Figure 1

Schematic depiction and sequence of pCathB-C29A-eGFP coding for inactive cathepsin B-eGFP chimeras. Schematic representation (A) and nucleotide sequence (B) of the mammalian expression vector pCathB-C29A-eGFP that codes for eGFP-tagged cathepsin B containing a cysteine-to-alanine substitution in its active site (yellow) which was introduced by site-directed mutagenesis of the cathepsin B coding sequence inserted into pEGFP-N1 by using EcoR1 and BamH1 restriction sites. The plasmid bears a CMV promotor (dark grey) as well as an origin of replication (pUC ori, light gray). The eGFP coding sequence (green) is located down-stream of the cathepsin B encoding DNA minus the nucleotides encoding the C-terminal pro-peptide extension of the enzyme. In the resulting chimeric protein, cathepsin B will be fused to eGFP by a 6-amino-acids linker peptide (light red). The sequences of pCathB-eGFP [27] and the active-site mutant pCathB-C29A-eGFP were aligned by means of ClustalW (version 1.82) multi alignment tool. The sequences are identical, except for the point mutations inserted through site-directed mutagenesis, and resulting in a GGG to GGC ‘silent’ mutation (red), as well as a TGT to GCT codon exchange (orange), causing a cysteine to alanine exchange within the active-site of cathepsin B.