Schematic diagram of the constructs used for TAT-Apoptin protein expression. (A) Schematic representation of the TAT-Apoptin protein fused with different affinity tags together with the expression vectors used in this study. The designations of the TAT-Apoptin protein and its expression vectors are indicated, (a), (b), (c) and (d). The constructs, (a) and (b), contain the full-length TAT-VP3 gene cloned into the vectors pET28a and pGEX-4 T-1; these were used for expression of TAT-Apoptin protein with either a six-histidine (6 × His) tag or a glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs (c) and (d) containing the TAT-VP3 gene that was codon-optimized; this was derived from construct (b) by replacing rare codons without altering the amino acid sequence. The codon-optimized TAT-VP3gene, TAT-VP3opt, was then cloned into pET28a and pGEX-4 T-1. (B) Sequence comparison between the TAT-VP3 gene and the TAT-VP3opt gene. The nucleotide sequences were compared between the original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized TAT-VP3 gene (TAT-VP3Opt) over the whole coding region. An asterisk (*) represents the fact that the aligned nucleotides are identical.