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Pina A.S.,New University of Lisbon | Pina A.S.,IBET Institute Biologia Experimental Tecnologica | Lowe C.R.,University of Cambridge | Roque A.C.A.,New University of Lisbon
Biotechnology Advances | Year: 2014

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. © 2013 Elsevier Inc. Source


Pina A.S.,New University of Lisbon | Pina A.S.,IBET Institute Biologia Experimental Tecnologica | Pina A.S.,University of Cambridge | Lowe C.R.,University of Cambridge | Roque A.C.A.,New University of Lisbon
Separation Science and Technology | Year: 2010

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support. © Taylor & Francis Group, LLC. Source


Pina A.S.,New University of Lisbon | Pina A.S.,IBET Institute Biologia Experimental Tecnologica | Pina A.S.,University of Cambridge | Dias A.M.G.C.,New University of Lisbon | And 7 more authors.
Journal of Chromatography A | Year: 2015

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. © 2015 Elsevier B.V. Source

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