title: Soluble expression of active recombinant human tissue plasminogen activator derivative (K2S) in Escherichia coli.
abstract: BACKGROUND
  The kringle 2 plus serine protease domains (K2S) of human tissue plasminogen activator (tPA) is an efficacious thrombolytic drug, which has been used to treat heart attacks and strokes by breaking up the clots that cause them. It has nine disulfide bridges, which are needed for proper folding and be the bottleneck in improving the production in the Escherichia coli system. So far, few reports have described the production of soluble active K2S from E. coli.
  
  
  OBJECTIVE
  To achieve high-level expression of active K2S in the E. coli system.
  
  
  METHODS
  The DNA fragment coding for K2S was fused with the E. coli disulfide isomerase DsbC. The constructed fusion protein was expressed in E. coli, and then purified with the Ni(2+)-chelating affinity chromatography. K2S was released by cleavage with Factor Xa protease, and the thrombolytic activity was determined using the fibrin plate assay.
  
  
  RESULTS
  The fusion protein DsbC-K2S was found in the culture supernatant of recombinant E. coli as a soluble form of ~40%. The result of fibrinolysis fibrin plate assay showed that the purified recombinant K2S exhibited significant fibrinolysis activity in vitro.
  
  
  CONCLUSIONS
  These works provided a novel approach for the production of active K2S in E. coli without the requirements of in vitro refolding process, and might establish a significant foundation for the following production of K2S.
authors.affiliation: Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin. xuegangluo@yahoo.com.cn
authors.forename: Xue-Gang
authors.initials: XG
authors.lastname: Luo
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Tian
authors.forename: Meng
authors.initials: M
authors.lastname: Ni
authors.forename: Xiao-Lan
authors.initials: XL
authors.lastname: Jing
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Lv
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.forename: Tong-Cun
authors.initials: TC
authors.lastname: Zhang
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D018082:Kringles / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics*
chemical_list: D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D019704:Protein Disulfide-Isomerases