A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells

PLoS One. 2014 May 7;9(5):e96635. doi: 10.1371/journal.pone.0096635. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell-Free System
  • Cells, Cultured
  • Insecta
  • Protein Biosynthesis / physiology*
  • Proteins / analysis*

Substances

  • Proteins

Grant support

This research is supported by the German Ministry of Education and Research (BMBF, No. 0315942 and No. 0312039). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.