Site-Specific Chemoselective Pyrrolysine Analogues Incorporation Using the Cell-Free Protein Synthesis System.

Cell-free protein synthesis (CFPS) is a fast and convenient way to synthesize proteins for analytical studies and applications. CFPS, when equipped with a suitable orthogonal pair, allows for protein-site-directed labeling with desired functionalities such as fluorescent dyes or therapeutic groups that are needed to tailor proteins for analytical applications. In this context, chemoselective reactive pyrrolysine analogues (CR-OAs) are of particular value, as this class of unnatural amino acids, among other useful properties, covers a wide range of different chemoselective reactions.

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing.

G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes.

Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems.

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system.

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets.

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates.

Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes.

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase.