Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• This invention relates to insecticidally effective proteins. More particularly, the invention relates, inter alia , to a family of insecticidally effective proteins which may be isolated from Filistata spider venom as well as methods for controlling invertebrate pests.
• the most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (hereinafter B . t . ) .
• This bacterial agent is used to control a variety of pests, including leaf-eating caterpillars, beetles and mosquitos.
• U.S. Patent No. 4,797,279 issued January 10, 1989 to Kara ata et al. discloses hybrid bacterial cells comprising the gene coding for B . t . kurstaki delta-endotoxin and the gene coding for B . t . tenebrionis delta-endotoxin and their preparation.
• the B . t . hybrids are active against pests susceptible to B . t .
• hybrids have useful insecticidal properties which are superior to those observed by physical mixtures of the parent strains in terms of level of insecticidal activity, or in terms of spectrum of activity, or both.
• the insecticidal compositions comprising such microorganisms may be used to combat insects by applying the hybrids in an insecticidally effective amount to the insects or to their environment.
• Another derivation from the bacterium B . t . was disclosed in European Patent Application, Publication No. 0 325 400 Al, issued to Gilroy and Wilcox.
• This invention relates to a hybrid toxin gene which is toxic to lepidopteran insects.
• the invention comprises a hybrid delta endotoxin gene comprising part of the B . t . var. kurstaki HD- 73 toxin gene and part of the toxin gene from B . t . var. kurstaki strain HD-1.
• the hybrid toxin gene (DNA) encoding a protein having activity against lepidopteran insects was disclosed.
• the bacterium B. t . was also utilized for its insecticidal properties in European Patent Application, Publication No. 0 340 948, issued to Wilcox, et al.
• This invention concerns hybrid pesticidal toxins which are produced by the fusion of an insect gut epithelial cell recognition region of a B . t . gene to diphtheria toxin B chain to prepare a hybrid B . t . toxin which is active against lepidopteran insects. It was suggested that the hybrid B . t . gene may be inserted into a plant or cloned into a baculovirus to produce a toxin which can be recovered. Alternatively, the host containing the hybrid B . t .
• scorpion venom was identified as a possible source of compounds providing insecticidal properties.
• Two insect selective toxins isolated from the venom of the scorpion Leirus guinguestriatus guinguestriatus were revealed in Zlotkin, et al. , "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Biochem and Biophysics, 240:877-87 (1985) .
• Zlotkin, et al. "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Biochem and Biophysics, 240:877-87 (1985) .
• U.S. Patent No. 4,879,236 issued November 7, 1989 to Smith and Summers relates to a method for incorporating a selected gene coupled with a baculovirus promoter into a baculovirus genome to produce a recombinant baculovirus expression vector capable of expression of the selected gene in an insect cell.
• the method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter.
• a recombinant transfer vector To prepare a recombinant transfer vector, the DNA fragment is inserted into a cloning vehicle and then a selected gene is inserted into this modified cloning vehicle such that it is under the control of the polyhedrin promoter. The recombinant transfer vector is then contacted in insect cells with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome.
• the baculovirus Autographa californica (AcMNPV) and its associated polyhedrin promoter were found to be useful in producing a viral expression vector capable of extremely high levels of expression of a selected gene in an insect host cell.
• the inventors suggest that the expression vector might be used in a system for controlling insects by selecting a gene which produces a protein which is toxic to a specific insect or to a spectrum of insects and cloning that gene into the AcMNPV expression vector. They suggest that the vector could be applied to the plant or animal to be protected. The recombinant virus could invade the cells of the intestinal wall following ingestion by the insect and begin replication. A method for producing insecticidal genes and introducing them to the target to be protected was disclosed in Cutler, "Electroporation Being Developed to Transform Crops: Success with Model Crop Confirmed," AG Biotech . News vol. 7(5) :3 & 17 (1990).
• the process consists of collecting pollen, germinating it in a germinating medium for 30-60 minutes after which the pollen tube will start to come out of the pollen grain, adding the desired DNA to the liquid suspension containing the pollen, administering an electric shock to open the pores of the pollen, washing the excess DNA away, and putting the altered pollen under the stigma of a plant and waiting until seeds are formed.
