Li Shuang, Cai Zhen, Chen Yong, Lin Zhanglin
Laboratory of Directed Evolution, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
Tsinghua Sci Technol. 2006 Aug;11(4):490-494. doi: 10.1016/S1007-0214(06)70222-X. Epub 2006 Jul 26.
The spike protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat (HR) region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins (subunits) or linear short peptides.
严重急性呼吸综合征冠状病毒（SARS-CoV）的刺突蛋白通过与靶细胞表面受体结合介导细胞融合。本文报道了一种简单的方法，用于剖析病毒蛋白并以随机但系统的方式寻找可折叠片段。该方法包括用DNA酶I消化以产生一组短DNA片段，随后进行这些片段的重新组装步骤，以产生具有随机末端但长度可控的DNA片段文库。为了快速筛选离散的可折叠多肽片段，将重新组装的基因片段进一步克隆到载体中，作为与折叠报告基因的N端融合，该报告基因是绿色荧光蛋白的变体。鉴定出了SARS-CoV刺突蛋白的两个可折叠片段，它们与源自刺突蛋白的肝重复（HR）区域2的各种抗SARS肽一致。该方法应适用于其他病毒蛋白，以分离抗原或疫苗候选物，从而为全长蛋白（亚基）或线性短肽提供替代方案。
