Abstract
In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.
Highlights
We demonstrate that representative model proteins, including membrane proteins, proteins with posttranslational modifications and cytosolic proteins, can be produced in the novel eukaryotic continuous-exchange cell-free (CECF) system with significantly increased protein yields compared to batch-based reactions
Translation efficiency was evaluated by the measurement of protein fluorescence intensity in translation reactions. These initial experiments resulted in a twofold increase in fluorescence intensity of eYFP synthesized in the insect CECF mode compared to the batch mode after 24 h of incubation
EYFP was expressed in insect lysate containing endoplasmic reticulum-derived vesicles as well as in lysate which was depleted of endogenous vesicles prior to its use in the cell-free reaction
Summary
Cell-free methods have proven themselves as a valuable platform allowing the synthesis of many different protein classes including membrane proteins [1,2,3,4,5,6,7], proteins with posttranslational modifications [8,9,10,11,12,13,14] and even toxic proteins [15,16,17]. Expression in E. coli and wheat germ systems has their limitations when it comes to the synthesis of complex proteins and proteins which require co-translational and posttranslational modifications [9,22]. Covalent posttranslational modifications such as glycosylation and disulfide bond formation are very common among eukaryotic proteins and it is well-known that they have a great impact on protein folding, localization and activity [23]. It is of highest interest to develop cell-free translation systems that ensure the formation of posttranslational modifications while providing a sufficient amount of protein for further functional and structural analysis
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