System-wide optimization of an orthogonal translation system with enhanced biological tolerance.

Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically relevant non-standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post-translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems-level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system-wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS-mediated host toxicity.

Revealing the amino acid composition of proteins within an expanded genetic code.

The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes.

Cell type-independent profiling of interactions between intracellular pathogens and the human phosphoproteome.

Interactions between proteins from intracellular pathogens and host proteins in an infected cell are often mediated by post-translational modifications encoded in the host proteome. Identifying protein modifications, such as phosphorylation, that dictate these interactions remains a defining challenge in unraveling the molecular mechanisms of pathogenesis. We have developed a platform in engineered bacteria that displays over 110,000 phosphorylated human proteins coupled to a fluorescent reporter system capable of identifying the host-pathogen interactome of phosphoproteins (H-PIP). This resource broadly enables cell-type independent interrogation and discovery of proteins from intracellular pathogens capable of binding phosphorylated human proteins. As an example of the H-PIP platform, we generated a unique, high-resolution SARS-CoV-2 interaction network which expanded our knowledge of viral protein function and identified understudied areas of host pathology.

Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine.

Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions.

Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. We developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. We genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. We designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. We then integrated our phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, we found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

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

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.

Robust production of recombinant phosphoproteins using cell-free protein synthesis.

Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signalling networks and kinase inhibitor drugs.

Expanded cellular amino acid pools containing phosphoserine, phosphothreonine, and phosphotyrosine.

Adding nonstandard amino acids to the genetic code of E. coli expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular phosphoamino acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in E. coli. We show that deletion of the phosphoserine phosphatase serB elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and serB deficient cells were grown in low phosphate media with supplemented phosphotyrosine, respectively. These findings remove a major barrier for further expansion of the genetic code with additional phosphorylated amino acids.

Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion.

Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151). However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.