Young Carissa L, Yuraszeck Theresa, Robinson Anne S
Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA.
Methods Enzymol. 2011;491:235-60. doi: 10.1016/B978-0-12-385928-0.00014-6.
Recombinant antibody fragments, for example, the classic monovalent single-chain antibody (scFv), are emerging as credible alternatives to monoclonal antibody (mAb) products. scFv fragments maintain a diverse range of potential applications in biotechnology and can be implemented as powerful therapeutic and diagnostic agents. As such, a variety of hosts have been used to produce antibody fragments resulting in varying degrees of success. Yeast, Saccharomyces cerevisiae, is an attractive host due to quality control mechanisms of the secretory pathway that ensure secreted proteins are properly folded. However, the expression of a recombinant protein in yeast is not trivial; neither are the quality control mechanisms the cell initiates to respond to overwhelming stress, such as an increased protein load, simplistic. The endoplasmic reticulum (ER) is a dynamic organelle, capable of sensing and adjusting its folding capacity in response to increased demand. When protein abundance or terminally misfolded proteins overwhelm the ER's capacity, the unfolded protein response (UPR) is activated. In the guidelines presented here, we discuss varying aspects of quality control, its modulation, and ways to design appropriate constructs for yeast recombinant protein expression. Furthermore, we have provided protocols and methods to monitor intracellular protein expression and trafficking as well as evaluation of the UPR, with essential controls. The latter part of this chapter will review considerations for the experimental design of microarray and quantitative polymerase chain reaction (q-PCR) techniques while suggesting appropriate means of data analysis.
重组抗体片段，例如经典的单价单链抗体（scFv），正成为单克隆抗体（mAb）产品可靠的替代品。scFv片段在生物技术领域有着广泛的潜在应用，可作为强大的治疗和诊断试剂。因此，人们使用了多种宿主来生产抗体片段，取得了不同程度的成功。酵母，即酿酒酵母，是一种有吸引力的宿主，因为其分泌途径的质量控制机制可确保分泌的蛋白质正确折叠。然而，在酵母中表达重组蛋白并非易事；细胞启动的应对巨大压力（如蛋白质负载增加）的质量控制机制也并非简单。内质网（ER）是一个动态细胞器，能够感知并根据需求增加调整其折叠能力。当蛋白质丰度或终末错误折叠的蛋白质超过内质网的能力时，未折叠蛋白反应（UPR）就会被激活。在本文给出的指南中，我们讨论了质量控制的不同方面、其调节以及为酵母重组蛋白表达设计合适构建体的方法。此外，我们还提供了监测细胞内蛋白质表达和运输以及评估UPR的方案和方法，并配有基本对照。本章后半部分将回顾微阵列和定量聚合酶链反应（q-PCR）技术实验设计的注意事项，同时建议合适的数据分析方法。
