title: Expression and purification of soluble human cystatin C in Escherichia coli with maltose-binding protein as a soluble partner.
abstract: Human cystatin C (CYSC) is a 13-kDa endogenous cysteine proteinase inhibitor and was investigated as a replacement for creatinine as a marker of renal function. However, expressing recombinant CYSC is difficult in Escherichia coli because of resulting low yield and insufficient purity and insolubility. Here, we cloned and fused CYSC to the C-terminus of three soluble partners - maltose-binding protein (MBP), glutathione S-transferase (GST) and translation initiation factor 2 domain I (IF2) - to screen for their ability to improve the solubility of recombinant CYSC when expressed in E. coli. MBP was best at enhancing the soluble expression of CYSC, with soluble fractions accounting for 92.8±3.11% of all proteins. For scaled production, we purified the de-tagged CYSC by using a 3C protease-cleaved MBP-T3-CYSC fused protein with immobilized metal affinity chromatography and cation-affinity purification. The molecular weights of the de-tagged CYSC and human natural CYSC were similar, and the former could react specifically with CYSC polyclonal antibody. Moreover, the de-tagged CYSC displayed full biological activity against papain and cathepsin B, which was very similar to that of the human natural CYSC protein standard. We provide a method to produce large amounts of soluble recombinant human CYSC in E. coli.
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Xu
authors.affiliation: Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Zhenglei
authors.initials: Z
authors.lastname: Zha
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Mingxin
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Dongwei
authors.initials: D
authors.lastname: Yao
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
authors.forename: Xuhong
authors.initials: X
authors.lastname: Wu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China; Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China. Electronic address: caolin@njau.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.affiliation: Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China. Electronic address: dr.guohongqian@gmail.com.
authors.forename: Hongqian
authors.initials: H
authors.lastname: Guo
mesh_terms: D002401:Cathepsin B / Q000378:metabolism; D002846:Chromatography, Affinity; D055316:Cystatin C / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D058619:Maltose-Binding Proteins / Q000235:genetics*; D010206:Papain / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D055316:Cystatin C; D058619:Maltose-Binding Proteins; D039881:Prokaryotic Initiation Factor-2; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002401:Cathepsin B; D010206:Papain
keywords: Cystatin C; Fusion expression; Soluble expression