Sortase-mediated fluorescent labeling of CRISPR complexes.

Autor:	Dillard KE; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States., Schaub JM; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States., Brown MW; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States., Saifuddin FA; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States., Xiao Y; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States., Hernandez E; Department of Chemistry, University of Texas at Austin, Austin, TX, United States., Dahlhauser SD; Department of Chemistry, University of Texas at Austin, Austin, TX, United States., Anslyn EV; Department of Chemistry, University of Texas at Austin, Austin, TX, United States., Ke A; Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States., Finkelstein IJ; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, United States. Electronic address: ifinkelstein@cm.utexas.edu.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2019; Vol. 616, pp. 43-59. Date of Electronic Publication: 2018 Dec 17.
DOI:	10.1016/bs.mie.2018.10.031
Abstrakt:	Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit. (© 2019 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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