WO2004094630A1 - Insect chymotrypsin and inhibitors thereof - Google Patents
Insect chymotrypsin and inhibitors thereof Download PDFInfo
- Publication number
- WO2004094630A1 WO2004094630A1 PCT/AU2004/000524 AU2004000524W WO2004094630A1 WO 2004094630 A1 WO2004094630 A1 WO 2004094630A1 AU 2004000524 W AU2004000524 W AU 2004000524W WO 2004094630 A1 WO2004094630 A1 WO 2004094630A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- chymotrypsin
- seq
- plant
- sequence
- hpch5
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- Nicotiana alata amass high levels of serine proteinase inhibitors (Pis) for protection against pests and pathogens (Atkinson et al, The Plant Cell 5: 203-213, 1993).
- These 6 kDa Pis accumulate in the vacuole (Miller et al, Plant Cell 11: 1499-1508, 1999) and are derived in vivo from the post-translational modification of a 40.3kDa precursor protein.
- the precursor of the PI protein (referred to as "NaPI”) is composed of six repeated regions of high sequence identity ( Figure 1) each with a potential PI reactive site. Processing of the six-repeat precursor protein unexpectedly occurs at sites located within, rather than between the repeated regions.
- Nicotiana alata also has a second gene related to NaPI that encodes a closely related precursor protein with four rather than six repeated domains (Miller et al, Plant Mol. Biol 42: 329-333, 2000). This precursor is also processed in vivo resulting in the release of three contiguous 6 kDa inhibitors (CI, T4 and T5) and the two-chain inhibitor C2 ( Figure 1). Three ofthe inhibitors (CI, C2 and T4) are identical to those released from the six-domain precursor.
- Related multidomain precursors have been described for other solanaceous plants including N. tabacum (Balandin et al, Plant Mol Biol. 27: 1197-1204, 1995), N. glutinosa (Choi et al, Biochim.
- the midgut proteases of several Lepidoptera, Coleoptera and Orthoptera have been partially characterized.
- the endoproteinase activity is due primarily to serine proteinases (trypsin, chymotrypsin and/or elastase) and cysteine and metalloproteinases are not detectable (Christeller et al, Insect Biochem. Molecul. Biol. 22: 735-746, 1992; Terra and Ferreira, Comp. Biochem. Physiol. 109: 1-62, 1994; Xu and Qin, J. Econ. Entomol. 87: 334-338, 1994; Lee and Anstee, Insect. Biochem. Molec. Biol.
- chymotrypsins were assumed to contribute relatively little to protein digestion in Lepidoptera and consequently most biochemical studies focused on characterization of the trypsins. This problem arose due to the initial use of synthetic substrates that worked well with mammalian chymotrypsins, but not at all or poorly with the Lepidopteran enzymes.
- Lepidopteran chymotrypsins prefer synthetic substrates with at least four amino acids to occupy the S1-S4 binding subsites on the enzyme, whereas mammalian trypsins are active on shorter substrates with one amino acid that is specific for the SI binding subsite.
- SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
- the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
- a summary of the sequence identifiers is provided in Table 1.
- a sequence listing is provided at the end ofthe specification.
- HpCh5 novel chymotrypsin from Helicoverpa spp. referred to herein as "HpCh5".
- Reference to "HpCh5" includes all variants, derivatives, homologs and analogs as well as members of a HpCh5 family of chymotrypsins.
- variants of HpCh5 include proteinase inhibitor (PI) sensitive forms. Such sensitive forms may carry inter alia a substitution of the arginine at position 192 to an asparagine or glutamine. This substitution is referred to herein as "R192N/Q” using single amino acid nomenclature or "Arg 192 Asn/Gln” using three letter amino acid code.
- Other derivatives of HpF5 include the signal sequence of HpF5.
- HpCh5 chymotrypsin is encoded by a nucleotide sequence referred to as "HpF5".
- HpF5 includes variants, homologs and analogs thereof.
- the term “HpF5" encompasses both a genomic sequence as well as a cDNA sequence.
- the amino acid sequence of HpCh5 is set forth in SEQ ID NO:2.
- the amino acid sequence of the N- terminal activation peptide is shown in SEQ ID NO:3.
- the nucleotide sequence of the coding region of HpF5 is set forth in SEQ ID NO:4 with the nucleotide sequence encoding the activation peptide is shown in SEQ ID NO: 5 and its entire 5 '-3' sequence shown in SEQ ID NO: 6.
- HpCh5 is generally characterized by being substantially insensitive to inhibition by a PI from N alata.
- Variants and homologs of HpCh5 include molecules having at least 75% amino acid identity to SEQ ID NO:2 after optimal alignment.
- Variants and homologs of HpF5 include nucleotide sequences having at least about 75% similarity to SEQ ID NO:4 or SEQ ID NO: 6 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions.
- one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a chymotrypsin from Helicoverpa ssp. or a variant, derivative, homolog or analog of said chymotrypsin, wherein said chymotrypsin exhibits resistance to a PI from N. alata.
- Another aspect of the present invention provides an isolated chymotrypsin from Helicoverpa ssp. wherein said chymotrypsin exhibits resistance to a PI from N alata or a variant, derivative, homolog or analog of said chymotrypsin.
- the antagonists include compounds which bind to and inhibit HpCh5 as well as antisense or sense nucleic acid molecules generated by a plant cell and then ingested by an insect.
- Reference to an "antagonist” includes reference to an inhibitor.
- the present invention further provides genetically modified plants which are engineered to produce a HpCh5 or HpF5 antagonist.
- Reference to a "plant” includes a monocotyledonous or dicotyledonous plant and may be a plant regenerated from genetically transformed callus or tissue or progeny of such a plant.
- the present invention further provides seeds and other reproductive material from the genetically modified plants of the present invention.
- Plants contemplated herein include cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant) and geraniums, cape gooseberry, maize, flax and linseed, alfalfa, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum (an ornamental plant), wheat, barley, oats, triticale, carrots, onions orchids, roses and/or petunias.
- Figure 1 is a graphical representation showing temperature stability of baculovirus expressed H. punctigera chymotrypsin.
- Bovine chymotrypsin ( ⁇ ) and HpF5 (A) in 50 mM Na acetate were compared by incubating 100 ⁇ L aliquots at 5°C intervals between 40°C and 70°C. After chilling the heated samples on ice, duplicate activity assays were performed by incubating 10 ⁇ L of be or 50 ⁇ L of HpF5 with substrate at 30°C. Residual activity was presented as a percentage ofthe activity ofthe untreated control.
- Figure 3 is a graphical representation showing the effect of various proteinase inhibitors on the chymotrypsin activity in unfractionated gut extracts from H. punctigera. Proteinase inhibitors were mixed with 1 ⁇ g of protein from an unfractionated gut extract before incubation with the chymotrypsin substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Inhibition is expressed as a percentage ofthe total activity in the control samples.
- FIG. 5 is a diagrammatic representation showing purification and N-terminal sequence of an NaPI-insensitive chymotrypsin from H. punctigera.
- A PVDF blot of chymotrypsin (i) eluted from potato inhibitor column. Potato Inhibitor II (ii) and potato inhibitor I (iii) both co-eluted from the matrix under denaturing conditions.
- B N-terminal amino acid sequence obtained from PVDF blot. Rechla was the most abundant of the four sequences obtained.
- Figure 8 is a diagrammatic representation showing oligonucleotide sequences used in RT-PCR amplification of Helicoverpa chymotrypsins.
- Figure 10 is a diagrammatic representation showing alignment of predicted amino acid sequence of chymotrypsins from H. punctigera.
- the catalytic residues are marked by a solid triangle (T).
- T The highly conserved active site motifs are highlighted with grey.
- the dipeptide R-I (J ) conserved among all chymotrypsins is the site for the proposed cleavage of the activation peptide by trypsin.
- the residues that lie in the substrate binding pocket and confer substrate specificity are indicated by the symbols ⁇ , ⁇ ,#.
- the cysteine (•) residues are highly conserved among all chymotrypsins.
- Figure 11 is a diagrammatic representation showing design of oligonucleotide primers for amplification of cDNA encoding the NaPI-insensitive chymotrypsins from H. punctigera.
- A Comparison of the N-terminal sequence of two NaPI-insensitive chymotrypsins with Helicoverpa chymotrypsins predicted from the cDNA clones. The unique regions FI and F2 are shaded.
- Figure 12 is a diagrammatic representation showing nucleotide sequence and deduced amino acid sequence from the cDNA encoding the insensitive chymotrypsin.
- the nucleotide sequence of the insensitive chymotrypsin cDNA and deduced amino acid sequence The amino acid sequence obtained from N-terminal sequence of purified protein is shaded in grey. The putative site for endoproteolytic cleavage by trypsin is shown by the arrow. The double underlined regions in the nucleotide sequence refer to the positions of the degenerate primers used for PCR amplification. The polyadenylation signal sequence is single underlined and an asterisk marks the stop codon.
- the deduced amino acid sequence of the putative activation peptide is numbered -40 to -1 followed by the mature domain (+1). The three amino acids that correspond to the catalytic residues are marked by the symbol #.
- the chymotrypsin substrate specificity residue, serine, located at the base of the primary substrate-binding pocket is marked with the symbol ⁇ .
- Figure 13 is a representation showing alignment of H. punctigera chymotrypsin families showing sequence identity. ClustalW alignment of members from the five families of H. punctigera chymotrypsins. Protein sequence is given in single letter code. Identical amino acids are coloured black, similar amino acids are grey. Amino acids are numbered on the right and gaps have been introduced to maximize the alignment.
- the NaPI- insensitive chymotrypsins are members of family 5 and are characterized by a unique arginine residue (arrowed) at position 185. Human trypsin IV also contains an arginine residue (arrowed) in a similar position.
- Figure 14 is a representation showing alignment of the H. punctigera NaPI-insensitive chymotrypsin with a homolog from H armigera that also has an arginine residue at position 185.
- Figure 15 is a representation showing the deduced protein sequences for the insensitive (HpCh5) and sensitive (HpCh2A) chymotrypsins from H punctigera aligned to the bovine chymotrypsin isoforms A and B.
- H. punctigera chymotrypsins HpCh2A and HpCh5 were aligned to the bovine chymotrypsin isoforms A and B using ClustalW.
- the numbering system (excluding gaps) is according to the nomenclature of Greer, Proteins 7: 317-34, 1990 used for bovine chymotrypsin. Dots throughout the sequences represent conserved residues.
- the regions shaded in grey designate residues that form surface loops that are involved in recognition and binding of substrates or inhibitors.
- the primary substrate- binding pocket is formed by the regions labeled SI A, SIB and SIC.
- the SI' site is formed by loops 35 and 60.
- Black boxes mark residues in the HpF5 sequence that differ significantly to amino acids in the corresponding positions in other chymotrypsins. Using the Greer, 1990, supra nomenclature these residues are Asp36, Arg 63, Thr72, Pro 83, Gly 109, lieu 120, Glu insertion between 129 and 130, Glu 134, Serl45, Arg 192 and Pro 207.
- the boxed amino acids are removed from bovine chymotrypsins by autocatalytic cleavage that results in the formation of ⁇ -chymotrypsin.
- Figure 16 is a diagrammatic representation showing several surface loops in the structural model ofH. punctigera chymotrypsin ⁇ pCh2A are larger than the cognate loops in bovine chymotrypsin.
- the structural model of H punctigera F2A chymotrypsin (grey) was superimposed onto the structure of alpha-chymotrypsin from Bos taurus (black).
- Surface loops 60, 35, and 142 that are implicated in substrate recognition are larger in the insect chymotrypsin model.
- the chymotrypsin substrate specificity residue, serine 189, positioned at the base of the primary substrate-binding pocket is viewed as a space filled representation ofthe van der Waals radius.
- Figure 17 is a representation showing glutamine 192 (Greer, 1990, supra nomenclature,
- Figure 18 is a diagrammatic representation showing comparison of the environment surrounding residue 192 of the sensitive and insensitive chymotrypsin complexed to CI. Enlarged view of the boxed area shown in Figure 17. Arginine 192 of the insensitive chymotrypsin (B) appears to clash with residues of the CI inhibitor, in contrast there is no apparent conflict with glutamine 192 ofthe sensitive chymotrypsin (A).
- Figure 19 is a diagrammatic representation showing the environment surrounding arginine 192 when the insensitive chymotrypsin is complexed to the Type 1 potato proteinase inhibitor (PotIB, Fig24).
- A Structural model of the insensitive chymotrypsin HpCh5 (grey) in complex with the potato type I proteinase inhibitor Potl (black).
- B Enlarged view of the region around Arg 192 (boxed area in B). The side chain of arginine 192 is labeled. The residues of Potl in the vicinity of Arg 192 are represented in stick configuration (black).
