An over-expression plasmid for Escherichia coli primase

Abstract

To facilitate the overexpression of Escherichia coli primase, the dnaG gene has been reconstructed using polymerase chain reaction to remove the 5' transcription terminator and the 3' RNA processing site. This construct was cloned into the T7 polymerase-transcribed expression vector, pET-3d. Cells containing the resulting plasmid (pGNG1) express up to 30% of the cellular protein as primase. The pGNG1-encoded primase has normal activity in synthesizing primer RNA on a single-stranded DNA template in vitro. Plasmid pGNG1 can also be used to synthesize [35S]methionine-labelled primase in in vitro transcription-translation systems. In addition, the small amount of transcription in the absence of T7 polymerase is sufficient to complement temperature-sensitive and amber dnaG chromosomal mutations in vivo. Plasmid pGNG1 can therefore be used not only to overproduce wild-type primase, but to change and manipulate the primase structure in vivo and in vitro. These mutant proteins can be overproduced and used for structural and functional studies.