Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an Escherichia coli thioredoxin gene fusion system.
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 periplasm 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 recovered after anion exchange chromatography of lysates 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 cytoplasm.
Watt
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Department of Chemistry and Biochemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Ontario, Canada.
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Mellors
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D004765:Enteropeptidase / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D016978:Mannheimia haemolytica / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D004765:Enteropeptidase; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase