Fig. 4.

Expression, purification, and activity of ttRNAP. (A) Expression levels of ttRNAP using expression vectors developed in this study (Fig. 3). (B and C) A one-step purification of ttRNAP using the His-tag (B) or CL7/Im7 (C) approaches. (D) Transcription elongation assays for the purified, overexpressed ttRNAP enzyme (Left) and the reference WT enzyme isolated from the host without overexpression (Right). 60C, lysate (LYS) heated at 60 °C for ∼45 min; β’ωH8/β’ωCL7, β’ω fusion with the His8 or CL7 tags; EL, eluate; FT, flow through; LD, molecular mass standards in kilodaltons; RNA18, synthetic 18-mer RNA with fluorescein (FLU) at the 5′-end; T/NT, DNA template/nontemplate strands.