Fig. 5
The expression of Tpases by IS1447. a Schematic representation of the pET21a-derived plasmids constructed for Tpase expression. Green arrows and red double lines indicate the potential initiation and termination codons for Tpase translation, respectively. Red triangles indicate the His6-tag. The orange box indicates the IRL sequence of IS1447, and the putative RBS in IRL is indicated by a blue arrow. The frameshift region is highlighted in yellow. The constructed E. coli BL21(DE3) strains, their predicted products with (+ 1) or without (N) programmed slippage and theoretical molecular weights are listed in the dashed boxes below. Green boxes indicate the eGFP gene. All lanes and bands indicated by arrows in b, c, and d are named according to the strains and products shown in the dashed boxes. b SDS-PAGE analysis of the crude extracts of the E. coli strains expressing IS1447 Tpases. BL21(DE3)::pET21a was used as the negative control. Black, green, and red arrows indicate the bands corresponding to the products a, d, and e, respectively, as shown in a. The ~ 18.8 kDa protein produced by BL21(DE3)::pET21a-OrfABt-A8 was further confirmed by mass spectrometry (Additional file 1: Figure S3). c Fluorescent imaging of strains expressing the eGFP-bearing IS1447 Tpases. Control, strain 6 and strain 7 refer to those listed in a. d SDS-PAGE (SP) and immunoblotting (WB) analyses of the expression of IS1447 Tpases-eGFP fusion proteins. Green and red arrows indicate the bands of approximate 74 and 62 kDa corresponding to the products g and f/h, respectively, as shown in a. e SDS-PAGE (SP) and immunoblotting (WB) analyses of the expressed OrfB proteins (OrfB1, OrfB2, and OrfB3) in E. coli BL21(DE3). Black arrows indicate the bands referring to the putative OrfB proteins expressed with different initiation codons as shown in a. M, protein standards