IIIUS005756459A United States Patent (19) 11 Patent Number: 5,756,459 Jackson et al. 45 Date of Patent: May 26, 1998

54 INSECTICDALLY EFFECTIVE PEPTIDES Dunwiddie, T.V., 'The Use of In Vitro Brain Slices in SOLATABLE FROM PHDPPUS Neuropharmacology". Electrophysiological Techniques in VENOM Pharmacology, H.M. Geller, 25ed. Alan R. Liss, Inc., New York, pp. 65-90 (1986). 75) Inventors: John Randolph Hunter Jackson; Eric Fuqua, et al., "A simple PCR Method for detection and DelMar; Janice Helen Johnson; cloning low abundant transcript". Biotechniques, 9:206-211 Robert Marden Kral, Jr., all of Salt (1990). Lake City, Utah Hink, et al., "Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines". Biotechnol. 73) Assignees: FMC Corporation, Philadelphia, Pa.; Prog, 7:9-14 (1991). NPS Pharmaceuticals, Inc., Salt Lake Jackson and Parks, "Spider Toxins: Recent Applications in City, Utah Neurobiology". Ann Rev Neurosci. 12:405-414 (1989). Jones, et al., "Molecular Cloning Regulation and Complete (21) Appl. No.: 484,357 Sequence of a Hemocyanin-Related Juvenile Hormone (22 Filed: Jun. 7, 1995 Suppressible Protein From Insect Hemolymphs". J. Biol. Chem, 265:8596-8602 (1990). (51) Int. Cl...... A61K 38/16; C07K 14/435 Miller, et al. "Bacterial. Viral and Fungal Insecticides". (52) U.S. Cl...... 514/12: 530/350 Science, 219:715–721 (1983). 58) Field of Search ...... 514/12; 530/350 Rossi, et al. "An Alternate Method for Synthesis of Dou ble-Stranded DNA Segments", J. Biol. Chem, 56) References Cited 257:9226-9229 (1982). U.S. PATENT DOCUMENTS Saiki, et al., "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase". Science, 4,683.195 7/1987 Mullis et al...... 435/6 239:487-491, 1988. 4,683.202 7/1987 Mullis ...... 435/9 4,703,008 10/987 Lin ...... 435/2402 Scopes, "Measurement of Protein by Spectrophotometry at 4,797.279 1/1989 Karamata et al...... 424/93 205 nm". Anal. Biochem, 59:277-282, 1974. 4.855.405 8/1989 Yoshioka et al...... 530/300 Skinner, et al. "Purification and characterization of two 4,861,595 8/1989 Barnes et al...... 424/195. classes of neurotoxins from the funnel web spider. Agele

4,879,236 11/1989 Smith et al...... 435/235 nopsis aperta”, J. Biol. Chem, 264:2150-2155 (1989). 4,918,107 4/1990 Nakajima et al...... 514/616 Stapleton, et al., "Curtatoxins, neurotoxic insecticidal 4,925,664 5/1990 Jackson et al...... 424/537 polypeptides isolated from the funnel web spider FOREIGN PATENT DOCUMENTS curta', J. Biol. Chem. 265:2054-2059 (1990). 2005658 6/1990 Canada. Stewart, et al., "Construction of an improveded baculovirus 0 325 400A1 7/1989 European Pat. insecticide containing an insect-specific toxin gene'. O 340 948 11/1989 European Pat. Nature, 352:85-88 (1991). O 374940 6/1990 European Pat. Summers and Smith, "A Manual of Methods for Baculovirus O 395 357. 10/1990 European Pat. Vectors and Insect Cell Culture Procedures". Texas Agricul O 431829 6/1991 O 505207 9/1992 tural Experiment Bulletin, No. 1555. 1, 1988. WO 89/O7608 8/1989 s Tomalski and Miller, "Insect paralysis by baculovirus-me WO 92/16637 10/1992 diated expression of a mite neurotoxin gene", Nature, WO 93/23428 11/1993 352:82-85 (1991). OTHER PUBLICATIONS (List continued on next page.) Adams, et al., "Isolation and Biological Activity of Synaptic Primary Examiner-Robert A. Wax Toxins from the Venom of The Funnel Web Spider, Agele Assistant Examiner-Kawai Lau nopsis aperta", in Insect Neurochemistry and Neurophysi Attorney, Agent, or Firm-Woodcock Washburn Kurtz ology 1986, Borkevec and Gelman, eds. Humana Press, Mackiewicz & Norris LLP New Jersey, 1986. Carbonell, et al. "Synthesis of a gene coding for an 57 ABSTRACT insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors", Gene, 73:409-418 (1988). This invention provides a family of insecticidally effective Chomczynski, et al., "Single-Step Method of RNAIsolation proteins and particular members of that family which may by Acid Guanidinium. Thiocyanate-Phenol-Chloroform be isolated from the venom of the spider Phidippus audax, Extraction". Analytical Biochemistry, 162:156-159 (1987). DNA encoding such proteins, insecticidal compositions of Cutler, et al., "Electroporation: Being Developed to Trans these proteins or the DNA encoding them, and methods for form Crops, (success with model crop confirmed)", AG controlling invertebrate pests. Recombinant expression vec Biotech. News, (5):3 & 17 (1990). tors and host cells and methods for producing insecticidally Davies, et al., "Recombinant baculovirus vectors expressing effective peptides are also provided. gluthathione-S-transferase fusion proteins", Biotech, 11:933-936 (1993). 3 Claims, 5 Drawing Sheets 5,756,459 Page 2

