Control of mosquito larvae with TMOF and 60 kDa Cry4Aa expressed in Pichia pastoris

• Autorzy: Borovsky D. , Nauwelaers S. , Van Mileghem A. , Meyvis Y. , Laeremans A. , Theunis C. , Bertier L. , Boons E.
• Języki publikacji: EN

Abstrakty

EN

Cry4Aa 678 amino acids fragment (60 kDa) was cloned into Escherichia coli. After induction with IPTG the 60 kDa Cry4Aa fragment was purified by Ni chromatography, separated by SDS PAGE and identified by mass spectrometry (MS/MS). The 60 kDa Cry4Aa fragment exhibited high toxicity towards Ae. aegypti larvae. The earlier results [1] show that Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with E. coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) are synergistic. Therefore, P. pastoris, which synthesizes high amounts of heterologous proteins was genetically engineered to produce TMOF and Cry4Aa. Codon-optimized synthetic genes, cry4Aa-tmfA, gst-cry4Aa-tmfA, tmfA and gfptmfA that were expressed by P. pastoris and fed to Ae. aegypti larvae caused 90% mortality. GST (glutathione-S-transferase) enhanced the activity of Cry4Aa-TMOF and protected it from heat denaturation and GFP (Green Fluorescent Protein)- TMOF allowed us to follow yeast cells consumption by individual larva using fluorescent microscopy. This report shows for the first time that 60 kDa Cry4Aa and TMOF expressed together in P. pastoris are highly toxic to Ae. aegypti larvae.

Słowa kluczowe

EN

mosquito; mosquito control; larva; Pichia pastoris; genetic engineering; biological control; Cry4Aa 678 fragment; human disease; malaria; insecticide; trypsin modulating oostatic factor; Bacillus thuringiensis ssp.israelensis; Cry toxin

• Wydawca: -
• Czasopismo: Pestycydy
• Rocznik: 2011
• Numer: 1-4
• Opis fizyczny: p.5-15,fig.,ref.

Twórcy

autor

Borovsky D.

• University of Florida-IFAS, Florida Medical Entomology Laboratory, 200 9th St SE, Vero Beach Florida, 32962 USA

autor

Nauwelaers S.

autor

Van Mileghem A.

autor

Meyvis Y.

autor

Laeremans A.

autor

Theunis C.

autor

Bertier L.

autor

Boons E.

Bibliografia

• [1] Borovsky D., Khasdan V., Nauwelaers S., Theunis C., Bertier L., Ben Dov E. Zaritsky A., Open Toxinol. J., 2010, 3, 116-125.
• [2] Borovsky D., Carlson D.A., Griffin P.R., Shabanowitz R., Hunt D.F., FASEB J., 1990, 4, 3015-3020.
• [3] Borovsky D., Janssen I., Vanden Broeck J., Huybrechts R., Verhaert P., DeBondt H.L., Bylemans D., DeLoof A., Eur. J. Biochem., 1996, 237, 279-287.
• [4] Borovsky D., Rabindran S., Dawson W.O., Powell C.R., Iannotti D., Morris T., Shabanowitz J., Hunt D.F., De Bondt H.L., De Loof A., Proc. Nat. Acad Sci. USA., 2006,103, 18963-18968.
• [5] Borovsky D., J. Exptl. Biol., 2003, 206, 3869-3875.
• [6] Borovsky D., Mahmood F., Regul. Peptides, 1995, 57, 273-281.
• [7] Borovsky D., Powell C. A., Nayar J.K., Blalock J.E., Hayes T.K., FASEB J., 1994, 8, 350-355.
• [8] Nauwelaers S., Borovsky D., Cloning and expression of trypsin modulating oostaticfactor (Aea-TMOF) and green fluorescent protein (GFP) in Saccharomyces cerevisiaein Arthropods 2001, (Konopinska D., Coast G., Goldsworthy G., Nachman R.J., Rosinski G., Eds.), Wydawnictwa Uniwersytetu Wrocławskiego, 2002, pp. 138-140.
• [9] Borovsky D., Meola S.M., Arch. Insect Biochem. Physiol., 2004, 55, 124-39.
• [10] Clements A.N., The biology of mosquitoes: development, nutrition and reproduction, Chapman and Hall, New York, 1992, vol. 1.
• [11] Goldberg L.J., Margalit J., Mosq. News, 1977, 37, 355-358.
• [12] Margalith Y., Ben-Dov E., Biological control by Bacillus thuringiensis subsp. Israelensis I, in Insect pest management: techniques for environmental protection, (Rechcigl J.E., Rechcigl N.A., Eds.), Lewis Publishers and CRC Press LLC. Boca Raton, FL, 2000, pp. 243-301.
• [13] Becker N., Ludwig M., J. Am. Mosq. Control Assoc., 1993, 9, 221-224.
• [14] Charles J.F., Nielsen-LeRoux C., Memorias Do Instituto Oswaldo Cruz, 2000,95, 201-206.
• [15] Manasherob R., Ben-Dov E., Zaritsky A., Barak Z., Appl. Environ. Microbiol., 1998, 64, 1750-1758.
• [16] Manasherob R., Ben-Dov E., Wu X., Boussiba S., Zaritsky A., Curr. Microbiol., 2002, 45, 217-220.
• [17] Boussiba S., WuX-Q., Ben-Dov E., ZarkaA., Zaritsky A., J. Appl. Phycol., 2000, 12, 461-467.
• [18] Wu X., Vennison S.J., Liu H., Ben-Dov E., Zaritsky A., Boussiba S .,Appl. Environ. Microbiol., 1997, 63, 1533-1537.
• [19] Lluisma A.O., Karmacharya N., Zarka A., Ben-Dov E., Zaritsky A., Boussiba S., Appl. Microbiol. Biotechnol., 2001, 57, 161-166.
• [20] Khasdan V., Ben-Dov E., Manasherob R., Boussiba S., Zaritsky A., FMES Microbiol. Lett., 2003,227, 189-195.
• [21] Sneh B., Schuster S., Gross S., Z. Angew. Entomol.,1983, 96, 7-83.
• [22] Regev A., Keller M., Striznov N., Sneh B., Prudovsky E., Chet I., Ginzberg I., Koncz-Kalman Z., Koncz C., Schell J., Zilberstein A., Appl. Environ. Microbiol., 1996,62, 3581-3586.
• [23]Sirichotpakorn N., Rongnoparut P., Choosang K., Panbangred W., J. Invertebr. Pathol., 2001, 78, 160-169.
• [24]Yamagiwa M., Esaki M., Otake K., Imagaki M., Komano T., Amachi T., Sakai H., Appl. Environm. Microbiol., 1999, 65, 3464-3469.
• [25] Laemmli U.K., Nature (London), 1970, 227, 680-685.
• [26] Nauwelaers S., Bioengineering of Chlorella species, Saccharomyces cerevisiae and Pichia pastoris with Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF), Ph. D. Thesis, Belgium, Catholic University of Leuven, 2002.
• [27] Porter A.G., Davidson E.W., Liu J-W., Microbiol. Rev., 1993, 57, 838-861.
• [28] Malakhov M.P., Mattern M.R., Malakhova O.A., Drinker M., Weeks S.D., Butt T.R., J. Structural Functional Genomics, 2004, 5, 75-86.
• [29] Paul A., Harrington L.C., Zhang L., Scott J.G., J.Am. Mosq. Control Assoc., 2005, 213, 05-309.
• [30] Wirth M.C., Georghiou G.P., Federici B.A., Proc. Natl. Acad. Sci. USA, 1997, 94, 10536-10540.