Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.
Protein Design and Immunotherapy Group, Departament de Bioquímica i Biologia Molecular. Facultat de Biociències, Universitat Autònoma de Barcelona, Barcelona, Spain.
Methods Mol Biol. 2022;2313:151-167. doi: 10.1007/978-1-0716-1450-1_8.
The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production.
近年来，人们对将单克隆抗体用作治疗分子的兴趣日益浓厚。由于抗体对其他生物分子具有高亲和力和特异性，因此被广泛用于治疗各种人类疾病，如癌症、风湿和心血管疾病。目前，IgG 样抗体的生产主要来自稳定或瞬时的哺乳动物表达系统，这些系统允许适当的折叠和翻译后修饰。尽管过去十年取得了技术进步，但这些系统的使用仍然具有相当高的生产成本和较长的处理时间。出于这些原因，研究人员越来越关注替代抗体生产方法和替代抗体形式。细菌系统，如大肠杆菌，被广泛用于重组蛋白生产，因为它们易于操作且成本低廉。然而，在已经分馏的重组蛋白中存在脂多糖 (LPS) 痕迹，这使得这些系统不适合制备治疗性分子。酵母系统，如巴斯德毕赤酵母，具有微生物系统易于操作的特点，但也表现出真核表达系统的一些关键优势，如改进的折叠机制和不存在 LPS。它们特别适合生产不需要人源化糖基化的抗体片段，从而避免了使用哺乳动物系统的高成本。本文详细提供了在巴斯德毕赤酵母中表达和纯化单链抗体片段 (scFv) 的方案，包括实验室操作的详细步骤和 5L 生物反应器生产的简要步骤。
