Expression, purification and crystallisation of the C-terminal domain of human HSP90

Abstract

The HSP90 proteins are a family of molecular chaperones that assist in protein folding, maturation and re-folding of denatured proteins. This HSP family is associated with several pathological conditions including different types of cancers and neurodegenerative diseases. There are two major mammalian isoforms of HSP90 localized in the cytoplasm, HSP90β which is consecutively expressed and HSP90α which is the inducible form. This study focused on the expression, purification and characterisation of the C-terminal domain of both HSP90 isoforms, as the functional characterisation of HSP90 C-terminal domain is still currently limited. In this project, subcloning of the alpha isoform was carried out, followed by expression of HSP90α-C (513- 732) in competent E.coli cells. Subsequently, an IMAC purification protocol was implemented, and the eluted protein was characterised using Native-PAGE and circular dichroism (CD) spectroscopy. The Hsp90β-C (530-724) was successfully expressed in BL21(DE3) cells, purified by IMAC and also characterised by Native-PAGE and CD. In addition, crystallisation trials were initiated with the pure Hsp90β-C (530-724) sample. A number of different conditions were set up and optimised over time and some promising conditions were identified by the end of this project.