USOO6114138A United States Patent (19) 11 Patent Number: 6,114,138 Adang (45) Date of Patent: Sep. 5, 2000 54 INSECTICIDAL PROTEIN FRAGMENTS Wong et al 1983 J Biol Chem v 258 1960-67. Yamamoto et al 1981 Bioch Biophy's Res Comm v. 103 75 Inventor: Michael J. Adang, Madison, Wis. 414-21. Schnepf et al 1985 “Delineation of a Toxin-Encoding Seg 73 Assignee: Mycogen Plant Science, Inc., San ment of a Bacillus thuringiensis Crystal Protein Gene' v 260 Diego, Calif. 6273-80. Chestukhina et al 1982 “The Main Features of Bacillus 2 1. Appl. No.: 06/617,321 thuringiensis of O-Endotoxin Molecular Strucutre' Arch 22 Filed: Jun. 4, 1984 Micro V 132 159-162. 5 1. Int. Cl." ........................... C12P 21/06; CO7H 21/04; Primary Examiner Karen Cochrane Carlson C12N 1/20; C12N 15/00 ASSistant Examiner-Devesh Srivastava 52 U.S. Cl. .................. 435/69.1; 536/23.71; 435/252.3; Attorney, Agent, or Firm-Saliwanchik, Lloyd & 435/252.33; 435/252.5; 435/419; 435/320.1 Saliwanchik 58 Field of Search .................................... 435/172.3, 68, 57 ABSTRACT 435/317, 252.31, 252.53, 69.1, 320.1; 935/9, 10, 29, 56, 74, 73; 536/23.71 The Bacillus thuringiensis var. kurStaki HD-73 crystal pro tein gene was cloned into pBR322. E. coli cells harboring 56) References Cited this recombinant plasmid produced a 130 kD protoxin that was toxic to Manduca Sexta (tobacco hornworm) larvae. U.S. PATENT DOCUMENTS Plasmids having the 3'-end of the protoxin gene deleted 4,448.885 5/1984 Schnepf et al. ......................... 435/253 where also constructed. E. coli cells harboring these deleted plasmids produced an active, Soluble 68 kD toxin, provided OTHER PUBLICATIONS that the 3'-deletion had not removed Sequences encoding the U.S. Ser. No. 535,354, Adang (application). 68 kD toxin. The invention provides methods to produce 68 Chang 1983 Trends Biotech. v1 100–1. kD toxin protein by constructing partial protoxin genes Huber et al 1981 Pathogenesis of Invertebrate Microbiol encoding the toxin followed by expression of the genes in DiS. 209-34. living cells. Useful plasmids and cells are also provided. Whitely et al 1982 Mol. Cloning & Gene Regulation in bacili pp. 131-144. 20 Claims, 9 Drawing Sheets U.S. Patent Sep. 5, 2000 Sheet 1 of 9 6,114,138 - HindIII - BamHI Acci Acc O O. HDa -- inc HincI 1OO t E. Hinc HincII 200 Xmn - 400 k Xmni - Clal - Cla 500 EcoRI - EcoRI 600 Xba 700 800 900 g Xba EcoRV ECORV 9. Xmn EcoRV EcoRV re- - 1200 CD5 - - - 1300 ECOR EcoRI O - 1400 - a U 1500 go AccI Acc 1600 g Bg I Cla Cla d 1700 Pst Pst 1800 o - 1900 ECORV EcoRV is 2000 PSI 2100 £ E-T EXA XhoI - 2200 Acc Acci if 2300 d 2400 t Kpni Kpn Kipni g 2500 Cla - Cla Clal 2600 Ca Clal Cia o 2700 2800 w w 2900 31 OO HindIII HindIII HindIII 3200 3300 3400 - 3500 - Pvull PWII 5. Acc gi Acc 3800 9. &- 3900 Nde I la Nde 4000 Clae V o ClaYCC -- 4100 O 4200 Risa O RsaI 4300 (up S. PSI in U.S. Patent Sep. 5, 2000 Sheet 2 of 9 6,114,138 FIG. 2A pBT73-10 5 (Bam) pBT73-161 FIG. 2B Hybridization PSt library with 6.7 kbp HindIII fragment 1 Kbp -->Aval -->ligate U.S. Patent Sep. 5, 2000 Sheet 3 of 9 6,114,138 OT OZ 0€ OQ! 09 O9 0£, O8 06 V£ºOIH U.S. Patent Sep. 5, 2000 Sheet 5 of 9 6,114,138 O8T 06T 02 OIZ OZ 0£Z OÏZ 0GZ +–"~º +–º–+ +-–+– +––+ +––+ +–º–+ –+“-– U.S. Patent Sep. 5, 2000 Sheet 6 of 9 6,114,138 O9Z OÁLZ O8Z 062 O£ OT£ OZ£ 6,114,138 1 2 INSECTICIDAL PROTEIN FRAGMENTS In vitro the protoxin may be solubilized by extremely high pH (e.g. pH 12), by reducing agents under moderately basic FIELD conditions (e.g. pH 10), or by Strong denaturants (guanidium, urea) under neutral conditions (pH 7). Once The present invention is in the fields of genetic engineer solubilized, the crystal protein may be activated in vitro by ing and bacterial bio-affecting compositions, especially the action of the protease Such as trypsin (R. M. Faust et al. those derived from the genus Bacillus. (1974) J. Invertebr. Pathol. 24:365–373). Activation of the BACKGROUND protoxin has been reviewed by H. E. Huber and P. Luthy (1981) in Pathogenesis of Invertebrate Microbiol. Diseases, The following are publications disclosing background 1O ed.: E. W. Davidson, pp. 209-234. The crystal protein is information related to the present invention: G. A. Held et al. reported to be antigenically related to proteins within both (1982) Proc. Natl. Acad. Sci. USA 77:6065-6069; A. Klier the Spore coat and the vegetative cell wall. Carbohydrate is et al. (1982) EMBO J. 1:791-799; A. Klier et al. (1983) not involved in the toxic properties of the protein. Nucl. Acids Res. 11:3973–3987; H. E. Schnepf and H. R. Whitely (1981) Proc. Natl. Acad. Sci. USA 78:2893–2897; 15 Toxicology H. E. Schnepf and H. R. Whitely, European Pat. application 63,949; H. R. Whitely et al. (1982) in Molecular Cloning B. thuringiensis and its crystalline endotoxin are useful and Gene Regulation in Bacilli, eds: A. T. Ganesan et al., pp. because the crystal protein is an insecticidal protein known 131-144; H. C. Wong et al. (1983) J. Biol. Chem. to be poisonous to the larvae of over a hundred of Species of 258:1960–1967. R. M. Faust et al. (1974) J. Invertebr. insects, most commonly those from the orders Lepidoptera Pathol. 24:365-373, T. Yamamoto and R. E. McLaughlin and Diptera. Insects Susceptible to the action of the B. (1981) Biochem. Biophys.Res. Commun. 103:414–421, and thuringiensis crystal protein include, but need not be limited to, those listed in Table 1. Many of these insect Species are H. E. Huber and P. Luthy (1981) in Pathogenesis of Inver economically important pests. Plants which can be protected tebrate Microbiol. Diseases, ed.: E. W. Davidson, pp. by application of the crystal protein include, but need not be 209-234, report production of activated toxin from crystal 25 protein protoxin. None of the above publications report that limited to, those listed in Table 2. Different varieties of B. partial protoxin genes when transcribed and translated pro thuringiensis, which include, but need not be limited to, duced insecticidal proteins as disclosed herein. These pub those listed in Table 3, have different host ranges (R. M. lications are discussed in the Background Section on Faust et al. (1982) in Genetic Engineering in the Plant Molecular Biology. S. Chang (1983) Trends Biotechnol. Sciences, ed. N. J. Panapolous, pp. 225-254); this probably 1:100-101, reported that the DNA sequence of the HD-1 reflects the toxicity of a given crystal protein in a particular gene had been publicly presented, (ref. 5 therein), and that host. The crystal protein is highly Specific to insects, in over the HD-1 toxin moiety resides in the amino-terminal 68kD two decades of commercial application of Sporulated B. peptide. M. J. Adang and J. D. Kemp, U.S. patent application thuringiensis cells to crops and ornamentals there has been Ser. No. 535,354, which is hereby incorporated by reference, 35 no known case of effects to plants or noninsect animals. The described a plasmid, p123/58-10 therein, pBt73-10 herein, efficacy and safety of the endotoxin have been reviewed by containing a partial protoxin gene that, when transformed R. M. Faust et al., Supra. Other useful reviews include those into E. coli, directed Synthesis of an insecticidal protein. M. by P. G. Fast (1981) in Microbial Control of Pests and Plant J. Adang and J. D. Kemp, Supra, and R. F. Barker and J. D. Diseases, 1970-1980, ed.: H. D. Burges, pp. 223-248, and Kemp, U.S. patent application Ser. No. 553,786, which is 40 H. E. Huber and P. Luthy (1981) in Pathogenesis of Inver hereby incorporated by reference, both teach expression of tebrate Microbial Diseases, ed.: E. W. Davidson, pp. the same p3t73-10 partial protoxin Structural gene in plants 209-234 cells. Detailed comparisons of results disclosed as part of the present application with published reports are also detailed Molecular Biology herein in the Examples, especially Example 5. 45 The crystal protein gene usually can be found on one of Several large plasmids that have been found in Bacillus Chemistry thuringiensis, though in Some Strains it may be located on Bacillus thuringiensis, a species of bacteria closely the chromosome (J. W. Kronstad et al. (1983) J. Bacteriol. related to B. cereus, forms a proteinacious crystalline inclu 154:419–428; J. M. Gonzalez Jr. et al. (1981) Plasmid Sion during Sporulation. This crystal is parasporal, forming 50 5:351-365). Crystal protein genes have been cloned into within the cell at the end opposite from the developing plasmids that can grow in E. coli by Several laboratories. Spore. The crystal protein, often referred to as the Whiteley’s group (H.R. Whiteley et al. (1982) in Molecu Ö-endotoxin, has two forms: a nontoxic protoxin of approxi lar Cloning and Gene Regulation in Bacilli, eds. A. T. mate molecular weight (MW) of 130 kilodaltons (kD), and Ganesan et al., pp. 131-144, H. E. Schnepf and H. R. a toxin having an approx. MW of 68 kD. The crystal 55 Whiteley (1981) Proc. Natl. Acad. Sci.