NDLI: Expression, purification, and characterization of recombinant human hypoxia inducible factor 1α in E.coli

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Expression, purification, and characterization of recombinant human hypoxia inducible factor 1α in E.coli

Content Provider	Semantic Scholar
Author	Yu, Rutong Li, Xueyuan Zhou, Xiuping Shi, Qiong Tang
Copyright Year	2011
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.
Starting Page	453
Ending Page	458
Page Count	6
File Format	PDF HTM / HTML
DOI	10.1007/s11274-010-0453-0
Volume Number	27
Alternate Webpage(s)	http://library.ibp.ac.cn/html/slwj/000286113200030.pdf
Alternate Webpage(s)	https://doi.org/10.1007/s11274-010-0453-0
Language	English
Access Restriction	Open
Content Type	Text
Resource Type	Article

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