Elise D Ficaretta, Soumya Jyoti Singha Roy, Lena Voss, Abhishek Chatterjee
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引用次数: 0
Abstract
Site-specific noncanonical amino acid (ncAA) mutagenesis in living cells has traditionally relied on heterologous, nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that do not cross-react with their endogenous counterparts. Such heterologous pairs often perform suboptimally in a foreign host cell since they were not evolutionarily optimized to function in the foreign environment. This suboptimal performance restricts the number of ncAAs that can be simultaneously incorporated into a protein. Here, we show that the use of an endogenous aaRS/tRNA pair to drive ncAA incorporation can offer a potential solution to this limitation. To this end, we developed an engineered Escherichia coli strain (ATMY-C321), wherein the endogenous tyrosyl-tRNA synthetase (TyrRS)/tRNA pair has been functionally replaced with an archaeal counterpart, and the release factor 1 has been removed to eliminate competing termination at the UAG nonsense codons. The endogenous TyrRS/tRNACUATyr pair exhibits remarkably efficient nonsense suppression in the resulting cell, relative to established orthogonal ncAA-incorporation systems in E. coli, allowing the incorporation of an ncAA at up to 10 contiguous sites in a reporter protein. Our work highlights the limitations of orthogonal translation systems using heterologous aaRS/tRNA pairs and offers a potential alternative involving the use of endogenous pairs.
期刊介绍:
ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology.
The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies.
We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.