- Figure 20 is a photographic representation showing expression of the chymotrypsin clone HpF2B in E. coli cells for the production of a polyclonal antibody.
- A The separation of total cell lysates taken at time points 0-5 hr after induction of HpCh2B on a 12.5% (w/v) SDS-PAGE gel stained with Coomassie Blue. The lanes are marked by the number of hours after induction and the arrow indicates the position of the induced protein with the correct predicted molecular mass.
- Panel 1 Bacterially expressed HpCh2B purified on Talon resin (BD Biosciences Clontech), separated on a 15% (w/v) SDS-PAGE gel and stained with Coomassie Blue.
- Panel 2 Identical sample to Panel 1 transferred to nitrocellulose and immunostained with anti-HpCh2B antibodies.
- C Decreasing amounts (200, 150, 100, 75, 50, 25, 20, 10, 0 ng) of bacterially expressed chymotrypsin H ⁇ Ch2B separated by SDS-PAGE and stained with silver and a protein blot of an identical gel probed with anti-chymotrypsin HpCh2B antibodies (1/2500).
- the H punctigera antibody had a detection limit of 20 ng of bacterially expressed protein.
- Figure 21 is a photographic representation showing specificity of antibodies raised against bacterially expressed NaPI-insensitive (HpCh5) and sensitive (HpCh2B) chymotrypsins from H punctigera.
- Bacterially expressed NaPI-insensitive (R) and sensitive (C) chymotrypsins were separated by SDS-PAGE on 12.5% (w/v) polyacrylamide gels and (A) stained with Coomassie Blue, (B) immunoblotted with an ⁇ -His tag antibody, (C) immunoblotted with the antibody to the HpCh2B chymotrypsin ( ⁇ -RC), and (D) immunoblotted with the antibody to the HpCh5 chymotrypsin ( ⁇ -SC).
- FIG. 22 are representations showing purification of Potl from potato tubers. Potl was purified from potato tubers (Russet Burbank) by acid extraction, ammonium sulphate precipitation and gel filtration.
- A SDS-PAGE stained with silver, lane 1 : molecular size markers (kDa), lane 2: pooled Potl containing fractions from G-75 column, lane 3: immunoblot of lane 2 using an antibody raised in rabbits to a commercial preparation of Potl (Calbiochem) linked to keyhole limpet hemocyanin. Potl was identified as a single band with an approximate mass of 6 kDa.
- B RP-HPLC of pooled G-75 fractions from A. Peaks 1, 2 and 3 are Potl isoforms, peak 4 is a contaminating protein.
- Figure 23 is a graphical representation showing growth of H. armigera larvae on artificial diet containing NaPI and Potl. Growth ofH. armigera. larvae fed on a cotton leaf artificial diet in the presence or absence of 0.26% (w/v) NaPI or 0.26% (w/v) NaPI plus 0.26% (w/v) Potl.
- the Potl was purified from potato tubers (var Russet Burbank), see Figure 22. Twenty five larvae were used on each diet. The weight of the larvae was measured at days 7, 10, 12, 14 and 17 post egg hatch. At day 17, larvae fed NaPI alone were 84% of the control and larvae fed NaPI and Potl were 34% of the control. Two larvae fed on the control diet died, seven larvae fed the NaPI diet died and six larvae fed on the NaPI plus Potl diet died.
- Figure 24 is a representation showing the alignment of predicted amino acid sequence of StPotIA and StPotIB with members ofthe potato Inhibitor I family. ClustalW alignment of several members of the Potato Inhibitor I family.
- X67950 potato cDNA, (Beuning and Christeller, Plant Physiol 102: 1061, 1993), P01052: potato protein (Richardson and Cossins, FEBS Letters, 52: 161, 1975), M17108: potato genomic sequence (Cleveland et al, Plant Mol. Biol. 8: 199-207, 1987), K03290: tomato (Graham et al, J. Biol.
- StPotlA has an additional four amino acids at position 41 to 44 that are also found in a wound induced Potl from tomato (K03290).
- StPotlB has a methionine at the PI site which is common for potato isolates.
- StPotlA has an alanine at the PI site which has not been reported for Potl isolates from potato, but is present in a Potl isolate from maize (X78988).
- Figure 26 is a graphical representation showing inhibition of NaPI-insensitive chymotrypsin by bacterially expressed StPotlA and StPotlB. Inhibition of the NaPI- insensitive chymotrypsin from the gut of H. punctigera with purified Potl.
- A substrate SA 2 PFpNA, development time 30 min
- B substrate SA 2 PLpNA, 30 min incubation.
- StPotlA, StPotlB and the Potl from potato tubers were good inhibitors of the NaPI- insensitive chymotrypsin.
- FIG 27 is a graphical representation showing growth of H armigera larvae on transgenic cotton expressing NaPI and Potl.
- Transgenic cotton cv Coker 315 was used in bioassays with H armigera.. Thirty larvae were fed leaves of either the control unfransformed Coker 315, transgenic line 1 (NaPI), transgenic line 2 (StPotlA) or transgenic plant 3 (NaPI X StPotlA). The weight ofthe larvae was measured at day 7 post- egg hatch. (A) Growth of larvae. At day 7, larvae fed leaves expressing NaPI were 86% of the weight of the control larvae fed unfransformed leaves.
- Larvae fed leaves expressing StPotlA were 92% of the control and larvae fed leaves expressing both NaPI and StPotlA were 46% of the control.
- B The effect of ingestion of NaPI and Potl on gut trypsin and chymotrypsin activity. Gut from the larvae in each experiment were pooled and extracts prepared. All assays were performed in duplicate. Trypsin activity (black) was determined using BApNA substrate and chymotrypsin activity (grey) with SA 2 PFpNA substrate. Units of activity are expressed as change in absorbance at 405 nm/min/ug gut extract protein.
- Trypsin activity was reduced, relative to the control, in the extracts from larvae fed leaves expressing NaPI and NaPI+StPotlA. Chymotrypsin activity was elevated in extracts from larvae fed leaves expressing NaPI or NaPI + StPotlA and reduced in extracts from larvae fed StPotlA alone.
- Figure 28 diagrammatic representation showing the nucleotide sequence and deduced amino acid sequence from the HpF5 cDNA encoding the NaPI-insensitive chymotrypsin and the location of the oligonucleotide primers used to add an endoplasmic reticulum sequence to HpCh5.
- the FwBacRECHl primer was used to add the first half of the ER signal sequence as well as a silent mutation, changing A to G to destroy the BamHl cut site.
- the FwBacRECH2 primer added the remainder of the coding sequence for the ER signal as well as a BamHl cut site to the 3' end of the sequence.
- the ER signal was added before the hexahistidine tag to enable purification of the expressed protein by metal affinity chromatography after cellular processing. The added amino acids are shaded in grey.
- Figure 29 is a photographic representation showing expression of the chymotrypsin clone HpF5 in baculovirus infected insect cells. Expressed proteins were separated on 12.5% (w/v) SDS-PAGE gels and subjected to immunoblots with the ⁇ -HpCh5-antibody (A) RCDNA. Production of HpCh5 by HIGH FIVE (trademark) insect cells transfected with 20 ⁇ l of bacmid DNA.
- HpCh5 novel insect chymotrypsin molecule termed "HpCh5".
- HpF5 novel insect chymotrypsin molecule
- the isolation of this molecule permits the identification and design of a range of products which are useful in controlling the growth, development and/or overall biological fitness of Helicoverpa spp. and other insects. These products generally act as antagonists of HpCh5 function or HpF5 gene expression and are useful as insect control agents.
- the amino acid sequence of HpCh5 is set forth in SEQ ID NO:2.
- the nucleotide sequence of HpF5 is set forth in SEQ ID NOs:4 and 6.
- HpCh5 should be understood as a reference to all forms of HpCh5 including, for example, any peptide isoforms which arise from alternative splicing of HpF5 mRNA, mutants or polymorphic variants of HpCh5, any post-translation modified forms of HpCh5 or any non-post-translational modified forms of HpCh5 as well as any homolog in other insect species or strains.
- HpCh5 also encompasses members in a HpCh5 family of chymotrypsin molecules. To the extent that it is not specified, reference herein to HpCh5 includes derivatives, homologs, analogs, chemical equivalents and mimetics thereof.
- HpCh5 also refers to any variant having at least 75% amino acid identity to SEQ ID NO: 2 after optimal alignment.
- variants of HpCh5 include Pi-sensitive variants such as those inter alia having an Arg 192 Gin or Arg 192 Asn substitution.
- Other variants include the N-terminal signal sequence of HpCh5 as defined in SEQ ID NO: 3 and which is encoded by the nucleotide sequence set forth in SEQ ID NO:5.
- Such variants include a signal sequence comprising an amino acid sequence having at least about 75% similarity to SEQ ID NO:3 after optimal alignment or encoded by a nucleotide sequence having at least about 75% identity to SEQ ID NO:5 or a nucleotide sequence capable of hybridizing to SEQ ID NO: 5 after optimal alignment.
- HpF5 should be understood as reference to all forms of HpF5 including any cDNA isoform, genomic forms, mutants and polymorphic variants of HpF5 as well as any homologs from other species or strains of insect.
- HpF5 also encompasses members of a HpF5 family of genes which encode HpCh5 or HpCh5-type chymotrypsins.
- HpF5 includes derivatives of HpF5 as well as a nucleotide sequence having at least about 75% identity to SEQ ID NO:4 or SEQ ID NO:6 or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO: 6 or its complementary form under low stringency conditions.
- the signal sequence of HpF5 as defined in SEQ ID NO: 3 and encoded by SEQ ID NO: 5 also encompasses variants thereof.
- compound used interchangeably herein to refer to a chemical compound which inhibits the activity of HpCh5 or the expression of a genomic gene corresponding to HpF5.
- the terms also encompass agriculturally or horticultural active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
- compound used, then it is to be understood that this includes the agent per se as well as agriculturally or horticulturally acceptable, physiologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
- compound is not to be construed as a chemical compound only but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and chemical analogs thereof.
- the plant may produce a PI capable of inhibiting HpCh5.
- the HpCh5 inhibitor is a non- proteinaceous chemical applied to the surface of a plant or taken up by the root system of a plant. Reference herein to a "plant” is not to exclude trees or cultured tissues (e.g. callus) from a plant (or tree).
- Reference to compounds, agents and actives also includes combinations of compounds, agents or actives. Such combinations may be formulated in multi-part agricultural or horticultural compositions which are admixed together prior to dispersement or given sequentially.
- an agent as used herein mean a sufficient amount of the agent to reduce or retard insect growth and development and/or to kill or inhibit the insect.
- an agent for reducing or retard insect growth and development and/or to kill or inhibit the insect.
- agriculturally acceptable or “horticulturally acceptable” carrier excipient or diluent is meant a vehicle comprised of a material that is not environmentally or otherwise undesirable to a plant or non-target insect. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
- treating and “treatment” as used herein in relation to plants or eradication of insects refer to reduction in severity of symptoms of insect infestation of a plant or the application of the agents to a group of insects resulting in retardation of their growth, development or biological fitness or wellbeing.
- nucleic acid molecule is also encompassed by the expression “genetic molecule” and includes hairpin constructs such as those which include RNAi-mediated post-transcriptional gene silencing or methylation-mediated silencing.
- one aspect of the present invention provides an isolated nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding a novel chymotrypsin protein or a derivative, homolog or mimetic thereof wherein said chymotrypsin is insensitive to a PI of N. alata.
- the present invention is directed to a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding, an amino acid sequence substantially as set forth in SEQ ID ⁇ O:2 or a derivative, homolog or mimetic thereof or having at least about 75% or greater identity to SEQ ID NO:2 after optimal alignment or a nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 or a nucleotide sequence having at least about 75% similarity or greater to SEQ ID NO:4 or SEQ ID NO:6 or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions.
- Another aspect of the present invention provides an isolated chymotrypsin Helicoverpa ssp. wherein said chymotrypsin exhibits resistance to a PI from N. alata or a variant, derivative, homolog or analog of said chymotrypsin.
- the isolated chymotrypsin comprises an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least about 75% similarity to SEQ ID NO:2 after optimal alignment.
- similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.
- sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
- a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g.
- Reference to at least about 75% identity or 75% similarity includes percentage identities and similarities greater than 75% such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
- a low stringency means from at least about 0 to at least about 15%) (v/v) formamide (including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13% and 14%) (v/v) formamide) and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
- low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
- nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
- the nucleic acid molecule encoding HpCh5 is preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence.
- a genomic sequence may also comprise exons or introns.
- a genomic sequence may also include a promoter region or other regulatory regions.
- the present invention further contemplates isolated introns and exons of HpF5 such as those involved in genetic networking within a plant cell.
- the nucleic acid molecule according to this aspect of the invention corresponds herein to HpF5.
- This cDNA has been determined, in accordance with the present invention, to encode a protein that defines a new family of chymotrypsins, family 5, within the group of chymotrypsin gene families, and this protein is referred to herein as HpCh5.
- Reference to "HpF5" also includes a genomic form of the gene. Within the Helicoverpa punctigera chymotrypsin gene families, there are varying levels of homology as shown in Table 2. Family 5 is exemplified by HpF5, and is most similar to family 2A at 73% and least similar to family 4 at ⁇ 20%.
- HpCh5 is exemplified by two unique stretches of sequence in the N-terminal (FI and F2) and by six amino acid substitutions relative to NaPI sensitive chymotrypsins. Five of these substitutions did not appear to fall into functionally significant regions, whereas the sixth substitution is associated with one of the ⁇ -strands that forms a wall of the primary substrate-binding pocket. The location of this substitution and conversion to an arginine, from glutamine, is highly unusual for the SI domain that is predominantly lined with non- polar residues that define chymotrypsin specificity
- the present invention provides, therefore, an isolated protein having chymotrypsin activity which is not substantially inhibited by a PI from N alata. Accordingly, another aspect of the present invention is directed to an isolated protein selected from the list consisting of:
- the present invention discloses the amino acid, and corresponding cDNA sequence of a novel chymotrypsin that is insensitive to the Type II serine proteinase inhibitors produced by solanaceous species such as N. alata. Therefore, this may be used as a target for agents to control insects carrying this insensitive proteinase.
- a number of compounds have been shown to inhibit the activity of HpCh5, and a list of these compounds as preferred embodiments is found in Table 3. TABLE 3
- SBTI soybean trypsin inhibitor
- PMSF phenylmethyl sulphonyl fluoride
- Bowman Birk soybean Bowman Birk inhibitor
- lima bean lima bean trypsin inhibitor (Sigma)
- Pot I potato proteinase inhibitor Type I. Bovine chymotrypsin (BC).
- the deduced amino acid sequences from the cDNA clones HpF2A (NaPI-sensitive) and HpF5 (NaPI-insensitive) were modeled on the structures ofthe Solenopsis invicta (fire ant) and Bos taurus (cow) chymotrypsins obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank.
- the Helicoverpa chymotrypsins are predicted to adopt similar structures to those reported for all the chymotrypsin structures available in the data bank.
- the modeled structures have the classic serine protease fold consisting of two, six-stranded anti-parallel beta barrels with the catalytic triad located between the two domains.
- chymotrypsin inhibitor CI was modeled in complex with NaPI-sensitive and NaPI-insensitive chymotrypsins to investigate what residues in the NaPI-insensitive chymotrypsin might be involved in the loss of inhibitor binding.
- the structure of the chymotrypsin inhibitor CI was previously determined by 1H NMR (Nielson et ah, 1994, supra) but has not been determined in a proteinase complex. Therefore the related proteinase inhibitor PCI-1 from Solanum tuberosum in complex with Proteinase B from Streptomyces griseus (Greenblatt et al, J. Mol. Biol. 205: 201, 1989) provided an appropriate basis guiding the alignment of the complexes.
- Figure 17 shows a close up view of the binding region surrounding Glnl92 in the Cl-HpF2A chymotrypsin complex. It is clear that Glnl92 is not in conflict with any regions on the inhibitor molecule. However, comparison to the cognate Arg residue in the Cl-HpCh5 chymotrypsin model demonstrates there is not enough space to accommodate this much larger residue (Figure 18) making contact with Thr5 and Ala9 in CI. Furthermore, modeling the StPotlA inhibitor into HpCh5 revealed that the NaPI-insensitive chymotrypsin could accommodate the Argl92 residue consistent with the inhibition of this chymotrypsin by StPotlA ( Figure 19).
- the NaPI-insensitive chymotrypsin from Helicoverpa species has an arginine in place of an asparagine or glutamine at position 192 that extends into the SI binding pocket and appears to interfere with CI binding. Furthermore, it is clear that this Arg residue does not interfere with Potl binding, consistent with the observation that Potl is a much more efficient inhibitor of insect chymotrypsins than the NaPI inhibitors. Large quantities ofthe Potl inhibitor were purified from potato tubers (Figure 22) to evaluate the combined effect of NaPI and Potl on the growth of H. armigera larvae ( Figure 23). Bioassays confirmed that Potl significantly enhances the activity of the NaPI inhibitors.
- Caterpillars fed NaPI and Potl in combination (0.26 and 0.34% (w/v), respectively) were 34%» the size of control larvae at the fifth instar stage of development whereas caterpillars feeding on NaPIs alone were about 84% the size ofthe controls.
- another aspect of the present invention provides a method for modulating activity ofthe ⁇ pCh5 or a homolog or variant thereof in an insect, said method comprising contacting the HpCh5 protein or its homolog or variant with an effective amount of an agent for a time and under conditions sufficient to decrease or increase HpCh5 activity.
- Yet another aspect of the present invention provides a method for modulating expression of HpF5 or homolog or variant in an insect, said method comprising contacting HpF5 or its homolog or variant with an effective amount of an agent for a time and under conditions sufficient to decrease or increase HpF5 expression.
- plants to be protected include those sensitive to H. armigera, H punctigera, H. zea andH virescens.
- Such plants include the H armigera sensitive plants such as cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant), geraniums, cape gooseberry, maize, flax and linseed, alfalfa, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds and Helichrysum (an ornamental plant)
- plants contemplated herein include cereals (such as wheat, barley, oats, triticale, etc.), horticultural plants (e.g. apples, carrots, onions, etc.), ornamental plants (such as orchids, roses, petunias, etc.) and trees.
- cereals such as wheat, barley, oats, triticale, etc.
- horticultural plants e.g. apples, carrots, onions, etc.
- ornamental plants such as orchids, roses, petunias, etc.
- the present invention contemplates, therefore, methods of screening for compounds which inhibit or act as antagonists of HpCh5 activity or HpF5 gene expression.
- one method involves contacting a candidate compound with HpCh5.
- the screening procedure includes assaying (i) for the presence of a complex between the compound and HpCh5, or (ii) an alteration in the expression levels of HpF5 cDNA or genomic DNA.
- One form of assay involves competitive binding assays. In such competitive binding assays, HpCh5 is typically labeled. Free HpCh5 is separated from any putative complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to bind to HpCh5.
- Another technique for agent screening provides high throughput screening for compounds having suitable binding affinity to HpCh5 and is described in detail in Geysen (International Patent Publication No. WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HpCh5 and washed. Bound HpCh5 molecules are then detected by methods well known in the art. This method may be adapted for screening for non-peptide, chemical entities. This aspect, therefore, extends to combinatorial approaches including phage display to screen for HpCh5 antagonists.
- Purified HpCh5 can be coated directly onto plates for use in the aforementioned agent screening techniques. However, non-neutralizing antibodies to HpCh5 may also be used to immobilize HpCh5 on the solid phase. Live animals such as H armigera and/or H. punctigera may also be used in feeding trials to find potential inhibitors.
- the present invention also contemplates the use of competitive agent screening assays in which neutralizing antibodies capable of specifically binding ⁇ pCh5 compete with a test compound for binding to HpCh5 or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of HpCh5.
- Yet another useful source of analogs of compounds which are chemically modified may be used to induce feed-back inhibition of biochemical or genetic pathways for generating authentic HpCh5.
- HpCh5 contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on HpCh5.
- side chain modifications of HpCh5 contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .
- modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid
- the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
- the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
- Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
- Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides.
- Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
- Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate .
- Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
- a list of unnatural amino acid, contemplated herein is shown in Table 4. TABLE 4 Codes for non-conventional amino acids
- Non-conventional Code Non-conventional Code amino acid amino acid
- D- ⁇ -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D- ⁇ -methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
- D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
- peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
- Such analogs especially if they retain activity or even the HpCL5 molecule itself may have indistinct applications such as in washing powder or as in a stain removal formulation.
- a compound antagonist includes a variant of HpCh5 such as a variant comprising an analog amino acid residue as indicated above.
- the target is the HpCh5 polypeptide.
- polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as those given in Table 4) or polypeptides with substituted linkages.
- a substance identified as an antagonist of HpCh5 function or HpF5 gene activity may be a peptide or non-peptide in nature.
- Non-peptide "small molecules" are often preferred for many agricultural or horticultural purposes due to their perceived stability.
- agrichemicaphore Once the agrichemicaphore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a agrichemicaphore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
- HpCh5 the three-dimensional structure of HpCh5 and a compound binding it. This can be especially useful where HpCh5 or its antagonist change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate inhibitors which interact with the linear sequence or a three-dimensional configuration.
- a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
- the template molecule and the chemical groups grafted onto it can conveniently be selected so that the agrichemicaphore is easy to synthesize and is likely to be agriculturally or horticulturally acceptable.
- the agrichemicaphore is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity.
- the agrichemicaphore or agrichemicaphores found by this approach can then be screened to see whether they have HpCh5 antagonistic property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final agents for testing.
- Yet another aspect of the present invention provides a method for detecting an agent capable of binding or otherwise associating with a HpCh5 binding site or functional equivalent thereof said method involving the use of in-silico 3-D modeling to identify compounds that bind to HpCh5 and specifically, are not interfered with by Arginine 192.
- HpCh5 antagonist design is to produce structural analogs of HpCh5 or of small molecules with which HpCh5 interacts (e.g. an antagonist or inhibitor) in order to fashion agents which are, for example, more inhibitory of HpCh5.
- HpCh5 e.g. an antagonist or inhibitor
- one first determines the three- dimensional structure of HpCh5 by x-ray crystallography, by computer modeling or, most typically, by a combination of approaches.
- Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of chymotrypsins.
- Target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol. 202: 2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide 's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions ofthe peptide.
- compounds including antagonists may be directed to particular locations or regions or domains or HpCh5.
- Chymotrypsin clone HpF2B is expressed in E. coli fused to a six histidine (6.H) tag at the C-terminus and is purified to homogeneity on Talon metal affinity resin ( Figure 20) for injection into a rabbit for production of polyclonal antibodies. N-terminal sequencing of the purified product confirmed the expression of the chymotrypsin HpCh2B.
- the serum is collected and tested on protein blots of bacterially expressed protein and unfractionated gut extracts. The antibody detected the full-length recombinant chymotrypsin at a dilution of 1 in 2500 as well as several break-down products. Unfractionated gut extract and a sample of protein bound to the CI affinity column were also stained with the anti-HpCh2B antibody which detected the mature native form ofthe enzyme.
- Purified 6H.HpCh2B is used to test the detection limit of anti-HpCh2B antibody by comparison of immunoblots to silver stained SDS-PAGE gels.
- the antibody detected 20 ng of bacterially expressed chymotrypsinogen and also recognized the mature form of the native chymotrypsin isolated from gut ofH. punctigera.
- the cDNA ( ⁇ pF5) encoding the NaPI-insensitive chymotrypsin (HpCh5) is expressed in E. coli in a similar manner except the six-histidine tag is fused to the N-terminus of the expressed protein.
- the polyclonal antiserum that is raised against the bacterially expressed chymotrypsin HpCh2B did not cross react with bacterially expressed NaPI- insensitive chymotrypsin (HpCh5) on protein blots ( Figure 21). Likewise the antiserum raised against HpCh5 did not bind to HpCh2B. This indicates that these antisera can be used to specifically distinguish between and monitor levels of the NaPI -insensitive and sensitive chymotrypsins in unfractionated gut extracts
- Still another aspect of the present invention is directed to antibodies to HpCh5 and HpCh2B including catalytic antibodies.
- a method for the isolation of and separation of individual isoforms of chymotrypsin consisting of:
- the present invention extends to a genetic approach to down-regulating expression of an HpF5 or its homologs or variants.
- Such an approach uses nucleic acid molecules or molecules having a genetic component (e.g. R ⁇ Ai) to induce pre- or post-transcriptional gene silencing.
- nucleic acids include R ⁇ A, cD ⁇ A, genomic D ⁇ A, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
- modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g.
- synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
- morpholinos are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development 7: 187-195, 1997). Such compounds are injected into embryos and the effect of interference with mRNA is observed.
- the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding HpCh5, i.e. the oligonucleotides induce transcriptional or post-transcriptional gene silencing.
- This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the inhibitor.
- the oligonucleotides may be provided directly to a cell or generated within the cell.
- target nucleic acid and “nucleic acid molecule encoding HpCh5" have been used for convenience to encompass DNA encoding the inhibitor, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
- RNA including pre-mRNA and mRNA or portions thereof
- cDNA derived from such RNA.
- antisense The hybridization of a compound ofthe subject invention with its target nucleic acid is generally referred to as "antisense”.
- antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
- the functions of DNA to be interfered with can include replication and transcription.
- Replication and transcription for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise.