OTHER PUBLICATIONS Raymond, "Presentation d'un programme Basic d'analyse log-probit pour micro-ordinateur", Set. Ent. med et Para Vialard, et al. "Synthesis of the Membrane Fusion and sitol., 23:117-121 1985. Hemagglutinin Proteins of Measles Virus Using a Novel Baculovirus Vector Containing the B-Galactosidase Gene". Luria, et al. In General Virology, John Wiley and Sons: New J. Virology, 64:37-50 (1990). York. Chapter 2, pp. 21-32, 1978. Waddel and Hill, "A Simple Ultraviolet Spectrophotometric Kuroda, et al., "Synthesis of Biologically Active Influenza Method for the Determination of Protein", J. Lab. Clin. Med. Virus Hemagglutinin in Insect Larvae", J. of Virology, 48:311-314 (1956). 63:1677-1685, 1989. Zlotkin, et al., "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Wood, "Protease Degradation of Autographa californica Possess a Common Binding Site". Arch, Biochem, and Nuclear Polyhedrosis Virus Proteins”. Virology, Biophysics, 240:877-887 (1985). 103:392-399, 1980. McCutchen, et al., “Development of a Recombinant Bacu Price, et al., "Complementation of recombinant baculovi lovirus Expressing an Insect-Selective Neurotoxin: Poten ruses by coinfection with wild-type facilitates production in tial for Pest Control". Biotechnology, 9:848-851, 1991. insect larvau of antigenic proteins of hepatitis B virus and Bentzien, "Biology of the Spider Diguetia imperiosa", The influenza virus", Proc. Natl. Acad. Sci., USA, 86:1453-1456, Pan-Pacific Entomologist, 49:110-123, 1973. 1989, Quicke, et al., "Extended summaries Pesticides Group and Bers, et al., "Protein and Nucleic Acid Blotting and Immu Physicochemical and Biophysical Panel Symposium Novel nobiochemical Detection". BioTechniques, 3:276-288, Approaches in Agrochemical Research". Pestic. Sci., 1985. 20:315-317, 1987. Quistad, et al., “Insecticidal Activity of Spider (Araneae), Bronstein, et al. "Rapid and Sensitive Detection of DNA in Centipede (Chilopoda), Scorpion (Scorpionida), and Snake Southern with Chemiluminescence". BioTechniques, (Serpentes) Venoms". J. Econ. Entom, 85:33-39. 8:310-314, 1990. Nentwig, et al. "Comparative Investigations on the Effect of Quistad, et al., "Paralytic and Insecticidal Toxins from the the Venoms of 18 Spider Species onto the Cockroach Blatta Funnel Web Spider, ”, Toxicon, 29:329-336, orientalis(Blattodea)", Zool. Jb. Physiol. 96:279-290 1991. (1992). Friedel, et al., "Immobilizing and Lethal Effects of Spider Bowers, et al., "Identification and purification of an irre Venoms on the Cockroach and the Common Mealbeetle". versible presynaptic neurotoxin from the venom of spider Todicon, 27:305-316, 1989. Hololena curta', Proc. Natl. Acad. Sci. USA, Roth, "The Spider Tegenaria in the Western Hemi 84:3506-3510, 1987. sphere ()". Amer: Museum Novitates, 2323:1-33, Geren, "Neurotoxins and Necrotoxins of Spider Venoms". J. 1968, Toxicol. -Toxin Reviews, 5:161-170, 1986. West, "Necrotic Arachnidism in the Northwest United States Grishin, "Toxic components from Buthus eupeus and Lycosa and Its Probably Relationship to Tegenaria agrestis (Wal singoriensis venoms". Front. Bioorg. Chem. Mol. Biol. ckenaer) ". Toxicon, 25:175-184, 1987. Proc. Int. Symp. (1980). Ed: Ananchenko. Pergamon: Creighton, "Modification of the Amino and Carboxyl Ter Oxford, England, pp. 93-96. minal Groups". Proteins. Structure and Molecular Proper ties, W.H. Freeman and Company: New York, pp. 75-76, Hermann and Frischauf, "Isolation of Genomic DNA", 1983. Methods in Enzymology, 152:180-183, 1987. Blobel, "Intracellular Protein Topogenesis". Proc. Natl. Kaneda, et al., "Scorpion toxin prolongs an inactivation Acad. Sci. USA, 77:1496-1500, 1980. phase of the voltage dependent sodium current in rat isolated Raineri, et al., "Agrobacterium-Mediated Transformation of single hippocampal neurons", Brain Res., 487: 192-195, Rice (Oryza satiya L.)". Biotechnology, 8:33-38, 1990. 1989. Fromm, et al. "Inheritance and Expression of Chimeric Genes in the Progeny of Transgenic Maize Plants", Biotech McKnight, et al., "Transcriptional Control Signals of an nology, 8:833-839, 1990. Eucaryotic Protein-Coding Gene". Science, 217, 316-324, Smith et al. "M, 26,000 antigen of Schistosoma japonicum 1982. recognized by resistant WEHI 129/J mice is a parasite Barrett, S.M.et al. (1993) "Passive hemagglutination inhi gluthathione S-transferase". Proc. Natl. Acad. Sci. USA, bition test for diagnosis of brown recluse spider bite enveno 83:8703-8707, 1986. mation" Clin. Chem. 39(10):2104-2107, Oct. 1993. U.S. Patent May 26, 1998 Sheet 1 of 5 5,756,459

O :

3OO

O.OO OO 2OO 3OO 4OO Time (min) F. G. U.S. Patent May 26, 1998 Sheet 2 of 5 5,756,459

len 9, . U.S. Patent May 26, 1998 Sheet 3 of 5 5,756,459

CN N

is dN N

leyn 8 U.S. Patent May 26, 1998 Sheet 4 of 5 5,756,459

o O C. MO

C. 8RE VN. > CO L

O 9

C O C O O do S3 S. S. & O leyn 99% U.S. Patent May 26, 1998 Sheet 5 of 5 5,756,459

