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Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N‐LR‐M‐C) domains
Author(s) -
Thongboonkerd Visith,
Aluksanasuwan Siripat,
Plumworasawat Sirikanya,
Manissorn Juthatip,
Peerapen Paleerath
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03751
Subject(s) - hsp90 , enteropeptidase , escherichia coli , lac operon , recombinant dna , chemistry , heat shock protein , nitrilotriacetic acid , expression vector , biochemistry , microbiology and biotechnology , biology , fusion protein , gene , organic chemistry , chelation
Heat shock protein 90 (HSP90) plays essential roles in normal physiology to maintain homeostasis and in many human diseases. It comprises four distinct domains, including NH 2 ‐terminal (N), charged linker region (LR), middle (M), and COOH‐terminal (C) domains, all of which are important for regulating 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). After rebinding to Ni‐NTA resin, 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). Our expression and purification protocols would facilitate further structural and functional studies of human HSP90. Support or Funding Information This study was supported by Mahidol University research grant and the Thailand Research Fund (IRN60W0004).