Abstract 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 developing a cell-free synthesis (CFPS) platform that employs crude extracts from genomically recoded strain Escherichia coli for site-specific, co-translational incorporation phosphoserine into proteins. We apply system robust production up milligram quantities human MEK1...

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness transcriptional and translational capacity of cells within test tube. By obviating need maintain viability cell, by eliminating cellular barrier, CFPS been foundational emerging applications in biomanufacturing traditionally challenging proteins, well rapid prototyping for metabolic engineering, functional genomics. Our methods implementing an E. coli-based platform allow new users...

ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and required for full virulence an animal model acute pneumonia. It comprised N-terminal domain with GTPase activating (GAP) activity towards Rho family GTPases C-terminal ADP ribosyltransferase (ADPRT) minimal synthetic substrate vitro. Consistent its as GTPase, has been shown to inhibit P. internalization into epithelial cells macrophages, disrupt the actin cytoskeleton through Rho-dependent...

Cell-free protein synthesis (CFPS) is a platform biotechnology that enables breadth of applications. However, field applications remain limited due to the poor shelf-stability aqueous cell extracts required for CFPS. Lyophilization E. coli improves shelf life but remains insufficient extended storage at room temperature. To address this limitation, we mapped chemical space ten low-cost additives with four distinct mechanisms action in combinatorial manner identify formulations capable...

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, and education. The Escherichia coli cell extract-based platform has been broadly adopted due its affordability versatility. upstream processing of cells generate crude lysate remains time-intensive technically nuanced, representing one the largest sources cost associated with biotechnology. To overcome these limitations, we improved...

Cell-free protein synthesis (CFPS) is an

Teaching the processes of transcription and translation is challenging due to intangibility these concepts a lack instructional, laboratory-based, active learning modules. Harnessing genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for direct manipulation reaction conditions biological machinery enable inquiry-based learning. Here, we report our efforts transform research-based CFPS biotechnology into hands-on module called "Genetic Code...

Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand of proteins for variety applications. Numerous advances have improved productivity CFPS to result in high-yielding reactions; however, many applications remain limited due long reaction times. To overcome this limitation, we first established benchmarks times across in-house E. coli extracts and commercial kits. We then set out fine-tune our extract systems improve Through optimization composition...

DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways gram‐negative bacteria, and specifically regulating the expression of virulence genes several organisms. Actinobacillus actinomycetemcomitans an important role pathogenesis juvenile adult periodontal disease, yet little is known about its mechanisms gene regulation. DAM shown here to directly or indirectly affect well‐known A. factors. A mutant strain lacking dam was created by homologous recombination...

The fragility of biological systems during storage, transport, and utilization necessitates reliable cold-chain infrastructure limits the potential biotechnological applications. In order to unlock broad applications existing emerging technologies, we report development a novel solid-state storage platform for complex biologics. resulting biologics (SSB) meets four key requirements: facile rehydration solid materials, activation biochemical activity, ability support downstream...

Salmonella enterica serovar Typhimurium was previously shown to be virulent in Caenorhabditis elegans. Here we demonstrate that DNA adenine methyltransferase (DAM) modulates virulence the nematode, as it does mice. After 5 days of continual exposure bacteria, twice many worms died when exposed wild-type than dam-mutant strain Salmonella. Similar trends were observed strains for h and transferred avirulent Escherichia coli OP50. While a 10-fold attenuation absence DAM, dam-strain still able...

Hydrogenotrophic methanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that two inactive Methanocaldococcus jannaschii, HmdII and HmdIII, form binary ternary complexes with several components translation apparatus. but not active Hmd, bind micromolar binding affinities to a number tRNAs tRNA(Pro) prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments suggest...

Fluorescent reporters have revolutionized modern applications in the fields of molecular and synthetic biology, enabling ranging from education to point-of-care diagnostics. Past advancements these primarily focused on improving reaction conditions, development new applications, broad dissemination technologies. However, field classroom-based remained limited part due nature fluorescent signal detection, which often requires use costly lab equipment observe quantify fluorescence readouts....

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS more accessible to new users expanded range of applications. For lysate based systems, cell extracts can be generated from variety organisms, harnessing unique biochemistry host augment synthesis. Within last 20 years, Escherichia coli (E. coli) become one most widely used organisms for supporting due its...

Abstract Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point-of-care detection, therapeutics, and education using affordable laboratory equipment reagents. The Escherichia coli cell extract-based platform, being one of the most versatile CFPS platforms, has been broadly adopted. In spite promise simplicity, cell-free platform remains technically nuanced, posing challenges reproducible...

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness transcriptional and translational capacity of cells within test tube. By obviating need maintain viability cell, by eliminating cellular barrier, CFPS been foundational emerging applications in biomanufacturing traditionally challenging proteins, well rapid prototyping for metabolic engineering, functional genomics. Our methods implementing an E. coli-based platform allow new users...

MEK1 is a protein kinase in the MAPK cellular signaling pathway that notable for its dual specificity and potential as drug target variety of cancer therapies. While much known about key role events, understanding structural features sustain function remains limited because absence crystal or NMR insights into phosphorylated activated form MEK1. In this work, homology modeling was used to overcome limitation generate computational models doubly active conformation. A were generated using...

We cloned and sequenced the DNA adenine-N6 methyltransferase gene of human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that enzyme methylates adenine in sequence GATC. Expression a DAM− background vivo activity. A PSI-BLAST search revealed M.AacDAM is most related to M.HindIV, M.EcoDAM, M.StyDAM, M.SmaII. The ClustalW alignment highly conserved regions characteristic for type MTases. Phylogenetic tree analysis cluster enzymes recognizing GATC, within...