Paragraph: Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH) dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2) oxygenase superfamily protein) were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS) analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests. obvious swelling of the abdomen, and, thus far, no other modes of insecticidal action have been recorded [8] . Fluorescence localization analysis indicated that periplocoside NW (PSNW) can bind to the midgut cells of Mythimna separata larvae [9] . Ultrastructural observations showed that microvilli, organelle, and cytomembrane are destructed in the midgut cells of M. separata larvae [10] . Research on histopathological effects indicated that brush-border membrane vesicles (BBMVs) from the midgut epithelium cells of M. separata larvae are the initial action site; one or more binding sites may also exist in the larvae [11] . However, the specific binding sites and insecticidal mechanism of periplocosides in M. separata larval midgut cells remain unclear. The cellular targets of bioactive compounds can be identified using several approaches. Affinity purification is a classic and commonly used technique to identify receptors of insecticidal active substances [12] . In contrast to other approaches, affinity-based approaches are employed to identify directly small-molecule binding proteins. In this research, although periplocoside E has relatively low insecticidal activity or is inactive compared with periplocoside P and T [13], periplocoside E was selected based on the structure and high consumption of experimental materials to determine the binding protein of periplocosides compounds. The chemical structure of periplocoside E is similar to those of periplocoside P and T in terms of the common features except for one or two substituents ( Figure 1 ). Thus, we separated the binding protein of periplocoside E from the BBMVs from the midgut epithelium of M. separata larvae through affinity chromatography to elucidate the mode of action of the compound. 
 Title: Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae) through Affinity Chromatography
Output: True