title: Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants.
abstract: Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPO(M)) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPO(P) from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPO(P). Using this process, up to 31% of the asialo-rhuEPO(P) could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO(P) expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Farooqahmed S
authors.initials: FS
authors.lastname: Kittur
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Arthur
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Maikhanh
authors.initials: M
authors.lastname: Nguyen
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Chiu-Yueh
authors.initials: CY
authors.lastname: Hung
authors.affiliation: Carilion Clinic and Virginia Tech Carilion School of Medicine, Roanoke, VA 24014, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Sane
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA. Electronic address: jxie@nccu.edu.
authors.forename: Jiahua
authors.initials: J
authors.lastname: Xie
mesh_terms: D001212:Asialoglycoproteins / Q000302:isolation & purification*; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018515:Plant Leaves / Q000737:chemistry; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014026:Nicotiana / Q000235:genetics*
chemical_list: D001212:Asialoglycoproteins; D011994:Recombinant Proteins; C500605:asialoerythropoietin; D004921:Erythropoietin
keywords: Basic recombinant human protein; Immunoaffinity chromatography; Ion-exchange chromatography; Plant-produced asialoerythropoietin