• This may be an easy method to move any gene into crop plants.
• the protein-producing microbial cell itself is used as the delivery system so no purification of the produced compound is necessary.
• Any protein, polypeptide, amino acid, or compound, including insecticides, that may be produced by microbial means may be the starting material of the invention.
• U.S. Patent No. 4,925,664 issued to Jackson and Parks on May 15, 1990, discloses methods of treating heart and neurological diseases by applying toxins derived from the spiders Agelenop ⁇ is aperta and Hololena curta .
• the toxins are also effective as specific calcium channel or excitatory amino acid receptor blockers that may be used against insects and related pests.
• spider toxins were discussed in Jackson and Parks, "Spider Toxins: Recent Applications in Neurobiology," Ann Rev Neurosci 12:405-14 (1989). This article teaches that there is great heterogeneity in the toxins of different taxa. It recognizes that experiments have suggested species-specific properties of calcium channels and the spider venoms might provide calcium channel antagonists. The spider venoms discussed are found to affect vertebrates. The article also identifies spider venoms as possible sources of insect-specific toxins for agricultural applications.
• Yoshioka et al. discloses a receptor inhibitor obtained from Joro spider (Nephila clavata) venom, and its manufacturing method. Yoshioka demonstrates that their toxins show glutamate receptor inhibitory activity in an insect electrophysiological assay.
• U.S. Patent No. 4,918,107 issued April 17, 1990 to Nakajima et al. relates to a compound which has glutamate receptor inhibitor activity, a process for preparing the same, and an insecticidal composition containing the same. Accordingly, due to a combination of problems associated with conventional chemical insecticides, including pest resistance and injurious effects on non-target organisms, there exists a continuing need for the development of novel means of invertebrate pest control.
• novel insecticidally effective proteins derived from, for example, a spider of the genus Fili ⁇ tata .
• Four insecticidal proteins were isolated from the fractionation of Filistata venom.
• the proteins are designated FIL-376, FIL-377, FIL-501 and FIL-502.
• the invention further provides a family of structurally related proteins.
• novel recombinant expression vectors and genetically engineered insecticidal microbes and methods of controlling invertebrate pests comprising contacting said pests with a recombinant baculovirus capable of expressing an effective amount of an insecticidally effective peptide substantially isolatable from Filistata spider venom and agriculturally or horticulturally acceptable salts thereof.
• FIG. 1 Chromatography of 8 ⁇ l of Filistata hibernalis venom by immobilized metal ion affinity chromatography (IMAC) .
• Bioassay of the fractions in tobacco budworm (TBW) are as follows :
• NF not feeding Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG . 2 Anion exchange chromatography of 50 ⁇ l of
• Controls 0 0/5 FI feeding inhibition Controls were inj ected with 10 ⁇ l of PBS , pH 6 . 5 .
• FIG. 3 Chromatography of 400 ⁇ l of Filista ta hibernalis venom by IMAC. Bioassay of the fractions in TBW are as follows:
• Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 4 Anion exchange chromatography of Metal 3 and Metal 4 fractions (Figure 3) from IMAC of Filistata hibernalis whole venom. Bioassay in TBW of combined, like fractions from two AX chromatographies are as follows :
• N/F not feeding. Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 5 Anion exchange chromatography of FIL-376 sample (AX 3 from Figure 4) .
• FIG. 6 Hydrophobic interaction chromatography of FIL-376 fraction from anion exchange chromatography shown in Figure 5. Bioassay of the fractions in TBW are as follows:
• Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 7 Hydrophobic interaction chromatography of the FIL-377-containing fraction (AX 1) from the anion exchange chromatography shown in Figure 4.
• Bioassay results in TBW are as follows:
• Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 8 A second anion exchange chromatography of the FIL-502-containing fraction (AX 3) from the anion exchange chromatography shown in Figure 4.
• FIG. 9 Hydrophobic interaction chromatography of FIL-502 from the anion exchange chromatography shown in Figure 8. Bioassay of the sample in TBW are as follows:
• N/F not feeding. Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 10 Anion exchange chromatography of the FIL- 501-containing fraction (Metal 2) from the IMAC fractionation shown in Figure 2. Bioassay results in TBW are as follows:
• N/F not feeding. Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 11 Hydrophobic interaction chromatography of the FIL-501-containing fraction (AX 2) from the chromatography shown in Figure 10.
