Expression Of PHD2 In Escherichia Coli And Analysis Of Its Invitro Activity

Prolyl hydroxylase domain protein 2 (PHD2) is the key oxygen sensor that controls the steady-state levels of hypoxia-inducible factor HIF-la in vivo.Under normoxic condition, PHD2 hydroxylated the specific proline residues in HIF-1? ODD domain with conserved LXXLAP motifs, leading to HIF-la degradation via ubiquitin-proteasome pathway.When exposed to hypoxic environment, the hydroxylation was inhibited, and HIF-1? was accumulated and then migrated to the nucleus, where it formed a heterodimer with HIF-1? and activated the hypoxia-responsive genes.The regulation of HIF-1 a by PHD2 can provide protential therapies for HIF-related hypoxic ischemic diseases and cancers.The aim of this study was to construct the prokaryotic expression vector of PHD2 by In-Fusion technology and achieve soluble expression of PHD2 in Escherichia coli by Nus·Tag.The catalytic activity of the recombinant protein was determined and analyzed.Firstly, the pET-43.1b(+) vector was digested by Sac I and specific In-Fusion PCR primers were designed. PHD2 gene was amplified from pCMV6-Entry-EGLN1 plasmid and the PCR products contained ends which were homologous to the linearized vector. Then the prokaryotic expression vector pET-43.1b(+)-PHD2 was constructed by In-Fusion technology. Secondly, pET-43.1b(+)-PHD2 was transformed into Escherichia coli, and both the host bacteria and expression conditions were optimized.Protein samples were analyzed and identified by SDS-PAGE and Western blot technology,respectively.Finally, Ni-NTA affinity chromatography was used to purify the fusion protein,and the catalytic activity on HIF-1? peptides of the purified proteins was studied by a hydroxylation reaction. The reaction products were detected by liquid chromatography-mass spectrography (LC-MS).In conclusion,we successfully constructed the pET-43.1b(+)-PHD2 prokaryotic expression vector. E.coli BL21(DE3) was chosen as the suitable host strain and soluble Nus-PHD2 fusion protein was obtained. In the Western Blot experiment, it was indicated that the recombinant protein could specifically bind to the PHD2 monoclonal antibody. The optimal expression conditions for Nus-PHD2 was to induce it with 0.8 mmol/L IPTG at 30? for 12 h.LC-MS assay results showed that the expressed Nus-PHD2 had the catalytic activity which could hydroxylate HIF-1? peptides and produce hydroxylation products. This thesis laid a good foundation for the further study of the biological functions of PHD2.