Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins (313 views)
Org Biomol Chem (ISSN: 1477-0520, 1477-0539, 1477-0539electronic), 2012; 10(2): 273-280.
Export to BibTeX Export to EndNote
Paper type: Journal Article,
Impact factor: 3.568, 5-year impact factor: 3.49
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-83455214139&partnerID=40&md5=382816b5a59ec4243b242ccf47d74a7e
Keywords: Chemical Biology, Desired Position, Experimental Setup, Expressed Protein Ligation, High Purity, Indispensable Tools, Labeled Proteins, Life-Sciences, Molecular Probes, Peptide Synthesis, Protein Sequences, Single Molecule, Site-Specific, Synthetic Methodology, Synthetic Strategies, Wide Spectrum, Biosynthesis, Article, Chemistry, Fluorescence Resonance Energy Transfer, Protein Engineering, Staining, Staining And Labeling,
Affiliations:
*** IBB - CNR ***
Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134, Napoli, Italy
Department of Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT-06511, United States
Dipartimento Delle Scienze Biologiche, Università di Napoli Federico II, via Mezzocannone 16, 80134, Napoli, Italy
IMDEA-Nanociencia, Centro Nacional de Biotecnología (CNB-CSIC), Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain
References:
Ratner, V., Kahana, E., Eichler, M., Haas, E., A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies (2002) Bioconjugate Chem., 13, pp. 1163-117
Kao, M.W.-P., Yang, L.-L., Lin, J.C.-K., Lim, T.-S., Fann, W., Chen, R.P.-Y., Strategy for efficient site-specific FRET-dye labeling of ubiquitin (2008) Bioconjugate Chemistry, 19 (6), pp. 1124-1126. , DOI 10.1021/bc700480j
Smith, J.J., Conrad, D.W., Cuneo, M.J., Hellinga, H.W., Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms (2005) Protein Science, 14 (1), pp. 64-73. , DOI 10.1110/ps.04965405
Jager, M., Michalet, X., Weiss, S., Protein-protein interactions as a tool for site-specific labeling of proteins (2005) Protein Science, 14 (8), pp. 2059-2068. , http://www.proteinscience.org/cgi/reprint/14/8/2059, DOI 10.1110/ps.051384705
Brustad, E.M., Lemke, E.A., Schultz, P.G., Deniz, A.A., A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (2008) J. Am. Chem. Soc., 130, pp. 17664-17665
Miyake-Stoner, S.J., Miller, A.M., Hammill, J.T., Peeler, J.C., Hess, K.R., Mehl, R.A., Brewer, S.H., Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan (2009) Biochemistry, 48, pp. 5953-5962
Antos, J.M., Chew, G.L., Guimaraes, C.P., Yoder, N.C., Grotenbreg, G.M., Popp, M.W., Ploegh, H.L., Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity (2009) J. Am. Chem. Soc., 131, pp. 10800-10801
Crochet, A.P., Kabir, M.M., Francis, M.B., Paavola, C.D., Site-selective dual modification of periplasmic binding proteins for sensing applications (2010) Biosens. Bioelectron., 26, pp. 55-61
Dawson, P.E., Muir, T.W., Clark-Lewis, I., Kent, S.B.H., Synthesis of proteins by native chemical ligation (1994) Science, 266 (5186), pp. 776-779
Muir, T.W., Sondhi, D., Cole, P.A., Expressed protein ligation: A general method for protein engineering (1998) Proceedings of the National Academy of Sciences of the United States of America, 95 (12), pp. 6705-6710. , DOI 10.1073/pnas.95.12.6705
Deniz, A.A., Laurence, T.A., Beligere, G.S., Dahan, M., Martin, A.B., Chemla, D.S., Dawson, P.E., Weiss, S., Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2 (2000) Proceedings of the National Academy of Sciences of the United States of America, 97 (10), pp. 5179-5184. , DOI 10.1073/pnas.090104997
Cotton, G.J., Muir, T.W., Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation (2000) Chemistry and Biology, 7 (4), pp. 253-261. , DOI 10.1016/S1074-5521(00)00100-9
Yang, J., Yang, W.Y., Site-specific two-color protein labeling for FRET studies using split inteins (2009) J. Am. Chem. Soc., 131, pp. 11644-11645