• Bioassay of the fractions in TBW are as follows:
• Controls were injected with 10 ⁇ l of PBS, pH 6.5.
• FIG. 12 The effect of FIL-377 on synaptic transmission (evoked population spike) at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampal slices. These data represent the time-averaged population spike recordings (a) for 5 minutes prior to FIL-377 addition and (b) during the 15-20 minute interval following FIL-377 addition at 0.25 and 1 ⁇ M. These recordings are superimposable, indicating that, at these concentrations, FIL- 377 has no activity in the rat CNS that can be detected in this assay.
• Spiders in the genus Filistata are members of the family Filistatidae.
• the genus Filistata is the most common in the family, and Filistata hibernalis is perhaps the most widely distributed species in the genus.
• Filistata hibernalis is a common house spider in the southern United States, spinning large, flat webs which are frequently seen on the outside walls of buildings. The spider generally hides in a crevice at the center of the web and waits for a disturbance of the web to indicate that prey has been captured.
• insecticidally effective proteins of this invention The mechanism of action of the insecticidally effective proteins of this invention is unknown. It has been found that these toxins produce a unique set of symptoms in various species of insects. The toxins cause a distinctive,
• Spider venom can be removed from Fili ⁇ tata by any method known such as venom gland extraction from cephalothorax.
• the spider venom preferably is obtained by electrical stimulation of the spiders to cause release of the venom and subsequent suction to collect the released venom and prevent contamination of the venom by regurgitate or hemolymph as described in U.S. 4,925,664.
• the spider venom Once the spider venom is obtained by electrical milking techniques, it can be fractionated into its protein (toxin) components by high performance liquid chromatography (HPLC) with a variety of separation modes such as hydrophobic interaction, ion exchange and immobilized metal ion affinity (IMAC) chromatography.
• HPLC high performance liquid chromatography
• IMAC immobilized metal ion affinity
• This invention in one of its aspects, provides a family of insecticidally effective proteins, and insecticidally effective fragments thereof and agriculturally or horticulturally acceptable salts thereof.
• amino acid sequence determination can be performed in any way known to those in the art such as N- ter inal amino acid sequencing and use of an automated amino acid sequencer.
• insecticidally effective proteins are expected to be within the scope of the invention. That is, it is believed other insecticidally effective proteins in the family exist and may be isolatable from Fili ⁇ tata as well as other sources in addition to the four detailed herein. The following relates to a family of insecticidally effective proteins. Members of this family of insecticidally effective proteins are believed to share the following characteristics:
• FIL-376 has a mass of about 22,850.20 ⁇ 0.76 amu as determined by mass spectroscopy.
• FIL-377 has a mass of about 27,704.05 ⁇ 0.85 amu as determined by mass spectroscopy.
• the N-terminal amino acid sequence of FIL-377 is presented in SEQ ID NO:4.
• FIL-501 has a mass of about 22,629.0 ⁇
• a substantially isolated DNA sequence encoding a protein of this invention may be determined by methods known to those in the art.
• the genes responsible for the production of proteins from a source can be isolated and identified. Numerous methods are available to obtain the gene responsible for the production of a protein. Examples include Fuqua, S. et al., "A simple PCR method for detection and cloning low abundant transcript", Biotechnique, Vol. 9, No. 2 (Aug 1990); Frohman, M.A. , "RACE: Rapid amplification of cDNA ends", PCR protocol ⁇ , ed. Innis
• a DNA molecule is synthesized which encodes the determined amino acid sequence or which represents the complementary DNA strand to such a DNA molecule which encodes the determined amino acid sequence.
• This synthetic DNA molecule may then be used to probe for DNA sequence homology in cell clones containing recombinant DNA molecules comprising, in part, DNA sequences derived from the genomic DNA of an organism such as a spider or derived from cDNA copies of mRNA molecules isolated from cells or tissues of an organism such as a spider.