Jen 9 8. A. 5,756,459 1 2 NSECTICDALLY EFFECTIVE PEPTDES quinquestriatus were revealed in Zlotkin, et al. Arch Bio SOLATABLE FROM PHDIPPUS SPIDER chem and Biophysics, 1985,240, 877-87. In a study related WENOM to their chemical and pharmacological properties, it was revealed that one toxin induced fast excitatory contractive FIELD OF THE INVENTION paralysis of fly larvae and the other induced slow depressant flaccid paralysis. Both affected sodium conductance in neu This invention relates to insecticidally effective proteins. OS. More particularly, the invention relates, interalia, to a family Canadian Patent 2,005,658 issued Jun. 19, 1990 to of insecticidally effective proteins which may be isolated Zlotkin, et al., discloses an insecticidally effective protein from Phidippus spider venom as well as methods for con derived from the Scorpion Leiurus quinquestriatus hebraeus. trolling invertebrate pests. 10 In this invention, the venomis lyophilized and separated into fractions. The fraction with the highest toxicity to blowfly BACKGROUND OF THE INVENTION larvae and the lowest toxicity to mice was subjected to In recent years, scientists and the general public have further purification and the final product is that referred to as become increasingly aware that the use of conventional “LqhP35". chemical insecticides may have undesirable environmental 15 Corresponding with the research and developments consequences. These include groundwater contamination, related to various compositions having insecticidal toxicity to non-target organisms such as birds and fish, and properties, researchers worked to develop methods for pro potential human health hazards arising from acute or chronic ducing insecticidal genes and introducing these to the target exposure. However, the need for effective insect control has 20 to be protected, or into microbial delivery systems. A not diminished. This has prompted researchers to develop method for producing insecticidal genes and introducing novel agents for insect control, including improved micro them to the target to be protected was disclosed in Cutler, AG bial insecticides. Biotech, News, 1990 vol. 7(5):3 & 17. This article teaches The most widely used microbial pesticides are derived that DNA may be electroporated directly into germinating from the bacterium Bacillus thuringiensis (hereinafter B.t.). pollen and that pollen may be put back on the flower to form This bacterial agent is used to control a variety of pests, seeds which then grow into transformed plants. This method including leaf-eating caterpillars, beetles and mosquitos. has been employed successfully in tobacco plants and may U.S. Pat. No. 4,797.279 issued Jan. 10, 1989 to Karamata, be successful in corn and alfalfa as well. This method may et al... discloses hybrid bacterial cells comprising the gene be easier than the electroporation of protoplasts because the coding for B. t. kurstaki delta-endotoxin and the gene coding 30 ultimate goal is to pollinate the flowers and "let the flowers for B.t. tenebrionis delta-endotoxin and their preparation. do the work” rather than to regenerate the plant. The process The B.t. hybrids are active against pests susceptible to B. t. consists of collecting pollen, germinating it in a germinating kurstaki strains as well as against pests susceptible to B.t. medium for 30-60 minutes after which the pollen tube will tenebrionis strains. Generally, these hybrids have useful start to come out of the pollen grain, adding the desired DNA insecticidal properties which are superior to those observed 35 to the liquid suspension containing the pollen, administering by physical mixtures of the parent strains in terms of level an electric shock to open the pores of the pollen, washing the of insecticidal activity, or in terms of spectrum of activity, or excess DNA away, and putting the altered pollen under the both. The insecticidal compositions comprising such micro stigma of a plant and waiting until seeds are formed. This organisms may be used to combat insects by applying the may be an easy method to move any gene into crop plants. hybrids in an insecticidally effective amount to the insects or An additional delivery system was disclosed in U.S. Pat. to their environment. No. 4,861.595 issued Aug. 29, 1989 to Barnes and Edwards. Another derivation from the bacterium B.t. was disclosed This invention concerns the use of treated, substantially in European Patent Application. Publication No. intact, microbial cells as a delivery system of protein com 0325400A1, issued to Gilroy and Wilcox. This invention pounds to and humans. The microbial cells initially relates to a hybrid toxin gene which is toxic to lepidopteran 45 produce a protein intracellularly via a homologous gene. The insects. Specifically, the invention comprises a hybrid delta protein-producing microbe is treated by chemical or physi endotoxin gene comprising part of the B.t. var. kurstaki cal means while the cell is substantially intact. Manipulation HD-73 toxin gene and part of the toxin gene from B.t. var. of the treatment process produces a nonproliferative treated kurstaki strain HD-1. The hybrid toxin gene (DNA) encod microbial cell without significant loss of the activity of the ing a protein having activity against lepidopteran insects was 50 intracellular compound. Since the cell will not replicate and disclosed. will have a stable cell wall which may then be broken down The bacterium B. t. was also utilized for its insecticidal in a desired area of the digestive system of the or properties in European Patent Application. Publication No. human, it allows the timed or targeted release of the products 0340948 issued to Wilcox, et al. This invention concerns encapsulatable by the subject invention. After suitable hybrid pesticidal toxins which are produced by the fusion of 55 treatment, the protein-producing microbial cell itself is used an insect gut epithelial cell recognition region of a B.t. gene as the delivery system so no purification of the produced to diphtheria toxin B chain to prepare a hybrid B.t. toxin compound is necessary. Any protein, polypeptide, amino which is active against lepidopteran insects. It was sug acid, or compound, including insecticides, that may be gested that the hybrid B.t. gene may be inserted into a plant produced by microbial means may be the starting material of or cloned into abaculovirus to produce a toxin which can be the invention. recovered. Alternatively, the host containing the hybrid B.t. U.S. Pat No. 4879,236 issued Nov. 7, 1989 to Smith and gene can be used as an insecticide by direct application to Summers. relates to a method for incorporating a selected the environment of the targeted insect. gene coupled with a baculovirus promoter into a baculovirus In the search for insecticidal compounds, scorpion venom genome to produce a recombinant baculovirus expression was identified as a possible source of compounds providing 65 vector capable of expression of the selected gene in an insect insecticidal properties. Two insect selective toxins isolated cell. The method involves cleaving baculovirus DNA to from the venom of the scorpion Leirus quinquestriatus produce a DNA fragment comprising a polyhedrin gene or 5,756,459 3 4 portion thereof, including a polyhedrin promoter. To prepare molecular weight factors reversibly bind to calcium chan a recombinant transfer vector, the DNA fragment is inserted nels with sufficient specificity and affinity to extinguish into a cloning vehicle and then a selected gene is inserted calcium conductance in neurons and to permit isolation and into this modified cloning vehicle such that it is under the purification of calcium channel structures. These venoms control of the polyhedrin promoter. The recombinant trans were found to be toxic to mammals. fer vector is then contacted in insect cells with a baculovirus Other applications of spider toxins were discussed in DNA so as to effect recombination and incorporation of the Jackson and Parks, Ann. Rey. Neurosci., 1989, 12, 405-14. selected gene into the baculovirus genome. The baculovirus This article describes the great heterogeneity in the toxins of Autographa californica (AcMNPV) and its associated poly different taxa. It recognizes that experiments have suggested hedrin promoter were found to be useful in producing a viral 10 species-specific properties of calcium channels and the expression vector capable of extremely high levels of spider venoms might provide calcium channel antagonists. expression of a selected gene in an insect host cell. The spider venoms discussed are found to affect vertebrates. The inventors suggest that the expression vector might be The article also identifies spider venoms as possible sources used in a system for controlling insects by selecting a gene of insect-specific toxins for agricultural applications. which produces a protein which is toxic to a specific insect 15 Adams, et al., "Isolation and Biological Activity of Syn or to a spectrum of insects and cloning that gene into the aptic Toxins from the Venom of the Funnel Web Spider. AcMNPV expression vector. They suggest that the vector Agelenopsis aperta,” in Insect Neurochemistry and Neuro could be applied to the plant or animal to be protected. The physiology 1986. Borkovec and Gelman (Eds.). Humana recombinant virus could invade the cells of the intestinal Press. New Jersey, 1986, teaches that multiple peptide toxins wall following ingestion by the insect and begin replication. 