Mild and cost-effective green fluorescent protein purification employing small synthetic ligands

Ana Sofia Pina, Ana Margarida G C Dias, Fatma Isik Ustok, Graziella El Khoury, Cláudia S M Fernandes, Ricardo Jorge Flores Branco, Christopher R Lowe, A Cecília A Roque

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)83-93
Number of pages11
JournalJournal Of Chromatography A
Volume1418
DOIs
Publication statusPublished - 30 Oct 2015

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Keywords

  • Adsorption
  • Combinatorial Chemistry Techniques
  • Costs and Cost Analysis
  • Escherichia coli
  • Green Fluorescent Proteins
  • Ligands
  • Models, Molecular
  • Pyrenes
  • Recombinant Fusion Proteins
  • Journal Article
  • Research Support, Non-U.S. Gov't

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