- the functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
- hybridization means the pairing of complementary strands of oligomeric compounds.
- the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
- nucleobases complementary nucleoside or nucleotide bases
- adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
- Hybridization can occur under varying circumstances.
- An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired.
- oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
- “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
- compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
- these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops.
- the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
- RNAse H a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
- antisense compound is a single-stranded antisense oligonucleotide
- dsRNA double-stranded RNA
- oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units.
- oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
- oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those herein described.
- the open reading frame (ORF) or "coding region” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is a region which may be effectively targeted. Within the context of the present invention, one region is the intragenic region encompassing the translation initiation or termination codon ofthe open reading frame (ORF) of a gene.
- the 5' cap site of an mRNA comprises an N7 -methylated guanosine residue joined to the 5 '-most residue of the mRNA via a 5 '-5' triphosphate linkage.
- the 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.
- eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns", which are excised from a transcript before it is translated. The remaining (and, therefore, translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence.
- Targeting splice sites i.e. intron- exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites.
- fusion transcripts mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
- nucleoside is a base-sugar combination.
- the base portion of the nucleoside is normally a heterocyclic base.
- the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
- Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
- the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
- the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
- linear compounds are generally preferred.
- linear compounds may have internal nucleobase complementarity and may, therefore, fold in a manner as to produce a fully or partially double-stranded compound.
- the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
- the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
- these oligonucleotides may contain modified backbones or non-natural internucleoside linkages.
- oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
- modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
- Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most intemucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
- Various salts, mixed salts and free acid forms are also included.
- genetic constructs including DNA "vaccines” are used to generate antisense or sense molecules in plant cells. Furthermore, many of the preferred features described above are appropriate for sense nucleic acid molecules.
- a further aspect of the present invention relates to a method for control of insect populations, said method comprising administering to insects an effective amount of an agent for a time and under conditions sufficient to inhibit the expression of HpF5 or sufficient to inhibit the activity of H ⁇ Ch5, wherein said modulation results in reduction of the biological fitness of said insects.
- the agent is one that can bind to the primary substrate binding pocket of HpCh5, and not be interfered with by the arginine residue found at position 192.
- Reference to a "reduction of biological fitness” should be understood to be changes in the insect including, but not limited to, changes in body mass and/or viability. Preferably, these changes are understood as reductions in both body mass and/or viability. A reduction in biological fitness, therefore, includes a reduction in their growth and development.
- the present invention provides a method for detecting an agent capable of modulating the function of HpCh5 or functional equivalent or derivative thereof, said method comprising administering to an insect containing said HpCh5 or functional equivalent or derivative thereof with a putative agent and detecting an altered activity phenotype associated with modulation of function of HpCh5 or its functional equivalent or derivative.
- references to "administration” of the modulator to HpCh5 refers to delivery of the modulating agent in any convenient means. In the agricultural setting this is likely to include, but not be limited to:-
- the administration of the agent is via the introduction of a nucleic acid encoding said agent into a plant for subsequent expression and production ofthe agent.
- Another preferred embodiment is the formulation of a spray or powder with said agent as the active ingredient.
- nucleic acid molecules encoding an antisense or sense form of HpF5 or encoding an inhibitor of HpCh5 activity or HpF5 expression is operably linked to a promoter, generally in a vector or other suitable medium for introduction to a plant genome.
- a promoter generally in a vector or other suitable medium for introduction to a plant genome.
- an existing PI may be cloned and modified to render it active against HpCh5.
- the present invention further provides a genetically modified plant comprising cells which are capable of producing an antagonist of HpCh5 or HpF5 gene expression.
- cotton or other crop plants are engineered to produce Potl or a combination of Potl and NaPI.
- Reference herein to "Potl” and “NaPI” includes reference to derivatives, variants and homologs including modifications to one or more domains in Potl or NaPI.
- Reference herein to a "promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e.
- upstream activating sequences which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
- a promoter is usually, but not necessarily, positioned upstream or 5', or a structural gene region, the expression of which it regulates.
- the regulatory elements comprising a promoter are usually positioned within 2 kb ofthe start site of transcription ofthe gene.
- Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule.
- regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecules.
- Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
- the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e. the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
- the promoter may regulate the expression of HpF5 or its variant or homolog constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst others.
- the promoter is capable of regulating expression of a nucleic acid molecule in a plant cell, tissue or organ, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression ofthe HpF5gene in said cell, tissue or organ.
- strong constitutive promoters are particularly useful for the purposes of the present invention or promoters which may be induced by virus infection or the commencement of HpF5 gene expression.
- Plant-operable promoters are particularly preferred for use in the construct of the present invention.
- suitable promoters include pCaMV 35S (Fang et al, Plant Cell 1: 141-150, 1989), PGEL1 (Hajdukiewicz et al, Plant Mol. Biol. 25: 989-994, 1994), class III chitinase (Samac and Shah, Plant Cell 3: 1063-1072, 1991), pin2 (Keil et al, EMBO J. 8: 1323-1330, 1989), PEP carboxylase (Pathirana et al, Plant J. 12: 293-304, 1997; MAP kinase (Schoenbeck et al, Molec.
- the terms "in operable connection with” or “operably under the control” or similar shall be taken to indicate that expression ofthe nucleic acid molecule is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole plant.
- the construct preferably contains additional regulatory elements for efficient transcription, for example, a transcription termination sequence.
- Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences generally containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.
- the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
- terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYC1 terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the cauliflower mosaic virus (CaMV) 35 S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any r/zo-independent E. coli terminator, or the lacZ alpha terminator, amongst others.
- SSU Rubisco small subunit gene
- SCSV subclover stunt virus
- the terminator is the SV40 polyadenylation signal or the HSV TK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissue or organs, or the lacZ alpha terminator which is active in prokaryotic cells.
- OCS octopine synthase
- NOS nopaline synthase
- lacZ alpha terminator which is active in prokaryotic cells.
- constructs described supra are capable of being modified further, for example, by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed.
- the marker nucleotide sequences will be present in a translatable format and be expressed.
- the constructs ofthe present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others.
- a nucleic acid e.g. HpF5
- a suitable vector or episome molecule such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
- a further aspect of the invention provides a genetic construct which at least comprises a genetic element as herein described and one or more origins of replication and/or selectable marker gene sequences.
- an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a plant cell, or integrated into the genome of a plant cell.
- selectable marker gene includes any gene which confers a phenotype on a cell on which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
- Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Amp r ), tetracycline-resistance gene (Tc 1 ), bacterial kanamycin-resistance gene (Kan 1 ), the zeocin resistance gene (Zeocin is a drug ofthe bleomycin family which is trade mark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gen (npt ⁇ l), hygromycin-resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the luciferase gene, amongst others.
- Amicillin-resistance gene Amicillin-resistance gene
- Tc 1 tetracycl
- the selectable marker gene is the nptll gene or Kan r gene or green fluorescent protein (GFP)-encoding gene.
- GFP green fluorescent protein
- the present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.
- Standard methods described supra may be used to introduce the constructs into the cell, tissue or organ, for example, liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells, cell mating, transformation or transfection procedures known to those skilled in the art.
- Additional means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCl 2 and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, elecfroporation, microinjection of DNA, microparticle bombardment of tissue explant or cells, vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA- mediated transfer from Agrobacterium to the plant tissue.
- a microparticle is propelled into a cell to produce a transformed cell.
- Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050).
- the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
- microparticles suitable for use in such systems include 1 to 5 ⁇ m gold spheres.
- the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
- the genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed.
- Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will generally be required.
- the present invention further extends to an isolated cell, tissue or organ comprising the constructs or parts thereof.
- the present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof as well as seeds and other reproductive material.
- plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g. all cells transformed to contain the expression cassette); grafts of transformed and unfransformed tissue (e.g. a transformed root stock grafted to an unfransformed scion in citrus species).
- Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
- first generation (or TI) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
- Plants contemplated herein include cotton, sweet com, tomato, tobacco, piniento, potato, sunflower, citms, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant), geraniums, cape gooseberry, maize, flax and linseed, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum (an ornamental plant), wheat, barley, oats, triticale, carrots, onions, orchids, roses and petunias
- Another aspect of the present invention relates to the insensitive chymotrypsin as a selectable marker for the transformation of insects.
- methods for the germ-line transformation of insects involves injection of insect embryos with a genetic constract comprising a transposable element, the gene of interest and a selectable marker.
- Somatic transformation of insects can also be achieved using viral vectors that include the gene of interest and a selectable marker (Peloquin et al., J. Cot. Sci. 5: 114-120, 2001).
- selectable markers are genes that complement the white-eye mutation or Enhanced Green Fluorescent Protein (EGFP).
- EGFP Enhanced Green Fluorescent Protein
- the white eye mutation is caused by mutant alleles such as the ra 1118 allele.
- mutant alleles such as the ra 1118 allele.
- These individuals can be returned to normal (red) eye pigmentation via the introduction of an allele conferring normal eye pigmentation such as white (Lidholm et al. , Genetics 134: 859-868, 1993) or miniwhite (Lozovskaya et al, Genetics 142: 173-177, 1996).
- the reversion of ro 1118 mutants to normal eye pigmentation acts as the marker for introduction of the vector.
- EGFP has been used to indicate the presence of a vector. Again, insects with non-pigmented eyes are used, and EGFP expression is detected in the eyes of these insects (Hediger et al, Insect Mol. Biol 10: 113-119, 2001).
- the selectable markers commonly used in the art require dissection of the insects to examine the eyes for either pigmentation or EGFP fluorescence, which is time consuming and requires destructive sampling of the insects.
- the present invention provides a means for the selection of transformed individuals without the need for insect dissection or inspection of individual insects. This would allow the recovery of live transformants and provides a non-laborious means of screening large numbers of putative transformants at one time.
- the present invention provides a means for the selection of transformants that does not rely on the availability of white-eye mutants.
- the present invention contemplates the use of HpF5 or a derivative, homolog or analog thereof encoding a NaPI-insensitive chymotrypsin, as a selectable marker in an insect transformation vector.
- This vector comprising HpF5
- the contemplated insects may be naturally resistant to CI, or may be ⁇ aPI- susceptible mutants or genetically modified ⁇ aPI susceptible strains of naturally ⁇ aPI- resistant insects.
- the insect host of the said vector would be Lepidopteran.
- HpF5 or a derivative, homolog or analog thereof encoding an NaPI-insensitive chymotrypsin could be incorporated into any insect transformation vector using common molecular biology techniques known to those in the art. Upon transformation the insect would transcribe HpF5 and produce HpCh5, the NaPI-insensitive chymotrypsin. Transformed individuals could then be selected by incorporation of the CI proteinase inhibitor of N. alata into the diet of the insects. In this case individuals that did not carry the insensitive chymotrypsin encoded by HpF5 in the vector would die, and those that did carry the vector encoding HpF5 would be insensitive to CI .
- insect transformation vectors including baculovirus vectors comprising HpF5 or a derivative, homolog or analog thereof, as a selectable marker.
- the vector may be used for any purpose in the insect. ⁇ on-limiting examples include: gene cloning, gene expression and gene knockouts.
- Specific examples of insect transformation vectors into which HpF5 could be incorporated as a selectable marker include, but are not limited to: those that utilize the piggyBac mobilizable element (Hediger et al, 2001, supra); P-element based vectors (Cripps et al, J. Cell Biol.
- hobo element based vectors Lobo element based vectors (Lozovskaya et al, 1996, supra); mariner element based vectors (Lidholm et al, 1993, supra); and viral vectors such as pTE/3'2J (Peloquin et al, 2001 , supra).
- Haricot bean diet Helicoverpa punctigera larvae were raised on artificial diets based on Haricot beans (Teakle et al, Journal of Invertebrate Pathology 46: 166-173, 1985).
- One litre of diet was composed of 58.5 g Haricot beans, 14 g agar, 700 ml water, 35 g Tortula yeast, 50 g wheatgerm, 3.5 g ascorbic acid, 1.1 g sorbic acid, 2.2 g p-hydroxybenzoic acid methyl ester, 0.2 g ampicillin, 0.2 g streptomycin, 16 mg prochloraz.
- the beans were soaked overnight in water, drained and homogenized to a fine paste.
- the larvae were reared in 1.5 ml eppendorf microfuge tubes (one larva/tube) until day eight when they were transferred to individual plastic containers with lids (SOLO [trademark] plastic portion cups, 28 mL). Larvae were fed small amounts of diet (40 mg) initially that was replaced as required to provide a continuous supply. The larvae were kept in a temperature controlled room at 25 ⁇ 1°C, 16:8 (L:D).
- Cotton leaf artificial diet was prepared from fresh young leaves from cotton plants (cultivar Coker 315) which were grown in an insect-free and insecticide-free temperature controlled cabinet at 26°C ( ⁇ 2°C) with a light regime of 16:8 (L:D). Following picking, the leaves were immediately frozen in liquid nitrogen and freeze dried. After drying, the leaves were ground to a fine powder in a mortar and pestle.
