title: [Expression of vWF73 and VWF114 fragments of von Willebrand factor A2 domain and their utilization in detecting ADAMTS13 activity].
abstract: OBJECTIVE
  To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.
  
  
  METHODS
  The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.
  
  
  RESULTS
  Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.
  
  
  CONCLUSIONS
  Two GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.
authors: The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
authors: Jing-yu
authors: JY
authors: Zhang
authors: Zhen-ni
authors: ZN
authors: Ma
authors: Ning-zheng
authors: NZ
authors: Dong
authors: Lu-ping
authors: LP
authors: Hu
authors: Jian
authors: J
authors: Su
authors: Zhao-yue
authors: ZY
authors: Wang
authors: Chang-geng
authors: CG
authors: Ruan
mesh_terms: D051722:ADAM Proteins / Q000097:blood* / Q000235:genetics; D000071120:ADAMTS13 Protein; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008297:Male; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor; D005982:Glutathione Transferase; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human