2002) PNAS 99 (Suppl. 4): 16499-16506; Mette et al. (2000) EMBO J
19(19):5194-5201).
[0167] Expression cassettes that are designed to express an RNA
molecule that forms a hairpin structure are referred to herein as
RNAi expression cassettes. In some embodiments, the RNAi expression
cassette is designed in accordance with a strategy outlined in FIG.
28. In such embodiments, an RNAi expression cassette can be
designed to suppress the expression of the individual FucT and XylT
genes (i.e., each cassette provides a single gene knockout), or can
be designed to suppress the expression of both the FucT and XylT
genes (i.e., a single RNAi expression cassette expresses an
inhibitory molecule that provides for suppression of expression of
both of these genes). Where the RNAi expression cassette suppresses
expression of both the FucT and XylT genes, it is referred to
herein as a "chimeric" RNAi expression cassette. The single-gene
and chimeric RNAi expression cassettes can be designed to express
larger hpRNA structures or, alternatively, small hpRNA structures,
as noted herein below.
[0168] Thus, in some embodiments, the RNAi expression cassette is
designed to express larger hpRNA structures having sufficient
homology to the target mRNA transcript to provide for
post-transcriptional gene silencing of one or both of a FucT and
XylT gene. For larger hp RNA structures, the sense strand of the
RNAi expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest, a forward fragment of the FucT or XylT gene sequence
comprising about 500 to about 800 nucleotides (nt) of a sense
strand for FucT or XylT, respectively, a spacer sequence comprising
about 100 to about 700 nt of any sequence as noted herein below,
and a reverse fragment of the XylT or FucT gene sequence, wherein
the reverse fragment comprises the antisense sequence complementary
to the respective (i.e., FucT or XylT) forward fragment. Thus, for
example, if a forward fragment is represented by nucleotides " . .
. acttg . . . ", the corresponding reverse fragment is represented
by nucleotides " . . . caagt . . . ", and the sense strand for such
an RNAi expression cassette would comprise the following sequence:
"5'- . . . acttg . . . nnnn . . . caagt . . . -3', where "nnnn"
represents the spacer sequence.
[0169] It is recognized that the forward fragment can comprise a
nucleotide sequence that is 100% identical to the corresponding
portion of the sense strand for the target FucT or XylT gene
sequence, or in the alternative, can comprise a sequence that
shares at least 90%, at least 95%, or at least 98% sequence
identity to the corresponding portion of the sense strand for the
target FucT or XylT gene to be silenced. In like manner, it is
recognized that the reverse fragment does not have to share 100%
sequence identity to the complement of the forward fragment; rather
it must be of sufficient length and sufficient complementarity to
the forward fragment sequence such that when the inhibitory RNA
molecule is expressed, the transcribed regions corresponding to the
forward fragment and reverse fragment will hybridize to form the
base-paired stem (i.e., double-stranded portion) of the hp RNA
structure. By "sufficient length" is intended a length that is at
least 10%, at least 15%, at least 20%, at least 30%, at least 40%
of the length of the forward fragment, more frequently at least
50%, at least 75%, at least 90%, or least 95% of the length of the
forward fragment. By "sufficient complementarity" is intended the
sequence of the reverse fragment shares at least 90%, at least 95%,
at least 98% sequence identity with the complement of that portion
of the forward fragment that will hybridize with the reverse
fragment to form the base-paired stem of the hp RNA structure.
Thus, in some embodiments, the reverse fragment is the complement
(i.e., antisense version) of the forward fragment.
[0170] In designing such an RNAi expression cassette, the lengths
of the forward fragment, spacer sequence, and reverse fragments are
chosen such that the combined length of the polynucleotide that
encodes the hpRNA construct is about 650 to about 2500 nt, about
750 to about 2500 nt, about 750 to about 2400 nt, about 1000 to
about 2400 nt, about 1200 to about 2300 nt, about 1250 to about
2100 nt, or about 1500 to about 1800. In some embodiments, the
combined length of the expressed hairpin construct is about 650 nt,
about 700 nt, about 750 nt, about 800 nt, about 850 nt, about 900
nt, about 950 nt, about 1000 nt, about 1050 nt, about 1100 nt,
about 1150 nt, about 1200 nt, about 1250 nt, about 1300 nt, about
1350 nt, about 1400 nt, about 1450 nt, about 1500 nt, about 1550
nt, about 1600 nt, about 1650 nt, about 1700 nt, about 1750 nt,
about 1800 nt, about 1850 nt, about 1900 nt, about 1950 nt, about
2000 nt, about 2050 nt, about 2100 nt, about 2150 nt, about 2200
nt, about 2250 nt, about 2300 nt, or any such length between about
650 nt to about 2300 nt.
[0171] In some embodiments, the forward fragment comprises about
500 to about 800 nt, for example, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, or 800 nt of a sense strand for FucT
or XylT, for example, of the sense strand set forth in SEQ ID NO:1
or 2 (FucT) or SEQ ID NO:4, 5, 19, or 20 (XylT); the spacer
sequence comprises about 100 to about 700 nt, for example, 100,
125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,
450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 nt of any
sequence as noted below, and the reverse fragment comprises the
antisense strand for the forward fragment sequence, or a sequence
having sufficient length and sufficient complementarity to the
forward fragment sequence.
[0172] The spacer sequence can be any sequence that has
insufficient homology to the target gene, i.e., FucT or XylT, and
insufficient homology to itself such that the portion of the
expressed inhibitory RNA molecule corresponding to the spacer
region fails to self-hybridize, and thus forms the loop of the
hairpin RNA structure. In some embodiments, the spacer sequence
comprises an intron, and thus the expressed inhibitory RNA molecule
forms an ihpRNA as noted herein above. In other embodiments, the
spacer sequence comprises a portion of the sense strand for the
FucT or XylT gene to be silenced, for example, a portion of the
sense strand set forth in SEQ ID NO:1 or 2 (FucT) or SEQ ID NO:4,
5, 19, or 20 (XylT), particularly a portion of the sense strand
immediately downstream from the forward fragment sequence.
[0173] The operably linked promoter can be any promoter of interest
that provides for expression of the operably linked inhibitory
polynucleotide within the plant of interest, including one of the
promoters disclosed herein below. The regulatory region can
comprise additional regulatory elements that enhance expression of
the inhibitory polynucleotide, including, but not limited to, the
5' leader sequences and 5' leader sequences plus plant introns
discussed herein below.
[0174] In one embodiment, the RNAi expression cassette is designed
to suppress expression of the FucT polypeptide of SEQ ID NO:3, a
biologically active variant of the FucT polypeptide of SEQ ID NO:3,
or a FucT polypeptide encoded by a sequence having at least 75%
sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2,
for example, at least 75%, at least 80%, at least 85%, at least
90%, or at least 95% sequence identity to the sequence of SEQ ID
NO:1 or SEQ ID NO:2. In this manner, the sense strand of the RNAi
expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a forward fragment of the FucT gene sequence, wherein the
forward fragment comprises nt 255-985 of SEQ ID NO:1; a spacer
sequence comprising about 100 to about 700 nt of any sequence as
noted above; and a reverse fragment of the FucT gene sequence,
wherein the reverse fragment comprises the complement (i.e.,
antisense version) of nt 255-985 of SEQ ID NO:1. In one such
embodiment, the spacer sequence is represented by nt 986-1444 of
SEQ ID NO:1, and the total length of that portion of the sense
strand of the RNAi expression cassette corresponding to the coding
sequence for the hpRNA structure is 1918 nt. Stably transforming a
plant with a nucleotide construct comprising this RNAi expression
cassette, for example, the vector shown in FIG. 8, effectively
inhibits expression of FucT within the plant cells of the plant in
which the hpRNA structure is expressed. In one embodiment, the
plant of interest is a member of the duckweed family, for example,
a member of the Lemnaceae, and the plant has been stably
transformed with the vector shown in FIG. 8.
[0175] In other embodiments of the invention, the RNAi expression
cassette is designed to suppress expression of the XylT polypeptide
of SEQ ID NO:6 or SEQ ID NO:21, a biologically active variant of
the XylT polypeptide of SEQ ID NO:6 or SEQ ID NO:21, or a XylT
polypeptide encoded by a sequence having at least 75% sequence
identity to the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19,
or SEQ ID NO:20, for example, at least 75%, at least 80%, at least
85%, at least 90%, or at least 95% sequence identity to the
sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID
NO:20. In this manner, the sense strand of the RNAi expression
cassette is designed to comprise in the 5'-to-3' direction the
following operably linked elements: a promoter of interest; a
forward fragment of the XylT gene sequence, wherein the forward
fragment comprises nt 318-1052 of SEQ ID NO:4; a spacer sequence
comprising about 100 to about 700 nt of any sequence as noted
above; and a reverse fragment of the XylT gene sequence, wherein
the reverse fragment comprises the complement (i.e., antisense
version) of nt 318-1052 of SEQ ID NO:4. In one such embodiment, the
spacer sequence is represented by nt 1053-1599 of SEQ ID NO:4, and
the total length of that portion of the sense strand of the RNAi
expression cassette corresponding to the coding sequence for the
hpRNA structure is 2015 nt. Stably transforming a plant with a
nucleotide construct comprising this RNAi expression cassette, for
example, the vector shown in FIG. 9, effectively inhibits
expression of FucT within the plant cells of the plant in which the
hpRNA structure is expressed. In one embodiment, the plant of
interest is a member of the duckweed family, for example, a member
of the Lemnaceae, and the plant has been stably transformed with
the vector shown in FIG. 9.
[0176] In other embodiments, the sense strand of the RNAi
expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a forward fragment of the XylT gene sequence, wherein the
forward fragment comprises nt 1-734 of SEQ ID NO:19; a spacer
sequence comprising about 100 to about 700 nt of any sequence as
noted above; and a reverse fragment of the XylT gene sequence,
wherein the reverse fragment comprises the complement (i.e.,
antisense version) of nt 1-734 of SEQ ID NO:19. In one such
embodiment, the spacer sequence is represented by nt 735-1282 of
SEQ ID NO:19, and the total length of that portion of the sense
strand of the RNAi expression cassette corresponding to the coding
sequence for the hpRNA structure is 2015 nt. Stably transforming a
plant with a nucleotide construct comprising this RNAi expression
cassette, for example, the vector shown in FIG. 9, effectively
inhibits expression of FucT within the plant cells of the plant in
which the hpRNA structure is expressed. In one embodiment, the
plant of interest is a member of the duckweed family, for example,
a member of the Lemnaceae, and the plant has been stably
transformed with the vector shown in FIG. 9.
[0177] In yet other embodiments, larger hpRNA structures can be
designed such that the antisense and sense sequences are in
opposite orientation. In this manner, the sense strand of the RNAi
expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest, the full-length FucT or XylT gene sequence in the
antisense orientation, and a forward fragment of the FucT or XylT
gene sequence comprising the 3'-half of the sequence in the sense
orientation (see FIG. 28, design 1). In this type of construct, the
3'-half of the sequence forms the base-paired (i.e.,
double-stranded) stem of the hpRNA, and the 5'-half of the sequence
acts as a spacer sequence. Without being bound by any theory or
mechanism, the 3' region of the FucT or XylT sequence is chosen to
form the double-stranded region of the hpRNA for for this construct
because this region is relatively conserved among different plant
species compared to the 5' region.
[0178] In one such embodiment, the RNAi expression cassette is
designed to suppress expression of the FucT polypeptide of SEQ ID
NO:3, a biologically active variant of the FucT polypeptide of SEQ
ID NO:3, or a FucT polypeptide encoded by a sequence having at
least 75% sequence identity to the sequence of SEQ ID NO:1 or SEQ
ID NO:2, for example, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% sequence identity to the sequence of SEQ
ID NO:1 or SEQ ID NO:2. In this manner, the sense strand of the
RNAi expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; nucleotides 1-1865 of SEQ ID NO:1 in antisense
orientation, and nucleotides 950-1865 of SEQ ID NO:1 in the sense
orientation. Stably transforming a plant with a nucleotide
construct comprising this RNAi expression cassette effectively
inhibits expression of FucT within the plant cells of the plant in
which the hpRNA structure is expressed. In one embodiment, the
plant of interest is a member of the duckweed family, for example,
a member of the Lemnaceae.
[0179] In another such embodiment, the RNAi expression cassette is
designed to suppress expression of the XylT polypeptide of SEQ ID
NO:6 or SEQ ID NO:21, a biologically active variant of the XylT
polypeptide of SEQ ID NO:6 or SEQ ID NO:21, or a XylT polypeptide
encoded by a sequence having at least 75% sequence identity to the
sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID
NO:20, for example, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% sequence identity to the sequence of SEQ
ID NO:5, SEQ ID NO:19, or SEQ ID NO:20. In this manner, the sense
strand of the RNAi expression cassette is designed to comprise in
the 5'-to-3' direction the following operably linked elements: a
promoter of interest, nucleotides 1-1860 of SEQ ID NO:4 in
antisense orientation, and nucleotides 950-1860 of SEQ ID NO:4 in
the sense orientation. Stably transforming a plant with a
nucleotide construct comprising this RNAi expression cassette
effectively inhibits expression of XylT within the plant cells of
the plant in which the hpRNA structure is expressed. In one
embodiment, the plant of interest is a member of the duckweed
family, for example, a member of the Lemnaceae.
[0180] In another such embodiment, the sense strand of the RNAi
expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest, nucleotides 1-1282 of SEQ ID NO:19 in antisense
orientation, and nucleotides 652-1282 of SEQ ID NO:19 in the sense
orientation. Stably transforming a plant with a nucleotide
construct comprising this RNAi expression cassette effectively
inhibits expression of XylT within the plant cells of the plant in
which the hpRNA structure is expressed. In one embodiment, the
plant of interest is a member of the duckweed family, for example,
a member of the Lemnaceae.
[0181] Where suppression of both the FucT and XylT proteins is
desired, it can be achieved by introducing these single-gene RNAi
expression cassettes into the plant in a single transformation
event, for example, by assembling these single-gene RNAi expression
cassettes within a single transformation vector, for example, a
vector similar to that shown in FIG. 11, or as separate
co-transformation events, for example, by assembling these
single-gene RNAi expression cassettes within two transformation
vectors, for example, vectors similar to those shown in FIGS. 8 and
9, using any suitable transformation method known in the art,
including but not limited to the transformation methods disclosed
elsewhere herein.
[0182] Alternatively, suppression of both the FucT and XylT
proteins can be achieved by introducing into the higher plant of
interest a chimeric RNAi expression cassette as noted herein above.
Thus, in some embodiments of the invention, the sense strand of a
chimeric RNAi expression cassette is designed to comprise in the
5'-to-3' direction the following operably linked elements: a
promoter of interest; a chimeric forward fragment, comprising about
500 to about 650 nucleotides (nt) of a sense strand for FucT and
about 500 to about 650 nt of a sense strand for XylT, wherein the
FucT sequence and XylT sequence can be in either order; a spacer
sequence comprising about 100 to about 700 nt of any sequence; and
a reverse fragment of the chimeric forward fragment, wherein the
reverse fragment comprises the antisense sequence complementary to
the respective chimeric forward fragment.
[0183] As previously noted for the individual RNAi expression
cassettes, it is recognized that the individual FucT or XylT
sequence within the chimeric forward fragment can comprise a
nucleotide sequence that is 100% identical to the corresponding
portion of the sense strand for the target FucT and XylT gene
sequence, respectively, or in the alternative, can comprise a
sequence that shares at least 90%, at least 95%, or at least 98%
sequence identity to the corresponding portion of the sense strand
for the target FucT or XylT gene to be silenced. In like manner, it
is recognized that the reverse fragment does not have to share 100%
sequence identity to the complement of the chimeric forward
fragment; rather it must be of sufficient length and sufficient
complementarity to the chimeric forward fragment sequence, as
defined herein above, such that when the inhibitory RNA molecule is
expressed, the transcribed regions corresponding to the chimeric
forward fragment and reverse fragment will hybridize to form the
base-paired stem (i.e., double-stranded portion) of the hpRNA
structure.
[0184] In designing such a chimeric RNAi expression cassette, the
lengths of the forward fragment, spacer sequence, and reverse
fragments are chosen such that the combined length of the
polynucleotide that encodes the hpRNA structure is about 1200 to
about 3300 nt, about 1250 to about 3300 nt, about 1300 to about
3300 nt, about 1350 to about 3300 nt, about 1400 to about 3300 nt,
about 1450 nt to about 3300 nt, about 1500 to about 3300 nt, about
2200 to about 3100 nt, about 2250 to about 2800 nt, or about 2500
to about 2700 nt. In some embodiments, the combined length of the
expressed hairpin construct is about 1200 nt, about 1250 nt, about
1300 nt, about 1350 nt, about 1400 nt, about 1450 nt, about 1500
nt, about 1800 nt, about 2200 nt, about 2250 nt, about 2300 nt,
about 2350 nt, about 2400 nt, about 2450 nt, about 2500 nt, about
2550 nt, about 2600 nt, about 2650 nt, about 2700 nt, about 2750
nt, about 2800 nt, about 2850 nt, about 2900 nt, about 2950 nt,
about 3000 nt, about 3050 nt, about 3100 nt, about 3150 nt, about
3200 nt, about 3250 nt, about 3300 nt, or any such length between
about 1200 nt to about 3300 nt.
[0185] In some embodiments, the chimeric forward fragment comprises
about 500 to about 650 nt, for example, 500, 525, 550, 575, 600,
625, or 650 nt, of a sense strand for FucT, for example, of the
sense strand set forth in SEQ ID NO:1 or 2, and about 500 to about
650 nt, for example, 500, 525, 550, 575, 600, 625, or 650 nt, of a
sense strand for XylT, for example, of the sense strand set forth
in SEQ ID NO:4, 5, 19, or 20, where the FucT and XylT sequence can
be fused in either order; the spacer sequence comprises about 100
to about 700 nt, for example, 100, 200, 225, 250, 275, 300, 325,
350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,
675, or 700 nt of any sequence of interest; and the reverse
fragment comprises the antisense strand for the chimeric forward
fragment sequence, or a sequence having sufficient length and
sufficient complementarity to the chimeric forward fragment
sequence.
[0186] As noted above for the single-gene RNAi expression
cassettes, the spacer sequence can be any sequence that has
insufficient homology to the target gene, i.e., FucT or XylT, and
insufficient homology to itself such that the portion of the
expressed inhibitory RNA molecule corresponding to the spacer
region fails to self-hybridize, and thus forms the loop of the
hpRNA structure. In some embodiments, the spacer sequence comprises
an intron, and thus the expressed inhibitory RNA molecule forms an
ihpRNA as noted herein above. In other embodiments, the spacer
sequence comprises a portion of the sense strand for the FucT or
XylT gene to be silenced, for example, a portion of the sense
strand set forth in SEQ ID NO:1 or 2 (FucT) or SEQ ID NO:4, 5, 19,
or 20 (XylT). In one embodiment, the chimeric forward fragment
comprises the FucT and XylT sequence fused in that order, and the
spacer sequence comprises a portion of the XylT sense strand
immediately downstream from the XylT sequence contained within the
chimeric forward fragment. In another embodiment, the chimeric
forward fragment comprises the XylT and FucT sequence fused in that
order, and the spacer sequence comprises a portion of the FucT
sense strand immediately downstream from the FucT sequence
contained within the chimeric forward fragment.
[0187] In some embodiments, the chimeric RNAi expression cassette
is designed to suppress expression of the FucT polypeptide of SEQ
ID NO:3, a biologically active variant of the FucT polypeptide of
SEQ ID NO:3, or a FucT polypeptide encoded by a sequence having at
least 75% sequence identity to the sequence of SEQ ID NO:1 or SEQ
ID NO:2, for example, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% sequence identity to the sequence of SEQ
ID NO:1 or SEQ ID NO:2, and to suppress expression of the XylT
polypeptide of SEQ ID NO:6 or SEQ ID NO:21, a biologically active
variant of the XylT polypeptide of SEQ ID NO:6 or SEQ ID NO:21, or
a XylT polypeptide encoded by a sequence having at least 75%
sequence identity to the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:19, or SEQ ID NO:20, for example, at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% sequence identity to
the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID
NO:20. For some of these embodiments, the FucT sequence within the
chimeric forward fragment is chosen such that it corresponds to nt
700 to nt 1400 of SEQ ID NO:1 or SEQ ID NO:2, and/or the XylT
sequence within the chimeric forward fragment is chosen such that
it corresponds to nt 700 to nt 1400 of SEQ ID NO:4 or SEQ ID NO:5,
or nt 383 to 1083 of SEQ ID NO:19 or 20. Without being bound by
theory, it is believed that this region (particularly for FucT) is
relatively conserved among different plant species, and therefore
is a potentially good target.
[0188] In other embodiments, the sense strand of the chimeric RNAi
expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a chimeric forward fragment comprising nt 254-855 of SEQ
ID NO:1 (FucT sequence) and nt 318-943 of SEQ ID NO:4 (XylT
sequence); a spacer sequence comprising about 100 to about 700 nt
of any sequence as noted above; and a reverse fragment comprising
the complement (i.e., antisense version) of the chimeric forward
fragment, i.e., comprising the complement of nt 318-943 of SEQ ID
NO:4 and the complement of nt 254-855 of SEQ ID NO:1. In a
particular embodiment, the spacer sequence within this chimeric
RNAi expression cassette is represented by nt 944-1443 of SEQ ID
NO:4, and the total length of that portion of the sense strand of
the RNAi expression cassette corresponding to the coding sequence
for the hpRNA structure is 2956 nt.
[0189] In another such embodiment, the sense strand of the chimeric
RNAi expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a chimeric forward fragment comprising nt 318-943 of SEQ
ID NO:4 (XylT sequence) and nt 254-855 of SEQ ID NO:1 (FucT
sequence); a spacer sequence comprising about 100 to about 700 nt
of any sequence as noted above; and a reverse fragment comprising
the complement (i.e., antisense version) of the chimeric forward
fragment, i.e., comprising the complement of nt 254-855 of SEQ ID
NO:1 and the complement of nt 318-943 of SEQ ID NO:4. In a
particular embodiment, the spacer sequence within this chimeric
RNAi expression cassette is represented by nt 856-1355 of SEQ ID
NO:1, and the total length of that portion of the sense strand of
the RNAi expression cassette corresponding to the coding sequence
for the hpRNA structure is 2956 nt.
[0190] In yet other embodiments, the sense strand of the chimeric
RNAi expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a chimeric forward fragment comprising nt 254-855 of SEQ
ID NO:1 (FucT sequence) and nt 1-626 of SEQ ID NO:19 (XylT
sequence); a spacer sequence comprising about 100 to about 700 nt
of any sequence as noted above; and a reverse fragment comprising
the complement (i.e., antisense version) of the chimeric forward
fragment, i.e., comprising the complement of nt 1-626 of SEQ ID
NO:19 and the complement of nt 254-855 of SEQ ID NO:1. In a
particular embodiment, the spacer sequence within this chimeric
RNAi expression cassette is represented by nt 627-1126 of SEQ ID
NO:19, and the total length of that portion of the sense strand of
the RNAi expression cassette corresponding to the coding sequence
for the hpRNA structure is 2956 nt.
[0191] In another such embodiment, the sense strand of the chimeric
RNAi expression cassette is designed to comprise in the 5'-to-3'
direction the following operably linked elements: a promoter of
interest; a chimeric forward fragment comprising nt 1-626 of SEQ ID
NO:19 (XylT sequence) and nt 254-855 of SEQ ID NO:1 (FucT
sequence); a spacer sequence comprising about 100 to about 700 nt
of any sequence as noted above; and a reverse fragment comprising
the complement (i.e., antisense version) of the chimeric forward
fragment, i.e., comprising the complement of nt 254-855 of SEQ ID
NO:1 and the complement of nt 1-626 of SEQ ID NO:19. In a
particular embodiment, the spacer sequence within this chimeric
RNAi expression cassette is represented by nt 856-1355 of SEQ ID
NO:1, and the total length of that portion of the sense strand of
the RNAi expression cassette corresponding to the coding sequence
for the hpRNA structure is 2956 nt.
[0192] Stably transforming a plant with a nucleotide construct
comprising a chimeric RNAi expression cassette described herein,
for example, stable transformation with the vector shown in FIG.
10, effectively inhibits expression of both FucT and XylT within
the plant cells of the plant in which the hpRNA structure is
expressed. In one embodiment, the plant of interest is a member of
the duckweed family, for example, a member of the Lemnaceae, and
the plant has been stably transformed with the vector shown in FIG.
10.
[0193] It is recognized that the plant can be stably transformed
with at least two of these chimeric RNAi expression cassettes to
provide for very efficient gene silencing of the FucT and XylT
proteins, including silencing of any isoforms of these two
proteins. See, for example, the two orientations provided in
"possible design 2" of FIG. 28. In this manner, the plant can be
stably transformed with a first chimeric RNAi expression cassette
wherein the chimeric forward fragment comprises the FucT and XylT
sequence fused in that order, and the spacer sequence comprises a
portion of the XylT sense strand immediately downstream from the
XylT sequence contained within the chimeric forward fragment; and
with a second chimeric RNAi expression cassette wherein the
chimeric forward fragment comprises the XylT and FucT sequence
fused in that order, and the spacer sequence comprises a portion of
the FucT sense strand immediately downstream from the FucT sequence
contained within the chimeric forward fragment.
[0194] The operably linked promoter within any of the RNAi
expression cassettes encoding large hpRNA structures, or large
ihpRNA structures can be any promoter of interest that provides for
expression of the operably linked inhibitory polynucleotide within
the plant of interest, including one of the promoters disclosed
herein below. The regulatory region can comprise additional
regulatory elements that enhance expression of the inhibitory
polynucleotide, including, but not limited to, the 5' leader
sequences and 5' leader sequences plus plant introns discussed
herein below.
[0195] In yet other embodiments, the RNAi expression cassette can
be designed to provide for expression of small hpRNA structures
having a base-paired stem region comprising about 200 base pairs or
less. Expression of the small hpRNA structure is preferably driven
by a promoter recognized by DNA-dependent RNA polymerase III. See,
for example, U.S. Patent Application No. 20040231016, herein
incorporated by reference in its entirety.
[0196] In this manner, the RNAi expression cassette is designed
such that the transcribed DNA region encodes an RNA molecule
comprising a sense and antisense nucleotide region, where the sense
nucleotide sequence comprises about 19 contiguous nucleotides
having about 90% to about 100% sequence identity to a nucleotide
sequence of about 19 contiguous nucleotides from the RNA
transcribed from the gene of interest and where the antisense
nucleotide sequence comprises about 19 contiguous nucleotides
having about 90% to about 100% sequence identity to the complement
of a nucleotide sequence of about 19 contiguous nucleotides of the
sense sequence. The sense and antisense nucleotide sequences of the
RNA molecule should be capable of forming a base-paired (i.e.,
double-stranded) stem region of RNA of about 19 to about 200
nucleotides, alternatively about 21 to about 90 or 100 nucleotides,
or alternatively about 40 to about 50 nucleotides in length.
However, the length of the base-paired stem region of the RNA
molecule may also be about 30, about 60, about 70 or about 80
nucleotides in length. Where the base-paired stem region of the RNA
molecule is larger than 19 nucleotides, there is only a requirement
that there is at least one double-stranded region of about 19
nucleotides (wherein there can be about one mismatch between the
sense and antisense region) the sense strand of which is
"identical" (allowing for one mismatch) with 19 consecutive
nucleotides of the target FucT or XylT polynucleotide of interest.
The transcribed DNA region of this type of RNAi expression cassette
may comprise a spacer sequence positioned between the sense and
antisense encoding nucleotide region. The spacer sequence is not
related to the targeted FucT or XylT polynucleotide, and can range
in length from 3 to about 100 nucleotides or alternatively from
about 6 to about 40 nucleotides. This type of RNAi expression
cassette also comprises a terminator sequence recognized by the RNA
polymerase III, the sequence being an oligo dT stretch, positioned
downstream from the antisense-encoding nucleotide region of the
cassette. By "oligo dT stretch" is a stretch of consecutive
T-residues. It should comprise at least 4 T-residues, but obviously
may contain more T-residues.
[0197] It is recognized that in designing the short hpRNA, the
fragments of the targeted gene sequence (i.e., fragments of FucT or
XylT gene sequence) and any spacer sequence to be included within
the hpRNA-encoding portion of the RNAi expression cassette are
chosen to avoid GC-rich sequences, particularly those with three
consecutive G/C's, and to avoid the occurrence of four or more
consecutive T's or A's, as the string "TTTT . . . " serves as a
terminator sequence recognized by the RNA polymerase III.
[0198] Thus, where gene silencing with a short hpRNA is desired,
the RNAi expression cassette can be designed to comprise in the
5'-to-3' direction the following operably linked elements: a
promoter recognized by a DNA dependent RNA polymerase III of the
plant cell, as defined herein below; a DNA fragment comprising a
sense and antisense nucleotide sequence, wherein the sense
nucleotide sequence comprises at least 19 contiguous nucleotides
having about 90% to about 100% sequence identity to a nucleotide
sequence of at least 19 contiguous nucleotides from the sense
strand of the FucT or XylT gene of interest, and wherein the
antisense nucleotide sequence comprises at least 19 contiguous
nucleotides having about 90% to about 100% sequence identity to the
complement of a nucleotide sequence of at least 19 contiguous
nucleotides of the sense sequence, wherein the sense and antisense
nucleotide sequence are capable of forming a double-stranded RNA of
about 19 to about 200 nucleotides in length; and an oligo dT
stretch comprising at least 4 consecutive T-residues.
[0199] In some embodiments of the invention, the RNAi expression
cassette is designed to express a small hpRNA that suppresses
expression of the FucT polypeptide of SEQ ID NO:3, a biologically
active variant of the FucT polypeptide of SEQ ID NO:3, or a FucT
polypeptide encoded by a sequence having at least 90% sequence
identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2. In this
manner, the RNAi expression cassette can be designed to comprise in
the 5'-to-3' direction the following operably linked elements: a
promoter recognized by a DNA dependent RNA polymerase III of the
plant cell, as defined herein below; a DNA fragment comprising a
sense and antisense nucleotide sequence, wherein the sense
nucleotide sequence comprises at least 19 contiguous nucleotides
having about 90% to about 100% sequence identity to a nucleotide
sequence of at least 19 contiguous nucleotides of SEQ ID NO:1, and
wherein the antisense nucleotide sequence comprises at least 19
contiguous nucleotides having about 90% to about 100% sequence
identity to the complement of a nucleotide sequence of at least 19
contiguous nucleotides of the sense sequence, wherein the sense and
antisense nucleotide sequence are capable of forming a
double-stranded RNA of about 19 to about 200 nucleotides in length;
and an oligo dT stretch comprising at least 4 consecutive
T-residues.
[0200] In other embodiments of the invention, the RNAi expression
cassette is designed to express a small hpRNA that suppresses
expression of the XylT polypeptide of SEQ ID NO:6 or SEQ ID NO:21,
a biologically active variant of the XylT polypeptide of SEQ ID
NO:6 or SEQ ID NO:21, or a XylT polypeptide encoded by a sequence
having at least 90% sequence identity to the sequence of SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20. In this manner,
the RNAi expression cassette can be designed to comprise in the
5'-to-3' direction the following operably linked elements: a
promoter recognized by a DNA dependent RNA polymerase III of the
plant cell, as defined herein below; a DNA fragment comprising a
sense and antisense nucleotide sequence, wherein the sense
nucleotide sequence comprises at least 19 contiguous nucleotides
having about 90% to about 100% sequence identity to a nucleotide
sequence of at least 19 contiguous nucleotides of SEQ ID NO:4, and
wherein the antisense nucleotide sequence comprises at least 19
contiguous nucleotides having about 90% to about 100% sequence
identity to the complement of a nucleotide sequence of at least 19
contiguous nucleotides of the sense sequence, wherein the sense and
antisense nucleotide sequence are capable of forming a double
stranded RNA of about 19 to about 200 nucleotides in length; and an
oligo dT stretch comprising at least 4 consecutive T-residues.
[0201] Amplicon-Mediated Interference
[0202] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or
antisense relative to the target sequence (i.e., the messenger RNA
for FucT or XylT, or both). Methods of using amplicons to inhibit
the expression of endogenous plant genes are described, for
example, in Angell and Baulcombe (1997) EMBO J. 16:3675-3684,
Angell and Baulcombe (1999) Plant J. 20:357-362, and U.S. Pat. No.
6,646,805, each of which is herein incorporated by reference.
[0203] Ribozymes
[0204] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of FucT or XylT,
or both. Thus, the polynucleotide causes the degradation of the
endogenous messenger RNA, resulting in reduced expression of the
FucT or XylT, or both. This method is described, for example, in
U.S. Pat. No. 4,987,071, herein incorporated by reference.
[0205] Small Interfering RNA or Micro RNA
[0206] In some embodiments of the invention, inhibition of the
expression of FucT or XylT, or both, may be obtained by RNA
interference by expression of a gene encoding a micro RNA (miRNA).
miRNAs are regulatory agents consisting of about 22
ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes. See, for example Javier et al.
(2003) Nature 425: 257-263, herein incorporated by reference.
[0207] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). For suppression of FucT
or XylT expression, the 22-nucleotide sequence is selected from a
FucT or XylT transcript sequence and contains 22 nucleotides of
said FucT or XylT sequence in sense orientation and 21 nucleotides
of a corresponding antisense sequence that is complementary to the
sense sequence. miRNA molecules are highly efficient at inhibiting
the expression of endogenous genes, and the RNA interference they
induce is inherited by subsequent generations of plants.
[0208] Polypeptide-Based Inhibition of Gene Expression
[0209] In one embodiment, the polynucleotide encodes a zinc finger
protein that binds to a gene encoding a FucT or XylT, or both,
resulting in reduced expression of the gene. In particular
embodiments, the zinc finger protein binds to a regulatory region
of a FucT or XylT gene. In other embodiments, the zinc finger
protein binds to a messenger RNA encoding a FucT or XylT and
prevents its translation. Methods of selecting sites for targeting
by zinc finger proteins have been described, for example, in U.S.
Pat. No. 6,453,242, and methods for using zinc finger proteins to
inhibit the expression of genes in plants are described, for
example, in U.S. Patent Publication No. 20030037355; each of which
is herein incorporated by reference.
[0210] Polypeptide-Based Inhibition of Protein Activity
[0211] In some embodiments of the invention, the polynucleotide
encodes an antibody that binds to at least one FucT or XylT, and
reduces the activity of the FucT or XylT. In another embodiment,
the binding of the antibody results in increased turnover of the
antibody-FucT or XylT complex by cellular quality control
mechanisms. The expression of antibodies in plant cells and the
inhibition of molecular pathways by expression and binding of
antibodies to proteins in plant cells are well known in the art.
See, for example, Conrad and Sonnewald (2003) Nature Biotech.
21:35-36, incorporated herein by reference.
[0212] Gene Disruption
[0213] In some embodiments of the present invention, the activity
of FucT or XylT, or both, is reduced or eliminated by disrupting
the gene encoding the FucT or XylT, or both. The gene encoding the
FucT or XylT, or both, may be disrupted by any method known in the
art. For example, in one embodiment, the gene is disrupted by
transposon tagging. In another embodiment, the gene is disrupted by
mutagenizing plants using random or targeted mutagenesis, and
selecting for plants that have reduced FucT and/or XylT
activity.
[0214] Transposon Tagging
[0215] In one embodiment of the invention, transposon tagging is
used to reduce or eliminate the activity of FucT or XylT, or both.
Transposon tagging comprises inserting a transposon within an
endogenous FucT or XylT gene to reduce or eliminate expression of
the FucT or XylT. "FucT" or "XylT" gene is intended to mean the
gene that encodes a FucT or XylT, respectively, according to the
invention.
[0216] In this embodiment, the expression of FucT or XylT is
reduced or eliminated by inserting a transposon within a regulatory
region or coding region of the gene encoding the FucT or XylT. A
transposon that is within an exon, intron, 5' or 3' untranslated
sequence, a promoter, or any other regulatory sequence of a FucT or
XylT, or both, gene may be used to reduce or eliminate the
expression and/or activity of the encoded FucT or XylT.
[0217] Methods for the transposon tagging of specific genes in
plants are well known in the art. See, for example, Maes et al.
(1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti (1999) FEMS
Microbiol. Lett. 179:53-59; Meissner et al. (2000) Plant J.
22:265-274; Phogat et al. (2000) J. Biosci. 25:57-63; Walbot (2000)
Curr. Opin. Plant Biol. 2:103-107; Gai et al. (2000) Nucleic Acids
Res. 28:94-96; Fitzmaurice et al. (1999) Genetics 153:1919-1928).
In addition, the TUSC process for selecting Mu insertions in
selected genes has been described in Bensen et al. (1995) Plant
Cell 7:75-84; Mena et al. (1996) Science 274:1537-1540; each of
which is herein incorporated by reference.
[0218] The invention encompasses additional methods for reducing or
eliminating the activity of FucT or XylT. Examples of other methods
for altering or mutating a genomic nucleotide sequence in a plant
are known in the art and include, but are not limited to, the use
of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair
vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA
oligonucleotides, and recombinogenic oligonucleobases. Such vectors
and methods of use are known in the art. See, for example, U.S.
Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972;
and 5,871,984; each of which are herein incorporated by reference.
See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al.
(1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is
herein incorporated by reference.
[0219] Thus inhibition of expression of FucT and/or XylT in a
higher plant of interest can be accomplished by any of the
foregoing methods in order to alter the N-glycosylation pattern of
endogenous and heterologous glycoproteins produced within that
plant such that these glycoproteins comprise complex N-glycans that
have a reduction in the amount of .beta.1,2-linked xylose residues
and/or .alpha.1,3-linked fucose residues. The extent to which
attachment of the .beta.1,2-linked xylose residue and/or
.alpha.1,3-linked fucose residue to glycoprotein N-glycans is
reduced is governed by the degree of inhibition of expression of
the respective XylT and FucT enzymes.
[0220] In some embodiments of the invention, recombinant
glycoproteins produced in a plant host that is stably transformed
using the methods described herein to target XylT expression have
N-linked glycans comprising less than 50%, less than 40%, less than
30% of the .beta.1,2-linked xylose residues occurring in the
respective N-linked glycans of glycloproteins produced in a plant
host that has not been genetically modified to inhibit expression
of the XylT enzyme and isoforms thereof. In other embodiments,
these recombinant glycoproteins have N-linked glycans comprising
less than 25%, less than 20%, less than 15%, less than 10%, less
than 5%, or less than 1% of the .beta.1,2-linked xylose residues
occurring in the respective N-linked glycans of glycloproteins
produced in a plant host that has not been genetically modified to
inhibit expression of the XylT enzyme and isoforms thereof. In yet
other embodiments, the methods of the invention provide for
complete silencing of the XylT gene and any isoforms thereof within
the stably transformed plant, such that the recombinant
glycoproteins produced within the plant have N-linked glycans that
are devoid of .beta.1,2-linked xylose residues.
[0221] In like manner, where a plant host has been stably
transformed using the methods described herein to target FucT
expression, recombinant glycoproteins produced within the plant
have N-linked glycans comprising less than 50%, less than 40%, less
than 30% of the .alpha.1,3-linked fucose residues occurring in the
respective N-linked glycans of glycoproteins produced in a plant
host that has not been genetically modified to inhibit expression
of the FucT enzyme and isoforms thereof. In other embodiments,
these recombinant glycoproteins have N-linked glycans comprising
less than 25%, less than 20%, less than 15%, less than 10%, less
than 5%, or less than 1% of the .alpha.1,3-linked fucose residues
occurring in the respective N-linked glycans of glycoproteins
produced in a plant host that has not been genetically modified to
inhibit expression of the FucT enzyme and isoforms thereof. In yet
other embodiments, the methods of the invention provide for
complete silencing of the FucT gene and any isoforms thereof within
the stably transformed plant, such that the recombinant
glycoproteins produced within the plant have N-linked glycans that
are devoid of .alpha.1,3-linked fucose residues.
[0222] Where a plant host has been stably transformed using the
methods described herein to target expression of both the XylT and
FucT enzymes, and any isoforms thereof, recombinant glycoproteins
produced within the plant have N-linked glycans comprising less
than 50%, less than 40%, less than 30% of the .beta.-1,2-linked
xylose residues and less than 50%, less than 40%, less than 30% of
the .alpha.1,3-linked fucose residues occurring in the respective
N-linked glycans of glycoproteins produced in a plant host that has
not been genetically modified to inhibit expression of the XylT and
FucT enzymes and isoforms thereof. In other embodiments, these
recombinant glycoproteins have N-linked glycans comprising less
than 25%, less than 20%, less than 15%, less than 10%, less than
5%, or less than 1% of the .beta.1,2-linked xylose residues and
25%, less than 20%, less than 15%, less than 10%, less than 5%, or
less than 1% of the .alpha.1,3-linked fucose residues occurring in
the respective N-linked glycans of glycoproteins produced in a
plant host that has not been genetically modified to inhibit
expression of the XylT and FucT enzymes and isoforms thereof. In
yet other embodiments, the methods of the invention provide for
complete silencing of the XylT and FucT gene and any isoforms
thereof within the stably transformed plant, such that the
recombinant glycoproteins produced within the plant have N-linked
glycans that are devoid of .beta.1,2-linked xylose residues and
.alpha.1,3-linked fucose residues.
[0223] In some embodiments of the present invention, a plant host
that has been stably transformed using the methods described herein
to target expression of both the XylT and FucT enzymes, and any
isoforms thereof, is capable of producing recombinant glycoproteins
wherein the N-linked glycans are substantially homogenous. By
"substantially homogenous" is intended that the glycosylation
profile reflects the presence of a single major peak corresponding
to a desired N-glycan species, more particularly, the G0 glycan
species, wherein at least 90% of the N-glycan structures present on
said glycoproteins are of the G0 glycan species.
[0224] Methods for monitoring changes in the N-glycosylation
pattern of glycoproteins, also referred to as glycosylation
profiles, are well known in the art and include, but are not
limited to, matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry, for example, using
the modified MALDI-TOF assay disclosed in Example 3 herein below,
liquid chromatograph mass spectrometry (LC-MS), gas chromatography,
anion-exchange chromatography, size-exclusion chromatography,
high-concentration polyacrylamide gel electrophoresis, nuclear
magnetic resonance spectroscopy, and capillary electrophoresis and
capillary gel electrophoresis, fluorescence labeling and detection
by high-performance liquid chromatography (HPLC) and QTOF, and the
like. In this manner, changes in the N-glycosylation pattern due to
inhibition of the expression or function of XylT and/or FucT in a
stably transformed plant of the invention can be monitored by
subjecting a sample (for example, a leaf tissue sample) obtained
from the stably transformed plant to total N-glycan analysis by
MALDI-TOF mass spectrometry, and comparing the results with those
obtained for a comparable sample from a control plant, wherein the
control plant has not been genetically modified to inhibit
expression or function of XylT and/or FucT. A reduction in the
amount of xylose and/or fucose residues in the N-glycans can be
monitored by a reduction of the mass of the respective peaks. See,
for example, Strasser et al. (2004) FEBS Letters 561:132-136.
[0225] Similarly, the glycosylation profile of any given
recombinantly produced glycoprotein is readily determined using
standard techniques well known to those in the art. See, for
example, the review provided in Morelle and Michalski (2005) Curr.
Anal. Chem. 1:29-57; herein incorporated by reference in its
entirety. Thus, glycoproteins that have been recombinantly produced
in a host organism, including a plant host, can be analyzed for the
ratio of the particular N-linked glycan structures attached
thereto. In this manner, a sample comprising isolated recombinantly
produced glycoprotein can be subjected to enzymatic or chemical
reaction to release the individual glycan structures from the
glycoprotein. Following this deglycosylation step, analysis of the
glycosylation profile can be carried out using any of the
analytical assays described herein above.
[0226] Recombinantly produced glycoprotein products typically exist
as a diverse population of glycoforms carrying between one and
several dozen different glycans in variable molar amounts at
glycosylation sites with varying degrees of site occupancy.
Depending upon the glycoprotein, different glycoforms can yield
different functional profiles. Thus, in some embodiments of the
invention, it is desirable to determine the glycosylation profile
of the glycoprotein having the N-glycans intact. Any technique
known in the art for determining the glycosylation profile of an
intact glycoprotein can be used, including the mass spectrometry
methods noted above and in the examples herein below.
[0227] By reducing or eliminating the expression or function of
fucosyltransferase and/or xylosyltransferase in the manner set
forth herein, either transiently or stably, it is possible to
produce a transgenic higher plant having the ability to produce
glycoproteins having an N-glycosylation profile with reduced
heterogeneity relative to that normally observed for glycoproteins
produced by this plant when expression or function of these enzymes
has not been altered (i.e., the plant has the native or wild-type
glycosylation machinery). Where expression or function of one or
both of these enzymes is stably reduced or eliminated using one or
more of the methods described herein above, the reduction in the
heterogeneity of the N-glycosylation profile of glycoproteins
produced by the transgenic plant can be maintained from plant
generation to plant generation, including with asexual or sexual
reproduction, and can be maintained across cultural conditions and
with scale-up in production.
[0228] In this manner, the present invention provides a method for
reducing heterogeneity of the N-glycosylation profile of a
glycoprotein produced in a higher plant, for example, a
dicotyledonous or monocotyledonous plant, for example, a duckweed
plant. The method comprises introducing into the plant a nucleotide
construct described herein such that the expression or function of
fucosyltransferase and/or xylosyltransferase is reduced or
eliminated within the plant. In some embodiments of the invention,
the method for reducing heterogeneity of the N-glycosylation
profile of a glycoprotein produced in a higher plant comprises
introducing into the higher plant of interest at least one
nucleotide construct described herein above, where the nucleotide
construct(s) provides for suppression of the expression of
fucosyltransferase and/or xylosyltransferase in the plant, for
example, using one or more of the methods described herein
above.
[0229] By "reducing heterogeneity of the N-glycosylation profile"
it is intended that the N-glycosylation profile is characterized by
a reduction in the total number of distinct N-glycan species that
appear in the profile. Thus, for example, where a glycoprotein
produced by a higher plant having the native or wild-type
glycosylation machinery (and thus which has not been genetically
modified to reduce or eliminate expression of fucosyltransferase
and xylosyltransferase) produces a glycoprotein with an
N-glycosylation profile characterized by the presence of a mixture
of 5 N-glycan species, the methods of the invention can be used to
reduce the number of N-glycan species appearing in the
N-glycosylation profile. In this manner, when that higher plant is
genetically modified in the manner set forth herein to reduce or
eliminate expression or function of fucosyltransferase and/or
xylosyltransferase, the N-glycosylation profile of this
glycoprotein would be characterized by a reduction in the number of
N-glycan species appearing in the profile, for example, a mixture
of fewer than 5 N-glycan species, for example, 4, 3, or 2 N-glycan
species, or even a single N-glycan species. Where heterogeneity of
the N-glycosylation profile is reduced such that the profile is
characterized by the presence of a single predominant N-glycan
species, the N-glycosylation profile would be substantially
homogeneous for that N-glycan species.
[0230] In some embodiments, the methods for reducing the
heterogeneity of the N-glycosylation profile of a glycoprotein
produced in a higher plant result in the produced glycoprotein
having an N-glycosylation profile that is substantially homogeneous
for the G0 glycan species. In such embodiments, the methods for
reducing the heterogeneity of the N-glycosylation profile of a
glycoprotein produced in a higher plant result in the produced
glycoprotein having a substantially homogeneous N-glycosylation
profile, wherein at least 80%, at least 85%, at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the total
amount of N-glycan species appearing in the N-glycosylation profile
for the glycoprotein is represented by the G0 glycan species. In
these embodiments, a trace amount of precursor N-glycan species may
appear in the N-glycosylation profile as noted elsewhere herein,
where any given precursor N-glycan species that is present in the
N-glycosylation profile is present at less than 5%, preferably less
than 4%, less than 3%, less than 2%, less than 1%, and even less
than 0.5% or even less than 0.1% of the total amount of N-glycan
species appearing in the profile.
[0231] The glycoprotein can be an endogenous glycoprotein of
interest, or can be a heterologous glycoprotein that is produced by
the higher plant of interest, for example, a mammalian
glycoprotein, including, for example, the glycoproteins described
elsewhere herein. In some embodiments, the glycoprotein is a
monoclonal antibody. In other embodiments, the glycoprotein is
selected from the group consisting of an interferon, erythropoietin
(EPO), tissue plasminogen activator (tPA), plasminogen,
granulocyte-macrophage colony stimulating factor (GM-CSF), and
therapeutic immunoglobulins.
[0232] Using the methods of the present invention, it is possible
to maintain the reduced heterogeneity within the N-glycosylation
profile for a glycoprotein produced in the transgenic plant with
scale-up in production, and thus the plant continues to produce the
glycoprotein such that its N-glycosylation profile is characterized
by a reduction in the number of N-glycan species appearing in the
profile. By "scale-up in production" or "increase in production
scale" is intended an increase in the amount of plant biomass that
is present within a culture system (i.e., a culture vessel or
culture container within which the plant is cultured) that is being
used to produce a protein of interest, in this case, a glycoprotein
of interest. Thus, scale-up in production occurs, for example, when
scaling up production from a scale that is suitable for research
purposes to one that is suitable for pilot production, and further
up to a scale that is suitable for commercial production of the
glycoprotein of interest.
[0233] In some embodiments, the transgenic higher plant is a
monocotyledonous plant, for example, a duckweed plant, that serves
as a host for recombinant production of a glycoprotein, and the
reduced heterogeneity of the N-glycosylation profile of the
recombinantly produced glycoprotein is maintained with an increase
in production scale, where the production scale is increased by at
least 300-fold, at least 500-fold, at least 700-fold, at least
1,000-fold, at least 1,500-fold or greater over the initial
starting biomass. In some of these embodiments, the transgenic
higher plant is a duckweed plant that recombinantly produces a
glycoprotein of interest, and the reduced heterogeneity of the
N-glycosylation profile is maintained with an increase in
production scale, where the production scale is increased by at
least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at
least 5,000-fold, at least 6,000-fold, at least 6,500-fold, or
greater over the initial starting biomass. In one such embodiment,
the higher plant is a duckweed plant that recombinantly produces a
glycoprotein of interest, and the reduced heterogeneity of the
N-glycosylation profile for that glycoprotein is maintained with an
increase in production scale, where the production scale is
increased by at least 7,000-fold, 8,000-fold, 9,000-fold,
10,000-fold, 12,500-fold, 15,000-fold, 17,500-fold, 20,000-fold,
23,000-fold, 26,000-fold, or greater over the initial starting
biomass.
[0234] Furthermore, when the transgenic plant of interest is to be
maintained by continuous clonal culture, the resulting transgenic
line continues to produce glycoproteins that exhibit the reduced
heterogeneity within their N-glycosylation profile. Continuous
clonal culture can be achieved using any suitable method known in
the art. In some embodiments, continuous clonal culture is achieved
by periodically taking one or more subsamples of the plant culture
and transferring the subsample(s) to fresh culture medium for
further culture. Thus, for example, in some embodiments, the
transgenic plant line that is maintained by continuous clonal
culture is a duckweed transgenic plant line that has been
genetically modified to reduce or eliminate expression or function
of fucosyltransferase and/or xylosyltransferase. In this manner,
the reduced heterogeneity of the N-glycosylation profile of
glycoproteins produced in the transgenic plant line is maintained
with continuous clonal culture of the transgenic plant line for at
least 8 months, at least 10 months, at least 1 year, at least 1.5
years, at least 2 years, at least 2.5 years, at least 3 years, at
least 3.5 years, at least 4 years, at least 4.5 years, at least 5
years, or longer, and can be maintained for as long as the
transgenic plant line is maintained.
Heterologous Polypeptides and Glycoproteins
[0235] Higher plants, particularly higher plants that serve as
expression systems for recombinant proteins for pharmaceutical use,
that have been stably transformed to produce glycoproteins with an
altered N-glycosylation pattern using the methods described herein
may be genetically modified to produce any recombinant protein of
interest. Where the recombinant protein is one in which
post-translational glycosylation is applicable, the methods of the
invention advantageously provide a means to produce these
glycoproteins with an N-glycosylation pattern that more closely
reflects that of mammalian hosts, particularly a glycosylation
pattern that is "humanized." Examples of recombinant proteins of
interest include, but are not limited to, insulin, growth hormone,
plasminogen, .alpha.-interferon, .beta.-interferon,
.beta.-glucocerebrosidase, .beta.-glucoronidase, retinoblastoma
protein, p53 protein, angiostatin, leptin, monoclonal antibodies
and fragments thereof, cytokines, for example, erythropoietin
(EPO), granulocyte macrophage colony stimulating factor, tissue
plasminogen activator, blood coagulation factors, for example,
Factor VII, Factor VIII, Factor IX, and activated protein C, alpha
1-antitrypsin, receptors, hormones, human vaccines, animal
vaccines, peptides, and serum albumin. Furthermore, the transgenic
higher plants of the invention are capable of producing a
glycoprotein product that has a substantially homogenous
glycosylation profile for the G0 glycan species, and which is
characterized by its substantial homogeneity for the G0 glycoform.
This advantageously results in plant host expression systems that
have increased production consistency, as well as reduced chemical,
manufacturing, and control (CMC) risk associated with the
production of these glycoprotein compositions.
[0236] The glycoprotein compositions produced in accordance with
the methods of the present invention may be contained in a
composition comprising a pharmaceutically acceptable carrier. Such
compositions are useful in a method of treating a subject for a
disease or disorder for which treatment with the glycoprotein will
provide a therapeutic benefit. In this manner, glycoproteins that
have been produced in a plant, for example, a duckweed plant,
stably transformed in accordance with the methods of the present
invention can be administered to a subject in need thereof.
[0237] The glycoprotein compositions of the invention comprise
N-linked glycans that are predominately of the G0 glycan structure.
In this manner, the present invention provides glycoprotein
compositions that have glycosylation profiles that are
"substantially homogeneous" or "substantially uniform" or have
"substantial homogeneity" as defined herein above. Thus, in some
embodiments, the glycoprotein compositions are substantially
homogeneous for the G0 glycan species, and thus have a
substantially homogeneous glycosylation profile, wherein at least
80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or at least 99% of the total amount of N-glycan species
appearing in the glycosylation profile for the composition is
represented by the G0 glycan species, with a trace amount of
precursor N-glycan species appearing in the glycosylation profile,
i.e., any given precursor N-glycan species that is present in the
glycosylation profile is present at less than 5%, preferably less
than 4%, less than 3%, less than 2%, less than 1%, and even less
than 0.5% or even less than 0.1% of the total amount of N-glycan
species appearing in the profile. For such a composition, a
representative precursor N-glycan species appearing in its
glycosylation profile would be the Man3GlcNAc2, MGn
(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose
arm), and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to
the 1,6 mannose arm) precursor N-glycan species described above,
where any single one or any combination of these precursor N-glycan
species can be present.
[0238] In this manner, the invention provides "substantially
homogeneous" or "substantially uniform" glycoprotein compositions
or glycoprotein compositions having "substantial homogeneity" as
defined herein above. In some embodiments, the invention provides
substantially homogeneous glycoprotein compositions, wherein at
least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or at least 99% of the glycoprotein present in the
composition is represented by the G0 glycoform, wherein all
anticipated glycosylation sites are occupied by the G0 glycan
species, with a trace amount of precursor or undesired glycoforms
being present in the composition, i.e., the precursor glycoforms
represent less than 5%, less than 4%, less than 3%, less than 2%,
less than 1%, or even less than 0.5%, or less than 0.1% of the
total glycoforms present within the composition. In such a
composition, a representative precursor glycoform would be one in
which glycosylation sites are unoccupied, and an exemplary
undesired glycoform would be a glycoform having a mixture of G0
glycan and G0X or G0XF3 glycan species attached to its
glycosylation sites.
[0239] In some embodiments of the invention, the plant host
comprises one or more polynucleotides that provide for expression
of an antibody that specifically binds to a mammalian protein of
interest, particularly a human protein of interest. Thus, in one
aspect, the invention provides methods for producing monoclonal
antibodies in higher plants, wherein the monoclonal antibodies have
an N-glycosylation pattern that reflects a reduction in the amount
of .beta.1,2-linked xylose residues and .alpha.1,3-linked fucose
residues within the N-linked glycans, and compositions comprising
recombinant monoclonal antibodies produced using plant hosts
genetically modified in the manner set forth herein. In some
embodiments, the plant host of interest is a member of the duckweed
family.
[0240] Monoclonal antibodies are increasingly being used as
therapeutic agents to treat human disease, including, but not
limited to, cancer and diseases having an autoimmune or
inflammatory component. See, for example, King (1999) Curr. Opin.
Drug Discovery Dev. 2:110-17; Vaswani and Hamilton (1998) Ann.
Allergy Asthma Immunol. 81:105-19; and Holliger and Hoogenboom,
Nat. Biotechnology 16:1015-16; each of which is herein incorporated
by reference. Although some of these antibodies have therapeutic
effects that result solely from antigen binding, for example
antibodies that bind to a receptor or ligand to prevent
ligand-receptor interactions, other antibodies need effector
functions such as the recruitment of the immune system to kill
target cells in order to be therapeutically active. See, for
example, Clynes et al. (2000) Nat. Med. 6:443-46; Clynes et al.
(1998) Proc. Natl. Acad. Sci. U.S.A. 95:652-56, and Anderson et al.
(1997) Biochem. Soc. Trans. 25:705-8; each of which is herein
incorporated by reference.
[0241] The antigen-recognition activities and effector functions of
antibodies reside in different portions of the antibody molecule.
The Fab' portion of the antibody provides antigen recognition
activity, while the Fc portion provides effector functions such as
the activation of accessory effector cells including phagocytic
cells (macrophages and neutrophils), natural killer cells, and mast
cells. Antibodies bind to cells via the Fc region, with an Fc
receptor site on the antibody Fc region binding to an Fc receptor
(FcR) on a cell. There are a number of Fc receptors that are
specific for different classes of antibodies, including IgG (gamma
receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu
receptors). Binding of antibody to Fc receptors on cell surfaces
triggers a number of important and diverse biological responses
including engulfment and destruction of antibody-coated particles,
clearance of immune complexes, lysis of antibody-coated target
cells by killer cells (called antibody-dependent cell-mediated
cytotoxicity, or ADCC), initiation of complement-dependent
cytotoxicity (CDC), release of inflammatory mediators, and control
of immunoglobulin production. Methods for assaying effector
function of antibodies are well known in the art and include those
assaying for CDC, ADCC, and apoptosis. See, for example,
Subbramanian et al. (2002) J. Clin. Microbiol. 40:2141-2146; Ahman
et al. (1994) J. Immunol. Methods 36:243-254; Brezicka et al.
(2000) Cancer Immunol. Immunother. 49:235-242; Gazzano-Santoro et
al. (1997) J. Immunol. Methods 202:163-171; Prang et al. (2005)
British J. Cancer 92:342-349; Shan et al. (1998) Blood
92:3756-3771; Ghetie et al. (2001) Blood 97:1392-1398; and, Mathas
et al. (2000) Cancer Research 60:7170-7176; all of which are herein
incorporated by reference.
[0242] It is known in the art that the glycosylation status of the
Fc portion of an antibody molecule plays a key role in determining
whether an antibody will have effector function. See, for example,
Tao and Morrison (1987) J. Immunol. 143:2595-601; Wright and
Morrison (1997) Trends in Biotech. 15:26-32; Wright and Morrison
(1998) J. Immunol. 160:3393-402; Mimura et al. (2000) Mol. Immunol.
37:697-706; Jefferis and Lund (2002) Immunol. Lett. 82:57-65; Krapp
et al. (2003) J. Mol. Biol. 325:979-89; and Jefferis (2005)
Biotechnol. Prog. 21:11-16; each of which is herein incorporated by
reference. Glycosylation of recombinantly produced antibodies
varies depending on the expression system used. See, for example,
Raju et al. (2000) Glycobiology 10:477-86; Wright and Morrison
(1997) Trends in Biotech. 15:26-32. Further, where the
N-glycosylation pattern of a mammalian-produced monoclonal antibody
is altered to reduce or deplete the .alpha.(1,6)-linked core fucose
residue, the monoclonal antibody exhibits increased effector
function, particularly increased ADCC activity. See, for example,
U.S. Pat. No. 6,946,292. For some mammalian-produced monoclonal
antibodies, where the N-glycosylation pattern is altered to reduce
or deplete the .beta.(1,4)-galactose residues attached to the 1,3
and/or 1,6 mannose arms, activation of complement-dependent
cytotoxicity (CDC) against antigen-bearing target cells may be
reduced without altering other functional activities of the
antibody, including ADCC activity. See, for example, Boyd et al.
(1995) Mol. Immunol. 32:1311-1318.
[0243] Antibodies having antigen recognition activity, and in some
embodiments improved effector function, may be produced by a higher
plant host, such as duckweed, that has been stably transformed in
the manner set forth herein to alter its glycosylation machinery.
Accordingly, the present invention provides methods for producing a
recombinant monoclonal antibody, including a monoclonal antibody
having improved effector function, wherein the antibody is
recombinantly produced within a plant having an altered
N-glycosylation pattern of endogenous and heterologous
glycoproteins produced therein such that these glycoproteins
exhibit a reduction in the amount of the plant-specific
.beta.1,2-linked xylose residues and/or .alpha.1,3-linked fucose
residues attached to the N-glycans thereof. Where the antibodies
have reduced amounts .alpha.1,3-linked fucose residues attached to
the N-glycans thereof, the antibodies may have increased ADCC
activity relative to antibodies produced in a control plant that
has not been genetically modified to inhibit expression or function
of FucT.
[0244] Also encompassed are recombinant monoclonal antibodies
having effector function, and in some embodiments, improved
effector function, where the antibodies are produced in a duckweed
expression system that has been genetically modified to inhibit
expression or function of the FucT of SEQ ID NO:3 and/or the XylT
of SEQ ID NO:6, and any isoforms thereof, for example, the XylT
isoform #2 comprising the sequence set forth in SEQ ID NO:21
(encoded by SEQ ID NO:20). Thus, in some embodiments, the plant
serving as the host for recombinant production of the monoclonal
antibody is a member of the Lemnaceae as noted elsewhere herein,
for example, a Lemna plant, comprising, for example, a XylT RNAi
expression cassette and/or a FucT RNAi expression cassette
described above stably integrated within its genome. In this
manner, the present invention provides a method for producing a
recombinant monoclonal antibody having an N-glycosylation pattern
that more closely resembles that found in a mammalian host
expression system, and with improved effector function, where the
method comprises expressing one or more chains of the antibody in a
duckweed plant, or duckweed cell or duckweed nodule, that has been
genetically modified to alter the glycosylation machinery such that
the recombinantly produced monoclonal antibody exhibits a reduction
in the attachment of the plant .beta.1,2-linked xylose residue
and/or .alpha.1,3-linked fucose residue to the N-glycans thereof,
and culturing the duckweed plant, or duckweed cell or duckweed
nodule, under conditions suitable for expression of the monoclonal
antibody.
[0245] Thus the present invention provides novel antibody
compositions wherein the antibody comprises N-linked glycans that
are predominately of the G0 glycan structure. In this manner, the
present invention provides antibody compositions, for example,
monoclonal antibody compositions, that have glycosylation profiles
that are "substantially homogeneous" or "substantially uniform" or
have "substantial homogeneity" as defined herein above. Thus, in
some embodiments, the antibody compositions are substantially
homogeneous for the G0 glycan species, and thus have a
substantially homogeneous glycosylation profile, wherein at least
80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or at least 99% of the total amount of N-glycan species
appearing in the glycosylation profile for the composition is
represented by the G0 glycan species, with a trace amount of
precursor N-glycan species appearing in the glycosylation profile,
i.e., any given precursor N-glycan species that is present in the
glycosylation profile is present at less than 5%, preferably less
than 4%, less than 3%, less than 2%, less than 1%, and even less
than 0.5% or even less than 0.1% of the total amount of N-glycan
species appearing in the profile. For such a composition, a
representative precursor N-glycan species appearing in its
glycosylation profile would be, for example, the Man3GlcNAc2, MGn
(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose
arm), and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to
the 1,6 mannose arm) precursor N-glycan species described above,
where any single one or any combination of these precursor N-glycan
species can be present.
[0246] In one such embodiment, the monoclonal antibody composition
has a substantially homogeneous glycosylation profile, wherein
95.8% of the total amount of N-glycan species appearing in the
glycosylation profile for the composition is represented by the G0
glycan species (GlcNAc.sub.2Man.sub.3GlcNAc.sub.2), with the
following precursor N-glycan species appearing in the glycosylation
profile: Man.sub.3GlcNAc.sub.2 (0.67%), GlcNAcMan.sub.3GlcNAc.sub.2
(1.6%), GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (1.2%),
Man.sub.6GlcNAc.sub.2 (0.21%), Man.sub.7GlcNAc.sub.2 (0.30%), and
Man.sub.8GlcNAc.sub.2 (0.28%). This can be compared with the
monoclonal antibody composition obtained from the "wild-type"
duckweed plant expression system wherein the same monoclonal
antibody is expressed but where the glycosylation machinery of the
duckweed plant has not been genetically modified to inhibit
expression of XylT and FucT. Such a "wild-type"-derived monoclonal
antibody composition has a more heterogeneous glycosylation profile
that is characterized by two predominant N-glycan species, i.e.,
G0XF.sup.3 and G0X, with several precursor N-glycan species
represented in trace amounts. In one such embodiment, the
"wild-type"-derived monoclonal antibody composition has a
glycosylation profile with the following N-glycan species
represented therein: G0 (GlcNAc.sub.2Man.sub.3GlcNAc.sub.2) (8.4%);
G0X (GlcNAc.sub.2[Xyl]Man.sub.3GlcNAc.sub.2) (17.2%); G0XF.sup.3
(GlcNAc.sub.2[Xyl]Man.sub.3[Fuc]GlcNAc.sub.2) (67.4%);
Man.sub.3GlcNAc.sub.2 (0.26%); GlcNAcMan.sub.3GlcNAc.sub.2 (0.40%);
(Xyl)Man.sub.3(Fuc)GlcNAc.sub.2 (0.76%);
GlcNAc.sub.2Man.sub.3(Fuc)GlcNAc.sub.2 (2.1%);
GlcNAc(Xyl)Man.sub.3(Fuc)GlcNAc.sub.2 (1.4%); Man.sub.6GlcNAc.sub.2
(0.21%); Man.sub.7GlcNAc.sub.2 (0.63%);
Gal(Fuc)GlcNAc.sub.2(Xyl)Man.sub.3(Fuc)GlcNAc.sub.2 (0.26%);
Man.sub.8GlcNAc.sub.2 (0.61%); and Man.sub.9GlcNAc.sub.2
(0.40%).
[0247] In this manner, the invention provides "substantially
homogeneous" or "substantially uniform" antibody compositions or
antibody compositions having "substantial homogeneity" as defined
herein above. In some embodiments, the invention provides
substantially homogeneous antibody compositions, for example,
monoclonal antibody compositions, wherein at least 80%, at least
85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
least 99% of the antibody present in the composition is represented
by the G0 glycoform, wherein all anticipated glycosylation sites
(for example, each of the Asn-297 residues of the C.sub.H2 domains
of the heavy chains of an IgG-type antibody) are occupied by the G0
glycan species, with a trace amount of precursor glycoforms being
present in the composition. In one such composition, the precursor
glycoforms are selected from the group consisting of an antibody
having an Fc region wherein the C.sub.H2 domain of one heavy chain
has a G0 glycan species attached to Asn 297, and the C.sub.H2
domain of the other heavy chain is unglycosylated; an antibody
having an Fc region wherein the C.sub.H2 domain of one heavy chain
has a G0 glycan species attached to Asn 297, and the C.sub.H2
domain of the other heavy chain has the GnM or MGn precursor glycan
attached to Asn 297; and an antibody having an Fc region wherein
the Asn 297 glycosylation site on each of the C.sub.H2 domains has
a G0 glycan species attached, with a third G0 glycan species
attached to an additional glycosylation site within the mAb
structure; wherein a trace amount of these precursor glycoforms is
present, i.e., the precursor glycoforms represent less than 5%,
less than 4%, less than 3%, less than 2%, less than 1%, or even
less than 0.5%, or less than 0.1% of the total glycoforms present
within the antibody composition.
[0248] The substantially homogeneous antibody compositions of the
invention wherein at least 80%, at least 85%, at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the antibody
present in the composition is represented by the G0 glycoform
represent "glycan-optimized antibodies." By "glycan-optimized
antibodies" is intended the antibodies of the invention have been
genetically engineered in their glycosylation pattern such that
they are substantially homogeneous for the G0 glycoform, which
yields an antibody having improved Fc effector function. By
"improved Fc effector function" is intended these antibodies have
increased ADCC activity relative to same-sequence antibodies (i.e.,
antibodies that have the same amino acid sequence) that, as a
result of the production process, have a more heterogeneous
glycosylation profile. Thus, for example, antibodies produced in
mammalian host expression systems, for example CHO cells, in insect
host cells, in yeast cells, or in other plant host expression
systems that have not been genetically altered to inhibit XylT and
FucT expression tend to have more heterogeneous glycosylation
profiles, and thus a mixture of glycoforms, that can effect overall
effector function of the antibody product. The G0 glycoform of the
antibody compositions of the present invention advantageously
provides an antibody composition that has increased ADCC activity
in association with the absence of fucose residues. In some
embodiments, ADCC activity is increased by 25-fold, 50-fold,
75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold,
400-fold, 500-fold, or even 1000-fold relative to same-sequence
antibodies having a heterogeneous glycosylation profile (i.e., with
multiple glycoforms present as major glycoforms in the antibody
composition). Furthermore, the G0 glycoform lacks the terminal Gal
residues present in antibodies having the G2 glycoform. As such,
these substantially homogeneous antibody compositions of the
invention having predominately the G0 glycoform have increased
ADCC/CDC ratios. In addition, the substantially homogeneous
antibody compositions of the invention having predominately the G0
glycoform have increased binding to the Fc.gamma.RIII, for example,
Fc.gamma.RIIIa, wherein binding affinity is increased about
20-fold, 30-fold, 40-fold, 50-fold, 75-fold, up to 100-fold over
that observed for same-sequence antibody compositions having a
heterogeneous glycosylation profile, and thus a mixture of
glycoforms. For oncology and autoimmune diseases, therapeutic
antibodies having increased binding affinity for Fc receptors, for
example, Fc.gamma.RIII, has been strongly correlated with increased
efficacy and improved response to treatment.
[0249] In some embodiments of the invention, the substantially
homogeneous antibody compositions of the invention having
predominately the G0 glycoform have altered CDC activity when
compared to that observed for same-sequence antibody compositions
having a heterogeneous glycosylation profile, and thus a mixture of
glycoforms. For example, in one such embodiment, the substantially
homogeneous antibody compositions of the invention have
predominately the G0 glycoform and have decreased CDC activity when
compared to that observed for same-sequence antibody compositions
having a heterogeneous glycosylation profile, and thus a mixture of
glycoforms. Thus, in some embodiments, the present invention
provides substantially homogeneous antibody compositions having
predominately the G0 glycoform and CDC activity that is decreased
by as much as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% when compared
to same-sequence antibody compositions having a heterogeneous
glycosylation profile. In some of these embodiments, the
substantially homogeneous antibody compositions having
predominately the G0 glycoform and decreased CDC activity are
further characterized by having increased ADCC activity when
compared to same-sequence antibody compositions having a
heterogeneous glycosylation profile.
[0250] Without being bound by any theory or mechanism of action, a
substantially homogeneous G0 glycoform antibody composition of the
present invention having decreased CDC activity, and similar or
increased ADCC activity in the manner described above, may
advantageously provide for increased cytotoxicity against target
cells while reducing potential adverse side effects that may be
associated with complement activation following its administration.
By reducing the potential for these adverse side effects, the
substantially homogeneous antibody compositions having
predominately the G0 glycoform can advantageously be administered
at faster infusion rates, thereby reducing dosing time at any given
administration, and/or can be dosed at higher initial
concentrations if warranted, with reduced concern for triggering
adverse side affects associated with complement activation.
[0251] For example, complement activation plays a pivotal role in
the pathogenesis of moderate to severe first-dose side effects of
treatment with the chimeric anti-CD20 monoclonal antibody IDEC-C2B8
(IDEC Pharmaceuticals Corp., San Diego, Calif.; commercially
available under the tradename Rituxan.RTM., also referred to as
rituximab). See, for example, van der Kolk et al. (2001) British J.
Haematol. 115:807-811. The rituximab antibody within the
Rituxan.RTM. product is expressed within Chinese hamster ovary
(CHO) cells, and thus the antibody composition comprises a
heterogeneous glycosylation profile (i.e., a mixture of
glycoforms). CDC activity of rituximab has been shown to be
correlated with galactose content. In this manner, as the number of
galactose residues increases from 0-2 moles/mole of heavy chain,
the level of CDC activity increases from 80% (.beta.-galactosidase
treated to remove all .beta.(1,4)-galactose residues from the 1,3
and 1,6 mannose arms of the N-glycans attached to Asn 297 of the
C.sub.H2 domains of the heavy chains) to 150% (UDP galactosyl
transferase treated to ensure .beta.(1,4)-galactose residues are
attached to both the 1,3 and 1,6 mannose arms of the N-glycans
attached to Asn 297 sites) of the maximum observed for the antibody
having 1 mole galactose/mole of heavy chain (see, IDEC BLA 97-0260
at the website fda.gov/Cder/biologics/review/ritugen112697,
available on the worldwide web). A substantially homogeneous
anti-CD20 antibody composition comprising anti-CD20 antibody having
the same sequence as rituximab and having predominately the G0
glycoform would advantageously have decreased CDC activity, thereby
reducing the potential for adverse side effects normally associated
with complement activation upon antibody administration when the
antibody composition comprises a heterogenous glycosylation profile
(i.e., a mixture of glycoforms).
[0252] Thus, a substantially homogeneous G0 glycoform antibody
composition of the present invention having decreased CDC activity,
and the same or increased ADCC activity, can advantageously be used
in therapeutic applications that have heretofore been unsuitable,
inadvisable, or inefficacious for one or more patient populations
as a result of complications due to adverse side effects normally
associated with complement activation upon administration of the
same-sequence antibody composition that comprises a heterogeneous
glycosylation profile (i.e., a mixture of glycoforms). Such side
effects include, but are not limited to, moderate to severe side
effects that can be associated with first-time and/or rapid
administration of an antibody, including, for example, fever and/or
chills, nausea, dyspnea, flushes, and the like. See, for example,
van der Kolk et al. (2001) British J. Haematol. 115:807-811 and the
references cited therein; Winkler et al. (1999) Blood
94:2217-2224). In this manner, the present invention provides a
method for reducing one or more adverse side effects related to
complement activation upon administration of an antibody, for
example, a monoclonal antibody, the method comprising administering
the antibody as a substantially homogeneous antibody composition as
defined herein above, and thus at least 80%, at least 85%, at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the
antibody present in the composition is represented by the G0
glycoform, with a trace amount of precursor glycoforms being
present in the composition. In some embodiments, at least 90%, at
least 95%, or at least 99% of the antibody present in the
composition is represented by the G0 glycoform, with a trace amount
of precursor glycoforms being present in the composition.
[0253] As a result of their increased Fc effector function, the
substantially homogenous antibody compositions of the invention
having the predominately G0 glycoform provide the opportunities for
new and improved routes of administration, for example, extending
the possible routes of administration for known therapeutic
antibodies to other routes of administration beyond infusion and
intravenous administration, for example, to subcutaneous
administration. Furthermore, as a result of their increased
potency, the antibody compositions of the invention can be dosed at
lower concentrations, or dosed at lower volumes, and dosed with
less frequency. A reduction in the volume of the administered
antibody composition is particularly advantageous in those
instances where adverse events resulting from infusion reactions
with a monoclonal antibody are volume-related. The increased
potency of the antibody compositions of the invention also opens up
new clinical indications for existing mAb targets that may not have
been responsive to antibody therapy with more heterogenous
glycoform antibody compositions, and provides the ability to
revisit previously undeveloped mAb targets.
[0254] The monoclonal antibodies produced in accordance with the
methods of the present invention may be contained in a composition
comprising a pharmaceutically acceptable carrier. Such compositions
are useful in a method of treating a subject in need of an antibody
having effector function, and in some embodiments, improved
effector function where FucT expression has been targeted for
inhibition. In this manner, monoclonal antibodies produced in a
plant, for example, a duckweed plant, stably transformed in
accordance with the methods of the present invention can be
administered to a subject in need thereof.
[0255] Any antibody may be produced within a plant host genetically
modified according to the methods of the invention. In one
embodiment, the antibody is a therapeutic antibody. Antibodies have
been approved for the treatment of a number of disorders, and many
additional therapeutic antibodies are in development. See, Brekke
and Sandlie (2003) Nat. Rev. Drug. Discov. 2(3):240, herein
incorporated by reference. Examples of therapeutic antibodies that
may be expressed by the methods of the invention include, but are
not limited to, anti-CD3 antibodies targeting the CD3 antigen
(e.g., OKT.RTM.3); anti-platelet gpIIb/IIIa antibodies (e.g.
abciximab (previously known as c7E3 Fab), distributed as
ReoPro.RTM.); anti-EpCAM antibodies (e.g., Panorex.RTM.); anti-CD20
antibodies targeting the CD20 antigen (e.g. rituximab, distributed
as Rituxan.RTM.; see U.S. Pat. No. 5,736,137, herein incorporated
by reference); anti IL-2 receptor (e.g. Zenapax.RTM.,
Simulect.RTM.); anti-ERBB2 (e.g. trastuzumab, distributed as
Herceptin.RTM.; see U.S. Pat. No. 6,165,464, herein incorporated by
reference); anti-TNF-.alpha. (e.g., infliximab, distributed as
Remicade.RTM.), anti-F-protein (e.g. Synagis.RTM.); anti-CD30
antibodies targeting the CD30 antigen (see, for example, the 5F11
antibody described in Borchmann et al. (2003) Blood 102:3737-3742
and in Example 6 herein below, see also WO 03/059282 and U.S.
Patent Application Publication No. 2004/0006215, herein
incorporated by reference; the SGN-30 antibody described in Wahl et
al. (2002) Cancer Res. 62:3736-3742, a chimeric version of the AC10
anti-CD30 antibody, see also U.S. Patent Application Publication
No. 20040018194 and U.S. Pat. No. 7,090,843, herein incorporated by
reference); anti-CD33 antibodies targeting the CD33 antigen (e.g.
Mylotarg.RTM.; see U.S. Pat. No. 5,733,001, herein incorporated by
reference); anti-CD52 antibodies targeting the CD52 antigen (e.g.,
alemtuzumab, distributed as Campath.RTM.; see U.S. Pat. No.
5,846,534, herein incorporated by reference); anti-CD20 (e.g.,
Zevalin.RTM.); anti-IgE Fc (e.g., omalizumab, marketed under
Xolair.RTM.); anti-VEGF (e.g., bevacizumab, distributed as
Avastin.RTM.; see, Presta et al. (1997) Cancer Res. 57:4593-9;
herein incorporated by reference); anti-EGF-R (e.g., cetuximab,
distributed as Erbitux.RTM.)); anti-CD11a (e.g., efalizumab,
distributed under Raptiva.RTM.), anti-IL-8; anti-C5;
anti-TNF-.alpha. (e.g., adalimumab, distributed as Humira.RTM.);
anti-TGF-.beta.2; anti-IL2 receptor (e.g., daclizumab (Zenapax) and
basiliximab (Simulect)); anti-.alpha.-4-integrin; anti-CD4;
anti-CD2; anti-CD19 (e.g., MT103, a bispecific antibody); anti-CD22
antibody targeting the CD22 antigen (e.g., the monoclonal antibody
BL-22); anti-CD23 antibody targeting the CD23 antigen on malignant
B cells (e.g., IDEC-152); anti-CD80 antibody targeting the CD80
antigen (e.g., IDEC-114); anti-CD38 antibody targeting the CD38
antigen on malignant B cells; .alpha.-M-CSF antibody targeting
macrophage colony stimulating factor; antibodies targeting the
receptor activator of nuclear factor-kappaB (RANK) and its ligand
(RANKL), which are overexpressed in multiple myeloma; anti-CD40
antibodies (e.g., SGN-40) targeting the CD40 antigen on malignant B
cells; antibodies targeting tumor necrosis factor-related
apoptosis-inducing ligand receptor 1 (TRAIL-R1) (e.g., the
agonistic human monoclonal antibody HGS-ETR1) and TRAIL-R2
expressed on a number of solid tumors and tumors of hematopoietic
origin) anti-HBV antibodies; or anti-MHC class II antibodies. Many
of these antibodies are known to require effector activity for
function, either CDC and/or ADCC, for example, Herceptin.RTM.,
Rituxan.RTM., Mylotarg.RTM., and Campath.RTM..
[0256] In some embodiments, the plant host that has been
genetically modified to alter its glycosylation machinery in the
manner set forth herein, for example, duckweed, may express
biologically active .alpha.-2b-interferon, for example human
.alpha.-2b-interferon precursor (NCBI Protein Accession No.
AAB59402) or mature human .alpha.-2b-interferon (amino acids 24-188
of NCBI Protein Accession No. AAB9402) or biologically active
variants thereof. Examples of biologically active variants of human
.alpha.-2b-interferon are known in the art. See, for example, WO
2005/035767, disclosing truncated versions of
.alpha.-2b-interferon; European patent EP211148B1; and U.S. Pat.
Nos. 4,748,233, 4,801,685, 4,816,566, 4,973,479, 4,975,276,
5,089,400, 5,098,703, 5,231,176, and 5,869,293; herein incorporated
by reference. In other embodiments, the plant host, for example,
duckweed, may express biologically active mature human growth
hormone with or without its accompanying signal peptide. The plant
host may also express biologically active variants of human growth
hormone. See, for example, WO 02/10414, directed to expression of
biologically active polypeptides using a duckweed expression
system.
[0257] In yet other embodiments, the plant host, for example,
duckweed, may express plasminogen, microplasminogen, or
biologically active variants thereof. The plasminogen or
microplasminogen to be expressed in the plant host may be from any
mammalian source. In some embodiments, the plasminogen or
microplasminogen is human or porcine. See, for example, WO
2005/078109, directed to expression of these proteins using a
duckweed expression system.
[0258] By "biologically active variant" of these heterologous
polypeptides is intended a polypeptide derived from the native
polypeptide by deletion (so-called truncation) or addition of one
or more amino acids to the N-terminal and/or C-terminal end of the
native protein; deletion or addition of one or more amino acids at
one or more sites in the native protein; or substitution of one or
more amino acids at one or more sites in the native protein.
Variants of a heterologous polypeptide of interest encompassed by
the present invention retain the biological activity of the native
polypeptides.
[0259] For example, biologically active variants of
.alpha.-2b-interferon continue to possess the desired biological
activity of the native .alpha.-2b-interferon including the ability
to increase resistance to viral infection, or the ability to
modulate the transcription of .alpha.-2b-interferon-regulated gene
targets. Such biologically active variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a heterologous protein of interest
will have at least about 50%, 60%, 65%, 70%, generally at least
about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or
more sequence identity to the amino acid sequence for the
heterologous protein of interest. Thus, for example, a biologically
active variant of native .alpha.-2b-interferon will have at least
about 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,
preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
and more preferably at least about 98%, 99% or more sequence
identity to the amino acid sequence for human .alpha.-2b-interferon
precursor (NCBI Protein Accession No. AAB59402) or mature human
.alpha.-2b-interferon (amino acids 24-188 of NCBI Protein Accession
No. AAB9402), where sequence identity is determined using the
parameters identified herein above. Thus, a biologically active
variant of a native heterologous protein of interest may differ
from that protein by as few as 1-15 amino acid residues, as few as
1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino
acid residue.
[0260] In one embodiment, the plant host that has been genetically
modified to alter its glycosylation machinery in the manner set
forth herein is a stably transformed duckweed plant or duckweed
cell or duckweed nodule that expresses biologically active
polypeptides that cannot effectively be commercially produced by
existing gene expression systems, because of cost or logistical
constraints, or both. For example, some proteins cannot be
expressed in mammalian systems because the protein interferes with
cell viability, cell proliferation, cellular differentiation, or
protein assembly in mammalian cells. Such proteins include, but are
not limited to, retinoblastoma protein, p53, angiostatin, and
leptin. The present invention can be advantageously employed to
produce mammalian regulatory proteins; it is unlikely given the
large evolutionary distance between higher plants and mammals that
these proteins will interfere with regulatory processes in
duckweed. Transgenic duckweed can also be used to produce large
quantities of proteins such as serum albumin (in particular, human
serum albumin), hemoglobin, and collagen, which challenge the
production capabilities of existing expression systems.
[0261] Finally, higher plant systems can be engineered to produce
biologically active multimeric proteins (e.g., monoclonal
antibodies, hemoglobin, P450 oxidase, and collagen, and the like)
far more easily than can mammalian systems. One exemplary approach
for producing biologically active multimeric proteins in duckweed
uses an expression vector containing the genes encoding all of the
polypeptide subunits. See, e.g., During et al. (1990) Plant Mol.
Biol. 15:281 and van Engelen et al. (1994) Plant Mol. Biol.
26:1701. The expression cassette comprising the XylT and/or FucT
inhibitory polynucleotide can be introduced into such a vector.
This vector is then introduced into duckweed cells using any known
transformation method, such as a ballistic bombardment or
Agrobacterium-mediated transformation. This method results in
clonal cell lines that express all of the polypeptides necessary to
assemble the multimeric protein, as well as the XylT and/or FucT
inhibitory sequences that alter the glycosylation pattern of the
N-glycans of glycoproteins. Accordingly, in some embodiments, the
transformed duckweed contains one or more expression vectors
encoding a heavy and light chain of a monoclonal antibody or Fab'
antibody fragment, and an expression cassette comprising the XylT
and/or FucT inhibitory polynucleotide, and the monoclonal antibody
or antibody fragment is assembled in the duckweed plant from the
expressed heavy and light chain.
[0262] A variation on this approach is to make single gene
constructs, mix DNA from these constructs together, then deliver
this mixture of DNAs into plant cells using ballistic bombardment
or Agrobacterium-mediated transformation. As a further variation,
some or all of the vectors may encode more than one subunit of the
multimeric protein (i.e., so that there are fewer duckweed clones
to be crossed than the number of subunits in the multimeric
protein). In an alternative embodiment, each duckweed clone has
been genetically modified to alter its glycosylation machinery and
expresses at least one of the subunits of the multimeric protein,
and duckweed clones secreting each subunit are cultured together
and the multimeric protein is assembled in the media from the
various secreted subunits. In some instances, it may be desirable
to produce less than all of the subunits of a multimeric protein,
or even a single protein subunit, in a transformed duckweed plant
or duckweed nodule culture, e.g., for industrial or chemical
processes or for diagnostic, therapeutic, or vaccination
purposes.
[0263] In some embodiments of the invention, the transgenic plant
host of interest is a "high expressor" of a glycoprotein described
herein, including, for example, the glycoproteins comprising
N-linked glycans that are predominately of the G0 glycan structure.
