Affitins for protein purification by affinity magnetic fishing
| Autor: | A. Cecília A. Roque, Raquel Santos, Frédéric Pecorari, Aline Canani Viecinski, Barbara Mouratou, Stella Ottengy, Ghislaine Béhar, Cláudia S. M. Fernandes |
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| Přispěvatelé: | UCIBIO-REQUIMTE, Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), This work was supported by Unidade de Ciências Biomoleculares Aplicadas, UCIBIO, financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728), contracts PTDC/EBB-BIO/118317/2010, fellowships PD/BD/105871/2014 and PD/BD/105753/2014 for C. Fernandes and R. dos Santos, respectively., Bernardo, Elizabeth |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Phage display Affitin Archaeal Proteins Lysozyme Magnetic particles [SDV.CAN]Life Sciences [q-bio]/Cancer Ligands 01 natural sciences Biochemistry Chromatography Affinity Antibodies Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound [SDV.CAN] Life Sciences [q-bio]/Cancer Affinity chromatography Magnetic fishing Protein purification Escherichia coli Animals Humans Tandem affinity purification Chromatography 010401 analytical chemistry Organic Chemistry General Medicine Protein engineering Affinity purification 0104 chemical sciences DNA-Binding Proteins 030104 developmental biology chemistry Immunoglobulin G Ribosome display Magnets Muramidase Chickens Protein Binding |
| Zdroj: | Journal of Chromatography A Journal of Chromatography A, Elsevier, 2016, 1457, pp.50-8. ⟨10.1016/j.chroma.2016.06.020⟩ Journal of Chromatography A, 2016, 1457, pp.50-8. ⟨10.1016/j.chroma.2016.06.020⟩ |
| ISSN: | 0021-9673 1873-3778 |
| DOI: | 10.1016/j.chroma.2016.06.020⟩ |
| Popis: | International audience; 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 " 7 kDa 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 58 mg lysozyme/g support and 165 mg IgG/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. |
| Databáze: | OpenAIRE |
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