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Expression and purification of Nb35

JD Jia Duan
DS Dan-dan Shen
XZ X. Edward Zhou
PB Peng Bi
QL Qiu-feng Liu
YT Yang-xia Tan
YZ You-wen Zhuang
HZ Hui-bing Zhang
PX Pei-yu Xu
SH Si-Jie Huang
SM Shan-shan Ma
XH Xin-heng He
KM Karsten Melcher
YZ Yan Zhang
HX H. Eric Xu
YJ Yi Jiang
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Nanobody-35 (Nb35) with a C-terminal His6 tag, was expressed in the periplasm of E. coli strain BL2119. Cultures of 2 L cells were grown to OD600 = 1.0 at 37 °C in TB media containing 0.1% glucose, 2 mM MgCl2, and 100 μg mL−1 ampicillin. Then, 1 mM IPTG was added to the medium to induce protein expression for another 4.5 h at 37 °C. Cells were harvested by centrifugation and lysed in ice-cold buffer (50 mM Tris pH 8.0, 12.5 mM EDTA, and 0.125 M sucrose), then centrifuged to remove cell debris. Nb35 was purified by nickel affinity chromatography, followed by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column, and finally spin concentrated to ~2.5 mg mL−1.

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