title: [Prokaryotic expression,  purification and  identification of  recombinant staphylokinase-HC fusion protein].
abstract: OBJECTIVE
  To construct the prokaryotic expression plasmid pEGX-6P-1-SAK-HC,  express it in E.coli,  and identify its biological activity.
  
  
  METHODS
  The fusion gene (SAK-HC) was obtained by overlap-extension PCR and then inserted into prokaryotic soluble pEGX-6P-1 vector with GST tag to construct expression plasmid (pEGX-6P-1-SAK-HC). GST-SAK-HC was expressed by E.coli B834 (DE3) under the induction of IPTG and purified by Glutathion-Sepharose 4B (GST) affinity chromatography and negative-ion exchange column (DEAE) chromatography. PreScission protease was used to remove the GST tag. The purity of the fusion protein was analyzed by SDS-PAGE and the fibrinolytic activity of SAK-HC in vitro was characterized by soluble fibrin plate method.
  
  
  RESULTS
  PCR,  sequencing and restriction enzyme digestion analysis demonstrated that the recombinant plasmid was constructed successfully. The fusion protein was expressed in E.coli B834 (DE3),  M(r); being 36 000 as shown by SDS-PAGE. After purified by GST affinity and DEAE chromatography,  SAK-HC fusion protein of high purity was obtained from the cell supernantants. In vitro experiments showed that the fibrinolytic activity of the recombinant SAK-HC was about 9.4×10();4 IU/mg.
  
  
  CONCLUSIONS
  The SAK-HC fusion protein we obtained was successfully expressed in E.coli and exhibited a fibrinolytic activity as high as the urokinase standard,  which offers a base for the identification of immunogenicity of the fusion protein.
authors.affiliation: Department of Cardiology, First Affiliated Hospital, Chongqing Medical University, Chongqing, China. chenjian19861122@126.com
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Ke
authors.initials: K
authors.lastname: Yang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Guo
authors.forename: Yang-li
authors.initials: YL
authors.lastname: Zhang
authors.forename: Shao-cheng
authors.initials: SC
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Sha-sha
authors.initials: SS
authors.lastname: Zhao
authors.forename: Quan
authors.initials: Q
authors.lastname: He
authors.forename: De-qiang
authors.initials: DQ
authors.lastname: Wang
authors.forename: Jian-zhong
authors.initials: JZ
authors.lastname: Zhou
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus