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Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm.

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机构: [1]State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China [2]The Gastroenterology Tumor and Microenvironment Laboratory, Department of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, PR China [3]Chengdu Institute of Biological Products Co., Ltd., PR China
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关键词: Human cystatin C Escherichia coli Periplasmic expression

摘要:
Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

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出版当年[2015]版:
大类 | 4 区 生物
小类 | 4 区 生化研究方法 4 区 生化与分子生物学 4 区 生物工程与应用微生物
最新[2023]版:
大类 | 4 区 生物学
小类 | 4 区 生化研究方法 4 区 生化与分子生物学 4 区 生物工程与应用微生物
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第一作者机构: [1]State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China
通讯作者:
通讯机构: [1]State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China [*1]No. 17, Section 3rd, South Renmin Road, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China.
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