Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. 
THERM
Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.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 an Escherichia coli expression system.


 When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction.  However, by expressing oANG at 37 °C from a tac 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 purified tac-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 of E. 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 in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively.  The K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG.


 CONCLUSIONS
 Recombinant oANG expressed in E. coli functions as a human renin substrate.  This study presents an E. 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.
C
To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system.



Escherichia coli expression system
When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction.  However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. 
T7 promoter
However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. 
tac promoter
Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. 
auto-induction culture medium