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首页> 外文期刊>Protein Expression and Purification >A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E-coli
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A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E-coli

机译：逐步方法可显着提高大肠杆菌中合理设计的激动剂抗体片段的蛋白质产量

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Fab59 is a rationally-designed antibody fragment (Fab) that mimics the activity of the cytokine thrombopoietin (TPO). Fab59 activity was initially detected directly from bacterial supernatants in a cell-based assay and was subsequently estimated to be equipotent to TPO using purified material. However, the expression of Fab59 was insufficient to support in vivo characterization of the Fab due to extremely low expression levels from its initial phage display expression vector. To boost expression, a new expression vector was designed and constructed, and Fab59 light chain codons were optimized for bacterial expression. However, from this a new challenge arose, in that the codon-optimized Fab59 was more toxic to Escherichia coli cells than parental Fab59. Co-expression of the bacterial chaperon protein Skp alleviated this toxicity. A two-step purification method was used to isolate monomeric Fab59 from the periplasm. Although Fab59 was prone to form aggregates during the purification process, buffer modification efficiently eliminated this problem. Overall, optimization of Fab59 expression and purification achieved a 100-fold increase in Fab59 production in E coli relative to the starting yield. The yield of purified monomeric Fab59 from a shake flask reached up to 3.5 mg/L, which was sufficient to support testing of the agonist activity of purified monomeric Fab59 in vivo. Even higher yields may be achieved by purification of Fab present in the culture media, as Skp most significantly increased accumulation of Fab59 in that location. (C) 2008 Elsevier Inc. All rights reserved.
机译：Fab59是经过合理设计的抗体片段（Fab），可模拟细胞因子血小板生成素（TPO）的活性。 Fab59活性最初是在基于细胞的分析中直接从细菌上清液中检测到的，随后使用纯化的材料估计与TPO等价。然而，由于来自其初始噬菌体展示表达载体的极低表达水平，Fab59的表达不足以支持Fab的体内表征。为了增强表达，设计并构建了新的表达载体，并针对细菌表达优化了Fab59轻链密码子。但是，由此产生了新的挑战，因为密码子优化的Fab59对大肠杆菌细胞的毒性比亲代Fab59高。细菌伴侣蛋白Skp的共表达减轻了这种毒性。使用两步纯化方法从周质中分离单体Fab59。尽管Fab59在纯化过程中易于形成聚集体，但缓冲液修饰有效地消除了该问题。总体而言，相对于起始产量，Fab59表达和纯化的优化使大肠杆菌中Fab59的产量提高了100倍。从摇瓶中纯化的单体Fab59的产量达到3.5mg / L，这足以支持对纯化的单体Fab59的激动剂活性进行体内测试。通过纯化存在于培养基中的Fab，甚至可以获得更高的产量，因为Skp最明显地增加了Fab59在该位置的积累。 （C）2008 Elsevier Inc.保留所有权利。

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