Journal: Protein expression and purification

Article Title: Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

doi: 10.1016/j.pep.2017.03.013

Figure Lengend Snippet: (A) Elution profile of the E. coli whole cell lysate containing recombinant MtbRNAP subunits from Ni2+ affinity column with imidazole gradient and a stepwise increase in [NaCl]. The fraction numbers are shown on top of the horizontal axis in red. (B) SDS-PAGE analysis of the eluted fractions.

Article Snippet: E. coli BL21 (DE3) strain (New England Biolabs) was used for expression of the recombinant RNAP of M. tuberculosis and the transcription factor CarD.

Techniques: Recombinant, Affinity Column, SDS Page

Journal: Protein expression and purification

Article Title: Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

doi: 10.1016/j.pep.2017.03.013

Figure Lengend Snippet: (A) Elution profile of Ni2+-affinity purified MtbRNAP from MonoQ column with a NaCl gradient. The odd fraction numbers are indicated on top of the horizontal axis. (B) SDS-PAGE analysis of the fractions eluted from MonoQ column. L (for Loading) is the material eluted from the Ni2+ affinity column (F2); FT and Wash are the fractions collected before application of the salt gradient; Peak1, Peak2, and Peak3 corresponding to the peaks in the A280 profile in (A); E. coli RNAP holoenzyme was used as reference. (C) Evaluation of the transcriptional activity of the fractions from MonoQ column. 12 pmol of RNA7FL-TDS76 hybrid (Table 2) in 8 μl TB40 containing 20 μM each ATP and GTP were used for the TEC assembly with 3 μl of the F2 fraction of the imidazole eluate (L) or fractions 17, 27, and 37 from MonoQ; 15 pmol NDS79 were added to each reaction. The TEC was chased with 1 mM NTPs for up to 40s. (D) Quantification of MtbRNAP activity. The TECs were assembled with 0.5, 1, or 2 pmol of RNA9Cre-TDS57 hybrid, 0.4, 0.2, 0.1, and 0.05 pmol MtbRNAP, and a 2-fold molar excess of NDS57 in 10 μl TB. The fraction of the active TEC was quantified as the % of the RNA extended after 5 min incubation with 10 μM each GTP + UTP and the [TEC] was quantified based on the total [RNA] and plotted vs [RNAP]. Each series represents the results obtained with a certain concentration of the RNA-DNA hybrid. The inset shows a gel for the “100 nM” (1 pmol of RNA9Cre-TDS57 hybrid) series.

Article Snippet: E. coli BL21 (DE3) strain (New England Biolabs) was used for expression of the recombinant RNAP of M. tuberculosis and the transcription factor CarD.

Techniques: Affinity Purification, SDS Page, Affinity Column, Activity Assay, Incubation, Concentration Assay