Research Article: Production and purification of human Hsp90β in Escherichia coli

Date Published: June 26, 2017

Publisher: Public Library of Science

Author(s): Martina Radli, Dmitry B. Veprintsev, Stefan G. D. Rüdiger, Didier Picard.

http://doi.org/10.1371/journal.pone.0180047

Abstract

The molecular chaperone Hsp90 is an essential member of the cellular proteostasis system. It plays an important role in the stabilisation and activation of a large number of client proteins and is involved in fatal disease processes, e.g. Alzheimer disease, cancer and cystic fibrosis. This makes Hsp90 a crucial protein to study. Mechanistic studies require large amounts of protein but the production and purification of recombinant human Hsp90 in Escherichia coli is challenging and laborious. Here we identified conditions that influence Hsp90 production, and optimised a fast and efficient purification protocol. We found that the nutrient value of the culturing medium and the length of induction had significant effect on Hsp90 production in Escherichia coli. Our fast, single-day purification protocol resulted in a stable, well-folded and pure sample that was resistant to degradation in a reproducible manner. We anticipate that our results provide a useful tool to produce higher amount of pure, well-folded and stable recombinant human Hsp90β in Escherichia coli in an efficient way.

Partial Text

The cellular proteostasis system evolved to maintain cellular health and stability and to protect cells from continuously occurring stress through tight control of protein production, quality control, folding, trafficking, aggregation and degradation [1,2]. Chaperones are crucial elements of the proteostasis system, they prevent protein misfolding and aggregation by various mechanisms and thereby contribute to cellular integrity [3].

Human Hsp90β is often produced in baculovirus system in insect Sf9 cells that is expensive, requires long preparation steps and is often difficult to scale-up [33–35]. Our optimised recombinant human Hsp90β production protocol in E. coli provides an attractive alternative to the baculovirus system.

 

Source:

http://doi.org/10.1371/journal.pone.0180047

 

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