Plasmids and Transfections
All DNA manipulations, mutation, cloning, and transformation experiments in Escherichia coli DH5α were performed according to standard protocols. The pcDNA3.1 vector carrying the cDNA sequence of the human p53R2 gene (pcDNA3-hp53R2) was a generous gift from Prof. Hirofumi Tanaka (Tokyo Medical and Dental University, Japan). The pcDNA3-hp53R2mut plasmid encoding p53R2-D342E was generated by PCR from pcDNA3-hp53R2 using the Phusion Site-Directed Mutagenesis kit (Thermo Scientific) and the following primers: 5′-CCAAGGTGAAGACGTTTTCTGTGGTTTCTGCCATAACTGCA-3′ and 5′-GGCAGAAACCACAGAAAACGTCTTCACCTTGGATGCAGATT-3′ (mutated nucleotide underlined). All steps were performed according to the manufacturer's instructions. The presence of the A to T point mutation in the p53R2 sequence was confirmed by sequencing. The pcDNA3-p53R2-ΔC9 plasmid was created by PCR amplification from the pcDNA3-hp53R2 plasmid using the following primers: 5′-TGGAATTCCAGACCGGCTAGCATGGGCGACCCGGGA-3′ and 5′-CTCGAGTTAATCTGTGGTTTCTGCCATAACTGC-3′. The PCR fragment digested with NheI and XhoI was ligated into the pcDNA3.1 expression vector opened with the same enzymes. Bacterial expression vectors expressing N-terminally His6-tagged wild-type, D342E, and p53R2-ΔC9 proteins were constructed as follows. A DNA fragment containing the sequence of the WT human p53R2 gene was amplified by PCR from the pcDNA3-hp53R2 plasmid using the primers 5′-GTGGTGGAATTCCAGACCGGCTAGCATGGGCGACCCGGAA-3′ and 5′-AAGCCACAGTGGAGGCTGATCA-3′. The fragment was then cleaved by NheI and XhoI and inserted in the pET28b vector opened by the same enzymes. NheI and XhoI were used to release p53R2-D342E and p53R2-ΔC9 fragments from pcDNA3-p53R2mut and pcDNA-p53R2-ΔC9, respectively. These sequences were then ligated into the pET28b vector opened by the same enzymes to create pET28b-p53R2-D342E and pET28b-p53R2-ΔC9. The pET28b-R1His plasmid expressing a N-terminally His6-tagged human R1 protein was built by PCR amplification of the R1 cDNA sequence contained in the pET3a-hR1 plasmid (kindly given by Pr. Lars Thelander, Umeå University, Sweden) using primers 5′-CTTTAAGAAGGAGATGCTAGCATGCATGTGATCAAG-3′ and 5′-GGGCTTTGTCTCGAGCCGGATCCAC, subsequent digestion with NheI and XhoI, and ligation into the pET28b vector cleaved by the same enzymes.
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