Expression, purification, and biophysical analysis of a part of the C-terminal domain of human hypoxia inducible factor-2α (HIF-2α)

Hypoxia inducible factor 2α (HIF-2α) is a member of the basic helix-loop-helix(bHLH)-Per-Arnt-Sim (PAS) family of transcription factors. It is overexpressed in several cancers, associated with poor prognosis of the patients and resistance to treatment. Here, we study the residues 366-704 of the C-terminal end of human HIF-2α, which contains the N-transcriptional activation domain (NTAD), the oxygen-dependent degradation domain (ODD), and a part of the inhibitory domain (IH). An efficient protocol was developed to produce the 366-704 domain of human HIF-2α protein. Subsequently, we analyzed its biophysical characteristics using circular dichroism spectroscopy and size exclusion chromatography showing that the protein forms an antiparallel beta sheet conformation, and a computational model of the HIF-2α structure was produced. Our data offer new structural information for the unique biological properties of HIF-2α. •Isolation of high purity and stability protein of HIF-2α (366-704).•Identification of the appropriate conditions for HIF-2α (366-704) protein refolding.•Analysis with circular dichroism spectroscopy and SEC-MALS of the refolded HIF-2α (366-704) protein.•Determination of antiparallel beta sheet conformation as the secondary structure of HIF-2α (366-704) protein.•Construction of a model of the HIF-2α 366-704 domains including the ODD, the N-TAD and a part of the inhibitory domain.