A Tailor-Made "Tag-Receptor" Affinity Pair for the Purification of Fusion Proteins

被引：9

作者：

Pina, Ana S. ^{[1
,2
,3
]}

Guilherme, Marcia ^{[1
]}

Pereira, Alice S. ^{[1
]}

Fernandes, Claudia S. F. M. ^{[1
]}

Branco, Ricardo J. F. ^{[1
]}

El Khoury, Graziella ^{[3
]}

Lowe, Christopher R. ^{[3
]}

Roque, A. Cecilia A. ^{[1
,2
]}

机构：

[1] Univ Nova Lisboa, Fac Ciencias & Tecnol, Dept Quim, REQUIMTE, P-2829516 Caparica, Portugal

[2] IBET, P-2780157 Oeiras, Portugal

[3] Univ Cambridge, Dept Chem Engn & Biotechnol, Inst Biotechnol, Cambridge CB2 1QT, England

来源：

CHEMBIOCHEM | 2014年 / 15卷 / 10期

关键词：

affinity chromatography; affinity tags; fusion proteins; ligand design; protein expression; Ugi reaction; UGI REACTION; LIGANDS; DESIGN; ADSORBENT; IMMUNOGLOBULINS; CHROMATOGRAPHY; EXPRESSION; STRATEGIES; SOLVATION; FRAGMENTS;

D O I：

10.1002/cbic.201400018

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 x 10(5)m(-1)). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

引用

页码：1423 / 1435

页数：13

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