title: Affitins for protein purification by affinity magnetic fishing.
abstract: Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing.
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: CS
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: Raquel
authors.initials: R
authors.lastname: Dos Santos
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Stella
authors.initials: S
authors.lastname: Ottengy
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Aline Canani
authors.initials: AC
authors.lastname: Viecinski
authors.affiliation: CRCNA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Béhar
authors.affiliation: CRCNA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mouratou
authors.affiliation: CRCNA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pecorari
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. Electronic address: cecilia.roque@fct.unl.pt.
authors.forename: A Cecília A
authors.initials: AC
authors.lastname: Roque
mesh_terms: D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry*; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification; D008024:Ligands; D059346:Magnets; D009113:Muramidase / Q000302:isolation & purification; D011485:Protein Binding
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D008024:Ligands; D009113:Muramidase
keywords: Affinity purification; Affitin; Antibodies; Lysozyme; Magnetic fishing; Magnetic particles