The expression of tomato prosystemin in Escherichia coli: A structural challenge

Prosystemin is the 200-amino-acid prohormone of the 18-amino-acid polypepti de called systemin, a systemic mobile signal that activates the synthesis o f defense genes in solanaceous plants in response to herbivore attacks. The unusual primary structural features of the tomato prosystemin cDNA and pro tein provided an extraordinary challenge in devising an expression system t o obtain the fall-length protein. Prosystemin expression inhibited the grow th of a eukaryotic and several prokaryotic hosts used. Prosystemin was init ially synthesized as a truncated protein of 185 amino acids in length using a T7 RNA polymerase expression system in E. coli strain BL21[DE3]. The tru ncation was found to be due to two factors: (1) the intramolecular associat ions of the 5' coding region of the prosystemin sequence with the expressio n vector's ribosome binding site and (2) the presence of a translation star t site just prior to the amino acid methionine at position 15. Mutations th at permitted the synthesis of the full-length prosystemin protein were intr oduced into the amino-terminal 5' coding region of the prosystemin cDNA. A 199-amino-acid recombinant prosystemin lacking the N-terminal methionine wa s purified from lysates and confirmed by N-terminal amino acid sequence and immunoblot analysis. (C) 1999 Academic Press.