猪促肾上腺皮质激素在大肠杆菌中的克隆、表达与纯化。
Cloning, expression, and purification of porcine adrenocorticotropic hormone in Escherichia coli.
机构信息
Department of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, Shanghai, 201203, China; Shanghai Duomirui Biotechnology Ltd, China State Institute of Pharmaceutical Industry, Shanghai, 201203, China.
Shanghai Duomirui Biotechnology Ltd, China State Institute of Pharmaceutical Industry, Shanghai, 201203, China.
出版信息
Protein Expr Purif. 2020 Dec;176:105731. doi: 10.1016/j.pep.2020.105731. Epub 2020 Aug 29.
Adrenocorticotropic hormone (ACTH) is an old medicine derived from porcine pituitary gland that has been marketed for more than 60 years. In this study, we present a recombinant approach to produce ACTH in Escherichia coli (E. coli). The SUMO-tagged fusion protein was cloned and expressed after induction with isopropyl-β-d-thiogalactopyranoside (IPTG) at 25 °C for 8 h. The fusion protein was extracted and purified by anion exchange chromatography, and the SUMO tag was subsequently removed by digestion with ubiquitin-like protease 1 (ULP1). Approximately 95.3 mg of recombinant ACTH with 94.2% purity was obtained after cation exchange purification performed on a 5 mL column, from 286 mL fermentation broth based on the amount of pellets homogenized. The molecular mass of the recombinant ACTH was confirmed by mass spectrometry to equal 4567.32 Da.
促肾上腺皮质激素（ACTH）是一种源自猪垂体的老药，已经上市 60 多年。在这项研究中，我们提出了一种在大肠杆菌（E. coli）中生产 ACTH 的重组方法。在 25°C 下用异丙基-β-D-硫代半乳糖苷（IPTG）诱导 8 小时后，克隆并表达了带有 SUMO 标签的融合蛋白。融合蛋白通过阴离子交换色谱提取和纯化，然后用泛素样蛋白酶 1（ULP1）消化去除 SUMO 标签。从基于匀浆沉淀量的 286 mL 发酵液中，通过在 5 mL 柱上进行阳离子交换纯化，获得了约 95.3 mg 纯度为 94.2%的重组 ACTH。通过质谱确认重组 ACTH 的分子量为 4567.32 Da。
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