Analysis of the expression of cloned genes using an Escherichia coli cell-free system

Abstract

An Escherichia coli coupled transcription–translation cell-free system, which is efficient in the synthesis of proteins directed by exogenously added DNA, is described. These cell-free extracts direct protein synthesis against a low background of endogenous protein synthesis providing a means for analyzing the expression of isolated genes. This is especially important when using restriction enzyme-linearized DNAs which are less efficient templates than circular DNAs. This cell-free system has been used to study the expression of the proteins coded by plasmids pBR322 and pBL101.