Binary polypeptide system for permanent and oriented protein immobilization

Journal of Nanobiotechnology

Research

Frédéric Darios¹  Enrico Ferrari¹  Fan Zhang¹  Dhevahi Niranjan¹  Bazbek Davletov¹  Mikhail Soloviev²  Julian Bailes² 

[1] MRC Laboratory of Molecular Biology, Hills Road, CB2 0QH, Cambridge, UK;School of Biological Sciences, Royal Holloway University of London, Egham, TW20 0EX, Surrey, UK;

关键词: Sodium Dodecyl Sulphate;    Gold Nanoparticles;    Snare Complex;    Snare Protein;    Affinity System;   

DOI  :  10.1186/1477-3155-8-9

 received in 2010-01-11, accepted in 2010-05-12,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundMany techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.ResultsHere we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution.ConclusionsIPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

【 授权许可】

Unknown   
© Ferrari et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.