Design and Engineering of Enhanced Ribosomes with Universal Protein-Folding Capabilities

如果蛋白质产生过多，它们可能折叠不正确。这通常导致形成一种称为包涵体（IB）的不溶性蛋白质团块。从肠易激综合症中获取功能蛋白是困难的。避免它们的形成是很重要的，可以通过修改蛋白质合成机制——核糖体来实现。计算机模拟将提示核糖体的有效变化，这些变化将被制造和评估。研究生和本科生将进行模拟和实验。PI和一些研究生还将积极参与一个促进墨西哥裔和拉丁裔美国人参与科学的会议。细菌中大量可溶性生物活性蛋白的产生仍然存在问题。本研究项目的目标是通过重组细菌（大肠杆菌）核糖体机制来克服蛋白质折叠问题。具体来说，减缓翻译终止的速度，从而降低完全合成的蛋白质从核糖体出口通道射出的速度。细菌核糖体的内在伴侣样特性将得到增强。将采用计算机模拟、crispr - cas9辅助重组、荧光和比色溶解度测定相结合的方法。完成这个项目的目标对科学和工程都有好处。它将为培训下一代基因工程师和化学生物学家提供载体。他们将能够结合理性和随机方法，并整合动力学模拟，计算设计和实验基因工程。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

If proteins are overproduced, they may fold incorrectly. This often results in the formation of an insoluble protein mass called an inclusion body (IB). Harvesting functioning protein from IBs is difficult. Avoiding their formation is important and might be achieved by modifying the protein synthesis machinery, the ribosome. Computer simulations will suggest effective changes in the ribosome, and those changes will be made and evaluated. Graduate and undergraduate students will perform simulations and experiments. The PI and some graduate students will also be heavily involved in a conference which fosters participation of Chicanos and Latino Americans in science. The production of large quantities of soluble bioactive proteins in bacteria is still problematic. The goal of this research project is to overcome the protein folding problem by reengineering the bacterial (E. coli) ribosomal machinery. Specifically, slowing the rate of translation termination, thereby reducing the rate of fully-synthesized protein ejected from the ribosomal exit tunnel. Intrinsic chaperone-like properties of the bacterial ribosome will be enhanced. A combination of computer simulations, CRISPR-Cas9-assisted recombineering, and fluorescence-based and colorimetric solubility assays will be employed. Accomplishing the goals of this project will benefit science and engineering alike. It will provide a vector for training the next generation of genetic engineers and chemical biologists. They will be able to combine rational and stochastic approaches, and integrate kinetic simulations, computational design and experimental genetic engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.