Abstract
The Hypoxia-inducible factor 1 (HIF-1) heterodimeric transcription factor which is composed of a regulated α subunit and a constitutively expressed β subunit is a critical regulatory factor that orchestrate the cellular responses to hypoxia. It plays an important role in the pathology of many human diseases. The oxygen-sensing and transactivating functions of HIF-1 are contained within the α subunit, which is up-regulated in most human cancers. To study the function of HIF-1α more intensively, we designed a system to express and purify functional recombinant human HIF-1α (rhHIF-1α) protein from E. coli. The expressed His-tagged rhHIF-1α which forms inclusion bodies at 28°C was washed, dissolved and refolded firstly. Then the refolding His-tagged rhHIF-1α fusion protein was purified by diethylaminoethyl ion-exchange chromatography. After cleaved by thrombin, rhHIF-1α was thoroughly separated from tags by using Ni–NTA affinity chromatography, which was confirmed by SDS–PAGE and western blotting analysis. Thereafter, rhHIF-1α was further purified by using molecular sieve and its function was identified by electrophoretic mobility shift assay and the result indicated that the purified rhHIF-1α has DNA binding activity. The present expression and purification procedure enabled us to obtain biologically active rhHIF-1α from E. coli for in vitro function studies.
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