PROCESSING BY OMPT OF FUSION PROTEINS CARRYING THE HLYA TRANSPORT SIGNAL DURING SECRETION BY THE ESCHERICHIA-COLI HEMOLYSIN TRANSPORT-SYSTEM

A fusion gene (ces-hlyA(s)) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyA(s)) of Escherichia coli hemolysin (HlyA) to the ces gene for a cholesterol esterase/lipase (CE) from a Pseudomonas species. Part (about 30%) of the expressed fusion protein CE-HlyA(s) was secreted in E. coli carrying hlyB and hlyD genes. Following the insertion between the reporter gene and hlyA(s) of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-HlyA(s) and CE-HlyA(s)) were shown to be cleaved by OmpT between the two parts during HlyB/HlyD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increased ompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.