A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins

Ana S. Pina; Márcia Guilherme; Alice S. Pereira; Cláudia S. F. M. Fernandes; Ricardo J. F. Branco; Graziella El Khoury; Christopher R. Lowe; Ana Cecília A. Roque

doi:10.1002/cbic.201400018

A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins

Ana S. Pina, Márcia Guilherme, Alice S. Pereira, Cláudia S. F. M. Fernandes, Ricardo J. F. Branco, Graziella El Khoury, Christopher R. Lowe, Ana Cecília A. Roque

Research output: Contribution to journal › Article › peer-review

12 Citations (Scopus)

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 (K_a of 2.45×10⁵ M^-1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

Original language	English
Pages (from-to)	1423-1435
Number of pages	13
Journal	Chembiochem
Volume	15
Issue number	10
DOIs	https://doi.org/10.1002/cbic.201400018
Publication status	Published - 7 Jul 2014

Keywords

affinity chromatography
affinity tags
fusion proteins
ligand design
protein expression
Ugi reaction

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10.1002/cbic.201400018

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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 2.45×105 M-1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.",

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author = "Pina, {Ana S.} and M{\'a}rcia Guilherme and Pereira, {Alice S.} and Fernandes, {Cl{\'a}udia S. F. M.} and Branco, {Ricardo J. F.} and {El Khoury}, Graziella and Lowe, {Christopher R.} and Roque, {Ana Cec{\'i}lia A.}",

note = "The authors thank the financial support from Fundacao para a Ciencia e a Tecnologia through grant no. PEst-C/EQB/LA0006/2013, contracts PTDC/BIO/65383/2006, /EBB-BIO/102163/2008, PTDC/EBB-BIO/098961/2008, PTDC/EBB-BIO/118317/2010, fellowships SFRH/BD/48804/2008 for A.S.P. and SFRH/BPD/69163/2010 for R.J.F.B.",

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doi = "10.1002/cbic.201400018",

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AU - Pina, Ana S.

AU - Guilherme, Márcia

AU - Pereira, Alice S.

AU - Fernandes, Cláudia S. F. M.

AU - Branco, Ricardo J. F.

AU - El Khoury, Graziella

AU - Lowe, Christopher R.

AU - Roque, Ana Cecília A.

N1 - The authors thank the financial support from Fundacao para a Ciencia e a Tecnologia through grant no. PEst-C/EQB/LA0006/2013, contracts PTDC/BIO/65383/2006, /EBB-BIO/102163/2008, PTDC/EBB-BIO/098961/2008, PTDC/EBB-BIO/118317/2010, fellowships SFRH/BD/48804/2008 for A.S.P. and SFRH/BPD/69163/2010 for R.J.F.B.

PY - 2014/7/7

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N2 - 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 2.45×105 M-1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

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A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins

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