WO 2007/137329 A2 - Multi-gene Expression Vehicle - The Lens

WO 2007/137329 A2

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Abstract

A multigene expression vehicle (MGEV) consisting essentially of a polynucleotide comprising 2 to 8 domain segments, D, each domain encoding a functional protein, each domain being joined to the next in a linear sequence by a Linker (L) segment encoding a Linker peptide, the D and L segments all being in the same reading frame, and at least one of the domains is not a type two protease inhibitor.

Claims

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A muitigene expression vehicle (MGEV) consisting essentially of a polynucleotide comprising 2 to 8 domain segments, D, each domain encoding a functional protein, each domain being joined to the next in a linear sequence by a Linker (L) segment encoding a Linker peptide, the D and L segments all being in the same reading frame, and at least one of the domains is not a type two protease inhibitor.
The MGEV of claim 1 wherein each Linker has the sequence of Sequence I. D. No. 17.
The MGEV of claim 1 further comprising a segment encoding a signal peptide (S) at the N-terminus of a functional protein.
The MGEV of claim 3 further comprising a segment encoding a vacuole targeting signal peptide V.
The MGEV of claim 2 further comprising a segment encoding an N-terminal clasp peptide, CN and a segment encoding a C-terminal clasp peptide, C0 , the N- terminal and C-terminal clasp peptides being joined by disulfide bonds to one another after translation to form a single protein having protease inhibitor activity.
A MGEV according to claim 2, wherein the D and L coding segments are joined in translational order, designated as (DkLj), where k is an ordinal number for each Domain numbered from 1 to k and k is in the range from 2 to 8, and j is an ordinal number for each Linker numbered from 1 to k-1.
A MGEV according to claim 5, wherein the CN, D, L and Cc segments are joined in translational order, designated as CN-LJ-DICLK+I- Cc, where k is an ordinal - 70 -
number for each domain numbered from 1 to k and k is in the range from 2 to 7, and j is an ordinal number for each Linker, numbered from 1 to k+1.
The MGEV of claim 6 further comprising a coding segment, S, encoding a signal peptide being combined in the order S-DK-LJ .
The MGEV of claim 7 further comprising a coding segment, S, encoding a signal peptide, being combined in the order S-CN-Lj-Dk-Lk+r C0.
The MGEV of claim 6, further comprising a coding segment V, wherein V is combined with the coding segment of any of D1-Dk, at either end of the segment encoding any of Di-Dk.
The MGEV of claim 7, further comprising a coding segment, V, wherein V is combined with the coding segment of any of D-i-Dk+i,CN or Cc, at either end of the segment encoding any of DrDk+i,CN θr Cc.
The MGEV of claim 7 further comprising a coding segment, V, encoding a vacuole transport peptide, wherein V is combined with any of Dk, at either end of the segment encoding any of Dk,CN or Cc-
A MGEV expression vector comprising a plant transformation vector carrying and replicating a MGEV according to claim 6 or 7, the MGEV being inserted at a locus in the vector that is under expression control of a plant-active promoter and a plant-active terminator.
A plant cell containing and expressing proteins encoded by a MGEV according to claim 6 or 7. - 71 -
A transgenic plant, transformed by, and concurrently expressing proteins encoded by a MGEV according to claim 6 or 7.
A multigene expression vehicle (MGEV) according to claim 11, having segments in the translation order:
S-CN-L1-D1-L2-D2-L3-D3-L4- C0-V
Where S encodes a signal peptide,
CN encodes a N-terminal clasp peptide,
C0 is a C-terminal clasp peptide,
L1, L2, L3, L4 each encodes a Linker peptide,
Di encodes a type two trypsin inhibitor,
D2 encodes Pot 1A
D3 encodes a type two chymotrypsin inhibitor, and;
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 16 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2-L2-D3-V
Where S encodes a signal peptide,
Di encodes a type two trypsin inhibitor,
