Patent Application: US-20585708-A

Abstract:
improved methods and compounds to control insects , involving a biological control method to induce toxicity in targeted insects using iridoptin . the present invention induces high levels of apoptosis and inhibition of host protein synthesis in insect cells . it is the first viral toxin against non - lepidopteran insects and is distinct from existing bacterial toxins , such as bacillus thuringiensis toxins , which are not effective against most beetles , including the boll weevil , and the baculoviridae , which is the main group of viruses currently used as biological control agents . iridoptin will have use in the control of agricultural pests . it will increase productivity and reduce disease transfer by vectors and household pests . by extension it has application in cancer therapy and other medical treatments where apoptosis is critical to removal of certain cells .

Description:
disclosed herein is an improved method of inducing toxicity in insect cells by protein engineering , wherein insect cells are exposed to iridoptin which then induces apoptosis , inhibits host protein synthesis in insect cells as well as mortality in aphid populations . the numerous innovative teachings of the present invention will be described with particular reference to several embodiments ( by way of example , and not of limitation ). reference is first made to fig1 a , 1 b , and 1 c wherein a region of the istk gene in chilo iridescent virus ( civ ; new zealand strain ) dna was mapped and sequenced and an open reading frame for a serine / threonine kinase was identified . the open reading frame encoding the putative istk gene was identified by primer walking and mapped to a 2 - kbp region ( shown in fig1 a by a filled - in grey block ) spanning the eco ri sites (“ e ”) separating fragments b and u in the civ genome . this gene was shown to be active and was designated istk ( iridovirus serine threonine kinase ). fig1 b depicts the nucleotide sequence of the 2 - kbp region encoding the complete 1236 - bp istk gene ( shown underlined in fig1 b ) which was determined . the gene product ( istk ) is a 49 - kda polypeptide . the start and stop sites for this gene are indicated in bold in fig1 b . reference is now made to fig2 , wherein a cleaved 37 - kda polypeptide resulting from istk was analyzed for c - terminal sequence . as indicated above , prior u . s . pat . no . 6 , 200 , 561 identified a chilo iridescent virus capsid protein extract that killed neonate boll weevil larvae , inhibited host expression and induced apoptosis . further research has shown that civ contains a serine - threonine kinase enzyme . the gene has now been cloned and expressed for this enzyme and it has been demonstrated that the 49 - kda product ( istk ) induces apoptosis in 63 % of treated insect cells and necrosis in the remaining cells . apoptosis was detected by dna fragmentation , blebbing , and with the tunel assay . the inventor has now made the significant finding that a 37 - kda cleavage product of the istk 49 - kda protein induces an apoptotic effect in nearly all cells in the population . thus , no additional factors or gene products are required for a complete effect , and post - translational processing of the 49 - kda gene product is important for activity . the new polypeptide was necessary and sufficient for full toxic effect . dose response studies using blebbing assays in cf and ag cells indicated that 100 ng / ml and 900 ng / ml , respectively , of the 37 - kda polypeptide were required to induce apoptosis in 50 % of the cell population . in the present invention , the cleavage site on istk has been identified and this information has been used to tailor a subgenic fragment of the istk gene ( shown underlined in fig1 c ). the positions of the s / t kinase domain and the atp binding site are shown highlighted in fig1 c . the subgenomic fragment ( underlined ) from the istk gene sequence was cloned in - frame with the c - terminal polyhistidine tag of pichia expression vector ( ppicz c ). the stop codon after polyhistidine tag in the expression vector was utilized for termination . the present invention further discloses that the modified gene codes for a 37 - kda polypeptide now designated iridoptin , which is more efficient in inducing apoptosis and inhibition of host protein synthesis than the product from the original gene . fig2 depicts the c - terminal sequencing of 37 - kda polypeptide following carboxypeptidase y digest which was carried out by the macromolecular structure , sequencing and synthesis facility , department of biochemistry at michigan state university . the c - terminal amino acids were asparagine - glycine ( asn - gly , or n - g ). fig2 shows that n - g was detected at two sites ( underlined in fig2 ) in the amino acid sequence derived from