Fig. 1From: Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migrationa Schematic representation of Bcl-2 and TeNT proteins. The active TeNT is composed of a light chain zinc protease (L, 50Â kDa) and a heavy chain (TTH, 100Â kDa) that is linked to the L chain during translation and requires activation and reduction of disulfides to dissociate. The TTH governs neuronal cell binding, uptake, and transport. The TTH chain is composed of two domains: an N-terminal 407-amino-acid domain (H N , 45Â kDa) and a C-terminal 452-amino-acid domain (TTC, 55Â kDa). The TTC domain alone retains neuronal binding and migration abilities. b PCR procedure. The cDNAs for Bcl-2, TTC and TTH were cloned using primers designed to introduce a start codon (ATG) or stop codon, FLAG sequence or restriction enzyme sites (Table 1). The selected restriction enzymes share compatible cohesive ends, e.g., XbaI and NheI, to facilitate construction of fusion protein cDNA. c Fusion proteins. Along with the Bcl-2 protein and the TTC fragment alone, TTC-Bcl2 and Bcl2-TTC fusion proteins were produced with the Bcl-2 fused at the C-terminus and N-terminus of TTC, respectively. A Bcl2-TTH fusion was also generated. Each fusion protein contained an N-terminal FLAG sequence. The estimated molecular weights were: Bcl-2: 26Â kDa, TTC: 55Â kDa, TTC-Bcl2: 81Â kDa, Bcl2-TTC: 81Â kDa, Bcl2-TTH: 126Â kDa. d Fusion proteins expressed in Cos7 cells. Fusion proteins were detected with anti-FLAG antibody. Only Bcl-2 expression was confirmed. e In vitro transcription/translation (TnT). During in vitro expression, Bcl-2 was expressed with an appropriate molecular size, whereas Bcl2-TTC and Bcl2-TTH were smaller than expected, likely due to premature termination of protein synthesis. When the TTC fragment was located at the N-terminus of the fusion proteins (e.g. TTC and TTC-Bcl2), no protein expression was detectedBack to article page