A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

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.

Cell-Free Synthesis Meets Antibody Production: A Review

“…Until now, eukaryotic cell-free translation systems based on insect cell extracts, human cell extracts and lysates from CHO cells could not reach such high production yields, although significant improvements have been made in recent years [68,110,111]. Nevertheless, for the future we anticipate an increase in productivity as more advanced reaction formats [68] can be combined with an improved template design [111].…”

“…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]. In addition, the de novo synthesis of membrane proteins and their subsequent embedding into the vesicular membrane of the insect vesicles have been reported [66][67][68]. …”

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

“…The difference becomes even more dramatic, when productivities are considered (Table 3), i.e. the continuous exchange cell-free system (CECF) [52][53][54], which in principle could be adapted to CHO lysates and would allow a scale up of the production, the high DNA consumption remains an unsolved problem. The performance of the cell-free system thus approaches overall productivities of bacterial hosts, where the active protein has to be renatured from inclusion bodies [51], but with full post-translational processing.…”

“…Established eukaryotic systems include yeast [18], wheat germ [19,20], insect [21,22], tobacco [23] and mammalian [24,25] cell lysates. The wheat germ system is highly popular due to its high yields, but limited in regard to the correct post-translational modification of human proteins.…”

Addressing biological uncertainties in engineering gene circuits

“…This dependence has spurred research into engineering cell-free systems. Cell free systems utilize enzymes or metabolites isolated from microorganisms to simulate cellular reactions [52, 53]. They also serve as the foundation for efforts to develop minimal cells – encapsulated cell-free gene expression systems.…”