in which an E. coli autotransporter
Antigen 43 (Ag43) was engineered to secrete an alginate lyase (Aly), an enzyme catalyzing the depolymerization
of alginate to oligomers. This autotransporter system is
unique in that the passenger domain Ag43a is self-cleaved yet
the secreted a domain is non-covalently attached to the bdomain,
forming an integral outer-membrane protein. In order
to secrete the recombinant Aly to culture medium, the researchers
replaced the majority of the passenger domain with
the catalytic domain of Aly whereas the segment of Ag43a
containing the cleavage mechanism was retained within the
chimera gene. This engineering strategy worked very well.
Aly enzyme activities were high, reaching about 1000 U/liter,
with almost all activities found in the culture medium, indicating
successful cleavage upon translocation of the protein
across the outer membrane