Figure 2
The marked Pch PRP8 inteins splice at high efficiency when expressed in E. coli. SDS-PAGE and Western blots of whole cell lysates of E. coli carrying plasmids expressing the modified inteins. The N-and C-terminal exteins are GST (29 kD) and 6 × His (3 kD), respectively. The markers inserted within the intein are indicated above each lane in the gel. Plasmid pGPch-1 is the starting vector with an unmodified Pch PRP8 intein, and CS-linker refers to pGPch-1 with a cloning site and linkers. The predicted molecular weights (kDa) of the inteins are: Pch PRP8 (18), Pch PRP8-cloning site + linker (19); Pch PRP8-G418R (49); Pch PRP8-His5 (42); Pch PRP8-HygBR (57); Pch PRP8-LexA-VP16 (51). Pch PRP8-LexA-VP16 migrates anomalously. GST-6 × His is ~30 kD. Expression of the inteins was driven by a tac promoter and was induced in lanes 2-7 with 0.2 mM IPTG. Western blot analysis was performed as described in Methods. The strong band at ~30 kD is the spliced extein, GST-6 × His. Plasmids in the host E. coli strain: Lanes 1 and 2, pGPch-1; Lane 3, pRR01; Lane 4, pRR02; Lane 5, pRR03; Lane 6, pRR04; Lane 7, pRR05.