corresponding regions of the civ istk gene . based on this sequence , cleavage should occur at a glycine - aspartate ( gly - asp , g - d ) site . cleavage at the amino - terminal g - d residues should result in the formation of a 26 - kda polypeptide containing only the atp binding site . cleavage at the carboxy - terminal g - d site should yield the predicted 37 - kda polypeptide with both atp binding site and s / t kinase motif . maldi - tof analysis ( matrix - assisted laser desorption / ionization - time of flight mass spectrometry ) conducted at the institute for cellular and molecular biology core facility , university of texas , austin ) confirmed the identity of the 37 - kda polypeptide and the presence of the atp - binding and s / t kinase motifs . the subgenic dna sequence coding for the 37 - kda polypeptide was amplified by pcr using specifically designed primers and civ genomic dna and expressed in the pichia system ( invitrogen ) to yield 6 × his - tagged product . reference is now made to fig3 , wherein it is shown that the yeast lysates from pichia expression contained his - tagged 37 - kda product as detected by silver staining of polyacrylamide gels and western blotting . purification of this material through nickel columns ( probond , invitrogen , ca ) that bind 6 × his - tagged polypeptides yielded pure 37 - kda polypeptide ; i . e ., iridoptin as detected with 6 × his antibody probe and silver staining for total protein . fig3 shows analysis of iridoptin product expressed in the pichia system and purified by affinity chromatography with the silver - stained gel and western blot ( using 6 × his antibody probe ). as shown in fig3 , the columns under “ u ” show that uninduced yeast lysate did not express iridoptin . the columns under “ l ” show that the induced yeast lysate reveals the predicted iridoptin band . “ e ” shows iridoptin purified on his - binding nickel affinity column ( probond ) retained the 6 × his tag and was detectable as a pure polypeptide by silver staining and western blotting . column “ m ” shows the molecular weight markers , wherein the numbers represent molecular mass in kilo - daltons . reference is now made to fig4 a , 4 b , 4 c , and 4 d wherein it is shown that iridoptin induces apoptosis in spruce budworm ( cf124t ) cells . cells in nunc 60 - well trays ( 5 . 6 × 10 3 cells per well in 7 μl medium ) were treated and incubated at 28 ° c . and examined for apoptotic blebbing by phase - contrast microscopy at 24 hr post treatment . the iridoptin product induced apoptotic blebbing in more than 90 % of cf cells compared to 98 % in positive controls treated with actinomycin d . results are demonstrated in fig4 a showing iridoptin : 37 - kda polypeptide expressed from subgenic istk ( pichia ; 10 μg / ml ); 92 % blebbing , sd 4 . 7 . fig4 b showing actinomycin d ( 4 μg / ml ; positive control ); 98 % blebbing , sd 1 . 1 . fig4 c showing δ iridoptin ( heat - inactivated iridoptin 10 μg / ml , 65 ° c ., 30 min ) 5 % blebbing . fig4 d showing mock treatment with buffer ( 1 × rinaldini &# 39 ; s balanced salt solution ; rbss ), 7 % blebbing . magnification : 800 × in fig4 a - 4d . reference is now made to fig5 a - 5d , wherein it is shown that iridoptin induces apoptosis in boll weevil cells ( ag3a ) as detected by cellular blebbing . ag3a cells were seeded in 60 - well terasaki plates and incubated at 28 ° c . for 24 hours . the highest dilution inducing blebs in 50 % of the cell population was approximately 0 . 9 μg / ml . the blebbing assay shows in fig5 a that iridoptin at 20 μg / ml induced 87 % blebbing in a boll weevil cell line , ag3a . fig5 b shows that heat - inactivated iridoptin ( 20 μg / ml , 65 ° c ., 30 min ) induced 7 % blebbing . fig5 c shows that actinomycin d ( 4 μg / ml ; positive control ) induced 99 % blebbing . fig5 d shows that mock treatment with rbss buffer induced negligible blebbing ( 2 . 7 %). reference is now made to fig5 e - 5h , wherein a tunel assay confirms iridoptin - induced apoptosis . fig5 e depicts ag3a cells treated with 10 μg / ml iridoptin showed nuclear diaminobenzidine ( dab ) signal confirming apoptosis in 53 % of cell populations . fig5 f depicts ag3a cells treated with 20 μg / ml iridoptin showed 84 % nuclear dab . fig5 g depicts ag3a cells treated with actinomycin d ( act d ) induced 96 % nuclear dab signal . fig5 h depicts ag3a mock treated with rbss had 1 % nuclear dab . reference is now made to fig5 j , wherein a dose - response analysis of iridoptin - induced apoptosis against boll weevil cells ( ag3a ) as detected by blebbing is shown . the results show that 0 . 5 μg iridoptin is sufficient to induce apoptosis in 50 % of the ag cell population . similar results were obtained for cf cells . results are demonstrated in fig5 j wherein the dose - response of iridoptin against boll weevil cells ( ag3a ) is shown according to percent blebbing . ag3a cells were seeded in 60 - well terasaki plates at 4 . 