'In vitro' expression and crystallization of C-terminal domain of Hsp90

Abstract

Cytosolic Hsp90 including both α and β isoforms are involved in several types of diseases including cancer. Hsp90 aids in the folding and maturation of cancer proteins such as receptor tyrosine kinases, telomerases, interleukin-6 (IL-6), TP53, Fanconi anaemia group A protein (FANCA) and Cyclin-dependent kinase 4 (CDK4). The help from Hsp90 enables these proteins to carry out their function which include escaping immune destruction, angiogenesis, immortalisation, invasion and metastasis. Only the N-terminal domain of Hsp90 has been fully crystallised and analysed. Therefore, obtaining the molecular structure of the C-terminal domain of Hsp90 would shed more light on the mechanism of action of Hsp90 as a chaperone. This will also permit the design of small molecule inhibitors that will interrupt these interactions and slow down cell proliferation. In this study, the C-domain of the α isoform was successfully subcloned in the pTH-1 vector through PCR. The C-domain of the β isoform, whose gene was pre-inserted in the pET28a vector, was successful expressed in E.coli BL21(DE3) competent cells and analysed using SDS-PAGE. The protein was purified using a cOMPLETETM His-tag Purification Column via an IMAC procedure up to 93% purity. The H6-Hsp90β-C (513-724) protein was characterised using circular dichroism and Native-PAGE. Crystallisation trials were set up under various conditions and some promising formulations were identified.