Biosynthesis and Genetic Encoding of Phosphothreonine Through Parallel Selection and Deep Sequencing

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2017 May 30

PMID 28553966

Citations 80

Authors

Michael Shaofei Zhang

Simon F Brunner

Nicolas Huguenin-Dezot

Alexandria D Liang

Wolfgang H Schmied

Daniel T Rogerson

Jason W Chin

Affiliations

Soon will be listed here.

Abstract

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel positive selections combined with deep sequencing and statistical analysis and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNA pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure determination and synthetic protein kinase activation.

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