(The) fusion between the signal sequence of ribose- binding protein and the ligand-binding domain of ribose repressor

Abstract

The DNA-binding domain of RbsR protein, the repressor for rbs operon in Escherichia coli, was replaced by the leader sequence of the periplasmic ribose-binding protein (RBP). The fusion protein was expressed under the $P_tac$ promoter. Upon induction, cells stop growth and become lysed as indicated by a decrease in culture density. After being translated, the fusion protein was targetted to the cytoplasmic membrane and then stayed there with its leader sequence processed. It seems that the lethal effect by the fusion protein results from the jamming of secretory machinery leading to an accumulation of precursor proteins destined to be translocated inside the cytoplasm. This lethal effect was releaved by the GroEL/GroES over-prouction that guided the fusion protein to the degradation pathway. The fusion protein made two bands in SDS-PAGE that became one in urea SDS-PAGE, which implies the fusion protein may be modified in the cell. We checked the fusion protein can be phosphorylated in vivo.