title: Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm.
abstract: 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.
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Yongjun
authors: Y
authors: Zhou
authors: The Gastroenterology Tumor and Microenvironment Laboratory, Department of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, PR China.
authors: Yan
authors: Y
authors: Zhou
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Jun
authors: J
authors: Li
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Jian
authors: J
authors: Chen
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Yuqin
authors: Y
authors: Yao
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Lin
authors: L
authors: Yu
authors: Chengdu Institute of Biological Products Co., Ltd., PR China.
authors: Desheng
authors: D
authors: Peng
authors: Chengdu Institute of Biological Products Co., Ltd., PR China.
authors: Mingrong
authors: M
authors: Wang
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
authors: Dan
authors: D
authors: Su
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China. Electronic address: heyong01@scu.edu.cn.
authors: Yong
authors: Y
authors: He
authors: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China. Electronic address: goulantu@scu.edu.cn.
authors: Lantu
authors: L
authors: Gou
mesh_terms: D055316:Cystatin C / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C525030:CST3 protein, human; D055316:Cystatin C; D011994:Recombinant Proteins
keywords: Escherichia coli; Human cystatin C; Periplasmic expression