Overproduction, purification, and transcriptional activity of recombinant Acinetobacter baylyi ADP1 RNA polymerase holoenzyme

Abstract

Acinetobacter baylyi is an interesting model organism to investigate bacterial metabolism due to its vast repertoire of metabolic enzymes and ease of genetic manipulation. However, the study of gene expression in vitro is dependent on the availability of its RNA polymerase (RNAp), an essential enzyme in transcription. In this work, we developed a convenient method of producing the recombinant A. baylyi ADP1 RNA polymerase holoenzyme (RNApholo) in E. coli that yields 22 mg of a >96% purity protein from a 1-liter shake flask culture. We further characterized the A. baylyi ADP1 RNApholo kinetic profile using T7 Phage DNA as template and demonstrated that it is a highly transcriptionally active enzyme with an elongation rate of 24 nt/s and a termination efficiency of 94%. Moreover, the A. baylyi ADP1 RNApholo has a substantial sequence identity (∼95%) with the RNApholo from the human pathogen Acinetobacter baumannii. This protein can serve as a source of material for structural and biological studies towards advancing our understanding of genome expression and regulation in Acinetobacter species.