A chemical genetics approach to improved IMP expression in E. coli

描述（由申请人提供）：正确折叠的完整膜蛋白（IMP）的高水平生产仍然是其生化和生物物理表征的主要障碍。原核IMP'S在大肠杆菌中的表达是一个“偶然性”的命题，有些在天然形式下表达良好，但大多数表达不良或根本不表达。不幸的是，在特定情况下成功或失败的原因尚不清楚，无论是依赖于目标IMP的序列和结构特征，还是控制生物发生过程效率的生理系统。真核IMP的情况甚至更糟，在大肠杆菌中成功表达同样神秘，但频率大大降低，领先的替代表达系统在后勤上繁重且通常非常昂贵。在这种情况下，改进的IMP在大肠杆菌中的表达方法具有很大的实用价值。我们建议使用“化学遗传学”来探索提高大肠杆菌IMP生产的药理学方法。一组有限的法医生理实验将在表达imp的细胞上进行，以提供活性化合物作用机制的初步数据。

DESCRIPTION (provided by applicant): High-level production of correctly folded integral membrane proteins (IMP's) remains a major obstacle to their biochemical and biophysical characterization. Expression of prokaryotic IMP'S in E. coli is a "hit-or-miss" proposition, with some expressing welt in native form but most expressing poorly or not at all. Unfortunately, the reasons for success or failure in specific cases are not understood, either in terms of the dependency on the sequence and structural features of the target IMP or in terms of the physiological systems controlling the efficiency of the biogenesis process. The situation for eukaryotic IMP's is even worse, with successful expression in E. coli being equally mysterious but substantially less frequent and the leading alternative expression systems being logistically onerous and generally very costly. In this context, improved methods for IMP expression in E. coli would have great practical value. We propose to use "chemical genetics" to explore a pharmacological approach to improving IMP production in E. coli. A limited set of forensic physiological experiments will be performed on IMP-expressing cells to provide preliminary data on the mechanism of action of active compounds.