Kurpiers, T., Mootz, H.D., Regioselective cysteine bioconjugation by appending a labeled cystein tag to a protein by using protein splicing in trans (2007) Angewandte Chemie - International Edition, 46 (27), pp. 5234-5237. , DOI 10.1002/anie.200700719
Volkmann, G., Liu, X., Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein (2009) PLoS ONE 4, p. 12
Ludwig, C., Pfeiff, M., Linne, U., Mootz, H.D., Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing (2006) Angew. Chem., Int. Ed., 45, pp. 5218-5221
Main, E.R.G., Xiong, Y., Cocco, M.J., D'Andrea, L., Regan, L., Design of stable α-helical arrays from an idealized TPR motif (2003) Structure, 11 (5), pp. 497-508. , DOI 10.1016/S0969-2126(03)00076-5
D'Andrea, L.D., Regan, L., TPR proteins: The versatile helix (2003) Trends in Biochemical Sciences, 28 (12), pp. 655-662. , DOI 10.1016/j.tibs.2003.10.007
Warrens, A.N., Jones, M.D., Lechler, R.I., Splicing by over-lap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest (1997) Gene, 186 (1), pp. 29-35. , DOI 10.1016/S0378-1119(96)00674-9, PII S0378111996006749
Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.-Q., Perler, F.B., Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein (1999) BioTechniques, 27 (1), pp. 110-120
Peroza, E.A., Freisinger, E., Tris is a non-innocent buffer during intein-mediated protein cleavage (2008) Protein Expression and Purification, 57 (2), pp. 217-225. , DOI 10.1016/j.pep.2007.10.003, PII S1046592807002422
Shafer, D.E., Inman, J.K., Lees, A., Reaction of tris(2-carboxyethyl)phosphine (TCEP) with maleimide and α- haloacyl groups: Anomalous elution of TCEP by gel filtration (2000) Analytical Biochemistry, 282 (1), pp. 161-164. , DOI 10.1006/abio.2000.4609
Tyagarajan, K., Pretzer, E., Wiktorowicz, J.E., Thiol-reactive dyes for fluorescence labeling of proteomic samples (2003) Electrophoresis, 24 (14), pp. 2348-2358. , DOI 10.1002/elps.200305478
Kao, M. W. -P., Yang, L. -L., Lin, J. C. -K., Lim, T. -S., Fann, W., Chen, R. P. -Y., Strategy for efficient site-specific FRET-dye labeling of ubiquitin (2008) Bioconjugate Chemistry, 19 (6), pp. 1124-1126. , DOI 10. 1021/bc700480j
Smith, J. J., Conrad, D. W., Cuneo, M. J., Hellinga, H. W., Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms (2005) Protein Science, 14 (1), pp. 64-73. , DOI 10. 1110/ps. 04965405
Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A., A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (2008) J. Am. Chem. Soc., 130, pp. 17664-17665
Miyake-Stoner, S. J., Miller, A. M., Hammill, J. T., Peeler, J. C., Hess, K. R., Mehl, R. A., Brewer, S. H., Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan (2009) Biochemistry, 48, pp. 5953-5962
Antos, J. M., Chew, G. L., Guimaraes, C. P., Yoder, N. C., Grotenbreg, G. M., Popp, M. W., Ploegh, H. L., Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity (2009) J. Am. Chem. Soc., 131, pp. 10800-10801
Crochet, A. P., Kabir, M. M., Francis, M. B., Paavola, C. D., Site-selective dual modification of periplasmic binding proteins for sensing applications (2010) Biosens. Bioelectron., 26, pp. 55-61
Dawson, P. E., Muir, T. W., Clark-Lewis, I., Kent, S. B. H., Synthesis of proteins by native chemical ligation (1994) Science, 266 (5186), pp. 776-779
Muir, T. W., Sondhi, D., Cole, P. A., Expressed protein ligation: A general method for protein engineering (1998) Proceedings of the National Academy of Sciences of the United States of America, 95 (12), pp. 6705-6710. , DOI 10. 1073/pnas. 95. 12. 6705
Deniz, A. A., Laurence, T. A., Beligere, G. S., Dahan, M., Martin, A. B., Chemla, D. S., Dawson, P. E., Weiss, S., Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2 (2000) Proceedings of the National Academy of Sciences of the United States of America, 97 (10), pp. 5179-5184. , DOI 10. 1073/pnas. 090104997
