Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.
Methods Mol Biol. 2022;2446:181-203. doi: 10.1007/978-1-0716-2075-5_9.
Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.
单域抗体（sdAb）是由单个免疫球蛋白结构域组成的结合物。sdAb 作为治疗药物、诊断试剂和研究工具的重要性日益增加。功能性 sdAb 通常在大肠杆菌中产生，大肠杆菌是生产重组蛋白的简单且广泛使用的宿主。然而，大肠杆菌表达系统存在一些缺点，包括重组蛋白可能错误折叠和内毒素污染有毒脂多糖。毕赤酵母是生产异源蛋白的替代宿主，因为它可以产生高重组蛋白产量，并且能够产生无内毒素污染的可溶性、正确折叠的蛋白质。在这里，我们描述了在毕赤酵母中生产 sdAb 的方法。我们介绍了将 sdAb 编码基因克隆到毕赤酵母表达载体、生产和纯化 sdAb 以及测量 sdAb 结合动力学的方法。使用这些方法可以轻松、常规地获得功能性 sdAb。