- the cotton leaf artificial diet was prepared in the same manner as haricot bean artificial diet using a recipe modified from potato leaf artificial diet (Gatehouse et al, J. Insect Physiol. 45 (6), 545-558, 1999).
- One hundred grams of cotton leaf artificial diet contained 3 g of cotton leaf powder, 0.08 mL linseed oil, 2 g yeast, 0.016 mL wheatgerm oil, 2.4 g wheat germ, 0.028 g ampicillin, 3.2 g ascorbic acid, 0.028 g streptomycin, 0.08 g sorbic acid, 3.2 g agar, 0.16 g paraben (mould inhibitor) plus NaPI or casein to required % ((w/v)).
- Gut proteinase activity was determined at pH 10 in 50 mM 3-(cyclohexylamino)-l- propanesulfonic acid (CAPS buffer) using the chromogenic substrate N-benzoyl-DL- arginine-p-nitroanilide (BApNA) for trypsin and N-succinyl-L-alanine-alanine-proline- phenylalanine-p-nitroanilide (SA 2 PFpNA) for chymotrypsin activity. Substrates were freshly prepared as 1 mM solutions in 10% ((w/v)) N,N-dimethylformamide (DMF), 50 mM CAPS buffer, pH 10.
- DMF N,N-dimethylformamide
- Trypsin and chymotrypsin inhibition assays were conducted using the standard trypsin and chymotrypsin assays described above except samples were pre-incubated with TI or CI inhibitor (80nM) for 30 min at 30°C prior to the addition of substrate to initiate the reaction.
- TI or CI inhibitor 80nM
- NaPI monomers TI and CI were HPLC purified as described by Heath et al, 1999, supra.
- Unfractionated gut extract containing approximately 1 ⁇ g of buffer soluble protein was added to 10 ⁇ L CAPS buffer (0.5 M, 3-[cyclohexylamino]-l-propane-sulfonic acid, pH 10) and made to a final volume of 100 ⁇ L in individual wells of a 96 well microtiter plate. Proteinase inhibitors were added over a range of concentrations and incubated for 15 min at 25°C. Bovine chymotrypsin (100 ng) was used as a positive control, both for activity and inhibition.
- Potato Inhibitor I was purchased from Calbiochem-Novabiochem or purified from potato tubers (Example 6). A crude mixture of Potato inhibitor I and II was also obtained from potatoes. N. alata proteinase inhibitors ( ⁇ aPIs) were purified as described by Atkinson et al., 1993, supra and Heath et ah, 1995, supra. The chymotrypsin inhibitor CI was purified from bacterial expression cultures. The purity of the inhibitors was assessed by SDS- PAGE and silver staining.
- the midgut was dissected from 80 fourth instar larvae and buffer soluble extracts were prepared.
- the gut extract was depleted of trypsins by repeated passage through a benzamidine-Sepharose affinity column.
- the unbound protein was collected and applied to an affinity column composed ofthe immobilized chymotrypsin inhibitor CI ( Figure 1) that had been produced using a bacterial expression system. This column was expected to specifically bind NaPI-sensitive chymotrysins. Proteins that did not bind to this column were applied to a third affinity column composed of either immobilized Potato Type I (Potl) and Type II inhibitors (Potll) or chymostatin.
- This column was designed to capture the chymotrypsins that did not bind to the CI column, that is, the NaPI-insensitive chymotrypsins. Proteins that bound to the affinity columns were eluted with 8 M urea and were subjected to electrophoresis through an SDS-polyacrylamide gel before transfer to a PVDF membrane for N-terminal sequencing ( Figures 4 and 5).
- the midgut was dissected from 100 fourth instar larvae and buffer soluble extracts were prepared.
- the gut extract was depleted of NaPI-sensitive proteases by passage through an affinity column composed of immobilized NaPI protein (CI, C2, T1-T4). All trypsins and NaPI-sensitive chymotrypsins bound to the column and the NaPI-insensitive chymotrypsin was unbound. This preparation of unbound material was used to study the pH optimum, substrate preference and effect of a range of proteinase inhibitors on the activity of the NaPI-insensitive proteinase. The effect of pH on activity of the insensitive chymotrypsins is illustrated in Figure 6.
- the enzyme is inactive below pH6 and is most active at pH10-12 consistent with its role in the alkaline midgut of larvae.
- the best substrate for enzyme assays was determined using seven different commercial substrates. The best substrate was N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (sAAPF-pNA) followed by N-succinyl-Ala- Ala-Pro-Leu-p-nitroanilide (sAAPL-pNA) and N-methoxysuccinyl-Ala-Ala-Pro-Met-p- nitroanilide (mAAPM-pNA).
- N-succinyl-Ala-Ala-Ala-p-nitroanilide sAAA-pNA
- benzoyl-tyr- p-NA Ac-Pro-Leu-Ser-p-NA
- Ac-Asn-Gly-Ile-Pro-p-NA were not substrates.
- the inhibitors were preincubated with 100 ng of bovine chymotrypsin or the amount of the gut chymotrypsin required to produce the same absorbance as 100 ng of bovine chymotrypsin (note that the Helicoverpa enzyme has been depleted of trypsins and sensitive chymotrypsins, but still contains other gut proteins) for 30 min at 30°C before the addition of substrate. The incubation was continued for a further 30 min at room temperature before absorbance was measured at 405 nm. Potl was the best ofthe proteinaceous inhibitors and NaPI did not inhibit (Table 3).
- Benzamidine-agarose (1 mL; was purchased from ICN Biomedicals) and contained 35 umoles benzamidine per ml of gel.
- the PCR product was sub-cloned into the pCR (registered trademark) 2.1-TOPO vector (Invitrogen) then excised with BamHl and Hindlll and ligated into the pQE-30 vector.
- the expression vector incorporated a hexahistidine tag at the N-terminus of the expressed protein for metal affinity purification.
- the Cl/pQE-30 constract was transformed into the chemically competent E. coli strain Ml 5 (Qiagen) prepared according to the method of Inoue et ⁇ l, Gene 96: 23-28, 1990. Bacterial expression cultures were grown and induced according to the procedures outlined in the QiaExpress manual (Qiagen).
- Potato Inhibitor I and potato Inhibitor II affinity column Cyanogen bromide-activated Sepharose 4B (1 g) was swollen and washed according to the manufacturer's protocol. A mixture of potato I and II Inhibitors (10 mg) was dissolved in coupling buffer [0.1 M NaHC0 3 , 0.5 M NaCI, pH 8.3], combined with the gel suspension and incubated over night at 4°C in an end-over-end mixer. The gel was rinsed several times in blocking buffer [0.1 M Tris-HCI, pH 8.0] to remove excess ligand. Following the washes the conjugated Sepharose was mixed with fresh blocking buffer and incubated overnight at 4°C.
- the gel slurry was transferred to a column (Amersham Biosciences) and washed alternately (x3) with five column volumes of coupling buffer then five column volumes of rinse buffer [0.1 M NaOAc, 0.5 M NaCI, pH 4.0]. Finally the matrix was extensively washed with coupling buffer and stored in 20% (v/v) ethanol at 4°C.
- Chymostatin (Sigma-Aldrich) was immobilized on EAH Sepharose-4B (1.5 ml; Sigma- Aldrich) according to the manufacturer's instructions. Chymostatin (10 mg) was dissolved in 500 ⁇ L of glacial acetic acid then 1 mL of distilled water was added and the pH adjusted to 4.5 with dilute NaOH. The gel suspension and chymostatin solution were combined before EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide] was slowly added to a final concentration of 0.1 M.
- the filtered gut extract was passed through the benzamidine column five times to remove the trypsins.
- the unbound fraction (10 mL) was passed through the CI affinity column (x3) before the column was washed with 20mL of extraction buffer and bound proteins were eluted with 8 M urea, pH 8.0 (5 mL). Proteins that did not bind to the CI column were applied to the Potato Inhibitor I and II affinity column prior to washing with 10 column volumes of buffer [20 mM CAPS pH 10, 0.5 M NaCI] and elution of bound proteins with 8 M urea, pH 8.0 (5 mL).
- transfer buffer 192 mM glycine, 48 mM Tris-base, 20% (v/v) methanol
- transfer buffer 192 mM glycine, 48 mM Tris-base, 20% (v/v) methanol
- transfer buffer 192 mM glycine, 48 mM Tris-base, 20% (v/v) methanol
- transfer buffer 192 mM glycine, 48 mM Tris-base, 20% (v/v) methanol
- Membranes were briefly washed in TBS (20 mM Tris-HCI, 150 mM NaCI, pH 7.5) then stained with amido black (1:50 dilution of 0.1% (w/v) amido black, 40% (v/v) methanol, 10% (v/v) acetic acid) to confirm transfer of the proteins and to visualize the molecular size markers.
- Blots were then blocked by incubation with 3% (w/v) skim milk powder (Dutchjug) in TBST [0.1% (w/v) Tween-20 in TBS] for 1 h at RT, followed by a 1 h incubation with the ⁇ -chymotrypsin antibody (H ⁇ CH2B; 1 :5000 dilution in 3% (w/v) skim milk powder in TBST).
- the nitrocellulose blots were then rinsed three times (5 min) in TBST and incubated for 1 hour at RT with the secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:5000 in TBST; Amersham Biosciences).
- FIG. 7 shows the predicted proteins and the regions complementary to the oligonucleotides ( Figure 8) chosen for PCR amplification of chymotrypsin cDNAs from H. punctigera.
- the PCR products were cloned and sequenced, and five distinct chymotrypsin sequences were obtained (FlApcr, FlBpcr, F2Bpcr, F3pcr and F4pcr, Figure 9). These PCR products were used to screen a cDNA library prepared from midgut mRNA isolated from late fourth instar and early fifth instar larvae.
- the N-terminal sequence obtained from the NaPI inhibitable chymotrypsin ( Figure 4) matched the N-terminal sequence predicted from the cDNA clones encoding chymotrypsins from family 2.
- the N- terminal sequence of the NaPI -insensitive chymotrypsins was not represented in the 4 families of chymotrypsins represented by the cD A clones.
- Protein sequences most similar to H punctigera were obtained using the BLAST search engine. Genbank accession numbers are listed. The mature activated proteins were compared and percentage of protein sequence identity determined.
- the N-terminal region of the insensitive chymotrypsins had two unique stretches of sequence, designated FI and F2 that were used to design oligonucleotides for PCR amplification of DNA encoding one ofthe insensitive chymotrypsins ( Figure 11)
- FI and F2 Two unique stretches of sequence
- F2 oligonucleotides for PCR amplification of DNA encoding one ofthe insensitive chymotrypsins
- Figure 11 A 641 bp fragment of DNA was obtained with the FI oligonucleotide that encompassed most of the protein-coding region.
- This PCR product was used to screen for a full length clone in the H punctigera midgut library. Approximately 0.2% of the 50,000 plaques screened hybridized strongly to the PCR product. Ten plaques were selected and and a full length clone was identified and sequenced (Figure 12).
- the 921 bp clone had an open reading frame of 828 bp and residues 41-76 of the deduced protein were an identical match to the N-terminal sequence obtained from the purified protein ( Figure 12).
- the clone lacked the 5 'signal peptide sequence, but comparison to the other chymotrypsin clones indicated the activation peptide was full length.
- the active enzyme is predicted to be 236aa in length, with a mass of 24.2 kDa. Protein sequence homology was determined by comparing the mature domains of each of the H. punctigera cDNAs. Family 2 and family 3 were most similar to ⁇ pCh5 (Rechla), with about 72 and 70% identity respectively.
- Bovine chymotrypsins A and B and the NaPI-sensitive chymotrypsin (HpCh2A) were compared to the insensitive chymotrypsin (HpCh5) to identify regions of variability that may be involved in low affinity binding to CI ( Figure 15).
- Chymotrypsin genes from Helicoverpa armigera were aligned using ClustalW and regions of high conservation were identified for design of chymotrypsin specific primers. Primers were individually designed to the H. armigera clone (CAA72951) due to the high level of divergence to the other chymotrypsin sequences. Forward primers were used in combination with RVG4 to amplify the gene fragments using reverse transcriptase polymerase chain reaction (RT-PCR; Superscript Preamplification System, Gibco BRL) with the protocols supplied.
- RT-PCR reverse transcriptase polymerase chain reaction
- PCR products were gel purified (Qiagen gel extraction kit), sub-cloned into TOPO PCR2.1 TA cloning vector (Invitrogen) and transformed into chemically competent E. coli XL 1 -Blue cells (Stratagene) prepared according to the method of (Inoue et al, 1990, supra). Plasmid DNA was prepared using the QIAprep (registered trademark) Spin Miniprep Kit (Qiagen).
