Purification of the sea urchin mtRNAP from baculovirus-infected insect cells and functional assay

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system"</p><p></p><p>Nucleic Acids Research 2007;35(7):2413-2427.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874651.</p><p>© 2007 The Author(s)</p> () Purification of mtRNAP by metal chelate affinity chromatography. The soluble portion of the insect cell lysate expressing the sea urchin mtRNAP was purified by Ni-NTA chromatography; cleared lysate, C.lys, flow-through, FT, wash, W, 3–5, fractions eluted at 250 mM imidazole, were separated on a 10% SDS–PAGE and revealed by immunoblotting as described in ‘Materials and Methods’ section. () Purification profile of mtRNAP as obtained by Heparin–Sepharose chromatography. Peak fractions from Ni-NTA column were pooled and subjected to Heparin–Sepharose chromatography. Input to the column (I) and fractions eluting between 0.75 and 0.9 M NaCl were analyzed by 7.5% SDS–PAGE and Coomassie Brilliant Blue stained. The molecular weight marker Precision Plus Protein Standards (Bio-Rad) is shown (M). The arrow inside the picture indicates the mtRNAP-containing band, as assessed by MALDI-TOF analysis. () Immunoblotting assay of input to the column (I) and Heparin–Sepharose eluted fractions. () Transcriptional activity of purified mtRNAP. The indicated Heparin–Sepharose fractions were assayed in the presence of [α-]PUTP, as described in ‘Materials and Methods’ section. On the top it is shown the diagram of the 71-bp tailed template, named 71bpDNA, with the open bar referring to the duplex DNA portion and the thin line to the 3′-tail. Run-off transcripts are indicated by arrowed line. Radiolabeled transcripts were separated on a 12% polyacrylamide/7M urea mini-gel followed by phosphorimaging analysis. 15 + R, fraction 15 treated with RNase A. RNA markers corresponding to the 10 nt ladder are indicated on the left