title: Generation of bioactive recombinant Ancylostoma caninum anticoagulant peptide c2.
abstract: Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction. Recently, several anticoagulant serine protease inhibitors have been identified from adult Ancylostoma caninum hookworms. One of them, A. caninum anticoagulant peptide c2 (AcAPc2), can inhibit the activity of factor VIIa/tissue factor complex to exert its antithrombotic effect. However, it is difficult to adopt traditional expression and purification systems to yield high-purity recombinant AcAPc2 (rAcAPc2). Here, we employed a simple method to produce high-yield and high-purity rAcAPc2. We obtained the full-length double-stranded cDNA encoding AcAPc2 by overlapping PCR and cloned it into an intein-based expression vector. The AcAPc2 cDNA was expressed in Escherichia coli and comprised 30% of the total bacterial proteins. The expressed rAcAPc2 was purified by cleaving the fused chitin-binding domain at pH 7.2. Finally, we produced a high yield of rAcAPc2 at a purity of greater than 98%. Importantly, the generated rAcAPc2 prolonged the prothrombin time (PT) and activated partial thromboplastin time (aPTT) of human plasma in vitro in a dose-dependent manner. Therefore, this method to generate the high-purity and bioactive rAcAPc2 may contribute to the scientific research on its biological function and the treatment of thrombotic diseases.
authors: Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children's Health, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China; Department of Pediatrics, West China Second University Hospital, Sichuan University, PR China; Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, PR China.
authors: Yu
authors: Y
authors: Tong
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, PR China.
authors: Jing
authors: J
authors: Zhou
authors: Laboratory of Early Developmental and Injuries, West China Institute of Woman and Children's Health, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China; Department of Pediatrics, West China Second University Hospital, Sichuan University, PR China; Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, PR China.
authors: Meng
authors: M
authors: Mao
authors: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China.
authors: Ju
authors: J
authors: Gao
authors: Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, PR China; Open laboratory, West China Institute of Woman and Children's Health, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China. Electronic address: xing.yuan7811@163.com.
authors: Lixing
authors: L
authors: Yuan
mesh_terms: D000722:Ancylostoma / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D015153:Blotting, Western; D002686:Chitin; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D047668:Inteins; D010314:Partial Thromboplastin Time; D019154:Protein Splicing; D011517:Prothrombin Time; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C094339:anticoagulant protein, Ancylostoma caninum; D002686:Chitin