Magnetic fishing of recombinant green fluorescent proteins and tagged proteins with designed synthetic ligands

ABSTRACT

Biomimetic ligands have emerged to overcome disadvantages inherent in biological ligands. In particular, the Ugi reaction can generate scaffolds where molecular diversity can be introduced, allowing the synthesis and screening of ligand libraries in a high-throughput manner against a variety of biological targets. Two adsorbents bearing Ugi-based synthetic ligands, coined A4C7 and A7C1, were previously developed for the selective recovery of green fluorescent protein (GFP) and RKRKRK-tagged GFP directly from Escherichia coli crude extracts. This work describes, for the first time, the in situ synthesis of Ugi-based ligands on magnetic beads and their application in the magnetic recovery of cognate proteins.

KEYWORDS:

• GFP purification
• magnetic particles
• magnetic fishing
• synthetic ligands
• tagged-proteins purification

Acknowledgments

This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728), contracts PTDC/EBB-BIO/118317/2010, fellowships SFRH/BPD/97585/2013 for A.S.P., SFRH/BD/64427/2009 for I.L.B., PD/BD/105871/2014 for C.S.F.