Protein Expression and Purification

该核心的主要目标是为各个项目和核心提供持续的支持 供应高度纯化的蛋白质，其数量是完成每个项目和核心的具体目标以及整个计划项目的长期目标所必需的。具体目标是 (1) 与项目 1 和 2 合作克隆 SCLpro 和 PLpro 转化为过表达载体，然后确定每种酶的最佳表达条件； (2) 为SCLpro和PLpro酶构建体开发快速有效的纯化程序，并确定其稳定和储存的最佳条件； (3) 生产大量高度纯化的酶，供项目 1-3 和计算核心使用。最终目标是为项目 1-3 和计算核心提供连续的蛋白质供应，用于生化、动力学和生物物理表征、X 射线结构阐明以及高通量酶促和配体结合和生物测定。

The major objective of this Core is to supply the various Projects and Cores with a continuous supply of highly purified protein in amounts necessary to complete the individual specific aims for each Project and Core, and therefore the long-term objectives of the overall Program Project. The specific aims are to (1) collaborate with Projects 1 & 2 in the cloning of SCLpro and PLpro into over-expression vectors, and to then determine their optimum expression conditions for each enzyme; (2) develop rapid and efficient purification procedures for SCLpro and PLpro enzyme constructs and determine the optimum conditions for their stabilization and storage; and (3) produce large amounts of highly purified enzyme that will be utilized by Projects 1-3, and by computational Core. The ultimate goal is to provide Projects 1-3, and the computational Core with a continuous supply of protein for the purposes of biochemical, kinetic, and biophysical characterization, x-ray structure elucidation, and high-throughput enzymatic and ligand-binding and bioassays.