Cell-free synthesis of proteins with selectively 13C-labelled methyl groups from inexpensive precursors.

The novel eCell system maintains the activity of the entire repertoire of metabolic Escherichia coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively 13C-labelled amino acids from inexpensive 13C-labelled precursors. The system is demonstrated with selective 13C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis-trans isomerase B. Starting from 3-13C-pyruvate, 13C-HSQC cross-peaks are obtained devoid of one-bond 13C-13C scalar couplings. Starting from 2-13C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows us to produce perdeuterated proteins with protonated methyl groups in a residue-selective manner. The system uses the isotope-labelled precursors sparingly and is readily scalable.

eCell Technology for Cell-Free Protein Synthesis, Biosensing, and Remediation.

The eCell technology is a recently introduced, specialized protein production platform with uses in a multitude of biotechnological applications. This chapter summarizes the use of eCell technology in four selected application areas. Firstly, for detecting heavy metal ions, specifically mercury, in an in vitro protein expression system. Results show improved sensitivity and lower limit of detection compared to comparable in vivo systems. Secondly, eCells are semipermeable, stable, and can be stored for extended periods of time, making them a portable and accessible technology for bioremediation of toxicants in extreme environments. Thirdly and fourthly, applications of eCell technology are shown to facilitate expression of correctly folded disulfide-rich proteins and incorporate chemically interesting derivatives of amino acids into proteins which are toxic to in vivo protein expression. Overall, eCell technology presents a cost-effective and efficient method for biosensing, bioremediation, and protein production.

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

In Vitro Protein Synthesis in Semipermeable Artificial Cells.

A novel cell free protein synthesis (CFPS) system utilizing layer-by-layer (LbL) polymer assembly was developed to reduce the operational cost of conventional CFPS. This yielded an encapsulated cell system, dubbed "eCells", that successfully performs in vitro CFPS and allows cost-effective incorporation of noncanonical amino acids into proteins. The use of eCells in CFPS circumvents the need for traditional cell lysate preparation and purification of amino acyl-tRNA synthetases (aaRS) while still retaining the small scale of an in vitro reaction. eCells were found to be 55% as productive as standard dialysis CFPS at 13% of the cost. The reaction was shown to be scalable over a large range of reaction volumes, and the crowding environment in eCells confers a stabilizing effect on marginally stable proteins, such as the pyrrolysl tRNA synthetase (PylRS), providing a means for their application in in vitro protein expression. Photocaged-cysteine (PCC) and Nε-(tert-butoxycarbonyl)-l-lysine (Boc-lysine) were incorporated into Peptidyl-prolyl cis-trans isomerase B (PpiB) using small amounts of ncAA with an adequate yield of protein. Fluorescent activated cell sorting (FACS) was used to demonstrate the partition of the lysate within the eCells in contrast to standard one pot cell lysate-based methods.