20 which antagonize synaptic transmission in insects have been The possibility of using DNA technology to incorporate a isolated from the spider Agelenopsis aperta. synthetic gene which encodes a neurotoxin found in scor U.S. Pat. No. 4.855.405 issued Aug. 8, 1989 to Yoshioka, pion venom was explored in Carbonell, et al., Gene, 1988, et al., discloses a receptor inhibitor obtained from Joro 73, 409-18. This article describes the possibility of using spider (Nephila clavata) venom, and its manufacturing DNA technology to incorporate a synthetic gene which 25 method. Yoshioka demonstrates that their toxins show encodes a neurotoxin found in the venom of the scorpion, glutamate receptor inhibitory activity in an insect electro Buthus eupeus, into the baculovirus genome to improve physiological assay. baculovirus pesticides. Three methods of expression using U.S. Pat. No. 4,918,107 issued Apr. 17, 1990 to Nakajima. the polyhedrin promoter-based AcMNPV expression system 30 et al., relates to a compound which has glutamate receptor to effect toxin production were studied. Expression of the 36 inhibitor activity, a process for preparing the same, and an codon gene alone provided minuscule production of the insecticidal composition containing the same. toxin. Some success was found with the attachment of a signal peptide to the toxin. Significant levels of protein were Accordingly, due to a combination of problems associated produced when the toxin gene was fused to the N-terminus with conventional chemical insecticides, including pest of polyhedrin. However, production was ten to twenty-fold 35 resistance and injurious effects on non-target organisms, less than that observed for polyhedrin itself. The limitation there exists a continuing need for the development of novel to expression was not believed to be at the level of tran means of invertebrate pest control. scription but at the post-transcriptional level including trans SUMMARY OF THE INVENTION lation and protein stability. Paralytic activity of the toxin products was not detected. However, Carbonell's failure to There are provided by this invention novel insecticidally express a synthetic gene encoding a specific neurotoxin in a effective proteins derived from, for example, a spider of the baculovirus vector is not predictive of the state of the art. To genus Phidippus. Two proteins, exhibiting insecticidal the contrary, several publications indicate the opposite. activity, were isolated from the chromatographical separa Davies, et al., Biotech., 1993, 11,933–36.in describing a tion of Phidippus whole venom toxin. The proteins are method for recombinant protein expression using baculovi 45 designated BW2 and BW5. rus vectors, noted that the baculovirus system may be used Further provided by this invention are methods for con to express almost any heterologous gene of interest, includ trolling invertebrate pests with insecticidal compositions ing functional recombinant proteins. Other publications containing insecticidal proteins or the genes encoding these describing a method for recombinant protein expression proteins and insecticidal compositions. using baculovirus vectors include Tomalski, et al., Nature, Still further provided by this invention are novel recom 1991, 352, 82-85:Stewart, et al., Nature, 1991, 352, binant expression vectors and genetically engineered insec 85-88;and McCutchen, et al., Biotechnology, 1991, 9. ticidal microbes and methods of controlling invertebrate 848-851. pests comprising contacting said pests with a recombinant Researchers have also been able to isolate toxins extracted 55 baculovirus capable of expressing an effective amount of an from the venom of spiders. U.S. Pat. No. 4925,664 issued insecticidally effective peptide substantially isolatable from to Jackson and Parks on May 15, 1990, discloses methods of Phidippus spider venom and agriculturally or horticulturally treating heart and neurological diseases by applying toxins acceptable salts thereof. derived from the spiders Agelenopsis aperta and Hololena curta. The toxins are also effective as specific calcium DETALED DESCRIPTION OF THE DRAWTNGS channel or excitatory amino acid receptor blockers that may FIG. 1 depicts a chromatogram representing the separa be used against insects and related pests. tion of the C-100 retentate of Phidippus audar whole venom Another study related to the properties of isolated spider by immobilized metal ion chromatography (MAC) on a venom toxins revealed the ability of low molecular weight TSK Chelate 5 PW column loaded with Cu". factors isolated from funnel-web spider venoms to revers 65 FIG. 2 depicts a chromatogram representing the separa ibly bind to calcium channels. WO 89/07608 issued Aug. 24, tion of fraction 2 (from FIG. 1) by anion exchange chro 1989 to Cherksey, et al... discloses that these active low matography on a Mono-Q column. 5,756.459 5 6 FIG. 3 depicts a chromatogram representing the separa It will be understood from this disclosure that additional tion of fraction 5 (from FIG. 1) by anion exchange chro insecticidally effective proteins are expected to be within the matography on a Mono-Q column. scope of the invention. That is, it is believed other insecti FIGS. 4A and 4B depict chromatograms representing the cidally effective proteins in the family exist and may be separation of fractions 2 (FIG. 4B) and 5 (FIG. 4B) isolatable from Phidippus as well as other sources in addi (obtained from FIGS. 2 and 3, respectively) by chromato tion to the two detailed herein. focusing on a Mono-P column. C. Identification of the Coding Sequence of Insecticidally DETAILED DESCRIPTION OF THE Effective Proteins of this Invention INVENTION A substantially isolated DNA sequence encoding a protein Phidippus audax is a member of the Family Salticidae. of this invention may be determined by methods known to better known as the jumping spiders. It is widely distributed those in the art. in the United States, and is especially common in the Employing partial amino acid sequence data, the genes southern states. Paudax is one of the larger members of the responsible for the production of proteins from a source can family, sometimes exceeding half an inch in length. P. be isolated and identified. Numerous methods are available audax, like most other salticids. is a hunting spider, relying 15 to obtain the gene responsible for the production of a on agility and visual acuity to detect and capture prey. protein. Examples include Fuqua, et al. Biotechnique. Vol. The mechanism of action of the insecticidally effective 9,No. 2 (Aug 1990); Frohman, "RACE: Rapid amplification proteins of this invention is unknown. It has been found that of cDNA ends", in PCR protocols, Innis, et al. (Eds.). these toxins produce a characteristic set of symptoms in Academic Press, San Diego, Calif. (1990) and U.S. Pat. No. lepidopteran larvae. These include a rapidly developing 4,703,008.which patent is incorporated herein by reference. flaccid paralysis, complete cessation of dorsal vessel con Briefly, a "degenerate” series of DNA oligonucleotides tractions (heartbeats), and in some cases a gradually spread are synthesized which encode the determined amino acid ing Zone of necrotic or cyanotic discoloration. Paralysis sequence or which represents the complementary DNA generally develops in less than ten minutes, and was (cDNA) strand to such a DNA molecule which encodes the observed in some cases to develop in less than two minutes. 25 determined amino acid sequence. These synthetic DNA These effects are identical to those caused by whole Phid oligonucleotides may then be used to probe for DNA ippus venom, strongly implying that the toxic proteins sequence homology in cell clones containing recombinant described in this invention are primarily responsible for the DNA molecules comprising, in part, DNA sequences insecticidal effects of this venom. derived from the genomic DNA of an organism such as a A. The Isolation of Proteins from Phidippus Venom 30 spider or derived from cDNA copies of mRNA molecules One source of insecticidally effective proteins is Phidip isolated from cells or tissues of an organism such as a spider. pus venom. Spider venom can be removed from Phidippus Generally, DNA molecules of fifteen (15) nucleotides or by any method known such as venom gland extraction from more are required for unique identification of an homolo cephalothorax. However, in order to avoid impurities within gous DNA, said number requiring unique determination of the spider venom and the isolated toxins, the spider venom 35 at least five (5) amino acids in sequence. It will be appre preferably is obtained by electrical stimulation of the spiders ciated that the number of different DNA oligonucleotides to cause release of the venom and subsequent suction to which can encode the determined amino acid sequence may collect the released venom and prevent contamination of the be very large since each amino acid may be encoded by up venom by regurgitate or hemolymph as described in U.S. to six (6) unique trinucleotide DNA sequences or codons. Pat. No. 4,925.664. Therefore, it is impractical to test all possible synthetic DNA Once the spider venom is obtained by electrical milking probes individually, and pools of several such DNA oligo techniques, it can be fractionated into its protein (toxin) nucleotides are used concomitantly as probes. The produc components by high performance liquid chromatography tion of such pools which are referred to as "degenerate" (HPLC) with a variety of separation modes such as ion probes is well known in the art. It will also be appreciated exchange and immobilized metal ion affinity chromatogra 45 that while only one DNA oligonucleotide in the probe phy (IMAC), and