Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

摘要: Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine (oANG) from sheep plasma has been shown be better substrate for human it used in clinical renin assays. To expand availability oANG, we aimed produce milligram levels recombinant oANG using Escherichia coli expression system. When was expressed T7 promoter various E. strains at 37 °C, accumulated insoluble fraction. However, expressing 37 °C tac promoter, weaker transcriptional activity than significantly elevated ratio soluble oANG. Using novel culturing system auto-induction culture medium, purified tac-expressed homogeneity, with yield 4.0 mg per liter culture. Based on size-exclusion gel filtration analysis dynamic light scattering analysis, resulting monomer solution. The circular dichroism spectrum coli-expressed similar that CHO cells. Differential scanning fluorimetry showed both preparations undergo two-state transition during thermal denaturation, melting temperatures cells were 49.4 ± 0.16 °C 51.6 ± 0.19 °C, respectively. K m values similar; k cat value slightly higher CHO-expressed Recombinant functions as substrate. This study presents coli-based rapid production quantities substrate, will useful fundamental studies hypertension.