Control of mosquito larvae with TMOF and 60kDa Cry4Aa expressed in Pichia pastoris
Borovsky, Dov × Nauwelaers, Sabine Van Mileghem, Anje Meyvis, Yves Laeremans, Annelies Theunis, Clara Bertier, Lien Boons, Eline #
Instytut Przemyslu Organicznego
Pestycydy vol:1 issue:4 pages:5-15
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  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.