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Complex preparation and crystallization

ST Shuguang Tan
HF Hui Feng
JY Jinghua Yan

Last updated date: Jul 5, 2021 Views: 773 Forks: 0

original An abbreviated version of this protocol was published in mAbs in Apr, 2019
Glycosylation-independent binding of monoclonal antibody toripalimab to FG loop of PD-1 for tumor immune checkpoint therapy
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The mammalian cell expressed PD-1 protein and toripalimab-Fab fragment were mixed at a molar ration of 1:1. The mixture was incubated on ice for 30 min and further purified by SuperdexTM 200 10/300 GL (GE Healthcare). 10 mg/mL of toripalimab/PD-1 proteins were used for crystal screening by vapour-diffusion sitting-drop method at 4°C. Diffractable crystals were obtained in a condition consisting of 0.09 M Halogens consisting of NaF, NaBr and NaI additives, 0.1 M Tris-Base (pH8.5), 37.5% (v/v) MPD-P1K-P3350 consisting of MPD (racemic), PEG 1K and PEG 3350 (Morpheus® MD1–46 kit, Molecular Dimensions).

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Tan, S, Feng, H and Yan, J(2021). Complex preparation and crystallization. Bio-protocol Preprint. bio-protocol.org/prep1258.
  2. Liu, H., Guo, L., Zhang, J., Zhou, Y., Zhou, J., Yao, J., Wu, H., Yao, S., Chen, B., Chai, Y., Qi, J., Gao, G. F., Tan, S., Feng, H. and Yan, J.(2019). Glycosylation-independent binding of monoclonal antibody toripalimab to FG loop of PD-1 for tumor immune checkpoint therapy. mAbs 11(4). DOI: 10.1080/19420862.2019.1596513

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