Purification and characterization of ovine angiotensinogen

Abstract

The two major forms of ovine angiotensinogen (renin substrate) have been purified to homogeneity from plasma and a third form has been partially purified. The purification procedure involved ammonium sulphate fractionation, gel filtration, ion‐exchange chromatography and chromatofocusing. The pure proteins have apparent relative molecular masses of 56000 as determined by sodium dodecyl sulphate gel electrophoresis. The amino acid compositions of the two major forms appear to be the same; however, they do have different carbohydrate compositions. Antibodies raised against the a form showed complete cross‐reactivity with the b and c forms. Both major forms have the same amino‐terminal sequence, which includes that of ovine angiotensin I: Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe‐His‐Leu. Thus ovine angiotensin is the Ile5 form and not the Val5 form as had previously been suggested.

Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris

“…The lack of glycosidic attachment at the 14th residue of AGT justifies the high affinity of human renin for oAGT and clearly indicates oAGT could be used as potential substrate to measure the PRAC level. The conventional method of purifying native oAGT from nephrectomized sheep plasma yielded a low titer of 35 mg/L of plasma . The existing bacterial and mammalian expression systems are associated with several disadvantages namely, lack of posttranslational modifications, formation of inclusion bodies, presence of endotoxins, high production cost, low yield, batch to batch variation, and viral contamination .…”

“…The conventional method of purifying native oAGT from nephrectomized sheep plasma yielded a low titer of 35 mg/L of plasma. 13 The existing bacterial and mammalian expression systems are associated with several disadvantages namely, lack of posttranslational modifications, formation of inclusion bodies, presence of endotoxins, high production cost, low yield, batch to batch variation, and viral contamination. 14 Conversely, yeast cells have proven to be a suitable platform for the expression of various heterologous proteins, which overcame the disadvantages associated with bacterial and mammalian expression systems.…”

Analysis on serum proteins from rainbow trout Oncorhynchus mykiss exposed to high temperature

“…2 shows only a few samples for both selected and non‐selected groups, specimens with the changed serum protein composition when treated at high temperature were eight out of 12 for non‐selected fish and no fish from a total of 13 for the pre‐selected group. The N‐terminal amino acid sequencing determined 10 amino acid residues for the 58, 44, and 36 kDa proteins on the SDS‐PAGE slab gel which were highly homologous to those of angiotensinogen, 9,10 creatine kinase, 11,12 and glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH), 13,14 respectively (Fig. 3).…”

“… The N‐terminal amino acid sequences of 58, 44, and 36 kDa proteins. (a) The 58‐kDa protein compared with angiotensinogen from bovine 9 and sheep; 10 (b) the 44‐kDa protein compared with creatine kinase from coho salmon 11 and rat; 12 (c) the 36‐kDa protein compared with GAPDH from bovine 13 and chicken 14 . Only different amino acids are indicated.…”

Sequencing and Expression of Sheep Angiotensinogen cDNA

“…1). The amino acid residue at position 14 in sheep angiotensinogen was serine, although Fernley et al 5 ) predicted that the residue was aspargine. These results indicated t To whom correspondence should be addressed.…”