Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli
Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli
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DOI：
10.1016/j.pep.2005.04.015
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发表时间：
2005-08-01
影响因子：
1.6
通讯作者：
Feldhaus, MJ
中科院分区：
文献类型：
--
作者：
Miller, KD;Weaver-Feldhaus, J;Feldhaus, MJ
单链（scFv）抗体用作诊断、治疗和蛋白质组学分析的亲和试剂。我们用于鉴定新型抗原结合剂的抗体发现平台涉及发现、表征和生产。发现和表征组分先前已经被表征，但是为了充分利用来自我们的酵母表面展示文库的亲和试剂的能力，努力集中于开发生产组分以获得纯化的、可溶的和活性的scFv。不是优化条件以实现最大产量，而是将努力集中在使用可以快速轻松地生产和处理数百种scFv抗体的系统上。评估了在酿酒酵母、巴斯德毕赤酵母和大肠杆菌中的异源蛋白表达快速、有效和一致地产生用于下游蛋白质组学应用的scFv抗体的能力。纯化后，使用新的Biacore测定法定量几种scFv抗体的结合活性。所有三种系统产生可溶性scFv抗体，其活性范围为0至99%。来自酵母属、毕赤酵母属和大肠杆菌的scFv抗体产量分别为1.5-4.2、0.4-7.3和0.63-16.4 mg L-1培养物。为了我们的目的，在大肠杆菌中的表达被证明是获得和表征用于下游应用的纯化的scFv的最快和最一致的方式。大肠杆菌表达系统随后用于研究三种scFv变体，其经工程改造以确定结构-功能关系。(c)2005年爱思唯尔公司All rights reserved.
Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships. (c) 2005 Elsevier Inc. All rights reserved.