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Methods Enzymol. 2014;539:3-15. doi: 10.1016/B978-0-12-420120-0.00001-3.

In vitro synthesis of proteins in bacterial extracts.

Author information

1
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
2
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: ragreen@jhmi.edu.

Abstract

This protocol describes the methods used to generate protein in a cell-free system derived from E. coli. The in vitro synthesis of protein has been used in studying many ribosome-based gene regulation steps (Gong and Yanofsky, 2001). Such techniques have also been used to study protein-protein, protein-DNA, and protein-RNA interactions, and to produce radiolabeled protein species. More recently, such approaches have been utilized to produce large quantities of toxic proteins for proteomic and structural studies (Yokoyama et al., 2000).

KEYWORDS:

Cell-free protein synthesis; Centrifugation; E. coli; French press; Growth and harvest E. coli; In vitro protein synthesis; Optimization of coupled transcription and translation reaction; S30 preparation; SDS-PAGE gel; Solutions and buffers; TCA precipitation

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