By "high expressor" is intended the transgenic plant host that has
been engineered to produce the glycoproteins described herein is
capable of producing the glycoprotein of interest at a level such
that the glycoprotein of interest represents at least 5% or more of
the total soluble protein produced in the transgenic plant host. In
some embodiments, a "high expressor" is a transgenic plant host
that has been engineered to produce the glycoproteins described
herein such that the glycoprotein of interest represents at least
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or more of the
total soluble protein produced in the transgenic plant host. Thus,
for example, in one embodiment, the transgenic plant host is a
duckweed that has been modified to inhibit expression of XylT and
FucT, and the transgenic duckweed is a high expresser of a
glycoprotein described herein. In some of these embodiments, the
transgenic duckweed is a high expressor of a glycan-optimized
monoclonal antibody having the predominate G0 glycoform described
herein above. In yet other embodiments, the transgenic duckweed
expresses the glycan-optimized monoclonal antibody such that this
glycoprotein represents about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, or greater, of the total soluble protein.
Further Humanization of Glycoproteins
[0264] In some embodiments, it may be desirable for the
glycoproteins of the present invention to comprise complex
N-glycans having the terminal .beta.(1,4)-galactose residues
attached to the 1,3 and/or 1,6 mannose arms. Without being bound by
theory, these terminal galactose residues may contribute to the
therapeutic function and/or pharmacokinetic activity of a
glycoprotein. It is recognized that the methods of the present
invention can be paired with other methods known in the art to
further modify the glycoproteins of the invention such that one or
more of the N-glycans attached thereto comprises one or more
terminal galactose residues, i.e., wherein one or more of the
N-glycans is represented by the G1 or G2 glycan species. In this
manner, the glycoprotein compositions of the present invention,
including the monoclonal antibodies described herein, can be
modified, for example, enzymatically with use of a
glycosyltransferase enzyme to obtain glycoproteins having a
substantially homogenous glycosylation profile for the G1,
preferably for the G2 glycan species. See, for example, U.S. Patent
Application Publication No. 2004/0191256, herein incorporated by
reference in its entirety, teaching galactosyltransferase
modification of a substrate glycoprotein to obtain a glycoprotein
wherein substantially all of the N-linked glycan species are of the
G2 form. In this manner, the glycoprotein of interest can be
reacted with an activated galactose in the presence of a
galactosyltransferase and a metal salt. The galactosyltransferase
can be a mammalian .beta.1,4 galactosyltransferase (GalT), for
example, human GalT, and the activated galactose can be, for
example, UDP-galactose.
[0265] Alternatively, the transgenic plants of the invention having
FucT and XylT expression silenced in the manner set forth herein
can be further modified in their glycosylation machinery such that
they express a galactosyltransferase and efficiently attach the
terminal galactose residue to the N-glycans of endogenous and
heterologous glycoproteins produced therein. In this manner, the
transgenic plants of the invention can be further modified by
introducing a nucleotide construct, for example, an expression
cassette, that provides for the expression of a
galactosyltransferase. The galactosyltransferase can be a mammalian
.beta.1,4 galactosyltransferase (GalT), for example, human GalT
(see, for example, U.S. Pat. No. 6,998,267, herein incorporated by
reference in its entirety) or a hybrid GalT (see, for example, WO
03/078637, herein incorporated by reference in its entirety)
comprising at least a portion of a cytoplasmic
tail-transmembrane-stem region of a first glycosyltransferase
(e.g., a plant glycosyltransferase such as xylosyltransferase,
N-acetylglycosaminlytransferase or fucosyltransferase) and at least
a portion of a catalytic region of a second glycosyltransefersae
(e.g., mammalian glycosyltransferase, for example, human GalT). By
silencing expression of XylT and FucT in a plant, for example, a
duckweed, and providing for expression of GalT, for example, human
GalT, or a hybrid enzyme comprising a portion of the catalytic
domain of GalT, for example, human GalT, in this plant, for
example, duckweed, it is possible to obtain transgenic plants
producing glycoproteins, both endogenous and heterologous, that
have an altered glycosylation pattern, wherein the N-linked glycans
attached thereto have a reduction in the attachment of
plant-specific xylose and plant-specific fucose residues and which
comprise the terminal galactose residues (i.e., G2 glycan species).
In this manner, glycoproteins that have a substantially homogeneous
profile for the G2 glycan species, and/or which are substantially
homogenous for the G2 glycoform can be obtained from transgenic
plants of the invention.
[0266] In other embodiments, it may be desirable to further modify
the glycosylation pattern of the glycoproteins of the invention,
wherein the N-linked glycans attached thereto further comprise a
terminal sialic acid residue attached to one or both of the
galactose residues attached to the 1,3 and 1,6 mannose arms. The
addition of the terminal sialic acid residue(s) may be required for
the sustained stability, and in some cases function, of some
therapeutic proteins.
[0267] Depending upon the transgenic plant system, natural
sialylation of glycoproteins may occur. Thus, there have been
reports in the literature that cultured Arabidopsis, tobacco, and
Medicago cultured cells synthesize sialylated glycoproteins (Shah
et al. (2003) Nat. Biotech. 21(12):1470-1471; Joshi and Lopez
(2005) Curr. Opin. Plant Biol. 8(2):223-226). More recently, it was
reported that Japanese rice express active sialyltransferase-like
proteins (Takashima et al. (2006) J. Biochem. (Tokyo)
139(2):279-287). Hence, there are now orthogonal reports that
plants have the machinery required to sialylate glycoproteins.
[0268] Where further modification of the glycosylation pattern of
the glycoproteins of the invention, wherein the N-linked glycans
attached thereto further comprise a terminal sialic acid residue
attached to one or both of the galactose residues attached to the
1,3 and 1,6 mannose arms, is desired, the transgenic plants of the
invention can be modified to express a .beta.-1,4
galactosyltransferase, for example, human .beta.-1,4
galactosyltransferase, and to express or overexpress a
sialyltransferase. Thus, for example, the transgenic plants can be
further modified to express a sialyltransferase such as
.alpha.-2,3- and/or .alpha.-2,6-sialyltransferase. See, for example
WO 2004/071177; and Wee et al. (1998) Plant Cell 10: 1759-1768;
herein incorporated by reference in their entirety. Alternatively,
the transgenic plants of the invention can be modified to express a
.beta.-1,4 galactosyltransferase, for example, human .beta.-1,4
galactosyltransferase, and to express any other enzymes that are
deficient in the plant host's sialic acid pathway. The strategy(s)
employed can be determined after an initial investigation of
whether the particular plant host, for example, a duckweed,
naturally expresses sialic acid-containing N-glycans on native or
recombinantly produced glycoproteins. For example, if there is not
evidence for the presence of the terminal sialic acid residues on
N-glycans of glycoproteins produced within the transgenic plant
host, particularly a transgenic plant host engineered to express a
.beta.-1,4 galactosyltransferase, then one or both of these
strategies could be employed to achieve terminal sialylation of the
N-glycans of glycoproteins produced within the transgenic plant
host of interest.
[0269] Alternatively, the glycoprotein compositions of the
invention that are substantially homologous for G2 glycan species
or the G2 glycoform can be modified by in vitro enzymatic
processing; see, for example, U.S. Patent Application Publication
No. 20030040037; herein incorporated by reference in its
entirety.
[0270] It is also recognized that for some glycoproteins produced
in the transgenic plants of the invention, it may be desirable to
have the mammalian .alpha.1-6 fucose residue attached to the
trimannose core structure (Man.sub.3GlcNAc.sub.2) of the N-glycan
species attached thereto. In such embodiments, the transgenic
plants of the invention can be further genetically modified to
express an .alpha.1-6 fucosyltransferase, for example, human
.alpha.1-6 fucosyltransferase, using glycoengineering methods known
in the art.
[0271] It is recognized that the glycoprotein compositions of the
invention can be produced by engineering any host cell of interest,
including the plant host cells exemplified and described herein. In
this manner, other protein expression host systems in addition to
plant hosts, including animal, insect, bacterial cells and the like
may be used to produce glycoprotein compositions according to the
present invention. Such protein expression host systems may be
engineered or selected to express a predominant glycoform or
alternatively may naturally produce glycoproteins having
predominant glycan structures. Examples of engineered protein
expression host systems producing a glycoprotein having a
predominant glycoform include gene knockouts/mutations (Shields et
al. (2002) JBC 277:26733-26740); genetic engineering (Umana et al.
(1999) Nature Biotech. 17:176-180); or a combination of both.
Alternatively, certain cells naturally express a predominant
glycoform, for example, chickens, humans, and cows (Raju et al.
(2000) Glycobiology 10:477-486). Thus, the expression of a
glycoprotein, including an immunoglobulin such as a monoclonal
antibody, or composition having predominantly one specific glycan
structure according to the present invention can be obtained by one
skilled in the art by selecting at least one of many expression
host systems. Further expression host systems found in the art for
production of glycoproteins include: CHO cells (see, for example,
WO 9922764A1 and WO 03/035835A1); hybridoma cells (Trebak et al.
(1999) J. Immunol. Methods 230:59-70); insect cells (Hsu et al.
(1997) JBC 272:9062-970). See also, WO 04/074499A2 regarding
additional plant host systems.
[0272] The glycoproteins produced in accordance with the methods of
the present invention can be harvested from host cells in which
they are recombinantly produced in order to obtain them in their
isolated or purified form. In this manner, the recombinantly
produced glycoproteins of the invention are isolated from the host
cells using any conventional means known in the art and purified,
for example, by chromatography, electrophoresis, dialysis,
solvent-solvent extraction, and the like. Thus, the present
invention also provides for purified glycoproteins, including
monoclonal antibody compositions, where the glycoproteins have
substantially homogeneous glycosylation profiles, and are
substantially homogeneous for the G0 glycoform. These purified
glycoproteins are substantially free of host cellular material, and
include preparations of glycoprotein having less than about 30%,
20%, 10%, 5%, or 1% (by dry weight) of contaminating protein, as
noted herein above. In some embodiments, these purified
glycoproteins can include at least 0.001%, 0.005%, 0.1%, 0.5%, 1%,
1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%,
8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dry
weight) of contaminating protein. Furthermore, for the
recombinantly produced purified glycoproteins of the invention,
optimally culture medium represents less than about 30%, 20%, 10%,
5%, or 1% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals within the purified glycoprotein
preparation, as noted herein above. Thus, in some embodiments,
culture medium components within these purified glycoproteins can
represent at least 0.001%, 0.005%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%,
3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,
9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dry weight) of
chemical precursors or non-protein-of-interest chemicals within the
purified glycoprotein preparation. In some embodiments, isolation
and purification results in recovery of purified glycoprotein that
is free of contaminating host protein, free of culture medium
components, and/or free of both contaminating host protein and
culture medium components.
[0273] Thus, in some embodiments, the protein expression host
system is a plant, for example, a duckweed, and the purified
glycoprotein obtained from the plant host is substantially free of
plant cellular material, including embodiments where the
preparations of glycoprotein have less than about 30%, 20%, 10%,
5%, or 1% (by dry weight) of contaminating plant protein. In other
embodiments, the plant culture medium represents less than about
30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals within the purified
glycoprotein.
[0274] In some embodiments, these purified glycoproteins obtained
from the plant host can include at least 0.001%, 0.005%, 0.1%,
0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%,
7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about
30% (by dry weight) of contaminating plant protein. In other
embodiments, plant culture medium components within in these
purified glycoproteins can represent at least 0.001%, 0.005%, 0.1%,
0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%,
7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about
30% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals within the purified glycoprotein.
In some embodiments, isolation and purification from the plant host
results in recovery of purified glycoprotein that is free of
contaminating plant protein, free of plant culture medium
components, and/or free of both contaminating plant protein and
plant culture medium components.
Expression Cassettes
[0275] According to the present invention, stably transformed
higher plants, for example, stably transformed duckweed, are
obtained by transformation with a polynucleotide of interest
contained within an expression cassette. Depending upon the
objective, the polynucleotide of interest can be one encoding a
FucT or XylT polypeptide of interest, for example, encoding the
polypeptide set forth in SEQ ID NO:3 (FucT) or SEQ ID NO:6 or 21
(XylT), or a variant thereof, thus providing for expression of
these polypeptides in a cell, for example, a plant cell, or can be
a FucT or XylT inhibitory polynucleotide that is capable of
inhibiting expression or function of the FucT or XylT polypeptide
when stably introduced into a cell, for example, a plant cell of
interest.
[0276] Thus, in some embodiments, the FucT and/or XylT
polynucleotides of the invention, including those set forth in SEQ
ID NOS:1 and 2 (FucT) and SEQ ID NOS:4, 5, 19, and 20 (XylT) and
fragments and variants thereof, are used to construct expression
cassettes that comprise a FucT and/or XylT inhibitory
polynucleotide as defined herein above. Stably introducing such an
expression cassette into a plant or plant cell of interest can
provide for inhibition of expression or function of the FucT and/or
XylT polypeptides of the invention, including those set forth in
SEQ ID NO:3 (FucT) and SEQ ID NO:6 or 21 (XylT) and variants
thereof, thereby altering the N-glycan glycosylation pattern of
endogenous and heterologous glycoproteins within a plant or plant
cell stably transformed with the expression cassette.
[0277] In some embodiments, the plant or plant cell that is stably
transformed with an expression cassette comprising a FucT and/or
XylT inhibitory polynucleotide has also been stably transformed
with an expression cassette that provides for expression of a
heterologous polypeptide of interest, for example, a mammalian
protein of interest, including the mammalian proteins of
pharmaceutical interest as noted herein above. The expression
cassette providing for expression of a heterologous polypeptide of
interest can be provided on the same polynucleotide (for example,
on the same transformation vector) for introduction into a plant,
or on a different polynucleotide (for example, on different
transformation vectors) for introduction into the plant or plant
cell of interest at the same time or at different times, by the
same or by different methods of introduction, for example, by the
same or different transformation methods.
[0278] The expression cassettes of the present invention comprise
expression control elements that at least comprise a
transcriptional initiation region (e.g., a promoter) linked to the
polynucleotide of interest, i.e., a polynucleotide encoding a FucT
or XylT polypeptide of the invention, a FucT and/or XylT inhibitory
polynucleotide, or a polynucleotide encoding a heterologous
polypeptide of interest, for example, a mammalian protein. Such an
expression cassette is provided with a plurality of restriction
sites for insertion of the polynucleotide or polynucleotides of
interest (e.g., one polynucleotide of interest, two polynucleotides
of interest, etc.) to be under the transcriptional regulation of
the promoter and other expression control elements. In particular
embodiments of the invention, the polynucleotide to be transferred
contains two or more expression cassettes, each of which encodes at
least one polynucleotide of interest.
[0279] By "expression control element" is intended a regulatory
region of DNA, usually comprising a TATA box, capable of directing
RNA polymerase II, or in some embodiments, RNA polymerase III, to
initiate RNA synthesis at the appropriate transcription initiation
site for a particular coding sequence. An expression control
element may additionally comprise other recognition sequences
generally positioned upstream or 5' to the TATA box, which
influence (e.g., enhance) the transcription initiation rate.
Furthermore, an expression control element may additionally
comprise sequences generally positioned downstream or 3' to the
TATA box, which influence (e.g., enhance) the transcription
initiation rate.
[0280] The transcriptional initiation region (e.g., a promoter) may
be native or homologous or foreign or heterologous to the host, or
could be the natural sequence or a synthetic sequence. By foreign,
it is intended that the transcriptional initiation region is not
found in the wild-type host into which the transcriptional
initiation region is introduced. By "functional promoter" is
intended the promoter, when operably linked to a sequence encoding
a protein of interest, is capable of driving expression (i.e.,
transcription and translation) of the encoded protein, or, when
operably linked to an inhibitory sequence encoding an inhibitory
nucleotide molecule (for example, a hairpin RNA, double-stranded
RNA, miRNA polynucleotide, and the like), the promoter is capable
of initiating transcription of the operably linked inhibitory
sequence such that the inhibitory nucleotide molecule is expressed.
The promoters can be selected based on the desired outcome. Thus
the expression cassettes of the invention can comprise
constitutive, tissue-preferred, or other promoters for expression
in plants.
[0281] As used herein a chimeric gene comprises a coding sequence
operably linked to a transcription initiation region that is
heterologous to the coding sequence.
[0282] Any suitable promoter known in the art can be employed
according to the present invention, including bacterial, yeast,
fungal, insect, mammalian, and plant promoters. For example, plant
promoters, including duckweed promoters, may be used. Exemplary
promoters include, but are not limited to, the Cauliflower Mosaic
Virus 35S promoter, the opine synthetase promoters (e.g., nos, mas,
ocs, etc.), the ubiquitin promoter, the actin promoter, the
ribulose bisphosphate (RubP) carboxylase small subunit promoter,
and the alcohol dehydrogenase promoter. The duckweed RubP
carboxylase small subunit promoter is known in the art
(Silverthorne et al. (1990) Plant Mol. Biol. 15:49). Other
promoters from viruses that infect plants, preferably duckweed, are
also suitable including, but not limited to, promoters isolated
from Dasheen mosaic virus, Chlorella virus (e.g., the Chlorella
virus adenine methyltransferase promoter; Mitra et al. (1994) Plant
Mol. Biol. 26:85), tomato spotted wilt virus, tobacco rattle virus,
tobacco necrosis virus, tobacco ring spot virus, tomato ring spot
virus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic
virus, sugarcane baciliform badnavirus and the like.
[0283] Other suitable expression control elements are disclosed in
the commonly owned and copending provisional application entitled
"Expression Control Elements from the Lemnaceae Family," assigned
U.S. Patent Application No. 60/759,308, Attorney Docket No.
040989/243656, filed Jan. 17, 2006, herein incorporated by
reference in its entirety. The expression control elements
disclosed in this copending application were isolated from
ubiquitin genes for several members of the Lemnaceae family, and
are thus referred to as "Lemnaceae ubiquitin expression control
elements." SEQ ID NO:7 of the present application sets forth the
full-length Lemna minor ubiquitin expression control element,
including both the promoter plus 5' UTR (nucleotides 1-1625) and
intron (nucleotides 1626-2160). SEQ ID NO:8 sets forth the
full-length Spirodella polyrrhiza ubiquitin expression control
element, including both the promoter plus 5' UTR (nucleotides
1-1041) and intron (nucleotides 1042-2021). SEQ ID NO:9 sets forth
the full-length Lemna aequinoctialis ubiquitin expression control
element, including both the promoter plus 5' UTR (nucleotides
1-964) and intron (nucleotides 965-2068). SEQ ID NO:10 sets forth
the promoter plus 5' UTR portion of the L. minor ubiquitin
expression control element (designated "LmUbq promoter" herein).
SEQ ID NO:11 sets forth the promoter plus 5' UTR portion of the S.
polyrrhiza ubiquitin expression control element (designated "SpUbq
promoter" herein). SEQ ID NO:12 sets forth the promoter plus 5' UTR
portion of the L. aequinoctialis ubiquitin expression control
element (designated "LaUbq promoter" herein). SEQ ID NO:13 sets
forth the intron portion of the L. minor ubiquitin expression
control element (designated "LmUbq intron" herein). SEQ ID NO:14
sets forth the intron portion of the S. polyrrhiza ubiquitin
expression control element (designated "SpUbq intron" herein). SEQ
ID NO:15 sets forth the intron portion of the L. aequinoctialis
ubiquitin expression control element (designated "LaUbq intron"
herein). It is recognized that the individual promoter plus 5' UTR
sequences set forth in SEQ ID NOs:10-12, and biologically active
variants and fragments thereof, can be used to regulate
transcription of operably linked nucleotide sequences of interest
in plants. Similarly, one or more of the intron sequences set forth
in SEQ ID NOs:13-15, and biologically active fragments or variants
thereof, can be operably linked to a promoter of interest,
including a promoter set forth in SEQ ID NO:10, 11, or 12 in order
to enhance expression of a nucleotide sequence that is operably
linked to that promoter.
[0284] Fragments and variants of the disclosed expression control
elements can also be used within expression cassettes to drive
expression of the operably linked polynucleotide of interest. By
"fragment of an expression control element" is intended a portion
of the full-length expression control element, such as a portion of
any one of the expression control elements set forth in SEQ ID
NOs:7-9. Fragments of an expression control element retain
biological activity and hence encompass fragments capable of
initiating or enhancing expression of an operably linked
polynucleotide of interest. Thus, for example, less than the entire
expression control elements disclosed herein may be utilized to
drive expression of an operably linked polynucleotide of interest.
Specific, non-limiting examples of such fragments of an expression
control element include the nucleotide sequences set forth in any
one of SEQ ID NOs:10-12 (as described herein above), as well as 5'
truncations of the L. minor ubiquitin expression control element
(SEQ ID NO:7), such as nucleotides 1288-2160 of SEQ ID NO:7 (LmUbq
truncated promoter No. 1) and nucleotides 1132-2160 of SEQ ID NO:1
(LmUbq truncated promoter No. 2). See the copending provisional
application assigned U.S. Patent Application No. 60/759,308, herein
incorporated by reference in its entirety.
[0285] The nucleotides of such fragments will usually comprise the
TATA recognition sequence of the particular expression control
element. Such fragments can be obtained by use of restriction
enzymes to cleave the naturally occurring expression control
elements disclosed herein; by synthesizing a nucleotide sequence
from the naturally occurring sequence of the expression control
element DNA sequence; or can be obtained through the use of
polymerase chain reaction (PCR) technology. See particularly,
Mullis et al. (1987) Methods Enzymol. 155:335-350, and Erlich, ed.
(1989) PCR Technology (Stockton Press, New York).
[0286] Variants of expression control elements, such as those
resulting from site-directed mutagenesis, can also be used in the
expression cassettes of the present invention to provide expression
of the operably linked polynucleotide of interest. By "variant of
an expression control element" is intended sequences having
substantial similarity with an expression control element disclosed
herein (for example, the expression control element set forth in
SEQ ID NO:7, 9, or 9), or with a fragment thereof (for example, the
respective sequences set forth in SEQ ID NOs:10-15). Naturally
occurring variants of expression control elements can be identified
with the use of well-known molecular biology techniques, as, for
example, with PCR and hybridization techniques as outlined above.
Variant expression control elements also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis. Generally, variants of a
particular expression control element disclosed herein, including
variants of SEQ ID NOs:7-15, will have at least 40%, 50%, 60%, 65%,
70%, generally at least 75%, 80%, 85%, preferably about 90%, 91%,
92%, 93%, 94%, to 95%, 96%, 97%, and more preferably about 98%, 99%
or more sequence identity to that particular nucleotide sequence as
determined by sequence alignment programs described herein above
using default parameters.
[0287] Expression control elements, including promoters, can be
chosen to give a desired level of regulation. For example, in some
instances, it may be advantageous to use a promoter that confers
constitutive expression (e.g, the mannopine synthase promoter from
Agrobacterium tumefaciens). Alternatively, in other situations, for
example, where expression of a heterologous polypeptide is
concerned, it may be advantageous to use promoters that are
activated in response to specific environmental stimuli (e.g., heat
shock gene promoters, drought-inducible gene promoters,
pathogen-inducible gene promoters, wound-inducible gene promoters,
and light/dark-inducible gene promoters) or plant growth regulators
(e.g., promoters from genes induced by abscissic acid, auxins,
cytokinins, and gibberellic acid). As a further alternative,
promoters can be chosen that give tissue-specific expression (e.g.,
root, leaf, and floral-specific promoters).
[0288] The overall strength of a given promoter can be influenced
by the combination and spatial organization of cis-acting
nucleotide sequences such as upstream activating sequences. For
example, activating nucleotide sequences derived from the
Agrobacterium tumefaciens octopine synthase gene can enhance
transcription from the Agrobacterium tumefaciens mannopine synthase
promoter (see U.S. Pat. No. 5,955,646 to Gelvin et al.). In the
present invention, the expression cassette can contain activating
nucleotide sequences inserted upstream of the promoter sequence to
enhance the expression of the nucleotide sequence of interest. In
one embodiment, the expression cassette includes three upstream
activating sequences derived from the Agrobacterium tumefaciens
octopine synthase gene operably linked to a promoter derived from
an Agrobacterium tumefaciens mannopine synthase gene (see U.S. Pat.
No. 5,955,646, herein incorporated by reference).
[0289] Where the expression control element will be used to drive
expression of an operably linked DNA sequence encoding a small
hpRNA molecule, for example, within an RNAi expression cassette
described herein above, it is advantageous to use an expression
control element comprising a promoter recognized by the DNA
dependent RNA polymerase III. As used herein, "a promoter
recognized by the DNA dependent RNA polymerase III" is a promoter
which directs transcription of the associated DNA region through
the polymerase action of RNA polymerase III. These include genes
encoding 5S RNA, tRNA, 7SL RNA, U6 snRNA and a few other small
stable RNAs, many involved in RNA processing. Most of the promoters
used by Pol III require sequence elements downstream of +1, within
the transcribed region. A minority of pol III templates however,
lack any requirement for intragenic promoter elements. These are
referred to as type 3 promoters. By "type 3 Pol III promoters" is
intended those promoters that are recognized by RNA polymerase III
and contain all cis-acting elements, interacting with the RNA
polymerase III upstream of the region normally transcribed by RNA
polymerase III. Such type 3 Pol III promoters can be assembled
within the RNAi expression cassettes of the invention to drive
expression of the operably linked DNA sequence encoding the small
hpRNA molecule.
[0290] Typically, type 3 Pol III promoters contain a TATA box
(located between -25 and -30 in Human U6 snRNA gene) and a Proximal
Sequence element (PSE; located between -47 and -66 in Human U6
snRNA). They may also contain a Distal Sequence Element (DSE;
located between -214 and -244 in Human U6 snRNA). Type 3 Pol III
promoters can be found, e.g., associated with the genes encoding
7SL RNA, U3 snRNA and U6 snRNA. Such sequences have been isolated
from Arabidopsis, rice, and tomato. See, for example, SEQ ID
NOs:1-8 of U.S. Patent Application Publication No. 20040231016.
[0291] Other nucleotide sequences for type 3 Pol III promoters can
be found in nucleotide sequence databases under the entries for the
A. thaliana gene AT7SL-1 for 7SL RNA (X72228), A. thaliana gene
AT7SL-2 for 7SL RNA (X72229), A. thaliana gene AT7SL-3 for 7SL RNA
(AJ290403), Humulus lupulus H17SL-1 gene (AJ236706), Humulus
lupulus H17SL-2 gene (AJ236704), Humulus lupulus H17SL-3 gene
(AJ236705), Humulus lupulus H17SL-4 gene (AJ236703), A. thaliana
U6-1 snRNA gene (X52527), A. thaliana U6-26 snRNA gene (X52528), A.
thaliana U6-29 snRNA gene (X52529), A. thaliana U6-1 snRNA gene
(X52527), Zea mays U3 snRNA gene (Z29641), Solanum tuberosum U6
snRNA gene (Z17301; X60506; S83742), tomato U6 smal nuclear RNA
gene (X51447), A. thaliana U3C snRNA gene (X52630), A. thaliana U3B
snRNA gene (X52629), Oryza sativa U3 snRNA promoter (X79685),
tomato U3 small nuclear RNA gene (X14411), Triticum aestivum U3
snRNA gene (X63065), and Triticum aestivum U6 snRNA gene
(X63066).
[0292] Other type 3 Pol III promoters may be isolated from other
varieties of tomato, rice or Arabidopsis, or from other plant
species using methods well known in the art. For example, libraries
of genomic clones from such plants may be isolated using U6 snRNA,
U3 snRNA, or 7SL RNA coding sequences (such as the coding sequences
of any of the above mentioned sequences identified by their
accession number and additionally the Vicia faba U6snRNA coding
sequence (X04788), the maize DNA for U6 snRNA (X52315), or the
maize DNA for 7SL RNA (X14661)) as a probe, and the upstream
sequences, preferably the about 300 to 400 bp upstream of the
transcribed regions may be isolated and used as type 3 Pol III
promoters. Alternatively, PCR based techniques such as inverse-PCR
or TAIL.TM.-PCR may be used to isolate the genomic sequences
including the promoter sequences adjacent to known transcribed
regions. Moreover, any of the type 3 Pol III promoter sequences
described herein, identified by their accession numbers and SEQ ID
NOS, may be used as probes under stringent hybridization conditions
or as source of information to generate PCR primers to isolate the
corresponding promoter sequences from other varieties or plant
species.
[0293] Although type 3 Pol III promoters have no requirement for
cis-acting elements located with the transcribed region, it is
clear that sequences normally located downstream of the
transcription initiation site may nevertheless be included in the
RNAi expression cassettes of the invention. Further, while type 3
Pol III promoters originally isolated from monocotyledonous plants
can effectively be used in RNAi expression cassettes to suppress
expression of a target gene in both dicotyledonous and
monocotyledonous plant cells and plants, type 3 Pol III promoters
originally isolated from dicotyledonous plants reportedly can only
be efficiently used in dicotyledonous plant cells and plants.
Moreover, the most efficient gene silencing reportedly is obtained
when the RNAi expression cassette is designed to comprise a type 3
Pol III promoter derived from the same or closely related species.
See, for example, U.S. Patent Application Publication No.
20040231016. Thus, where the plant of interest is a
monocotyledonous plant, and small hpRNA interference is the method
of choice for inhibiting expression of FucT and/or XylT, the type 3
Pol III promoter preferably is from another monocotyledonous plant,
including the plant species for which the glycosylation pattern of
N-linked glycans of a glycoprotein of interest is to be
altered.
[0294] The expression cassette of the invention thus includes in
the 5'-3' direction of transcription, an expression control element
comprising a transcriptional and translational initiation region, a
polynucleotide of interest, for example, a sequence encoding a
heterologous protein of interest or a sequence encoding a FucT or
XylT inhibitory sequence that, when expressed, is capable of
inhibiting the expression or function of FucT and/or XylT, and a
transcriptional and translational termination region functional in
plants. Any suitable termination sequence known in the art may be
used in accordance with the present invention. The termination
region may be native with the transcriptional initiation region,
may be native with the nucleotide sequence of interest, or may be
derived from another source. Convenient termination regions are
available from the Ti-plasmid of A. tumefaciens, such as the
octopine synthetase and nopaline synthetase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141;
Proudfoot (1991) Cell 64:671; Sanfacon et al. (1991) Genes Dev.
5:141; Mogen et al. (1990) Plant Cell 2:1261; Munroe et al. (1990)
Gene 91:151; Ballas et al. (1989) Nucleic Acids Res. 17:7891; and
Joshi et al. (1987) Nucleic Acids Res. 15:9627. Additional
exemplary termination sequences are the pea RubP carboxylase small
subunit termination sequence and the Cauliflower Mosaic Virus 35S
termination sequence. Other suitable termination sequences will be
apparent to those skilled in the art, including the oligo dT
stretch disclosed herein above for use with type 3 Pol III
promoters driving expression of a FucT and/or XylT inhibitory
polynucleotide that forms a small hpRNA structure.
[0295] Alternatively, the polynucleotide(s) of interest can be
provided on any other suitable expression cassette known in the
art.
[0296] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells or
tissues. Selectable marker genes include genes encoding antibiotic
resistance, such as those encoding neomycin phosphotransferase II
(NEO) and hygromycin phosphotransferase (HPT), as well as genes
conferring resistance to herbicidal compounds. Herbicide resistance
genes generally code for a modified target protein insensitive to
the herbicide or for an enzyme that degrades or detoxifies the
herbicide in the plant before it can act. See DeBlock et al. (1987)
EMBO J. 6:2513; DeBlock et al. (1989) Plant Physiol. 91:691; Fromm
et al. (1990) BioTechnology 8:833; Gordon-Kamm et al. (1990) Plant
Cell 2:603. For example, resistance to glyphosphate or sulfonylurea
herbicides has been obtained using genes coding for the mutant
target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
and acetolactate synthase (ALS). Resistance to glufosinate
ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have
been obtained by using bacterial genes encoding phosphinothricin
acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate
monooxygenase, which detoxify the respective herbicides.
[0297] For purposes of the present invention, selectable marker
genes include, but are not limited to, genes encoding neomycin
phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in
Plant Science 4:1); cyanamide hydratase (Maier-Greiner et al.
(1991) Proc. Natl. Acad. Sci. USA 88:4250); aspartate kinase;
dihydrodipicolinate synthase (Perl et al. (1993) BioTechnology
11:715); bar gene (Toki et al. (1992) Plant Physiol. 100:1503;
Meagher et al. (1996) Crop Sci. 36:1367); tryptophan decarboxylase
(Goddijn et al. (1993) Plant Mol. Biol. 22:907); neomycin
phosphotransferase (NEO; Southern et al. (1982) J. Mol. Appl. Gen.
1:327); hygromycin phosphotransferase (HPT or HYG; Shimizu et al.
(1986) Mol. Cell. Biol. 6:1074); dihydrofolate reductase (DHFR;
Kwok et al. (1986) Proc. Natl. Acad. Sci. USA 83:4552);
phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J.
6:2513); 2,2-dichloropropionic acid dehalogenase
(Buchanan-Wollatron et al. (1989) J. Cell. Biochem. 13D:330);
acetohydroxyacid synthase (U.S. Pat. No. 4,761,373 to Anderson et
al.; Haughn et al. (1988) Mol. Gen. Genet. 221:266);
5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al.
(1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalker
et al.); acetyl-coenzyme A carboxylase (Parker et al. (1990) Plant
Physiol. 92:1220); dihydropteroate synthase (sulI; Guerineau et al.
(1990) Plant Mol. Biol. 15:127); and 32 kDa photosystem II
polypeptide (psbA; Hirschberg et al. (1983) Science 222:1346
(1983).
[0298] Also included are genes encoding resistance to: gentamycin
(e.g., aacC1, Wohlleben et al. (1989) Mol. Gen. Genet.
217:202-208); chloramphenicol (Herrera-Estrella et al. (1983) EMBO
J. 2:987); methotrexate (Herrera-Estrella et al. (1983) Nature
303:209; Meijer et al. (1991) Plant Mol. Biol. 16:807); hygromycin
(Waldron et al. (1985) Plant Mol. Biol. 5:103; Zhijian et al.
(1995) Plant Science 108:219; Meijer et al. (1991) Plant Mol. Bio.
16:807); streptomycin (Jones et al. (1987) Mol. Gen. Genet.
210:86); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic
Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.
7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio.
15:127); bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D
(Streber et al. (1989) BioTechnology 7:811); phosphinothricin
(DeBlock et al. (1987) EMBO J. 6:2513); spectinomycin
(Bretagne-Sagnard and Chupeau, Transgenic Research 5:131).
[0299] The bar gene confers herbicide resistance to
glufosinate-type herbicides, such as phosphinothricin (PPT) or
bialaphos, and the like. As noted above, other selectable markers
that could be used in the vector constructs include, but are not
limited to, the pat gene, also for bialaphos and phosphinothricin
resistance, the ALS gene for imidazolinone resistance, the HPH or
HYG gene for hygromycin resistance, the EPSP synthase gene for
glyphosate resistance, the Hm1 gene for resistance to the Hc-toxin,
and other selective agents used routinely and known to one of
ordinary skill in the art. See Yarranton (1992) Curr. Opin.
Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl. Acad. Sci.
USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff (1992) Mol.
Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu et
al. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et
al. (1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad.
Sci. USA 86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA
86:2549; Deuschle et al. (1990) Science 248:480; Labow et al.
(1990) Mol. Cell. Biol. 10:3343; Zambretti et al. (1992) Proc.
Natl. Acad. Sci. USA 89:3952; Baim et al. (1991) Proc. Natl. Acad.
Sci. USA 88:5072; Wyborski et al. (1991) Nuc. Acids Res. 19:4647;
Hillenand-Wissman (1989) Topics in Mol. And Struc. Biol. 10:143;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591;
Kleinschnidt et al. (1988) Biochemistry 27:1094; Gatz et al. (1992)
Plant J. 2:397; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA
89:5547; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913;
Hlavka et al. (1985) Handbook of Experimental Pharmacology 78; and
Gill et al. (1988) Nature 334:721. Such disclosures are herein
incorporated by reference.
[0300] The above list of selectable marker genes are not meant to
be limiting. Any selectable marker gene can be used in the present
invention.
Modification of Nucleotide Sequences for Enhanced Expression in a
Plant Host
[0301] Where the plant of interest is also genetically modified to
express a heterologous protein of interest, for example, a
transgenic plant host serving as an expression system for
recombinant production of a heterologous protein, the present
invention provides for the modification of the expressed
polynucleotide sequence encoding the heterologous protein of
interest to enhance its expression in the host plant. Thus, where
appropriate, the polynucleotides may be optimized for increased
expression in the transformed plant. That is, the polynucleotides
can be synthesized using plant-preferred codons for improved
expression. See, for example, Campbell and Gowri (1990) Plant
Physiol. 92:1-11 for a discussion of host-preferred codon usage.
Methods are available in the art for synthesizing nucleotide
sequences with plant-preferred codons. See, e.g., U.S. Pat. Nos.
5,380,831 and 5,436,391; Perlak et al. (1991) Proc. Natl. Acad.
Sci. USA 15:3324; Iannacome et al. (1997) Plant Mol. Biol. 34:485;
and Murray et al., (1989) Nucleic Acids. Res. 17:477, herein
incorporated by reference.
[0302] In some embodiments of the invention, the plant host is a
member of the duckweed family, and the polynucleotide encoding the
heterologous polypeptide of interest, for example, a mammalian
polypeptide, is modified for enhanced expression of the encoded
heterologous polypeptide. In this manner, one such modification is
the synthesis of the polynucleotide encoding the heterologous
polypeptide of interest using duckweed-preferred codons, where
synthesis can be accomplished using any method known to one of
skill in the art. The preferred codons may be determined from the
codons of highest frequency in the proteins expressed in duckweed.
For example, the frequency of codon usage for Lemna gibba is found
on the web page:
http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Lemna+gibba+[-
gbpln], and the frequency of codon usage for Lemna minor is found
on the web page
http://www.kazusa.or.jp/codon/cgibin/showcodon.cgi?species=Lemna-
+minor+[gbpln] and in Table 1. It is recognized that heterologous
genes that have been optimized for expression in duckweed and other
monocots, as well as other dicots, can be used in the methods of
the invention. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432;
Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324; Iannacome
et al. (1997) Plant Mol. Biol. 34:485; and Murray et al. (1989)
Nuc. Acids Res. 17:477, and the like, herein incorporated by
reference. It is further recognized that all or any part of the
polynucleotide encoding the heterologous polypeptide of interest
may be optimized or synthetic. In other words, fully optimized or
partially optimized sequences may also be used. For example, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons may be
duckweed-preferred codons. In one embodiment, between 90 and 96% of
the codons are duckweed-preferred codons. The coding sequence of a
polynucleotide sequence encoding a heterologous polypeptide of
interest may comprise codons used with a frequency of at least 17%
in Lemna gibba. In one embodiment, the modified nucleotide sequence
is the human .alpha.-2B-interferon encoding nucleotide sequence
shown in SEQ ID NO:16, which contains 93% duckweed preferred
codons.
TABLE-US-00001 TABLE 1 Lemna gibba-preferred codons from GenBank
Release 113 UUU 2.2 ( 4) UCU 0.5 ( 1) UAU 2.2 ( 4) UGU 0.0 ( 0) UUC
50.5 ( 92) UCC 31.9 ( 58) UAC 40.1 ( 73) UGC 17.6 ( 32) UUA 0.0 (
0) UCA 0.5 ( 1) UAA 3.8 ( 7) UGA 1.6 ( 3) UUG 2.7 ( 5) UCG 15.4 (
28) UAG 0.0 ( 0) UGG 24.2 ( 44) CUU 0.5 ( 1) CCU 6.6 ( 12) CAU 0.5
( 1) CGU 1.1 ( 2) CUC 39.0 ( 71) CCC 43.4 ( 79) CAC 6.6 ( 12) CGC
26.9 ( 49) CUA 1.1 ( 2) CCA 2.2 ( 4) CAA 4.4 ( 8) CGA 1.1 ( 2) CUG
22.5 ( 41) CCG 20.9 ( 38) CAG 26.9 ( 49) CGG 7.7 ( 14) AUU 0.0 ( 0)
ACU 3.3 ( 6) AAU 1.1 ( 2) AGU 0.0 ( 0) AUC 33.5 ( 61) ACC 26.4 (
48) AAC 37.9 ( 69) AGC 22.0 ( 40) AUA 0.0 ( 0) ACA 0.5 ( 1) AAA 0.0
( 0) AGA 4.9 ( 9) AUG 33.5 ( 61) ACG 9.3 ( 17) AAG 57.1 ( 104) AGG
6.0 ( 11) GUU 9.3 ( 17) GCU 7.1 ( 13) GAU 1.6 ( 3) GGU 1.1 ( 2) GUC
28.0 ( 51) GCC 73.6 ( 134) GAC 38.4 ( 70) GGC 46.7 ( 85) GUA 0.0 (
0) GCA 5.5 ( 10) GAA 2.2 ( 4) GGA 1.1 ( 2) GUG 34.0 ( 62) GCG 20.9
( 38) GAG 62.6 ( 114) GGG 27.5 ( 50)
[0303] Other modifications can also be made to the polynucleotide
encoding the heterologous polypeptide of interest to enhance its
expression in a plant host of interest, including duckweed. These
modifications include, but are not limited to, elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such well
characterized sequences which may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the polynucleotide encoding the heterologous polypeptide of
interest may be modified to avoid predicted hairpin secondary mRNA
structures.
[0304] There are known differences between the optimal translation
initiation context nucleotide sequences for translation initiation
codons in animals and plants and the composition of these
translation initiation context nucleotide sequences can influence
the efficiency of translation initiation. See, for example,
Lukaszewicz et al. (2000) Plant Science 154:89-98; and Joshi et al.
(1997); Plant Mol. Biol. 35:993-1001. In the present invention, the
translation initiation context nucleotide sequence for the
translation initiation codon of the polynucleotide nucleotide of
interest, for example, the polynucleotide encoding a heterologous
polypeptide of interest, may be modified to enhance expression in
duckweed. In one embodiment, the nucleotide sequence is modified
such that the three nucleotides directly upstream of the
translation initiation codon of the nucleotide sequence of interest
are "ACC." In a second embodiment, these nucleotides are "ACA."
[0305] Expression of a transgene in a host plant, including
duckweed, can also be enhanced by the use of 5' leader sequences.
Such leader sequences can act to enhance translation. Translation
leaders are known in the art and include, but are not limited to,
picornavirus leaders, e.g., EMCV leader (Encephalomyocarditis 5'
noncoding region; Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6126); potyvirus leaders, e.g., TEV leader (Tobacco Etch
Virus; Allison et al. (1986) Virology 154:9); human immunoglobulin
heavy-chain binding protein (BiP; Macajak and Sarnow (1991) Nature
353:90); untranslated leader from the coat protein mRNA of alfalfa
mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature 325:622);
tobacco mosaic virus leader (TMV; Gallie (1989) Molecular Biology
of RNA, 23:56); potato etch virus leader (Tomashevskaya et al.
(1993) J. Gen. Virol. 74:2717-2724); Fed-1 5' untranslated region
(Dickey (1992) EMBO J. 11:2311-2317); RbcS 5' untranslated region
(Silverthorne et al. (1990) J. Plant. Mol. Biol. 15:49-58); and
maize chlorotic mottle virus leader (MCMV; Lommel et al. (1991)
Virology 81:382). See also, Della-Cioppa et al. (1987) Plant
Physiology 84:965. Leader sequence comprising plant intron
sequence, including intron sequence from the maize alcohol
dehydrogenase 1 (ADH1) gene, the castor bean catalase gene, or the
Arabidopsis tryptophan pathway gene PAT1 has also been shown to
increase translational efficiency in plants (Callis et al. (1987)
Genes Dev. 1:1183-1200; Mascarenhas et al. (1990) Plant Mol. Biol.
15:913-920). See also copending provisional application U.S. Patent
Application No. 60/759,308, wherein leader sequence comprising a
duckweed intron sequence selected from the group consisting of the
introns set forth in SEQ ID NOs:13-15 provides for increased
translational efficiency in duckweed.
[0306] In some embodiments of the present invention, nucleotide
sequence corresponding to nucleotides 1222-1775 of the maize
alcohol dehydrogenase 1 gene (ADH1; GenBank Accession Number
X04049), or nucleotide sequence corresponding to the intron set
forth in SEQ ID NO:13, 14, or 15, is inserted upstream of the
polynucleotide encoding the heterologous polypeptide of interest or
the FucT and/or XylT inhibitory polynucleotide to enhance the
efficiency of its translation. In another embodiment, the
expression cassette contains the leader from the Lemna gibba
ribulose-bis-phosphate carboxylase small subunit 5B gene (RbcS
leader; see Buzby et al. (1990) Plant Cell 2:805-814; also see SEQ
ID NO:16, 17, or 18 of the present invention).
[0307] See also, by way of example only, the expression vectors
disclosed in the figures herein, wherein the RbcS leader and ADH1
intron are included as upstream regulatory sequences within an
expression cassette comprising the FucT inhibitory polynucleotide
(FIG. 8), the XylT inhibitory polynucleotide (FIGS. 9 and 11), an
expression cassette comprising the chimeric FucT/XylT inhibitory
molecule (FIG. 10), or an expression cassette comprising the coding
sequence for the heterologous polypeptide, the IgG1 heavy chain of
a monoclonal antibody (FIGS. 12, 13, and 14) or the light chain of
a monoclonal antibody (FIG. 14); wherein the LmUbq promoter and
LmUbq intron are included as upstream regulatory sequences within
an expression cassette comprising the FucT inhibitory
polynucleotide (FIG. 11), or an expression cassette comprising the
coding sequence for the heterologous polypeptide, the IgF1 light
chain of a monoclonal antibody (FIG. 13); wherein the SpUbq
promoter and SpUbq intron are included as upstream regulatory
sequences within an expression cassette comprising the FucT
inhibitory polynucleotide (FIG. 13), or an expression cassette
comprising the chimeric FucT/XylT inhibitory polynucleotide (FIG.
12); and wherein the LaUbq promoter and LaUbq intron are included
as upstream regulatory sequences in an expression cassette
comprising the XylT inhibitory polynucleotide (FIG. 13).
[0308] It is recognized that any of the expression-enhancing
nucleotide sequence modifications described above can be used in
the present invention, including any single modification or any
possible combination of modifications. The phrase "modified for
enhanced expression" in a plant, for example, a duckweed plant, as
used herein refers to a polynucleotide sequence that contains any
one or any combination of these modifications.
Signal Peptides
[0309] It is recognized that the heterologous polypeptide of
interest may be one that is normally or advantageously expressed as
a secreted protein. Secreted proteins are usually translated from
precursor polypeptides that include a "signal peptide" that
interacts with a receptor protein on the membrane of the
endoplasmic reticulum (ER) to direct the translocation of the
growing polypeptide chain across the membrane and into the
endoplasmic reticulum for secretion from the cell. This signal
peptide is often cleaved from the precursor polypeptide to produce
a "mature" polypeptide lacking the signal peptide. In an embodiment
of the present invention, a biologically active polypeptide is
expressed in the plant host of interest, for example, duckweed or
other higher plant, from a polynucleotide sequence that is operably
linked with a nucleotide sequence encoding a signal peptide that
directs secretion of the polypeptide into the culture medium. Plant
signal peptides that target protein translocation to the
endoplasmic reticulum (for secretion outside of the cell) are known
in the art. See, for example, U.S. Pat. No. 6,020,169 to Lee et al.
In the present invention, any plant signal peptide can be used to
target the expressed polypeptide to the ER.
[0310] In some embodiments, the signal peptide is the Arabidopsis
thaliana basic endochitinase signal peptide (amino acids 14-34 of
NCBI Protein Accession No. BAA82823), the extensin signal peptide
(Stiefel et al. (1990) Plant Cell 2:785-793), the rice
.alpha.-amylase signal peptide (amino acids 1-31 of NCBI Protein
Accession No. AAA33885), or a modified rice .alpha.-amylase signal
sequence (SEQ ID NO:17). In another embodiment, the signal peptide
corresponds to the signal peptide of a secreted duckweed
protein.
[0311] Alternatively, a mammalian signal peptide can be used to
target recombinant polypeptides expressed in a genetically
engineered plant of the invention, for example, duckweed or other
higher plant of interest, for secretion. It has been demonstrated
that plant cells recognize mammalian signal peptides that target
the endoplasmic reticulum, and that these signal peptides can
direct the secretion of polypeptides not only through the plasma
membrane but also through the plant cell wall. See U.S. Pat. Nos.
5,202,422 and 5,639,947 to Hiatt et al. In one embodiment of the
present invention, the mammalian signal peptide that targets
polypeptide secretion is the human .alpha.-2b-interferon signal
peptide (amino acids 1-23 of NCBI Protein Accession No.
AAB59402).
[0312] In one embodiment, the nucleotide sequence encoding the
signal peptide is modified for enhanced expression in the plant
host of interest, for example, duckweed or other higher plant,
utilizing any modification or combination of modifications
disclosed above for the polynucleotide sequence of interest.
[0313] The secreted biologically active polypeptide can be
harvested from the culture medium by any conventional means known
in the art and purified by chromatography, electrophoresis,
dialysis, solvent-solvent extraction, and the like. In this manner,
purified polypeptides, as defined above, can be obtained from the
culture medium.
[0314] Thus, in some embodiments, the protein expression host
system is a plant, for example, a duckweed or other higher plant,
and the secreted biologically active polypeptide is a glycoprotein
of the invention, where the glycoprotein has a substantially
homogeneous glycosylation profile, and is substantially homogeneous
for the G0 glycoform. In such embodiments, any such glycoprotein
that may remain within the plant material can optionally be
isolated and purified as described above. The secreted glycoprotein
can be obtained from the plant culture medium and purified using
any conventional means in the art as noted above. In this manner,
the purified glycoprotein obtained from the plant material is
substantially free of plant cellular material, and includes
embodiments where the preparations of glycoprotein have less than
about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating
plant protein. Where the purified glycoprotein is obtained from the
plant culture medium, the plant culture medium represents less than
about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical
precursors or non-protein-of-interest chemicals within the purified
glycoprotein preparation.
[0315] In some embodiments, these purified glycoproteins obtained
from the plant host can include at least 0.001%, 0.005%, 0.1%,
0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%,
7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about
30% (by dry weight) of contaminating plant protein. In other
embodiments, where the glycoprotein is collected from the plant
culture medium, the plant culture medium in these purified
glycoproteins can include at least 0.001%, 0.005%, 0.1%, 0.5%, 1%,
1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%,
8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dry
weight) of chemical precursors or non-protein-of-interest chemicals
within the purified glycoprotein preparation. In some embodiments,
isolation and purification from the plant host, and where secreted,
from the culture medium, results in recovery of purified
glycoprotein that is free of contaminating plant protein, free of
plant culture medium components, and/or free of both contaminating
plant protein and plant culture medium components.
Transformed Plants and Transformed Duckweed Plants and Duckweed
Nodule Cultures
[0316] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell or nodule, that is, monocot or
dicot, targeted for transformation. Suitable methods of introducing
nucleotide sequences into plants or plant cells or nodules include
microinjection (Crossway et al. (1986) Biotechniques 4:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat.
Nos. 5,563,055 and 5,981,840, both of which are herein incorporated
by reference), direct gene transfer (Paszkowski et al. (1984) EMBO
J. 3:2717-2722), ballistic particle acceleration (see, e.g., U.S.
Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782 (each of
which is herein incorporated by reference); and Tomes et al. (1995)
"Direct DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe
et al. (1988) Biotechnology 6:923-926). The cells that have been
transformed may be grown into plants in accordance with
conventional ways. See, for example, McCormick et al. (1986) Plant
Cell Reports 5:81-84.
[0317] The stably transformed duckweed utilized in this invention
can be obtained by any method known in the art. In one embodiment,
the stably transformed duckweed is obtained by one of the gene
transfer methods disclosed in U.S. Pat. No. 6,040,498 to Stomp et
al., herein incorporated by reference. These methods include gene
transfer by ballistic bombardment with microprojectiles coated with
a nucleic acid comprising the nucleotide sequence of interest, gene
transfer by electroporation, and gene transfer mediated by
Agrobacterium comprising a vector comprising the nucleotide
sequence of interest. In one embodiment, the stably transformed
duckweed is obtained via any one of the Agrobacterium-mediated
methods disclosed in U.S. Pat. No. 6,040,498 to Stomp et al. The
Agrobacterium used is Agrobacterium tumefaciens or Agrobacterium
rhizogenes.
[0318] It is preferred that the stably transformed duckweed plants
utilized in these methods exhibit normal morphology and are fertile
by sexual reproduction. Preferably, transformed plants of the
present invention contain a single copy of the transferred nucleic
acid, and the transferred nucleic acid has no notable
rearrangements therein. Also preferred are duckweed plants in which
the transferred nucleic acid is present in low copy numbers (i.e.,
no more than five copies, alternately, no more than three copies,
as a further alternative, fewer than three copies of the nucleic
acid per transformed cell).
[0319] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0320] Lemna, a small aquatic plant, is a scalable and economically
attractive expression platform for the manufacture of therapeutic
proteins free of human pathogens and with a clear path towards
regulatory approval. The Lemna expression system (LEX
System.sup.SM) enables rapid clonal expansion of transgenic plants,
secretion of transgenic proteins, high protein yields, ease of
containment that is comparable to mammalian cell culture systems
such as CHO cells, and has the additional advantage of low
operating and capital costs (Gasdaska et al. (2003) Bioprocessing
J. 50-56). In addition, this plant expression system offers the
advantage of high protein yields (in the range of 6-8% of the total
soluble protein (TSP)). These expression levels, in combination
with Lemna's high protein content and fast growth rate (36 hr
doubling time), enable production of >1 g of mAb per kg biomass
in a robust and well-controlled format.
[0321] The following examples demonstrate how humanization of the
glycosylation profile of a mAb was accomplished by coexpression of
the mAb with an interference RNA (RNAi) construct targeting the
endogenous expression of .alpha.1,3-fucosyltransferase and
.beta.1,2-xylosyltransferase genes. The resultant mAb contained a
single major N-glycan species (>95%) devoid of the plant
specific .alpha.-1,3-linked fucose and .beta.-1,2-linked xylose
sugars. In receptor binding assays, this glycan optimized mAb
exhibited enhanced effector cell receptor binding activity when
compared to mAb produced in wild-type Lemna having the native
glycosylation machinery and mAb produced in CHO cells.
Example 1
Isolation of Lemna minor Proteins Involved in N-Glycosylation of
Proteins
[0322] In order to generate recombinant proteins with remodeled
N-glycan, alpha 1-3 fucosyltransferase and .beta.1-2
xylosyltransferase were selected as targets for RNAi gene silencing
in L. minor. Initial results from cDNA sequencing efforts indicated
that two or more isoforms were present for each of the target
genes. Sequence homology between the isoforms was determined to be
between 90% and 95%. Full length cDNA sequences for both target
genes were retrieved and characterized. The full-length cDNA
sequence, including 5'- and 3'-UTR, for L. minor .alpha.1-3
fucosyltransferase (FucT) is set forth in FIG. 1; see also SEQ ID
NO:1 (open reading frame set forth in SEQ ID NO:2). The predicted
amino acid sequence encoded thereby is set forth in SEQ ID NO:3.
The encoded protein shares some similarity with other FucTs from
other higher plants. See FIG. 2. For example, the L. minor FucT
sequence shares approximately 50.1% sequence identity with the
Arabidopsis thaliana FucT shown in FIG. 2.
[0323] The full-length cDNA sequence, including 5'- and 3'-UTR, for
L. minor .beta.1-2 xylosyltransferase (XylT) (isoform #1) is set
forth in FIG. 3; see also SEQ ID NO:4 (ORF set forth in SEQ ID
NO:5). The predicted amino acid sequence encoded thereby is set
forth in SEQ ID NO:6. The encoded protein shares some similarity
with other XylTs from other higher plants. See FIG. 4. For example,
the L. minor XylT shares approximately 56.4% sequence identity with
the Arabidopsis thaliana XylT shown in FIG. 4. A partial-length
cDNA sequence, including 3'-UTR, for L. minor .beta.1-2
xylosyltransferase (XylT) (isoform #2) is set forth in FIG. 31; see
also SEQ ID NO:19 (ORF set forth in SEQ ID NO:20). The predicted
amino acid sequence encoded thereby is set forth in SEQ ID NO:21.
The partial-length XylT isoform #2 shares high sequence identity
with the corresponding region of the full-length XylT isoform #1,
as can be seen from the alignment shown in FIG. 32.
Example 2
RNAi Inhibition of Expression of L. minor FucT and XylT
[0324] Several RNAi strategies were undertaken to inhibit
expression of the L. minor FucT and XylT isoforms. FIGS. 5-7, 33,
and 34 outline these strategies. FIGS. 8-13 show maps of the
various constructs that were made to achieve the desired knockout
of expression of these two genes. A number of transgenic lines
comprising the various knockout RNAi constructs were generated
using standard transformation protocols described herein above.
[0325] The test antibody, designated herein as mAbI, was expressed
in wild-type Lemna having the native glycosylation machinery, and
transgenic Lemna lines expressing RNAi constructs designed to
inhibit expression of L. minor XylT and FucT isoforms. Generally,
three binary vectors were constructed for expression of mAbI in the
Lemna system. Expression vector mAbI01 contained codon optimized
genes encoding heavy (H) and light (L) chains of mAbI; vector
mAbI04 contained codon optimized genes encoding mAbI H and L chains
and a chimeric RNAi construct targeting expression of both XylT and
FucT isoforms; and vector mAbI05 contained codon optimized genes
encoding mAbI H and L chains, a single-gene RNAi construct
targeting FucT gene expression, and a single-gene RNAi construct
targeting XylT gene expression. Independent transgenic lines were
generated for the mABI01, mAbI04, and mAbI05 expression
vectors.
[0326] Optimized genes for mAbI H and L chains were designed to
have Lemna preferred codon usage (63%-67% GC content) and contain
the rice .alpha.-amylase signal sequence (GenBank M24286) fused to
the 5' end of their coding sequences. Restriction endonuclease
sites were added for cloning into Agrobacterium binary vectors
(EcoRI (5')/SacI (3'), H-chain) and (SalI (5')/HindIII (3'),
L-chain).
[0327] For the XF02 data presented in FIGS. 15-17 and the mAbI04
data presented in FIGS. 22-24 and 26, described herein below, the
RNAi strategy for inhibiting expression of the L. minor FucT and
XylT isoforms employed the chimeric RNAi design shown in FIG. 34.
For the mAbI05 data presented in FIG. 27, described herein below,
the RNAi strategy for inhibiting expression of the L. minor FucT
and XylT isoforms employed a double knockout of these genes using a
combination of the single gene RNAi designs shown in FIG. 5 (FucT
RNAi design) and FIG. 33 (XylT RNAi design).
[0328] Independent expression cassettes containing promoter, gene
of interest, and Nos terminator were created for the optimized mAbI
H and L chains and the single-gene or chimeric RNAi. Expression
cassettes were cloned into a modification of the Agrobacterium
binary vector pBMSP3 (obtained from Dr. Stan Gelvin, Purdue
University) with the appropriate restriction sites. Depending upon
the expression cassette, the L chain was fused to either the
modified chimeric octopine and mannopine synthase promoter with
Lemna gibba 5' RbcS leader (mAbI01, FIG. 14) or the high
expression, constitutive Lemna minor polyubiquitin promoter (LmUbq)
(mAbI04, FIG. 12; mAbI05; FIG. 13). The H-chain was fused to the
modified chimeric octopine and mannopine synthase promoter with
Lemna gibba 5' RbcS leader (mAbI04, mAbI05, and mAbI01). The
chimeric RNAi cassette, taken from plasmid XF02 in T7-4, was fused
to the high expression, constitutive Spirodela polyrhiza
polyubiquitin promoter (SpUbq). The single-gene RNAi cassette for
expression of the FucT inhibitory sequence was driven by the SpUbq
promoter; and the single gene RNAi cassette for expression of the
XylT inhibitory sequence was driven by an operably linked
expression control element comprising the Lemna aequinoctialis
ubiquitin promoter plus 5' UTR (LaUbq promoter). The H, L, and
chimeric RNAi expression cassettes were cloned into the modified
pBMSP3 binary vector in tandem orientation creating plasmid mAbI04.
The H, L, and single-gene RNAi expression cassettes targeting FucT
and XylT expression were cloned into the modified pBMSP3 binary
vector creating plasmid mAbI05. The H and L expression cassettes
were cloned into the modified pBMSP3 binary vector creating plasmid
mAbI01.
[0329] Though any transformation protocol can be used as noted
herein above, in some embodiments, the transformation protocol was
as follows. Using Agrobacterium tumefaciens C58Z707, a disarmed,
broad host range C58 strain (Hepburn et al. (1985) J. Gen.
Microbiol. 131:2961-2969), transgenic plants representing
individual clonal lines were generated from rapidly growing Lemna
minor nodules according to the procedure of Yamamoto et al. (2001)
In Vitro Cell Dev. Biol. Plant 37:349-353. For transgenic
screening, individual clonal lines were preconditioned for 1 week
at 150 to 200 .mu.mol m-2s-2 in vented plant growth vessels
containing SH media (Schenk and Hildebrandt (1972) Can. J. Botany
50:199-204) without sucrose. Fifteen to twenty preconditioned
fronds were then placed into vented containers containing fresh SH
media, and allowed to grow for two weeks. Tissue and media samples
from each line were frozen and stored at -70.degree. C.
[0330] A MALDI-TOF assay was developed to measure L. minor
.beta.-1,2-xylosyltransferase (XylT) and
.alpha.-1,3-fucosyltransferase (FucT) activities (Example 3
below).
[0331] FIGS. 15-17 represent primary screening data for the XF02,
mAbII04, and mAbI05 plants lines using the aforementioned assay. In
this assay, WT (wild-type) represents the FucT and XylT activity in
wild-type plants while BWT (boiled wild-type) represents their
activity in boiled plant extracts. Boiled wild-type (BWT) plant
extracts are representative of plant material in which FucT and
XylT activity has been deactivated. This data set shows that
several plant lines from each construct have a reduced level of
FucT and XylT activity compared with wild-type plant lines (WT) and
a comparable level of activity with boiled wild-type samples
(BWT).
[0332] Specifically, primary screening data for transgenic RNAi L.
minor plant lines comprising the XF02 construct of FIG. 10 are
shown in FIGS. 15 and 16. The XF02 construct expresses a chimeric
RNAi molecule that targets expression of both the L. minor FucT and
XylT proteins, including the various isoforms of the respective
proteins.
[0333] FIG. 17 shows primary screening data for transgenic RNAi L.
minor plant lines comprising the mAbI04 construct of FIG. 12 and
mAbI05 construct of FIG. 13.
Example 3
MALDI-TOF Assay for N-glycan .beta.-1,2-xylosyltransferase (XylT)
and .alpha.-1,3-fucosyltransferase (FucT) Activity
[0334] The following modified MALDI-TOF assay was used to determine
XylT and FucT activity in the transgenic plants described in
Example 2 above.
Materials
[0335] HOMOGENIZATION BUFFER: 50 mM HEPES, pH 7.5, 0.25 M sucrose,
2 mM EDTA, 1 mM DTT. [0336] REACTION BUFFER: 0.1 M Mes, pH 7.0, 10
mM MnCl.sub.2, 0.1% (v/v) Triton X-100. [0337]
URIDINE-5'-DIPHOSPHO-D-XYLOSE (UDP-Xyl) [0338]
GUANOSINE-5'-DIPHOSPHO-L-FUCOSE (GDP-Fuc) [0339]
N-ACETYLGLUCOSAMINE [0340] POLYETHYLENE GLYCOL (PEG) MIXTURE
1000-3000 (10 mg/mL PEG 1000, 2000, and 3000 (4:5:6 ratio) mixed
4:1 with 2 mg/mL sodium iodide). [0341] [Glu.sup.1]-FIBRINOPEPTIDE
B (GFP), HUMAN (1 pmol/.mu.L in water) [0342] DABSYLATED,
TETRAPEPTIDE, N-GLYCAN ACCEPTOR (EMD Biosciences) [0343] CHCA
(.alpha.-CYANO-4-HYDROXYCINNAMIC ACID) MATRIX (10 mg in 50% [v/v]
acetonitrile, 0.05% [v/v] trifluoroacetic acid).
Microsome Preparation
[0344] L. minor tissue (100 mg) was ground in 1 mL of cold
homogenization buffer in a bead mill at 5.times. speed for 40 s.
The homogenate was spun at 1,000 g for 5 min, 4.degree. C. The
supernatant was removed and spun at 18,000 g for 2 h, 4.degree. C.
The supernatant was then discarded. The pellet was resuspended in
20 .mu.L of cold reaction buffer and kept on ice or stored at
-80.degree. C. until use.
Reaction Conditions
[0345] The reaction mix contains 125 mM N-acetylglucosamine, 6.25
mM UDP-Xyl, 6.25 mM GDP-Fuc, 12.5 mM MnCl.sub.2, and 1.5 nmol of
dabsylated, tetrapeptide N-glycan acceptor. Microsomes (4 .mu.L)
were added to the reaction mix to start the reaction. The reaction
was incubated for 30 min at room temperature, and 90 min at
37.degree. C. The reaction was terminated by centrifugation at
18,000 g for 1 min and incubation at 4.degree. C.
MALDI-TOF Analysis
[0346] A portion of the supernatant from each reaction (0.5 .mu.L)
was mixed with 0.5 .mu.L of CHCA matrix on a MALDI target plate and
allowed to dry. The MALDI instrument was set to reflectron positive
ion mode and calibrated with PEG 1000-3000. Combined MS spectra
(200 shots) were taken from 1500-2500 Da using 0.5 .mu.mol GFP as
the lock mass. Ion counts of the reference peak (m/z=2222.865)
should be above 400. Ion counts of the XylT and FucT products
(m/z=2192.854 and 2206.870, respectively) were normalized to the
reference peak and the protein concentration of the microsome
fraction.
Example 4
Effect of RNAi Inhibition of Expression of L. minor FucT and XylT
on Glycosylation Profile of Monoclonal Antibodies
[0347] Monoclonal antibodies produced by wild-type (i.e., FucT and
XylT expression not silenced) L. minor comprising the mAbI01
construct (see FIG. 14) and L. minor lines transgenic for the
mAbI04 construct (see FIG. 12) or mAbI05 construct (see FIG. 13)
were analyzed for their N-glycosylation profile. The following
procedures were used.
Purification of mAb from Lemna.
[0348] Plant tissue was homogenized with 50 mM Sodium Phosphate,
0.3M Sodium Chloride, and 10 mM EDTA at pH 7.2 using a Silverson
High Shear Mixer at a tissue:buffer ratio of 1:8. The homogenate
was acidified to pH 4.5 with 1M Citric Acid, and centrifuged at
7,500.times.g for 30 minutes at 4.degree. C. The supernatant was
filtered through a 0.22 .mu.m filter and loaded directly on
mAbSelect SuRe resin (GE Healthcare) equilibrated with a solution
containing 50 mM Sodium Phosphate, 0.3M Sodium Chloride, and 10 mM
EDTA, pH 7.2. After loading, the column was washed to baseline with
the equilibration buffer followed by an intermediate wash with 5
column volumes of 0.1M Sodium Acetate, pH 5.0, and finally, bound
antibody was eluted with 10 column volumes of 0.1M Sodium Acetate,
pH 3.0. The eluate was immediately neutralized with 2M Tris
base.
Purification of N-linked Glycans.
[0349] Protein A-purified monoclonal antibodies (1 mg) from
wild-type and RNAi L. minor plant lines were dialyzed extensively
against water and lyophilized to dryness. Samples were resuspended
in 100 .mu.L of 5% (v/v) formic acid, brought to 0.05 mg/mL pepsin,
and incubated at 37.degree. C. overnight. The samples were heat
inactivated at 95.degree. C. for 10 min and dried. Pepsin digests
were resuspended in 100 .mu.L of 100 mM sodium acetate, pH 5.0 and
incubated with 1 mU of N-glycosidase A at 37.degree. C. overnight.
The released N-glycans were isolated using 4 cc Carbograph SPE
columns according to Packer et al. (1998) Glycoconj. J. 15:
737-747, and dried.
[0350] Dried N-glycans were further purified using 1 cc Waters
Oasis MCX cartridges. Columns were prepared by washing with 3
column volumes of methanol followed by 3 column volumes of 5% (v/v)
formic acid. N-glycans, resuspended in 1 mL of 5% (v/v) formic
acid, were loaded onto the prepared columns. The unbound fraction
as well as 2 additional column volume washes of 5% (v/v) formic
acid were collected, pooled and dried.
Derivatization of Oligosaccharides with 2-aminobenzoic acid
(2-AA).
[0351] Purified N-glycans or maltooligosaccharides were labeled
with 2-AA and purified using 1 cc Waters Oasis HLB cartridges
according to Anumula and Dhume (1998) Glycobiology 8: 685-694.
Labeled N-glycans and maltooligosaccharides were resuspended in 50
.mu.L of water and analyzed by MALDI-TOF MS and normal phase (NP)
HPLC-QTOF MS.
MALDI-TOF Mass Spectrometry.
[0352] MALDI-TOF MS was conducted using a Waters MALDI Micro MX
(Millford, Mass.). 2-AA labeled N-glycans (0.5 .mu.L) were properly
diluted with water, mixed with 0.5 .mu.L of 10 mg/mL DHB matrix in
70% (v/v) acetonitrile, spotted onto a target plate and analyzed in
negative reflectron mode.
NP-HPLC-Q-TOF MS Analysis of 2-AA Labeled N-glycans.
[0353] 2-AA labeled N-glycans or maltooligosaccharides were brought
to 80% (v/v) acetonitrile and separated on a Waters 2695 HPLC
system fitted with a TSK-Gel Amide-80 (2 mm.times.25 cm, 5 .mu.m)
column (Tosoh Biosciences, Montgomeryville, Pa.). 2-AA labeled
carbohydrates were detected and analyzed by fluorescence (230 nm
excitation, 425 nm emission) using a Waters 2475 fluorescence
detector and a Waters Q-TOF API US quadropole-time of flight
(Q-TOF) mass spectrometer (Millford, Mass.) fitted in-line with the
HPLC system.
[0354] Separations were conducted at 0.2 mL/min, 40.degree. C.,
using 10 mM ammonium acetate, pH 7.3 (solvent A) and 10 mM ammonium
acetate, pH 7.3, 80% (v/v) acetonitrile (solvent B). Sample elution
was carried out at 0% A isocratic for 5 min, followed by a linear
increase to 10% A at 8 min, and a linear increase to 30% A at 48
min. The column was washed with 100% A for 15 min and equilibrated
at 0% A for 15 min prior to the next injection.
[0355] Q-TOF analysis was conducted in negative ion mode with
source and desolvation temperatures of 100.degree. C. and
300.degree. C., respectively, and capillary and cone voltages of
2,100 and 30 V, respectively. Mass spectra shown are the result of
combining .gtoreq.50 individual scans per labeled N-glycan.
RP-HPLC-Q-TOF MS Analysis of Intact IgG.
[0356] Protein A purified IgG's (50 .mu.g) were desalted using the
Waters 2695 HPLC system fitted with a Poros R1-10 column (2
mm.times.30 mm; Applied Biosystems). IgG's were detected and
analyzed using a Waters 2487 dual wavelength UV detector (280 nm)
and the Waters Q-TOF API US. Separations were conducted at 0.15
mL/min, 60.degree. C., using 0.05% (v/v) trifluoroacetic acid (TFA;
solvent A) and 0.05% (v/v) TFA, 80% (v/v) acetonitrile (solvent B).
Sample elution was carried out using a linear increase from 30 to
50% B for 5 min, an increase to 80% B for 5 min. The solvent ratio
remained at 80% B for an additional 4 min, followed by a wash with
100% B for 1 min and equilibration of the column with 30% B for 15
min prior to the next run.
[0357] Q-TOF analysis was conducted in positive ion mode with
source and desolvation temperatures of 100.degree. C. and
300.degree. C., respectively, and capillary and cone voltages of
3.0 and 60 V, respectively. Data are the result of combining
.gtoreq.100 individual scans and deconvolution to the parent mass
spectrum using MaxEnt 1.
[0358] See also Triguero et al. (2005) Plant Biotechnol. J. 3:
449-457; Takahashi et al. (1998) Anal. Biochem. 255: 183-187;
Dillon et al. (2004) J. Chromatogr. A. 1053: 299-305.
Results.
[0359] FIG. 18 shows the structure and molecular weight of
derivatized wild-type L. minor monoclonal antibody N-glycans.
[0360] FIG. 19 shows that the wild-type mAbI01 construct (shown in
FIG. 15) providing for expression of the mAbI monoclonal IgG1
antibody in L. minor, without RNAi suppression of L. minor FucT and
XylT, produces an N-glycosylation profile with three major N-glycan
species, including one species having the .beta.1,2-linked xylose
and one species having both the .beta.1,2-linked xylose and core
.alpha.1,3-linked fucose residues; this profile is confirmed with
liquid chromatography mass spectrometry (LC-MS) (FIG. 20) and MALDI
(FIG. 21) analysis.
[0361] FIG. 22 shows an overlay of the relative amounts of the
various N-glycan species of the mAbI produced in the wild-type L.
minor line comprising the mAbI01 construct (no suppression of FucT
or XylT) and in the two transgenic L. minor lines comprising the
mAbI04 construct of FIG. 12. Note the enrichment of the GnGn (i.e.,
G0) glycan species, with no .beta.1,2-linked xylose or core
.alpha.1,3-linked fucose residues attached, and the absence of the
species having the .beta.1,2-linked xylose or both the
.beta.1,2-linked xylose and core .alpha.1,3-linked fucose residues.
This profile is confirmed with mass spec (LC-MS) (FIG. 23) and
MALDI (FIG. 24) analysis.
[0362] FIG. 25 shows intact mass analysis of the mAbI compositions
produced in wild-type L. minor (line 20) comprising the mAbI01
construct. When XylT and FucT expression are not suppressed in L.
minor, the recombinantly produced mAbI composition is
heterogeneous, comprising at least 9 different glycoforms, with the
G0XF.sup.3 glycoform being the predominate species present. Note
the very minor peak representing the G0 glycoform.
[0363] FIG. 26 shows intact mass analysis of the mAbI compositions
produced in transgenic L. minor (line 15) comprising the mAbI04
construct of FIG. 12. When XylT and FucT expression are suppressed
in L. minor using this chimeric RNAi construct, the intact mAbI
composition is substantially homogeneous for G0 N-glycans, with
only trace amounts of precursor N-glycans present (represented by
the GnM and MGn precursor glycan species). In addition, the mAbI
composition is substantially homogeneous for the G0 glycoform,
wherein both glycosylation sites are occupied by the G0 N-glycan
species, with three minor peaks reflecting trace amounts of
precursor glycoforms (one peak showing mAbI having an Fc region
wherein the C.sub.H2 domain of one heavy chain has a G0 glycan
species attached to Asn 297, and the C.sub.H2 domain of the other
heavy chain is unglycosylated; another peak showing mAbI having an
Fc region wherein the C.sub.H2 domain of one heavy chain has a G0
glycan species attached to Asn 297, and the C.sub.H2 domain of the
other heavy chain has the GnM or MGn precursor glycan attached to
Asn 297; and another peak showing mAbI having an Fc region wherein
the Asn 297 glycosylation site on each of the C.sub.H2 domains has
a G0 glycan species attached, with a third G0 glycan species
attached to an additional glycosylation site within the mAbI
structure).
[0364] FIG. 27 shows intact mass analysis of the mAbI compositions
produced in transgenic L. minor (line 72) comprising the mAbI05
construct of FIG. 13. When XylT and FucT expression are suppressed
in L. minor using this construct, the intact mAbI composition is
substantially homogeneous for G0 N-glycans, with only trace amounts
of precursor N-glycan species present (represented by the GnM and
MGn precursor glycan species). In addition, the mAbI composition is
substantially homogeneous (at least 90%) for the G0 glycoform, with
the same three minor peaks reflecting precursor glycoforms as
obtained with the mAbI04 construct.
[0365] The receptor binding activity of the mAbI produced in the
wild-type Lemna lines comprising the mAbI01 construct (i.e.,
without inhibition of XylT and FucT expression) and the transgenic
Lemna lines comprising the mAbI04 or mAbI05 construct (i.e., with
XylT and FucT expression inhibited) was compared to the receptor
binding activity of the mAbI produced in mammalian cell lines (CHO
and SP2/0).
[0366] Binding to FcFc.gamma.RIIIa on freshly isolated human NK
cells was assessed for the various mAbI products. Control data
collected for CHO-derived mAbI and SP2/0-derived mAbI is shown in
FIG. 35. Test data collected for wild-type Lemna-produced mAbI
having the normal plant N-glycan profile are designated as
mAbI01-15 and mabI01-20, wherein the mAbI product has N-linked
glycans that include .alpha.(1,3)-fucose residues (see FIGS. 36 and
37). Test data collected for transgenic Lemna-derived mAbI having
an optimized N-glycan profile (OPT) obtained with gene-silencing
RNAi constructs that target expression of
.alpha.1,3-fucosyltransferase are designated as mAbI05-72,
mAbI05-74, mAbI04-24, and mAbI04-15 (see FIGS. 36 and 37), wherein
the mAbI product has N-linked glycans that are devoid of
.alpha.(1,3)-fucose residues. Data comparing binding efficacy of
mAbI01-15, mAbI01-20, mAbI SP2/0, mAbI04-15, mAbI04-24, mAbI05-72,
and mAbI05-74 to recombinant mouse Fc.gamma.RIV, a receptor that is
sensitive to IgG fucose levels and which served as a surrogate for
human Fc.gamma.RIIIa, is shown in FIG. 38.
[0367] These data demonstrate that the transgenic Lemna-derived
mAbI product having the optimized glycan profile (OPT) shows
enhanced binding to Fc.gamma.RIIIa on freshly isolated human NK
cells (enhanced about 20 to 50-fold) as well as enhanced binding to
recombinant mouse Fc.gamma.RIV (enhanced about 10-fold) as compared
to the wild-type Lemna-derived mAbI product.
Example 6
Production of Anti-CD30 Monoclonal Antibody Having Improved
Receptor Binding and Increased ADCC Activity
[0368] This example outlines the expression of human anti-CD30 mAbs
in Lemna. Optimization of anti-CD30 mAb glycosylation was
accomplished by co-expression with an RNAi construct targeting the
endogenous expression of .alpha.-1,3-fucosyltransferase (FucT) and
.beta.-1,2-xylosyltransferase (XylT) genes in a manner similar to
that noted in the examples above for mAbI. The resultant anti-CD30
mAb produced in Lemna having its native glycosylation machinery
engineered to suppress FucT and XylT expression contained a single
major N-glycan species without any trace of plant-specific
N-glycans. In addition to the N-glycan homogeneity, glyco-optimized
anti-CD30 mAbs were also shown to have enhanced antibody-dependent
cell-mediated cytotoxicity (ADCC) and effector cell receptor
binding activity when compared to CHO-expressed anti-CD30 mAbs.
Methods
Strains and Reagents.
[0369] Novablue competent Escherichia coli cells were used for all
recombinant DNA work (EMD Biosciences, San Diego, Calif.).
Restriction endonucleases and DNA modification enzymes were
obtained from New England Biolabs (Ipswich, Mass.).
Oligonucleotides were obtained from Integrated DNA technologies
(Coralville, Iowa). Waters Oasis HLB and MCX columns (1 cc),
2,5-dihydroxybenzoic acid (DHB), and
.alpha.-cyano-4-hydroxycinnamic acid (CHCA) were from Waters
Corporation (Milford, Mass.). Purified dabsylated, tetrapeptide,
GnGn N-glycan acceptors (GnGn-dabsyl-peptide) and N-glycosidase A
were from EMD Biosciences. Carbograph SPE columns (4 cc) were from
Grace Davidson Discovery Sciences (Deerfield, Ill.).
Uridine-5'-diphospho-D-xylose (UDP-Xyl) was purchased from
Carbosource Services (Athens, Ga.). Acetonitrile (Optima grade) was
from Fisher Scientific (Summerville, N.Y.). Ammonium acetate was
from MP Biochemicals (Irvine, Calif.). Maltooligosaccharides
(MD6-1) were from V-Labs Inc. (Covington, Calif.). Monosaccharide
standards were from Dionex (Sunnyvale, Calif.). BATDA
(bis(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate) and
Europium solution were from Perkin-Elmer (Wellesley, Mass.).
Guanosine-5'-diphospho-L-fucose (GDP-Fuc), N-acetylglucosamine
(GlcNAc), 2-aminobenzoic acid (2-AA) and all other materials were
from Sigma (St. Louis, Mo.).
Construction of mAb and RNAi Expression Vectors.
[0370] The heavy (H) and light (L) chain variable region cDNA
sequences of fully human mAb 1 kappa antibody MDX-060 derived from
a transgenic Medarex HuMAb-Mouse.RTM. (Borchmann et al. (2003)
Blood 102:3737-3742) were determined and the full length MDX-060
human mAb 1 kappa antibody was produced recombinantly by a Chinese
hamster ovary cell line, CHO DG44 (Urlaub et al. (1986) Cell Mol.
Genet. 12:555-566), using standard techniques. Optimized genes for
H and L chains were designed to have Lemna-preferred codon usage
(63%-67% GC content) and contain the rice .alpha.-amylase signal
sequence (GenBank M24286) fused to the 5' end of their coding
sequences. Restriction endonuclease sites were added for cloning
into Agrobacterium binary vectors (EcoRI (5')/SacI (3'), H-chain)
and (SalI (5')/HindIII (3'), L-chain). Synthetic genes were
constructed and provided by Picoscript (Houston, Tex.).
[0371] A chimeric hairpin RNA (see FIG. 34) was designed to target
silencing of endogenous Lemna genes encoding
.alpha.-1,3-fucosyltransferase (based on the coding sequence for L.
minor FucT isoform #1, set forth in SEQ ID NO:2; see also GenBank
DQ789145) and .beta.-1,2-xylosyltransferase (based on the coding
sequence for L. minor XylT isoform #2, nt 1-1275 of SEQ ID NO:19;
set forth in SEQ ID NO:20; see also GenBank DQ789146). The chimeric
FucT+XylT hairpin RNA was designed to have 602 bp of double
stranded FucT sequence, 626 bp of double stranded XylT sequence,
and 500 bp of spacer sequence. The sense strand portion of the
hairpin RNA cassette encompasses the FucT forward fragment (nt
12-613 of SEQ ID NO:2; equivalent to nt 254-855 of SEQ ID NO:1) and
XylT forward fragment (nt 1-626 of SEQ ID NO:19), a spacer sequence
(nt 627-1126 of SEQ ID NO:19). The antisense strand portion of the
hairpin RNA encompasses the XylT reverse fragment (antisense
version of nt 1-626 SEQ ID NO:19) and FucT reverse fragment
(antisense version of nt 12-613 of SEQ ID NO:2 or nt 254-855 of SEQ
ID NO:1). The chimeric hairpin RNA was constructed by PCR
amplifying FucT and XylT forward and reverse gene fragments from
Lemna minor (8627) cDNA and sequentially cloning them into pT7blue
(EMD Biosciences) creating plasmid XF02 in T7-4. The FucT forward
gene fragment was amplified with DNA primers BLX 686
(5'-ATGGTCGACTGCTGCTGGTGCTC TCAAC-3') (SEQ ID NO:22) and BLX690
(5'-ATGTCTAGAATG CAGCAGCAAGTGCACC-3') (SEQ ID NO:23) producing a
620 bp product with terminal SalI (5') and XbaI (3') cloning sites.
The XylT forward gene fragment was amplified with DNA primers BLX
700 (5'-ATGACTAGTTGC GAAGCCTACTTCGGCAACAGC3') (SEQ ID NO:24) and
BLX694 (5'-ATGGGATCCGAATCTCAAGA ACAACTGTCG-3') (SEQ ID NO:25)
producing a 1144 bp product with terminal SpeI (5') and BamHI (3')
cloning sites. The XylT reverse gene fragment was amplified with
DNA primers BLX 695 (5'-ATGGGTACCTGCGAAGCCTACTTCGGCAA CAGC-3') (SEQ
ID NO:26) and BLX696 (5'-ATGGGA TCCACTGGCTGGGAGAAGTTCTT-3') (SEQ ID
NO:27) producing a 644 bp product with terminal BamHI (5') and KpnI
(3') cloning sites. The FucT reverse gene fragment was amplified
with DNA primers BLX 691 (5'-ATGGAGCTCTGCTGCTGGTGCT CTCAAC-3') (SEQ
ID NO:28) and BLX692 (5'-ATGGGTACCATGCAGCAGCAAGTGCACC-3') (SEQ ID
NO:29) producing a 620 bp product with terminal KpnI (5') and SacI
(3') cloning sites.
[0372] Independent expression cassettes containing promoter, gene
of interest, and Nos terminator were created for the optimized
MDX-060H and L chains and the chimeric RNAi. Expression cassettes
were cloned into a modification of the Agrobacterium binary vector
pBMSP3 (obtained from Dr. Stan Gelvin, Purdue University) with the
appropriate restriction sites. The H chain was fused to the
modified chimeric octopine and mannopine synthase promoter with
Lemna gibba 5' RbcS leader (Gasdaska et al. (2003) Bioprocessing J.
50-56). The L-chain was fused to the high expression, constitutive
Lemna minor polyubiquitin promoter (LmUbq). The chimeric RNAi
cassette, taken from plasmid XF02 in T7-4, was fused to the high
expression, constitutive Spirodela polyrhiza polyubiquitin promoter
(SpUbq). The three expression cassettes were cloned into the
modified pBMSP3 binary vector in tandem orientation creating
plasmid MDXA04.
Transformation and Plant Line Screening.
[0373] Using Agrobacterium tumefaciens C58Z707 (Hepburn et al.
(1985) J. Gen. Microbiol. 131:2961-2969), transgenic plants
representing individual clonal lines were generated from rapidly
growing Lemna minor nodules according to the procedure of Yamamoto
et al. (Yamamoto et al. (2001) In vitro Cell. Dev. Biol. 37). For
transgenic screening, individual clonal lines were preconditioned
for 1 week at 150 to 200 .mu.mol m.sup.-2s.sup.-2 in vented plant
growth vessels containing SH media (Schenk and Hildenbrandt (1972)
Can. J. Botany 50:199-204) without sucrose. Fifteen to twenty
preconditioned fronds were then placed into vented containers
containing fresh SH media, and allowed to grow for two weeks.
Tissue and media samples from each line were frozen and stored at
-70.degree. C.
ELISA Analysis of mAb Produced in Lemna.
[0374] Lemna tissue (100 mg) was homogenized using a FastPrep FP120
bead mill (Thermo Electron Corporation). Supernatants were diluted
to 1 .mu.g/mL and assayed using the IgG Quantitation ELISA kit
(Bethyl Laboratories). For the assay, microtiter plates were coated
with goat anti-human IgG at a concentration of 10 .mu.g/mL, and mAb
was detected by horseradish peroxidase (HRP)-conjugated goat
anti-human IgG diluted 1:100,000. Standard curves were created with
Human Reference IgG supplied with the ELISA kit. The sensitivity of
the ELISA was 7.8 ng/mL. All samples were analyzed in
duplicate.
Preparation of Lemna Microsomal Membranes and Assaying for Core
.beta.-1,2-xylosyltransferase and .alpha.-1,3-fucosyltransferase
Activities.
