Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Abstract

: The main goal of cell-free protein synthesis is to produce correctly folded and functional proteins in reasonable amounts for further downstream applications. Especially for eukaryotic proteins, functionality is often directly linked to the presence of posttranslational modifications. Thus, it is of highest interest to develop novel cell-free expression systems that enable the synthesis of posttranslationally modified proteins. Here we present recent advances for the synthesis of glycoproteins, proteins containing disulfide bridges, membrane proteins, and fluorescently labeled proteins. The basis for the expression of these difficult-to-express target proteins is a translationally active cell extract which can be prepared from eukaryotic cell lines such as Spodoptera frugiperda 21 (Sf21) and Chinese hamster ovary (CHO) cells. Due to a very mild lysate preparation procedure, microsomal vesicles derived from the endoplasmic reticulum (ER) can be maintained in the eukaryotic lysate. These vesicles are translocationally active and serve as functional modules facilitating protein translocation and enrichment as well as posttranslational modification of de novo synthesized proteins. In particular, for the synthesis of membrane proteins microsomal vesicles are the essential prerequisite for the insertion of the desired protein into a biologically active membrane scaffold providing a natural environment. We anticipate that the use of such translationally active eukaryotic cell lysates containing translocationally active vesicles may solve a large number of problems still persistent when expressing eukaryotic proteins in vitro.

Automated production of functional membrane proteins using eukaryotic cell-free translation systems

“…The determination of the optimal concentration of the previously described E. coli-derived mutant synthetase eAzFRS (Chin et al, 2003;Takimoto et al, 2009) in combination with a natural amber suppressor tRNA CUA from E. coli (Edwards and Schimmel, 1990) used in this study, was performed stochastically in batch-formatted reactions (data not shown). Based on earlier findings which indicated a significant influence of the cation concentration on the yield of cell-free synthesized proteins with incorporated AzF (Stech et al, 2013), we applied the automated workflow to an amber suppression screening varying the Mg 2+ concentration. Moreover, we also implemented a second dimension screening procedure using an iterative development process at different insect cell lysate concentrations.…”

“…On the one hand, fluorescent amino acid derivatives such as BODIPY-TMR-lysine can be directly incorporated into proteins in a statistical manner by sense codon reassignment using pre-charged tRNAs which allows for a sensitive detection of synthesized proteins . On the other hand, the site-directed incorporation of p-Azido-l-phenylalanine (AzF) based on amber suppression, mediated by an orthogonal tRNA/synthetase pair derived from E. coli, enabled us to fluorescently modify cell-free synthesized human erythropoietin in a selective manner by Staudinger ligation (Stech et al, 2013;Quast et al, 2014). The employment of amino acids with selective reactivity facilitates a subsequent modification of proteins to introduce a variety of different characteristics.…”

Cell-Free Synthesis Meets Antibody Production: A Review

“…Thus, protein synthesis is disconnected from cell fate and the protein synthesis reaction itself is not constrained by a cell wall, meaning that reaction conditions are accessible from the outside of the reaction vessel [5,7,42].…”

“…These features are made possible as the cell extract contains translationally active, endogenous microsomal vesicles having their origin in the ER of the insect cells. These vesicles are formed during the lysate preparation procedure, where the native structure of the ER is ripped, followed by its rearrangement and formation of smaller compartments designated as microsomal vesicles [5]. Due to the presence of these vesicles, target proteins can be translocated into the lumen of these vesicles, if they are fused to an appropriate signal sequence [65].…”

“…However, a cell-based production of target proteins is known to be rather time-consuming and cost-intensive, thus resulting in the development of efficient and powerful cell-free protein synthesis systems. Over the last decade, cellfree systems have proven themselves as reliable, flexible and potent protein production platform, providing proteins of varying origins, classes and properties [3][4][5][6][7]. In particular, cell-free systems have been successfully used for the synthesis of antibodies and antibody fragments [8][9][10].…”