Shuang Wu, Joni Tsukuda, Nancy Chiang, To Hao, Yongmei Chen, Isidro Hötzel, Sowmya Balasubramanian, Gerald Nakamura, Ryan L Kelly
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引用次数: 0
摘要
抗体发现过程在不断进步,越来越多的潜在结合序列被从体内、体外和硅学来源中识别出来。在这项工作中,我们描述了一种利用吉布森组装线性 DNA 片段(GLF)进行高产重组抗体(IgG 和 Fab)表达的快速系统。与以前的方法相比,该方法从 1 ml 表达中纯化的重组抗体产量大约高出五到十倍,这主要是由于在 GLF 的 5' 和 3' 端使用了新的侧翼保护序列。这种方法可用于小规模(1 mL)表达和纯化,以快速评估结合力和活性;也可用于大规模(30 mL)表达和纯化,以进行灵敏度更高的检测,这种检测需要通过两层色谱柱(蛋白质 A 和尺寸排阻色谱)纯化毫克量的抗体。与基于质粒的表达相比,这些方法可在多种应用中提供几乎等量的高质量材料,从而降低成本,缩短周转时间,提高抗体发现过程的效率。
High titer expression of antibodies using linear expression cassettes for early-stage functional screening.
Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.
期刊介绍:
Protein Engineering, Design and Selection (PEDS) publishes high-quality research papers and review articles relevant to the engineering, design and selection of proteins for use in biotechnology and therapy, and for understanding the fundamental link between protein sequence, structure, dynamics, function, and evolution.