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

• Published in: Protein expression and purification Vol. 176; p. 105722
• Main Authors: Polidano, Joseph, Vankadari, Naveen, Price, John T., Wilce, Jacqueline A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2020
• Subjects: Gene Expression; Heat shock factor; Heat Shock Transcription Factors - biosynthesis; Heat Shock Transcription Factors - chemistry; Heat Shock Transcription Factors - genetics; Heat Shock Transcription Factors - isolation & purification; Humans; Oligomerisation; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Stress response; Transcription factor; Heat shock factor; Transcription factor; Stress response; Oligomerisation
• ISSN: 1046-5928; 1096-0279; 1096-0279
• DOI: 10.1016/j.pep.2020.105722

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. [Display omitted] •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.