• DNA molecules of fifteen (15) nucleotides or more are required for unique identification of an homologous DNA, said number requiring unique determination of at least five (5) amino acids in sequence.
• PCR Polymerase Chain Reaction
• the resultant product represents an amplified cDNA which can be ligated to any of a number of known cloning vectors. Not withstanding this, it will be appreciated that "families" of proteins or peptides may exist in spider venoms which will have similar amino acid sequences and that in such cases, the use of mixed oligonucleotide primer sequences may result in the amplification of one or more of the related cDNAs encoding these related proteins. Genes encoding related proteins are
• the produced cDNA sequence can be cloned into an appropriate vector using conventional techniques, analyzed and the nucleotide base sequence determined. A direct amino acid translation of these PCR products will reveal that they corresponded to the complete coding sequence for the mature protein. The portion of the DNA sequence which might encode amino acids corresponding to precursor and or propeptide regions may not be obtained by this approach. Such sequences may be determined by isolation of genomic or cDNA clones using the cDNA clone produced in this approach as a hybridization probe which is within the scope of the art.
• insecticidally effective proteins of this invention are believed to be useful in controlling invertebrate pests such as those in the order Lepidoptera.
• Methods for using the insecticidally effective proteins of this invention may include contacting the pests with an effective amount of a protein of this invention.
• Methods of contacting an invertebrate pest with a protein to control said pests are known. Examples include the insertion of a gene encoding a toxic peptide or protein into the genome of a baculovirus, such as the Autographa calif ornica nuclear polyhedrosis virus. Of course, methods of controlling invertebrate pests using the proteins of this invention can be used in combination with other methods of controlling pests.
• expression vector includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operably linked to other
• expression vector is given a functional definition, and any DNA sequence which is capable of supporting expression of a specified DNA code disposed therein is included in this term as it is applied to the specified sequence.
• expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA which, in their vector form are not bound to the chromosome.
• Plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
• Recombinant host cells refers to cells which have been transformed with vectors constructed using recombinant DNA techniques.
• a recombinant expression vector comprising a DNA sequence which encodes an insecticidally effective peptide substantially isolatable from Filistata spider venom.
• the vector is capable of effecting the expression of the coding sequence in transformed cells.
• recombinant host cells with a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Fili ⁇ tata spider venom in a manner allowing the host cell to express the peptide.
• a recombinant expression vector transformed, transfected or otherwise applied in said host cells has a DNA sequence encoding the peptide and recovering the insecticidally effective peptide from the recombinant host cell culture or host organism.
• the vector is capable of supporting with host cell factors the expression of the coding sequence in the transformed cells.
• control sequences such as promoters, and preferably enhancers and termination controls, are readily available and known in the art for a variety of hosts. See e . g. , Sambrook et al., Molecular Cloning a Laboratory Manual , Second Ed. Cold Spring Harbor Press (1989) .
• the desired proteins can be prepared in both procaryotic and eucaryotic systems, resulting, in the case of many proteins, in a spectrum of processed forms.
• procaryotic system The most commonly used procaryotic system remains E. coli , although other systems such as B . ⁇ ubtili ⁇ and Pseudomonas are also expected to be useful.
• Suitable control sequences for procaryotic systems include both constitutive and inducible promoters including the lac promoter, the trp promoter, hybrid promoters such as tac promoter, the lambda phage PI promoter.
• foreign proteins may be produced in these hosts either as fusion or mature proteins. When the desired sequences are produced as mature proteins,
• the sequence produced may be preceded by a methionine which is not necessarily efficiently removed. Accordingly, the peptides and proteins claimed herein may be preceded by an N- terminal Met when produced in bacteria. Moreover, constructs may be made wherein the coding sequence for the peptide is preceded by an operable signal peptide which results in the secretion of the protein. When produced in procaryotic hosts in this matter, the signal sequence is removed upon secretion.
• eucaryotic hosts are also now available for production of recombinant foreign proteins. As in bacteria, eucaryotic hosts may be transformed with expression systems which produce the desired protein directly, but more commonly signal sequences are provided to effect the secretion of the protein.
• Eucaryotic systems have the additional advantage that they are able to splice introns which may occur in the messenger RNA encoding proteins of higher organisms. Eucaryotic systems also provide a variety of post-translational mechanisms which result in, for example, glycosylation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth.
