Pasteurella haemolytica Al secretes an O-sialoglycoprotein endopeptida
se (EC. 3.4.24.57) (glycoprotease: Cop) which is specific for O-linked
sialoglycoproteins. When the cloned gene is expressed in Escherichia
coli, the recombinant glycoprotease (rGcp) is secreted to the periplas
m where it is present as a disulfide-linked aggregate which lacks enzy
matic activity. In vitro refolding and activation of rGcp by mammalian
protein disulfide isomerase (PDI) or by the E. coli chaperones (DnaK,
DnaJ and GrpE) indicate that the redox environment of rGcp is critica
l in restoring biological activity. A fusion protein, rTrx-Gcp, was co
nstructed to investigate the role of thioredoxin (E. coli TrxA) in the
production of enzymatically active rGcp. This 47 kDa protein was expr
essed at a high level, in a soluble, monomeric form, in the cytoplasm
of E. coli. Cleavage of the fusion protein by enterokinase released th
e rGcp fragment (35 kDa) with glycoprotease activity. A higher recombi
nant glycoprotease activity was recovered after anion exchange chromat
ography of lysates of E. coli expressing rTrx-Gcp. Thus when E. coli T
rxA is combined in a recombinant fusion protein with P. haemolytica Al
Gcp, productive folding of the glycoprotease can occur as a result of
the chaperone action of the protein disulfide reductase coupled with
its ability to retain the fusion gene product in the E. coli cytoplasm
.