title: Mild and cost-effective green fluorescent protein purification employing small synthetic ligands.
abstract: The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of GFP or GFP fusion proteins, giving GFP a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered GFP with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand A4C7 maintained the selectivity to recover other proteins fused to GFP. The performance of A4C7 adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for GFP purification.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; IBET - Instituto de Biologia Experimental Tecnológica, Oeiras, Portugal; Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT Cambridge, UK. Electronic address: asp14526@campus.fct.unl.pt.
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Pina
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Ana Margarida G C
authors.initials: AMGC
authors.lastname: Dias
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT Cambridge, UK.
authors.forename: Fatma Isik
authors.initials: FI
authors.lastname: Ustok
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT Cambridge, UK.
authors.forename: Graziella
authors.initials: G
authors.lastname: El Khoury
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Cláudia S M
authors.initials: CSM
authors.lastname: Fernandes
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Ricardo J F
authors.initials: RJF
authors.lastname: Branco
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT Cambridge, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lowe
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; IBET - Instituto de Biologia Experimental Tecnológica, Oeiras, Portugal. Electronic address: cecilia.roque@fct.unl.pt.
authors.forename: A Cecília A
authors.initials: ACA
authors.lastname: Roque
mesh_terms: D000327:Adsorption; D020650:Combinatorial Chemistry Techniques; D003365:Costs and Cost Analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000191:economics / Q000235:genetics / Q000302:isolation & purification*; D008024:Ligands; D008958:Models, Molecular; D011721:Pyrenes / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008024:Ligands; D011721:Pyrenes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: Affinity tag; Chromatography; Fusion proteins; Green fluorescent protein; Protein purification; Synthetic ligands