- PolyA+ mRNA was isolated from the RNA (1.0 mg) using conventional protocols.
- Purified mRNA (5 ⁇ g) was used to constract a cDNA library with the Lambda ZAP-cDNA synthesis kit and the Zap- cDNA Gigapacklll Gold packaging extract (Stratagene) following the manufacturer's instructions.
- the amplified library titre was 2.8 x 10 10 pfu/rnL.
- RT-PCR products 50 ng were individually labelled with [ ⁇ - 32 P] dCTP (Amersham Life Sciences) using the MEGAPRIME (trademark) DNA labeling system labeling (Amersham Life Sciences). Unincorporated radiolabeled nucleotides were removed using the Micro Bio-spin P-30 chromatography columns (BioRad) according to the manufacturer's protocol. The double stranded labelled probes were denatured by boiling (5 min), cooled on ice, and then added to the hybridization solution (as below). Screening the cDNA library
- the primary screen was performed using ten (15 cm) Petri dishes per probe, with about 50,000 phage per plate. Preparation of the plates for subsequent plaque lifts and treatment of the membranes prior to hybridization was performed according to instructions provided with the Lambda Zap cDNA synthesis kit (Stratagene).
- the membranes (Hybond-N; Amersham Biosciences) were prehybridized in 50 ml of 2 x PIPES (0.8 M NaCI, 16 mM Piperazine-l,4-bis(2-ethanesulphonic acid) pH 6.5) 50% (v/v) formamide, 0.5% (w/v) SDS and 100 ⁇ g/mL Herring sperm DNA (Boehringer Manningham) at 42°C for 3 hours.
- the incubation solution was replaced with 50 ml of 50% (v/v) formamide, 2 x PIPES buffer, 0.5% (w/v) SDS and 100 ⁇ g/mL denatured herring sperm DNA containing the labeled probe and left overnight at 42°C.
- the hybridization membranes were washed three times in 2 X SSPE/0.1% (w/v) SDS at room temperature and twice in 0.2 X SSPE/0.1%) (w/v) SDS at room temperature, before they were blotted on 3 mm Whatman paper and exposed to X-ray film (Kodak XAR-5) for 48 hours at -70°C with intensifying screens.
- At least 50 positive plaques of varying intensities were selected from each screen probed with an individual RT-PCR product.
- Each plaque was transferred to a 1.5 mL microfuge tube containing 1 mL of SM buffer (0.1 M NaCI, 8 mM MgS0 4 .7H 2 0, 50 mM Tris-HCI, pH 7.5, 0.01% (w/v) gelatin) and chloroform (20 ⁇ L) and stored at 4°C.
- SM buffer 0.1 M NaCI, 8 mM MgS0 4 .7H 2 0, 50 mM Tris-HCI, pH 7.5, 0.01% (w/v) gelatin
- chloroform 20 ⁇ L
- cDNA clones were grouped on the basis of restriction fragment patterns obtained using combinations of the endonucleases BamHl, Xhol ⁇ , Kpnl, Sacl, Sac ⁇ , and Sail (Promega).
- RT-PCR products and cDNA inserts were sequenced in both directions using Ml 3 universal primers at either Micromon sequencing facility at Monash University (Melbourne) or SUPAMAC at the Royal Prince Alfred Hospital in Sydney.
- the sequence data was edited using the BioEdit v5.0.9.1 software written by Tom Hall, North Carolina State University freely available at the web address: www.mbio.ncsu.edu/BioEdit/ bioedit.html.
- PCR products were separated on 1% (w/v) agarose gel (SEAKEM (registered trademark); BioWhittaker Molecular Applications) and a band of approximately 650 bp was excised and purified using the Concert purification system (Gibco).
- SEAKEM registered trademark
- BioWhittaker Molecular Applications a band of approximately 650 bp was excised and purified using the Concert purification system (Gibco).
- the partial cDNA was cloned into the TOPO PCR2.1-TA vector (Invitrogen) and transformed into E. coli strain XL-BL1 (Stratagene).
- the modeled structure consists of the classic serine protease fold consisting of two, six-stranded anti-parallel ⁇ barrels with the catalytic triad located between the two domains.
- Two surface loops, 60 and 142 are considerably larger in the H punctigera chymotrypsins ( Figures 15 and 16). Due to the limitations of modelling, a small amount of ambiguity was present in several surface loops, some of which are cleaved in mammalian chymotrypsins (loop 142), but remain intact within insect chymotrypsins.
- CI was modeled in complex with sensitive and insensitive chymotrypsins to investigate whether substitution of glutamine (or asparagine) 192 (Greer nomenclature, Figure 15) with an arginine would affect the binding capacity of the Helicoverpa chymotrypsins.
- the stracture of the chymotrypsin inhibitor (CI) was previously determined by IH NMR (Nielson et al, 1994, supra). No stractures of CI complexes have been solved and therefore the related proteinase inhibitor PCI-1 from Solanum tuber osum in complex with Proteinase B from Streptomyces griseus (Greenblatt et al., 1989, supra) provided a basis for structural modeling.
- Figure 17 illustrates the binding region surrounding Gin 192 in the Cl-HpF2B chymotrypsin complex.
- the predicted model of HpF2B and CI shows that glutamine 192 is not in conflict with any regions on the inhibitor molecule. Comparison to the cognate arginine residue in the insensitive chymotrypsin however suggests there is limited space to accommodate this much larger residue ( Figure 18).
- the NaPI-insensitive chymotrypsin from Helicoverpa species has an arginine in place of an asparagine or glutamine at position 192 that extends into the SI binding pocket and appears to interfere with CI binding. Furthermore, it is clear that this arginine residue does not interfere with Potl binding, consistent with the observation that Potl is a much more efficient inhibitor of insect chymotrypsins than the NaPI inhibitors.
- Chymotrypsin clone HpF2B was expressed in E. coli fused to a six histidine (6.H) tag at the C-terminus and was purified to homogeneity on Talon metal affinity resin ( Figure 20) for injection into a rabbit for production of polyclonal antibodies. N-terminal sequencing of the purified product confirmed the expression of the NaPI inhibitable chymotrypsin (HpCh2B). After the fourth boost with antigen, the serum was collected and tested on protein blots of bacterially expressed protein and unfractionated gut extracts. The antibody detected the full-length recombinant chymotrypsin at a dilution of 1 in 2500 as well as several break-down products. Unfractionated gut extract and a sample of protein bound to the CI affinity column were also stained with the anti-HpCh2B antibody which detected the mature native form ofthe enzyme.
- Purified 6H.HpCh2B was used to test the detection limit of anti-HpCh2B antibody by comparison of immunoblots to silver stained SDS-PAGE gels.
- the antibody detected 20 ng of bacterially expressed chymotrypsinogen and also recognized the mature form of the native chymotrypsin isolated from gut ofH. punctigera.
- the cDNA ( ⁇ pF5) encoding the NaPI-insensitive chymotrypsin (HpCh5) was expressed in E. coli in a similar manner except the six.histidine tag was fused to the N-terminus of the expressed protein.
- the polyclonal antiserum that was raised against the bacterially expressed NaPI inhibitable chymotrypsin (HpCh2B) did not cross-react with bacterially expressed NaPI-insensitive chymotrypsin (HpCh5) on protein blots ( Figure 21). Likewise the antiserum raised against HpCh5 did not bind to HpCh2B. This indicates that these antisera can be used to specifically distinguish between and monitor levels of the NaPI- insensitive and sensitive chymotrypsins in unfractionated gut extracts.
- the amplified region consisted of the pro-peptide and mature domain of the chymotrypsinogen, but lacked the putative secretion signal.
- Digests using Ncol and Bgll ⁇ enzymes (Promega) were performed on the PCR amplified product and the pQE-60 expression vector (Qiagen).
- the pQE-60 vector provides a His-tag at the C-terminus ofthe expressed protein.
- Each restriction digest was purified using WIZARD (registered trademark) D ⁇ A clean up system (Promega) and the vector and chymotrypsin insert were subsequently ligated using standard molecular biology techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition 2001) The ligation mix was heated at 65°C for 10 min before being transformed into the E. coli strain XL 1 -Blue. Plasmid D ⁇ A was prepared for sequencing and subsequent transformation into the E. coli cell line Ml 5 (Qiagen) using the QIAPREP (registered trademark) Spin Miniprep Kit (Qiagen).
- chymotrypsinogen was performed under denaturing conditions according to the methods detailed in the QiaExpress manual (Qiagen). The purity of the expressed chymotrypsinogen was assessed by SDS-PAGE and the identity of the recombinant protein confirmed by N-terminal sequencing.
- Preparation of the chymotrypsin for injection consisted of removal of the urea by dialysis against 50 mM Tris-HCI pH 8.0. During this process most of the protein aggregated. The aggregated protein was collected by centrifugation and resuspended in 1 mL of 50 mM Tris-HCI, pH 8.0 for injection. The protein concentration was approximated by the comparison of a 10 ⁇ L sub-sample to a series of bovine trypsin standards (Sigma) using SDS-PAGE and Coomassie staining.
- the cDNA clone ⁇ pF5' which encodes the NaPI-insensitive chymotrypsin ( Figure 12) was PCR amplified essentially as described for clone HpF2B above except the forward primer (FwpMalRECH ) incorporated a Histidine tag at the N-terminus of the expressed protein and the reverse primer (RvRECH) contained a stop codon and thus prevented incorporation of a Histidine tag at the C-terminus from the pQE-60 expression vector.
- Potl genes (PotlA and PotIB) were isolated using mRNA isolated from potato tubers and wounded potato leaves. These cDNA clones were used to construct vectors for bacterial expression of the Potl proteins. The bacterially produced Potl proteins totally inhibited the
- Potato tubers (2 Kg, Solanum tuberosum var Russet Burbank) were diced and soaked overnight at 4°C in two litres of 50 mM ⁇ 2 SO .
- the tissue was homogenized in a blender and insoluble material was removed by filtration through two layers of Miracloth followed by centrifugation (13,000 rpm, 15 min, 4°C).
- the supernatant was adjusted to pH 7.8 with 10 M NaOH, heated for 30 min in boiling water and cooled before precipitated material was separated by centrifugation (13,000 rpm, 12 min, 4°C).
- Soluble proteins were precipitated with ammonium sulphate (80% saturation), collected by centrifugation and redissolved in gel filtration buffer (150 mM KC1, lOmM Tris-HCI, pH 8) before they were applied to a Sephadex G75 column (85 x 2.54cm). Elution fractions (50mL) containing Potl were identified using SDS-polyacrylamide gel electrophoresis and inhibition assays with bovine chymotrypsin. Potl containing fractions were pooled, dialysed and freeze dried. The protein in these pooled fractions was examined by reverse phase -HPLC on a system Gold HPLC (Beckman, Fullerton, CA) coupled to a 166 detector (Beckman).
- the analytical RP-HPLC was conducted on a Brownlee Aquapore RP300 C8 column (4.6 x 100mm; Perkin-Elmer).
- the protein was eluted with a linear gradient of 0-100% (v/v) buffer B (60%[v/v] acetonitrile in 0.089% [v/v] trifluoroacetic acid) at a flow rate of 1 mL min-1 over 40min ( Figure 22).
- the identity of the Potl proteins was confirmed by N- terminal sequencing and mass spectrometry and at least two Potl isoforms were identified.
- Potl cD ⁇ A sequences were subsequently amplified using gene specific primers 5' CGG-GAT-CCA- TGG-AGT-CAA-AGT-TTG-C-3* [SEQ ID NO: 14] (sense) and 5'-GCG-TCG-ACG-CTT- AAG-CCA-CCC-TAG-G-3' [SEQ ID NO: 15] (antisense) that were designed to anneal to the 5' and 3' ends of the open reading frame of the published Potl genomic sequence M17108 (Cleveland et al, Plant Mol. Biol. 8: 199-207, 1987) and included restriction sites Bam HI and Sal I respectively.
- DNA encoding Potl without the endoplasmic reticulum signal sequence (amino acids 37- 111 in StPotlA and 37-107 in StPotlB) was amplified by PCR.
- Primers used were 5'-CGG- GAT-CCA-AGG-AAT-CGG-AAT-CTG-3' [SEQ ID NO: 16] (StPotlA sense), 5'-CGG- GAT-CCA-AGG-AAT-TTG-AAT-GC-3' [SEQ ID NO: 17] (StPotlB sense) and 5'-CGA- GCT-CTT-AAG-CCA-CCC-TAG-G-3' [SEQ ID NO: 18] (StPotlA/B antisense).
- PCR products were initially cloned into the pGEM T-Easy vector (Promega) before they were excised with BamHl and S ⁇ cl and ligated into the bacterial expression vector pQE30 (Qiagen) which provides a 6x His-tag at the N-terminus ofthe expressed protein.
