Development of a Unidirectional Expression Vector : in a Search of Suppressor against a Cell Death-Inducing Protein, Jpk

Abstract

Jopock (Jpk) has previously been ascertained that induces both bacterial and mammalian cell death. The Escherichia coli cells expressing Glutathion S-transferase (GST) fused Jpk showed elongated phenotype and inhibited cell growth which led eventual cell death. In an attempt to search the genetic suppressor of the lethal protein Jpk in bacterial cells, we constructed a unidirectional protein expression vector inserting tac promoter next to the C-terminus Jpk in pGEX-Jpk. The function of additional tac promoter was confirmed by substituting lac promoter in Plac-TOPO plasmid. The cells harboring plac-TOPO, which regulates lacZα gene expression under lac promoter, formed blue colonies in 5-bromo-4-3 indolyo-β-D-galactoside (X-gal) plate. When lac promoter was changed tac promoter, same results were observed. Since the addition of tac promoter did not affect the toxic effect of Jpk, the pGEX-Jpk-ptac could be a useful vector for the screening of suppressor(s) for Jpk, in which GST-Jpk and a putative Jpk-suppressing protein are coexpressing from two unidirectional tac promoters, which response to the same inducer, isopropyl-β-D-thiogalactopyranoside(IPTG).