Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: selection of signal peptide and optimal growth conditions
Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: selection of signal peptide and optimal growth conditions
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DOI：
10.1186/s13568-018-0727-8
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发表时间：
2019-01-07
期刊：
影响因子：
3.7
通讯作者：
Overton, Tim W.
中科院分区：
文献类型：
--
作者：
Kasli, Ikhlaas M.;Thomas, Owen R. T.;Overton, Tim W.
在细菌大肠杆菌的周质中产生重组蛋白如抗体片段具有许多优点，包括形成二硫键的能力，有助于正确折叠，以及相对容易释放和随后的捕获和纯化。在本研究中，我们使用两个N端信号肽PelB和DsbA，分别通过Sec和SRP途径将重组scFv抗体（单链可变区片段）13R4引导至周质。使用实验设计（DoE）方法来优化影响细菌生理学和scFv的生产率、溶解度和位置的工艺条件（温度、诱导剂浓度和诱导点）。DoE研究表明，scFv的滴度和亚细胞位置取决于所用的温度和诱导剂浓度，并且还揭示了PelB信号肽在scFv溶解度和细胞生理学方面优于DsbA信号肽。随后使用带挡板的摇瓶来优化较高生物质浓度下的scFv生产。最小化应激（低温）的条件显示有利于周质scFv的产生。本研究强调了信号肽选择和工艺优化对于生产scFv抗体的重要性，并证明了DoE用于选择最佳工艺参数的实用性。
Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters.