A genetically engineered fusion protein M4/TNF with increased bifunctional activity refolded in the presence of protein disulfide isomerase.
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 mumol 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.
Yang
J
J
Saskatoon Cancer Center, University of Saskatchewan, Canada.
Raju
R
R
Sharma
R
R
Xiang
J
J
D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D007535:Isomerases; D051379:Mice; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D004274:DNA, Recombinant; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C047052:tumor-associated antigen 72; D007535:Isomerases; D019704:Protein Disulfide-Isomerases