Abstract
Cell-free DNA-dependent in vitro transcription/translation is a well-established procedure when working with the expression of circular closed DNA and with long linear DNA. Attempts of expression from short pieces of linear DNA were only partially successful. The smaller the DNA used, the more difficult it was to produce relevant amounts of protein. It was shown that these difficulties mainly resulted from the presence of exonucleases. During in vitro transcription and translation with S30 lysates from Escherichia coli, it was demonstrated that exonuclease V was responsible for the degradation of linear DNA. Exonuclease V consists of three subunits (the gene products of recB, recC, recD). This exonuclease cleaves linear DNA from its 3′-ends.
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