Kunapuli S P, Prasad G L, Kumar A
J Biol Chem. 1987 Jun 5;262(16):7672-5.
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.
从人肝脏文库中分离出人类血管紧张素原cDNA克隆。对这些cDNA克隆进行核苷酸序列分析发现，信使RNA中作为PstI识别序列一部分的第1075位与已发表序列（Kageyama, R., Ohkubo, H., and Nakanishi, S. (1984) Biochemistry 23, 3603 - 3609）不同。这种变化导致相应蛋白质序列中该位置的氨基酸改变，并提示可能存在限制性片段长度多态性。从部分cDNA克隆构建全长人类血管紧张素原cDNA，并将其连接到异丙基-1-硫代-β-D-吡喃半乳糖苷诱导型细菌表达载体pUC9中，构建表达质粒pUCHAG27。该质粒可在大肠杆菌中合成人类血管紧张素原。重组细菌过量产生一种53 kDa的蛋白质，该蛋白质可被抗人类血管紧张素原抗体识别。用异丙基-1-硫代-β-D-吡喃半乳糖苷诱导后，这种蛋白质的合成显著增加。通过硫酸铵分级分离和Sephadex G - 100凝胶过滤对几乎与人血管紧张素原相同的嵌合蛋白进行了部分纯化。已证明人肾素可酶解这种重组蛋白，产生去（血管紧张素I）血管紧张素原和一种小多肽。因此，我们提供了证据表明通过大肠杆菌合成的重组人血管紧张素原具有生物活性，可作为人肾素的底物。
