TY - JOUR
T1 - Detailed protocol for optimised expression and purification of functional monomeric human Heat Shock Factor 1
AU - Polidano, Joseph
AU - Vankadari, Naveen
AU - Price, John T.
AU - Wilce, Jacqueline A.
PY - 2020/12
Y1 - 2020/12
N2 - 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.
AB - 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.
KW - Heat Shock Factor
KW - transcription factor
KW - stress response
KW - oligomerisation
UR - http://www.scopus.com/inward/record.url?scp=85089491838&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2020.105722
DO - 10.1016/j.pep.2020.105722
M3 - Article
C2 - 32768454
AN - SCOPUS:85089491838
SN - 1046-5928
VL - 176
JO - Protein Expression and Purification
JF - Protein Expression and Purification
M1 - 105722
ER -