Shea Colleen, Bloedorn Laura, Sullivan Mark A
Department of Pediatrics and Center for Human Genetics and Molecular Pediatric Disease, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, NY 14642, USA.
J Struct Funct Genomics. 2005;6(2-3):171-5. doi: 10.1007/s10969-005-5246-6.
High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His(6) epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.
结构基因组学的高通量方法需要表达、纯化并结晶从克隆到宿主生物体（通常为大肠杆菌）中的预测开放阅读框衍生而来的蛋白质。这种方法的早期结果表明，从真核蛋白质中获得衍射良好的晶体的成功率低得令人失望。一种提高结晶几率的成熟方法是与已知结构且易于结晶的构象稳定伴侣形成复合物。这种复合物自身也能够形成与原始蛋白质不同的晶体接触。源自单克隆抗体的Fab片段已成功用于多种蛋白质的这一目的，然而从杂交瘤中分离单克隆抗体的传统方法既耗时又昂贵。我们正在探索利用噬菌体展示来产生针对靶蛋白的重组抗体，这些抗体可用于获得共复合物以促进结晶和结构测定。我们正在使用一个大型的人源单链Fv（scFv）文库来筛选与一组硕大利什曼原虫靶蛋白结合的抗体。在三轮富集后，16种靶蛋白中有13种产生了良好的结合物。总共鉴定出55种不同的scFv，其中5种靶蛋白各自至少产生了5种不同的scFv。对单个克隆进行结合特异性分析，并且可通过附加的His(6)表位标签轻松生产和纯化可溶性scFv。使用免疫亲和色谱法，鉴定出8对scFv-靶蛋白，它们表现出稳定的复合物形成，适用于共结晶试验。
