Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Generation and characterization of scR1antTNF-Fc protein. A, schematic structure modeling of scR1antTNF-Fc protein. Two TNFR1 antagonistic proteins were fused with human IgG-Fc. N, N-terminal. Amino acid sequences and domain information of scR1antTNF-Fc are described in Fig. S1A. B, X-ray structure modeling of the scR1antTNF-TNFR1 complex. scR1antTNF bound to the homotrimer of TNFR1. Red, TNFR1; green, TNFR1 interaction domain of scR1antTNF; orange, peptide linker for forming the single-chain structure. C, schematic pCAG-based mammalian expression vector for scR1antTNF-Fc protein. The cDNA was composed to express scR1antTNF-Fc whereby triple R1antTNF domains fused by peptide linkers (GGGSGGG) were further fused to a human–IgG Fc domain (Ch2 and Ch3). Signal sequence peptide genes derived from a mouse IgG Vh (Vhss) or human IL-2 (IL-2ss) were linked at the 5′-terminal of scR1antTNF-Fc cDNA. D, supernatants of cultured medium (left side) and purified proteins (right side) 7 days after transfection were assessed by SDS-PAGE following Coomassie Brilliant Blue staining. Arrowhead shows an ∼75-kDa band of the scR1antTNF-Fc monomer. E, each recombinant protein expressed with Vhss and IL-2ss was purified by size-exclusion chromatography. F, the molecular weight of monomeric scR1antTNF-Fc protein was confirmed by Western blotting with an anti-human IgG-Fc antibody.

Article Snippet: The resultant gene was inserted to a pCAG-Hyg human IgG-Fc fusion vector (Fujifilm Wako) to generate the scR1antTNF-Fc expression vector.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Cell Culture, Purification, Transfection, SDS Page, Staining, Recombinant, Size-exclusion Chromatography, Molecular Weight, Western Blot