Detailed protocol for optimised expression and purification of functional monomeric human Heat Shock Factor 1

Highlights

• A detailed protocol for the expression and purification of human monomeric HSF1.

• Simple strategies to overcome oligomerisation and degradation to heat sensitive proteins.

• A refined method of inducing HSF1 activation in vitro.

Abstract

Heat Shock Factor 1 (HSF1) is the master regulator of the heat shock response, a universal survival mechanism throughout eukaryotic species used to buffer potentially lethal proteotoxic conditions. HSF1's function in vivo is regulated by several factors, including post translational modifications and elevated temperatures, whereupon it forms trimers to bind with heat shock elements in DNA. Unsurprisingly, HSF1 is also extremely sensitive to elevated temperatures in vitro, which poses specific technical challenges when producing HSF1 using a recombinant expression system. Although there are several useful publications which outline steps taken for HSF1 expression and purification, studies that describe specific strategies and detailed protocols to overcome HSF1 trimerisation and degradation are currently lacking. Herein, we have reported our detailed experimental protocol for the expression and purification of monomeric human HSF1 (HsHSF1) as a major species. We also propose a refined method of inducing HsHSF1 activation in vitro, that we consider more accurately mimics HsHSF1 activation in vivo and is therefore more physiologically relevant.

Introduction

Heat Shock Factor 1 (HSF1) is a highly conserved transcription factor expressed ubiquitously in eukaryotic cells, that is responsible for a myriad of physiological processes [[1], [2], [3], [4], [5], [6]]. Crucially, it directs the so called ‘heat shock response’, that enables cells to maintain homeostasis in response to adverse conditions, such as thermal stress, hypoxia and acidosis that can lead to protein misfolding and potential cytotoxicity. This is mainly accomplished via transcriptional regulation of stress response genes that encode a class of molecular chaperones termed heat shock proteins (HSPs). The HSPs play an integral role in the stress response of a cell, assisting with protein folding, preventing protein aggregation thus averting cells from initiating events leading to stress induced cell death [7,8].

HSF1 is also central to several pathophysiological conditions. In cancer cells, it is intimately linked with oncogenic initiation, progression and metastasis, presumably through promoting stress tolerance in these cells [[9], [10], [11], [12]]. Consistent with this, HsHSF1 is strongly associated with poor outcomes in cancer patients (breast, prostate, lung, etc.) [11,13]. In neurodegenerative diseases (i.e. Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis, etc.) the formation of protein aggregates and loss of protein homeostasis has been linked with a compromised ability to activate HsHSF1 during stress and protein misfolding [14,15]. Hence, HSF1 in humans has been identified as an attractive therapeutic target for either downregulation in cancer, or upregulation in neurodegenerative disease. This point is highlighted by the numerous research programs seeking to develop HsHSF1 activators and inhibitors, as well as gaining a better understanding of HsHSF1 structure and function [[16], [17], [18]].

HSF1 activation is understood to be regulated by several intrinsic and external factors, including post translational modifications (hyperphosphorylation, acetylation, sumoylation) and elevated temperatures amongst others [[19], [20], [21], [22]]. Activated HsHSF1 forms trimers via its HR-A/B domain and is translocated to the nucleus where it engages with specific DNA promoter regions termed heat shock elements (HSE), while inactivated HsHSF1 is monomeric and repressed as part of the large multiprotein HSP90 complex [23] (Fig. 1). Under elevated temperatures (42 °C), recombinant HsHSF1 is also known to become activated, forming trimers and higher order oligomers [24,25].

To date, several publications have broadly reported on the expression and purification of HsHSF1 for its in vitro biophysical characterisation [24,26,27]. While these have provided useful information, detailed methods and descriptive protocols for optimal expression, purification and isolation of functional monomeric HsHSF1 and separation of other HsHSF1 conformers (i.e. dimer, trimer, higher order oligomers) from a heterogeneous mixture are lacking. In particular, full length HsHSF1 is found to be sensitive to temperature, resulting in the formation of different conformers, and is also highly susceptible to degradation independent of temperature changes. Production of stable, monomeric and functional HsHSF1 is essential for structural and functional studies and to understand the molecular interplay of HsHSF1 with itself and with other cell regulating factors. In order to overcome the problem of HsHSF1 trimerisation and degradation during recombinant expression and purification processes, more descriptive protocols and strategies are required.

Herein, we have detailed our experimental protocol and results with modified strategies for the expression and purification of functional and monomeric HsHSF1 using a simple Escherichia coli protein expression system. Specifically, we report our optimised protocol, including strategies of cell disruption to overcome HsHSF1 oligomerisation and degradation. This has allowed us to obtain monomeric HsHSF1 as a major conformer and to isolate different conformers of HsHSF1 (i.e. dimer, trimer, higher order oligomers). Our Native-PAGE analysis, SEC-MALS and other DNA binding studies allowed us to validate our monomeric HsHSF1 as a functional species capable of acquiring DNA binding competence. We anticipate that these detailed and optimised methods will also serve in the purification of other related or heat sensitive proteins.