CAREER: Bimolecular Engineering via Directed Evolution

以人雌激素受体（ER）为模型系统，CAREER项目主要有三个具体目标：（1）开发一种新的定向进化策略--体外共进化，创造新的ER配体特异性。 (2)采用定向进化和合理设计相结合的方法，设计基于ER配体结合结构域和合成化学物质的正交配体-受体对，并随后构建遗传开关。 (3)利用DNA测序、反式激活分析、配体结合亲和力分析、分子模拟和分子对接等多种生物化学和生物物理学方法，对上述两个特异性目标所导致的所有ER突变体进行表征，以揭示ER配体特异性的分子基础和核受体的分子进化机制。 这些研究将为ER的药理学提供见解，并有助于开发用于绝经期激素替代疗法和乳腺癌治疗等应用的雌激素药物。 由此产生的发现对于其他类型的核受体的研究也将是非常宝贵的，其中大多数与各种疾病有关，并代表主要的药物靶点。拟议的教育发展计划包括两个组成部分：在生物分子工程的校园研究和教育计划，和生物技术推广计划。

Huimin ZhaoBES-0348107Using the human estrogen receptor (ER) as a model system, the proposed CAREER project focuses on three specific aims: (1) To develop a novel directed evolution strategy - in vitro co-evolution to create novel ER ligand specificity. (2) To engineer an orthogonal ligand-receptor pair based on the ER ligand binding domain and a synthetic chemical using a combined directed evolution and rational design approach and subsequently construct a genetic switch. (3) To use various biochemical and biophysical methods including DNA sequencing, transactivation assay, ligand binding affinity assay, molecular modeling and molecular docking to characterize all the evolved ER mutants resulting from the above two specific aims in an effort to uncover the molecular bases for ER ligand specificity and the molecular evolution mechanisms of nuclear receptors. Such studies will provide insights into the pharmacology of the ER and assist in the development of estrogen pharmaceuticals for applications such as menopausal hormone replacement therapy and breast cancer treatment. The resulting findings will also be invaluable for the studies of other types of nuclear receptors, most of which are linked to various diseases and represent major drug targets. The proposed educational development plan comprises two components: an on-campus research and education program in biomolecular engineering, and a biotechnology outreach program.