INCORPORATION OF SEQUENCE LISTING

The file named “38_21_61965_0001_ST25.txt” containing a computer-readable form of the Sequence Listing was created on May 22, 2017. This file is 220,940 bytes in size (as measured in the MS-Windows® operating system), filed contemporaneously with this application by electronic submission (using the United States Patent Office EFS-Web filing system), and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitory proteins. A novel class of insecticidal proteins exhibiting insect inhibitory activity against agriculturally-relevant pests of crop plants and seeds is disclosed in this application. In particular, the disclosed class of proteins exhibits insecticidal activity against agriculturally-relevant pests of crop plants and seeds, particularly the Hemipteran order of insect pests. Plants, plant parts, and seeds containing a recombinant nucleic acid molecule encoding one or more of the disclosed toxin proteins are provided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally-significant plants including, among others, corn, soybean, sugarcane, rice, wheat, vegetables, and cotton, has become increasingly important. In addition to the growing need for agricultural products to feed, clothe and provide energy for a growing human population, climate-related effects and pressure from the growing population to use land other than for agricultural practices are predicted to reduce the amount of arable land available for farming. These factors have led to grim forecasts with respect to food security, particularly in the absence of major improvements in plant biotechnology and agronomic practices. In light of these pressures, environmentally sustainable improvements in technology, agricultural techniques, and pest management are vital tools to expand crop production on the limited amount of arable land available for farming.

Insects, particularly insects within the order Hemiptera, are considered a major cause of damage to field crops, thereby decreasing crop yields in infested areas. Hemipteran pest species which negatively impact agriculture include, but are not limited to, stink bug, Western tarnished plant bug and Tarnished plant bug.

Historically, the intensive application of synthetic chemical insecticides was relied upon as the pest control agent in agriculture. Concerns for the environment and human health, in addition to emerging resistance issues, stimulated the research and development of biological pesticides. This research effort led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

The biological control paradigm shifted when the potential of entomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest control agent. Strains of the bacteriumBacillus thuringiensis(Bt) have been used as a source for insecticidal proteins since it was discovered that Bt strains show a high toxicity against specific insects. Bt strains are known to produce delta-endotoxins that are localized within parasporal crystalline inclusion bodies at the onset of sporulation and during the stationary growth phase (e.g., Cry proteins), and are also known to produce secreted insecticidal protein. Upon ingestion by a susceptible insect, delta-endotoxins as well as secreted toxins exert their effects at the surface of the midgut epithelium, disrupting the cell membrane, leading to cell disruption and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including otherBacillusand a diversity of other bacterial species, such asBrevibacillus laterosporus, Lysinibacillus sphaericus(“Ls” formerly known asBacillus sphaericus) andPaenibacillus popilliae.

Crystalline and secreted soluble insecticidal protein toxins are highly specific for their hosts and have gained worldwide acceptance as alternatives to chemical insecticides. For example, insecticidal toxin proteins have been employed in various agricultural applications to protect agriculturally important plants from insect infestations, decrease the need for chemical pesticide applications, and increase yields. Insecticidal toxin proteins are used to control agriculturally-relevant pests of crop plants by mechanical methods, such as spraying to disperse microbial formulations containing various bacteria strains onto plant surfaces, and by using genetic transformation techniques to produce transgenic plants and seeds expressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal proteins has been globally adopted. For example, in 2012, 26.1 million hectares were planted with transgenic crops expressing Bt toxins (James, C., Global Status of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44). The global use of transgenic insect-protected crops and the limited number of insecticidal proteins used in these crops has created a selection pressure for existing insect alleles that impart resistance to the currently-utilized insecticidal proteins.

Hemipteran pests injure plants by puncturing tissues with their piercing and sucking mouthparts and then extracting plant fluids. The principle damage to tissues comes from loss of plant fluids and injection of digestive enzymes. The puncture sites can be predisposed to colonization by pathogenic organisms, causing significant yield and quality loss. One family in the order of Hemiptera, Pentatomidae, has many members commonly referred to as Stink Bugs. Certain species of Stink Bugs that are agricultural pests have become difficult to control with insecticidal chemicals due to the emergence of resistant populations.

Various characteristics of Stink Bugs result in a high susceptibility to resistance emergence. For example, Stink Bugs can have multiple generations in a single growing season. Further, they can overwinter as adults almost anywhere where they can be protected (including homes), and then begin feeding on various host plants as the temperatures rise. Once development begins, Stink Bugs have two to three generations, depending on the species and host plants.

On corn, Stink Bugs are most often found feeding on young plants in late spring and early summer. Feeding at this time results in holes in the leaf, and if severe, results in deformed plants. The Brown Stink Bug is the primary Stink Bug pest for corn crops during the early vegetative stages. The Brown Marmorated Stink Bug, another corn pest, is generally found in late summer on corn usually feeding on the ear, directly destroying the kernels. This is especially a concern on sweet corn and can be quite damaging.

On soybean, most of the injury from Stink Bugs occurs during second half of the growing season when significant feeding takes place on pods and developing seed. Darkish spots will occur where the mouthparts puncture the plant tissue, although these are difficult to see. This can cause deformation and abortion of the seeds, as well as provide a route for infection by pathogenic organisms. Stink Bug feeding on soybean often results in delayed leaf maturity and foliage retention. During seed formation, seeds will become shriveled, deformed, undersized, and even be aborted. Feeding on more developed seeds will result in minor shriveling and discoloration. This not only negatively affects yield, but also will result in lower market value or inhibit the sale of produced seed.

Due to warming temperatures, stink bugs are expanding their population size and geographical range in the United States. Due to the damage on agricultural crops inflicted by stink bugs, their high susceptibility to the emergence there is a need to find new and novel insect toxins that are effective against Hemipteran insect pests such as stink bugs.

SUMMARY OF THE INVENTION

The present invention provides a novel class of insect inhibitory polypeptides (insect toxic proteins) which are shown to exhibit inhibitory activity against several hemipteran pests of crop plants. Each of the proteins can be used alone or in combination with each other and with other Bt proteins and toxic agents in formulations and in planta, providing alternatives to known Bt proteins and insecticide chemistries.

In one embodiment, a recombinant nucleic acid molecule is disclosed comprising a heterologous promoter fragment operably linked to a polynucleotide segment encoding a pesticidal protein or fragment thereof, wherein said pesticidal protein or pesticidal fragment thereof (a) comprises the amino acid sequence of SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; or (b) comprises an amino acid sequence having at least from about 62% to about 100% amino acid sequence identity, or any fraction percentage point between 62% and 100%, to the proteins selected from the group consisting of SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28. In another embodiment, said polynucleotide segment hybridizes to a polynucleotide having the nucleotide sequence of SEQ ID NO:11, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27 under hybridization conditions of 68° C., followed by washing at 68° C., in 2×SSC containing 0.1% SDS; or (b) said recombinant nucleic acid molecule is in operable linkage with a vector, and said vector is selected from the group consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial or yeast artificial chromosome. The recombinant nucleic acid molecule can comprise a sequence that functions to express the pesticidal protein in a plant; or is expressed in a plant cell to produce a pesticidally effective amount of pesticidal protein.

In another embodiment, host cells are provided which contain at least one recombinant nucleic acid molecule disclosed herein, wherein the host cell is selected from the group consisting of a bacterial, a yeast, and a plant cell. Bacterial host cells include at least species such asAgrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, andErwinia. TheBacillusspecies is aBacillus cereusor aBacillus thuringiensis, theBrevibacillusis aBrevibacillus laterosperous, and saidEscherichiais anEscherichia coli. Yeast host cells include at leastPichyaandSaccharomycesspecies. Plant host cells include at least dicotyledonous plant cells and monocotyledonous plant cells, and as applicable, further include at least an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cells.

