title: Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli.
abstract: BACKGROUND
  Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.
  
  
  RESULTS
  We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.
  
  
  CONCLUSIONS
  This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. 582526247@qq.com.|Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 582526247@qq.com.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Long
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. 522689024@qq.com.|Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 522689024@qq.com.
authors.forename: Yeran
authors.initials: Y
authors.lastname: Gou
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 536513878@qq.com.|Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China. 536513878@qq.com.|Department of Clinical Laboratory, Yubei District People's Hospital, Chongqing, 401120, PR China. 536513878@qq.com.
authors.forename: Miao
authors.initials: M
authors.lastname: Luo
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 125425439@qq.com.|Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China. 125425439@qq.com.
authors.forename: Shaocheng
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 610627111@qq.com.|Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China. 610627111@qq.com.
authors.forename: Hongpeng
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. 411351709@qq.com.|Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 411351709@qq.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Bai
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 395480829@qq.com.|Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China. 395480829@qq.com.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. hequan822@yahoo.cn.
authors.forename: Quan
authors.initials: Q
authors.lastname: He
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. 378793028@qq.com.
authors.forename: Ke
authors.initials: K
authors.lastname: Chen
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. ahuang1964@163.com.
authors.forename: Ailong
authors.initials: A
authors.lastname: Huang
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. zjzcqu@163.com.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Disease (Ministry of Education), The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. wangd@ustc.edu.|Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China. wangd@ustc.edu.
authors.forename: Deqiang
authors.initials: D
authors.lastname: Wang
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005342:Fibrinolysis; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator