Publication: Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains
2
Issued Date
2020-02-01
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ISSN
10960309
00032697
00032697
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2-s2.0-85075748075
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Mahidol University
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SCOPUS
Bibliographic Citation
Analytical Biochemistry. Vol.590, (2020)
Suggested Citation
Siripat Aluksanasuwan, Paleerath Peerapen, Sirikanya Plumworasawat, Juthatip Manissorn, Visith Thongboonkerd Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains. Analytical Biochemistry. Vol.590, (2020). doi:10.1016/j.ab.2019.113518 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/49534
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Title
Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains
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Abstract
© 2019 Elsevier Inc. 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.