Expression of acetylcholinesterase from Atta sexdens on insect cells

• Grant number: 18/26632-7
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: March 01, 2019
• End date: June 30, 2019
• Field of knowledge: Physical Sciences and Mathematics - Chemistry
• Principal Investigator: João Batista Fernandes
• Grantee: Melissa Tomaz Soares
• Supervisor: Jonathan Gershenzon
• Host Institution: Centro de Ciências Exatas e de Tecnologia (CCET). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Institution abroad: Max Planck Society, Jena, Germany
• Associated to the scholarship: 17/16005-2 - Functional study of an unsaturated glucoronyl hydrolase (UGL) of Leucoagaricus gongylophorus, a symbiotic fungus of leaf-cutting ants, BP.IC

Abstract

Acetylcholinesterase (AChE) (EC 3.1.1.7) is the enzyme responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine leading to the release of acetate and choline. In insects, acetylcholine is one of the most important neurotransmitters, and inhibition of AChE produces a generalized synaptic collapse causing the insect death. AChE is, thus, an important target for the development of new insecticides. We have expressed an AChE from the leaf-cutting ant Atta sexdens in Pichia pastoris. The enzyme was expressed in the extracellular medium in a soluble and active form. However, it was not possible to characterize the AChE due to the low yield of the expression enzyme. To perform structural studies, it is necessary to increase the expression yield and to improve enzyme purification. Alternative recombinant protein expression system (such as insect cells) may be an ally for improved enzyme yields. Prof. Jonathan Gershenzon is the Director of the Department of Biochemistry of Max Planck Institute for Chemical Ecology, Jena, Germany. He has studied the biosynthesis of putative plant defense compounds to explore their ecological roles. Dr. Gershenzon is a renowned scientist, having published 19 articles only in 2018 in important journals. One of nine project groups of Biochemistry Department, coordinated by Dr. Daniel Giddings Vassao, has used insect cells cultures as a recombinant protein expression system. We hope that this system is good enough to express the AChE enzyme for the continuity of its characterization studies. The 4-month internship of Melissa T. Soares, proposed in this project, will provide the student with fantastic experience in a leading research group, such as the Dr. Gershenzon, and will undoubtedly contribute greatly to her academic career. (AU)