title: Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.
abstract: In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%.
authors: College of Life Science, Northwest University, Xi'an, 710069, China.
authors: Liujiao
authors: L
authors: Bian
authors: Xu
authors: X
authors: Ji
authors: Wei
authors: W
authors: Hu
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D011232:Chemical Precipitation; D002642:Chick Embryo; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000302:isolation & purification; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D010958:Plasminogen / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C491499:plasminogen kringle 5; D010958:Plasminogen; D000645:Ammonium Sulfate
keywords: ammonium sulfate salting-out; human plasminogen Kringle 5; isolation and purification; liquid chromatography