[0375] Lemna tissue (100 mg) from each line was homogenized in 1 mL
of cold homogenization buffer (50 mM
4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES], pH 7.5,
0.25 M sucrose, 2 mM ethylenediaminetetraacetic acid [EDTA], 1 mM
1,4-dithiothreitol [DTT]) for 40 s in a FastPrep FP120 bead mill
(Thermo Electron Corporation, Waltham, Mass.). The homogenate was
centrifuged at 1,000 g for 5 min at 4.degree. C. The supernatant
was removed and centrifuged at 18,000 g for 90 min at 4.degree. C.
The resulting pellet was resuspended in 20 .mu.L of cold reaction
buffer (0.1 M 2-[4-morpholino]ethanesulfonic acid [Mes], pH 7.0,
0.1% [v/v] Triton X-100, 10 mM MnCl.sub.2) and kept on ice or
stored at -80.degree. C. until use.
[0376] Core .beta.-1,2-xylosyltransferase and
.alpha.-1,3-fucosyltransferase activities were measured
simultaneously in 4 .mu.L of microsomal membranes prepared from
each RNAi line by incubating with 125 mM GlcNAc, 6.25 mM UDP-Xyl,
6.25 mM GDP-Fuc, 12.5 mM MnCl.sub.2, and 1.5 nmol of
GnGn-dabsyl-peptide acceptor for 2 h at 37.degree. C. as described
previously (Leiter et al. (1999) J. Biol. Chem. 274:21830-21839).
The reaction was terminated by a brief centrifugation and
incubation at 4.degree. C. and the products were analyzed by
positive reflectron mode MALDI-TOF MS.
Purification of MDX-060 LEX and LEX.sup.Opt mAbs.
[0377] Plant tissue was homogenized with 50 mM sodium phosphate,
0.3 M sodium chloride, and 10 mM EDTA, pH 7.2 using a Silverson
high shear mixer. The homogenate was acidified to pH 4.5 with 1 M
citric acid, and centrifuged at 7,500 g for 30 min at 4.degree. C.
The pH of the supernatant was adjusted to pH 7.2 with 2 M Tris,
prior to filtration using 0.22 .mu.m filters. The material was
loaded directly on mAbSelect SuRe protein A resin (GE Healthcare)
equilibrated with a solution containing 50 mM sodium phosphate, 0.3
M sodium chloride, and 10 mM EDTA, pH 7.2. After loading, the
column was washed to baseline with the equilibration buffer
followed by an intermediate wash with 5 column volumes of 0.1 M
sodium acetate, pH 5.0. Bound antibody was eluted with 10 column
volumes of 0.1 M sodium acetate, pH 3.0. The protein A eluate was
immediately neutralized with 2 M
2-amino-2-[hydroxymethyl]-1,3-propanediol (Tris). For aggregate
removal, the protein A eluate was adjusted to pH 5.5 and applied to
a ceramic hydroxyapatite type I (Bio-Rad) column equilibrated with
25 mM sodium phosphate, pH 5.5. After washing the column with 5
column volumes of equilibration buffer, the antibody was eluted in
a single step-elution using 0.25 M sodium phosphate, pH 5.5.
Fractions containing antibody by A.sub.280 were pooled and stored
at -80.degree. C.
[0378] Tissue extract and protein A flow through samples were
prepared for SDS-PAGE under reducing and non-reducing conditions by
addition of 2.times.SDS sample buffer .+-.5% [v/v]
2-mercaptoethanol. Protein A eluate and hydroxyapatite eluate
samples were diluted to a protein concentration of 0.5 mg/mL
followed by addition of 2.times.SDS sample buffer .+-.5% [v/v]
2-mercaptoethanol. Samples were incubated at 95.degree. C. for 2
minutes prior to electrophoresis using 4-20% Tris-Glycine gradient
gels (Invitrogen, Carlsbad, Calif.). Mark12 Molecular weight
markers (Invitrogen) and a MDX-060 reference standard were included
on the gels. Gels were stained with Colloidal Blue stain.
Purification of N-Linked Glycans.
[0379] Protein A purified monoclonal antibodies (1 mg) from
wild-type and RNAi Lemna plant lines were dialyzed extensively
against water and lyophilized to dryness. Samples were resuspended
in 100 .mu.L of 5% (v/v) formic acid, brought to 0.05 mg/ml pepsin,
and incubated at 37.degree. C. overnight. The samples were heat
inactivated at 95.degree. C. for 15 min and dried. Pepsin digests
were resuspended in 100 .mu.L of 100 mM sodium acetate, pH 5.0 and
incubated with 1 mU of N-glycosidase A at 37.degree. C. overnight.
The released N-glycans were isolated using 4 cc Carbograph SPE
columns (Packer et al. (1998) Glycoconj. J. 19:737-747) and
dried.
[0380] Dried N-glycans were further purified using 1 cc Waters
Oasis MCX cartridges. Columns were prepared by washing with 3
column volumes of methanol followed by 3 column volumes of 5% (v/v)
formic acid. N-glycans, resuspended in 1 mL of 5% (v/v) formic
acid, were loaded onto the prepared columns. The unbound fraction
as well as 2 additional column volume washes of 5% (v/v) formic
acid were collected, pooled, and dried.
Derivatization of Oligosaccharides with 2-aminobenzoic acid
(2-AA).
[0381] Purified N-glycans or maltooligosaccharides were labeled
with 2-AA and purified using 1 cc Waters Oasis HLB cartridges
according to Anumula and Dhume, 1998 (Anumula and Dhume (1998)
Glycobiology 8:685-694). Labeled N-glycans and
maltooligosaccharides were resuspended in 50 .mu.L of water and
analyzed by negative mode MALDI-TOF MS and NP-HPLC-QTOF MS.
MALDI-TOF Mass Spectrometry.
[0382] MALDI-TOF MS was conducted using a Waters MALDI Micro MX
(Millford, Mass.). Analysis of
.beta.-1,2-xylosyltransferase/.alpha.-1,3-fucosyltransferase
reaction products was conducted by mixing 0.5 .mu.L of each
reaction supernatant with 0.5 .mu.L of 10 mg/mL CHCA in 0.05% (v/v)
TFA, 50% (v/v) acetonitrile on a target plate. Xylosylated
([M+H].sup.+=2192.85 Da) or fucosylated ([M+H].sup.+=2206.87 Da)
GnGn-dabsyl-peptide products were detected in positive reflectron
mode. Ion counts of 200 combined spectra from each sample were
normalized against that of .beta.-1,4-galactosylated,
GnGn-dabsyl-peptide ([M+H].sup.+=2222.87 Da) present as a
contaminant (<5%) in the original GnGn-dabsyl-peptide mixture
from EMD Biosciences.
[0383] 2-AA labeled N-glycans or maltooligosaccharides (0.5 .mu.L)
were diluted with water, mixed with 0.5 .mu.L of 10 mg/ml DHB
matrix in 70% (v/v) acetonitrile, spotted onto a target plate and
analyzed in negative reflectron mode.
NP-HPLC-Q-TOF MS Analysis of 2-AA Labeled N-Glycans.
[0384] 2-AA labeled N-glycans or maltooligosaccharides were brought
to 80% (v/v) acetonitrile and separated on a Waters 2695 HPLC
system fitted with a TSK-Gel Amide-80 (2 mm.times.25 cm, 5 .mu.m)
column (Tosoh Biosciences, Montgomeryville, Pa.). 2-AA labeled
carbohydrates were detected and analyzed using a Waters 2475
fluorescence detector (230 nm excitation, 425 nm emission) and a
Waters Q-TOF API US quadropole-time of flight (QTOF) mass
spectrometer fitted on-line with the HPLC system.
[0385] Separations were conducted at 0.2 mL/min, 40.degree. C.,
using 10 mM ammonium acetate, pH 7.3 (solvent A) and 10 mM ammonium
acetate, pH 7.3, 80% (v/v) acetonitrile (solvent B). Sample elution
was carried out at 0% A isocratic for 5 min, followed by a linear
increase to 10% A at 8 min, and a linear increase to 30% A at 48
min. The column was washed with 100% A for 15 min and equilibrated
at 0% A for 15 min prior to the next injection.
[0386] QTOF analysis was conducted in negative ion mode with source
and desolvation temperatures of 100.degree. C. and 300.degree. C.,
respectively, and capillary and cone voltages of 2,100 and 30 V,
respectively. Mass spectra shown are the result of combining
.gtoreq.40 individual scans per labeled N-glycan.
Monosaccharide Analysis by HPAEC-PAD.
[0387] mAb samples (200 .mu.g) were subjected to acid hydrolysis
using 2 N TFA at 100.degree. C. for 3 h. Samples were dried by
vacuum centrifugation at ambient temperature and reconstituted in
150 .mu.L water prior to analysis by HPAE-PAD (Dionex). An aliquot
(25 .mu.L) of the reconstituted sample was applied to a CarboPac
PA10 column (4.times.250 mm) with a pre-column Amino Trap (Dionex).
Separation of monosaccharides was accomplished with a mobile phase
of 3.5 mM KOH, using an EG40 eluent generator. Monosaccharide peak
identity and relative abundance were determined using
monosaccharide standards.
Thermal Stability of mAb.
[0388] A MicroCal (Northampton, Mass.) VP-Capillary differential
scanning calorimetry (DSC) instrument was used to determine thermal
stability of glycol-optimized and wild-type mAbs. Purified mAb
samples were dialyzed in 20 mM NaH.sub.2PO.sub.4, pH 7.4, 150 mM
NaCl (PBS) overnight. Thermal denaturation data was collected by
heating the samples at a concentration of 300 .mu.g/mL from 35 to
95.degree. C. at a scan rate of 1.degree. C./min using PBS as the
reference buffer. The feedback and gain were set to low. The
baseline-corrected and normalized data was fit to a non-2-state
model using Origin v7.0 software.
FcR Binding Activity of mAb.
[0389] The experiment was conducted using a BIACORE (Biacore AB,
Uppsala, Sweden) instrument using surface plasmon resonance
technology. mAbs, 2 .mu.g/mL, were captured on the antigen coated
surface (recombinant human CD30). Several concentrations of both
the Val.sup.158 and Phe.sup.158 allotypes of FcR.gamma.IIIa,
starting from 6 .mu.M, were flowed over the captured antibodies for
3 min. The binding signal as a function of FcR.gamma.IIIa was fit
to a one-site binding model using GraphPad Prism (v4.01) software
to obtain the K.sub.D values. HBS-EP buffer (10 mM HEPES, 0.15 M
NaCl, 3 mM EDTA and 0.005% (v/v) P20 at pH 7.4) was used throughout
the experiment. Binding of the mAbs to buffer or FcR.gamma.IIIa to
blank surfaces were used as negative controls.
Assay for Antigen Binding Affinity.
[0390] CD30-expressing L540 cells (DSMZ Cell Culture Collection #
ACC 72) were used as antigen positive cells to assay for binding.
Aliquots of 2.times.10.sup.5 cells/well were incubated for 30 min
at 4.degree. C. with 100 .mu.L of primary antibody at the indicated
concentrations. Cells were washed twice in PBS with 2% (v/v) fetal
bovine serum (FBS) before addition of goat anti-human mAb,
FITC-labeled secondary antibody (Jackson ImmunoResearch, West
Grove, Pa.) at 1:500 dilution in 100 .mu.L/well for 30 min at
4.degree. C. Cells were washed twice in PBS with 2% (v/v) FBS and
assayed by flow cytometry using a FACS Calibur instrument (Becton
Dickinson, Franklin Lakes, N.J.). EC.sub.50 values of MDX-060 CHO,
LEX and LEX.sup.Opt mAb binding to CD30 on L540 cells were
determined from binding curves utilizing GraphPad Prism 3.0
software.
ADCC Assay.
[0391] Human peripheral blood mononuclear effector cells were
purified from heparinized whole blood by standard Ficoll-Paque
separation. Cells (2.times.10.sup.6) were washed in PBS and sent
for genotyping. The remaining effector cells were then resuspended
at 1.times.10.sup.6 cells/mL in RPMI 1640 medium containing 10%
(v/v) FBS and 50 U/mL of human IL-2 (Research Diagnostics, Concord,
Mass.) and incubated overnight at 37.degree. C. The effector cells
were washed once in culture medium and resuspended at
1.times.10.sup.7 cells/mL prior to use. L540 target cells at
1.times.10.sup.6 cells/mL in RPMI 1640 medium containing 10% (v/v)
FBS and 5 mM probenecid were labeled with 20 .mu.M BATDA
(bis(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate) for
20 min at 37.degree. C. Target cells were washed three times in PBS
supplemented with 20 mM HEPES and 5 mM probenecid, resuspended at
1.times.10.sup.5 cells/mL and added to effector cells in 96-well
plates (1.times.10.sup.4 target cells and 5.times.10.sup.5 effector
cells/well) at a final target to effector ratio of 1:50. Maximal
release was obtained by incubation of target cells in 3% (v/v)
Lysol and spontaneous release obtained by incubation in cell
culture medium alone. After 1 h incubation at 37.degree. C., 20
.mu.L of supernatant was harvested from each well and added to
wells containing 180 .mu.L of Europium solution. The reaction was
read with a Perkin Elmer Fusion Alpha TRF reader using a 400
.mu.sec delay and 330/80, 620/10 excitation and emission filters
respectively. The counts per second were plotted as a function of
antibody concentration and the data was analyzed by non-linear
regression, sigmoidal dose response (variable slope) using GraphPad
Prism 3.0 software. The percent specific lysis was calculated as:
(experimental release-spontaneous release)/(maximal
release-spontaneous release).times.100. In all studies, human mAb1
isotype control was included and compared to MDX-060 CHO, LEX, and
LEX.sup.Opt mAbs. Other controls included target and effector cells
with no mAb, target cells with no effector cells and target and
effector cells in the presence of 3% (v/v) Lysol.
Results
Expression of MDX-060 mAb in the LEX System.
[0392] MDX-060 is an anti-CD30 antibody (formally known as 5F11)
being developed for the treatment of Hodgkins lymphoma and
anaplastic large cell lymphoma (Borchmann et al. (2003) Blood
102:3737-3742). Two binary vectors were constructed for the
expression of MDX-060 in the LEX system. Expression vector MDXA01
contained codon optimized genes encoding heavy (H) and light (L)
chains of MDX-060 while vector MDXA04 contained genes encoding H
and L chains in addition to a chimeric FucT/XylT RNAi gene (FIG.
39). Independent transgenic lines were generated for both the
MDXA01 (165 lines) and MDXA04 (195 lines) expression vectors. For
simplicity, MDXA01 derived mAbs (wild-type N-glycosylation), and
MDXA04 derived mAbs (containing the FucT/XylT RNAi construct) will
be referred to as MDX-060 LEX and MDX-060 LEX.sup.Opt,
respectively, in the discussions below.
[0393] Transgenic plant lines were first screened for mAb
expression with an IgG ELISA. LEX.sup.Opt lines with high levels of
mAb expression were assayed further for FucT and XylT activity.
Transferase activities in the majority of the high expressing
MDX-060 LEX.sup.Opt lines were reduced to levels of the negative
control indicating effective silencing in the majority of the
assayed lines (FIG. 40). MDX-060 LEX.sup.Opt lines did not exhibit
any morphological or growth differences compared to wild-type Lemna
plants (data not shown).
[0394] Thermal stabilities of the MDX-060 CHO, LEX, and LEX.sup.Opt
mAbs were determined using differential scanning calorimetry (DSC).
All three mAbs exhibited similar melting curve kinetics (data not
shown) and melting transition point temperatures (Table 2 below),
further demonstrating the structural integrity of the
Lemna-produced MDX-060 LEX and LEX.sup.Opt mAbs compared to the
MDX-060 CHO mAb. SDS-PAGE analysis under non-reducing (FIG. 41A)
and reducing conditions (FIG. 41B) showed that mAbs from the
MDX-060 LEX.sup.Opt and MDX-060 CHO lines had similar protein
profiles with the mAb appearing as the major component in the
protein extract.
TABLE-US-00002 TABLE 2 Comparison of the thermal stabilities of
MDX-060 CHO, MDX-060 LEX, and glyco-optimized MDX-060 LEX.sup.Opt
mAbs by differential scanning calorimetry (DSC). Antibody T.sub.m1
(.degree. C.) T.sub.m2 (.degree. C.) T.sub.m3 (.degree. C.) MDX-060
CHO 72 75 84 MDX-060 LEX 71 75 84 MDX-060 LEX.sup.Opt 72 76 84
N-Glycan Structures of MDX-060 CHO, LEX, and LEX.sup.Opt mAbs.
[0395] The N-glycan profiles of recombinant MDX-060 CHO, MDX-060
LEX, and MDX-060 LEX.sup.opt derived mAbs were determined by
negative reflectron mode MALDI-TOF MS and normal phase (NP)
HPLC-QTOF MS. The structures of N-glycans referred to in the
following discussion are shown in FIG. 53.
[0396] MALDI-TOF MS analysis of N-glycans from MDX-060 CHO lines
indicated the presence of four major N-glycans with m/z values
corresponding to 2-AA labeled GnGnF.sup.6 (nomenclature derived
from http://www.proglycan.com), Man5, GnA.sub.isoF.sup.6, and
AAF.sup.6 (FIG. 42). NP-HPLC separated the GnA.sub.isoF.sup.6
N-glycan into its two isoforms (Gal attached to the .alpha.-1,6-Man
or .alpha.-1,3-Man arm) bringing the total number of major
N-glycans found on MDX-060 CHO to five (FIG. 43). MS/MS
fragmentation of the peaks was not conducted to confirm the
identity of each isoform; however, the higher abundance of the
earlier peak suggested that Gal was attached to the .alpha.-1,6-Man
arm of this N-glycan (Shinkawa et al. (2003) J. Biol. Chem.
278:3466-3473; Zhu et al. (2005) Nat. Biotechnol. 23:1159-1169).
On-line negative mode QTOF MS analysis gave m/z values
corresponding to doubly charged GnGnF.sup.6, Man5,
GnA.sub.isoF.sup.6 (both isoforms), and AAF.sup.6, confirming the
MALDI-TOF MS results (Table 3 below). Peak integration of the
fluorescent trace revealed that GnGnF.sup.6, Man5, AGnF.sup.6,
GnAF.sup.6, and AAF.sup.6 constituted 50.8, 2.5, 26.1, 10.7 and
6.8%, respectively, of the total N-glycan structures from MDX-060
CHO. The remaining 3.1% of N-glycans were found to be a mixture of
GnGn, GnM.sub.isoF.sup.6, GnM.sub.iso, and MM with no structure
higher than 1.2% of the total (data not shown).
TABLE-US-00003 TABLE 3 Summary of observed MALDI-TOF and QTOF MS
masses of the major 2-AA labeled N-glycans from MDXA-060 mAbs
produced by CHO cells (CHO), wild-type Lemna (LEX) or
glyco-optimized Lemna lines expressing the RNAi construct
(LEX.sup.Opt). Observed Observed % N-glycan Theoretical m/z
MALDI-TOF.sup.c Q-TOF.sup.c Peak name.sup.a Proposed
Structure.sup.b [M - H].sup.- [M - 2H].sup.2- [M - H].sup.- [M -
2H].sup.2- Area.sup.c CHO GnGnF.sup.6-2AA ##STR00002## 1582.590
790.7911 1582.455 790.7825 50.8 Man5-2AA ##STR00003## 1354.479
676.7436 1354.392 676.7343 2.50 GnA.sub.isoF.sup.6-2AA ##STR00004##
1744.642 871.8175 1744.492 871.7970 36.8 AAF.sup.6-2AA ##STR00005##
1906.695 952.8438 1906.567 952.8181 6.80 LEX GnGn-2AA ##STR00006##
1436.532 717.7622 1436.549 717.7894 8.40 GnGnX-2AA ##STR00007##
1568.574 783.7833 1568.581 783.8150 17.2 GnGnXF.sup.3-2AA
##STR00008## 1714.632 856.8122 1714.615 856.853 67.4 LEX.sup.Opt
GnGn-2AA ##STR00009## 1436.532 717.7622 1436.523 717.7993 95.8
.sup.aN-glycan names are based on Proglycan
(http://www.proglycan.com) nomenclature. 2AA, 2-aminobenzoic acid.
.sup.bThe symbols of the proposed N-glycan structures are as
follows: , N-acetylglucosamine; , mannose; , galactose; , xylose; ,
.alpha.-1,3-fucose; , .alpha.-1,6-fucose; , 2-aminobenzoic acid.
.sup.cThe m/z values and the % peak area of each N-glycan structure
were obtained from FIG. 2.
[0397] MALDI-TOF MS analysis of wild-type MDX-060 LEX mAb revealed
the presence of three major species with m/z values corresponding
to GnGnXF.sup.3, GnGnX and GnGn (FIG. 42). NP-HPLC-QTOF MS analysis
showed three major fluorescent peaks with m/z values corresponding
to doubly charged GnGnXF.sup.3, GnGnX and GnGn, again confirming
the MALDI-TOF MS results (FIG. 43; Table 3). Integration of the
fluorescent peaks indicated that GnGnXF.sup.3, GnGnX and GnGn
constituted 67.4, 17.2 and 8.4%, respectively, of the total
N-glycans derived from the MDX-060 LEX mAb. The remaining 7% of
N-glycans were determined to be a mixture of MM, GnM.sub.iso,
MMXF.sup.3, GnGnF.sup.3, GnM.sub.isoXF.sup.3, Man6, Man7,
Gn(FA).sub.isoXF.sup.3, Man8 and Man9 with no N-glycan greater than
2% of the total. Similar results were seen with mAbs isolated from
two independently transformed MDX-060 LEX lines (data not shown).
The simple array of N-glycans on LEX mAbs demonstrated here
provides an amenable starting point for glyco-optimization.
[0398] In contrast to the MDX-060 LEX mAb, N-glycans from the
MDX-060 LEX.sup.Opt mAb possessed GnGn as the major N-glycan
species by both MALDI-TOF MS and NP-HPLC-QTOF MS analysis (FIGS. 42
and 43; Table 3). GnGn comprised 95.8% of the total N-glycans with
the remaining 4.2% of N-glycans determined to be MM, GnM.sub.iso,
GnA.sub.iso, Man6, Man7 and Man8 with no one structure greater than
1.2% of the total N-glycans. None of the LEX.sup.Opt N-glycans
contained fucose (Fuc) or xylose (Xyl). These results demonstrated
that co-expression of an RNAi construct targeting Lemna FucT and
XylT resulted in the complete elimination of Fuc and Xyl-containing
N-glycans from MDX-060 LEX.sup.Opt mAbs and produced highly
homogeneous mAb glycoforms. The same results were obtained for
MDX-060 LEX.sup.Opt mAb harvested from an independent transgenic
line (line 225) comprising the MDXA04 expression vector, at a
different growth scale (300 g tissue for transgenic line 225 versus
1 g tissue for transgenic line 52, which produced the MDX-060
LEX.sup.Opt mAb having the N-glycan profile shown in FIGS. 42 and
43. Unlike mammalian cell culture systems where N-glycan
heterogeneity can change with culture conditions, growth scale, and
growth period (Kanda et al. (2006) Biotechnol. Bioeng. 94:680-688),
the glycan uniformity observed with LEX.sup.Opt mAbs was shown to
be consistent under a variety of growth conditions and scales.
[0399] The absence of Fuc or Xyl on MDX-060 LEX.sup.Opt mAb
N-glycans was further confirmed by monosaccharide analysis (Table 4
below). Monosaccharides were released from MDX-060 CHO, LEX and
LEX.sup.Opt mAbs by acid hydrolysis and analyzed by high
performance anion exchange chromatography (HPAEC) coupled to pulsed
amperometric detection (PAD). The monosaccharide ratios for Man and
GlcNAc residues were similar for CHO and wild-type LEX mAbs and
correlated well with expected values. LEX mAbs were significantly
decreased in Gal and Fuc content and had a significant increase in
Xyl when compared to CHO-derived mAbs. Monosaccharide analysis of
Lemna derived mAbs revealed that while Fuc and Xyl were present on
wild-type LEX N-glycans, they were not detected on LEX.sup.Opt
mAbs. Collectively, these results demonstrate that co-expression of
an RNAi construct targeting Lemna XylT and FucT results in the
elimination of Fuc and Xyl-containing N-glycans from MDX-060
LEX.sup.Opt mAbs and produce highly homogeneous mAb glycoforms. The
robustness of this glyco-optimization strategy has been confirmed
with multiple independent plant lines expressing the MDX-060
LEX.sup.Opt mAb as well as with other mAbs expressed in the LEX
System, for example, the mAbI monoclonal antibody discussed in the
examples herein above. In these subsequent transformations,
glyco-optimized mAb expression levels up to 6% of total soluble
protein (TSP) have been obtained.
TABLE-US-00004 TABLE 4 Monosaccharides released from MDX-060 CHO,
LEX and LEX.sup.Opt mAbs by acid hydrolysis and analyzed by
HPAEC-PAD. The monosaccharide content from each mAb was determined
by normalizing against carbohydrate controls. MDX-060 MDX-060 CHO
MDX-060 LEX LEX.sup.Opt Monosaccharide pmol (% total) pmol (%
total) pmol (% total) Fuc 254 (20) 232 (13) 0 GlcNAc 605 (47) 773
(45) 1,003 (67) Gal 75 (6) 0 0 Man 355 (27) 491 (29) 501 (33) Xyl 0
226 (13) 0 Total 1,289 (100) 1,722 (100) 1,504 (100)
Functional Activity of MDX-060 CHO, LEX and LEX.sup.Opt mAbs.
[0400] Antigen binding properties of the MDX-060 CHO, MDX-060 LEX,
and MDX-060 LEX.sup.Opt mAbs were determined using CD30 expressing
L540 cells. All three mAbs had nearly identical binding curves
(FIG. 43). EC.sub.50 concentrations were determined to be 0.180
.mu.g/mL, 0.227 .mu.g/mL, and 0.196 .mu.g/mL for MDX-060 CHO, LEX,
and LEX.sup.Opt, respectively (FIG. 44), indicating that antigen
binding for all three mAbs were similar.
[0401] Fc-receptor-mediated effector cell function has been shown
to be important for the in vivo activity of many therapeutic mAbs.
Since the FcR expressed on NK cells and macrophages responsible for
ADCC activity is Fc.gamma.RIIIa, binding of the various mAbs to
this receptor was compared. FcR binding of CHO, LEX and LEX.sup.Opt
mAbs was determined by equilibrium binding of the mAbs with
effector cells expressing two different human FcR.gamma.IIIa
allotypes (Phe.sup.158 or Val.sup.158). MDX-060 LEX had a 1.7-fold
increase in FcR.gamma.IIIaPhe.sup.158 and a 0.4-fold decrease in
FcR.gamma.IIIaVal.sup.158 binding compared to the CHO-derived mAb,
demonstrating that receptor binding for CHO and LEX mAbs were
similar. In contrast, LEX.sup.Opt mAbs had a 27 and 15-fold higher
affinity for FcR.gamma.IIIaPhe.sup.158 and
FcR.gamma.IIIaVal.sup.158, respectively, than CHO mAbs (FIG. 45).
These results suggested that RNAi silencing of the Lemna FucT and
XylT activities in LEX.sup.Opt lines produced mAbs with enhanced
FcR binding.
[0402] ADCC activities of the CHO, LEX and LEX.sup.Opt mAbs were
determined by incubating mAbs with either homozygous
(FcR.gamma.IIIaPhe.sup.158) or heterozygous
(FcR.gamma.IIIaPhe/Val.sup.158) human effector cells and BATDA
(bis(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate)
labeled L540 target cells (FIG. 45). MDX-060 LEX mAbs (31%) had the
same maximal percent cell lysis as CHO mAbs (31%) using
heterozygous FcR.gamma.IIIaPhe/Val.sup.158 human effector cells
(FIG. 46) with similar EC.sub.50 values. Maximal percent cell lysis
for LEX mAbs (27%) was slightly increased compared to CHO mAbs
(15%) using homozygous Fc.gamma.RIIIa Phe/Phe.sup.158 effector
cells. Importantly, LEX.sup.Opt mAbs had significantly increased
ADCC activity compared to MDX-060 CHO and LEX mAbs, irrespective of
the donor genotype. This was assessed by both an increase in
potency and efficacy. Maximal percent lysis for MDX-060 Lex.sup.Opt
was 45% for both experiments, while the EC.sub.50 value was 3 to 5
times lower than MDX-060 LEX and MDX-060 CHO mAbs, respectively,
for Fc.gamma.RIIIa Val/Phe.sup.158 effector cells and 2 to 3 times
lower for the Fc.gamma.RIIIa Phe/Phe.sup.158 effector cells. These
results demonstrate that removal of Fuc and Xyl-containing
N-glycans from MDX-060 LEX.sup.Opt mAbs caused an enhancement in
ADCC activity and hence can improve their therapeutic
potential.
RP-HPLC-Q-TOF MS Analysis of Intact IgG for MDX-060 LEX and MDX-060
LEX.sup.Opt.
[0403] Protein A purified IgG's (50 .mu.g) were desalted using the
Waters 2695 HPLC system fitted with a Poros R1-10 column (2
mm.times.30 mm; Applied Biosystems). IgG's were detected and
analyzed using a Waters 2487 dual wavelength UV detector (280 nm)
and the Waters Q-TOF API US. Separations were conducted at 0.15
mL/min, 60.degree. C., using 0.05% (v/v) trifluoroacetic acid (TFA;
solvent A) and 0.05% (v/v) TFA, 80% (v/v) acetonitrile (solvent B).
Sample elution was carried out using a linear increase from 30 to
50% B for 5 min, an increase to 80% B for 5 min. The solvent ratio
remained at 80% B for an additional 4 min, followed by a wash with
100% B for 1 min and equilibration of the column with 30% B for 15
min prior to the next run.
[0404] Q-TOF analysis was conducted in positive ion mode with
source and desolvation temperatures of 100.degree. C. and
300.degree. C., respectively, and capillary and cone voltages of
3.0 and 60 V, respectively. Data are the result of combining
.gtoreq.100 individual scans and deconvolution to the parent mass
spectrum using MaxEnt 1.
[0405] See also Triguero et al. (2005) Plant Biotechnol. J. 3:
449-457; Takahashi et al. (1998) Anal. Biochem. 255: 183-187;
Dillon et al. (2004) J. Chromatogr. A. 1053: 299-305.
[0406] FIG. 47 shows intact mass analysis of the MDX-060 LEX mAb
compositions produced in wild-type L. minor comprising the MDXA01
construct. When XylT and FucT expression are not suppressed in L.
minor, the recombinantly produced MDX-060 LEX mAb composition
comprises at least 7 different glycoforms, with the G0XF.sup.3
glycoform being the predominate species present. Note the absence
of a peak representing the G0 glycoform.
[0407] FIG. 48 shows glycan mass analysis of the heavy chain of the
MDX-060 LEX mAb produced in wild-type L. minor comprising the
MDXA01 construct. When XylT and FucT expression are not suppressed
in L. minor, the predominate N-glycan species present is
G0XF.sup.3, with additional major peaks reflecting the G0X species.
Note the minor presence of the G0 glycan species.
[0408] FIG. 49 shows intact mass analysis of the MDX-060
LEX.sup.Opt mAb compositions produced in transgenic L. minor
comprising the MDXA04 construct. When XylT and FucT expression are
suppressed in L. minor, the intact mAb composition contains only G0
N-glycans. In addition, the composition is substantially
homogeneous for the G0 glycoform (peak 2), wherein both
glycosylation sites are occupied by the G0 N-glycan species, with
two minor peaks reflecting trace amounts of precursor glycoforms
(peak 1, showing mAb having an Fc region wherein the C.sub.H2
domain of one heavy chain has a G0 glycan species attached to Asn
297, and the C.sub.H2 domain of the other heavy chain is
unglycosylated; and peak 3, showing mAb having an Fc region wherein
the Asn 297 glycosylation site on each of the C.sub.H2 domains has
a G0 glycan species attached, with a third G0 glycan species
attached to an additional glycosylation site within the mAb
structure).
[0409] FIG. 50 shows glycan mass analysis of the heavy chain of the
MDX-060 LEX.sup.Opt mAb produced in transgenic L. minor comprising
the MDXA04 construct. When XylT and FucT expression are suppressed
in L. minor, the only readily detectable N-glycan species attached
to the Asn 297 glycosylation sites of the C.sub.H2 domains of the
heavy chains is G0.
Discussion
[0410] Glyco-optimization of MDX-060 was accomplished by
co-expression with an RNAi cassette aimed at silencing the
endogenous Lemna FucT and XylT genes. This simultaneous silencing
of both FucT and XylT genes was achieved using a single RNAi
transcript. The absence of Fuc and Xyl on the LEX.sup.Opt mAb was
confirmed by MALDI-TOF, NP-HPLC-QTOF MS, and monosaccharide
analysis of N-glycans purified from the MDX-060 LEX.sup.Opt mAb.
These analyses corroborate the lack of transferase activity
observed in microsomal membranes. Importantly, >95% of the
N-glycans released from LEX.sup.Opt mAbs were of a single
structure, GnGn, indicating that this strategy had the added
benefit of producing mAbs with a homogeneous N-glycan profile.
MDX-060 LEX and LEX.sup.Opt mAbs were found to be indistinguishable
with regard to thermal stability and antigen binding compared to
MDX-060 CHO. Electrophoretic analysis was also found to be similar
for all three mAbs. In fact, the only structural differences
detected were in the mAb N-glycan profiles.
[0411] Without being bound by theory, the ability of the MDX-060
LEX.sup.Opt lines to produce mAbs with a single major N-glycan
species may be based on the more uniform mAb glycoform distribution
found in wild-type Lemna. N-glycans released from mAbs purified
from wild-type tobacco (Fujiyama et al. (2006) J. Biosci. Bioeng.
101:212-218; Elbers et al. (2001) Plant Physiol. 126:1314-1322;
Bakker et al. (2001) Proc. Natl. Acad. Sci. 98:2899-2904), alfalfa
(Bardor et al. (2003) Plant Biotechnol. J. 1:451-462), and moss
(Koprivova et al. (2003) Plant Bio. 5:582-591) show that mAb
glycoform heterogeneity in plants with wild-type N-glycosylation
can range from five (alfalfa) (Bardor et al. (2003) Plant
Biotechnol. J. 1:451-462) to eight (tobacco) (Fujiyama et al.
(2006) J. Biosci. Bioeng. 101:212-218) different major structures.
MDX-060 LEX possesses only three major N-glycan structures (GnGn,
GnGnX and GnGnXF.sup.3). This simple array of N-glycans on mAbs
produced by wild-type Lemna may provide a more amenable starting
point for glyco-optimization leading to greater homogeneity than
that observed in other systems.
[0412] Fc-receptor mediated effector cell function has been shown
to be important for the in vivo activity of many therapeutic mAbs.
In this study, the ADCC activity of MDX-060 CHO, MDX-060 LEX, and
MDX-060 LEX.sup.Opt mAbs was compared. Since the FcR expressed on
NK cells and macrophages responsible for ADCC activity is
Fc.gamma.RIIIa, the binding of the various mAbs to this receptor
was also compared. The results discussed above show that MDX-060
LEX.sup.Opt mAb has an increased binding affinity (15-25 fold) and
maximal binding (4-5 fold) to Fc.gamma.RIIIa as well as enhanced
ADCC activity compared to MDX-060 CHO and MDX-060 LEX mAbs. The
removal of .alpha.-1,6-linked Fuc from various mAbs produced in
other expression systems has been shown previously to increase FcR
binding and enhance ADCC function (Shinkawa et al. (2003) J. Biol.
Chem. 278:3466-3473; Shields et al. (2002) J. Biol. Chem.
277:26733-26740; Niwa et al. (2004) Clin. Cancer Res.
10:6248-6255). The results presented herein suggest that removal of
the .alpha.-1,3-linked Fuc from the MDX-060 LEX.sup.Opt mAbs has
the same effect on mAb function as the removal of
.alpha.-1,6-linked Fuc.
[0413] In this study, two naturally occurring polymorphic isoforms
of Fc.gamma.RIIIa at residue 158.sup.41, Val.sup.158 and
Phe.sup.158, were evaluated. MDX-060 LEX.sup.Opt shows higher
binding affinity to Fc.gamma.RIIIa-Val.sup.158 compared to
Fc.gamma.RIIIa-Phe.sup.158 as has been observed with other IgG1
mAbs (Shields et al. (2002) J. Biol. Chem. 277:26733-26740). The
fact that an increase in binding with MDX-060 LEX.sup.Opt was
observed with both isoforms is important since differential binding
to Val.sup.158 over Phe.sup.158 was found to be predictive of the
clinical and immunological responsiveness of certain patient groups
receiving anti-CD20 treatment (Cartron et al. (2002) Blood
99:754-758; Weng et al. (2003) J. Clin. Oncol. 21:3940-3947; Weng
et al. (2004) J. Clin. Oncol. 22:4717-4724). This increase in
binding has been hypothesized to result in a higher percentage of
patients responding to treatment that requires Fc
functionality.
[0414] A similar increase in ADCC activity was also observed. In
this study, the MDX-060 LEX.sup.Opt mAb showed an increase in cell
lysis and a decrease in the EC.sub.50 value, resulting in an
increase in efficacy and potency when compared to MDX-060 CHO. This
corresponds to a 20-fold increase in activity, determined by taking
the maximum percent lysis of MDX-060 CHO and calculating the
concentration of MDX-060 LEX.sup.Opt mAb giving rise to the same
percent cell lysis. As with the Fc.gamma.RIIIa binding study, the
increase in ADCC activity was observed with both a homozygous
Fc.gamma.RIIIaPhe/Phe.sup.158 and a heterozygous Fc.gamma.RIIIa
Phe/Val.sup.158 effector cell donor. The results presented here
suggest that removal of the .alpha.-1,3-linked Fuc from the MDX-060
LEX.sup.Opt mAbs has the same effect on mAb function as the removal
of .alpha.-1,6-linked Fuc.
[0415] The robustness of this glyco-optimization strategy has been
demonstrated with multiple independent Lemna plant lines expressing
the MDX-060 LEX.sup.Opt mAb as well as with other mAbs expressed in
the Lemna expression system (see, for example, Examples 2-4 above).
Furthermore, there is no apparent difference in plant phenotype or
growth rate compared with wild-type Lemna plants. Unlike mammalian
cell culture systems where N-glycan heterogeneity can change with
culture conditions, growth scale and growth period.sup.8, the
glycan uniformity observed with LEX.sup.Opt mAbs has been shown to
be consistent under a variety of growth conditions and scales (data
not shown).
[0416] In conclusion, an RNAi strategy was used to produce a
glyco-optimized anti-CD30 antibody in the Lemna expression system.
The resulting mAb consists of a single, major N-glycan structure,
without any evidence of the plant-specific Fuc and Xyl residues. In
addition, the resulting optimized mAb has increased ADCC activity
and Fc.gamma.RIIIa binding activity compared to a CHO-derived mAb.
The homogeneous glycosylation profile obtained on mAbs produced in
a Lemna expression system having this FucT+XylT gene knockout
strategy makes it is possible to express these mAbs with increased
production consistency.
Example 7
Scale-Up Production of Glycan-Optimized mAbI in Lemna minor
[0417] L. minor transgenic line 24 comprising the mAbI04 construct
of FIG. 12 (providing for suppression of FucT and XylT) was
generated in a manner similar to that described above. Following
its generation, transgenic line 24 was continuously maintained by
clonal culture, wherein periodically a subsample of the plant
culture was transferred to fresh culture medium for further
culturing. This transgenic line was analyzed for the
N-glycosylation pattern of the recombinantly produced mAbI antibody
following production scale-up from 1 g tissue up to 300 g (0.3 kg)
tissue, and further production scale-up to 6.5 kg tissue. The
process of scaling production up to 6.5 kg tissue occurred
approximately 8 months after transgenic line 24 was generated.
Results are shown in FIGS. 51A (MALDI-TOF analysis of N-glycans)
and 51B (HPLC fluorescence analysis of N-glycans).
[0418] The glycosylation profile for the mAbI antibody produced by
transgenic line 24 comprising the mAbI04 construct remained
homogeneous with scale-up in production from 1 g tissue to 0.3 kg
tissue, and further scale-up in production to 6.5 kg tissue, and
thus was characterized by the presence of a single predominant peak
corresponding to the GnGn (i.e., G0) glycan species. Thus, the
homogeneity of the glycosylation profile in transgenic L. minor
comprising the mAbI04 construct was consistently maintained with an
approximately 6,500-fold increase in production scale (i.e., from 1
g up to 6.5 kg). Furthermore, the homogeneity of the glycosylation
profile was consistently maintained in this transgenic line at 8
months following its generation.
[0419] These data demonstrate that the homogeneity of the
glycosylation profile in transgenic L. minor comprising the mAbI04
construct remains consistent with at least a 6,500-fold increase in
production scale. Furthermore, the homogeneity of the glycosylation
profile in transgenic L. minor comprising the mAbI04 construct is
maintained for at least 8 months after the transgenic line is
generated. The homogeneity of the glycosylation profile would be
expected to be maintained with further increase in production
scale, and thus, for example, would be expected to be maintained if
production scale was increased by another 4-fold beyond 6.5 kg
(e.g., scale-up from 6.5 kg to 26 kg). The homogeneity of the
glycosylation profile would also be expected to be maintained with
continuous clonal culture of the transgenic line well beyond 8
months after generation of the transgenic line.
Example 8
Glycosylation Pattern for Endogenous Glycoproteins in Lemna minor
Lines Transgenic for mAbI04 RNAi Construct
[0420] The L40 protease is a representative endogenous glycoprotein
produced in L. minor. In order to assess the impact of the mAbI04
RNAi construct (FIG. 12) on glycosylation of endogenous proteins,
the L40 protein was isolated from a L. minor line transgenic for
the mAbI04 RNAi construct using benzamidine affinity
chromatography. The N-glycosylation pattern for the isolated L40
protein was analyzed using MALDI-TOF analysis in the manner
described above. Results are shown in FIG. 52.
[0421] As can be seen from this analysis, suppression of FucT and
XylT expression using the chimeric RNAi mAbI04 construct results in
endogenous glycoproteins having a homogeneous glycosylation pattern
consistent with that observed for recombinant glycoproteins. Thus,
the heterogeneous N-glycan profile for the L40 glycoprotein
isolated from L. minor having the wild-type glycosylation machinery
is represented by a mixture of N-glycans species having the
.beta.1,2-linked xylose residue, or both the .beta.1,2-linked
xylose and core .alpha.1,3-linked fucose residues attached. In
contrast, the homogeneous N-glycan profile for L40 isolated from L.
minor transgenic for the mAbI04 RNAi construct is represented by a
single predominant peak corresponding to the G0 glycan species, and
is characterized by the absence of N-glycan species having the
.beta.1,2-linked xylose or both the .beta.1,2-linked xylose and
core .alpha.1,3-linked fucose residues attached.
Example 9
Production of Anti-CD20 and Anti-HER2 Monoclonal Antibody Having
Increased ADCC Activity
[0422] IDEC-C2B8 (IDEC Pharmaceuticals Corp., San Diego, Calif.;
commercially available under the tradename Rituxan.RTM., also
referred to as rituximab; see U.S. Pat. No. 5,736,137, herein
incorporated by reference) is a chimeric anti-CD20 monoclonal
antibody containing human IgG1 and kappa constant regions with
murine variable regions isolated from a murine anti-CD20 monoclonal
antibody, IDEC-2B8 (Reff et al. (1994) Blood 83:435-445). Rituximab
is licensed for treatment of relapsed B cell low-grade or
follicular non-Hodgkin's lymphoma (NHL). The anti-CD20 antibody
marketed as rituximab (Rituxan.RTM.) is recombinantly produced in
CHO cells. The glycosylation pattern of this CHO-expressed
anti-CD20 antibody reveals a heterogeneous mixture of
glycoforms.
