Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate. Issue 1 (December 2016) - Yamashita, Shinji
Abstract Background Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using anEscherichia coli expression system. Results When recombinant oANG was expressed from a T7 promoter in variousE. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from atac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purifiedtac -expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum ofE. coli -expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed inE. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. TheK m values of both oANG preparations were similar; thek cat value ofE. coli -expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. Conclusions Recombinant oANG expressed inE. coli functions as a human renin substrate. This study presents anE. coli -based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.