Synthesis and toxicity of full-length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells using a baculovirus vector

Abstract

Full-length (72K) and truncated (61K) CrylVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CrylVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The crylVD genes were inserted into the Autographa californica multinucleo- Infected cells and purified inclusions from the virus (AcCrylVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCrylVD-C) expressing the truncated protein with a 9-6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CrylVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic fulllength protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

“…This region of Cry proteins contains a higher concentration of cysteine residues, which are responsible for disulfide bond formation and are believed by some authors to contribute to crystal formation (Ge et al, 1998). The expression of cry genes in insect cells, such as cry11A, cry1Ab, cry1Ac and cry1Ca, using baculovirus as vectors have been shown in previous works (Pang et al, 1992;Ribeiro & Crook, 1998;Lima et al, 2008). Remarkably, in our work, cytoplasmic Cry4Aa crystals expressed by vAcCry4Aa seem to be two fold bigger than those expressed by vSynCry4Aa.…”

“…The expression of Cry toxins using the heterologous protein expression system based on baculovirus has been reported, as part of efforts to obtain isolated Cry toxins for toxicity studies or to potentialize the pathogenicity of such insect virus to crop pests (Aguiar et al, 2006;Martins et al, 2008;Lima et al, 2008;Merryweather et al, 1990;Pang et al, 1992;Ribeiro & Crook 1993;Ribeiro & Crook 1998;Chang et al, 2003). Baculoviruses are a very diverse group of viruses with double-stranded, circular, supercoiled genomes.…”

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

“…Full-length (72 kDa) and truncated (61 kDa) Cry11Aa proteins from B. thuringiensis were also expressed in insect cells by recombinant baculoviruses. These proteins formed cuboidal crystals and only the full-length protein was shown to be toxic to insects (10). The crystal protein from B. thuringiensis subsp.…”

“…This is probably due to contamination of the OBs with crystal proteins. The expression of B. thuringiensis crystal proteins in insect cells could be useful for the study on the mode of action of such proteins since they assemble in crystals similar to those found in bacteria and have been shown to be biologically active (6,10).…”

“…Bacillus thuringiensis delta endotoxins or crystal proteins have also been introduced into the genome of baculoviruses and, although highly expressed and immunologically similar to their native counterparts, they did not improve the insecticidal activity of the recombinant baculoviruses (6)(7)(8)(9)(10). Crys-tal proteins are potent insecticides and exibit a very high degree of insect specificity.…”