2 × 10 3 cells per well , treated with serial 10 - fold dilutions of iridoptin , and incubated at 28 ° c . for 24 hr . actinomycin d ( 4 μg / ml ) was the positive control and heat - inactivated iridoptin ( 20 μg / ml , 65 ° c ., 30 min ) was the negative control ; mock treatments were with rinadini &# 39 ; s balanced salt solution ( rbss ). data points represent percent blebbing for approximately 200 cells per field . the highest dilution inducing blebs in 50 % of the cell population was approximately 0 . 5 μg / ml . linear regression line was generated using microsoft excel : y = 17 . 5 log x + 55 , where x is the concentration of iridoptin in μg / ml and y is the percent of cell population with blebs ; r 2 = 0 . 94 . controls showed expected blebbing ( actinomycin d : 99 %, sd 1 . 5 ; heat - inactivated iridoptin : 7 %, sd 1 . 7 ; rbss : 3 %, sd 1 . 9 ). assays were performed in triplicate . reference is now made to fig6 a , wherein it is shown that iridoptin induced inhibition of protein synthesis in boll weevil ( ag3a ) cells . ag3a cells were seeded in costar 24 - well plates treated with iridoptin at the concentrations shown . controls included actinomycin d ( a , positive , 4 μg / ml ), heat - inactivated iridoptin ( a , negative , 10 μg / ml , 65 ° c ., 30 min . ), and mock ( m , rbss ). beginning at 3 hr post treatment , cells were pulsed with 35 s - methionine for one hour , lysed with sds - page sample buffer ( 2 % sds , 20 % glycerol , 20 mm tris - hcl ph 8 , 2 % β - mercaptoethanol , and 0 . 1 mg / ml bromophenol blue ) and fractionated using sds - page . the relative rate of protein synthesis was quantitated using a phosphorimager . the experiment was performed in triplicate . reference is now made to fig6 b , wherein iridoptin - induced inhibition of protein synthesis in boll weevil ( ag3a ) cells is quantified . iridoptin at 26 μg / ml inhibited more than 90 % host protein synthesis , whereas actinomycin d inhibited 68 % protein synthesis . ag3a cells were seeded in costar 24 - well plates treated with iridoptin at concentrations ( μg / ml .) shown in fig6 b . controls included actinomycin d ( actd , positive control , 4 μg / ml ), heat - inactivated iridoptin ( heated , negative control , 10 μg / ml , 65 ° c ., 30 min ), and cells that were mock - treated with rinaldini &# 39 ; s balanced salt solution ( mock ). beginning at 3 hr post treatment , cells were pulsed with 35 s - methionine for one hour , lysed with sds - page sample buffer and fractionated using sds - page . bands were visualized using a phosphorimager . the relative rate of protein synthesis was quantitated using functions of a phosphorimager . this experiment was performed in triplicate . all major bands from three independent experiments were used to determine inhibition . iridoptin at 26 μg / ml inhibited more than 90 % host protein synthesis . reference is now made to fig6 c , wherein a dose - response analysis of iridoptin - induced inhibition of protein synthesis in boll weevil ( ag3a ) cells is shown . the data from fig6 a was quantified using functions of the phosphorimager . the highest dilution to inhibit 90 % host protein synthesis was approximately 23 μg / ml . log of iridoptin concentrations was plotted against percent protein inhibition . a linear regression line was generated using microsoft excel 2003 with the following equation : y = 98 . 4x − 44 . 9 , where x is log of iridoptin dilutions in μg / ml and y is percent inhibition of host protein synthesis . the r 2 value for the fitted line was 0 . 99 . reference is now made to fig7 , wherein iridoptin - induced inhibition of protein synthesis in cf cells is shown . inhibition with iridoptin ( iridoptin ; 7 μg / ml ; 63 %) was 93 % of that observed with the positive control , actinomycin d ( act d ; 4 μg / ml ; 68 %); whereas inhibition with heated iridoptin ( δ iridoptin ; 7 μg / ml , 65 ° c ., 30 min ) was 18 % of positive controls . the optical density of equivalent areas from relevant lanes in sds - page gels was measured using mock lanes as control and converted to percent transmittance . inhibition values were determined from percent transmittance against mock lanes . reference is now made to table 1 , which depicts protein kinase activity of iridoptin . assays for protein kinase showed significant activity for iridoptin . gamma 32 p - atp was used as label and protamine as substrate . samples were spotted on phosphocellulose paper and radioactivity was counted after washing off excess label . specific activity of kinase was expressed as cpm per μg total protein in the enzyme preparation used . the specific activity of iridoptin was slightly higher than that of civ virion protein extract ( cvpe ). kinase activity was low in heated