应用翻译暂停理性重设计方法优化蓝藻抗病毒蛋白CVN的异源表达

项目名称： 应用翻译暂停理性重设计方法优化蓝藻抗病毒蛋白CVN的异源表达

项目编号： No.31300649

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 张弓

作者单位： 暨南大学

项目金额： 22万元

中文摘要： 我们前期发表在Nature系列杂志的研究成果表明，翻译暂停是指导蛋白质正确折叠的重要信息，不正确的翻译暂停位点将会干扰蛋白质的正常折叠与功能。但迄今为止对此理论的验证都局限在大肠杆菌内源蛋白。在大肠杆菌中表达的外源蛋白质是否也服从这一规律，目前尚无直接实验证据。本项目以来源于蓝藻的蓝藻抗病毒蛋白N（CVN）为例，验证其在大肠杆菌内异源表达时翻译暂停与折叠之间的关系。CVN具有广谱抗病毒活性，极具药用价值，但直接在大肠杆菌中克隆表达很难得到可溶性有功能的产物，多年来一直未能实用化。本研究首次应用翻译暂停理论，用计算机辅助设计算法对CVN的编码基因进行理性重设计，人工调整其翻译暂停以增强其折叠效率，大幅度提升CVN的可溶性表达量，达到生产实用水平。本研究的成果将揭示翻译暂停对外源蛋白质可溶性表达的规律，并建立全新、通用的翻译暂停理性重设计表达优化方法。

中文关键词： 翻译暂停；蛋白质折叠；可溶性表达；蛋白质工程；翻译组

英文摘要： Our previous work, published on Nature series journals, has discovered that translational pausing serves as important information for protein folding. Incorrect translational pausing sites can perturb the folding of proteins and thus impairing their biological functions. However, the experimental validation till now was limited on intrinsic E. coli proteins. It is not validated whether heterologous proteins expressed in E. coli still follow this law. In this study, we use Cyanovirin-N (CVN) as an example to validate the connection of translational pausing and heterologous protein folding. CVN has the activity inhibiting multiple viruses and is highly potential for medical applications. However it is hardly soluble when directly cloned and expressed E. coli, and thus impractical since decades. In this study, we apply translational pausing theory for the first time to rationally redesign the coding gene sequence of CVN aided by a novel algorithm, artificially manipulating its translational pausing sites to facilitate its folding, thus enhancing largely its soluble expression in E. coli to industrial production level. This study is expected to reveal the details of translational pausing and heterologous protein expression, establishing a novel and general optimization strategy using rational redesign of translation

英文关键词： translational pausing；protein folding；soluble expression；protein engineering；translatomics