Sellami Sameh, Cherif Marwa, Jamoussi Kaïs
Biopesticides Team LPAP, Centre of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia.
J Basic Microbiol. 2016 Jun;56(6):654-61. doi: 10.1002/jobm.201500712. Epub 2016 Feb 15.
To study the importance of N- and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 kDa), a number of mutants were generated. The addition of two (2R: RS) or eleven (11R: RSRPGHHHHHH) amino acid residues at the Vip3Aa16 (L121I) C-terminus allowed to an unappropriated folding illustrated by the abundant presence of the 62 kDa proteolytic form. The produced Vip3Aa16 (L121I) full length form was less detected when increasing the number of amino acids residues in the C-terminus. Bioassays demonstrated that the growth of the lepidopteran Ephestia kuehniella was slightly affected by Vip3Aa16 (L121I)-2R and not affected by Vip3Aa16 (L121I)-11R. Additionally, the fusion at the Vip3Aa16 (L121I) N-terminus of 39 amino acids harboring the E. coli OmpA leader peptide and the His-tag sequence allowed to the increase of protease sensitivity of Vip3Aa16 (L121I) full length form, as only the 62 kDa proteolysis form was detected. Remarkably, this fused protein produced in Escherichia coli (E. coli) was biologically inactive toward Ephestia kuehniella larvae. Thus, the N-terminus of the protein is required to the accomplishment of the insecticidal activity of Vip3 proteins. This report serves as guideline for the study of Vip3Aa16 (L121I) protein stability and activity.
为研究苏云金芽孢杆菌Vip3Aa16（L121I）毒素（88 kDa）的N端和C端的重要性，构建了多个突变体。在Vip3Aa16（L121I）的C端添加两个（2R：RS）或十一个（11R：RSRPGHHHHHH）氨基酸残基会导致不适当的折叠，表现为大量存在62 kDa的蛋白水解形式。当增加C端氨基酸残基数量时，所产生的Vip3Aa16（L121I）全长形式的检测量减少。生物测定表明，鳞翅目昆虫地中海粉螟的生长受到Vip3Aa16（L121I）-2R的轻微影响，而不受Vip3Aa16（L121I）-11R的影响。此外，在Vip3Aa16（L121I）的N端融合包含大肠杆菌OmpA前导肽和His标签序列的39个氨基酸，会增加Vip3Aa16（L121I）全长形式的蛋白酶敏感性，因为仅检测到62 kDa的蛋白水解形式。值得注意的是，在大肠杆菌中产生的这种融合蛋白对地中海粉螟幼虫没有生物活性。因此，该蛋白的N端是Vip3蛋白杀虫活性实现所必需的。本报告为研究Vip3Aa16（L121I）蛋白的稳定性和活性提供了指导。
