has retained the potential of neuronal differentiation into a dopaminergic phenotype [17,18,19,20]. Results Expression and characterization of human TNC-scTNFR2 A prototype of a TNFR2-selective highly bioactive TNF variant has been recently reported [16], comprised of the chicken TNC trimerization domain and a TNFR2-selective TNF mutant (D143N/A145R; [12]) in the single chain TNF format [13]. To obtain a fully human TNF reagent we here fused the trimerization domain of human TNC (AA 110–139) to the N-terminus of the TNFR2-selective scTNF variant (TNC-scTNFR2), which was further modified for improved production. Thus, peptide linkers connecting the three TNF monomers were reduced to GGGGS and overall codon usage was adapted for expression in mammalian cell systems. Bioactivity of TNC-scTNFR2 was compared to the monomeric TNFR2-selective scTNFR2 (sche- matic overview in Fig. 1a and b). Both scTNFR2 and TNC-scTNFR2 were expressed in HEK293T cells and isolated by immobilized metal ion chroma- tography (IMAC) in a single step using a N-terminal his-tag present in the molecule. Purity was confirmed by SDS-PAGE and Coomassie staining (Fig. 1C). Under reducing conditions the TNF variants exhibited an apparent molecular mass of approximately 51 kDa and 54 kDa, matching the calculated molecular mass of 53.5 kDa and 56.8 kDa for scTNFR2 and TNC-scTNFR2, respectively. Under non-reducing conditions additional bands of 110 kDa (dimer) and above 170 kDa (trimer) were observed for Neuroregeneration by a TNFR2 Agonist TNC-scTNFR2 (Fig. 1C and D). The oligomerization state of TNC-scTNFR2 was further characterized by size exclusion chromatography (SEC; Fig. 1E). Both scTNFR2 and TNC- scTNFR2 eluted as a single major peak and TNC-scTNFR2 with a higher molecular mass than scTNFR2, indicating that the TNC domain, as expected, causes stable oligomerization of scTNFR2 molecules. The fact that TNC-scTNFR2 elutes in a single peak while migrating in non-reducing SDS-PAGE as dimers and trimers is in good accordance with the finding that the TNC trimerization domain assembles into stable trimers without stabilization of disulfide bridges. Receptor selectivity and bioactivity of TNC-scTNFR2 TNF receptor selectivity of scTNFR2 and TNC-scTNFR2 was analyzed using TNFR12/2/TNFR22/2 mouse embryonic fibro- blasts (MEF) stably expressing chimeric TNFRecd-Fasicd receptors that bind human TNF and trigger Fas-associated signaling pathways, e.g. apoptosis induction [21]. In contrast to human soluble recombinant TNF (huTNF), the TNFR2-selective TNF variants generated here induced no apoptosis in MEF TNFR1-Fas cells (Fig. 2A), verifying that both variants had highly reduced affinities for TNFR1 due to the mutations D143N/A145R. In contrast, TNC-scTNFR2 induced a strong apoptotic response in MEF TNFR2-Fas cells. Treatment with huTNF and scTNFR2 caused cell death only at high concentrations, which could be enhanced by addition of the cross-linking TNFR2-specific monoclonal antibody 80M2 (Fig. 2A) to similar values as obtained for TNC-scTNFR2 alone, thus in concert mimicking the action of memTNF [15]. Similar results were obtained using the human Figure 1. Genetic engineering of the TNFR2-selective TNF muteins. (A,B) Schematic representation of the TNF variants used in this study. CT: cys-tag, HT: his-tag; TNF: huTNFR2-specific (D143N/A145R) TNF module (aa 80–233); L: GGGGS-linker; TNC: trimerization domain of human tenascin C (aa 110–139). (C) Coomassie staining and (D) immunoblot analysis of scTNFR2 (1) and TNC-scTNFR2 (2). Purified TNF variants were analyzed by 8% SDS-PAGE under reducing (r.) or non-reducing (n.r.) conditions and either stained with Coomassie or immunoblotted with anti-his-tag antibodies. (E) TNF muteins were analyzed by HPLC size exclusion chromatography using a BioSep-Sec-2000 column. Peak positions of relevant standard proteins are indicated (200 kDa; 67 kDa and 29 kDa). doi:10.1371/journal.pone.0027621.g001 PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27621