Production of alternatively-spliced proteins

选择性剪接为增加真核生物的功能多样性提供了一种机制。剪接变异体(亚型)普遍存在于人类基因组中。在这项拟议的研究中，人类基因异构体的氨基酸序列被映射到共享相同折叠的同源蛋白质的三维结构上。推断出的结构变化至少分为六类。根据选择性剪接的功能效应、选择性剪接在疾病中的作用以及结构在调节选择性功能中的作用来选择结构研究的靶点。我们将在五年内确定至少50个亚型的结构。该项目蛋白质生产部分的目标是为结构研究提供足够数量的高纯度蛋白质样本。对于亚型功能尚不清楚的情况，我们还将提供克隆和纯蛋白质给合作者，他们将研究它们的生物和生化功能。由于从基因到结构的过程中的磨损，将开始对数百种蛋白质进行研究。为了实现这一目标，我们将从外部来源或通过基因合成获得cDNA。为了应对生产大量可溶性人类蛋白所面临的挑战，我们将开发和实现一个灵活的克隆系统，用于探索多种表达载体。该系统基于本计划期间开发的高通量克隆和表达平台。我们将改变目标基因的结构域边界，测试可溶性蛋白的表达，并设计替代剪接版本。我们将进一步扩展该系统以利用融合蛋白，以探索 从细菌包涵体中体外折叠，并在真核系统中表达可溶性蛋白。可溶蛋白质将通过最有效的鉴定方案进行提纯。

Alternative splicing provides a mechanism for increasing function diversity in eukaryotic organisms. Splice variants (isoforms) are prevalent in the human genome. In the proposed study, the amino acid sequences of isoforms of human genes are mapped onto the three-dimensional structures of homologous proteins that share the same fold. The inferred structural modifications fall into at least six classes. Targets are selected for structural studies on the basis of functional effect of the alternative splicing, involvement of alternative splicing in disease, and the role of structure in mediating alternative function. We will determine the structures of at least 50 isoforms in the course of five years. The goal of the protein production component of the project is to supply sufficient amounts of highly purified protein samples for structural studies. For cases where the functions of the isoforms are still unknown, we will also supply clones and pure proteins to collaborators who will investigate their biological and biochemical functions. Because of the attrition when proceeding from genes to structures, work will be initiated on several hundreds of proteins. To achieve this goal, we will obtain cDNAs either from outside sources or by gene synthesis. To address the challenges associated with producing large amounts of soluble human proteins, we will develop and implement a flexible cloning system for exploring multiple expression vectors. The system is based on the high throughput cloning and expression platform developed during the current Program Project. We will vary the domain boundaries of the targeted genes, test for soluble protein expression, and engineer alternate splice versions. We will further expand the system to utilize fusion proteins, to explore in vitro folding from bacterial inclusion bodies, and to express soluble protein in eukaryotic systems. Soluble proteins will be purified by the most efficient protocols identified.