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Article type: Research Article
Authors: Yang, Junbaoa | Raju, Rajalab | Sharma, Rajendrab | Xiang, Jima; *
Affiliations: [a] Saskatoon Cancer Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada | [b] Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Correspondence: [*] Correspondence and reprint requests to: Dr Jim Xiang, Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada.
Abstract: Recombinant DNA techniques were used to clone, to construct and to express a fusion protein M4/TNF in Escherichia coli. The fusion protein includes the chimeric F(ab')2 fragment (M4) recognizing the human tumor-associated TAG72 antigen and the tumor necrosis factor alpha (TNF) moiety. The M4/TNF purified from inclusion bodies of the bacteria homogenates was further solubilized in a denaturing buffer containing 6 mol l−1 guanidine and refolded in a refolding buffer. Our results showed that the M4/TNF refolded in a buffer containing 6 mmol l−1 oxidized glutathione (GSSG), 0.2 mmol l−1 dithioerythione (DTE) and 0.5 μmol l−1 protein disulfide isomerase (PDI) displayed a 4-fold higher anti-TAG72 immunoreactivity and a 5-fold higher TNF activity than that refolded in the same refolding buffer but without PDI. Our data thus indicates that the protein disulfide isomerase not only facilitates the correct formation of disulfide-bonds of the antibody molecule, but also the correct refolding of the TNF moiety in vitro.
Keywords: Fusion protein, protein refolding, tumor necrosis factor, protein disulfide isomerase
DOI: 10.3233/HAB-1995-6402
Journal: Human Antibodies, vol. 6, no. 4, pp. 129-136, 1995
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