- the His-tagged Potl proteins were expressed in E. Coli (BR21 DE3 Cod ⁇ n Plus strain (Stratagene) for StPotlA and Ml 5 strain (Qiagen) for StPotlB).
- Cells were induced with 1 mM IPTG, harvested by centrifugation and lysed in 8 M urea, 0.1 M NaH 2 PO 4 , 0.01 M Tris-HCI, pH 8.0. Cell debris was removed by centrifugation at 10,000 g for 5 min and the His-tagged Potl was purified from the supernatant by metal-affinity chromatography on Talon resin (Clontech).
- Bound protein was eluted from the resin with 8 M urea, 0.1 M NaH 2 PO 4 , 0.01 M Tris-Cl, pH 4.0 and elution was monitored by SDS-PAGE.
- the StPotlA and SfPoffB proteins were purified further by RP-HPLC ( Figure 25) their identity was confirmed by N-terminal sequencing and mass spectrometry. Inhibition of the NaPI-insensitive chymotrypsins from H. punctigera by StPotlA and
- the inhibitory activity of StPotlA and StPotlB against the NaPI-insensitive chymotrypsins from H punctigera was determined by preincubating the NaPI-insensitive protease (10 ⁇ L) with varying amounts of StPotlA and StPotlB (0-600 nM) in 133 mM CAPS buffer, pH 10.0 at 30°C in 96 well microtitre plates. After the 30 min preincubation, the incubation was started by the addition of substrate (S A 2 PFpNA, S A 2 PLpNA or S A 2 PMpNA) to a final concentration of 1 mM in a final volume of 100 ⁇ L. Absorbance was measured at 405 nm after 30 or 60 min.
- pHEX2 Two gene constructs (pHEX2 and pHEX6) were prepared for transformation of cotton (Gossypium hirsutum).
- pHEX2 consists of a 35S promoter driving the NaPI gene with a 35S terminator, inserted into the binary vector pBIN 19 (Bevan, Nucl. Acids Research, 12: 8711-8721, 1984).
- pHEX6 consists of a 35S promoter driving the StPotlA gene with a 35S terminator inserted into the binary vector pBIN 19.
- Transgenic cotton was produced using the method of Umbek et al., Biotechnology, 5: 263- 266, 1987) with modifications. Hypocotyl sections of cotton Cv Coker 315 were co- cultivated with Agrobacterium tumefaciens strain LBA 4404 containing the required binary vector. Callus was induced on media consisting of MS salts, B5 vitamins, 3% glucose, 0.9 g/L MgCl 2 (hexahydrate), 1.9 g/L potassium nitrate, 2 g/L Gelrite, 0.1 mg/L Kinetin, 0.1 mg/L 2,4-D, 500 mg/L carbenicillin, 35 mg/L Kanamycin.
- Embryogenic callus was induced by growing the callus on the same media but without hormones. Embryos were excised and incubated on media in petri dishes (Stewart and Hsu, Planta 137: 113-117, 1977). Germinated embryos that had produced roots and true leaves were transferred to containers for further development and then transferred to soil and grown in a growth cabinet at 27°C.
- Leaves from plant 3 were used in a bioassay with H. armigera (Figure 27). While expression of either NaPI or StPotlA in the leaves only resulted in a small inhibition of larval growth compared to the control, expression of both proteins had a synergistic effect on larval growth.
- the amplification products were purified after electrophoresis on 0.7% (w/v) agarose gels before they were used for subsequent PCRs.
- HpF5 cDNA with the ER signal sequence was subcloned into the pCR (registered trademark)-2.1 TOPO vector (Invitrogen) and was sequenced at the Micromon, DNA sequencing facility, Monash University, Victoria, Australia. Recombinants with the correct sequence were digested with EcoRI and gel purified before they were digested with BamHl and Hwdlll and ligated into the pFastBac vector (Invitrogen) and transformed into E. coli XLl Blue cells.
- pCR registered trademark
- TOPO vector Invitrogen
- E. coli XLl Blue cells were screened for the presence of the ⁇ pF5/ ⁇ R cDNA in the pFastBac vector (pFastBac/HpF5/ER) by PCR and restriction digest. Minipreps were performed on positive transformants. E. coli DHlOBac competent cells containing bacmid DNA and the helper plasmid required for transposition of HpF5/ER to the bacmid DNA were thawed on ice.
- GENE PULSER registered trademark
- COLI trademark
- a sample was withdrawn and serially diluted (10 "1 , 10 "2 , and 10 "3 ) using LB medium before 100 ⁇ L of each dilution was spread evenly onto LB agar plates containing 50 ⁇ g/mL kanamycin, 7 ⁇ g/mL gentiamicin, 10 ⁇ g/mL tetracycline, 100 ⁇ g/mL 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside (X-Gal) and 40 ⁇ g/mL IPTG prior to incubation for 48 hours at 37°C.
- a pFastBac vector with no insert was treated the same way and used as a control.
- the supernatant was then transferred to a 1.5 mL microfuge tube containing 0.8 ml absolute isopropanol and was mixed by inversion. After 10 min on ice samples were centrifuged for 15 min at 14,000 x g at room temperature. The supernatant was removed and pellet was washed twice with 0.5 mL of 70% (v/v) ethanol. The sample was then centrifuged for 5 min at 14,000 x g at room temperature before the 70% (v/v) ethanol was removed and the DNA pellet was air dried. The DNA was then dissolved in 60 ⁇ L TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) and stored at -20°C until needed.
- TE buffer 10 mM Tris-HCI, 1 mM EDTA, pH 8.0
- the bacmid was examined by electrophoresis on a 0.5% (w/v) agarose gel prepared in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8) buffer at 23 V for 12 hours with ⁇ DNA/Hw ⁇ II Fragment markers (MBI Fragments).
- the bacmid was also screened using PCR with Ml 3 Forward and reverse primers.
- Solution A mini-prep of bacmid DNA into 100 ⁇ L EX-CELL (trademark) 405 media without antibiotics
- Solution B 6 ⁇ L of CELLFECTIN (registered trademark) Reagent (Gibco BRL) into 100 ⁇ L EX-CELL (trademark) 405 media without antibiotics.
- Solutions A and B were combined and incubated for 1 hour at room temperature followed by 0.8 mL of selection free media added. After the cells had attached to the plate, they were washed once with 2 mL media without antibiotics. The media was removed from cells and was replaced with the DNA containing solution. Cells were incubated for 5 hours in a 27°C incubator before the transfection mixture was aspirated and replaced with 2 mL of media containing antibiotics. Cells were incubated further at 27°C for up to 72 hours.
- ER signal sequence The original HpF5 cDNA clone did not encode an ER signal sequence. While the ER signal sequence was not required for bacterial expression it was essential for baculovirus expression. An ER signal sequence was thus constructed using the DNA sequence from chymotrypsin family 2 A which is most closely related to the chymotrypsin family 5.
- the sequence was added using PCR reactions with two primers encoding part of the ER signal sequence.
- the FwBacRECHl primer had a silent mutation to remove the BamHl restriction digest site and encoded half the ER signal sequence.
- the FwBacRECH2 primer encoded the remainder of the ER signal sequence and introduced a BamHl restriction site ( Figure 28).
- PCR using primers the FwBacRECHl and RvRECH and HpF5cDNA as the template yielded a product of ⁇ 900 bp.
- a second PCR using this PCR product together with FwBacRECH2 and RvRECH primers was performed to yield a product of -930 bp that encoded the HpCh5 protein with an ER signal sequence.
- the amplified product was subcloned into pCR (registered trademark)-2.1 TOPO vector (Invitrogen) before transfection into TOP 10 competent cells. Colonies were screened for the presence of insert using Ml 3 forward and reverse primers. Restriction digests of the isolated plasmids with EcoRI then subsequently with BamHl and Hindlll yielded a product ofthe expected size of -930 bp.
- the -930 bp fragment was subsequently ligated into the pFastBac vector to create pFastBac/HpF5/ ⁇ R.
- the presence of the HpF5 cDNA insert in the pFastBac vector was confirmed by restriction digests with BamHl and H dIII and PCR using FwBacREC ⁇ 2 and RVRECH primers (Table 9). Plasmids containing the insert were sent to Micromon for DNA sequencing. Analysis of Bacmid DNA
- the bacmid was checked for the HpF5 cDNA insert by PCR analysis with Ml 3 forward and reverse primers. The expected -3300 bp fragment was further analysed using the primers to HpF5/ER cDNA and Ml 3 to ensure the insert was in the correct orientation. The positive bacmid DNA was subsequently used for the transfection of insect cells.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Insects & Arthropods (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2004233354A AU2004233354B2 (en) | 2003-04-23 | 2004-04-23 | Insect chymotrypsin and inhibitors thereof |
BRPI0409638-0A BRPI0409638A (en) | 2003-04-23 | 2004-04-23 | isolated nucleic acid molecule, vector, genetically modified cell, isolated helicoveipa spp. chymotrypsin, antagonist, composition, genetically modified plant, seed or other reproductive material, methods for modulating hpch5 activity or a homologue or variant thereof in an insect , to modulate expression of hpf5 or homolog or variant in an insect, to isolate and separate individual chymotrypsin isoforms, to select a chymotrypsin antagonist insensitive to helicoverpa spp., and, isolated inhibitor |
US10/554,237 US7462695B2 (en) | 2003-04-23 | 2004-04-23 | Insect chymotrypsin and inhibitors thereof |
EP04729014A EP1620553A4 (en) | 2003-04-23 | 2004-04-23 | Insect chymotrypsin and inhibitors thereof |
NZ542729A NZ542729A (en) | 2003-04-23 | 2004-04-23 | Isolation of a chymotrypsin, HpCh5, from Helicoveipa spp, characterised by its resistance to the proteinase inhibitors derived from Nicotiana alata |
CA2521940A CA2521940C (en) | 2003-04-23 | 2004-04-23 | Insect chymotrypsin and inhibitors thereof |
US12/274,999 US20090188010A1 (en) | 2003-04-23 | 2008-11-20 | Insect Chymotrypsin and Inhibitors Thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US46505403P | 2003-04-23 | 2003-04-23 | |
US60/465,054 | 2003-04-23 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/274,999 Continuation US20090188010A1 (en) | 2003-04-23 | 2008-11-20 | Insect Chymotrypsin and Inhibitors Thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2004094630A1 true WO2004094630A1 (en) | 2004-11-04 |
Family
ID=33310988
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU2004/000524 WO2004094630A1 (en) | 2003-04-23 | 2004-04-23 | Insect chymotrypsin and inhibitors thereof |
Country Status (8)
Country | Link |
---|---|
US (2) | US7462695B2 (en) |
EP (1) | EP1620553A4 (en) |
CN (1) | CN1802432A (en) |
AU (1) | AU2004233354B2 (en) |
BR (1) | BRPI0409638A (en) |
CA (1) | CA2521940C (en) |
NZ (2) | NZ542729A (en) |
WO (1) | WO2004094630A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007137329A2 (en) | 2006-05-25 | 2007-12-06 | Hexima Limited | Multi-gene expression vehicle |
WO2012174478A3 (en) * | 2011-06-17 | 2013-04-11 | Halozyme, Inc. | Stable formulations of a hyaluronan-degrading enzyme |
JP2013192526A (en) * | 2012-03-22 | 2013-09-30 | Sanyo Chem Ind Ltd | Protein solution, method for restoring protease activity of protein solution, and detergent composition containing the same protein solution |
CN104400672A (en) * | 2014-10-29 | 2015-03-11 | 杨祝华 | Metallic bond diamond grinding wheel |
US9497908B2 (en) | 2011-02-07 | 2016-11-22 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
US9993529B2 (en) | 2011-06-17 | 2018-06-12 | Halozyme, Inc. | Stable formulations of a hyaluronan-degrading enzyme |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ523345A (en) | 2001-02-08 | 2004-04-30 | Hexima Ltd | Plant-derived molecules and genetic sequences encoding same and uses therefor |
US20080050776A1 (en) * | 2006-05-26 | 2008-02-28 | Kenneth Neet | Stable mutated pro nerve growth factors |
EP2762498A1 (en) * | 2007-04-20 | 2014-08-06 | Hexima Limited | Modified plant defensin |
AR075257A1 (en) | 2008-02-01 | 2011-03-23 | Hexima Ltd | PLANTS PROTECTION SYSTEM AGAINST INFECTION BY PATHOGEN AGENTS |
AR072910A1 (en) * | 2008-08-05 | 2010-09-29 | Hexima Ltd | ANTI-PATHOGEN SYSTEMS |
