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Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites.
Italia, James S; Addy, Partha Sarathi; Erickson, Sarah B; Peeler, Jennifer C; Weerapana, Eranthie; Chatterjee, Abhishek.
Afiliación
  • Italia JS; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
  • Addy PS; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
  • Erickson SB; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
  • Peeler JC; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
  • Weerapana E; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
  • Chatterjee A; Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.
J Am Chem Soc ; 141(15): 6204-6212, 2019 04 17.
Article en En | MEDLINE | ID: mdl-30909694
Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN de Transferencia / Proteínas de Escherichia coli / Escherichia coli / Aminoácidos / Aminoacil-ARNt Sintetasas Idioma: En Revista: J Am Chem Soc Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN de Transferencia / Proteínas de Escherichia coli / Escherichia coli / Aminoácidos / Aminoacil-ARNt Sintetasas Idioma: En Revista: J Am Chem Soc Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos