Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites

生物结合 化学 生物正交化学 氨酰tRNA合成酶 化学生物学 氨基酸 蛋白质工程 转移RNA 生物化学 计算生物学 组合化学 核糖核酸 点击化学 生物 基因
作者
James S. Italia,Partha Sarathi Addy,Sarah B. Erickson,Jennifer C. Peeler,Eranthie Weerapana,Abhishek Chatterjee
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:141 (15): 6204-6212 被引量:95
标识
DOI:10.1021/jacs.8b12954
摘要

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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