In another embodiment, the pesticidal protein exhibits activity against an insect species of the order Hemiptera, includingChinavia hilaris, Chinavia marginata, Chinavia pensylvanica, Chlorochroa granulose, Chlorochroa kanei, Chlorochroa ligata, Chlorochroa lineate, Chlorochroa opuntiae, Chlorochroa persimilis, Chlorochroa rossiana, Chlorochroa sayi, Chlorochroa uhleri, Chlorochroa belfragii, Chlorochroa faceta, Chlorochroa osborni, Chlorochroa saucia, Chlorochroa senilis, Nezara viridula, Edessa meditabunda, Edessa bifida, Edessa florida, Euschistus heros, Euschistus acuminatus, Euschistus biformis, Euschistus conspersus, Euschistus crenator, Euschistus egglestoni, Euschistus ictericus, Euschistus inflatus, Euschistus latimarginatus, Euschistus obscures, Euschistus politus, Euschistus quadrator, Euschistus sevus, Euschistus strenuous, Euschistus tristigmus, Euschistus variolarius Halyomorpha halys, Thyanta accerra, Thyanta calceata, Thyanta custator, Thyanta pallidovirens, Thyanta perditor, Thyanta maculate, Thyanta pseudocasta Dichelops melacanthus, Dichelops avilapiresi, Dichelops bicolor, Dichelops dimidatus, Dichelops furcatus, Dichelops furcifrons, Dichelops lobatus, Dichelops miriamae, Dichelops nigrum, Dichelops peruanus, Dichelops phoenix, Dichelops saltensis, Piezodorus guildinni, Piezodorus lituratus Megacopta cribraria, Lygus hesperus, andLygus lineolaris.

Also provided are plants comprising a recombinant nucleic acid molecule comprising a polynucleotide segment corresponding to a heterologous promoter operably linked to a segment encoding a pesticidal protein or pesticidal fragment thereof, wherein: (a) said pesticidal protein or pesticidal fragment thereof comprises the amino acid sequence of SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; or (b) said pesticidal protein or pesticidal fragment thereof comprises an amino acid sequence having at least from about 62% to about 100% amino acid sequence identity, or any fraction percentage point between 62% and 100%, to the proteins selected from the group consisting of SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28; or (c) said polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO:11, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27 under hybridization conditions of 68° C., followed by washing at 68° C., in 2×SSC containing 0.1% SDS; or (d) said plant exhibits a detectable amount of said pesticidal protein, wherein the pesticidal protein is chosen from the group consisting of SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

In one embodiment, the plant is either a dicotyledonous plant or a monocotyledonous plant. In another embodiment, the plant is further selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

In a further embodiment, seeds comprising the recombinant nucleic acid molecules are disclosed.

In another embodiment, an insect inhibitory composition is provided comprising the recombinant nucleic acid molecules as set forth herein. The insect inhibitory composition can further comprise a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. In certain embodiments, the at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, and an ancillary protein. The at least one other pesticidal agent in the insect inhibitory composition exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, Hemiptera, Homoptera, or Thysanoptera. The at least one other pesticidal agent in the insect inhibitory composition is selected from the group consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC3131, TIC2160, VIP3A, VIP3B, VIP3Ab, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, Axmi52, Axmi58, Axmi88, Axmi97, Axmi102, Axmi112, Axmi117, Axmi100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, Axmi171, AXMI-184, axmi196, axmi204, axmi207, axmi209, Axmi205, AXMI218, AXMI220, AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279, AXMI335, AXMI345, AXMI-R1, and variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-11, DIG-657 protein, PHI-4 variants, PIP-72 variants, PIP-45 variants, PIP-64 variants, PIP-74 variants, PIP-77 variants, DIG-305, PIP-47 variants, DIG-17, DIG-90, DIG-79, and DIG-303.

Commodity products are provided comprising a detectable amount of the recombinant nucleic acid molecules disclosed herein. Such commodity products include commodity corn which may be bagged by a grain handler, corn flakes, corn cakes, corn flour, corn meal, corn syrup, corn oil, corn silage, corn starch, corn cereal, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity products including, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such commodity products containing a detectable amount of such polynucleotides and or polypeptides of this application, whole or processed cotton seed, cotton oil, lint, seeds and plant parts processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts.

Also contemplated is a method of producing seed comprising one or more of the recombinant nucleic acid molecules disclosed herein. The method includes planting at least one such seed; growing a plant from the seed; and harvesting progeny seed from the plant, wherein the harvested seed comprises the one or more recombinant nucleic acid molecules.

In another embodiment, a plant resistant to insect infestation is provided. The cells of said plant optionally comprise: (a) a recombinant nucleic acid molecule encoding an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; or (b) an insecticidally effective amount of a protein comprising an amino acid sequence having at least 62%, or 65%, or 70%, or 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

Also disclosed are methods for controlling a Lepidopteran species pest, and controlling a Hemipteran species pest infestation of a plant, particularly a crop plant. The method will comprise contacting the pest with an insecticidally effective amount of a pesticidal proteins as set forth in SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; or contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 62%, or 65%, or 70%, or 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence encoding the TIC4747 toxin protein obtained fromBacillus thuringiensis(Bt) species CFB007452.

SEQ ID NO:2 is the amino acid sequence of TIC4747 protein.

SEQ ID NO:3 is the nucleotide sequence encoding the TIC7181 toxin protein obtained from Bt species EG9737.

SEQ ID NO:4 is the amino acid sequence of TIC7181 protein.

SEQ ID NO:5 is the nucleotide sequence encoding the TIC4904 toxin protein obtained from Bt species CFB007432.

SEQ ID NO:6 is the amino acid sequence of TIC4904 protein.

SEQ ID NO:7 is the nucleotide sequence encoding the TIC6547 toxin protein obtained from Bt species CFB231019.

SEQ ID NO:8 is the amino acid sequence of TIC6547 protein.

SEQ ID NO:9 is the native nucleotide sequence encoding the TIC4006 toxin protein obtained from Bt species WC12466.

SEQ ID NO:10 is the amino acid sequence of TIC4006 protein.

SEQ ID NO:11 is a synthetic coding sequence encoding a TIC4747PL pesticidal protein designed for expression in a plant cell wherein an additional alanine codon is inserted immediately following the initiating methionine codon.

SEQ ID NO:12 is the amino acid sequence of TIC4747PL encoded by a synthetic coding sequence designed for expression in a plant cell (SEQ ID NO:11), and wherein an additional alanine amino acid is inserted immediately following the initiating methionine.

SEQ ID NO:13 is a synthetic coding sequence encoding a TIC7181PL pesticidal protein designed for expression in a plant cell wherein an additional alanine codon is inserted immediately following the initiating methionine codon.

SEQ ID NO:14 is the amino acid sequence of TIC7181PL encoded by a synthetic coding sequence designed for expression in a plant cell (SEQ ID NO:13), and wherein an additional alanine amino acid is inserted immediately following the initiating methionine.

SEQ ID NO:15 is a synthetic coding sequence encoding a TIC4904PL pesticidal protein designed for expression in a plant cell wherein an additional alanine codon is inserted immediately following the initiating methionine codon.

SEQ ID NO:16 is the amino acid sequence of TIC4904PL encoded by a synthetic coding sequence designed for expression in a plant cell (SEQ ID NO:15), and wherein an additional alanine amino acid is inserted immediately following the initiating methionine.

SEQ ID NO:17 is a synthetic coding sequence encoding a TIC6547PL pesticidal protein designed for expression in a plant cell wherein an additional alanine codon is inserted immediately following the initiating methionine codon.

SEQ ID NO:18 is the amino acid sequence of TIC6547PL encoded by a synthetic coding sequence designed for expression in a plant cell (SEQ ID NO:17), and wherein an additional alanine amino acid is inserted immediately following the initiating methionine.

SEQ ID NO:19 is a synthetic coding sequence encoding a TIC4006PL pesticidal protein designed for expression in a plant cell wherein an additional alanine codon is inserted immediately following the initiating methionine codon.

SEQ ID NO:20 is the amino acid sequence of TIC4006PL encoded by a synthetic coding sequence designed for expression in a plant cell (SEQ ID NO:19), and wherein an additional alanine amino acid is inserted immediately following the initiating methionine.

SEQ ID NO:21 is a nucleotide sequence encoding TIC4747_His which is comprised of the TIC4747 coding sequence with a Histadine tag coding sequence operably linked 3′ to the TIC4747 coding sequence.

SEQ ID NO:22 is the amino acid sequence of TIC4747_His.

SEQ ID NO:23 is a nucleotide sequence encoding TIC4904_His which is comprised of the TIC4904 coding sequence with a Histadine tag coding sequence operably linked 5′ to the TIC4904 coding sequence.

SEQ ID NO:24 is the amino acid sequence of TIC4904_His.

SEQ ID NO:25 is a nucleotide sequence encoding TIC6547_His which is comprised of the TIC6547 coding sequence with a Histadine tag coding sequence operably linked 5′ to the TIC6547 coding sequence.

SEQ ID NO:26 is the amino acid sequence of TIC6547_His.

SEQ ID NO:27 is a nucleotide sequence encoding TIC4006_His which is comprised of the TIC4006 coding sequence with a Histadine tag coding sequence operably linked 5′ to the TIC4006 coding sequence.

SEQ ID NO:28 is the amino acid sequence of TIC4006_His.