Cotton, G. J., Muir, T. W., Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation (2000) Chemistry and Biology, 7 (4), pp. 253-261. , DOI 10. 1016/S1074-5521 (00) 00100-9
Yang, J., Yang, W. Y., Site-specific two-color protein labeling for FRET studies using split inteins (2009) J. Am. Chem. Soc., 131, pp. 11644-11645
Main, E. R. G., Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Design of stable -helical arrays from an idealized TPR motif (2003) Structure, 11 (5), pp. 497-508. , DOI 10. 1016/S0969-2126 (03) 00076-5
D'Andrea, L. D., Regan, L., TPR proteins: The versatile helix (2003) Trends in Biochemical Sciences, 28 (12), pp. 655-662. , DOI 10. 1016/j. tibs. 2003. 10. 007
Warrens, A. N., Jones, M. D., Lechler, R. I., Splicing by over-lap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest (1997) Gene, 186 (1), pp. 29-35. , DOI 10. 1016/S0378-1119 (96) 00674-9, PII S0378111996006749
Southworth, M. W., Amaya, K., Evans, T. C., Xu, M. -Q., Perler, F. B., Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein (1999) BioTechniques, 27 (1), pp. 110-120
Peroza, E. A., Freisinger, E., Tris is a non-innocent buffer during intein-mediated protein cleavage (2008) Protein Expression and Purification, 57 (2), pp. 217-225. , DOI 10. 1016/j. pep. 2007. 10. 003, PII S1046592807002422
Shafer, D. E., Inman, J. K., Lees, A., Reaction of tris (2-carboxyethyl) phosphine (TCEP) with maleimide and - haloacyl groups: Anomalous elution of TCEP by gel filtration (2000) Analytical Biochemistry, 282 (1), pp. 161-164. , DOI 10. 1006/abio. 2000. 4609
Org Biomol Chem (ISSN: 1477-0520, 1477-0539, 1477-0539electronic), 2012; 10(2): 273-280.
Export to BibTeX Export to EndNote
Paper type: Journal Article,
Impact factor: 3.568, 5-year impact factor: 3.49
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-83455214139&partnerID=40&md5=382816b5a59ec4243b242ccf47d74a7e
Keywords: Chemical Biology, Desired Position, Experimental Setup, Expressed Protein Ligation, High Purity, Indispensable Tools, Labeled Proteins, Life-Sciences, Molecular Probes, Peptide Synthesis, Protein Sequences, Single Molecule, Site-Specific, Synthetic Methodology, Synthetic Strategies, Wide Spectrum, Biosynthesis, Article, Chemistry, Fluorescence Resonance Energy Transfer, Protein Engineering, Staining, Staining And Labeling,
Affiliations:
*** IBB - CNR ***
Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134, Napoli, Italy
Department of Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT-06511, United States
Dipartimento Delle Scienze Biologiche, Università di Napoli Federico II, via Mezzocannone 16, 80134, Napoli, Italy
IMDEA-Nanociencia, Centro Nacional de Biotecnología (CNB-CSIC), Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain
References:
Ratner, V., Kahana, E., Eichler, M., Haas, E., A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies (2002) Bioconjugate Chem., 13, pp. 1163-117
Kao, M.W.-P., Yang, L.-L., Lin, J.C.-K., Lim, T.-S., Fann, W., Chen, R.P.-Y., Strategy for efficient site-specific FRET-dye labeling of ubiquitin (2008) Bioconjugate Chemistry, 19 (6), pp. 1124-1126. , DOI 10.1021/bc700480j
Smith, J.J., Conrad, D.W., Cuneo, M.J., Hellinga, H.W., Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms (2005) Protein Science, 14 (1), pp. 64-73. , DOI 10.1110/ps.04965405
Jager, M., Michalet, X., Weiss, S., Protein-protein interactions as a tool for site-specific labeling of proteins (2005) Protein Science, 14 (8), pp. 2059-2068. , http://www.proteinscience.org/cgi/reprint/14/8/2059, DOI 10.1110/ps.051384705
Brustad, E.M., Lemke, E.A., Schultz, P.G., Deniz, A.A., A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (2008) J. Am. Chem. Soc., 130, pp. 17664-17665
Miyake-Stoner, S.J., Miller, A.M., Hammill, J.T., Peeler, J.C., Hess, K.R., Mehl, R.A., Brewer, S.H., Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan (2009) Biochemistry, 48, pp. 5953-5962
