Zhao Qi, Chan Yin-Wah, Lee Susanna Sau-Tuen, Cheung Wing-Tai
School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Protein Expr Purif. 2009 Dec;68(2):190-5. doi: 10.1016/j.pep.2009.08.004. Epub 2009 Aug 13.
Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.
经过多轮噬菌体展示抗体库淘选，通常可获得数百万个候选克隆，而对这些筛选出的单链可变片段（scFv）进行表达，是进行二级功能筛选以鉴定阳性克隆所必需的。大规模功能筛选常常受到阻碍，因为将这些候选scFv克隆亚克隆到携带用于scFv纯化和检测的亲和标签的细菌表达载体中既耗时又费力。为了克服这些限制并开发一种多重方法，构建了一种改进的六聚组氨酸标签噬菌粒载体，用于一步法scFv表达和纯化。通过使用六聚组氨酸作为亲和标签，可从大肠杆菌周质提取物中快速且经济高效地捕获可溶性scFv。为了验证概念，针对两种scFv检测了改进的噬菌粒载体的可行性，这两种scFv分别是靶向细胞表面抗原的L17E4d和识别单克隆抗体（mAb）的L18Hh5。使用1 ml Ni-NTA琼脂糖，从1 L细菌培养物中获得了0.2 - 0.5 mg可溶性scFv，纯化后的scFv以高亲和力特异性结合其靶抗原。此外，使用两个随机选择的半抗原特异性scFv噬菌体克隆，证明scFv在噬菌体表面的展示不受六聚组氨酸亲和标签的影响。这些结果表明，改进的噬菌粒载体可实现噬菌体展示抗体库淘选与功能性scFv生产之间的转换。重要的是，改进的噬菌粒载体可轻松适用于多重筛选。