• insecticidally effective protein of this invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art.
• a “deletion” is defined as a polypeptide in which one or more internal amino acid residues are absent.
• An “addition” is defined as a polypeptide which has one or more additional internal amino acid residues as compared to the wild type.
• a “substitution” results from the replacement of one or more amino acid residues by other residues.
• a protein "fragment” is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related.
• substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
• naturally- occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar and/or aromatic. It is generally preferred that encoded peptides differing from the native form contain substituted codons for amino acids
• the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, lie, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lie and Leu are more closely related) , and the aromatic amino acids Phe, Trp, and Tyr are interchangeable.
• proteins of the invention can be made by recombinant techniques as well as by automated amino acid synthesizers. Because of the variety of post-translational characteristics conferred by various host cells, various modifications for the naturally-occurring proteins will also be obtained.
• a "modified" protein differs from the unmodified protein as a result of post-translational events which change the glycosylation, amidation or lipidation pattern, or the primary, secondary, or tertiary structure of the protein and are of course included within the scope of the invention as claimed.
• Phenylglycine for example, can be substituted for Trp, Tyr or Phe an aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic.
• the conformation conferring properties of the proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp) .
• insecticidally effective peptide alone or in combination with another insect toxin is expected to be useful in potentiating or enhancing the toxicity of microbes such as baculoviruses and hybrid bacteria.
• a recombinant expression vector expected to be particularly suitable for use in this invention is a baculovirus expression vector such as the type disclosed in U.S. Patent 4,879,236, which patent is incorporated by
• the recombinant expression vector virus could then be applied to the plant or animal upon which the insect is a pest, and when the virus is ingested by the pest insect, the recombinant virus will invade the cells of the intestinal wall and begin replication. During replication, the gene for the insecticidally effective protein will be expressed, resulting in the disablement or death of the insect in a shorter period than if the insect had ingested the wild type AcMNPV virus.
• hybrid virus also expected to be useful is taught in European Patent Application 0 340 948.
• the hybrid virus expressing the DNA of this invention is expected to yield a virus having an altered insect host range.
• fusion proteins could be expressed as a single polypeptide product of a hybrid gene consisting of DNA of this invention and a specific insect gut cell recognition protein to direct the expressed insecticidally effective peptide to the host insect target.
• prokaryotic and eukaryotic microbes can be transformed to express a hybrid toxin gene encoding an insecticidally effective protein by the method taught in European Patent Application 0 325 400.
• Hybrid bacterial cells comprising a plasmid with the gene coding for the protein of this invention are expected to be useful in the method of this invention. Insects would
• baculovirus that would be suitable for use in this invention are described in Tomalski et al., "Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene", Nature , 352: 82-85 (1991) and Stewart et al., "Construction of an improved baculovirus insecticide containing an insect-specific toxin gene", Nature , 352:85-88 (1991); McCutchen, et al., “Development of a recombinant Baculovirus expressing an insect seective Neurotoxin: Potential for Pest Control," Biotechnology, 9:848-851 (1991).
• An insecticidal composition comprising an insecticidally effective amount of a protein according to this invention and agriculturally or horticulturally acceptable salts thereof in an agriculturally or horticulturally acceptable carrier therefor is also provided.
• the spider venom is preferably obtained by electrical stimulation of the spiders to cause release of the venom and subsequent suction to collect the released venom and prevent contamination of the venom by regurgitate or hemolymph as described in U.S. Patent No. 4,925,664.
• the venom was fractionated by high performance liquid chromatography (HPLC) incorporating Beck an System Gold 126 solvent delivery and 168 photodiode array detector modules. The following columns and conditions were used in the purifications.
• Immobilized metal ion affinity chromatography (IMAC) was performed on a ProgelTMTSK Chelate 5PW column (7.5 x 75 mm, from Supelco) freshly loaded with Cu +2 ions (40 mM CuS0 4 (aq)) .
• the A buffer was 20 mM NaH 2 P0 4 , ImM imidazole, 0.5 M NaCl adjusted to pH 7.0 with 10 M NaOH.
• the B buffer was 20 mM NaH 2 P0 4 , 20 mM imidazole, 0.5 M NaCl adjusted to pH 7.0 with 10 M NaOH.
• the column was equilibrated in the A buffer and eluted at a flow rate of 1 ml/min with a 17 min linear gradient (begun 5 min after injection) from 0 to 43% B buffer, followed by a 3 min gradient from 43-100% B. After 5 min at 100% B the column was returned to 0% B over 2 min and equilibrated before the next injection was made.
• the effluent was monitored at 280 nm and fractions collected with a Gilson model 203 fraction collector.
• Anion exchange chromatography was performed on a MF- PLUSTM HEMA-IEC BIO Q column (4.6 x 150 mm, lO ⁇ particle size, from Alltech Associates) .
• the A buffer was 25 mM Tris base adjusted to pH 7.5 with 6 N HC1 and the B buffer was 25 mM Tris base, 1.0 M NaCl adjusted to pH 7.5 with 6 N HC1.
• the column was eluted at a flow rate of 1 ml/min with a 20 min linear gradient (begun 10 min after sample injection) from 0 to 12% B buffer. The column was then taken to 50% B over 2 min, held at 50% B for 4 min, returned to 0% B over 2 min and allowed to equilibrate before the next injection.
• This chromatography was monitored at 280 nm and fractions collected with a Gilson model 203 fraction collector.
• Example 1 Initial Fractionation of Filistata hibernalis Whole Venom and Identification of Insecticidal Fractions from IMAC and Anion Exchange Chromatography.
• PW column in the Cu +2 form was eluted at 1 ml/min with a 40 min linear gradient from 0% B to 100% B.
• Buffer B was 20 mM NaH 2 P0 4 , 0.5 M NaCl, 20 mM imidazole adjusted to pH 7.0 with 10 M NaOH.
• the column was monitored at 280 nm and fractions collected. The fractions were combined into 3 pools (Fig. 1) and concentrated in C-10 filters for testing in TBW as outlined in Methods. Only pool 2 showed appreciable insecticidal activity.
• Example 2 Separation of 4 Insecticidal Components from 400 ⁇ l of Filistata hibernalis Whole Venom.
• Example 3 Purification of FIL-376. Sample AX 3 from Example 2 was exchanged into 25 mM
• FIL-376 prepared by a similar route was desalted by ultrafiltration versus water in a C-10 filter. The sample was divided into two portions and sent for N- terminal sequence and mass spectral analysis. The N-terminal sequence was found to be:
• FIL-377 was purified from pool AX 1 described in Example 2 above.
• the sample 500 ⁇ l, was diluted to 2 ml with 50 mM NaH 2 P0 4 , 2 M NaCl, adjusted to pH 7.0 with 10 M NaOH. (Final NaCl concentration in the diluted sample was 1.5 M.)
• This was chromatographed in 2 x 1 ml portions on the Hl-Propyl hydrophobic interaction column as described in Methods (see Fig. 7) .
• Like fractions from both runs were combined as indicated and concentrated in C-10 filters.
• the concentrates were exchanged into PBS and reconcentrated to -400 ⁇ l for
• the N-terminal sequence was found to be: Leu-Glu-Asp-Pro-Tyr-Lys-Ser-Asp-Ser-Asn-Ser-Arg-Tyr-Ile-Glu- Val-Val-Val-Val-Asn-Asp-Asn-Ser-Met-Phe-Arg-Lys-Tyr-Asn- Arg.... (SEQ ID NO:4).
• the mass of FIL-377 was determined to be 27,704.05 ⁇ 0.85 amu.
• the Metal 2 fraction from Fig. 3 also showed insecticidal activity and was therefore subjected to anion exchange chromatography.
• One half of Metal 2 (-500 ⁇ l) was diluted to 5 ml with 25 mM Tris, pH 7.5, and chromatographed on the HEMA-BIO Q anion exchange column as described in Methods (Fig. 10) .
• the fractions were pooled as indicated, concentrated in C-10 filters, exchanged into PBS, pH 6.5, and reconcentrated to -350 ⁇ l for assay in TBW. Pool 2 was then subjected to hydrophobic interaction chromatography.
