Ferrari, Enrico and Darios, Frederic and Zhang, Fan and Niranjan, Dhevahi and Bailes, Julian and Soloviev, Mikhail and Davletov, Bazbek (2010) Binary polypeptide system for permanent and oriented protein immobilization. Journal of Nanobiotechnology, 8 (9). ISSN 1477-3155Full text not available from this repository.
Background Many 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. Results Here 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. Conclusions IPAS 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.
|Keywords:||protein immobilization, SNARE proteins, ref03, refdoi|
|Subjects:||C Biological Sciences > C700 Molecular Biology, Biophysics and Biochemistry|
|Divisions:||College of Sciences > Faculty of Science > School of Life Sciences|
|Depositing User:||Enrico Ferrari|
|Date Deposited:||15 Oct 2012 20:25|
|Last Modified:||08 May 2013 15:19|
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