Antos, J.M., Chew, G.L., Guimaraes, C.P., Yoder, N.C., Grotenbreg, G.M., Popp, M.W., Ploegh, H.L., Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity (2009) J. Am. Chem. Soc., 131, pp. 10800-10801
Crochet, A.P., Kabir, M.M., Francis, M.B., Paavola, C.D., Site-selective dual modification of periplasmic binding proteins for sensing applications (2010) Biosens. Bioelectron., 26, pp. 55-61
Dawson, P.E., Muir, T.W., Clark-Lewis, I., Kent, S.B.H., Synthesis of proteins by native chemical ligation (1994) Science, 266 (5186), pp. 776-779
Muir, T.W., Sondhi, D., Cole, P.A., Expressed protein ligation: A general method for protein engineering (1998) Proceedings of the National Academy of Sciences of the United States of America, 95 (12), pp. 6705-6710. , DOI 10.1073/pnas.95.12.6705
Deniz, A.A., Laurence, T.A., Beligere, G.S., Dahan, M., Martin, A.B., Chemla, D.S., Dawson, P.E., Weiss, S., Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2 (2000) Proceedings of the National Academy of Sciences of the United States of America, 97 (10), pp. 5179-5184. , DOI 10.1073/pnas.090104997
Cotton, G.J., Muir, T.W., Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation (2000) Chemistry and Biology, 7 (4), pp. 253-261. , DOI 10.1016/S1074-5521(00)00100-9
Yang, J., Yang, W.Y., Site-specific two-color protein labeling for FRET studies using split inteins (2009) J. Am. Chem. Soc., 131, pp. 11644-11645
Kurpiers, T., Mootz, H.D., Regioselective cysteine bioconjugation by appending a labeled cystein tag to a protein by using protein splicing in trans (2007) Angewandte Chemie - International Edition, 46 (27), pp. 5234-5237. , DOI 10.1002/anie.200700719
Volkmann, G., Liu, X., Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein (2009) PLoS ONE 4, p. 12
Ludwig, C., Pfeiff, M., Linne, U., Mootz, H.D., Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing (2006) Angew. Chem., Int. Ed., 45, pp. 5218-5221
Main, E.R.G., Xiong, Y., Cocco, M.J., D'Andrea, L., Regan, L., Design of stable α-helical arrays from an idealized TPR motif (2003) Structure, 11 (5), pp. 497-508. , DOI 10.1016/S0969-2126(03)00076-5
D'Andrea, L.D., Regan, L., TPR proteins: The versatile helix (2003) Trends in Biochemical Sciences, 28 (12), pp. 655-662. , DOI 10.1016/j.tibs.2003.10.007
Warrens, A.N., Jones, M.D., Lechler, R.I., Splicing by over-lap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest (1997) Gene, 186 (1), pp. 29-35. , DOI 10.1016/S0378-1119(96)00674-9, PII S0378111996006749
Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.-Q., Perler, F.B., Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein (1999) BioTechniques, 27 (1), pp. 110-120
Peroza, E.A., Freisinger, E., Tris is a non-innocent buffer during intein-mediated protein cleavage (2008) Protein Expression and Purification, 57 (2), pp. 217-225. , DOI 10.1016/j.pep.2007.10.003, PII S1046592807002422
Shafer, D.E., Inman, J.K., Lees, A., Reaction of tris(2-carboxyethyl)phosphine (TCEP) with maleimide and α- haloacyl groups: Anomalous elution of TCEP by gel filtration (2000) Analytical Biochemistry, 282 (1), pp. 161-164. , DOI 10.1006/abio.2000.4609
Tyagarajan, K., Pretzer, E., Wiktorowicz, J.E., Thiol-reactive dyes for fluorescence labeling of proteomic samples (2003) Electrophoresis, 24 (14), pp. 2348-2358. , DOI 10.1002/elps.200305478
Kao, M. W. -P., Yang, L. -L., Lin, J. C. -K., Lim, T. -S., Fann, W., Chen, R. P. -Y., Strategy for efficient site-specific FRET-dye labeling of ubiquitin (2008) Bioconjugate Chemistry, 19 (6), pp. 1124-1126. , DOI 10. 1021/bc700480j
Smith, J. J., Conrad, D. W., Cuneo, M. J., Hellinga, H. W., Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms (2005) Protein Science, 14 (1), pp. 64-73. , DOI 10. 1110/ps. 04965405
Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A., A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (2008) J. Am. Chem. Soc., 130, pp. 17664-17665
Miyake-Stoner, S. J., Miller, A. M., Hammill, J. T., Peeler, J. C., Hess, K. R., Mehl, R. A., Brewer, S. H., Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan (2009) Biochemistry, 48, pp. 5953-5962
Antos, J. M., Chew, G. L., Guimaraes, C. P., Yoder, N. C., Grotenbreg, G. M., Popp, M. W., Ploegh, H. L., Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity (2009) J. Am. Chem. Soc., 131, pp. 10800-10801
Crochet, A. P., Kabir, M. M., Francis, M. B., Paavola, C. D., Site-selective dual modification of periplasmic binding proteins for sensing applications (2010) Biosens. Bioelectron., 26, pp. 55-61
Dawson, P. E., Muir, T. W., Clark-Lewis, I., Kent, S. B. H., Synthesis of proteins by native chemical ligation (1994) Science, 266 (5186), pp. 776-779
Muir, T. W., Sondhi, D., Cole, P. A., Expressed protein ligation: A general method for protein engineering (1998) Proceedings of the National Academy of Sciences of the United States of America, 95 (12), pp. 6705-6710. , DOI 10. 1073/pnas. 95. 12. 6705
Deniz, A. A., Laurence, T. A., Beligere, G. S., Dahan, M., Martin, A. B., Chemla, D. S., Dawson, P. E., Weiss, S., Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2 (2000) Proceedings of the National Academy of Sciences of the United States of America, 97 (10), pp. 5179-5184. , DOI 10. 1073/pnas. 090104997
Cotton, G. J., Muir, T. W., Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation (2000) Chemistry and Biology, 7 (4), pp. 253-261. , DOI 10. 1016/S1074-5521 (00) 00100-9
Yang, J., Yang, W. Y., Site-specific two-color protein labeling for FRET studies using split inteins (2009) J. Am. Chem. Soc., 131, pp. 11644-11645
Main, E. R. G., Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Design of stable -helical arrays from an idealized TPR motif (2003) Structure, 11 (5), pp. 497-508. , DOI 10. 1016/S0969-2126 (03) 00076-5
D'Andrea, L. D., Regan, L., TPR proteins: The versatile helix (2003) Trends in Biochemical Sciences, 28 (12), pp. 655-662. , DOI 10. 1016/j. tibs. 2003. 10. 007
Warrens, A. N., Jones, M. D., Lechler, R. I., Splicing by over-lap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest (1997) Gene, 186 (1), pp. 29-35. , DOI 10. 1016/S0378-1119 (96) 00674-9, PII S0378111996006749
Southworth, M. W., Amaya, K., Evans, T. C., Xu, M. -Q., Perler, F. B., Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein (1999) BioTechniques, 27 (1), pp. 110-120
Peroza, E. A., Freisinger, E., Tris is a non-innocent buffer during intein-mediated protein cleavage (2008) Protein Expression and Purification, 57 (2), pp. 217-225. , DOI 10. 1016/j. pep. 2007. 10. 003, PII S1046592807002422
Shafer, D. E., Inman, J. K., Lees, A., Reaction of tris (2-carboxyethyl) phosphine (TCEP) with maleimide and - haloacyl groups: Anomalous elution of TCEP by gel filtration (2000) Analytical Biochemistry, 282 (1), pp. 161-164. , DOI 10. 1006/abio. 2000. 4609
Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
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. © 2012 The Royal Society of Chemistry.
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. © 2012 The Royal Society of Chemistry.
Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
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Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins
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3 Records (3 excluding Abstracts).
Total impact factor: 11.598 (11.598 excluding Abstracts).
Total 5 year impact factor: 7.38 (7.38 excluding Abstracts).
Total impact factor: 11.598 (11.598 excluding Abstracts).
Total 5 year impact factor: 7.38 (7.38 excluding Abstracts).
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