• N-terminal sequence was found to be: Gly-Gly-Ser-Asp-Pro-Glu-Tyr-Met-Glu-Leu-Val-Val-Ile-Asn-Asp- Asn-Lys-Met-Phe-Arg-Lys-Tyr-Gly-Ser-Xaa-Thr-Xaa-Xaa-Val-Ala- Glu-Xaa-Xaa-Xa-Gln-Xaa-Met-Asn-Ile-Ala....
• Example 7 Isolating the coding genes for insecticidally effective peptides isolated from Fili ⁇ tata hibernali ⁇ venom
• RNA is extracted from the cephalothoraxes using the protocol of Chomczynski and Sacchi, Analytical Biochemi ⁇ try, 162, 156 (1987) .
• Polyadenylated messenger RNA (mRNA) is purified using oligo d(T) cellulose (Pharmacia LKB, Sweden) chromatography. Step #2: cDNA Synthesis
• RNA is reverse transcribed to cDNA with murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, MD) using the manufacturer's protocol.
• the 20 ⁇ l reaction mixture contains the enzyme buffer as supplied in a cDNA synthesis kit (Boehringer Mannheim, IN) , 50 ng of mRNA, 2 units of RNase H, 30 ng of d(T) Not I primer (Promega, Madison, WI) , 1 mM each deoxynucleoside triphosphates, and 100 ⁇ g of reverse transcriptase.
• the reaction mixture is incubated for 1 h at 37° C and continued for 10 minutes at 42° C.
• the reaction mixture is ethanol precipitated and resuspended in 20 ⁇ l water.
• Step #4 Amplification Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase was initially described by Saikki et al.. Science , 239:487 (1988). For this application, 5 ⁇ l of the Filistata cephalothorax cDNA is used as template in a polymerase chain reaction containing reagents contained in the GeneAmpTM DNA amplification kit (Perkin Elmer Cetus, CA) .
• the amplification reaction contains the sense and antisense primers in a 2 ⁇ M concentration, 100 ⁇ M of each deoxynucleotide triphosphate, and 4 units of the thermostable recombinant Tag I polymerase.
• the reaction is run in a DNA Thermal Cycler manufactured by Perkin Elmer Cetus using both high and low stringency reactions. Step #5: Cloning of PCR Products
• the gel-purified PCR products are separated from unincorporated primers using a Centricon-100 (Amicon) molecular size separation unit.
• the retained products are then digested with the restriction enzyme Not I (MBR) , Milwaukee, WI) , which cleaves within the downstream (3' end) primer leaving a sticky end.
• MLR restriction enzyme
• the vector, pKS Stratagene, LaJolla, CA
• EcoR V US Biochemical
• Not I to generate sites specific for directional cloning.
• Vector and insert are ligated and transformed into compex DHS ⁇ F'. Colony lifts are screened with the 32 P labeled internal probe and candidate colonies are further characterized by sequencing (US Biochemical , s Sequenase Version 2.0) mini-prep DNA using the internal probe as primer.
• a lepidopteran signal sequence (Jones et al., Molecular Cloning Regulation and Complete Sequence of a Hemocyanin-Related Juvenile Hormone-Suppressible Protein From Insect Hemolymphs, J. Biol . Chem. 265:8596 (1990)), is constructed from two synthetic oligonucleotides using the method of Rossi, et al. (J. Biol . Chem . 257:9226 (1982)). Two 48mers are purified by ion exchange chromatography. These two oligonucleotides share eleven base pairs of complementary sequence at their 3' termini.
• DNA sequencing confirms an in-frame fusion between the two cDNA sequences.
• the entire synthetic gene construct is excised and adapted for cloning into the Nhel site of pBlueBac, a baculovirus transfer vector [Vialard, J. , et al., J. Virology 64:3-50 (1990)].
• Subclones are sequenced to confirm the correct insertion of the construct.
• the use of the pBlueBac vector expedites the screening process as insertion of the recombinant gene into the baculovirus genome is accompanied by co-expression of jS-galactodidase and detectable by a color change when grown on indicating media.