chromatofucusing. mixture will have an exact sequence homology to the gene Thus, using the technique of electrically milking the of interest, several of the synthetic DNA oligonucleotides in spider coupled with HPLC using IMAC. ion exchange and the pool may be capable of uniquely identifying said gene chromatofocusing columns, it is possible to obtain substan since only a high degree of homology is required. Therefore, tially purified spider toxins. It will be appreciated, however, 50 successful isolation of the gene of interest may be accom that other equivalent techniques may also be employed plished with synthetic DNA oligonucleotide probe pools within the scope of the present invention in order to isolate which do not contain all possible DNA sequences. In the spider toxins. The toxins thus isolated can be assayed for general, codons which are infrequently utilized by the organ insecticidal activity and the DNA and amino acid sequences ism need not be represented in the oligonucleotide probe determined by methods known to those in the art. 55 pool. In fact, a single sequence DNA probe may be produced B. Insecticidally Effective Proteins by including only the DNA codons most frequently utilized This invention, in one of its aspects, provides a family of by the organism for each amino acid, although, it will be insecticidally effective proteins, and insecticidally effective appreciated that this approach is not always successful. fragments thereof and agriculturally or horticulturally One technique to isolate a gene sequence employs the acceptable salts thereof. Polymerase Chain Reaction (PCR). See e.g., U.S. Pat. Nos. Once an insecticidally effective, protein-containing frac 4,683.195 and 4,683.202 which patents are incorporated by tion has been isolated from a source and purified as reference as if fully set forth herein. Essentially, PCR allows described herein, amino acid sequence determination can be the amplification of a selected DNA sequence when at least performed in any way known to those in the art such as one of the two terminal portions of the sequence are known. N-terminal amino acid sequencing and use of an automated 65 Primers, or oligonucleotide probes, are designed which amino acid sequencer. The entire amino acid sequence can correspond to the 5' ends of both complimentary strands of be determined. the sequence of interest. Using PCR with the addition of 5,756,459 7 8 deoxynucleotide bases and a heat stable polymerase, the implied, although not always explicitly stated, that these portion of the DNA sequence between the primers is then expression vectors must be replicable in the host organisms synthetically constructed. Numerous cycles of PCR thus either as episomes or as an integral part of the chromosomal allow a logarithmic amplification of even a single copy of a DNA. Clearly a lack of replicability would render them DNA sequence. effectively inoperable. In sum, "expression vector" is given In one such method of employing PCR to obtain the gene a functional definition, and any DNA sequence which is which encodes a unique spider venom toxin, RNA is isolated capable of supporting expression of a specified DNA code from the spider and purified by methods known in the art. disposed therein is included in this term as it is applied to the Reverse transcriptase and random primers are then used to specified sequence. In general, expression vectors of utility reverse transcribe the spider RNA into cDNA. Alternatively, in recombinant DNA techniques are often in the form of oligo(dT) can be used, instead of random primers, to spe "plasmids” which refer to circular double stranded DNA cifically reverse transcribe mRNA. The "degenerate" mix which, in their vector form are not bound to the chromo ture of synthetic DNA oligonucleotides encoding the some. "Plasmid" and "vector" are used interchangeably as 5'-termini of the cDNA, corresponding to the amino the plasmid is the most commonly used form of vector. terminus of the protein, and the 5'-termini of its complimen 15 However, the invention is intended to include such other tary strand, corresponding to the carboxy-terminus of the forms of expression vectors which serve equivalent func protein, is then designed with a restriction endonuclease site tions and which become known in the art subsequently on their 5'-termini. This restriction site allows for the inser hereto. tion of the cloned DNA fragment into a cloning vector. This 'Recombinant host cells' refers to cells which have been DNA oligonucleotide mixture is used to prime a PCR transformed with vectors constructed using recombinant reaction. Because the synthetic DNA oligonucleotide mix DNA techniques. ture used to prime the PCR reaction is specific to the desired Further provided by this invention is a recombinant sequence, only the desired cDNA will be effectively ampli expression vector comprising a DNA sequence which fied. The resultant product represents an amplified cDNA encodes an insecticidally effective peptide substantially iso which can be ligated to any of a number of known cloning 25 latable from Phidippus spider venom. The vector is capable vectors. Not withstanding this, it will be appreciated that of effecting the expression of the coding sequence in trans "families" of proteins or peptides may exist in spider ven formed cells. Also provided by the invention are recombi oms which will have similar amino acid sequences and that nant host cells with a DNA sequence encoding an insecti in such cases, the use of mixed oligonucleotide primer cidally effective peptide substantially isolatable from sequences may result in the amplification of one or more of 30 Phidippus spider venom in a manner allowing the host cell the related cDNAs encoding these related proteins. Genes to express the peptide. encoding related proteins are also within the scope of the Such recombinant expression vectors may be employed in invention as the related proteins also have useful insecticidal methods for producing insecticidally effective peptides. activities. Such methods comprise culturing recombinant host cells Finally, the produced cDNA sequence can be cloned into 35 wherein a recombinant expression vector transformed, trans an appropriate vector using conventional techniques, ana fected or otherwise applied in said host cells has a DNA lyzed and the nucleotide base sequence determined. A direct sequence encoding the peptide and recovering the insecti amino acid translation of these PCR products will reveal that cidally effective peptide from the recombinant host cell they corresponded to the complete coding sequence for the culture or host organism. In such methods the vector is mature protein. The portion of the DNA sequence which capable of supporting with host cell factors the expression of might encode amino acids corresponding to precursor and or the coding sequence in the transformed cells. propeptide regions may not be obtained by this approach. Provision of a suitable DNA sequence encoding the Such sequences may be determined by isolation of genomic desired protein permits the production of the protein using or cDNA clones using the cDNA clone produced in this recombinant techniques now known in the art. The coding approach as a hybridization probe which is within the scope 45 sequence can be obtained by retrieving a cDNA or genomic of the art. sequence from a native source of the protein or can be D. Application of the Proteins as Insecticides prepared synthetically using the accurate amino acid The insecticidally effective proteins of this invention are sequence determined from the nucleotide sequence of the believed to be useful in controlling invertebrate pests such as gene. When the coding DNA is prepared synthetically, those in the order Lepidoptera. Methods for using the 50 advantage can be taken of known codon preferences of the insecticidally effective proteins of this invention may intended host. include contacting the pests with an effective amount of a Expression systems containing the requisite control protein of this invention. sequences, such as promoters, and preferably enhancers and Methods of contacting an invertebrate pest with a protein termination controls, are readily available and known in the to control said pests are known. Examples include the 55 art for a variety of hosts. See e.g., Sambrook, et al., insertion of a gene encoding a toxic peptide or protein into Molecular Cloning a Laboratory Manual, Second Ed. Cold the genome of a baculovirus, such as the Autographa Spring Harbor Press, New York (1989). californica nuclear polyhedrosis virus. Of course, methods Thus, the desired proteins can be prepared in both pro of controlling invertebrate pests using the proteins of this caryotic and eucaryotic systems, resulting, in the case of invention can be used in combination with other methods of many proteins. in a spectrum of processed forms. controlling pests. The most commonly used procaryotic system remains E. E. Recombinant expression coli, although other systems such as B. subtilis and As used herein, "expression vector" includes vectors Pseudomonas are also expected to be useful. Suitable con which are capable of expressing the desired peptide or trol sequences for procaryotic systems include both consti protein from DNA sequences contained therein, where such 65 tutive and inducible promoters including the lac promoter, sequences are operably linked to other sequences capable of the trp promoter, hybrid promoters such as tac promoter, the effecting their expression, i.e., promoter sequences. It is lambda phage Pl promoter. In general. foreign proteins may

5,756,459 11 12 moth) have been registered in some countries and used as subsequent suction to collect the released venom and pre pesticides. Introduction of at least one insect-selective toxin vent contamination of the venom by regurgitate or into the genome is expected to significantly enhance the hemolymph as described in U.S. Pat. No. 4,925.664. potency of such pesticides. Crude venom (stored at -80° C.) was thawed and mixed A recombinant expression vector expected to be particu thoroughly with the starting solvent prior to chromatogra larly suitable for use in this invention is a baculovirus phy. The venom was fractionated by high performance expression vector such as the type disclosed in U.S. Pat. No. liquid chromatography (HPLC) incorporating Beckman 4.879,236, which patent is incorporated by reference as if System Gold 126 solvent delivery and 168 photodiode array fully set forth herein. Other publications describing a detector modules. Several columns and conditions were method for recombinant protein expression using baculovi 10 used in the purifications described below. rus vectors include Tomalski, et al., Nature, 1991, 352. Ultrafiltration of the Phidippus crude venom in CENTRI 82-85;Stewart, et al., Nature, 1991, 352, 85-88; and CONTM C-3.C-10.and C-100 microconcentrators (Amicon) McCutchen, et al., Biotechnology. 1991. 9, 848-851. The indicated that the insecticidal activity remained in the reten vector is expected to be useful in a system where a DNA tate. Therefore, HPLC column fractions were generally sequence encoding an insecticidally effective peptide sub 5 concentrated in CENTRICONTM C-10 or C-3 microconcen stantially isolatable from Phidippus spider venom can be trators for assay in TBW larvae as follows. Column fractions cloned into baculovirus such as Autographa californica were placed in C-3 filters and centrifuged at approximately (AcMNPV) expression vector as described in U.S. Pat. No. 4000xg for 70 min (this concentrated the samples to 4,879.236 and Miller, et al., Science, 1983, 219, 715-721. approximately 200 ul). Two ml of phosphate buffered saline, The recombinant expression vector virus could then be 20 pH 6.5 (PBS; 136.7 mM NaCl. 2.6 mM KCl, 1.46 mM applied to the plant or animal upon which the insect is a pest, KHPO, 8.0 mM NaHPO) was added to the concentrate and when the virus is ingested by the pest insect, the and samples again centrifuged to give a final volume of recombinant virus will invade the cells of the intestinal wall 150-200 ul. and begin replication. During replication, the gene for the Immobilized metal ion affinity chromatography (IMAC) insecticidally effective protein will be expressed, resulting in 25 was performed on a ProgelTM TSK Chelate 5PW column the disablement or death of the insect in a shorter period than (7.5x75 mm, from Supelco) freshly loaded with Cu" ions if the insect had ingested the wild type AcMNPV virus. (40 mM CuSO, (aq)). The Abuffer was 20 mM NaH2PO, A hybrid virus also expected to be useful is taught in 1 mM imidazole. 0.5M NaCl adjusted to pH 7.0 with 10M European Patent Application 0340948. The hybrid virus NaOH. The B buffer was 20 mM NaH2PO 20 mM expressing the DNA of this invention is expected to yield a 30 imidazole. 0.5M. NaCl adjusted to pH 7.0 with 10M NaOH. virus having an altered insect host range. For example, The column was equilibrated in the Abuffer and eluted at 1 fusion proteins could be expressed as a single polypeptide ml/min with a 40 min linear gradient from 0% B to 100% B product of a hybrid gene consisting of DNA of this invention after 5 min at 0%. B. After 5 min at 100% B the column was and a specific insect gut cell recognition protein to direct the returned to 0% B over 2 min and equilibrated with A buffer expressed insecticidally effective peptide to the host insect 35 before the next injection was made. The effluent was moni target. tored at 280 nm and fractions collected with a Gilson model Various prokaryotic and eucaryotic microbes can be trans 203 fraction collector. formed to express a hybrid toxin gene encoding an insecti Anion exchange chromatography (AEC) was performed cidally effective protein by the method taught in European on a Mono-Q column (5.0x50 mm). The A buffer was 25 Patent Application 0325400. mM Tris base adjusted to pH 7.5 with 6N HCl and the B Hybrid bacterial cells, comprising a plasmid with the gene buffer was 25 mM Tris base, 1.0M NaCl adjusted to pH 7.5 coding for the protein of this invention are expected to be with 6N HC. The column was eluted at a flow rate of 1 useful in the method of this invention. Insects would be ml/min with a linear gradient (begun 5 minutes after controlled by applying the hybrids to insects. See e.g., U.S. injection) from 0 to 100% B buffer over 75 min. The column Pat. No. 4.797.279 which patent is incorporated by reference 45 was taken to 50% B buffer over 2 min, held at 50% B buffer as if fully set forth herein. for 4 min, returned to 0% B buffer over 2 min and equili Other examples of employing baculovirus that would be brated before the next injection. Chromatography was moni suitable for use in this invention are described in Tomalski, tored at 280 nm and fractions collected with a Gilson model et al., Nature, 1991, 352, 82-85 and Stewart, et al., Nature, 203 fraction collector. 1991. 352, 85-88); and McCutchen, et al., Biotechnology, 50 Chromatofocusing was performed using a Mono-P col 1991, 9, 848-851. umn (5.0x50 mm). Buffer A was 2.5 mM Bis-Tris adjusted to An insecticidal composition comprising an insecticidally pH 7.2 and buffer B was Polybuffer 74 diluted 1:10 and effective amount of a protein according to this invention and adjusted to pH 5.0 with 6NHCl. The column was eluted at agriculturally or horticulturally acceptable salts thereof in an a flow rate of 0.5 ml/min with Ok B buffer for 5 min then agriculturally or horticulturally acceptable carrier therefor is 55 immediately switched to 100% B buffer. This chromatog also provided. raphy was monitored at 280 nm and fractions collected with a Gilson model 203 fraction collector. EXAMPLES Protein concentrations were determined using the Pierce BCA assay reagents and methods unless otherwise noted. The following examples are given to illustrate particular N-terminal amino acid sequence analysis was performed at compositions and methods within the scope of the present the Biotechnology Center at Utah State University in Logan, invention but they are not intended to limit the scope of the Utah. present invention. Insecticidal Activity Methods MATER ALS AND METHODS The insects tested were last instar. laboratory reared General Separation Methods 65 larvae of the tobacco budworm (TBW), Heliothis virescens, The spider venom is preferably obtained by electrical the cabbage looper (CL), Trichoplusia ni, and the beet stimulation of the spiders to cause release of the venom and armyworm (BAW), Spodoptera exigua. All samples. 5,756.459 13 14 whether whole venom or venom fractions, were prepared in filter-sterilized physiological saline (PBS), pH 6.5. Samples TABLE were administered by injection into the hemocoel at or near Ultrafiltration Fractions of Phidippus audar Whole Venom the lateral midline of the fourth abdominal segment; the Contains Paralytic Activity in TBW Larvae needle was inserted at a shallow angle to avoid injury to internal organs. After treatment the larvae were held in Sample Dose' Initial 24 hours individual Petri dishes with food and observed periodically Control (PBS) -- OW5 OWS C-3 retentate 23.5 4/5 4/5 for paralysis and other effects such as feeding inhibition. C-3 filtrate 24.7 OW5 OW5 Whole venom doses were calculated in terms of whole O C-100 retentate 10.0 5/5 5/5 venom equivalents (WVE). One WVE is the amount of any C-100 filtrate 16.2 W5 OWS material which is normally present in one microliter of "This value represents the dosage of the fraction injected in terms of g of whole milked venom. Doses of components from early rotein per g of larvae. fractionations were also calculated in terms of WWE. Doses E. his value represents the mumber of larvae paralyzed versus total number of of purified toxins were calculated by protein assay, or by 15 larvae injected. measuring short wavelength ultraviolet absorbances Example 3 (Scopes. Anal. Biochem., 1974. 59,277;and Waddell, J. Lab, Fractionation of C-100 Retentate of Phidippus audax Whole Clin. Med., 1956, 48. 311). Both methods gave similar Venom by Immobilized Metal Ion Affinity Chromatography results, with calculated concentrations varying by a factor of and Identification of Paralytic Fractions. less than 2. 20 The C-100 retentate of whole venom was separated by IMAC using a TSK Chelate 5 PW column (7.5x75 mm) Example 1 loaded with Cu'. The column was eluted at 1 ml/min with Phidippus audax Whole Venom Contains Paralytic Activity a 40 min linear gradient from 0% B to 100% B after 5 min Whole venom was diluted 1:10 or 1:100 in sterile-filtered 25 at 0%. B. Buffer Awas 20 mMNaHPO, 0.5M NaCl. 1 mM phosphate-buffered saline (PBS), pH 6.5. Three microliter imidazole adjusted to pH 7.0. Buffer B was 20 mM NaHPO, 0.5MNaCl. 20 mMimidazole adjusted to pH 7.0. aliquots of these venom solutions (providing doses of 0.3 or The column was monitored at 280 nm and fractions col 0.03 WWE/larva) were injected into TBW, BAW, and CL lected with a Gilson model 203 fraction collector. This larvae as described previously. The larvae were analyzed separation, depicted in the chromatogram shown in FIG. 1, initially and after 24 hours for paralysis and the results are resulted in the collection of eight fractions. These fractions indicated in Table I. As indicated in Table I, whole venom were injected into TBW larvae as described previously. The contained paralytic activity for all three species tested. larvae were analyzed after 24 and 48 hours and examined for Control larvae, which were injected with PBS alone, were paralysis and feeding inhibition and the results are indicated unaffected. in Table II. As indicated in Table II, fractions 2, 5, 6 and 35 8 contained paralytic activity for TBW. TABLE Phidippus audal Whole Venom Contains Paralytic Activity TABLE MAC Fractions of C-100 Retentate of Species Dose (WWE/larva) 24 hours' 48 hours Phidippus audar Whole Venom Contains Paralytic and TBW 0.3 5/5 5/5 Feeding Inhibitory Activity in TBW Larvae TBW 0.03 45 4/5 TBW Control 05 OW5 Sample Dose" 24 hours 48 hours BAW 0.3 66 66 BAW 0.03 66 6V6 Control (PBS) --- OW3 OW3 BAW Control OW6 OW6 45 Cu 21.5 OW3 OW3 CL 0.03 S/6 5/6 Cu2 20.4 1/3 OW3 CL Control OW6 OW6 Cu3 21.6 0/3 O/3 Cu4 20.8 0/3 OW3 This value represents the number of larvae paralyzed versus total number of Cu5 2O.O 2/3 23 larvae injected Cu6 21.4 3/3 113 50 C 19.7 O3 OW3 Cu3 22.7 3/3 23 Example 2 This value represents the dosage of the fraction injected in terms of ug of Initial Fractionation of Phidippus audar Whole Venom by Eiseis value perg represents of larvae. the number of larvae paralyzed versus total number of Ultrafiltration and Identification of Paralytic Fractions. larvae iniected. 55 Some of the larvae that were not paralyzed exhibited feeding inhibition at 48 Whole venom was filtered using CENTRICONTMC-3 and hours. C-100 microconcentrators, as described previously, and fractions consisting of the retentates and filtrates were Example 4 obtained. Various amounts of these fractions were injected Fractionation of C-100 Retentate of Phidippus audax Whole into TBW larvae as described previously. The larvae were Venom by Immobilized Metal Ion Affinity Chromatography analyzed initially and after 24 hours for paralysis and the Followed by Anion Exchange Chromatography and Identi results are indicated in Table II. As indicated in Table II, the fication of Paralytic Fractions. paralytic activity was retained in the retentate fractions of AEC was performed on fractions 2 and 5.depicted in FIG. both C-3 and C-100 microfiltrations. The specific activity of 1, from the IMAC preparation of C-100 retentate of whole the C-100 retentate was higher than that of the C-3 retentate, 65 venom using a Mono-Q column (5.0x50 mm). The Abuffer suggesting that the bulk of the activity resides in the C-100 was 25 mM Tris base adjusted to pH 7.5 and the B buffer was retentate fraction. 25 mM Tris base, 1.0M NaCl adjusted to pH 7.5. The

