title: Expression of human angiotensinogen cDNA in Escherichia coli.
abstract: Human angiotensinogen cDNA clones were isolated from a human liver library. Nucleotide sequence analysis of these cDNA clones revealed that position 1075 in the messenger RNA, which is part of a PstI recognition sequence, is different from the published sequence (Kageyama, R., Ohkubo, H., and Nakanishi, S. (1984) Biochemistry 23, 3603-3609). This change results in an altered amino acid at this position in the corresponding protein sequence and suggests possible restriction fragment length polymorphism. The full length human angiotensinogen cDNA was constructed from partial cDNA clones and ligated into an isopropyl-1-thio-beta-D-galactopyranoside inducible bacterial expression vector pUC9 to develop expression plasmid pUCHAG27. This plasmid permitted the synthesis of human angiotensinogen in Escherichia coli. The recombinant bacteria overproduced a 53-kDa protein which was recognized by anti-human angiotensinogen antibodies. The synthesis of this protein was greatly increased upon induction with isopropyl-1-thio-beta-D-galactopyranoside. The chimeric protein, almost identical to human angiotensinogen, was partially purified by ammonium sulfate fractionation and gel filtration on Sephadex G-100. Human kidney renin was shown to enzymatically cleave this recombinant protein to produce des-(angiotensin I)-angiotensinogen and a small polypeptide. Thus, we provide evidence that recombinant human angiotensinogen synthesized through E. coli is biologically active and serves as a substrate for human renin.
mesh_terms: D000595:Amino Acid Sequence; D000808:Angiotensinogen / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000201:enzymology; D010957:Plasmids; D012083:Renin / Q000378:metabolism
chemical_list: D000808:Angiotensinogen; D004247:DNA; D004262:DNA Restriction Enzymes; D012083:Renin