Kasli Ikhlaas M, Thomas Owen R T, Overton Tim W
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
AMB Express. 2019 Jan 7;9(1):5. doi: 10.1186/s13568-018-0727-8.
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.
在大肠杆菌周质中生产重组蛋白（如抗体片段）具有许多优势，包括能够形成二硫键，有助于正确折叠，以及相对容易释放、后续捕获和纯化。在本研究中，我们使用了两个N端信号肽PelB和DsbA，分别通过Sec和SRP途径将重组单链抗体片段（scFv）13R4导向周质。采用实验设计（DoE）方法优化影响细菌生理学以及scFv的生产力、溶解度和定位的工艺条件（温度、诱导剂浓度和诱导点）。DoE研究表明，scFv的滴度和亚细胞定位取决于所用的温度和诱导剂浓度，并且还揭示了在scFv溶解度和细胞生理学方面，PelB信号肽优于DsbA信号肽。随后使用 baffled摇瓶在更高生物量浓度下优化scFv的生产。已表明将压力降至最低的条件（低温）有利于周质scFv的生产。本研究强调了信号肽选择和工艺优化对scFv抗体生产的重要性，并证明了DoE在选择最佳工艺参数方面的实用性。