5,756,459 17 18 cleaves within the downstream (3' end) primer leaving a Bulletin No. 1555, 1988). Four days post-transfection, dilu sticky end. The vector, pKS (Stratagene, Lajolla, Calif.), is tions of the cell supernatant are plaqued on 100 mm plates double digested with EcoRV (US Biochemical) and Not I to seeded with 5x10 Sf9 cells and covered with agarose generate sites specific for directional cloning. Vector and containing Bluo-gal (Gibco BRL, Gaithersburg, Md.) as insert are ligated and transformed into complex DH5OF. substrate. Within 5 to 6 days. recombinants are detectable by Colony lifts are screened with the 'p labeled internal probe their pale blue color. Plaques are picked using a pasteur pipet and eluted in 1 ml of media. This eluent is used to re-infect and candidate colonies are further characterized by sequenc Sf9 cells seeded into a T-25 flask. Three days post-infection ing (US Biochemical's Sequenase Version 2.0) mini-prep a small volume of supernatant is collected from six different DNA using the internal probe as primer. isolates and used to prepare viral DNA. PCR amplification Example 7 using viral specific primers from the region surrounding the Recombinant Baculovirus Construction polyhedrin gene confirms that viral isolates contain an A lepidopteran signal sequence (Jones. et al., J. Biol. appropriately sized insert and lacked any wild-type contami Chem. 1990, 265.8596), is constructed from two synthetic nation. Titered stocks of the recombinant viruses are then oligonucleotides using the method of Rossi, et al., J. Biol. 15 prepared for in vivo and in vitro testing. Chem., 1982, 257. 9226. Two 48 mers are purified by ion exchange chromatography. These two oligonucleotides MAMMALIAN TOX COLOGY share eleven base pairs of complementary sequence at their 3' termini. When the sequences are annealed in the presence Whole venom and the C-100 retentate fraction of whole of the four deoxy-ribonucleoside triphosphates and the Kle venom were tested at doses of 5 and 10 WWE/mouse, now fragment of DNA polymerase I, a double-stranded respectively, in male Swiss-Webster mice (25-30 g) by product is synthesized. Reaction products are purified using intraperitoneal (i.p.) injection (n=1). Samples were diluted hydroxylapatite chromatography and the double-stranded in physiological saline prior to injection; control mice were DNA molecules are digested with Aat, the restriction injected with equivalent volumes of saline. Mice were enzymes appropriate for inserting this sequence upstream of 25 observed continuously for the first several hours after cDNA cloned into pKS-DK9c. Subclones are screened for treatment, and then periodically for 48 hours. The only the insertion of the signal sequence and evaluated by DNA visible effect of the C-100 retentate fraction was a slight sequencing. twitching of the subcutaneous muscles. This slowly sub DNA sequencing confirms an in-frame fusion between the sided over several hours; no other effects were noted at any two cDNA sequences. The entire synthetic gene construct is 30 time. No effects were noted from the saline injection or the excised and adapted for cloning into the Nhe site of whole venom injection.

