Heinzelman Pete, Low Alyssa, Simeon Rudo, Wright Gus A, Chen Zhilei
1Department of Microbial Pathogenesis & Immunology, Texas A&M University, College Station, Texas 77843 USA.
2Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843 USA.
J Biol Eng. 2019 Oct 16;13:76. doi: 10.1186/s13036-019-0203-2. eCollection 2019.
The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.
Fluorescence activated cell sorting (FACS) of 1.5 × 10 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in shake flask culture, with post-purification yields exceeding 10 mg/L.
FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.
包含一个或多个结合域的生物制药前景推动了从头分离病毒体结合域及亲和力成熟的新方法的发展。鉴于获取保留病毒体表面所展示构象的高纯度病毒相关蛋白存在困难，确定克服与使用病毒体作为筛选试剂相关挑战的途径至关重要。
对从天然酵母表面展示的人纤连蛋白结构域（Fn3）中获取的1.5×10个克隆进行针对完整病毒体的荧光激活细胞分选（FACS），得到了两种与寨卡病毒体独特结合的结合蛋白。基于其中一种结合蛋白构建定点结合环突变体文库并进行FACS，得到了多个与寨卡病毒结合亲和力增强的子代克隆。这些亲和力成熟的克隆在ELISA分析中以低至个位数或双位数纳摩尔的亲和力结合寨卡病毒体，并且在摇瓶培养中作为可溶性蛋白表达良好，纯化后产量超过10mg/L。
酵母展示结合域文库的FACS是从头分离病毒体结合域的有效方法。通过对突变子代文库进行FACS筛选，可轻松提高分离的病毒体结合克隆的亲和力。鉴于大多数结合域与酵母展示兼容，本研究采用的方法可广泛用于生成针对许多不同病毒的病毒体结合域，用于被动免疫治疗和预防病毒感染。
