Protocol for the purification of the plastid-encoded RNA polymerase from transplastomic tobacco plants

The plastid-encoded RNA polymerase (PEP) plays an essential role in the transcription of the chloroplast genome. Here, we present a strategy to purify the transcriptionally active protein complex from transplastomic tobacco ( Nicotiana tabacum ) lines in which one of the PEP core subunits is fused to an epitope tag. We describe experimental procedures for designing transformation constructs for PEP purification, selection, and analysis of transplastomic tobacco plants. We then detail the steps for purifying PEP from the transplastomic tobacco leaves. For complete details on the use and execution of this protocol, please refer to Wu et al. 1 • Steps for purifying large endogenous complexes in plants • Instructions for the efficient purification of plastid-encoded RNA polymerase • Guidance on inserting DNA fragments into the chloroplast genome Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The plastid-encoded RNA polymerase (PEP) plays an essential role in the transcription of the chloroplast genome. Here, we present a strategy to purify the transcriptionally active protein complex from transplastomic tobacco ( Nicotiana tabacum ) lines in which one of the PEP core subunits is fused to an epitope tag. We describe experimental procedures for designing transformation constructs for PEP purification, selection, and analysis of transplastomic tobacco plants. We then detail the steps for purifying PEP from the transplastomic tobacco leaves.