[0423] A humanized anti-ERBB2 antibody is commercially available
under the tradename Herceptin.RTM. (Genentech, Inc., San Francisco,
Calif.) (see U.S. Pat. No. 6,165,464, herein incorporated by
reference). This recombinant humanized monoclonal antibody has high
affinity for p185HER2. Early clinical trials with patients having
extensive metastatic breast carcinomas demonstrate the ability of
this monoclonal antibody to inhibit growth of breast cancer cells
that overexpress HER2 (Baselga et al. (1996) J. Clin. Oncol.
14(3):737-744).
[0424] A rituximab-sequence antibody and Herceptin.RTM.
anti-ERBB2-sequence antibody are recombinantly produced in Lemna
having a wild-type glycosylation pattern, using the mAbI01
construct described above, and in Lemna that is genetically
modified to suppress expression of both FucT and XylT, using the
mAbI04 chimeric RNAi construct, with the rituximab or anti-ERBB2
heavy and light chain coding sequences replacing those for the mAbI
in each of these constructs. The sequences encoding the heavy and
light chains are optionally both codon-optimized with
Lemna-preferred codons. The secreted rituximab-sequence mAb (i.e.,
having the amino acid sequence of the rituximab antibody) and
anti-ERBB2-sequence mAb (i.e., having the amino acid sequence of
the Herceptin.RTM. anti-ERBB2 mAb) are analyzed for glycosylation
pattern as described above in Example 1.
[0425] The glycosylation profile for intact rituximab-sequence mAb
or anti-ERBB2-sequence mAb produced in the wild-type Lemna
comprising the mAbI01-like construct shows a heterogeneous profile
with numerous peaks corresponding to multiple glycoforms. In
contrast, the glycoyslation profile for intact rituximab-sequence
mAb or intact anti-ERBB2-sequence mAb produced in transgenic Lemna
comprising the mAbI04-like construct shows a substantially
homogeneous glycoprotein composition, with three major glycoform
peaks, the largest of which corresponds to the G0 glycoform, and
two very minor peaks corresponding to trace amounts of precursor
glycoforms, wherein xylose and fucose residues are not
attached.
[0426] The rituximab-sequence and anti-ERBB2-sequence monoclonal
antibody compositions having a glycosylation profile that is
substantially homogeneous for the G0 glycoform are tested for ADCC
activity and Fc.gamma. receptor IIIa (Fc.gamma. RIIIa) binding on
freshly-isolated human NK cells. The rituximab-sequence and
anti-ERBB2-sequence mAbs produced from L. minor lines engineered
with the mAbI04-like construct exhibit the expected enhanced
binding in view of the lack of any fucose residues, relative to the
binding observed for the rituximab-sequence and anti-ERBB2-sequence
mAbs produced from L. minor lines having the wild-type
glycosylation machinery (i.e., no silencing of FucT or XylT).
Furthermore, binding affinity is at least strong as for the
corresponding mAb produced in CHO cell lines.
[0427] ADCC activity of the substantially homogeneous G0 glycoform
of rituximab-sequence mAb and of anti-ERBB2-sequence mAb produced
in the L. minor line engineered with the mAbI04-like construct is
assayed using purified human peripheral blood mononuclear cells as
effector cells (see, for example, Shinkawa et al. (2003) J. Biol.
Chem. 278(5):3466-3473; herein incorporated by reference in its
entirety). Activity of the G0 glycoform rituximab-sequence and
anti-ERBB2-sequence mAb compositions is improved 50-1000 fold over
that exhibited by the respective rituximab-sequence mAb or
anti-ERBB2-sequence mAb produced in the L. minor line having the
wild-type glycosylation machinery or produced in CHO cells.
[0428] CDC activity of the substantially homogeneous G0 glycoform
of rituximab-sequence mAb produced in the L. minor line engineered
with the mAbI04-like RNAi construct is assayed using standard
assays known in the art (see, for example, the complement
activation assays described in U.S. Patent Application Publication
No. 2004/0167319, herein incorporated by reference in its
entirety), and compared to that observed for rituximab.
Non-relevant antibody serves as the negative control. In one such
assay, CDC activity of the various antibodies against target Daudi
cells is measured by assessing elevated membrane permeability using
a propidium iodide (PI) exclusion assay, with serum from healthy
volunteers serving as a complement source. Serum for complement
lysis is prepared by drawing blood from healthy volunteers into
autosep gel and clot activator vacutainer tubes (BD Biosciences,
Rutherford, N.J.), which are held at room temperature for 30-60
minutes and then centrifuged at 3000 rpm for 5 minutes. Serum is
harvested and stored at -80.degree. C.
[0429] Briefly, for this CDC activity assay, Daudi cells are washed
and resuspended in RPMI-1% BSA at 1.times.10.sup.6/ml. Various
concentrations of the substantially homogeneous G0 glycoform of
rituximab-sequence mAb, rituximab, and negative control mAb are
added to the Daudi cells and allowed to bind for 10-15 minutes at
room temperature. Thereafter, serum as a source of complement is
added to a final concentration of 20% (v/v) and the mixtures are
incubated for 45 min at 37.degree. C. The cells are then kept at
4.degree. C. until analysis. Each sample (150 .mu.l) is then added
to 10 .mu.l of PI solution (10 .mu.g/ml in PBS) in a FACS tube. The
mixture is assessed immediately for cell lysis (number of
PI-positive cells) by a FACScalibur flow cytometer and analysed
using CellQuest pro software (BD Biosciences, Mountain View,
Calif.). At least 5000 events are collected for analysis with cell
debris excluded by adjustment of the forward sideward scatter (FCS)
threshold.
[0430] CDC activity of the substantially homogeneous G0 glycoform
rituximab-sequence mAb is decreased relative to that exhibited by
rituximab.
[0431] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
4111865DNALemna minorCDS(243)...(1715) 1acgcgggggg aagtggttga
gtagctcagt ggaaaattgg aaatgtctat tagaggggga 60agaggggagg gatccgaggg
gaacgaggaa ggtgtgccga attctcgtag atttcttcaa 120ttcctgcaga
tctcgtcttc tctctgattt cttcccgagc tccgcccgta ggaactcaat
180cggactcgat ccaagttgac gaggcctacy gaggaaggcg attttccgaa
gccctgcgat 240cg atg gcc acc tct gct gct ggt gct ctc aac gcc ggt
ggc agg gtc 287 Met Ala Thr Ser Ala Ala Gly Ala Leu Asn Ala Gly Gly
Arg Val 1 5 10 15ggg ggc agg agg agt tgg gtc aga ttg ctt ccc ttc
ttt gtg ttg atg 335Gly Gly Arg Arg Ser Trp Val Arg Leu Leu Pro Phe
Phe Val Leu Met 20 25 30ctg gtg gta ggg gag atc tgg ttc ctc ggg cgg
ctg gat gtg gtc aag 383Leu Val Val Gly Glu Ile Trp Phe Leu Gly Arg
Leu Asp Val Val Lys 35 40 45aac gcc gct atg gtt caa aac tgg act tcc
tcc cac ttg ttt ttc tta 431Asn Ala Ala Met Val Gln Asn Trp Thr Ser
Ser His Leu Phe Phe Leu 50 55 60cca gtt tct tcc tac acg tgg tcc gag
acc gtc aag gag gaa gag gat 479Pro Val Ser Ser Tyr Thr Trp Ser Glu
Thr Val Lys Glu Glu Glu Asp 65 70 75tgc aag gac tgg ctg gaa aga gta
gat gcg gtc gat tac aag aga gat 527Cys Lys Asp Trp Leu Glu Arg Val
Asp Ala Val Asp Tyr Lys Arg Asp80 85 90 95ttc cgt gtg gaa ccc gtt
ctg gta aat gac gct gaa cag gat tgg agt 575Phe Arg Val Glu Pro Val
Leu Val Asn Asp Ala Glu Gln Asp Trp Ser 100 105 110tca tgt tca gtg
ggc tgt aag ttc gga tca ttc ccc gga aga acg cct 623Ser Cys Ser Val
Gly Cys Lys Phe Gly Ser Phe Pro Gly Arg Thr Pro 115 120 125gat gct
aca ttt ggt ttc tct cag aat cca tca aca gtc agt gtc cat 671Asp Ala
Thr Phe Gly Phe Ser Gln Asn Pro Ser Thr Val Ser Val His 130 135
140cga tcc atg gaa tca tcc cat tat tat ttg gag aat aat ctt gat aat
719Arg Ser Met Glu Ser Ser His Tyr Tyr Leu Glu Asn Asn Leu Asp Asn
145 150 155gca cga cgg aaa ggc tat caa att gtg atg aca act agt ctc
ttg tca 767Ala Arg Arg Lys Gly Tyr Gln Ile Val Met Thr Thr Ser Leu
Leu Ser160 165 170 175gat gtg cct gtc ggt tat ttc tca tgg gct gaa
tat gat atc atg gcg 815Asp Val Pro Val Gly Tyr Phe Ser Trp Ala Glu
Tyr Asp Ile Met Ala 180 185 190cct ctt cag ccg aaa act gct ggt gca
ctt gct gct gca ttt ata tct 863Pro Leu Gln Pro Lys Thr Ala Gly Ala
Leu Ala Ala Ala Phe Ile Ser 195 200 205aat tgc gga gca cgt aat ttc
cgc ttg cag gcc ctt gat atg ctc gaa 911Asn Cys Gly Ala Arg Asn Phe
Arg Leu Gln Ala Leu Asp Met Leu Glu 210 215 220aag tcg aat att aag
att gat tca tat ggt gct tgc cat cgc aac caa 959Lys Ser Asn Ile Lys
Ile Asp Ser Tyr Gly Ala Cys His Arg Asn Gln 225 230 235gac ggt aaa
gtg gac aag gta caa act ttg aag cgg tat aag ttc agc 1007Asp Gly Lys
Val Asp Lys Val Gln Thr Leu Lys Arg Tyr Lys Phe Ser240 245 250
255tta gct ttt gaa aac tcg aac gag gat gac tat gtt act gag aag ttc
1055Leu Ala Phe Glu Asn Ser Asn Glu Asp Asp Tyr Val Thr Glu Lys Phe
260 265 270ttt caa tct ctt gtc gct gga gct att cct gtt gtc gtc gga
gcc ccc 1103Phe Gln Ser Leu Val Ala Gly Ala Ile Pro Val Val Val Gly
Ala Pro 275 280 285aac att caa aat ttt gcg cca tct tct gat tca att
ctg cac atc agg 1151Asn Ile Gln Asn Phe Ala Pro Ser Ser Asp Ser Ile
Leu His Ile Arg 290 295 300gag ccc aag gat gtc agt tca gtc gct gag
aga atg aaa ttt ctc gct 1199Glu Pro Lys Asp Val Ser Ser Val Ala Glu
Arg Met Lys Phe Leu Ala 305 310 315tca aat cca gaa gca tat aac caa
tca ctg agg tgg aag ttt gag ggc 1247Ser Asn Pro Glu Ala Tyr Asn Gln
Ser Leu Arg Trp Lys Phe Glu Gly320 325 330 335cct tct aac tcc ttc
aaa gcc ctg gtg gac atg gca gca gtt cac tcc 1295Pro Ser Asn Ser Phe
Lys Ala Leu Val Asp Met Ala Ala Val His Ser 340 345 350tcc tgc cgc
cta tgc att cac att gcc acc aag atc aga gag aag gaa 1343Ser Cys Arg
Leu Cys Ile His Ile Ala Thr Lys Ile Arg Glu Lys Glu 355 360 365gag
aga aac ccg aat ttc aag act cgc cct tgc aag tgc acc cgc aat 1391Glu
Arg Asn Pro Asn Phe Lys Thr Arg Pro Cys Lys Cys Thr Arg Asn 370 375
380ggg tct acc tta tat cac tta tac gcc cgc gaa aga ggc acc ttt gac
1439Gly Ser Thr Leu Tyr His Leu Tyr Ala Arg Glu Arg Gly Thr Phe Asp
385 390 395ttc tta tca atc ttc atg aga tcg gat aat cta tca ctg aaa
gcg ctg 1487Phe Leu Ser Ile Phe Met Arg Ser Asp Asn Leu Ser Leu Lys
Ala Leu400 405 410 415ggg tca aca gtt ctt gag aaa ttc agt tct ttg
aag cac gtg ccg att 1535Gly Ser Thr Val Leu Glu Lys Phe Ser Ser Leu
Lys His Val Pro Ile 420 425 430tgg aag aag gag agg cca gag agt ctg
aaa gga ggg agc aag ctg gat 1583Trp Lys Lys Glu Arg Pro Glu Ser Leu
Lys Gly Gly Ser Lys Leu Asp 435 440 445ctt tac aga atc tat cca gtg
ggc att act cag aga gaa gct ctc ttc 1631Leu Tyr Arg Ile Tyr Pro Val
Gly Ile Thr Gln Arg Glu Ala Leu Phe 450 455 460tct ttc cag ttc aac
act gac aaa gaa ctt caa atc tac ctt gaa tcc 1679Ser Phe Gln Phe Asn
Thr Asp Lys Glu Leu Gln Ile Tyr Leu Glu Ser 465 470 475cat cca tgt
gcg aag ttt gaa gtc atc ttt att tga tccctgaggt 1725His Pro Cys Ala
Lys Phe Glu Val Ile Phe Ile480 485 490aattaggtca cgaattcagc
taatttggtt aattatgctt caagcccaca tggtatttca 1785tatcattaat
tgaaggcata gttagttgat attgacattt tcgtctagga tcattctaaa
1845gtctatccca atgaacttaa 186521473DNALemna
minorCDS(1)...(1473)Encodes alpha-1, 3-fucosyltransferase 2atg gcc
acc tct gct gct ggt gct ctc aac gcc ggt ggc agg gtc ggg 48Met Ala
Thr Ser Ala Ala Gly Ala Leu Asn Ala Gly Gly Arg Val Gly1 5 10 15ggc
agg agg agt tgg gtc aga ttg ctt ccc ttc ttt gtg ttg atg ctg 96Gly
Arg Arg Ser Trp Val Arg Leu Leu Pro Phe Phe Val Leu Met Leu 20 25
30gtg gta ggg gag atc tgg ttc ctc ggg cgg ctg gat gtg gtc aag aac
144Val Val Gly Glu Ile Trp Phe Leu Gly Arg Leu Asp Val Val Lys Asn
35 40 45gcc gct atg gtt caa aac tgg act tcc tcc cac ttg ttt ttc tta
cca 192Ala Ala Met Val Gln Asn Trp Thr Ser Ser His Leu Phe Phe Leu
Pro 50 55 60gtt tct tcc tac acg tgg tcc gag acc gtc aag gag gaa gag
gat tgc 240Val Ser Ser Tyr Thr Trp Ser Glu Thr Val Lys Glu Glu Glu
Asp Cys65 70 75 80aag gac tgg ctg gaa aga gta gat gcg gtc gat tac
aag aga gat ttc 288Lys Asp Trp Leu Glu Arg Val Asp Ala Val Asp Tyr
Lys Arg Asp Phe 85 90 95cgt gtg gaa ccc gtt ctg gta aat gac gct gaa
cag gat tgg agt tca 336Arg Val Glu Pro Val Leu Val Asn Asp Ala Glu
Gln Asp Trp Ser Ser 100 105 110tgt tca gtg ggc tgt aag ttc gga tca
ttc ccc gga aga acg cct gat 384Cys Ser Val Gly Cys Lys Phe Gly Ser
Phe Pro Gly Arg Thr Pro Asp 115 120 125gct aca ttt ggt ttc tct cag
aat cca tca aca gtc agt gtc cat cga 432Ala Thr Phe Gly Phe Ser Gln
Asn Pro Ser Thr Val Ser Val His Arg 130 135 140tcc atg gaa tca tcc
cat tat tat ttg gag aat aat ctt gat aat gca 480Ser Met Glu Ser Ser
His Tyr Tyr Leu Glu Asn Asn Leu Asp Asn Ala145 150 155 160cga cgg
aaa ggc tat caa att gtg atg aca act agt ctc ttg tca gat 528Arg Arg
Lys Gly Tyr Gln Ile Val Met Thr Thr Ser Leu Leu Ser Asp 165 170
175gtg cct gtc ggt tat ttc tca tgg gct gaa tat gat atc atg gcg cct
576Val Pro Val Gly Tyr Phe Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro
180 185 190ctt cag ccg aaa act gct ggt gca ctt gct gct gca ttt ata
tct aat 624Leu Gln Pro Lys Thr Ala Gly Ala Leu Ala Ala Ala Phe Ile
Ser Asn 195 200 205tgc gga gca cgt aat ttc cgc ttg cag gcc ctt gat
atg ctc gaa aag 672Cys Gly Ala Arg Asn Phe Arg Leu Gln Ala Leu Asp
Met Leu Glu Lys 210 215 220tcg aat att aag att gat tca tat ggt gct
tgc cat cgc aac caa gac 720Ser Asn Ile Lys Ile Asp Ser Tyr Gly Ala
Cys His Arg Asn Gln Asp225 230 235 240ggt aaa gtg gac aag gta caa
act ttg aag cgg tat aag ttc agc tta 768Gly Lys Val Asp Lys Val Gln
Thr Leu Lys Arg Tyr Lys Phe Ser Leu 245 250 255gct ttt gaa aac tcg
aac gag gat gac tat gtt act gag aag ttc ttt 816Ala Phe Glu Asn Ser
Asn Glu Asp Asp Tyr Val Thr Glu Lys Phe Phe 260 265 270caa tct ctt
gtc gct gga gct att cct gtt gtc gtc gga gcc ccc aac 864Gln Ser Leu
Val Ala Gly Ala Ile Pro Val Val Val Gly Ala Pro Asn 275 280 285att
caa aat ttt gcg cca tct tct gat tca att ctg cac atc agg gag 912Ile
Gln Asn Phe Ala Pro Ser Ser Asp Ser Ile Leu His Ile Arg Glu 290 295
300ccc aag gat gtc agt tca gtc gct gag aga atg aaa ttt ctc gct tca
960Pro Lys Asp Val Ser Ser Val Ala Glu Arg Met Lys Phe Leu Ala
Ser305 310 315 320aat cca gaa gca tat aac caa tca ctg agg tgg aag
ttt gag ggc cct 1008Asn Pro Glu Ala Tyr Asn Gln Ser Leu Arg Trp Lys
Phe Glu Gly Pro 325 330 335tct aac tcc ttc aaa gcc ctg gtg gac atg
gca gca gtt cac tcc tcc 1056Ser Asn Ser Phe Lys Ala Leu Val Asp Met
Ala Ala Val His Ser Ser 340 345 350tgc cgc cta tgc att cac att gcc
acc aag atc aga gag aag gaa gag 1104Cys Arg Leu Cys Ile His Ile Ala
Thr Lys Ile Arg Glu Lys Glu Glu 355 360 365aga aac ccg aat ttc aag
act cgc cct tgc aag tgc acc cgc aat ggg 1152Arg Asn Pro Asn Phe Lys
Thr Arg Pro Cys Lys Cys Thr Arg Asn Gly 370 375 380tct acc tta tat
cac tta tac gcc cgc gaa aga ggc acc ttt gac ttc 1200Ser Thr Leu Tyr
His Leu Tyr Ala Arg Glu Arg Gly Thr Phe Asp Phe385 390 395 400tta
tca atc ttc atg aga tcg gat aat cta tca ctg aaa gcg ctg ggg 1248Leu
Ser Ile Phe Met Arg Ser Asp Asn Leu Ser Leu Lys Ala Leu Gly 405 410
415tca aca gtt ctt gag aaa ttc agt tct ttg aag cac gtg ccg att tgg
1296Ser Thr Val Leu Glu Lys Phe Ser Ser Leu Lys His Val Pro Ile Trp
420 425 430aag aag gag agg cca gag agt ctg aaa gga ggg agc aag ctg
gat ctt 1344Lys Lys Glu Arg Pro Glu Ser Leu Lys Gly Gly Ser Lys Leu
Asp Leu 435 440 445tac aga atc tat cca gtg ggc att act cag aga gaa
gct ctc ttc tct 1392Tyr Arg Ile Tyr Pro Val Gly Ile Thr Gln Arg Glu
Ala Leu Phe Ser 450 455 460ttc cag ttc aac act gac aaa gaa ctt caa
atc tac ctt gaa tcc cat 1440Phe Gln Phe Asn Thr Asp Lys Glu Leu Gln
Ile Tyr Leu Glu Ser His465 470 475 480cca tgt gcg aag ttt gaa gtc
atc ttt att tga 1473Pro Cys Ala Lys Phe Glu Val Ile Phe Ile 485
4903490PRTLemna minor 3Met Ala Thr Ser Ala Ala Gly Ala Leu Asn Ala
Gly Gly Arg Val Gly1 5 10 15Gly Arg Arg Ser Trp Val Arg Leu Leu Pro
Phe Phe Val Leu Met Leu 20 25 30Val Val Gly Glu Ile Trp Phe Leu Gly
Arg Leu Asp Val Val Lys Asn 35 40 45Ala Ala Met Val Gln Asn Trp Thr
Ser Ser His Leu Phe Phe Leu Pro 50 55 60Val Ser Ser Tyr Thr Trp Ser
Glu Thr Val Lys Glu Glu Glu Asp Cys65 70 75 80Lys Asp Trp Leu Glu
Arg Val Asp Ala Val Asp Tyr Lys Arg Asp Phe 85 90 95Arg Val Glu Pro
Val Leu Val Asn Asp Ala Glu Gln Asp Trp Ser Ser 100 105 110Cys Ser
Val Gly Cys Lys Phe Gly Ser Phe Pro Gly Arg Thr Pro Asp 115 120
125Ala Thr Phe Gly Phe Ser Gln Asn Pro Ser Thr Val Ser Val His Arg
130 135 140Ser Met Glu Ser Ser His Tyr Tyr Leu Glu Asn Asn Leu Asp
Asn Ala145 150 155 160Arg Arg Lys Gly Tyr Gln Ile Val Met Thr Thr
Ser Leu Leu Ser Asp 165 170 175Val Pro Val Gly Tyr Phe Ser Trp Ala
Glu Tyr Asp Ile Met Ala Pro 180 185 190Leu Gln Pro Lys Thr Ala Gly
Ala Leu Ala Ala Ala Phe Ile Ser Asn 195 200 205Cys Gly Ala Arg Asn
Phe Arg Leu Gln Ala Leu Asp Met Leu Glu Lys 210 215 220Ser Asn Ile
Lys Ile Asp Ser Tyr Gly Ala Cys His Arg Asn Gln Asp225 230 235
240Gly Lys Val Asp Lys Val Gln Thr Leu Lys Arg Tyr Lys Phe Ser Leu
245 250 255Ala Phe Glu Asn Ser Asn Glu Asp Asp Tyr Val Thr Glu Lys
Phe Phe 260 265 270Gln Ser Leu Val Ala Gly Ala Ile Pro Val Val Val
Gly Ala Pro Asn 275 280 285Ile Gln Asn Phe Ala Pro Ser Ser Asp Ser
Ile Leu His Ile Arg Glu 290 295 300Pro Lys Asp Val Ser Ser Val Ala
Glu Arg Met Lys Phe Leu Ala Ser305 310 315 320Asn Pro Glu Ala Tyr
Asn Gln Ser Leu Arg Trp Lys Phe Glu Gly Pro 325 330 335Ser Asn Ser
Phe Lys Ala Leu Val Asp Met Ala Ala Val His Ser Ser 340 345 350Cys
Arg Leu Cys Ile His Ile Ala Thr Lys Ile Arg Glu Lys Glu Glu 355 360
365Arg Asn Pro Asn Phe Lys Thr Arg Pro Cys Lys Cys Thr Arg Asn Gly
370 375 380Ser Thr Leu Tyr His Leu Tyr Ala Arg Glu Arg Gly Thr Phe
Asp Phe385 390 395 400Leu Ser Ile Phe Met Arg Ser Asp Asn Leu Ser
Leu Lys Ala Leu Gly 405 410 415Ser Thr Val Leu Glu Lys Phe Ser Ser
Leu Lys His Val Pro Ile Trp 420 425 430Lys Lys Glu Arg Pro Glu Ser
Leu Lys Gly Gly Ser Lys Leu Asp Leu 435 440 445Tyr Arg Ile Tyr Pro
Val Gly Ile Thr Gln Arg Glu Ala Leu Phe Ser 450 455 460Phe Gln Phe
Asn Thr Asp Lys Glu Leu Gln Ile Tyr Leu Glu Ser His465 470 475
480Pro Cys Ala Lys Phe Glu Val Ile Phe Ile 485 49041860DNALemna
minorCDS(63)...(1592) 4ggcttccaac cggaggatct cgagctgaag aatcttcatg
actgaagaat tcatgtgatc 60cc atg gct ttg gtg aac tcg cga ggg agc agg
gtc aga cgc atc gcg 107 Met Ala Leu Val Asn Ser Arg Gly Ser Arg Val
Arg Arg Ile Ala 1 5 10 15aag ccc acc ttc gtt ttc ctc ttg atc aac
gta gtc tgt ctc ctg tac 155Lys Pro Thr Phe Val Phe Leu Leu Ile Asn
Val Val Cys Leu Leu Tyr 20 25 30ttt ttc cgt cag aac cct aat ccc att
ccc gac gct tgt ctt cac ggg 203Phe Phe Arg Gln Asn Pro Asn Pro Ile
Pro Asp Ala Cys Leu His Gly 35 40 45gaa tgc gac aaa ccc ccg att tta
gtg act ccc cgg cga tgg aac ttg 251Glu Cys Asp Lys Pro Pro Ile Leu
Val Thr Pro Arg Arg Trp Asn Leu 50 55 60aag cca tgg ccg att ctt cct
tcc ttt ctg cca tgg gtg ccg agc tcc 299Lys Pro Trp Pro Ile Leu Pro
Ser Phe Leu Pro Trp Val Pro Ser Ser 65 70 75cac cct gcc cag ggc tcc
tgc gaa gcc tac ttc ggc aac agc ttc aac 347His Pro Ala Gln Gly Ser
Cys Glu Ala Tyr Phe Gly Asn Ser Phe Asn80 85 90 95cgc cgg acg gag
atg ctg aag aag gta gag gga aga gga tgg ttc cag 395Arg Arg Thr Glu
Met Leu Lys Lys Val Glu Gly Arg Gly Trp Phe Gln 100 105 110tgc ctg
tac agc gat act ctt cga agt tct gtt tgc cag gga ggg aat 443Cys Leu
Tyr Ser Asp Thr Leu Arg Ser Ser Val Cys Gln Gly Gly Asn 115 120
125ttg cgg atg gac ccg gaa agg att agg atg tcg aaa ggg ggg gaa gat
491Leu Arg Met Asp Pro Glu Arg Ile Arg Met Ser Lys Gly Gly Glu Asp
130 135 140cta gag gag gtg atg aag aga gag gag gaa gaa gaa ttg ccc
aaa ttc 539Leu Glu Glu Val Met Lys Arg Glu Glu Glu Glu Glu Leu Pro
Lys Phe 145 150 155gag gag ggg tcg ttc cag att gaa tct ggt tat gga
agc gga ggg gaa 587Glu Glu Gly Ser Phe Gln Ile Glu Ser Gly Tyr Gly
Ser Gly Gly Glu160 165 170 175gtt gga gag aga att gcg act gac gag
gtc ctc gat aat gtt gtg ccg 635Val Gly Glu Arg
Ile Ala Thr Asp Glu Val Leu Asp Asn Val Val Pro 180 185 190aaa ggc
gct gtt cat gta cat acc atg cgc aat ctc atc agt tcg att 683Lys Gly
Ala Val His Val His Thr Met Arg Asn Leu Ile Ser Ser Ile 195 200
205cag att gtt ggt ccc ggg cat ctt caa tgc tct cag tgg atc gac gaa
731Gln Ile Val Gly Pro Gly His Leu Gln Cys Ser Gln Trp Ile Asp Glu
210 215 220ccg gtt ctt ctt gtc aca cgc ttc gaa tac gcc aat ctc ttt
cac acc 779Pro Val Leu Leu Val Thr Arg Phe Glu Tyr Ala Asn Leu Phe
His Thr 225 230 235gtc acc gac tgg tac agc gcc tac gca agc tcg agg
att gcc aac ttg 827Val Thr Asp Trp Tyr Ser Ala Tyr Ala Ser Ser Arg
Ile Ala Asn Leu240 245 250 255cct tct cgc cct cac tta att ttc gtc
gat ggc cat tgc agg gcg gaa 875Pro Ser Arg Pro His Leu Ile Phe Val
Asp Gly His Cys Arg Ala Glu 260 265 270cag tta gag gac atg tgg aga
gcc ctg ttc tcg acc gtc cga tac tcc 923Gln Leu Glu Asp Met Trp Arg
Ala Leu Phe Ser Thr Val Arg Tyr Ser 275 280 285aag aac ttc tcc cag
cca atc tgc ttc cgc cac gtc gtc ctc tca cct 971Lys Asn Phe Ser Gln
Pro Ile Cys Phe Arg His Val Val Leu Ser Pro 290 295 300ctg ggc tat
gag acg gct ctc ttc aaa ggc cta tca gag agc ttc agc 1019Leu Gly Tyr
Glu Thr Ala Leu Phe Lys Gly Leu Ser Glu Ser Phe Ser 305 310 315tgt
gag gga gct ccg gcc aat cgg ctc aaa gtc aac ccc gat gac cag 1067Cys
Glu Gly Ala Pro Ala Asn Arg Leu Lys Val Asn Pro Asp Asp Gln320 325
330 335aag act gca aga ctg gct gaa ttc gga gag atg atc aga gcc gcc
ttt 1115Lys Thr Ala Arg Leu Ala Glu Phe Gly Glu Met Ile Arg Ala Ala
Phe 340 345 350gac ttt cct gtc gtt gac ccg tcc att gac ccg ttg acc
aaa tcc atc 1163Asp Phe Pro Val Val Asp Pro Ser Ile Asp Pro Leu Thr
Lys Ser Ile 355 360 365ctc ttc gtg cgg cgg gaa gat tac gtg gcg cac
cca cgc cac agt ggg 1211Leu Phe Val Arg Arg Glu Asp Tyr Val Ala His
Pro Arg His Ser Gly 370 375 380aga gtg gag tcg cgg ctg acc aac gag
caa gag gtg ttt gac ttt ctg 1259Arg Val Glu Ser Arg Leu Thr Asn Glu
Gln Glu Val Phe Asp Phe Leu 385 390 395cac aat tgg gca agt cat cac
aga ggc agg tgc aac atc agt atg gtc 1307His Asn Trp Ala Ser His His
Arg Gly Arg Cys Asn Ile Ser Met Val400 405 410 415aac ggg ctt ttc
gcg cac atg gga atg aag gaa cag ttg aag gcg att 1355Asn Gly Leu Phe
Ala His Met Gly Met Lys Glu Gln Leu Lys Ala Ile 420 425 430atg gaa
gct tcg gtg gtg gtg ggg gcc cac ggg gct ggt ttg acc cat 1403Met Glu
Ala Ser Val Val Val Gly Ala His Gly Ala Gly Leu Thr His 435 440
445ctg gtg gca gca agg tca acg aca gtt gtt ctt gag att ctg agt agt
1451Leu Val Ala Ala Arg Ser Thr Thr Val Val Leu Glu Ile Leu Ser Ser
450 455 460caa tac cgt aga ccg cac ttt caa ctg att tct cgg tgg aaa
ggg ttg 1499Gln Tyr Arg Arg Pro His Phe Gln Leu Ile Ser Arg Trp Lys
Gly Leu 465 470 475gac tac cat gca att aat ctt gcc ggg tca ttt gct
gac cct cgg gag 1547Asp Tyr His Ala Ile Asn Leu Ala Gly Ser Phe Ala
Asp Pro Arg Glu480 485 490 495gtg gtc gag aaa ttg act ggc ata gtt
gac agg ctt gga tgt tga 1592Val Val Glu Lys Leu Thr Gly Ile Val Asp
Arg Leu Gly Cys 500 505agagaagtga aagtcaacat ttggaatttt aactttaagg
ggtggttaac aattgagcgg 1652cattgtcaac gggtttggat gctgggaaaa
gtgaaaatca acacttggag ttctgacatt 1712gaaggcaaga cgtggaattt
tgatggtgtt gaggatattt ggatgtggag ttctgatgaa 1772ttaaagcagg
ggttgatcat ttgccagtgg aattatgttg gtgtaagaga gaagggggag
1832aataaacagt gttagagagc tatgctgg 186051530DNALemna
minorCDS(1)...(1530)XyIT isoform #1; Encodes beta-1,
2-xylosyltransferase 5atg gct ttg gtg aac tcg cga ggg agc agg gtc
aga cgc atc gcg aag 48Met Ala Leu Val Asn Ser Arg Gly Ser Arg Val
Arg Arg Ile Ala Lys1 5 10 15ccc acc ttc gtt ttc ctc ttg atc aac gta
gtc tgt ctc ctg tac ttt 96Pro Thr Phe Val Phe Leu Leu Ile Asn Val
Val Cys Leu Leu Tyr Phe 20 25 30ttc cgt cag aac cct aat ccc att ccc
gac gct tgt ctt cac ggg gaa 144Phe Arg Gln Asn Pro Asn Pro Ile Pro
Asp Ala Cys Leu His Gly Glu 35 40 45tgc gac aaa ccc ccg att tta gtg
act ccc cgg cga tgg aac ttg aag 192Cys Asp Lys Pro Pro Ile Leu Val
Thr Pro Arg Arg Trp Asn Leu Lys 50 55 60cca tgg ccg att ctt cct tcc
ttt ctg cca tgg gtg ccg agc tcc cac 240Pro Trp Pro Ile Leu Pro Ser
Phe Leu Pro Trp Val Pro Ser Ser His65 70 75 80cct gcc cag ggc tcc
tgc gaa gcc tac ttc ggc aac agc ttc aac cgc 288Pro Ala Gln Gly Ser
Cys Glu Ala Tyr Phe Gly Asn Ser Phe Asn Arg 85 90 95cgg acg gag atg
ctg aag aag gta gag gga aga gga tgg ttc cag tgc 336Arg Thr Glu Met
Leu Lys Lys Val Glu Gly Arg Gly Trp Phe Gln Cys 100 105 110ctg tac
agc gat act ctt cga agt tct gtt tgc cag gga ggg aat ttg 384Leu Tyr
Ser Asp Thr Leu Arg Ser Ser Val Cys Gln Gly Gly Asn Leu 115 120
125cgg atg gac ccg gaa agg att agg atg tcg aaa ggg ggg gaa gat cta
432Arg Met Asp Pro Glu Arg Ile Arg Met Ser Lys Gly Gly Glu Asp Leu
130 135 140gag gag gtg atg aag aga gag gag gaa gaa gaa ttg ccc aaa
ttc gag 480Glu Glu Val Met Lys Arg Glu Glu Glu Glu Glu Leu Pro Lys
Phe Glu145 150 155 160gag ggg tcg ttc cag att gaa tct ggt tat gga
agc gga ggg gaa gtt 528Glu Gly Ser Phe Gln Ile Glu Ser Gly Tyr Gly
Ser Gly Gly Glu Val 165 170 175gga gag aga att gcg act gac gag gtc
ctc gat aat gtt gtg ccg aaa 576Gly Glu Arg Ile Ala Thr Asp Glu Val
Leu Asp Asn Val Val Pro Lys 180 185 190ggc gct gtt cat gta cat acc
atg cgc aat ctc atc agt tcg att cag 624Gly Ala Val His Val His Thr
Met Arg Asn Leu Ile Ser Ser Ile Gln 195 200 205att gtt ggt ccc ggg
cat ctt caa tgc tct cag tgg atc gac gaa ccg 672Ile Val Gly Pro Gly
His Leu Gln Cys Ser Gln Trp Ile Asp Glu Pro 210 215 220gtt ctt ctt
gtc aca cgc ttc gaa tac gcc aat ctc ttt cac acc gtc 720Val Leu Leu
Val Thr Arg Phe Glu Tyr Ala Asn Leu Phe His Thr Val225 230 235
240acc gac tgg tac agc gcc tac gca agc tcg agg att gcc aac ttg cct
768Thr Asp Trp Tyr Ser Ala Tyr Ala Ser Ser Arg Ile Ala Asn Leu Pro
245 250 255tct cgc cct cac tta att ttc gtc gat ggc cat tgc agg gcg
gaa cag 816Ser Arg Pro His Leu Ile Phe Val Asp Gly His Cys Arg Ala
Glu Gln 260 265 270tta gag gac atg tgg aga gcc ctg ttc tcg acc gtc
cga tac tcc aag 864Leu Glu Asp Met Trp Arg Ala Leu Phe Ser Thr Val
Arg Tyr Ser Lys 275 280 285aac ttc tcc cag cca atc tgc ttc cgc cac
gtc gtc ctc tca cct ctg 912Asn Phe Ser Gln Pro Ile Cys Phe Arg His
Val Val Leu Ser Pro Leu 290 295 300ggc tat gag acg gct ctc ttc aaa
ggc cta tca gag agc ttc agc tgt 960Gly Tyr Glu Thr Ala Leu Phe Lys
Gly Leu Ser Glu Ser Phe Ser Cys305 310 315 320gag gga gct ccg gcc
aat cgg ctc aaa gtc aac ccc gat gac cag aag 1008Glu Gly Ala Pro Ala
Asn Arg Leu Lys Val Asn Pro Asp Asp Gln Lys 325 330 335act gca aga
ctg gct gaa ttc gga gag atg atc aga gcc gcc ttt gac 1056Thr Ala Arg
Leu Ala Glu Phe Gly Glu Met Ile Arg Ala Ala Phe Asp 340 345 350ttt
cct gtc gtt gac ccg tcc att gac ccg ttg acc aaa tcc atc ctc 1104Phe
Pro Val Val Asp Pro Ser Ile Asp Pro Leu Thr Lys Ser Ile Leu 355 360
365ttc gtg cgg cgg gaa gat tac gtg gcg cac cca cgc cac agt ggg aga
1152Phe Val Arg Arg Glu Asp Tyr Val Ala His Pro Arg His Ser Gly Arg
370 375 380gtg gag tcg cgg ctg acc aac gag caa gag gtg ttt gac ttt
ctg cac 1200Val Glu Ser Arg Leu Thr Asn Glu Gln Glu Val Phe Asp Phe
Leu His385 390 395 400aat tgg gca agt cat cac aga ggc agg tgc aac
atc agt atg gtc aac 1248Asn Trp Ala Ser His His Arg Gly Arg Cys Asn
Ile Ser Met Val Asn 405 410 415ggg ctt ttc gcg cac atg gga atg aag
gaa cag ttg aag gcg att atg 1296Gly Leu Phe Ala His Met Gly Met Lys
Glu Gln Leu Lys Ala Ile Met 420 425 430gaa gct tcg gtg gtg gtg ggg
gcc cac ggg gct ggt ttg acc cat ctg 1344Glu Ala Ser Val Val Val Gly
Ala His Gly Ala Gly Leu Thr His Leu 435 440 445gtg gca gca agg tca
acg aca gtt gtt ctt gag att ctg agt agt caa 1392Val Ala Ala Arg Ser
Thr Thr Val Val Leu Glu Ile Leu Ser Ser Gln 450 455 460tac cgt aga
ccg cac ttt caa ctg att tct cgg tgg aaa ggg ttg gac 1440Tyr Arg Arg
Pro His Phe Gln Leu Ile Ser Arg Trp Lys Gly Leu Asp465 470 475
480tac cat gca att aat ctt gcc ggg tca ttt gct gac cct cgg gag gtg
1488Tyr His Ala Ile Asn Leu Ala Gly Ser Phe Ala Asp Pro Arg Glu Val
485 490 495gtc gag aaa ttg act ggc ata gtt gac agg ctt gga tgt tga
1530Val Glu Lys Leu Thr Gly Ile Val Asp Arg Leu Gly Cys 500
5056509PRTLemna minor 6Met Ala Leu Val Asn Ser Arg Gly Ser Arg Val
Arg Arg Ile Ala Lys1 5 10 15Pro Thr Phe Val Phe Leu Leu Ile Asn Val
Val Cys Leu Leu Tyr Phe 20 25 30Phe Arg Gln Asn Pro Asn Pro Ile Pro
Asp Ala Cys Leu His Gly Glu 35 40 45Cys Asp Lys Pro Pro Ile Leu Val
Thr Pro Arg Arg Trp Asn Leu Lys 50 55 60Pro Trp Pro Ile Leu Pro Ser
Phe Leu Pro Trp Val Pro Ser Ser His65 70 75 80Pro Ala Gln Gly Ser
Cys Glu Ala Tyr Phe Gly Asn Ser Phe Asn Arg 85 90 95Arg Thr Glu Met
Leu Lys Lys Val Glu Gly Arg Gly Trp Phe Gln Cys 100 105 110Leu Tyr
Ser Asp Thr Leu Arg Ser Ser Val Cys Gln Gly Gly Asn Leu 115 120
125Arg Met Asp Pro Glu Arg Ile Arg Met Ser Lys Gly Gly Glu Asp Leu
130 135 140Glu Glu Val Met Lys Arg Glu Glu Glu Glu Glu Leu Pro Lys
Phe Glu145 150 155 160Glu Gly Ser Phe Gln Ile Glu Ser Gly Tyr Gly
