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A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins
Author(s) -
Pina Ana S.,
Guilherme Márcia,
Pereira Alice S.,
Fernandes Cláudia S. M.,
Branco Ricardo J. F.,
El Khoury Graziella,
Lowe Christopher R.,
Roque A. Cecília A.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201400018
Subject(s) - fusion protein , affinity chromatography , green fluorescent protein , ligand (biochemistry) , fusion , target protein , chemistry , combinatorial chemistry , protein purification , docking (animal) , biochemistry , receptor , computational biology , recombinant dna , biology , gene , medicine , linguistics , philosophy , nursing , enzyme
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 ( K a 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.