• Recombinant baculoviruses are produced by transfection of Spodoptera frugiperda strain Sf9 (ATCC# CRL1711) cells with a mixture of 1 ⁇ g AcMNPV viral DNA and 2 ⁇ g plasmid DNA using the protocol of Summers and Smith (in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures", Texas Agricultural Experiment Bulletin
• PCR amplification using viral specific primers from the region surrounding the polyhedrin gene confirms that viral isolates contain an appropriately sized insert and lacked any wild-type contamination. Tittered stocks of the recombinant viruses are then prepared for in vivo and in vitro testing.
• insects tested were last instar, laboratory reared larvae of the tobacco budwor , Heliothis vire ⁇ cen ⁇ (TBW) ; the beet armyworm, Spodoptera exigua (BAW) ; and the cabbage looper, Trichoplu ⁇ ia ni (CL) . All three species are in the family Noctuidae of the order Lepidoptera. All samples, whether whole venom or venom fractions, were prepared in filter-sterilized physiological saline (PBS), pH 6.5. Samples were administered by injection into the hemocoel at or near the lateral midline of the fourth abdominal segment; the needle was inserted at a shallow angle to avoid injury to internal organs.
• PBS filter-sterilized physiological saline
• WVE whole venom equivalents
• the LD 50 Of FIL-377 is 4.8 ⁇ g/g (0.17 nmol/g), while the LD 50 of FIL-376 is 3.6 ⁇ g/g (0.16 nmol/g).
• the LD 50 for FIL-376 is approximately 3.3 ⁇ g/g, while in CL this dose causes 75% mortality (table 2) .
• Both FIL-376 and FIL-377 cause a slowly developing flaccid paralysis and the localized, gradually spreading necrotic discoloration which is characteristic of whole Fili ⁇ tata venom.
• FIL-376 and FIL-377 are also insecticidally effective in larvae of the corn earworm (Heliothi ⁇ virescen ⁇ ) , fall armyworm (Spodoptera frugiperda) , soybean looper (Pseudoplu ⁇ ia includen ⁇ ) , European corn borer (O ⁇ trinia nubilali ⁇ ) , and diamondback moth (Plutella xylostella) .
• the estimated LD 50 of either FIL-376 or FIL-377 ranged from 1 to 10 ⁇ g/g (table 2) .
• the overall pattern of susceptibility to Filistata toxins among these species is similar to that among TBW, BAW, and CL.
• Toxin Dose ( ⁇ g/g) % paralysis % mortality (48 hr) (96 h)
• Trichoplu ⁇ ia FIL-376 10.0 48 hr 85 20 85 ni (cabbage 3.3 48 hr 75 20 75 looper) CONTROL 0 48 hr 0 20 0
• FIL-377 was also assessed for its effect on synaptic transmission (evoked population spike) at the Schaffer collateral-CAl pyramidal cell synapse in rat hippocampal slices. At 0.25 and 1 ⁇ M, it was without effect.
• the data depicted in Figure 12 represent the time-averaged population spike recordings (a) for 5 minutes prior to FIL-377 addition and (b) during the 15-20 min interval following FIL-377 addition. These recordings are superimposable, indicating that, at these concentrations, FIL-377 has no activity in the rat CNS that can be detected in this assay.
• the assay is capable of detecting a variety of effects on various mammalian ion channels and neurotransmitter receptors (T.V. Dunwiddie, "The Use of In Vitro Brain Slices in Neuropharmacology". In Electrophy ⁇ iological Techniques in Pharmacology (H.M. Geller, ed.), Alan R. Liss, Inc., New York (1986)).
• ATA AAG GAC AGA GTC GGT GCT ATA ATA AAC GGT GCA AGT GCA 126 lie Lys Asp Arg Val Gly Ala lie lie Asn Gly Ala Ser Ala 30 35 40
• TGT GGT AAT GGG AAA TTA GAA GAG GGC GAA GAA TGT GAT TGT 672 Cys Gly Asn Gly Lys Leu Glu Glu Gly Glu Glu Cys Asp Cys
• Asp Arg Val Gly Ala lie lie Asn Gly Ala Ser Ala Leu Leu Ser
• Ala lie Xaa Leu Xaa Asn

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