D2 encodes Pot 1A,
D3 encodes a type two chymotrypsin inhibitor, - 72 -
Li and L2 encode Linker peptides,
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 18 under expression control of a plant-active promoter.
A MGEV according to claim 11 having coding segments in the translational order:
S-CN-L1-D1-L2-D2-L3-D3-L4- C0-V
Where S encodes a signal peptide,
CN encodes a N-terminal clasp peptide,
L1, L2, L3 and L4 each encode a Linker peptide,
D1 encodes a type two trypsin inhibitor,
D2 encodes Pot 1A,
D3 encodes a type two chymotrypsin inhibitor,
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 20 under expression control of a plant-active promoter.
A MGEV according to claim 11 having coding segments in the translational order:
S-CN-L1-D1-L2-D2-L3-D3-L4- C0-V
Where S encodes a signal peptide, CN encodes a N-terminal clasp peptide, - 73 -
Li, L2, L3 and L4 each encode a Linker peptide, Di encodes a type two trypsin inhibitor, D2 encodes a green fluorescent protein, D3 encodes a type two chymotrypsin inhibitor, Cc encodes a C-terminal clasp peptide, and
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 22 under expression control of a plant-active promoter.
A MGEV according to claim 11 having coding segments in the translation order:
S-CN-LI-D1-L2-D2-L3-D3-L4-D4-L5-D5-L6- CC-V
Where S encodes a signal peptide,
CN encodes a N-terminal clasp peptide,
Li,L2,L3,L4,L5 and Le each encode a Linker peptide,
Di encodes a type-two trypsin inhibitor, D2 encodes a plant defensin, and;
D3 and D4 each encode Pot 1A,
D5 encodes a type-two chymotrypsin inhibitor,
Cc encodes a C-terminal clasp peptide, and;
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 24 under expression control of a plant-active promoter.
A MGEV according to claim 9 having coding segments in the translational order: - 74 -
S-CN-LI-D1-L2-D2-L3-D3-L4- C0
Where S encodes a signal peptide, and;
CN encodes a clasp peptide,.
Di encodes a type two trypsin inhibitor,
D2 encodes a green fluorescent protein,
D3 encodes a type two chymotrypsin inhibitor,
Cc encodes a clasp peptide, and;
L-i, L21L3 and L4 each encode a Linker peptide.
A plant transformation vector comprising a MGEV according to claim 26 under expression control of a plant-active promoter.
A MGEV according to claim 11 having coding segments in the translation order:
S-CN-L1-D1-L2-D2-L3-D3-L4- C0-V
Where S encodes a signal peptide,
CN encodes a clasp peptide,
D1 encodes a type two trypsin inhibitor,
D2 encodes a defensin having a C-terminal propeptide,
D3 encodes a type two chymotrypsin inhibitor,
Cc encodes a clasp peptide,
Li, L2, L3 and L4 each encode a Linker peptide, and;
V encodes a vacuole targeting peptide.
A MGEV expression vector comprising a MGEV according to claim 28 under expression control of a plant-active promoter. - 75 -
A MGEV according to claim 11 having coding segments in the translation order:
S-CN-LI-D1-L2-D2-L3-D3-L4-D4-L5- CC-V
Where S encodes a signal peptide, and;
CN encodes a clasp peptide, and;
Di encodes a type two trypsin inhibitor, and;
D2 and D3 each encode Pot 1A, and;
D4 encodes a chymotrypsin inhibitor, and;
Cc encodes a chymotrypsin inhibitor, and;
Li, L2, L3) L4 and L5 each encode a Linker peptide, and;
V encodes a vacuole targeting peptide.
A plant transformation vector comprising a MGEV according to claim 30 under expression control of a plant-active promoter.
A MGEV according to claim 11 having coding segments in the translation order:
S-CN-L1-D1-L2-D2-L3-D3-L4-D4-L5- CC-V
Where S encodes a signal peptide,
CN encodes a N-terminal clasp peptide,
Li1 L2, L3, L4, and L5 each encode a Linker peptide,
Cc encodes a C-terminal clasp peptide,
V encodes a vacuole targeting peptide, Di encodes a type-two trypsin inhibitor, D2 encodes a first plant defensin, - 76 -
D3 encodes a second plant defensin, and; D4 encodes a type-two chymotrypsin inhibitor.
A MGEV expression vector comprising a MGEV according to claim 32 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2-L2-D3-L3-D4-V
Where S encodes a signal peptide,
Di encodes a type-two trypsin inhibitor,
D2 and D3 each encode Pot 1A,
D4 encodes a type-two chymotrypsin inhibitor,
V encodes a vacuole targeting peptide, and;
L1, L2, and L3 each encodes a Linker peptide.
A MGEV expression vector comprising a MGEV according to claim 34 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2-L2-D3-V
Where S encodes a signal peptide, D1 encodes a type-two trypsin inhibitor, D2 encodes a first plant defensin, - 77 -
D3 encodes a type-two chymotrypsin inhibitor,
V encodes a vacuole targeting peptide, and; Li and L2 each encodes a Linker peptide.
A MGEV expression vector comprising a MGEV according to claim 36 under expression control of a plant-active promoter.
A MGEV according to claim 8 having coding segments in the translation order:
S-D1-L1-D2
Where S encodes a signal peptide, Li encodes a Linker peptide, D1 and D2 each encode a potato type one proteinase inhibitor.
A MGEV expression vector comprising a MGEV according to claim 38 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2-V
Where S encodes a signal peptide, Li encodes a Linker peptide D1 encodes a type-two trypsin inhibiter, D2 encodes a plant defensin, and;
V encodes a vacuole targeting peptide. - 78 -
A MGEV expression vector comprising a MGEV according to claim 40 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-DrLi-D2-V
Where S encodes a signal peptide, L1 encodes a Linker peptide, V encodes a vacuole targeting peptide, D-I encodes a first plant defensin, and; D2 encodes a second plant defensin.
A MGEV expression vector comprising a MGEV according to claim 42 under expression control of a plant-active promoter.
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2
Where S encodes a signal peptide, L1 encodes a Linker peptide, D1 encodes a first plant defensin, and; D2 encodes a second plant defensin.
A MGEV expression vector comprising a MGEV according to claim 44 under expression control of a plant-active promoter. - 79
A MGEV according to claim 10 having coding segments in the translation order:
S-D1-L1-D2-L2-D3-V
Where S encodes a signal peptide, V encodes a vacuole targeting peptide, Li and L2 each encode a Linker peptide, D1 encodes a type-two trypsin inhibitor, D2 encodes a beta-glucuronidase, and; D3 encodes a type-two chymotrypsin inhibitor.
A plant transformation vector comprising a MGEV according to claim 46 under expression control of a plant-active promoter.
A MGEV according to claim 4 selected from the group of MGEVs consisting of MGEV 5, MGEV 8, MGEV 6, MGEV 7, MGEV 9, MGEV 10, MGEV 11 , MGEV 12, MGEV 13, MGEV 14, MGEV 15, MGEV 16, MGEV 17, MGEV 18, MGEV 19, MGEV 20.
A MGEV according to claim 5 selected from the group of MGEVs consisting of MGEV 5, MGEV 6, MGEV 7, MGEV 9, MGEV 10, MGEV 11 , MGEV 12, MGEV 13.
MGEV expression vector selected from the group of MGEV expression vectors consisting of pHEX 29, pHEX 56, pHEX 31 , pHEX 46, pHEX 55, pHEX 45, pHEX 42, pHEX 33, pHEX 39, pHEX 48, pHEX 47, pHEX 35, pHEX 41 , pHEX 52, pHEX 51 , pHEX 58. - 80 -
A method of concurrently expressing from two to eight desired proteins in a plant cell comprising the steps of:
a. assembling a multi-gene expression vehicle (MGEV) consisting essentially of a polynucleotide segment comprising from 2 to 8 Domain segments, Dk, each Domain encoding a functional protein wherein at least one such protein is not a type-two protease inhibitor and each Domain is joined to the next in a linear sequence by a Linker segment, L, encoding a Linker peptide having a sequence of SEQ ID NO:17, all the D and L coding segments being joined in the same reading frame in translational order designated as DkLj, and where k is an ordinal number for each Domain numbered from 1 to k and k is in the range from 2 to 8, and j is an ordinal number for each Linker numbered from 1 to k-1.
b. combining the MGEV with a plant transformation vector, at a locus in the vector that is under expressive control of a plant-active promoter and a plant-active terminator, thereby providing a MGEV expression vector, and;
c. transforming a plant cell with the MGEV expression vector, thereby providing a MGEV-transformed cell, and;
