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

如果蛋白质过度生产，它们可能会错误折叠。这通常导致形成称为包涵体（IB）的不溶性蛋白质物质。从IBs中收获功能蛋白是困难的。避免它们的形成是重要的，可以通过修改蛋白质合成机器，核糖体来实现。计算机模拟将提出核糖体的有效变化，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和一些研究生也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。 在细菌中生产大量可溶性生物活性蛋白仍然是个问题。该研究项目的目标是通过重组细菌（E。coli）核糖体机器。具体地说，减慢翻译终止的速率，从而降低完全合成的蛋白质从核糖体出口通道排出的速率。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辅助重组工程以及基于荧光和比色的溶解度测定的组合。实现这个项目的目标将有利于科学和工程。它将为培养下一代遗传工程师和化学生物学家提供载体。他们将能够结合联合收割机的理性和随机的方法，并集成动力学模拟，计算设计和实验遗传工程。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.