title: Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins.
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.
authors.affiliation: Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134, Napoli, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: De Rosa
authors.forename: Aitziber L
authors.initials: AL
authors.lastname: Cortajarena
authors.forename: Alessandra
authors.initials: A
authors.lastname: Romanelli
authors.forename: Lynne
authors.initials: L
authors.lastname: Regan
authors.forename: Luca Domenico
authors.initials: LD
authors.lastname: D'Andrea
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D015202:Protein Engineering; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D011506:Proteins
references.citation: Angew Chem Int Ed Engl. 2006 Aug 4;45(31):5218-21
references.pmid: 16823791
references.citation: PLoS One. 2009 Dec 21;4(12):e8381
references.pmid: 20027230
references.citation: Electrophoresis. 2003 Jul;24(14):2348-58
references.pmid: 12874870
references.citation: J Am Chem Soc. 2009 Aug 12;131(31):10800-1
references.pmid: 19610623
references.citation: Gene. 1997 Feb 20;186(1):29-35
references.pmid: 9047341
references.citation: Biotechniques. 1999 Jul;27(1):110-4, 116, 118-20
references.pmid: 10407673
references.citation: Science. 1994 Nov 4;266(5186):776-9
references.pmid: 7973629
references.citation: Bioconjug Chem. 2002 Sep-Oct;13(5):1163-70
references.pmid: 12236801
references.citation: Trends Biochem Sci. 2003 Dec;28(12):655-62
references.pmid: 14659697
references.citation: Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6705-10
references.pmid: 9618476
references.citation: Angew Chem Int Ed Engl. 2007;46(27):5234-7
references.pmid: 17538921
references.citation: Structure. 2003 May;11(5):497-508
references.pmid: 12737816
references.citation: Biochemistry. 2009 Jun 30;48(25):5953-62
references.pmid: 19492814
references.citation: Proc Natl Acad Sci U S A. 2000 May 9;97(10):5179-84
references.pmid: 10792044
references.citation: Protein Sci. 2005 Aug;14(8):2059-68
references.pmid: 15987886
references.citation: Bioconjug Chem. 2008 Jun;19(6):1124-6
references.pmid: 18507427
references.citation: Chem Biol. 2000 Apr;7(4):253-61
references.pmid: 10780925
references.citation: J Am Chem Soc. 2009 Aug 26;131(33):11644-5
references.pmid: 19645470
references.citation: Protein Expr Purif. 2008 Feb;57(2):217-25
references.pmid: 18032065
references.citation: Protein Sci. 2005 Jan;14(1):64-73
references.pmid: 15576565
references.citation: Anal Biochem. 2000 Jun 15;282(1):161-4
references.pmid: 10860517
references.citation: Biosens Bioelectron. 2010 Sep 15;26(1):55-61
references.pmid: 20541393
references.citation: J Am Chem Soc. 2008 Dec 31;130(52):17664-5
references.pmid: 19108697