Herrero Salvador, Combes Eliette, Van Oers Monique M, Vlak Just M, de Maagd Ruud A, Beekwilder Jules
Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Centre, Wageningen, The Netherlands.
Insect Biochem Mol Biol. 2005 Oct;35(10):1073-82. doi: 10.1016/j.ibmb.2005.05.006.
A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.
描述了一种在鳞翅目昆虫甜菜夜蛾中肠表达的新型胰凝乳蛋白酶。这种酶被称为SeCT34，代表了一类新型的胰凝乳蛋白酶。其氨基酸序列具有肠道胰凝乳蛋白酶的共同特征，但可与昆虫肠道中表达的其他丝氨酸蛋白酶明显区分开来。最值得注意的是，SeCT34含有一个胰凝乳蛋白酶激活位点，并且催化结构域中围绕活性位点丝氨酸的高度保守基序DSGGP变为DSGSA。使用一种经过工程改造以优化蛋白质生产的特殊杆状病毒载体，在Sf21昆虫细胞中实现了SeCT34的重组表达。这是在鳞翅目消化道中起作用的活性丝氨酸蛋白酶重组表达的首个实例。纯化的重组SeCT34酶通过其水解各种合成底物的能力及其对蛋白酶抑制剂的敏感性来表征。它似乎对在切割位点带有苯丙氨酸残基的底物具有高度选择性。SeCT34表现出pH依赖性和对抑制剂的敏感性，这是半纯化的鳞翅目肠道蛋白酶的特征。表达分析表明，SeCT34仅在幼虫最后一龄末期、化蛹开始前的中肠中表达。这表明这种蛋白质在幼虫到蛹蜕皮期间肠道中发生的蛋白水解重塑中可能发挥作用。
