Figure 1.

Cartoon representation of the PI-SceI GFP-fusion protein structure, SDS-PAGE of purified proteins, and illustration of PCR steps performed to create and amplify the PI-SceI-GFP encoding gene. Panel A shows the ribbon structure of GFP and PI-SceI intein bound to its target sequence ¹⁰, ¹⁴. GFP was inserted into the loop containing G117 (the green ball-and-stick model indicated by arrows); this position does not interfere strongly with the intein's functions. Panel B illustrates the use of overlapping primers and the PCR based cloning technique as described in Material and Methods. After the three primary PCR products were combined, the primers VMA-1 and VMA-2 were used to amplify the entire recombinant gene. Panel C, Lane 1: Commassie blue stain of the purified PI-SceI protein, about 51kDa, from E. coli XL1B (DE3) transformed with pET-15b PI-SceI; lane 2: Commassie blue stain of the purified PI-SceI117GFP protein, about 78kDa, from E. coli XL1B (DE3) transformed with pET-28a PI-SceI 117GFP separated on 10% SDS-gel.