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Updated: Apr 28, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins
Ana S Pina1, Márcia Guilherme, Alice S Pereira
1REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica (Portugal); IBET-Instituto de Biologia Experimental e Tecnológica, Av. República, Qta. do Marquês, 2780-157 Oeiras (Portugal); Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge (UK).
Abstract:
A novel affinity "tag-receptor" pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×10(5) M(-1) ). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.
