Core D: Protein Production Core

点击翻译按钮获取中文摘要

The Protein Production Core's mission is to produce heterologously expressed, purified and reconstituted human neuronal receptors in the large quantities required for photolabeling and sequencing. This is a challenging resource-intensive objective best handled by a central facility. During the four years of its existence, the Core has thoroughly developed several inducible mammalian cell lines capable of expressing a number of Cys-loop ligand-gated ion channel receptors bearing accessible purification epitopes. With such cell lines the successive steps of solubilization, purification and reconstitution have been optimized. Two receptors are now in production in such cell lines, and it is proposed to developed three more. Using its Bioreactor facility, the Core is now in a position to routinely fulfill its overall specific aim of supplying Projects 1 & 2 with the milligram quantities of well characterized, purified receptors functionally reconstituted into defined lipid bilayers that are required for photolabeling studies. The specific receptors to be produced in this way are all human neuronal members of the Cys-loop superfamily. The primary focus is on the inhibitory receptors, specifically the following GABAA receptors: synaptic receptors composed of subunits aip3y2L: extrasynaptic receptors composed of subunits a4p36, and aipS receptors to act as controls. The representative excitatory receptors in the Cys-loop superfamily will be the neuronal nicotinic a4p2 receptor and the 5-HT3A receptor. A subsidiary goal is to support the needs of investigators involved in electrophysiological studies (Raines and Forman). They will be able to achieve efficiencies by using the Core's resources both for the more straightfonward establishment of stable cell lines and for the development of mutants, although the primary responsibility remains with the investigator thereafter. This latter service will not detract from the Core's main labor-intensive goal of developing stable, inducible cell lines and over- expressing human neuronal receptors for photolabeling and biochemical studies. The Core will be situated in the Mallinckrodt Pharmacology Laboratory at MGH. RELEVANCE (See instructions):