iridoptin ( 65 ° c ., 30 min ), and bsa controls . reference is now made to fig8 , which depicts iridoptin induced mortality in aphids . bioassays showed that iridoptin induced 72 % mortality in treated aphids compared to 14 % mortality in controls . the effect of iridoptin treatment on cotton aphids is shown in fig8 , wherein twelve aphids were placed on cotton leaves that were painted with the following preparations : 1 ) iridoptin : the iridoptin gene was expressed in the pichia system ; yeast lysates were purified on nickel columns ( probond ), eluates were desalted and exchanged with rbss and diluted to 50 μg / ml with rbss containing final concentrations of 20 μg / ml casein , 1 μg / ml pepstatin a , and 2 μg / ml leupeptin ; 2 ) mock : pichia strain ( x33 ) not containing the iridoptin gene was processed as above ; lysates were mock - purified and diluted with the above solvents at ratios utilized for the iridoptin preparation ; and 3 ) untreated : aphids were incubated at 28 ° c . for 3 days and examined for mortality with a dissecting microscope . the results are presented as the number of dead aphids above that on untreated leaves . aphid mortality in iridoptin -, mock -, and untreated leaves was 72 %, 14 % and 0 %, respectively . the experiment was performed in triplicate . the bars indicate standard error . the disclosed method and apparatus is generally described below , with the following examples incorporated as particular embodiments of the invention and to demonstrate the practice and advantages thereof . it is understood that the examples are given by way of illustration and are not intended to limit the specification or the claims in any manner . to facilitate the understanding of this invention , a number of terms may be defined below . terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention . terms such as “ a ”, “ an ”, and “ the ” are not intended to refer to only a singular entity , but include the general class of which a specific example may be used for illustration . the terminology herein is used to described specific embodiments of the invention , but their usage does not delimit the disclosed method , except as may be outlined in the claims . tunel : a staining assay that detects fragmented dna in the nuclei of apoptotic cells ; positive stain is diagnostic for apoptotic cells . apoptosis : programmed cell death in which cells shrink , undergo nuclear dna fragmentation , and develop blebs at the surface . by conducting tests of iridoptin for apoptosis activity , inhibition of host protein synthesis in cell culture , and mortality in aphids , it has been shown that iridoptin , the product of the modified istk gene from civ , induces a very high level of apoptosis in more than 90 % of treated insect cells , inhibits host protein synthesis , and kills 63 % of aphid populations over control treatments . these data strongly suggest that iridoptin will have toxic or inhibitory effects against other insects , including the cotton boll weevil , lygus bug , the whitefly , and noctuids . alternate applications of this invention include using the dna segment coding for iridoptin to engineer and produce : ( i ) cotton and other crop plants resistant to aphids , boll weevils , lygus bugs , the whitefly , noctuids and other insect pests , ( ii ) microorganisms for controlling agricultural pests as well as plant , animal , and human disease vectors and household pests , and ( iii ) large amounts of iridoptin for direct control of agricultural and household pests as well as disease vectors . by extension , iridoptin finds application in cancer therapy and other medical treatments where apoptosis is critical to removal of certain cells . it will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention . the principal features of this invention can be employed in various embodiments without departing from the scope of the invention . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , numerous equivalents to the specific procedures described herein . such equivalents are considered to be within the scope of this invention and are covered by the claims . all publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains . all publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference . in the claims , all transitional phrases such as “ comprising ,” “ including ,” “ carrying ,” “ having ,” “ containing ,” “ involving ,” and the like are to be understood to be open - ended , i . e ., to mean including but not limited to . only the transitional phrases “ consisting of ” and “ consisting essentially of ,” respectively , shall be closed or semi - closed transitional phrases . all of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . bernal , j ., gonzález , d , e . t . natwick , j . g . loya , r . león - 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