d. maintaining the MGEV-transformed cell and progeny thereof under conditions suitable for gene expression within the cell, whereby genes encoded within MGEV-transformed cells are concurrently expressed.
The method of claim 51 wherein proteins Di t0 k are individually selected from the group of proteins, consisting of a type-two trypsin inhibitor, a type-two - 81 -
chymotrypsin inhibitor, a Pot I protease inhibitor, a defensin, a defensin having a C- terminal propeptide, a green fluorescent protein, and an indicator enzyme.
The method of claim 52 wherein the MGEV expression vector is selected from the group of MGEV expression vectors consisting of pHEX 56, pHEX 48, pHEX 47, pHEX 17, pHEX 18, pHEX 19, pHEX 20.
The method of claim 52 further comprising the step of regenerating an adult transgenic plant from the MGEV-transformed cell.
The method of claim 54 wherein the wherein the adult transformed plant is selected from the group of plants consisting of cotton, soybean, corn and rice.
A method of concurrently expressing from 3 to 8 proteins in a plant cell comprising the steps of:
a. assembling a multi-gene expression vehicle (MGEV) consisting essentially of a polynucleotide segment comprising from 3 to 8 Domain segments, Dk, each Domain encoding a functional protein wherein at least one such protein is not a type-two protease inhibitor and each Domain is joined to the next in a linear sequence by a Linker segment, L, encoding a Linker peptide having a sequence of SEQ ID NO:17, all the D and L coding segments being joined in the same reading frame in translational order, designated as CN-Lj-Dk-Lk+i-Cc, where k is an ordinal number for each domain numbered from 1 to k and k is in the range from 3 to 7, and j is an ordinal number for each Linker, numbered from 1 to k+1. - 82 -
b. combining the MGEV with a plant transformation vector, at a locus in the vector that is under expression control of a plant-active promoter and a plant-active terminator, thereby providing a MGEV expression vector, and;
c. transforming a plant cell with the MGEV expression vector, thereby providing a MGEV-transformed cell, and;
d. maintaining the MGEV-transformed cell and progeny thereof under conditions suitable for gene expression within the cell, whereby genes encoded within MGEV transferred cells are concurrently expressed.
Plant transformation vector selected from the group of plant transformation vectors consisting of pHEX 10, pHEX 29, pHEX 31, pHEX 46, pHEX 55, pHEX 45, pHEX 42, pHEX 33 and pHEX 39.
The method of claim 56 further comprising the step of regenerating an adult transgenic plant from the MGEV-transformed cell.
The method of claim 56 wherein the wherein the adult transformed plant is selected from the group of plants consisting of cotton, soybean, corn and rice.
A method for concurrently expressing from two to eight proteins in a plant cell comprising transforming a plant cell with a MGEV according to claim 2, wherein the MGEV is under expression control of a single promoter.
The method of claim 60 wherein the linker has the sequence of SEQ ID NO:5.

Applicants

Hexima Ltd
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Anderson Marilyn Anne
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Heath Robyn Louise
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Inventors

Anderson Marilyn Anne
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Heath Robyn Louise
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CPC Classifications

C07K14/811
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C07K7/04
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C12N15/62
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C12N15/8216
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C12N15/8257
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C12N15/8279
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C12N15/8286
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C12N9/2402
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Document History

Publication: Dec 6, 2007
WO 2007/137329 A2
Application: May 24, 2007
AU 2007/000712 W
Priority: May 25, 2006
US 80320606 P