Protein expression, purification, and structural characterization

University Of Pennsylvania, Philadelphia PA

Investigators

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Abstract

This is a proposal for a core facility to support a Program Project application. The main roles of this core (Core B) are to express and purify the protein targets for which anesthetic binding will be studied, and to provide structural support to the program. Aims 1 and 2 of this core proposal focus on the production and purification of eukaryotic potassium channels; these proteins include both anesthetic-sensitive and - insensitive channels (with the latter acting as negative controls, for example for photolabeling experiments). Target proteins include the voltage-gated potassium channels Shaw2 and Kvl.2. Aim 3 focuses on the production and purification of prokaryotic homologs of eukaryotic anesthetic targets, including the sodium channel NaChBac. All of these proteins will be expressed in heterologous systems and supplied to our colleagues, either in purified, detergent-solubilized form or as crude membrane fractions. Structural support will be supplied to the program in the form of crystallographic expertise; this expertise will be devoted to exploring crystallization conditions for the membrane proteins being studied as anesthetic targets, so as to enable future high-resolution analysis of anesthetic binding. We will also crystallize and determine structures for anesthetics bound to model proteins such as apoferritin.