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Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins (Articolo in rivista)

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Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins (Articolo in rivista) (literal)

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De Rosa, L; Cortajarena, AL; Romanelli, A; Regan, L; D'Andrea, LD; (2012)
Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
in Organic & biomolecular chemistry; Royal Society of Chemistry, Cambridge (Regno Unito)
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1. CNR, Ist Biostrutture & Bioimmagini, I-80134 Naples, Italy 2. Yale Univ, Dept Biophys & Biochem, New Haven, CT 06511 USA 3. Univ Naples Federico 2, Dipartimento Sci Biol, I-80134 Naples, Italy 4. Univ Autonoma Madrid, IMDEA Nanociencia, E-28049 Madrid, Spain 5. Univ Autonoma Madrid, Ctr Nacl Biotecnol CNB CSIC, E-28049 Madrid, Spain (literal)

Titolo

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins (literal)

Abstract

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes. (literal)

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