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Covalent attachment of protein cargo to a computationally designed toroid scaffold
Author(s) -
Polkinghorn Caley Ray,
Hallinan Jazmine,
Doyle Lindsey,
Bradley Phil,
Stoddard Barry,
Kaiser Brett K.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.798.20
Subject(s) - scaffold protein , toroid , protein engineering , scaffold , fusion protein , biophysics , solenoid , chemistry , computational biology , biology , physics , computer science , biochemistry , recombinant dna , plasma , gene , database , signal transduction , quantum mechanics , enzyme
Recent developments in computational design have expanded the accessible protein structures to those beyond what was selected for through the evolutionary process. Closed alpha‐solenoid tandem repeat structures (alpha‐toroids) are one class of rationally designed proteins. Four designed toroids, each containing a central pore and a varied number of repeats, were previously validated by x‐ray crystallography. We envision using toroids as stabilizing scaffolds upon which other proteins can be attached. The goal of this study is to develop an approach for attaching protein cargo to the toroid using the Spycatcher/Spytag peptide tagging system. Spycatcher (protein) and Spytag (peptide) are split protein partners derived from the fibronectin binding protein of Streptococcus pyogenes that form an irreversible, stable isopeptide bond. We demonstrated that 24‐repeat toroids containing four Spycatcher domains embedded within loop regions (“toroid‐SpyCatcher”) properly fold and assemble by size exclusion chromatography. We then showed that a Spytag‐GFP fusion protein binds efficiently and irreversibly to the toroid‐SpyCatcher using fluorescent binding assays. This proof of principle experiment demonstrates that this approach is feasible for attaching proteins to the toroid scaffold and could be expanded to attach cargo proteins with diverse activities. Support or Funding Information Murdock Charitable Trust This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .