Engineering a new family of consensus repeat proteins based on nucleoporins

在我们所有的细胞中，DNA都被一层屏障保护着，它允许某些必要的分子通过毛孔，这些毛孔的打开和关闭调节着通道。最近，在细菌中产生了这些蛋白质孔，它们保留了调节分子通过的相同能力。为了理解毛孔是如何运作的，我们将从几种不同类型的细胞中选取这些例子进行比较和对比。一旦工作原理被确定，这些孔将被设计用于净化新的生物药物，从液体样品中去除毒素，甚至可能开发出评估药物在体内吸收速度的测试。通过PI参与ACCESS计划来促进URM研究生学习，以及CMSE社区学院研究计划来为社区学院学生提供暑期研究经验，从而实现了对多元化社区的推广。通过与巴西UNICAMP的国际推广和教学交流，研究将纳入新的教材。教材将以多种语言的数字形式编写，包括在线视频，供美国和拉丁美洲社区使用。许多来自蛋白质的最小共识重复（MCR）序列已经被确定，这导致了模块化蛋白质设计和刺激反应性药物递送材料和仿生组织工程基质等技术的进步。最近，从酵母核孔蛋白（一种调节进出细胞核运输的蛋白质）中获得了MCR。当聚合成凝胶时，该MCR表现出天然核孔蛋白的高速率和高选择性过滤。理性设计将用于探索突变，以了解功能和修改蛋白质的选择性。将对突变体的物理特性（扩散、溶解度和蛋白质结合能）进行表征，并使用定量转运模型对完全重组蛋白的转运和屏障特性进行基准测试。MCR序列也将从人类核孔蛋白中提取，以探索序列空间的宽度，这可以导致选择性转运特性，并为模拟核转运提供类似人类的体外工具。该项目由化学、生物工程和运输部门的细胞和生物化学工程项目以及分子和细胞生物科学部的分子生物物理学项目共同资助。

Inside all of our cells, DNA is protected by a barrier that allows certain necessary molecules to pass through pores that open and close to regulate passage. Recently, these protein pores have been produced in bacteria, where they retain the same ability to regulate the passage of molecules. To understand how the pores operate, examples of these will be taken from several different types of cells and compared and contrasted. Once the operating principles are identified, these pores will be engineered help purify new biological drugs, remove toxins from liquid samples, and possibly even develop tests to estimate how quickly drugs are taken up in the body. Outreach to a diverse community is accomplished through participation of the PI in the ACCESS Program to promote URM graduate study, and the CMSE community college research program to provide summer research experiences to community college students. Research will be integrated into new teaching materials through international outreach and teaching exchanges with UNICAMP in Brazil. Teaching materials will be prepared in digital form, including online videos, in multiple languages for U.S. and Latin American communities.Numerous minimal consensus repeat (MCR) sequences from proteins have been identified, leading to modular protein design and advances in technologies such as stimuli-responsive drug delivery materials and biomimetic tissue engineering matrices. Recently, a MCR has been derived from yeast nucleoporin, a protein that regulates transport into and out of the nucleus. When polymerized into a gel, this MCR exhibits the high rate and high selectivity filtration of the natural nucleoporin. Rational design will be used to explore mutations to understand function and modify the selectivity of the protein. The physical properties (diffusion, solubility, and protein binding energies) of mutants will be characterized, and transport and barrier properties will be benchmarked against full recombinant proteins using quantitative transport models. MCR sequences will also be extracted from human nucleoporins to explore the breadth of sequence space that can lead to selective transport properties and provide human-similar in vitro tools for modeling nuclear transport.This project is supported by the Cellular and Biochemical Engineering Program in the Chemical, Bioengineering and Transport Division with cofounding from the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences.