Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an Escherichia coli thioredoxin gene fusion system
AUTOR(ES)
Watt, Mary-Anne
FONTE
Cell Stress Society International
RESUMO
Pasteurella haemolytica A1 secretes an O-sialoglycoprotein endopeptidase (EC. 3.4.24.57) (glycoprotease: Gcp) which is specific for O-linked sialoglycoproteins. When the cloned gene is expressed in Escherichia coli, the recombinant glycoprotease (rGcp) is secreted to the peripalsm where it is present as a disulfide-linked aggregate which lacks enzymatic 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 critical in restoring biological activity. A fusion protein, rTrx-Gcp, was constructed to investigate the role of thioredoxin (E. coli TrxA) in the production of enzymatically active rGcp. This 47 kDa protein was expressed at a high level, in a soluble, monomeric form, in the cytoplasm of E. coli. Cleavage of the fusion protein by enterokinase released the rGcp fragment (35 kDa) with glycoprotease activity. A higher recombinant glycoprotease activity was recoveref after anion exchange chromatography of lystates of E. coli expressing rTrx-Gcp. Thus when E. coli TrxA is combined in a recombinant fusion protein with P. haemolytica A1 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 cytopalsm.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=312996Documentos Relacionados
- Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.
- A neutral glycoprotease of Pasteurella haemolytica A1 specifically cleaves O-sialoglycoproteins.
- Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.
- Extensive homology between the leukotoxin of Pasteurella haemolytica A1 and the alpha-hemolysin of Escherichia coli.
- Analysis of the Capsule Biosynthetic Locus of Mannheimia (Pasteurella) haemolytica A1 and Proposal of a Nomenclature System