Xiaoyun Meng; Lanjun Zhang; Hong Wei; Furong Li; Lihua Hu; Haiyun Ma; Qian Liu; Xiaoyu Li; Zhongchuan Liu
BioTechniques, 2020, 69, 69(1), 427-433
https://doi.org/10.2144/btn-2019-0170

Abstract

Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.
METHOD SUMMARY:
An Escherichia coli system was used for IL-17A expression. Two-way optimizations of the refolding conditions were implemented by exploring various ratios of oxidized and reduced forms of the oxido-shuffling reagents and the refolding duration. A detailed protocol for the optimized refolding has been submitted to protocols.io (doi 10.17504/protocols.io.bda3i2gn). Yield enhancement of anti-IL-17A Fab production was achieved after generating a stable HEK293-F cell line expressing the Fab fragment by G418 selection of transfected cells. The purified IL-17A/anti-IL-17A Fab complex was characterized by SDS-PAGE and western blotting. Single crystals of the quaternary complex, IL-17A/Fab/HAP (high affinity peptide) with small-molecule compounds derived from our DNA encoded library could be obtained using a batch method.