Evolved Orthogonal Ribosome Purification for in Vitro Characterization

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Feb 27

PMID 20185573

Citations 11

Authors

Oliver P T Barrett

Jason W Chin

Affiliations

Soon will be listed here.

Abstract

We developed orthogonal ribosome-mRNA pairs in which the orthogonal ribosome (O-ribosome) specifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, they have been used to provide new information about the ribosome, and they form a crucial part of foundational technologies for genetic code expansion and encoded and evolvable polymer synthesis in cells. The production of O-ribosomes in the cell makes it challenging to study the properties of O-ribosomes in vitro, because no method exists to purify functional O-ribosomes from cellular ribosomes and other cellular components. Here we present a method for the affinity purification of O-ribosomes, via tagging of the orthogonal 16S ribosomal RNA. We demonstrate that the purified O-ribosomes are pure by primer extension assays, and structurally homogenous by gel electrophoresis and sucrose gradients. We demonstrate the utility of this purification method by providing a preliminary in vitro characterization of Ribo-X, an O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to amber codons. Our data suggest that the basis of Ribo-X's in vivo activity is a decreased affinity for RF1.

Citing Articles

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Direct measurements of mRNA translation kinetics in living cells.

Metelev M, Lundin E, Volkov I, Gynna A, Elf J, Johansson M Nat Commun. 2022; 13(1):1852.

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Strategies for in vitro engineering of the translation machinery.

Hammerling M, Kruger A, Jewett M Nucleic Acids Res. 2019; 48(3):1068-1083.

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Controlling orthogonal ribosome subunit interactions enables evolution of new function.

Schmied W, Tnimov Z, Uttamapinant C, Rae C, Fried S, Chin J Nature. 2018; 564(7736):444-448.

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Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

Gan R, Perez J, Carlson E, Ntai I, Isaacs F, Kelleher N Biotechnol Bioeng. 2016; 114(5):1074-1086.

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