title: [The functional expression of humanized ScFv-urokinase fusion protein in Escherichia coli].
abstract: The fusion protein of Humanized mouse anti-human fibrin ScFv and the low molecular weight urokinase (IIn-UK) contained seven disulfide bonds and formed inclusion body while expressing in normal E. coli strain. By coexpressing DsbC and using the special E. coli strain Origami(DE3) which was trxB/gor double mutant, the fusion protein IIn-UK was functionally expressed in the cytoplasm of E. coli. The expressed fusion protein in the soluble fraction was purified by using affinity chromatography specific against urokinase. The purified fusion protein could combine the thrombus in vitro, and the specific activity of urokinase reached 80,000 IU/mg fusion protein. The result showed that the fusion protein retained the activity of two moieties, and this study laid a foundation for further research of targeting thrombolytic agent.
authors.affiliation: Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China. liuzg@nic.bmi.ac.cn
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Liu
authors.forename: Jian-Bo
authors.initials: JB
authors.lastname: Lin
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Yuan
authors.forename: Tie-Jun
authors.initials: TJ
authors.lastname: Kang
authors.forename: Wei-Yuan
authors.initials: WY
authors.lastname: Yu
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
chemical_list: D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D014568:Urokinase-Type Plasminogen Activator