SEQUENCELISTING

( 1 GENERAL INFORMATION: ( i i i NUMBER OF SEQUENCES: 1

( 2 INFORMATION FOR SEQID No:1: ( i SEQUENCE CHARACTERISTICs: ( A ) LeNGTH: 13 amino acid residues (B) TYPE: amino acid ( D TOPOLOGY: unknown ( x i SEQUENCE DESCRIPTION: SEQDD No:1: A s p G 1 y 1 e V a 1 G1 y Lys A 1 a Ser Ser Ty r A 1 a. A a eu 1. 5 1 0 pBlueBac, a baculovirus transfer vector (Vialard, et al., J. What is claimed: Virology, 1990, 64, 3-50). Subclones are sequenced to 1. A substantially purified, insecticidally effective protein confirm the correct insertion of the construct. DNA sequenc from Phidippus spider venom characterized as causing flac ing of plasmid WR9 confirms the insertion of the synthe 55 cid paralysis when injected into larvae of at least one sized gene in the baculovirus transfer vector pblueBac. The member of the class Insecta, wherein said protein comprises use of the pblueBac vector expedites the screening process an N-terminal amino acid sequence having SEQ ID NO:1. as insertion of the recombinant gene into the baculovirus 2. The protein of claim 1 wherein the spider venom is genome is accompanied by co-expression off-galactosidase from Phidippus auda. 3. An insecticidal composition comprising an insecticid and detectable by a color change when grown on indicating 60 ally effective amount of a substantially purified, protein from media. Phidippus spider venom, characterized by causing flaccid Recombinant baculoviruses are produced by transfection paralysis when injected into larvae of at least one member of of Spodoptera frugiperda strain Sf9 (ATCC# CRL1711) the class Insecta, in an agriculturally or horticulturally cells with a mixture of 1 Jug AcMNPV viral DNA and 2 pig acceptable carrier, wherein said protein comprises an plasmid DNA using the protocol of Summers and Smith (in 65 "A Manual of Methods for Baculovirus Vectors and Insect N-terminal amino acid sequence having SEQ ID NO:1. Cell Culture Procedures", Texas Agricultural Experiment x: *k k k k