Fig. (10) Expression of HT7-Rluc with optimized linker. HT7-Rluc (linker N-HT7; see the Supplementary Material), HT2-Rluc (linker N-3; see Supplementary Material), and Rluc were overexpressed in E. coli KRX at 25 °C and then lysate fractions containing total (T) or soluble (S) protein were labeled to completion with the TMR-ligand and resolved by SDS-PAGE. TMR-labeled HT7-Rluc was also incubated with TEV protease (S+) for 30 min at 30 °C. Gels were imaged for both total protein (SimplyBlue, panel A) and the amount of functional fusion (TMR fluorescence, panel B). Relative amounts of soluble and functional (TMR-labeled) protein (full length and proteolytically cleaved) could be quantitated from the fluorescence scan (Eex/Eem=532/580 nm). Overlaid arrows indicate bands of interest. Note the ~34 kDa band in panel B which presumably represents truncation of the fusion.