US9889184B2 (en) | 2008-08-05 | 2018-02-13 | Hexima Limited | Anti-pathogen systems |
PL3538665T3 (en) * | 2016-11-14 | 2024-05-06 | PPB Technology Pty Ltd | Protease sensor molecules |
WO2020021041A1 (en) | 2018-07-25 | 2020-01-30 | Invaio Sciences International Gmbh | Injection systems, injection tools and methods for same |
EP3864036A1 (en) * | 2018-10-10 | 2021-08-18 | Novozymes A/S | Chymotrypsin inhibitor variants and the use thereof |
CN110551707A (en) * | 2019-10-17 | 2019-12-10 | 山西大学 | Method for purifying neutral or alkaline protease |
BR112022015027A2 (en) | 2020-01-29 | 2022-09-20 | Invaio Sciences Int Gmbh | INJECTION SYSTEMS, INJECTION TOOLS AND METHODS THEREOF |
WO2021245115A1 (en) | 2020-06-02 | 2021-12-09 | Invaio Sciences International Gmbh | Tip setters and tip adapters for installing injection tools to plant parts |
US20240081195A1 (en) | 2021-01-29 | 2024-03-14 | Invaio Sciences International Gmbh | Plant injection systems and uses thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3135541A1 (en) * | 1981-09-08 | 1983-03-24 | Bayer Ag, 5090 Leverkusen | Derivatives of kallikrein-trypsin inhibitor (BPTI), carrier-bound BPTI derivatives, their preparation and their use for preparing the enzymes trypsin, chymotrypsin and kallikrein in pure form |
WO1984003564A1 (en) | 1983-03-08 | 1984-09-13 | Commw Serum Lab Commission | Method of determining antigenically active amino acid sequences |
WO1997002048A1 (en) | 1995-06-30 | 1997-01-23 | Millennium Pharmaceuticals, Inc. | Compositions for the treatment and diagnosis of body weight disorders, including obesity |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4945050A (en) | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5122466A (en) | 1989-06-13 | 1992-06-16 | North Carolina State University | Ballistic transformation of conifers |
EP0674712B1 (en) | 1992-12-16 | 2005-03-23 | Hexima Limited | A proteinase inhibitor, precursor thereof and genetic sequences encoding same |
-
2004
- 2004-04-23 CA CA2521940A patent/CA2521940C/en not_active Expired - Fee Related
- 2004-04-23 US US10/554,237 patent/US7462695B2/en not_active Expired - Fee Related
- 2004-04-23 CN CNA2004800128599A patent/CN1802432A/en active Pending
- 2004-04-23 NZ NZ542729A patent/NZ542729A/en not_active IP Right Cessation
- 2004-04-23 EP EP04729014A patent/EP1620553A4/en not_active Withdrawn
- 2004-04-23 AU AU2004233354A patent/AU2004233354B2/en not_active Ceased
- 2004-04-23 WO PCT/AU2004/000524 patent/WO2004094630A1/en active Application Filing
- 2004-04-23 BR BRPI0409638-0A patent/BRPI0409638A/en not_active Application Discontinuation
- 2004-04-23 NZ NZ572316A patent/NZ572316A/en not_active IP Right Cessation
-
2008
- 2008-11-20 US US12/274,999 patent/US20090188010A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3135541A1 (en) * | 1981-09-08 | 1983-03-24 | Bayer Ag, 5090 Leverkusen | Derivatives of kallikrein-trypsin inhibitor (BPTI), carrier-bound BPTI derivatives, their preparation and their use for preparing the enzymes trypsin, chymotrypsin and kallikrein in pure form |
WO1984003564A1 (en) | 1983-03-08 | 1984-09-13 | Commw Serum Lab Commission | Method of determining antigenically active amino acid sequences |
WO1997002048A1 (en) | 1995-06-30 | 1997-01-23 | Millennium Pharmaceuticals, Inc. | Compositions for the treatment and diagnosis of body weight disorders, including obesity |
Non-Patent Citations (39)
Title |
---|
ANTCHEVA ET AL., PROTEIN SCI., vol. 10, 2001, pages 2280 - 2290 |
ATKINSON ET AL., THE PLANT CELL, vol. 5, 1993, pages 203 - 213 |
BALANDIN ET AL., PLANT MOL. BIOL., vol. 27, 1995, pages 1197 - 1204 |
BOWN, D.P. ET AL.: "Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 27, no. 7, 1997, pages 625 - 638, XP008075527 * |
BROADWAY, J INSECT. PHYSIOL., vol. 41, 1995, pages 107 - 116 |
BROADWAY; VILLANI, ENTOMOL. EXPO. APPL., vol. 76, 1995, pages 303 - 312 |
CHOI ET AL., BIOCHIM. ET BIOPHYS. ACTA, vol. 1492, 2000, pages 211 - 215 |
CHRISTELLER ET AL., INSECT BIOCHEM. MOLECUL. BIOL., vol. 22, 1992, pages 735 - 746 |
CRIPPS ET AL., J CELL BIOL., vol. 126, 1994, pages 689 - 699 |
ERICKSON ET AL., SCIENCE, vol. 249, 1990, pages 527 - 533 |
GATEHOUSE ET AL.: "Biotech. in Agriculture", 1992, article "Plant Genetic Manipulation for Crop Protection", pages: 155 - 181 |
GATEHOUSE, L.N. ET AL.: "Characterisation of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to four proteinase inhibitors in the diet", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 27, no. 11, 1997, pages 929 - 944, XP008075528 * |
HEATH ET AL., EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 230, no. 1, 1995, pages 25 - 257 |
HEATH, R.L. ET AL: "Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests", JOURNAL OF INSECT PHYSIOLOGY, vol. 43, no. 9, 1997, pages 833 - 842, XP008075526 * |
HEDIGER ET AL., INSECT MOL. BIOL., vol. 10, 2001, pages 113 - 119 |
HJELMELAND, K.: "Characteristics of two trypsin type isozymes isolated from the arctic fish capelin (Mallotus villosus)", COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B,, vol. 71B, no. 4, 1982, pages 557 - 562, XP009071291 * |
HODGSON, BIOLTECHHOLOGY, vol. 9, 1991, pages 19 - 21 |
JOHNSTON ET AL., INSECT BIOCHEM. MOLEC. BIOL., vol. 25, no. 3, 1995, pages 375 - 383 |
JOHNSTON ET AL., INSECT BIOCHEM., vol. 21, 1991, pages 389 - 397 |
JONGSMA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, no. 17, 1995, pages 8041 - 8045 |
LEE ET AL., NATURE STRUCTURAL BIOLOGY, vol. 6, no. 6, 1999, pages 526 - 530 |
LEE; ANSTEE, INSECT. BIOCHEM. MOLEC. BIOL., vol. 25, 1995, pages 63 - 71 |
LIDHOLM ET AL., GENETICS, vol. 134, 1993, pages 859 - 868 |
LOZOVSKAYA ET AL., GENETICS, vol. 142, 1996, pages 173 - 177 |
MAZUMDAR-LEIGHTON, S.: "Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 31, 2001, pages 633 - 644, XP008075529 * |
MILLER ET AL., PLANT MOL. BIOL., vol. 42, 2000, pages 329 - 333 |
MILLER ET AL., PLANT T CELL, vol. 11, 1999, pages 1499 - 1508 |
MOURA; RYAN, PLANT PHYSIOL., vol. 126, 2001, pages 289 - 298 |
NIELSON ET AL., BIOCHEMISTRY, vol. 34, 1995, pages 14304 - 14311 |
NIELSON ET AL., J MOL. BIOL., vol. 242, 1994, pages 231 - 243 |
PELOQUIN ET AL., J COT. SCI., vol. 5, 2001, pages 114 - 120 |
RYAN, ANNU. REV. PHYTOPATHOL., vol. 28, 1990, pages 425 - 449 |
SAKAL, E. ET AL.: "Purification and characterization of trypsins from the digestive tract of Locusta migratoria", INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH, vol. 34, no. 6, 1989, pages 498 - 505, XP009071290 * |
See also references of EP1620553A4 |
SUMMERTON; WELLER, ANTISENSE AND NUCLEIC ACID DRUG DEVELOPMENT, vol. 7, 1997, pages 187 - 195 |
TAYLOR ET AL., PLANT MOL. BIOL., vol. 23, 1993, pages 1005 - 1014 |
TERRA; FERREIRA, COMP. BIOCHEM. PHYSIOL., vol. 109, 1994, pages 1 - 62 |
WELLS, METHODS ENZYMOL., vol. 202, 1991, pages 2699 - 2705 |
XU; QIN, J. ECON. ENTOMOL., vol. 87, 1994, pages 334 - 338 |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007137329A2 (en) | 2006-05-25 | 2007-12-06 | Hexima Limited | Multi-gene expression vehicle |
US9497908B2 (en) | 2011-02-07 | 2016-11-22 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
US10174339B2 (en) | 2011-02-07 | 2019-01-08 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
WO2012174478A3 (en) * | 2011-06-17 | 2013-04-11 | Halozyme, Inc. | Stable formulations of a hyaluronan-degrading enzyme |
US9993529B2 (en) | 2011-06-17 | 2018-06-12 | Halozyme, Inc. | Stable formulations of a hyaluronan-degrading enzyme |
JP2013192526A (en) * | 2012-03-22 | 2013-09-30 | Sanyo Chem Ind Ltd | Protein solution, method for restoring protease activity of protein solution, and detergent composition containing the same protein solution |
CN104400672A (en) * | 2014-10-29 | 2015-03-11 | 杨祝华 | Metallic bond diamond grinding wheel |
Also Published As
Publication number | Publication date |
---|---|
US7462695B2 (en) | 2008-12-09 |
EP1620553A4 (en) | 2007-01-03 |
US20090188010A1 (en) | 2009-07-23 |
CA2521940C (en) | 2010-01-26 |
NZ572316A (en) | 2010-06-25 |
BRPI0409638A (en) | 2006-04-25 |
AU2004233354B2 (en) | 2008-02-21 |
US20070219147A1 (en) | 2007-09-20 |
AU2004233354A1 (en) | 2004-11-04 |
NZ542729A (en) | 2009-04-30 |
CA2521940A1 (en) | 2004-11-04 |
AU2004233354A2 (en) | 2004-11-04 |
EP1620553A1 (en) | 2006-02-01 |
CN1802432A (en) | 2006-07-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20090188010A1 (en) | Insect Chymotrypsin and Inhibitors Thereof | |
Liu et al. | An atypical thioredoxin imparts early resistance to sugarcane mosaic virus in maize | |
Ruegger et al. | The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p | |
ES2390919T3 (en) | Plants that have improved performance-related traits and a method to make them | |
Qu et al. | Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice | |
AU2016202110A1 (en) | Methods of controlling plant seed and organ size | |
EP1991685B1 (en) | Compositions and methods for increasing plant tolerance to high population density | |
CN1555414B (en) | Plant-derived resistance gene | |
JP2007530063A (en) | Plant having improved growth characteristics and method for producing the same | |
Wang et al. | A Medicago truncatula EF-Hand family gene, MtCaMP1, is involved in drought and salt stress tolerance | |
KR20120125225A (en) | Plants having enhanced yield-related traits and a method for making the same | |
KR101957550B1 (en) | Antifungal plant proteins, peptides, and methods of use | |
Baba et al. | Exon/intron organization of the gene encoding the mouse epithelin/granulin precursor (acrogranin) | |
HU228701B1 (en) | Novel nucleic acid sequences and their use in methods for achieving pathogen resistance in plants | |
CN101157920B (en) | Method for cultivating drought-resistant and /or growth-delaying plant in hostile environment | |
WO2019201059A1 (en) | Gene for regulating anti-aluminum toxicity transcription factor stop1 protein and application thereof | |
US20040209325A1 (en) | Mitogen-activated protein kinase and method of use to enhance biotic and abiotic stress tolerance in plants | |
AU2008202250B2 (en) | Insect chymotrypsin and inhibitors thereof | |
AU2008255131B2 (en) | Modification of plant responses to salt (2) | |
MXPA05011445A (en) | Insect chymotrypsin and inhibitors thereof | |
JPWO2006126294A1 (en) | Muginate iron complex selective transporter gene | |
KR100328507B1 (en) | Recombinant Plant Expression Vector Comprising cDNA Encoding Antimicrobial Peptide Derived from Seed of Pharbitis nil L. | |
EP1029923A1 (en) | Method for conveying BNYVV resistance to sugar beet plants | |
CN101157921B (en) | Method for cultivating drought-resistant and /or growth-delaying plant in hostile environment | |
WO2004016775A2 (en) | Plants having modified growth and a method for making the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DPEN | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2004233354 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 542729 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2521940 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2004233354 Country of ref document: AU Date of ref document: 20040423 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2004233354 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2005/011445 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 20048128599 Country of ref document: CN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 5343/DELNP/2005 Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004729014 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2004729014 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: PI0409638 Country of ref document: BR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007219147 Country of ref document: US Ref document number: 10554237 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 10554237 Country of ref document: US |