Ser Gly Gly Glu Val 165 170 175Gly Glu Arg Ile Ala Thr Asp Glu Val
Leu Asp Asn Val Val Pro Lys 180 185 190Gly Ala Val His Val His Thr
Met Arg Asn Leu Ile Ser Ser Ile Gln 195 200 205Ile Val Gly Pro Gly
His Leu Gln Cys Ser Gln Trp Ile Asp Glu Pro 210 215 220Val Leu Leu
Val Thr Arg Phe Glu Tyr Ala Asn Leu Phe His Thr Val225 230 235
240Thr Asp Trp Tyr Ser Ala Tyr Ala Ser Ser Arg Ile Ala Asn Leu Pro
245 250 255Ser Arg Pro His Leu Ile Phe Val Asp Gly His Cys Arg Ala
Glu Gln 260 265 270Leu Glu Asp Met Trp Arg Ala Leu Phe Ser Thr Val
Arg Tyr Ser Lys 275 280 285Asn Phe Ser Gln Pro Ile Cys Phe Arg His
Val Val Leu Ser Pro Leu 290 295 300Gly Tyr Glu Thr Ala Leu Phe Lys
Gly Leu Ser Glu Ser Phe Ser Cys305 310 315 320Glu Gly Ala Pro Ala
Asn Arg Leu Lys Val Asn Pro Asp Asp Gln Lys 325 330 335Thr Ala Arg
Leu Ala Glu Phe Gly Glu Met Ile Arg Ala Ala Phe Asp 340 345 350Phe
Pro Val Val Asp Pro Ser Ile Asp Pro Leu Thr Lys Ser Ile Leu 355 360
365Phe Val Arg Arg Glu Asp Tyr Val Ala His Pro Arg His Ser Gly Arg
370 375 380Val Glu Ser Arg Leu Thr Asn Glu Gln Glu Val Phe Asp Phe
Leu His385 390 395 400Asn Trp Ala Ser His His Arg Gly Arg Cys Asn
Ile Ser Met Val Asn 405 410 415Gly Leu Phe Ala His Met Gly Met Lys
Glu Gln Leu Lys Ala Ile Met 420 425 430Glu Ala Ser Val Val Val Gly
Ala His Gly Ala Gly Leu Thr His Leu 435 440 445Val Ala Ala Arg Ser
Thr Thr Val Val Leu Glu Ile Leu Ser Ser Gln 450 455 460Tyr Arg Arg
Pro His Phe Gln Leu Ile Ser Arg Trp Lys Gly Leu Asp465 470 475
480Tyr His Ala Ile Asn Leu Ala Gly Ser Phe Ala Asp Pro Arg Glu Val
485 490 495Val Glu Lys Leu Thr Gly Ile Val Asp Arg Leu Gly Cys 500
50572160DNALemna minor 7agtcgagtga tatgaaatct tggtgaagaa ggatcggaga
acggaccggg tgaggcaagg 60ataattctgc tgttaaattc gagagcaaga cacctgcaat
tcaagaatcg agtggcaatt 120aatatagcag gatgatctgg aaggtagatc
ctgcccatcg aatgatccaa acatcaacac 180taggatcata ccgttaacaa
taatgaatga aaaagtagaa gatgacgaag ttgaagtgat 240gaccaaaaac
tttgaaaatt ccaaccgtat ggccggaatc agtgtgaaga aaatcgaaat
300caaatactct aatggatcgg attgttattc tggaggcaaa tctgaaactt
cgaggatagg 360atttaatcca cgcaagtaat aatttgaaac tcagaaggag
aaaaaaaaac taaaatagag 420aagaagagat ctcaaagaag ccgtgagcac
gagacgaacg agaagaggta aagcaccagt 480cagaggaaaa caccaaaatt
agagaaatag cacgaacatt aaagcacaga tccgcgccgc 540aaacccgaaa
gacgaaaaat agagccaaac gaaaccctaa taatcgatct gcacaaaaaa
600aaaaaaaaaa aactttgaga agagccgcga aattacccta gaatcctcag
aactggccgg 660acgagagaag cgctcgatcg aaacccaaca taaaacccct
tccaacggca aattactccg 720caaaacccga aaaataaaca aaatcaacga
tcacgagaag gtgcaagggc aaaaagaggc 780agtgcgatcg agagtctacc
tgaatcgtcg gcgcaaaagg cgagcccacc gacgaacgct 840ccctctagaa
cctggagatg cggcgagaga gaaggaaaga tcttcggtgg gtgatgctcg
900ctatttatcg caagagagtt agagagatct tcttcggcgg cggatttctg
gcatctagcg 960tttaacctca ccgcccagtg ctcacatcct tcttctcata
tttgaatatt taattaacaa 1020atgaatcagt catttttctt taatttttaa
ttcccggaga gggcaatgtt ggtatcaaaa 1080attatttagg aaaaattaat
tacacgaata atcggatttt tccctttttt taattaattt 1140ctaattttgg
aaaaggaaag aaaaatttta ggggtatgga gggcaagaat gaaatattac
1200aaaattaggg gtttttgcgt aatttattat atttaataaa gaaagtcgaa
tattcccatc 1260cgattggtag ttgaaagggg ccgaaaggcc tcggggtttc
tagagatttc tacattattc 1320tcgtttttgt cgccaagaag gtgggcaatt
atgtttcatg ccttaacttc ttctttttgt 1380gggaatactc ttattcttag
tacaaaagaa aagagtatat gcataaataa gatgaaaaat 1440gggtttattc
gagatttcta cgtcatgtgt gactcgctta ggaaatatcg ccgaaaccta
1500acaaaggcgg tacgctcctc tcccccgacc tataaataga gacctttgcc
tcgtctttct 1560caactcaagc atttctgtat gatccttctc tttccgcgga
agctctcgcg ccagttgatc 1620gcaaggtatg cgtctttcct ccttgtgatt
cgatctttct gttggctaga tctggtctat 1680tgatctgctc tattgatctg
gtctatttat cgctgcatcg ggatctattg atccgtatgt 1740tgatttggga
tccgtaggtt ggtttggatc ggagactgcg atttgattct tgtgatttcg
1800cttggatttc ggaaatcggt gtggttgaag tcgtgcgatc ttttagatct
gctccttttt 1860ttatttgcta ttttatattt acgttgttta tgatcgcgga
ttattttgat tcgtttattc 1920gagatccatg ccgtttaact cgttctttgt
gctccgatct ttgcgatacg tcggtcgttc 1980tagatccgtt cactaggtta
gttttaagtt ctttgagctt gatttatatg gatttgctgt 2040tttccaggaa
aaatttatgc gcgattctta cgcccgtttc cccattttac tttaggtcgt
2100gaattctttt gatctgagaa tgatgaatct gacatgtacc ttccggtttg
taatttgcag 216082021DNASpirodela polyrrhiza 8caaataaaga gatggacaga
taatgagatg aattagaaaa aaaaaattcg tgttgtaaga 60tagaatactt gctatctact
gatgaatgca gttcagtttt cctcacgatc ttaaagatcg 120cgcactatcc
tcagcttcac tctggaaatt ttgattctct tcttctgctc agcagcctcg
180actctgtcta gggtttcgta caatcggacg ccattctaca tgaatcgagc
acagggaatg 240aagacaatta ggagatcctc gatgtcctcc gacttacttg
catgacttga cggggaagat 300ctcgagcagg gaagcgacgc ctctccggag
gactcgcctc gccgagagga cctcctccgc 360gacacggacc atggcctcca
cggggtagaa gctggccctg ttctttattc tcttgaggat 420catcggccga
agcctccgca aatccatccc cgaggagtag aatctcgcct gcaggaagca
480tctgtcgaga tcctcgccga ggcggcggag atacctcgcc ggcgccgcca
tggcgccggg 540gacggagcac caccacggag aagaagaacc ctaacccaag
gcattaacga agttgcgcag 600attatacaaa agccctcaaa tatctttcat
tttctatttc actgatacat tttcattatt 660gtatatgagt gtttatttaa
attattccgt attagaaaag cacctccaga acccgacaaa 720atagggtgac
gtcatcatgg tgtcatgacc gcccaacagc cgcagattta aaatcggtgg
780atgagtgcgg ccacgccacg aaagcgatgg gccttcgtcg atgccgtgag
aatccatctg 840acataaagta aacggcgccg tcagtattga cggcgtatga
cacgtggaaa gaagctattg 900gttcacgcat cggtggttcc gctagcctcc
gtcgaccgct agtactataa atacggtccc 960gaggcctcct caccactcgc
acatatcctc tttgttttcc tctccgtgaa agaagcgagg 1020aagcgcgtcg
tctctcccaa ggtaaggagc agatctcttt gatcgttttt gttcttcttt
1080tgttttgttt tttttttctg cggatcttcg gttgcatcat gccttggctg
tttttattag 1140tttaggatat cctcgtttgg atctgagccg atcatatatg
ttaaaggttg tgttcgatct 1200ctttgttcat
tttcgcatga aaaggatgta tccttttgat gtgaggcgat cttctatggt
1260taagactttg ttcggtctat tgatcatttc tgttcttcgt ttttgagttt
ttttctgcgg 1320atatcgcatc atccctaggt ttttgctttg gttaggatgc
atcctttgga tttgagccga 1380tctcccttgg ttaaggctgt gtctgttgca
gaggagaaag tctgtcgagg tccttatgca 1440ggctttgtcc agatgcgcgt
gctctctcat gctatgaatt tatgttttga gaactcctcc 1500cggtttttct
agatccggat ttgaagtatt cattgcggtt ccccttcggt tttatgtatt
1560tctcgagttg atttggtcca tgatcgtgtt ctgtccagat ctctcttgat
atggatgaga 1620tattcgttac ctctttcaaa catcggtgga tgttcttttt
agtcttggct cacctttatc 1680tagaaattaa ttttcggttt gaaacccctg
cttgttaagg tgatgtattc cttctttata 1740gatttcggtg tgttatttct
taacggtgat ctgtccgatc catgtgttgc acctcttgtt 1800ttctgtgtaa
tcctctgtga attataatta tgttttgaaa acgtacttaa gtaaggggca
1860tgttccccgt ttaaaacttt tgttctatca atttgtggtt aatagatcct
gatttgtggt 1920cgccttattc tgtctttaat cgtggatttt atttatcttg
agcgcgtcct tttcttttaa 1980aatcatgtgt ttaacctttc agtcgtcata
tgttccatca g 202192068DNALemna aequinoctialis 9agtgtaccaa
tattttaaac cctacattta tcattcttta ttcattattg ccataagtta 60atgaatattg
aaattcaaat acgcgcaaga tgtcaatatc gatcgaatat gaataccaga
120tataaaatca aaaatcaaat atcaaattaa taaagatata aaatattgaa
tccaaaagca 180ataaagaata tcactattaa tatcaaaata tcgatttgaa
gttcaaaaat tgggtccatt 240aggagccaag accgatcatg atccgatact
gatatcaata tctgtagctc agtggctagg 300cccctcaatt tgcctggccg
aaggcagtgt acaaaacctg gctctcgcaa gggcaaagaa 360agagtctttc
ccaaaaaaaa aaaaatcgaa cccatttgta gtatccaata tttggattga
420cataagatac caaaacataa agtactaacc acccaatctt ataattaatc
aagatttata 480tcacatccaa tatcaagatc cgatatcaat acctagaccg
gtaaacccta atttactctt 540cccccctcta aaaatttcca ataaatatct
ccacatattt aactattaaa aaattgataa 600gagataggcc ctagccctaa
gtcctaacat ataaccactc tctatgaaaa gtcctattaa 660atgacgtcat
ttatttattt attgccggtt ggctgctcca cagccgcaat ttaatggatg
720gctgacacgg cacgaaaccg acgggcggtg ccgtgggaat aattctagag
taaacctaac 780ggcgccgtta actttgacgg tggcgaagac gcgtggggat
aggtggttgg tccgcgtgac 840ggcggcggtt cagcccgtcg accttgagcc
gagactataa atcgaggcga agggatgagc 900tttgccattg cgttcttctt
ctgttcatct ctgaaattcg ggcggaatcc ttcttcttct 960caaggtatgg
gcctcgatct ttctgtttca atcgagtttt gatcttcgtt ttggcggcga
1020tcggtgtttt ctttgtattg tgaataaatc cttgataaga aaaccctagg
ttttgtgacc 1080tgttgacgga tgcgtgcgga tctgttattt gtcttttagg
cgattttctc ttgtttgtaa 1140tagtttatca taaccagatg aacatggatc
aagtcgattt gacttatttt ttctgtgaaa 1200ttaggccgaa atcctttttt
ttggtttgag ccttgatatt tctatataat tcgatttgat 1260tttttgtttt
cttctgcgtc tgatgctttc tcttgactcc tgattaaatt tttgctacgg
1320aaaccctaga tgtcgagatc tgttgacaga ttctggcaaa tctgttttta
tcataatcag 1380atgaacgcaa attaagtcga tttggttttt ctctgaaatt
aggggggaaa ctccttatag 1440tatgagcctc gatatttcta taatagtcga
tttgattttc tcttgcctcc tgattcaatt 1500tttggtgcgg aaaccctaga
tattgtaatc tgtttacgga tgcttgcgga tctgattttt 1560aatattgtga
tctattgacg gatgctcgta gatctggttg ttttgatttc ttcatgcctt
1620atacggcgat ttgattcggc gattaaaaat tttcaattct tttaaaaaaa
atattaagat 1680tttcaacgtt tcaaattatt tcatagatcg gcacaaatac
ttttcatcag attcctcctg 1740atgtgatggt ttgtgtttaa aatctgttga
agatatcaga ttctattagg tcaccgatat 1800aatcttctct gtttattctg
cgatcggtgc ttacaaaccc tatttcctac ggtgattaat 1860tatttttaat
ctcctagcta gcgtaaatat atattttttt aatttgatct ttgcattagt
1920ttcctccttt tatttgctat taattgtaac cgatgctaca aaacatcaga
ttttttttcc 1980caattcgttg tcatcattat agaaaacttt tatctgatat
ttttaatcgt cattaatata 2040attttcaatt tattattttc ccttgcag
2068101625DNALemna minor 10agtcgagtga tatgaaatct tggtgaagaa
ggatcggaga acggaccggg tgaggcaagg 60ataattctgc tgttaaattc gagagcaaga
cacctgcaat tcaagaatcg agtggcaatt 120aatatagcag gatgatctgg
aaggtagatc ctgcccatcg aatgatccaa acatcaacac 180taggatcata
ccgttaacaa taatgaatga aaaagtagaa gatgacgaag ttgaagtgat
240gaccaaaaac tttgaaaatt ccaaccgtat ggccggaatc agtgtgaaga
aaatcgaaat 300caaatactct aatggatcgg attgttattc tggaggcaaa
tctgaaactt cgaggatagg 360atttaatcca cgcaagtaat aatttgaaac
tcagaaggag aaaaaaaaac taaaatagag 420aagaagagat ctcaaagaag
ccgtgagcac gagacgaacg agaagaggta aagcaccagt 480cagaggaaaa
caccaaaatt agagaaatag cacgaacatt aaagcacaga tccgcgccgc
540aaacccgaaa gacgaaaaat agagccaaac gaaaccctaa taatcgatct
gcacaaaaaa 600aaaaaaaaaa aactttgaga agagccgcga aattacccta
gaatcctcag aactggccgg 660acgagagaag cgctcgatcg aaacccaaca
taaaacccct tccaacggca aattactccg 720caaaacccga aaaataaaca
aaatcaacga tcacgagaag gtgcaagggc aaaaagaggc 780agtgcgatcg
agagtctacc tgaatcgtcg gcgcaaaagg cgagcccacc gacgaacgct
840ccctctagaa cctggagatg cggcgagaga gaaggaaaga tcttcggtgg
gtgatgctcg 900ctatttatcg caagagagtt agagagatct tcttcggcgg
cggatttctg gcatctagcg 960tttaacctca ccgcccagtg ctcacatcct
tcttctcata tttgaatatt taattaacaa 1020atgaatcagt catttttctt
taatttttaa ttcccggaga gggcaatgtt ggtatcaaaa 1080attatttagg
aaaaattaat tacacgaata atcggatttt tccctttttt taattaattt
1140ctaattttgg aaaaggaaag aaaaatttta ggggtatgga gggcaagaat
gaaatattac 1200aaaattaggg gtttttgcgt aatttattat atttaataaa
gaaagtcgaa tattcccatc 1260cgattggtag ttgaaagggg ccgaaaggcc
tcggggtttc tagagatttc tacattattc 1320tcgtttttgt cgccaagaag
gtgggcaatt atgtttcatg ccttaacttc ttctttttgt 1380gggaatactc
ttattcttag tacaaaagaa aagagtatat gcataaataa gatgaaaaat
1440gggtttattc gagatttcta cgtcatgtgt gactcgctta ggaaatatcg
ccgaaaccta 1500acaaaggcgg tacgctcctc tcccccgacc tataaataga
gacctttgcc tcgtctttct 1560caactcaagc atttctgtat gatccttctc
tttccgcgga agctctcgcg ccagttgatc 1620gcaag 1625111041DNASpirodela
polyrrhiza 11caaataaaga gatggacaga taatgagatg aattagaaaa aaaaaattcg
tgttgtaaga 60tagaatactt gctatctact gatgaatgca gttcagtttt cctcacgatc
ttaaagatcg 120cgcactatcc tcagcttcac tctggaaatt ttgattctct
tcttctgctc agcagcctcg 180actctgtcta gggtttcgta caatcggacg
ccattctaca tgaatcgagc acagggaatg 240aagacaatta ggagatcctc
gatgtcctcc gacttacttg catgacttga cggggaagat 300ctcgagcagg
gaagcgacgc ctctccggag gactcgcctc gccgagagga cctcctccgc
360gacacggacc atggcctcca cggggtagaa gctggccctg ttctttattc
tcttgaggat 420catcggccga agcctccgca aatccatccc cgaggagtag
aatctcgcct gcaggaagca 480tctgtcgaga tcctcgccga ggcggcggag
atacctcgcc ggcgccgcca tggcgccggg 540gacggagcac caccacggag
aagaagaacc ctaacccaag gcattaacga agttgcgcag 600attatacaaa
agccctcaaa tatctttcat tttctatttc actgatacat tttcattatt
660gtatatgagt gtttatttaa attattccgt attagaaaag cacctccaga
acccgacaaa 720atagggtgac gtcatcatgg tgtcatgacc gcccaacagc
cgcagattta aaatcggtgg 780atgagtgcgg ccacgccacg aaagcgatgg
gccttcgtcg atgccgtgag aatccatctg 840acataaagta aacggcgccg
tcagtattga cggcgtatga cacgtggaaa gaagctattg 900gttcacgcat
cggtggttcc gctagcctcc gtcgaccgct agtactataa atacggtccc
960gaggcctcct caccactcgc acatatcctc tttgttttcc tctccgtgaa
agaagcgagg 1020aagcgcgtcg tctctcccaa g 104112964DNALemna
aequinoctialis 12agtgtaccaa tattttaaac cctacattta tcattcttta
ttcattattg ccataagtta 60atgaatattg aaattcaaat acgcgcaaga tgtcaatatc
gatcgaatat gaataccaga 120tataaaatca aaaatcaaat atcaaattaa
taaagatata aaatattgaa tccaaaagca 180ataaagaata tcactattaa
tatcaaaata tcgatttgaa gttcaaaaat tgggtccatt 240aggagccaag
accgatcatg atccgatact gatatcaata tctgtagctc agtggctagg
300cccctcaatt tgcctggccg aaggcagtgt acaaaacctg gctctcgcaa
gggcaaagaa 360agagtctttc ccaaaaaaaa aaaaatcgaa cccatttgta
gtatccaata tttggattga 420cataagatac caaaacataa agtactaacc
acccaatctt ataattaatc aagatttata 480tcacatccaa tatcaagatc
cgatatcaat acctagaccg gtaaacccta atttactctt 540cccccctcta
aaaatttcca ataaatatct ccacatattt aactattaaa aaattgataa
600gagataggcc ctagccctaa gtcctaacat ataaccactc tctatgaaaa
gtcctattaa 660atgacgtcat ttatttattt attgccggtt ggctgctcca
cagccgcaat ttaatggatg 720gctgacacgg cacgaaaccg acgggcggtg
ccgtgggaat aattctagag taaacctaac 780ggcgccgtta actttgacgg
tggcgaagac gcgtggggat aggtggttgg tccgcgtgac 840ggcggcggtt
cagcccgtcg accttgagcc gagactataa atcgaggcga agggatgagc
900tttgccattg cgttcttctt ctgttcatct ctgaaattcg ggcggaatcc
ttcttcttct 960caag 96413535DNALemna minor 13gtatgcgtct ttcctccttg
tgattcgatc tttctgttgg ctagatctgg tctattgatc 60tgctctattg atctggtcta
tttatcgctg catcgggatc tattgatccg tatgttgatt 120tgggatccgt
aggttggttt ggatcggaga ctgcgatttg attcttgtga tttcgcttgg
180atttcggaaa tcggtgtggt tgaagtcgtg cgatctttta gatctgctcc
tttttttatt 240tgctatttta tatttacgtt gtttatgatc gcggattatt
ttgattcgtt tattcgagat 300ccatgccgtt taactcgttc tttgtgctcc
gatctttgcg atacgtcggt cgttctagat 360ccgttcacta ggttagtttt
aagttctttg agcttgattt atatggattt gctgttttcc 420aggaaaaatt
tatgcgcgat tcttacgccc gtttccccat tttactttag gtcgtgaatt
480cttttgatct gagaatgatg aatctgacat gtaccttccg gtttgtaatt tgcag
53514980DNASpirodela polyrrhiza 14gtaaggagca gatctctttg atcgtttttg
ttcttctttt gttttgtttt ttttttctgc 60ggatcttcgg ttgcatcatg ccttggctgt
ttttattagt ttaggatatc ctcgtttgga 120tctgagccga tcatatatgt
taaaggttgt gttcgatctc tttgttcatt ttcgcatgaa 180aaggatgtat
ccttttgatg tgaggcgatc ttctatggtt aagactttgt tcggtctatt
240gatcatttct gttcttcgtt tttgagtttt tttctgcgga tatcgcatca
tccctaggtt 300tttgctttgg ttaggatgca tcctttggat ttgagccgat
ctcccttggt taaggctgtg 360tctgttgcag aggagaaagt ctgtcgaggt
ccttatgcag gctttgtcca gatgcgcgtg 420ctctctcatg ctatgaattt
atgttttgag aactcctccc ggtttttcta gatccggatt 480tgaagtattc
attgcggttc cccttcggtt ttatgtattt ctcgagttga tttggtccat
540gatcgtgttc tgtccagatc tctcttgata tggatgagat attcgttacc
tctttcaaac 600atcggtggat gttcttttta gtcttggctc acctttatct
agaaattaat tttcggtttg 660aaacccctgc ttgttaaggt gatgtattcc
ttctttatag atttcggtgt gttatttctt 720aacggtgatc tgtccgatcc
atgtgttgca cctcttgttt tctgtgtaat cctctgtgaa 780ttataattat
gttttgaaaa cgtacttaag taaggggcat gttccccgtt taaaactttt
840gttctatcaa tttgtggtta atagatcctg atttgtggtc gccttattct
gtctttaatc 900gtggatttta tttatcttga gcgcgtcctt ttcttttaaa
atcatgtgtt taacctttca 960gtcgtcatat gttccatcag 980151104DNALemna
aequinoctialis 15gtatgggcct cgatctttct gtttcaatcg agttttgatc
ttcgttttgg cggcgatcgg 60tgttttcttt gtattgtgaa taaatccttg ataagaaaac
cctaggtttt gtgacctgtt 120gacggatgcg tgcggatctg ttatttgtct
tttaggcgat tttctcttgt ttgtaatagt 180ttatcataac cagatgaaca
tggatcaagt cgatttgact tattttttct gtgaaattag 240gccgaaatcc
ttttttttgg tttgagcctt gatatttcta tataattcga tttgattttt
300tgttttcttc tgcgtctgat gctttctctt gactcctgat taaatttttg
ctacggaaac 360cctagatgtc gagatctgtt gacagattct ggcaaatctg
tttttatcat aatcagatga 420acgcaaatta agtcgatttg gtttttctct
gaaattaggg gggaaactcc ttatagtatg 480agcctcgata tttctataat
agtcgatttg attttctctt gcctcctgat tcaatttttg 540gtgcggaaac
cctagatatt gtaatctgtt tacggatgct tgcggatctg atttttaata
600ttgtgatcta ttgacggatg ctcgtagatc tggttgtttt gatttcttca
tgccttatac 660ggcgatttga ttcggcgatt aaaaattttc aattctttta
aaaaaaatat taagattttc 720aacgtttcaa attatttcat agatcggcac
aaatactttt catcagattc ctcctgatgt 780gatggtttgt gtttaaaatc
tgttgaagat atcagattct attaggtcac cgatataatc 840ttctctgttt
attctgcgat cggtgcttac aaaccctatt tcctacggtg attaattatt
900tttaatctcc tagctagcgt aaatatatat ttttttaatt tgatctttgc
attagtttcc 960tccttttatt tgctattaat tgtaaccgat gctacaaaac
atcagatttt ttttcccaat 1020tcgttgtcat cattatagaa aacttttatc
tgatattttt aatcgtcatt aatataattt 1080tcaatttatt attttccctt gcag
11041664DNALemna gibba 16aagcacgagc tgagcgagaa ttcggggagg
ctgagtcgaa gaggaagaga gaagtaggta 60cgcc 641758DNALemna gibba
17actcgcaagt ggagagagga tccgagcgtc cagtgagagg aagagagagg gaggcgcg
581862DNALemna gibba 18aaactcccga ggtgagcaag gatccggagt cgagcgcgaa
gaagagaaag agggaaagcg 60cg 62191282DNALemna minorCDS(1)...(1275)
19tgc gaa gcc tac ttc ggc aac agc ttc aac cgc cgg acg gag atg ctg
48Cys Glu Ala Tyr Phe Gly Asn Ser Phe Asn Arg Arg Thr Glu Met Leu1
5 10 15aag aag gta gag gga aga gga tgg ttc cag tgc ctg tac agc gat
act 96Lys Lys Val Glu Gly Arg Gly Trp Phe Gln Cys Leu Tyr Ser Asp
Thr 20 25 30ctt cga agt tct gtt tgc cag gga ggg aat ttg cgg atg gac
ccg gaa 144Leu Arg Ser Ser Val Cys Gln Gly Gly Asn Leu Arg Met Asp
Pro Glu 35 40 45agg att agg atg tcg aaa ggg ggg gaa gat cta gag gag
gtg atg aag 192Arg Ile Arg Met Ser Lys Gly Gly Glu Asp Leu Glu Glu
Val Met Lys 50 55 60aga gag gag gaa gaa gaa ttg ccc aaa ttc gag gag
ggg tcg ttc cag 240Arg Glu Glu Glu Glu Glu Leu Pro Lys Phe Glu Glu
Gly Ser Phe Gln65 70 75 80att gaa tct ggt tat gga agc gga ggg gaa
gtt gga gag aga att gcg 288Ile Glu Ser Gly Tyr Gly Ser Gly Gly Glu
Val Gly Glu Arg Ile Ala 85 90 95act gac gag gtc ctc gat aat gtt gtg
ccg aaa ggc gct gtt cat gta 336Thr Asp Glu Val Leu Asp Asn Val Val
Pro Lys Gly Ala Val His Val 100 105 110cat acc atg cgc aat ctc atc
agt tcg att cag att gtt ggt ccc ggg 384His Thr Met Arg Asn Leu Ile
Ser Ser Ile Gln Ile Val Gly Pro Gly 115 120 125cat ctt caa tgc tct
cag tgg atc gac gaa ccg gtt ctt ctt gtc aca 432His Leu Gln Cys Ser
Gln Trp Ile Asp Glu Pro Val Leu Leu Val Thr 130 135 140cgc ttc gaa
tac gcc aat ctc ttt cac acc gtc acc gac tgg tac agc 480Arg Phe Glu
Tyr Ala Asn Leu Phe His Thr Val Thr Asp Trp Tyr Ser145 150 155
160gcc tac gca agc tcg agg att gcc aac ttg ccc tct cgc cct cac ttg
528Ala Tyr Ala Ser Ser Arg Ile Ala Asn Leu Pro Ser Arg Pro His Leu
165 170 175att ttc gtc gat ggc cat tgc agg gca gaa cag tta gag gac
acg tgg 576Ile Phe Val Asp Gly His Cys Arg Ala Glu Gln Leu Glu Asp
Thr Trp 180 185 190cga gcc ctg ttc tca acc gtc cga tac gcc aag aac
ttc tcc cag cca 624Arg Ala Leu Phe Ser Thr Val Arg Tyr Ala Lys Asn
Phe Ser Gln Pro 195 200 205gtc tgc ttc cgc cac gcc gtc ctc tcc cct
ctt ggc tat gag aca gct 672Val Cys Phe Arg His Ala Val Leu Ser Pro
Leu Gly Tyr Glu Thr Ala 210 215 220ctc ttc aaa ggc cta tca gag agc
ttc agc tgt gag gga gtg ccg gcc 720Leu Phe Lys Gly Leu Ser Glu Ser
Phe Ser Cys Glu Gly Val Pro Ala225 230 235 240aat cag ctc aaa gtc
aac cct gat gac cag aag act gcg aga ctg gct 768Asn Gln Leu Lys Val
Asn Pro Asp Asp Gln Lys Thr Ala Arg Leu Ala 245 250 255gaa ttc gga
gag atg atc agg gct gcc ttt gac ttt cct gtc gtt gac 816Glu Phe Gly
Glu Met Ile Arg Ala Ala Phe Asp Phe Pro Val Val Asp 260 265 270ccg
ccc gtt gac ccg ttg acc aaa tcc atc ctc ttt gtg cgg cgg gaa 864Pro
Pro Val Asp Pro Leu Thr Lys Ser Ile Leu Phe Val Arg Arg Glu 275 280
285gat tac gtg gcg cac cca cgc cac agt ggg aga gtg gag tcg cgg ttg
912Asp Tyr Val Ala His Pro Arg His Ser Gly Arg Val Glu Ser Arg Leu
290 295 300acc aat gag caa gag gtg ttt gac ttt ctg cac aaa tgg gca
agt caa 960Thr Asn Glu Gln Glu Val Phe Asp Phe Leu His Lys Trp Ala
Ser Gln305 310 315 320cac aga agc agg tgc aac gtc agt gtg gtc aac
ggg ctt ttc gcg cac 1008His Arg Ser Arg Cys Asn Val Ser Val Val Asn
Gly Leu Phe Ala His 325 330 335atg gga atg aag gaa cag gtg aag gca
att atg gaa gct tcg gtg gtg 1056Met Gly Met Lys Glu Gln Val Lys Ala
Ile Met Glu Ala Ser Val Val 340 345 350gtc ggg gcc cac ggg gct ggt
ttg act cat ctg gtg gca gca agg tca 1104Val Gly Ala His Gly Ala Gly
Leu Thr His Leu Val Ala Ala Arg Ser 355 360 365acg aca gtt gtt ctt
gag att ctg agc agt caa tat cgt aga ccg cac 1152Thr Thr Val Val Leu
Glu Ile Leu Ser Ser Gln Tyr Arg Arg Pro His 370 375 380ttt caa ctg
att tca cgg tgg aaa ggg ttg gac tac cac gca att aat 1200Phe Gln Leu
Ile Ser Arg Trp Lys Gly Leu Asp Tyr His Ala Ile Asn385 390 395
400ctt gcc ggg tcg tat gct gat cct cgg gag gtg gtc gag aaa ttg act
1248Leu Ala Gly Ser Tyr Ala Asp Pro Arg Glu Val Val Glu Lys Leu Thr
405 410 415ggc ata gtc gat ggg ctt gga tgt tga a gataag 1282Gly Ile
Val Asp Gly Leu Gly Cys 420201275DNALemna
minorCDS(1)...(1275)misc_feature(0)...(0)Xy1T isoform #2; Encodes
partial-length beta-1, 2-xylosyltransferase 20tgc gaa gcc tac ttc
ggc aac agc ttc aac cgc cgg acg gag atg ctg 48Cys Glu Ala Tyr Phe
Gly Asn Ser Phe Asn Arg Arg Thr Glu Met Leu1 5 10 15aag aag gta gag
gga aga gga tgg ttc cag tgc ctg tac agc gat act 96Lys Lys Val Glu
Gly Arg Gly Trp Phe Gln Cys Leu Tyr Ser Asp Thr 20 25 30ctt cga agt
tct gtt tgc cag gga ggg aat ttg cgg atg gac ccg gaa 144Leu Arg Ser
Ser Val Cys Gln Gly Gly Asn Leu Arg Met Asp Pro Glu 35 40 45agg att
agg atg tcg aaa ggg ggg gaa gat cta gag gag gtg atg aag 192Arg Ile
Arg Met Ser Lys Gly Gly Glu Asp Leu Glu Glu Val Met Lys 50 55 60aga
gag gag gaa gaa gaa ttg ccc aaa ttc gag gag ggg tcg ttc cag 240Arg
Glu Glu Glu Glu Glu Leu Pro Lys Phe Glu Glu Gly Ser Phe Gln65
70 75 80att gaa tct ggt tat gga agc gga ggg gaa gtt gga gag aga att
gcg 288Ile Glu Ser Gly Tyr Gly Ser Gly Gly Glu Val Gly Glu Arg Ile
Ala 85 90 95act gac gag gtc ctc gat aat gtt gtg ccg aaa ggc gct gtt
cat gta 336Thr Asp Glu Val Leu Asp Asn Val Val Pro Lys Gly Ala Val
His Val 100 105 110cat acc atg cgc aat ctc atc agt tcg att cag att
gtt ggt ccc ggg 384His Thr Met Arg Asn Leu Ile Ser Ser Ile Gln Ile
Val Gly Pro Gly 115 120 125cat ctt caa tgc tct cag tgg atc gac gaa
ccg gtt ctt ctt gtc aca 432His Leu Gln Cys Ser Gln Trp Ile Asp Glu
Pro Val Leu Leu Val Thr 130 135 140cgc ttc gaa tac gcc aat ctc ttt
cac acc gtc acc gac tgg tac agc 480Arg Phe Glu Tyr Ala Asn Leu Phe
His Thr Val Thr Asp Trp Tyr Ser145 150 155 160gcc tac gca agc tcg
agg att gcc aac ttg ccc tct cgc cct cac ttg 528Ala Tyr Ala Ser Ser
Arg Ile Ala Asn Leu Pro Ser Arg Pro His Leu 165 170 175att ttc gtc
gat ggc cat tgc agg gca gaa cag tta gag gac acg tgg 576Ile Phe Val
Asp Gly His Cys Arg Ala Glu Gln Leu Glu Asp Thr Trp 180 185 190cga
gcc ctg ttc tca acc gtc cga tac gcc aag aac ttc tcc cag cca 624Arg
Ala Leu Phe Ser Thr Val Arg Tyr Ala Lys Asn Phe Ser Gln Pro 195 200
205gtc tgc ttc cgc cac gcc gtc ctc tcc cct ctt ggc tat gag aca gct
672Val Cys Phe Arg His Ala Val Leu Ser Pro Leu Gly Tyr Glu Thr Ala
210 215 220ctc ttc aaa ggc cta tca gag agc ttc agc tgt gag gga gtg
ccg gcc 720Leu Phe Lys Gly Leu Ser Glu Ser Phe Ser Cys Glu Gly Val
Pro Ala225 230 235 240aat cag ctc aaa gtc aac cct gat gac cag aag
act gcg aga ctg gct 768Asn Gln Leu Lys Val Asn Pro Asp Asp Gln Lys
Thr Ala Arg Leu Ala 245 250 255gaa ttc gga gag atg atc agg gct gcc
ttt gac ttt cct gtc gtt gac 816Glu Phe Gly Glu Met Ile Arg Ala Ala
Phe Asp Phe Pro Val Val Asp 260 265 270ccg ccc gtt gac ccg ttg acc
aaa tcc atc ctc ttt gtg cgg cgg gaa 864Pro Pro Val Asp Pro Leu Thr
Lys Ser Ile Leu Phe Val Arg Arg Glu 275 280 285gat tac gtg gcg cac
cca cgc cac agt ggg aga gtg gag tcg cgg ttg 912Asp Tyr Val Ala His
Pro Arg His Ser Gly Arg Val Glu Ser Arg Leu 290 295 300acc aat gag
caa gag gtg ttt gac ttt ctg cac aaa tgg gca agt caa 960Thr Asn Glu
Gln Glu Val Phe Asp Phe Leu His Lys Trp Ala Ser Gln305 310 315
320cac aga agc agg tgc aac gtc agt gtg gtc aac ggg ctt ttc gcg cac
1008His Arg Ser Arg Cys Asn Val Ser Val Val Asn Gly Leu Phe Ala His
325 330 335atg gga atg aag gaa cag gtg aag gca att atg gaa gct tcg
gtg gtg 1056Met Gly Met Lys Glu Gln Val Lys Ala Ile Met Glu Ala Ser
Val Val 340 345 350gtc ggg gcc cac ggg gct ggt ttg act cat ctg gtg
gca gca agg tca 1104Val Gly Ala His Gly Ala Gly Leu Thr His Leu Val
Ala Ala Arg Ser 355 360 365acg aca gtt gtt ctt gag att ctg agc agt
caa tat cgt aga ccg cac 1152Thr Thr Val Val Leu Glu Ile Leu Ser Ser
Gln Tyr Arg Arg Pro His 370 375 380ttt caa ctg att tca cgg tgg aaa
ggg ttg gac tac cac gca att aat 1200Phe Gln Leu Ile Ser Arg Trp Lys
Gly Leu Asp Tyr His Ala Ile Asn385 390 395 400ctt gcc ggg tcg tat
gct gat cct cgg gag gtg gtc gag aaa ttg act 1248Leu Ala Gly Ser Tyr
Ala Asp Pro Arg Glu Val Val Glu Lys Leu Thr 405 410 415ggc ata gtc
gat ggg ctt gga tgt tga 1275Gly Ile Val Asp Gly Leu Gly Cys
42021424PRTLemna minor 21Cys Glu Ala Tyr Phe Gly Asn Ser Phe Asn
Arg Arg Thr Glu Met Leu1 5 10 15Lys Lys Val Glu Gly Arg Gly Trp Phe
Gln Cys Leu Tyr Ser Asp Thr 20 25 30Leu Arg Ser Ser Val Cys Gln Gly
Gly Asn Leu Arg Met Asp Pro Glu 35 40 45Arg Ile Arg Met Ser Lys Gly
Gly Glu Asp Leu Glu Glu Val Met Lys 50 55 60Arg Glu Glu Glu Glu Glu
Leu Pro Lys Phe Glu Glu Gly Ser Phe Gln65 70 75 80Ile Glu Ser Gly
Tyr Gly Ser Gly Gly Glu Val Gly Glu Arg Ile Ala 85 90 95Thr Asp Glu
Val Leu Asp Asn Val Val Pro Lys Gly Ala Val His Val 100 105 110His
Thr Met Arg Asn Leu Ile Ser Ser Ile Gln Ile Val Gly Pro Gly 115 120
125His Leu Gln Cys Ser Gln Trp Ile Asp Glu Pro Val Leu Leu Val Thr
130 135 140Arg Phe Glu Tyr Ala Asn Leu Phe His Thr Val Thr Asp Trp
Tyr Ser145 150 155 160Ala Tyr Ala Ser Ser Arg Ile Ala Asn Leu Pro
Ser Arg Pro His Leu 165 170 175Ile Phe Val Asp Gly His Cys Arg Ala
Glu Gln Leu Glu Asp Thr Trp 180 185 190Arg Ala Leu Phe Ser Thr Val
Arg Tyr Ala Lys Asn Phe Ser Gln Pro 195 200 205Val Cys Phe Arg His
Ala Val Leu Ser Pro Leu Gly Tyr Glu Thr Ala 210 215 220Leu Phe Lys
Gly Leu Ser Glu Ser Phe Ser Cys Glu Gly Val Pro Ala225 230 235
240Asn Gln Leu Lys Val Asn Pro Asp Asp Gln Lys Thr Ala Arg Leu Ala
245 250 255Glu Phe Gly Glu Met Ile Arg Ala Ala Phe Asp Phe Pro Val
Val Asp 260 265 270Pro Pro Val Asp Pro Leu Thr Lys Ser Ile Leu Phe
Val Arg Arg Glu 275 280 285Asp Tyr Val Ala His Pro Arg His Ser Gly
Arg Val Glu Ser Arg Leu 290 295 300Thr Asn Glu Gln Glu Val Phe Asp
Phe Leu His Lys Trp Ala Ser Gln305 310 315 320His Arg Ser Arg Cys
Asn Val Ser Val Val Asn Gly Leu Phe Ala His 325 330 335Met Gly Met
Lys Glu Gln Val Lys Ala Ile Met Glu Ala Ser Val Val 340 345 350Val
Gly Ala His Gly Ala Gly Leu Thr His Leu Val Ala Ala Arg Ser 355 360
365Thr Thr Val Val Leu Glu Ile Leu Ser Ser Gln Tyr Arg Arg Pro His
370 375 380Phe Gln Leu Ile Ser Arg Trp Lys Gly Leu Asp Tyr His Ala
Ile Asn385 390 395 400Leu Ala Gly Ser Tyr Ala Asp Pro Arg Glu Val
Val Glu Lys Leu Thr 405 410 415Gly Ile Val Asp Gly Leu Gly Cys
4202228DNAArtificial Sequenceprimer 22atggtcgact gctgctggtg
ctctcaac 282328DNAArtificial Sequenceprimer 23atgtctagaa tgcagcagca
agtgcacc 282433DNAArtificial Sequenceprimer 24atgactagtt gcgaagccta
cttcggcaac agc 332530DNAArtificial Sequenceprimer 25atgggatccg
aatctcaaga acaactgtcg 302633DNAArtificial Sequenceprimer
26atgggtacct gcgaagccta cttcggcaac agc 332729DNAArtificial
Sequenceprimer 27atgggatcca ctggctggga gaagttctt
292828DNAArtificial Sequenceprimer 28atggagctct gctgctggtg ctctcaac
282928DNAArtificial Sequenceprimer 29atgggtacca tgcagcagca agtgcacc
2830487PRTTriticum aestivum 30Met Lys Gly Ser His Ser Gln Ser Gln
Ala Ala Ser Arg Arg Arg Arg1 5 10 15Cys Gly Trp Leu Leu Pro Leu Leu
Val Gly Val Ala Phe Val Gly Glu 20 25 30Ile Ala Phe Leu Gly Arg Leu
Asp Met Ser Lys Asn Ala Ala Ala Val 35 40 45Glu Ser Trp Thr Thr Ser
Phe Tyr Arg Leu Ser Ser Thr Trp Gly Ala 50 55 60Asp Ala Pro Pro Gly
Ser Gly Asp Asp Asp Asp Glu Cys Glu Glu Arg65 70 75 80Leu Glu Arg
Glu Asp Ala Val Pro Tyr Asp Arg Asp Phe Glu Arg Asp 85 90 95Pro Val
Leu Val Gly Gly Ala Ala Lys Asp Trp Asn Arg Cys Ser Val 100 105
110Gly Cys Glu Phe Gly Phe Pro Ala Ser Lys Thr Pro Asp Ala Thr Phe
115 120 125Gly Ile Ala Pro Asp Pro Ser Val Glu Ser Ile Leu Arg Ser
Met Glu 130 135 140Ser Ser Gln Tyr Tyr Ser Glu Asn Asn Ile Asn Ala
Ala Arg Gly Arg145 150 155 160Gly Tyr Gln Ile Val Met Thr Thr Ser
Leu Ser Ser Asp Val Pro Val 165 170 175Gly Tyr Phe Ser Trp Ala Glu
Tyr Asp Ile Met Ala Pro Val Pro Pro 180 185 190Lys Thr Glu Glu Ala
Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala 195 200 205Arg Asn Phe
Arg Leu Gln Ala Leu Glu Met Leu Glu Ser Leu Asp Val 210 215 220Lys
Ile Asp Ser Tyr Gly Ser Cys His Arg Asn Arg Asp Gly Lys Val225 230
235 240Asp Lys Val Glu Thr Leu Lys Arg Tyr Lys Phe Ser Leu Ala Phe
Glu 245 250 255Asn Ser Gly Glu Glu Asp Tyr Val Thr Glu Lys Phe Phe
Gln Ser Leu 260 265 270Val Thr Gly Ala Ile Pro Val Val Val Gly Ala
Pro Asn Ile Gln Glu 275 280 285Phe Ser Pro Gly Glu Gly Ala Ile Leu
His Ile Lys Glu Leu Asp Asp 290 295 300Val Ile Ser Val Ala Lys Thr
Met Lys His Ile Ala Ser Asn Pro Asp305 310 315 320Ala Phe Asn Gln
Ser Leu Arg Trp Lys Tyr Asp Gly Pro Ser Asp Ser 325 330 335Phe Lys
Ala Leu Ile Asp Met Ala Ala Val His Ser Ser Cys Arg Leu 340 345
350Cys Ile His Ile Ala Thr Lys Ile His Glu Lys Glu Glu Arg Thr Pro
355 360 365Lys Phe Met Asn Arg Ser Cys Ser Cys Ser Ser Lys Arg Gly
Thr Val 370 375 380Tyr His Leu Phe Val Arg Glu Arg Gly Arg Phe Lys
Thr Glu Ser Ile385 390 395 400Tyr Leu Arg Ser Asp Gln Leu Thr Leu
Gly Ala Leu Glu Ser Ala Val 405 410 415His Gly Lys Phe Arg Ser Leu
Lys His Val Pro Val Trp Lys Asp Glu 420 425 430Arg Pro Ser Ser Ile
Arg Gly Gly Asp Glu Leu Lys Val Tyr Lys Ile 435 440 445Tyr Pro Ile
Gly Leu Thr Glu Arg Gln Ala Leu Tyr Lys Phe Gln Phe 450 455 460Ser
Asp Asp Ala Glu Val Ala Arg Tyr Ile Lys Gly His Pro Cys Ala465 470
475 480Lys Leu Glu Val Ile Phe Val 48531486PRTHordeum vulgare 31Met
Gly Ser His Ser Leu Thr Gln Ala Ala Ser Arg Arg Arg Arg Cys1 5 10
15Gly Trp Leu Leu Pro Leu Val Val Gly Val Ala Phe Leu Gly Glu Ile
20 25 30Ala Phe Leu Gly Arg Leu Asp Met Ser Lys Asn Ala Ala Ala Val
Glu 35 40 45Ser Trp Thr Thr Ser Phe His Arg Leu Ser Ser Thr Trp Gly
Ala Asp 50 55 60Ala Pro Pro Gly Ser Gly Asp Asp Asp Glu Glu Cys Glu
Glu Arg Leu65 70 75 80Glu Arg Asp Asp Ala Val Pro Tyr Asp Arg Asp
Phe Glu Arg His Pro 85 90 95Val Leu Val Gly Gly Ala Ala Lys Asp Trp
Asn Arg Cys Ser Val Gly 100 105 110Cys Glu Phe Gly Phe Pro Ala Ser
Lys Thr Pro Asp Ala Thr Phe Gly 115 120 125Ile Ala Pro Asp Pro Ser
Val Glu Ser Ile Leu Arg Ser Met Glu Ser 130 135 140Ser Gln Tyr Tyr
Ser Glu Asn Asn Ile Asn Ala Ala Arg Gly Arg Gly145 150 155 160Tyr
Gln Ile Val Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly 165 170
175Tyr Phe Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro Val Pro Pro Lys
180 185 190Thr Glu Glu Ala Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly
Ala Arg 195 200 205Asn Phe Arg Leu Gln Ala Leu Glu Met Leu Glu Ser
Leu Asp Val Lys 210 215 220Ile Asp Ser Tyr Gly Ser Cys His Arg Asn
Arg Asp Gly Lys Val Asp225 230 235 240Lys Val Glu Thr Leu Lys Gly
Tyr Lys Phe Ser Leu Ala Phe Glu Asn 245 250 255Ser Asn Glu Glu Asp
Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val 260 265 270Thr Gly Ala
Ile Pro Val Val Val Gly Ala Pro Asn Ile Gln Glu Phe 275 280 285Ser
Pro Gly Glu Asp Ala Ile Leu His Ile Lys Glu Leu Asp Asp Val 290 295
300Ile Ser Val Ala Lys Thr Met Lys His Ile Ala Ser Asn Pro Asp
Ala305 310 315 320Phe Asn Gln Ser Leu Arg Trp Lys Tyr Asp Gly Pro
Ser Asp Ser Phe 325 330 335Lys Ala Leu Ile Asp Met Ala Ala Val His
Ser Ser Cys Arg Leu Cys 340 345 350Ile His Ile Ala Thr Lys Ile His
Glu Lys Glu Glu Lys Thr Pro Lys 355 360 365Phe Met Asn Arg Ser Cys
Ser Cys Ser Ser Lys Arg Gly Thr Val Tyr 370 375 380His Leu Phe Val
Arg Glu Arg Gly Arg Phe Lys Thr Glu Asn Ile Tyr385 390 395 400Leu
Arg Ser Asp Gln Leu Thr Leu Gly Ala Leu Lys Ser Ala Val His 405 410
415Asp Lys Phe Ser Ser Leu Lys His Val Pro Ile Trp Lys Asp Glu Arg
420 425 430Pro Ser Ser Ile Arg Gly Gly Asp Glu Leu Lys Val Tyr Lys
Ile Tyr 435 440 445Pro Ile Gly Leu Thr Glu Arg Gln Ala Leu Tyr Lys
Phe Gln Phe Ser 450 455 460Asp Asp Ala Glu Val Ala Arg Tyr Ile Lys
Gly His Pro Cys Ala Lys465 470 475 480Leu Glu Val Ile Phe Val
48532513PRTOryza sativa 32Met Lys Gly Ser His Ser Gln Ser Gln Ala
Gln Ala Gln Ser Gln Ala1 5 10 15Gly Arg Arg Arg Arg Cys Gly Trp Leu
Leu Pro Leu Leu Val Gly Ala 20 25 30Ala Phe Leu Ala Glu Ile Ala Phe
Leu Gly Arg Leu Asp Met Ala Lys 35 40 45Asn Ala Ala Ala Val Glu Ser
Trp Thr Thr Ser Phe Tyr Ala Arg Ser 50 55 60Ser Ala Pro Ala Arg Asp
Gly Lys Ala Ala Val Val Val Pro Gly Ala65 70 75 80Asp Ala Asp Asp
Ala Pro Pro Gly Gly Gly Glu Val Val Glu Glu Asp 85 90 95Asp Gly Asp
Ile Arg Leu Cys Glu Glu Arg Leu Glu Arg Glu Asp Gly 100 105 110Val
Pro His Asp Arg Asp Phe Asp Lys Asp Pro Val Leu Val Gly Gly 115 120
125Ala Ala Lys Asp Trp Asn Lys Cys Ser Val Gly Cys Glu Phe Gly Phe
130 135 140Ser Ala Thr Lys Thr Pro Asp Ala Thr Phe Gly Ile Ala Pro
Asp Pro145 150 155 160Thr Val Glu Ser Ile Leu Arg Ser Met Glu Ser
Ser Gln Tyr Tyr Ser 165 170 175Glu Asn Asn Ile Ala Val Ala Arg Gly
Arg Gly Tyr Lys Ile Val Met 180 185 190Thr Thr Ser Leu Ser Ser Asp
Val Pro Val Gly Tyr Phe Ser Trp Ala 195 200 205Glu Tyr Asp Ile Met
Ala Pro Val Pro Pro Lys Thr Glu Glu Ala Leu 210 215 220Ala Ala Ala
Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu Gln225 230 235
240Ala Leu Glu Met Leu Glu Ser Leu Asp Val Lys Ile Asp Ser Tyr Gly
245 250 255Ser Cys His Arg Asn His Asp Gly Lys Val Asp Lys Val Glu
Thr Leu 260 265 270Lys Arg Tyr Lys Phe Ser Leu Ala Phe Glu Asn Ser
Asn Glu Glu Asp 275 280 285Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu
Val Thr Gly Ala Ile Pro 290 295 300Val Val Ile Gly Ala Pro Asn Ile
Gln Glu Phe Ser Pro Gly Glu Gly305 310 315 320Ala Ile Leu His Ile
Lys Glu Leu Asp Asp Val Pro Ser Ile Ala Lys 325 330 335Thr Met Lys
His Ile Ala Ser Asn Gln Glu Ala Phe Asn Gln Ser Leu 340 345 350Arg
Trp Lys Tyr Asp Gly Pro Ser Asp Ser Phe Lys Ala Leu Ile Asp 355 360
365Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile His Val Ala Thr
370 375 380Lys Ile His Glu Lys
Glu Glu Arg Thr Pro Lys Phe Met Asn Arg Pro385 390 395 400Cys Ser
Cys Ser Ser Lys Arg Gly Lys Val Tyr His Leu Phe Val Arg 405 410
415Glu Arg Gly Arg Phe Lys Thr Glu Ser Ile Phe Leu Arg Ser Asp Gln
420 425 430Leu Thr Met Gly Ala Leu Glu Ser Ala Val Leu Ala Lys Phe
Arg Ser 435 440 445Leu Asn His Val Pro Val Trp Lys Asp Glu Arg Pro
Pro Ser Ile Arg 450 455 460Gly Gly Asp Glu Leu Lys Val Tyr Lys Ile
Tyr Pro Ile Gly Leu Thr465 470 475 480Gln Arg Gln Ala Leu Tyr Gln
Phe Arg Phe Arg Asp Asp Ala Asp Leu 485 490 495Asp Lys Tyr Ile Lys
Asp His Pro Cys Ala Lys Leu Glu Val Ile Phe 500 505
510Val33506PRTMedicago sativa 33Met Gly Leu Val Ser Arg Thr Thr Thr
Thr Thr Thr Gln Glu Gly Leu1 5 10 15Pro Val Ser Val Ser Val Ser Thr
Thr Val Pro Lys Lys Lys Trp Ser 20 25 30Asn Leu Met Pro Leu Phe Val
Ala Leu Val Val Ile Ala Glu Ile Ala 35 40 45Phe Leu Gly Arg Leu Asp
Met Ala Lys Asn Ala Ala Met Val Ala Asp 50 55 60Leu Phe Tyr Arg Ser
Arg Ala Val Val Glu Gly Asp Asp Phe Gly Leu65 70 75 80Glu Thr Val
Gly Gly Asp Lys Asn Leu Glu Leu Glu Arg Glu Thr Cys 85 90 95Glu Glu
Trp Leu Glu Arg Glu Asp Ala Val Thr Tyr Ser Arg Asn Phe 100 105
110Asn Lys Glu Pro Val Phe Val Ser Gly Ala Glu Gln Glu Trp Lys Ser
115 120 125Cys Ser Val Gly Cys Lys Phe Gly Phe Asn Gly Asp Arg Lys
Pro Glu 130 135 140Ala Ala Phe Gly Leu Pro Gln Gln Ala Gly Thr Ala
Ser Val Leu Arg145 150 155 160Ser Met Glu Ser Ala Gln Tyr Tyr Ala
Glu Asn Asn Leu Ala Met Ala 165 170 175Arg Arg Arg Gly Tyr His Ile
Val Met Thr Thr Ser Leu Ser Ser Asp 180 185 190Val Pro Val Gly Tyr
Phe Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro 195 200 205Ile Lys Pro
Lys Thr Glu Lys Ala Leu Ala Ala Ala Phe Ile Ser Asn 210 215 220Cys
Gly Ala Arg Asn Phe Arg Leu Gln Ala Leu Glu Ala Leu Glu Lys225 230
235 240Thr Asn Ile Ser Ile Asp Ser Tyr Gly Ser Cys His Arg Asn Arg
Asp 245 250 255Gly Arg Val Asp Lys Leu Glu Thr Leu Thr Arg Tyr Lys
Phe Ser Leu 260 265 270Ala Phe Glu Asn Ser Asn Glu Glu Asp Tyr Val
Thr Glu Lys Phe Phe 275 280 285Gln Ser Leu Val Ala Gly Thr Ile Pro
Val Val Val Gly Pro Pro Asn 290 295 300Ile Gln Asp Phe Ala Pro Ser
Pro Asp Ser Phe Leu Tyr Ile Lys Glu305 310 315 320Leu Glu Asp Val
Glu Ser Val Ala Lys Ser Met Arg Tyr Leu Ala Glu 325 330 335Asn Pro
Glu Ala Tyr Asn His Ser Leu Arg Trp Lys Tyr Glu Gly Pro 340 345
350Ser Asp Ser Phe Lys Ala Leu Val Asp Met Ala Ala Val His Ser Ser
355 360 365Cys Arg Leu Cys Ile His Leu Ala Thr Lys Ser Arg Glu Lys
Glu Glu 370 375 380Lys Ser Pro Asp Phe Lys Lys Arg Pro Cys Lys Cys
Thr Arg Gly Ser385 390 395 400Glu Thr Val Tyr His Ile Tyr Val Arg
Glu Arg Gly Thr Phe Glu Met 405 410 415Glu Ser Ile Tyr Leu Arg Ser
Ser Asn Leu Thr Leu Glu Ser Phe Lys 420 425 430Thr Ala Val Leu Thr
Lys Phe Thr Ser Leu Asn His Val Pro Val Trp 435 440 445Lys Pro Glu
Arg Pro Glu Ile Leu Lys Gly Gly Asp Lys Leu Lys Val 450 455 460Tyr
Lys Ile Ile Pro Ala Gly Leu Thr Gln Arg Gln Ala Leu Tyr Thr465 470
475 480Phe Gln Phe Asn Gly Asp Val Asp Phe Arg Ser His Leu Glu Ser
Asn 485 490 495Pro Cys Ala Lys Phe Glu Val Ile Phe Val 500
50534513PRTArabidopsis thaliana 34Met Gly Val Phe Ser Asn Leu Arg
Gly Pro Arg Ala Gly Ala Thr His1 5 10 15Asp Glu Phe Pro Ala Thr Asn
Gly Ser Pro Ser Ser Ser Ser Ser Pro 20 25 30Ser Ser Ser Ile Lys Arg
Lys Leu Ser Asn Leu Leu Pro Leu Cys Val 35 40 45Ala Leu Val Val Ile
Ala Glu Ile Gly Phe Leu Gly Arg Leu Asp Lys 50 55 60Val Ala Leu Val
Asp Thr Leu Thr Asp Phe Phe Thr Gln Ser Pro Ser65 70 75 80Leu Ser
Gln Ser Pro Pro Ala Arg Ser Asp Arg Lys Lys Ile Gly Leu 85 90 95Phe
Thr Asp Arg Ser Cys Glu Glu Trp Leu Met Arg Glu Asp Ser Val 100 105
110Thr Tyr Ser Arg Asp Phe Thr Lys Asp Pro Ile Phe Ile Ser Gly Gly
115 120 125Glu Lys Asp Phe Gln Trp Cys Ser Val Asp Cys Thr Phe Gly
Asp Ser 130 135 140Ser Gly Lys Thr Pro Asp Ala Ala Phe Gly Leu Gly
Gln Lys Pro Gly145 150 155 160Thr Leu Ser Ile Ile Arg Ser Met Glu
Ser Ala Gln Tyr Tyr Pro Glu 165 170 175Asn Asp Leu Ala Gln Ala Arg
Arg Arg Gly Tyr Asp Ile Val Met Thr 180 185 190Thr Ser Leu Ser Ser
Asp Val Pro Val Gly Tyr Phe Ser Trp Ala Glu 195 200 205Tyr Asp Ile
Met Ser Pro Val Gln Pro Lys Thr Glu Arg Ala Ile Ala 210 215 220Ala
Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu Gln Ala225 230
235 240Leu Glu Ala Leu Met Lys Thr Asn Ile Lys Ile Asp Ser Tyr Gly
Gly 245 250 255Cys His Arg Asn Arg Asp Gly Lys Val Asp Lys Val Glu
Ala Leu Lys 260 265 270Arg Tyr Lys Phe Ser Leu Ala Phe Glu Asn Thr
Asn Glu Glu Asp Tyr 275 280 285Val Thr Glu Lys Phe Phe Gln Ser Leu
Val Ala Gly Ser Val Pro Val 290 295 300Val Val Gly Pro Pro Asn Ile
Glu Glu Phe Ala Pro Ala Ser Asp Thr305 310 315 320Phe Leu His Ile
Lys Thr Met Glu Asp Val Glu Pro Val Ala Lys Arg 325 330 335Met Lys
Tyr Leu Ala Ala Asn Pro Ala Ala Tyr Asn Gln Thr Leu Arg 340 345
350Trp Lys Tyr Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val Asp Met
355 360 365Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala
Thr Arg 370 375 380Val Arg Glu Gln Glu Glu Glu Ser Pro Asn Phe Lys
Lys Arg Pro Cys385 390 395 400Lys Cys Ser Arg Gly Gly Ser Asp Thr
Val Tyr His Val Phe Val Arg 405 410 415Glu Arg Gly Arg Phe Glu Met
Glu Ser Val Phe Leu Arg Gly Lys Ser 420 425 430Val Thr Gln Glu Ala
Leu Glu Ser Ala Val Leu Ala Lys Phe Lys Ser 435 440 445Leu Lys His
Glu Ala Val Trp Lys Lys Glu Arg Pro Gly Asn Leu Lys 450 455 460Gly
Asp Lys Glu Leu Lys Ile His Arg Ile Tyr Pro Leu Gly Leu Thr465 470
475 480Gln Arg Gln Ala Leu Tyr Asn Phe Lys Phe Glu Gly Asn Ser Ser
Leu 485 490 495Ser Ser His Ile Gln Asn Asn Pro Cys Ala Lys Phe Glu
Val Val Phe 500 505 510Val35475PRTArtificial SequenceConsensus
Sequence 35Gly Ser His Ser Ser Ser Ala Ala Ala Ser Arg Arg Arg Arg
Cys Gly1 5 10 15Trp Leu Leu Pro Leu Leu Val Gly Leu Ala Val Ile Ala
Glu Ile Ala 20 25 30Phe Leu Gly Arg Leu Asp Met Ala Lys Asn Ala Ala
Ala Val Glu Ser 35 40 45Trp Thr Thr Ser Phe Tyr Ala Leu Ser Ser Gly
Ala Asp Ala Pro Pro 50 55 60Gly Ser Gly Asp Asp Glu Glu Cys Glu Glu
Trp Leu Glu Arg Glu Asp65 70 75 80Ala Val Pro Tyr Asp Arg Asp Phe
Glu Lys Asp Pro Val Leu Val Gly 85 90 95Gly Ala Glu Lys Asp Trp Asn
Arg Cys Ser Val Gly Cys Glu Phe Gly 100 105 110Phe Ala Ser Lys Thr
Pro Asp Ala Thr Phe Gly Ile Ala Gln Asp Pro 115 120 125Ser Val Glu
Ser Ile Leu Arg Ser Met Glu Ser Ser Gln Tyr Tyr Ser 130 135 140Glu
Asn Asn Ile Ala Ala Arg Arg Arg Gly Tyr Gln Ile Val Met Thr145 150
155 160Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp Ala
Glu 165 170 175Tyr Asp Ile Met Ala Pro Val Pro Pro Lys Thr Glu Glu
Ala Leu Ala 180 185 190Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn
Phe Arg Leu Gln Ala 195 200 205Leu Glu Met Leu Glu Ser Leu Asn Ile
Lys Ile Asp Ser Tyr Gly Ser 210 215 220Cys His Arg Asn Arg Asp Gly
Lys Val Asp Lys Val Glu Thr Leu Lys225 230 235 240Arg Tyr Lys Phe
Ser Leu Ala Phe Glu Asn Ser Asn Glu Glu Asp Tyr 245 250 255Val Thr
Glu Lys Phe Phe Gln Ser Leu Val Thr Gly Ala Ile Pro Val 260 265
270Val Val Gly Ala Pro Asn Ile Gln Glu Phe Ala Pro Gly Glu Asp Ala
275 280 285Ile Leu His Ile Lys Glu Leu Asp Asp Val Ser Val Ala Lys
Thr Met 290 295 300Lys His Ile Ala Ser Asn Pro Glu Ala Phe Asn Gln
Ser Leu Arg Trp305 310 315 320Lys Tyr Asp Gly Pro Ser Asp Ser Phe
Lys Ala Leu Ile Asp Met Ala 325 330 335Ala Val His Ser Ser Cys Arg
Leu Cys Ile His Ile Ala Thr Lys Ile 340 345 350Arg Glu Lys Glu Glu
Arg Thr Pro Lys Phe Met Asn Arg Pro Cys Ser 355 360 365Cys Ser Ser
Lys Arg Gly Thr Val Tyr His Leu Phe Val Arg Glu Arg 370 375 380Gly
Arg Phe Lys Thr Glu Ser Ile Phe Leu Arg Ser Asp Gln Leu Thr385 390
395 400Leu Gly Ala Leu Glu Ser Ala Val Leu Ala Lys Phe Ser Ser Leu
Lys 405 410 415His Val Pro Val Trp Lys Asp Glu Arg Pro Ser Ile Lys
Gly Gly Asp 420 425 430Glu Leu Lys Val Tyr Lys Ile Tyr Pro Ile Gly
Leu Thr Gln Arg Gln 435 440 445Ala Leu Tyr Phe Gln Phe Asp Asp Ala
Glu Leu Ala Arg Tyr Ile Lys 450 455 460His Pro Cys Ala Lys Leu Glu
Val Ile Phe Val465 470 47536511PRTMedicago sativa 36Met Asn Lys Lys
Val Gln Ile Leu Leu Phe Leu Phe Ile Phe Asn Ser1 5 10 15Phe Ser Leu
Cys Leu Tyr Phe Ile His His Pro Ser Phe Leu Asn Pro 20 25 30Thr Pro
Ser Pro Asn Pro Asp Ile Lys Ile Phe Thr Lys Asp Ser Pro 35 40 45Phe
Arg Arg Pro Phe Ile Glu Lys His Lys Thr His Ile Leu Lys Pro 50 55
60Trp Pro Ile Leu Pro Ser Tyr Ser Pro Trp Ser Gln Pro Ser Asn Ser65
70 75 80Thr Pro Leu Arg Ser Cys Glu Gly Tyr Phe Gly Asn Gly Phe Thr
Arg 85 90 95Leu Val Asp Val Phe Ser Ser Arg Ala Glu Gly Arg Gly Gly
Gly Trp 100 105 110Phe Arg Cys Trp Tyr Ser Glu Thr Leu Arg Ser Ser
Val Cys Glu Gly 115 120 125Gly Arg Val Arg Met Val Val Glu Arg Ile
Lys Met Ala Lys Gly Gly 130 135 140Glu Ser Leu Gly Asp Val Ile Gly
Arg Ser Glu Asp Glu Glu Leu Pro145 150 155 160Leu Phe Glu Asp Gly
Ala Phe Glu Val Asp Gly Gly Val Gly Phe Lys 165 170 175Gly Gly Lys
Met Val Val Asp Glu Gly Phe Leu Asp Arg Tyr Val Pro 180 185 190Arg
Gly Glu Ile Met Arg His Thr Met Arg Asp Leu Ile Gly Lys Met 195 200
205Lys Val Val Gly Arg Lys Glu Phe Lys Cys Asp Lys Trp Ile Glu Glu
210 215 220Pro Thr Leu Leu Val Thr Arg Phe Glu Tyr Ala Asn Leu Phe
His Thr225 230 235 240Val Thr Asp Trp Tyr Ser Ala Tyr Val Ser Ser
Arg Val Thr Gly Leu 245 250 255Pro Ser Arg Pro His Leu Ile Phe Val
Asp Gly His Cys Lys Ala Pro 260 265 270Leu Glu Glu Thr Trp Lys Ala
Leu Phe Ser Ser Val Arg Tyr Ala Lys 275 280 285Asn Phe Thr Gly Thr
Val Cys Phe Arg His Ala Ile Leu Ser Pro Leu 290 295 300Gly Tyr Glu
Thr Ala Leu Phe Lys Gly Leu Thr Glu Asp Ile Phe Cys305 310 315
320Asp Gly Ala Ser Ala Gln Glu Leu Trp Arg Lys Pro Asp Asn Glu Lys
325 330 335Thr Ala Arg Ile Ser Glu Phe Gly Glu Met Ile Arg Ala Ser
Phe Gly 340 345 350Leu Pro Leu Asn Leu Asn His Val Gly Lys Pro Ile
Phe Gly Gly His 355 360 365Asn Ala Leu Phe Val Arg Arg Glu Asp Tyr
Leu Ala His Pro Arg His 370 375 380Gly Gly Lys Val Glu Ser Arg Leu
Thr Asn Glu Ala Glu Val Phe Glu385 390 395 400Ser Leu Lys Ser Trp
Ala Ala Asn Tyr Lys Gly Cys Lys Ile Asn Leu 405 410 415Val Asn Gly
Leu Phe Ala His Met Ser Met Lys Glu Gln Val Arg Ala 420 425 430Ile
Gln Asp Ala Ser Val Ile Ile Gly Ala His Gly Ala Gly Leu Thr 435 440
445His Ile Val Ser Ala Leu Pro Lys Thr Val Ile Leu Glu Ile Ile Ser
450 455 460Ser Gln Phe Arg Arg Pro His Phe Ala Tyr Ile Ala Arg Trp
Lys Gly465 470 475 480Leu Glu Tyr His Ala Ile Asn Leu Asp Gly Ser
Tyr Ala Asn Pro Glu 485 490 495Thr Val Ile Asn Ala Leu Val Ser Ile
Met Lys Ser Leu Arg Cys 500 505 51037533PRTOryza sativa 37Met Met
Pro Val Arg Thr Tyr His His His His His His Asn Asn Ser1 5 10 15Asn
Asn His Arg Leu Arg Arg Ile Ile Pro Arg Val Leu Leu Ala Val 20 25
30Phe Ala Ile Tyr Ala Val Ser Phe Ala Ala Tyr Leu Leu Arg His Gln
35 40 45Ser Pro His Pro His Pro His Pro Ala Ala Asp Pro Glu Arg Asp
Ala 50 55 60Val Asp Ala Ala Gly Gly Gly Gly Gly Gly Gly Ala Val Asp
Arg Val65 70 75 80Arg Val Glu Ala Pro Ser Ser Gln Lys Pro Trp Pro
Arg Leu Pro Ser 85 90 95Phe Leu Pro Trp Thr Ser Ala Ser Val Arg Pro
Pro Pro Lys His Ser 100 105 110Cys Glu Gly Tyr Phe Gly Asn Gly Phe
Ser Arg Leu Val Asp Val Leu 115 120 125Pro Ala Arg Gly Gly Gly Gly
Gly Gly Trp Phe Arg Cys His His Ser 130 135 140Glu Thr Leu Arg Ser
Ser Ile Cys Glu Gly Gly Arg Val Arg Leu Asp145 150 155 160Pro Gly
Leu Ile Ala Met Ser Arg Gly Gly Glu Pro Leu Asp Gln Val 165 170
175Met Gly Arg Ala Glu Glu Glu Glu Leu Pro Lys Tyr Glu Pro Gly Ala
180 185 190Leu Gln Val Glu Ala Ala Ala Lys Arg Thr Gly Pro Leu Val
Glu Ala 195 200 205Gly Phe Leu Asp Ala Tyr Val Pro Thr Gly Gly Ile
Gly Met His Thr 210 215 220Met Arg Ser Leu Leu Asp Ser Gly Arg Val
Val Pro Pro Gly Glu Leu225 230 235 240His Cys Ser Gln Trp Val Glu
Glu Pro Thr Leu Leu Val Thr Arg Phe 245 250 255Glu Tyr Ala Asn Leu
Phe His Thr Ile Thr Asp Trp Tyr Ser Ala Tyr 260 265 270Val Ser Ser
Arg Val Thr Asp Leu Pro Asn Arg Pro Asn Val Val Phe 275 280 285Val
Asp Gly His Cys Lys Ala Gln Leu Glu Gln Thr Trp Glu Ala Leu 290 295
300Phe Ser Asn Val Thr Tyr Val Lys Asn Phe Ser Gly Pro Val Cys
Phe305 310 315
320Arg His Ala Ile Leu Ser Pro Leu Gly Tyr Glu Thr Ala Leu Phe Lys
325 330 335Gly Leu Ser Glu Ser Phe Ser Cys Glu Gly Ala Ser Ala Glu
Ser Leu 340 345 350Arg Glu Lys Pro Asp His Gln Lys Thr Ala Arg Leu
Ser Glu Phe Gly 355 360 365Glu Met Ile Leu Ala Ser Phe Asp Leu Leu
Arg Asp Asp Ile Leu Ser 370 375 380Ser Lys Thr Ser Asn Gly Leu Asn
Val Leu Phe Val Arg Arg Glu Asp385 390 395 400Tyr Leu Ala His Pro
Arg His Ser Gly Lys Val Glu Ser Arg Leu Ser 405 410 415Asn Glu Lys
Glu Val Tyr Asp Ala Ile Glu Gly Trp Ala Lys Gly Gln 420 425 430Lys
Cys Lys Ile Asn Val Ile Asn Gly Leu Phe Ala His Met Asn Met 435 440
445Lys Glu Gln Leu Arg Ala Ile Gln Glu Ala Ser Val Val Ile Gly Ala
450 455 460His Gly Ala Gly Leu Thr His Leu Val Ser Ala Thr Pro Asp
Thr Lys465 470 475 480Val Leu Glu Ile Ile Ser Ser Met Tyr Arg Arg
Pro His Phe Ala Leu 485 490 495Ile Ser His Trp Lys Ser Leu Glu Tyr
His Ala Ile Asn Leu Pro Gly 500 505 510Ser Tyr Ala Arg Val Thr Asp
Val Ile Asn Glu Leu Ser Asn Ile Leu 515 520 525Lys Gly Phe Gly Cys
53038534PRTArabidopsis thaliana 38Met Ser Lys Arg Asn Pro Lys Ile
Leu Lys Ile Phe Leu Tyr Met Leu1 5 10 15Leu Leu Asn Ser Leu Phe Leu
Ile Ile Tyr Phe Val Phe His Ser Ser 20 25 30Ser Phe Ser Pro Glu Gln
Ser Gln Pro Pro His Ile Tyr His Val Ser 35 40 45Val Asn Asn Gln Ser
Ala Ile Gln Lys Pro Trp Pro Ile Leu Pro Ser 50 55 60Tyr Leu Pro Trp
Thr Pro Pro Gln Arg Asn Leu Pro Thr Gly Ser Cys65 70 75 80Glu Gly
Tyr Phe Gly Asn Gly Phe Thr Lys Arg Val Asp Phe Leu Lys 85 90 95Pro
Arg Ile Gly Gly Gly Gly Glu Gly Ser Trp Phe Arg Cys Phe Tyr 100 105
110Ser Glu Thr Leu Gln Ser Ser Ile Cys Glu Gly Arg Asn Leu Arg Met
115 120 125Val Pro Asp Arg Ile Val Met Ser Arg Gly Gly Glu Lys Leu
Glu Glu 130 135 140Val Met Gly Arg Lys Glu Glu Glu Glu Leu Pro Ala
Phe Arg Gln Gly145 150 155 160Ala Phe Glu Val Ala Glu Glu Val Ser
Ser Arg Leu Gly Phe Lys Arg 165 170 175His Arg Arg Phe Gly Gly Gly
Glu Gly Gly Ser Ala Val Ser Arg Arg 180 185 190Leu Val Asn Asp Glu
Met Leu Asn Glu Tyr Met Gln Glu Gly Gly Ile 195 200 205Asp Arg His
Thr Met Arg Asp Leu Val Ala Ser Ile Arg Ala Val Asp 210 215 220Thr
Asn Asp Phe Val Cys Glu Glu Trp Val Glu Glu Pro Thr Leu Leu225 230
235 240Val Thr Arg Phe Glu Tyr Ala Asn Leu Phe His Thr Val Thr Asp
Trp 245 250 255Tyr Ser Ala Tyr Val Ser Ser Arg Val Thr Gly Leu Pro
Asn Arg Pro 260 265 270His Val Val Phe Val Asp Gly His Cys Thr Thr
Gln Leu Glu Glu Thr 275 280 285Trp Thr Ala Leu Phe Ser Gly Ile Arg
Tyr Ala Lys Asn Phe Thr Lys 290 295 300Pro Val Cys Phe Arg His Ala
Ile Leu Ser Pro Leu Gly Tyr Glu Thr305 310 315 320Ala Leu Phe Lys
Gly Leu Ser Gly Glu Ile Asp Cys Lys Gly Asp Ser 325 330 335Ala His
Asn Leu Trp Gln Asn Pro Asp Asp Lys Arg Thr Ala Arg Ile 340 345
350Ser Glu Phe Gly Glu Met Ile Arg Ala Ala Phe Gly Leu Pro Val Asn
355 360 365Arg His Arg Ser Leu Glu Lys Pro Leu Ser Ser Ser Ser Ser
Ser Ala 370 375 380Ser Val Tyr Asn Val Leu Phe Val Arg Arg Glu Asp
Tyr Leu Ala His385 390 395 400Pro Arg His Gly Gly Lys Val Gln Ser
Arg Leu Ile Asn Glu Glu Glu 405 410 415Val Phe Asp Ser Leu His His
Trp Val Ala Thr Gly Ser Thr Gly Leu 420 425 430Thr Lys Cys Gly Ile
Asn Leu Val Asn Gly Leu Leu Ala His Met Ser 435 440 445Met Lys Asp
Gln Val Arg Ala Ile Gln Asp Ala Ser Val Ile Ile Gly 450 455 460Ala
His Gly Ala Gly Leu Thr His Ile Val Ser Ala Thr Pro Asn Thr465 470
475 480Thr Ile Phe Glu Ile Ile Ser Val Glu Phe Gln Arg Pro His Phe
Glu 485 490 495Leu Ile Ala Lys Trp Lys Gly Leu Glu Tyr His Ala Met
His Leu Ala 500 505 510Asn Ser Arg Ala Glu Pro Thr Ala Val Ile Glu
Lys Leu Thr Glu Ile 515 520 525Met Lys Ser Leu Gly Cys
53039515PRTSaccharum officinarum 39Met Met Ala Gly Arg Ala His Gly
His Arg Asn Arg Leu Arg Arg Leu1 5 10 15Ile Pro Arg Leu Leu Leu Leu
Val Phe Ala Val Tyr Ala Ala Ser Phe 20 25 30Ala Ile Tyr Leu Leu Leu
Gln Ser Pro Gln Ser Glu Ser His His Gln 35 40 45Ser Pro Pro Asp Pro
Thr Pro Arg Thr Glu Ala Arg Asp Gly Val Gly 50 55 60Ala Pro Pro Tyr
Ser His Lys Pro Trp Pro Arg Leu Pro Ser Cys Leu65 70 75 80Pro Trp
Val Tyr Ser Ala Ala Pro Pro His Thr Cys Glu Ala Tyr Phe 85 90 95Gly
Asn Gly Phe Ser Arg Arg Val Asp Val Leu Pro Ala Gly Arg Gly 100 105
110Gly Gly Gly Gly Trp Phe Arg Cys His His Ser Glu Thr Leu Gly Ser
115 120 125Ser Ile Cys Glu Gly Ala Arg Val Arg Leu Asp Pro Ala Leu
Ile Ala 130 135 140Met Ser Arg Gly Gly Glu Pro Leu Glu Gln Val Met
Gly Arg Ala Glu145 150 155 160Glu Glu Glu Leu Pro Lys Tyr Glu Pro
Ser Ala Leu Gln Val Glu Gly 165 170 175Pro Ala Ala Gly Lys Thr Ala
Pro Leu Val Asp Ala Gly Phe Leu Asn 180 185 190Asp Tyr Val Pro Asn
Gly Gly Ile Arg Met His Thr Met Arg Ala Leu 195 200 205Leu Asp Ser
Ala Arg Val Val Pro Pro Gly Glu Leu His Cys Ser Gln 210 215 220Trp
Val Glu Glu Pro Thr Leu Leu Val Thr Arg Phe Glu Tyr Ala Asn225 230
235 240Leu Phe His Thr Ile Thr Asp Trp Tyr Ser Ala Tyr Val Ser Ser
Arg 245 250 255Val Thr Asn Leu Pro Asp Arg Pro Asn Val Val Phe Val
Asp Gly His 260 265 270Cys Lys Ala Pro Leu Glu Gln Thr Trp Glu Ala
Leu Phe Ser Asn Val 275 280 285Thr Tyr Ile Lys Ser Phe Ser Gly Pro
Val Cys Phe Arg Arg Ala Val 290 295 300Leu Ser Pro Leu Gly Tyr Glu
Thr Ala Leu Phe Lys Gly Leu Ser Glu305 310 315 320Ser Phe Ser Cys
Glu Gly Ala Ser Ala Gln Ser Leu Arg Asn Arg Pro 325 330 335Asp His
Gln Lys Thr Ala Arg Leu Ser Glu Phe Gly Glu Met Ile Ile 340 345
350Ala Ser Phe Asp Leu Leu Glu Asp Gly Ile Val Pro Ser Lys Lys Thr
355 360 365Ser Thr Gly Leu Lys Val Leu Phe Val Arg Arg Glu Asp Tyr
Leu Ala 370 375 380His Pro Arg His Ser Gly Lys Val Glu Ser Arg Leu
Ser Asn Glu Gln385 390 395 400Glu Val Phe Gly Ala Val Glu Lys Trp
Ala Lys Lys Gly Gln Lys Cys 405 410 415Lys Ile Asn Val Val Asn Gly
Leu Phe Ala His Met Ser Met Lys Glu 420 425 430Gln Leu Gln Ala Ile
Leu Glu Ala Ser Val Ile Ile Gly Ala His Gly 435 440 445Ala Gly Leu
Thr His Leu Val Ser Ala Thr Pro Asp Thr Lys Val Leu 450 455 460Glu
Ile Ile Ser Ser Phe Tyr Arg Arg Pro His Phe Ala Leu Ile Ser465 470
475 480His Trp Lys Ala Leu Glu Tyr His Ala Ile Asn Leu Pro Gly Ser
Tyr 485 490 495Ala Ser Ile Pro Asp Val Thr Ser Glu Leu Ser Asn Ile
Leu Lys Gly 500 505 510Leu Gly Cys 51540523PRTNicotiana tabacum
40Met Asn Lys Lys Lys Leu Lys Phe Leu Val Ser Leu Phe Ala Leu Asn1
5 10 15Ser Ile Thr Leu Tyr Leu Tyr Phe Ser Ser His Ser Asp His Phe
Arg 20 25 30His Lys Ser Pro Gln Asn His Phe Pro Asn Thr Gln Asn His
Tyr Ser 35 40 45Leu Ser Glu Asn His His Asp Asn Phe His Ser Ser Val
Thr Ser Gln 50 55 60Tyr Thr Lys Pro Trp Pro Ile Leu Pro Ser Tyr Leu
Pro Trp Ser Gln65 70 75 80Asn Pro Asn Val Ser Leu Arg Ser Cys Glu
Gly Tyr Phe Gly Asn Gly 85 90 95Phe Thr Leu Lys Val Asp Leu Leu Lys
Thr Ser Pro Glu Leu His Gln 100 105 110Lys Phe Gly Glu Asn Thr Val
Ser Gly Asp Gly Gly Trp Phe Arg Cys 115 120 125Phe Phe Ser Glu Thr
Leu Gln Ser Ser Ile Cys Glu Gly Gly Ala Ile 130 135 140Arg Met Asn
Pro Asp Glu Ile Leu Met Ser Arg Gly Gly Glu Lys Leu145 150 155
160Glu Ser Val Ile Gly Arg Ser Glu Asp Asp Glu Leu Pro Val Phe Lys
165 170 175Asn Gly Ala Phe Gln Ile Lys Val Thr Asp Lys Leu Lys Ile
Gly Lys 180 185 190Lys Leu Val Asp Glu Lys Ile Leu Asn Lys Tyr Leu
Pro Glu Gly Ala 195 200 205Ile Ser Arg His Thr Met Arg Glu Leu Ile
Asp Ser Ile Gln Leu Val 210 215 220Gly Ala Asp Glu Phe His Cys Ser
Glu Trp Ile Glu Glu Pro Ser Leu225 230 235 240Leu Ile Thr Arg Phe
Glu Tyr Ala Asn Leu Phe His Thr Val Thr Asp 245 250 255Trp Tyr Ser
Ala Tyr Val Ala Ser Arg Val Thr Gly Leu Pro Ser Arg 260 265 270Pro
His Leu Val Phe Val Asp Gly His Cys Glu Thr Gln Leu Glu Glu 275 280
285Thr Trp Lys Ala Leu Phe Ser Ser Leu Thr Tyr Ala Lys Asn Phe Ser
290 295 300Gly Pro Val Cys Phe Arg His Ala Val Leu Ser Pro Leu Gly
Tyr Glu305 310 315 320Thr Ala Leu Phe Lys Gly Leu Thr Glu Thr Ile
Asp Cys Asn Gly Ala 325 330 335Ser Ala His Asp Leu Trp Gln Asn Pro
Asp Asp Lys Arg Thr Ala Arg 340 345 350Leu Ser Glu Phe Gly Glu Met
Ile Arg Ala Ala Phe Gly Phe Pro Val 355 360 365Asp Arg Gln Asn Ile
Pro Arg Thr Val Thr Gly Pro Asn Val Leu Phe 370 375 380Val Arg Arg
Glu Asp Tyr Leu Ala His Pro Arg His Gly Gly Lys Val385 390 395
400Gln Ser Arg Leu Ser Asn Glu Glu Gln Val Phe Asp Ser Ile Lys Ser
405 410 415Trp Ala Leu Asn His Ser Glu Cys Lys Leu Asn Val Ile Asn
Gly Leu 420 425 430Phe Ala His Met Ser Met Lys Glu Gln Val Arg Ala
Ile Gln Asp Ala 435 440 445Ser Val Ile Val Gly Ala His Gly Ala Gly
Leu Thr His Ile Val Ser 450 455 460Ala Ala Pro Lys Ala Val Ile Leu
Glu Ile Ile Ser Ser Glu Tyr Arg465 470 475 480Arg Pro His Phe Ala
Leu Ile Ala Gln Trp Lys Gly Leu Glu Tyr His 485 490 495Pro Ile Tyr
Leu Glu Gly Ser Tyr Ala Asp Pro Pro Val Val Ile Asp 500 505 510Arg
Leu Ser Ser Ile Leu Arg Ser Leu Gly Cys 515 52041443PRTArtificial
SequenceConsensus Sequence 41Arg Leu Arg Lys Ile Leu Lys Ile Leu
Leu Leu Phe Ala Ile Asn Ser1 5 10 15Val Ser Leu Ile Tyr Phe Leu His
Ser Ser Pro Ser Ser Lys Pro Trp 20 25 30Pro Ile Leu Pro Ser Tyr Leu
Pro Trp Ser Ser Ser Pro Ser Cys Glu 35 40 45Gly Tyr Phe Gly Asn Gly
Phe Thr Arg Arg Val Asp Val Leu Lys Ala 50 55 60Gly Gly Gly Gly Gly
Trp Phe Arg Cys Phe Tyr Ser Glu Thr Leu Arg65 70 75 80Ser Ser Ile
Cys Glu Gly Gly Arg Val Arg Met Asp Pro Asp Arg Ile 85 90 95Met Ser
Arg Gly Gly Glu Leu Glu Glu Val Met Gly Arg Ala Glu Glu 100 105
110Glu Glu Leu Pro Lys Phe Glu Gly Ala Phe Gln Val Glu Gly Ala Lys
115 120 125Gly Lys Arg Leu Val Asp Asp Gly Phe Leu Asn Tyr Val Pro
Gly Gly 130 135 140Ile Arg His Thr Met Arg Asp Leu Ile Asp Ser Ile
Arg Val Val Gly145 150 155 160Pro Gly Glu Leu His Cys Ser Gln Trp
Ile Glu Glu Pro Thr Leu Leu 165 170 175Val Thr Arg Phe Glu Tyr Ala
Asn Leu Phe His Thr Val Thr Asp Trp 180 185 190Tyr Ser Ala Tyr Val
Ser Ser Arg Val Thr Gly Leu Pro Ser Arg Pro 195 200 205His Leu Val
Phe Val Asp Gly His Cys Lys Ala Gln Leu Glu Glu Thr 210 215 220Trp
Lys Ala Leu Phe Ser Ser Val Thr Tyr Ala Lys Asn Phe Ser Gly225 230
235 240Pro Val Cys Phe Arg His Ala Ile Leu Ser Pro Leu Gly Tyr Glu
Thr 245 250 255Ala Leu Phe Lys Gly Leu Ser Glu Ser Ile Ser Cys Glu
Gly Ala Ser 260 265 270Ala Gln Leu Trp Gln Asn Pro Asp Asp Gln Lys
Thr Ala Arg Leu Ser 275 280 285Glu Phe Gly Glu Met Ile Arg Ala Ala
Phe Gly Leu Pro Val Asp Ile 290 295 300Lys Lys Thr Ser Ser Gly Asn
Val Leu Phe Val Arg Arg Glu Asp Tyr305 310 315 320Leu Ala His Pro
Arg His Ser Gly Lys Val Glu Ser Arg Leu Ser Asn 325 330 335Glu Glu
Val Phe Asp Ser Leu Trp Ala Ala Gly Lys Cys Lys Ile Asn 340 345
350Val Val Asn Gly Leu Phe Ala His Met Ser Met Lys Glu Gln Leu Arg
355 360 365Ala Ile Gln Asp Ala Ser Val Ile Ile Gly Ala His Gly Ala
Gly Leu 370 375 380Thr His Ile Val Ser Ala Thr Pro Thr Val Ile Leu
Glu Ile Ile Ser385 390 395 400Ser Tyr Arg Arg Pro His Phe Ala Leu
Ile Ala Arg Trp Lys Gly Leu 405 410 415Glu Tyr His Ala Ile Asn Leu
Gly Ser Tyr Ala Asp Pro Asp Val Ile 420 425 430Glu Lys Leu Ser Ile
Leu Lys Ser Leu Gly Cys 435 440
* * * * *
References