热休克蛋白90
大肠杆菌
肠肽酶
化学
重组DNA
紫胶操纵子
热休克蛋白
氨基三乙酸
生物化学
表达式向量
分子生物学
色谱法
生物
融合蛋白
基因
有机化学
螯合作用
作者
Siripat Aluksanasuwan,Paleerath Peerapen,Sirikanya Plumworasawat,Juthatip Manissorn,Visith Thongboonkerd
标识
DOI:10.1016/j.ab.2019.113518
摘要
Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5–1 U) and urea (0–3 M). Each HSP90 construct was highly purified and approximately 0.1–1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection – Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.
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