Construction,expression and purification of pTAT-HIF-1?-PAS-B in prokaryotic expression system / 第三军医大学学报

ABSTRACT

Objective To construct a prokaryotic expression vector for a fusion protein, TAT protein transduction domain (PTD) and the PAS-B domain of hypoxia inducible factor 1?(HIF-1?), and then express and purifr the fusion protein. Methods The expression plasmids pTAT-PAS-B, pET-PAS-B and pTAT-EGFP were constructed respectively, and transformed into E. coli. BL21(DE3)pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE and Western blotting. The fusion protein were purified with Ni-NTA-His affinity chromatography. Results The three recombinant plasmids were constructed successfully. The objective fusion proteins were obtained by optimizing the conditions for expression and purification. Conclusion The successful expression and purification of the fusion protein TAT-HIF-1?PAS-B has laid the foundation for using it to modulate the activity of HIF-1? in vivo.