A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation

Abstract

Biochemical investigation of protein phosphorylation events is limited by inefficient production of the phosphorylated and non-phosphorylated forms of full-length proteins. Here, using a genomically recoded strain of E. coli with a flexible UAG codon we produce site-specific serine- or phosphoserine-containing proteins, with purities approaching 90%, from a single recombinant DNA. Specifically, we synthesize human MEK1 kinase with two serines or two phosphoserines, from one DNA template, and demonstrate programmable kinase activity. Programmable protein phosphorylation is poised to help reveal the structural and functional information encoded in the phosphoproteome.

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

“…Our goal in this study was not to necessarily maximize the yield of phosphoprotein. We [17] (Table S1) and others [11,22,26] have already produced phosphoproteins in sufficient amounts for biochemical and structural studies. Rather, we were concerned with demonstrating unambiguously and in a more rigorous fashion than previous reports that we indeed obtained pure phosphoprotein.…”

“…1). Although the RF1-deficient E. coli produces phosphoprotein efficiently per cell, contamination with natural amino acids impedes pure phosphoprotein synthesis [17,22].…”

The ABCs of PTMs

“…2a). For instance, phosphorylation within an activation loop can act as an on/off switch for protein kinases, and phosphoserine incorporation as an ncAA can yield constitutively active kinase preparations 9,14 . Nonstandard amino acid incorporation can also be used to prepare recombinant modified proteins for crystallization.…”

The central role of tRNA in genetic code expansion

“…Although in some cases expressing o-tRNAs from several gene copies increases the ncAA-containing protein yield [20], endoribonucleases RNase E and RNase III may be required to cleave long precursors into smaller pieces prior to RNase P processing [18]. Thus, an increased in transcribed tRNA in the cell and the number of properly processed molecules may not correlate accordingly [21]. Both RNase E and RNase III have broad specificities and process the 3′ ends of the precursors leaving a few extra bases downstream the CCA end [18].…”