Tsukio Masegi

Extracellular production of human immunoglobulin G Fc region (hIgG-Fc) by Escherichia coli

SummaryWe have constructed the recombinant plasmid for the extracellular production of human immunoglobulin G Fc region (hIgG-Fc) in Escherichia coli. The excretion vector pEXFC10 contained the weakly activated kil gene of plasmid pMB9 and the DNA fragment encoding a fused protein, in which the codons for the alkalophilic Bacillus sp. No. 170 penicillinase signal peptide and the hIgG-Fc were fused through the one additional amino acid Ser, which was identical with the N-terminus of alkalophilic Bacillus mature penicillinase. By cultivating E. coli carrying pEXFC10, about 40% of hIgG-Fc was excreted into the culture medium. The N-terminal amino acid sequence of the extracellular hIgG-Fc indicated that processing occurred correctly between Ala and Ser. From the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) in the nonreducing condition, it was suggested that most of the extracellular hIgG-Fc proteins took the dimeric form via disulfide bonds.

Characterization of a Novel Human Tumor Necrosis Factor‐α Mutant with Increased Cytotoxic Activity

Various novel recombinant human tumor necrosis factor‐α (TNF) mutants were prepared using protein engineering techniques, and their cytotoxic activity was compared with that of the intact form of TNF (intact TNF). Mutant 471 (a TNF mutant molecule with the deletion of 7 amino acids at the amino‐terminal and the substitution of Pro8Ser9Asp10 by ArgLysArg) had a 6‐fold higher cytotoxic activity against murine L929 cells. The mutant TNF had an increased ability to bind to TNF receptor on murine L929 fibroblasts cells. A cross‐linking study revealed that mutant 471 had an increased ability to form an active trimer. Mutant 471 also showed higher cytotoxic activity against human KYM myosarcoma cells and human MIA PaCa‐2 pancreatic carcinoma cells. The possible cachectin activity of the mutant was almost the same as that of intact TNF. These results suggest that mutant 471 might be a more promising candidate as an anticancer agent than intact TNF.

Production of the human immunoglobulin γ1 chain constant region polypeptides in Escherichia coli

Abstract We have determined the nucleotide sequence of the constant region exons of the rearranged human immunoglobulin γ1 chain gene cloned from a human plasma cell leukemia line, ARH-77. The amino acid sequence deduced from the nucleotide sequence revealed that the allotype of the ARH-77 γ1 chain was Glm (−1, −2, 3). Recombinant plasmids were then constructed in order to express the human γ1 chain constant region genes (the Fc region gene and the CH2-CH3 domains gene) in Escherichia coli. The human γ1 chain constant region genes without introns were derived from the genomic gene using synthetic DNA fragments. E. coli carrying each expression plasmid produced antigenically active constant region polypeptides as soluble proteins. In the case of the E. coli-derived Fc region polypeptides, they were generated as monomeric forms in the cytoplasm.

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

SummaryWe prepared various TNF-α derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-α. The additional substitution of Ala156 or Leu157 by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF-α are effective for the improvement of antitumor activity.