REFOLDING OF RECOMBINANT PASTEURELLA-HAEMOLYTICA A1 GLYCOPROTEASE EXPRESSED IN AN ESCHERICHIA-COLI THIOREDOXIN GENE FUSION SYSTEM

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 .