Vermeulen Jan-G, Burt Felicity, van Heerden Esta, Cason Errol, Meiring Muriel
Department of Haematology and Cell Biology, Faculty of Health Sciences, University of the Free State, South Africa.
National Health Laboratory Service, Universitas, Bloemfontein, South Africa; Division of Virology, Faculty of Health Sciences, University of the Free State, South Africa.
Protein Expr Purif. 2018 Nov;151:62-71. doi: 10.1016/j.pep.2018.06.005. Epub 2018 Jun 9.
The development of therapeutic antibodies in their various forms has been a constant challenge since the development of the first monoclonal antibodies in 1975. This is especially true for the development of therapeutic single chain variable (scFv) fragments in Escherichia coli. In a previous study the selection of a tissue factor inhibiting single chain variable fragment (TFI-scFv) isolated from the Thomlinson I + J phage libraries was described. Although the initial findings were promising, additional characterization of the antibody fragment and subsequent application was hampered due low protein yield. This study reports on: i) the improved expression of a previously low yielding TFI-scFv in the cytoplasm of E. coli BL21 (DE3) through modifications to the expression systems in conjunction with codon optimization ii) evaluation of two commercial methods of protein recovery: in vitro refolding and the utilization of cold shock expression systems in conjunction with E. coli SHuffle. Results showed that TFI-scFv could be expressed at higher levels in the cytoplasm of E. coli than previously achieved in the periplasm. Both the in vitro refolding and cold shock strategies were capable of producing functional TFI-scFv with varying degrees of success. These procedures could be applied to improve the production of other problematic low yielding scFv isolated from phage display repositories in order to facilitate their characterization.
自1975年首个单克隆抗体问世以来，各种形式治疗性抗体的研发一直是一项持续的挑战。对于在大肠杆菌中研发治疗性单链可变片段（scFv）而言尤其如此。在之前的一项研究中，描述了从Thomlinson I + J噬菌体文库中分离出的组织因子抑制单链可变片段（TFI-scFv）的筛选过程。尽管最初的研究结果很有前景，但由于蛋白质产量低，抗体片段的进一步表征及后续应用受到了阻碍。本研究报告了：i）通过对表达系统进行修饰并结合密码子优化，提高了先前在大肠杆菌BL21（DE3）细胞质中低产的TFI-scFv的表达水平；ii）评估了两种商业蛋白质回收方法：体外重折叠以及结合大肠杆菌SHuffle使用冷休克表达系统。结果表明，TFI-scFv在大肠杆菌细胞质中的表达水平高于之前在周质中的表达水平。体外重折叠和冷休克策略都能够不同程度地成功产生功能性TFI-scFv。这些方法可用于提高从噬菌体展示文库中分离出的其他有问题的低产scFv的产量，以便于对其进行表征。
