The PCR mixtures for the MT Ivan mutants contained 6 mM Tris-HCl, pH 8.5, 2.5 mM KCl, 1 mM 2-mercaptoethanol, 0.01% Triton X-100, 1.5 mM selleck products MgCl2, 100 ng DNA, 25 pmol of each primer, 200 μM dNTPs and 2.5 U Taq polymerase in a final volume of 25 μL. After an initial denaturation step of 2.5 min at 96 °C, 35 cycles of 1 min at 96 °C, 30 s at 55 °C (MT Ivan) or 50 °C (MT Iver) and 1 min at 72 °C were performed. After the last cycle, a final elongation step of 15 min at 72 °C was carried out. The purified fragments were used as templates in the second PCR step. The PCR mixtures for the second step were the same as those used for the first PCR, but without adding DNA or primers. After the addition of 50 ng of each fragment, the reaction was started with a first denaturation step of 2.5 min at 96 °C, which was followed by 20 cycles of 30 s at 96 °C and 20 s at 72 °C. Thereafter, the primers were HSP cancer added (MT Ivan primers 1 and 5, MT Iver primers 3 and 5, see Table 1). Following a first denaturation step of 30 s at 96 °C, 35 cycles of 20 s at 96 °C, 30 s at 55 °C (MT Ivan)

or 50 °C (MT Iver) and 2 min at 72 °C were performed. In a last step, 20 cycles of 30 s at 96 °C and 1 min at 72 °C are followed by a last elongation step of 15 min at 72 °C. The mutated genes and the vector pET11a were digested with NdeI and BamHI according to the manufacturer’s protocol (Fermentas, St. Leon-Rot, Germany) and ligated in 1 × ligation buffer and 1 U T4 ligase (1 h, 22 °C). Escherichia coli TB 1 (New England Biolabs, Frankfurt/Main, Germany) was transformed with the plasmids using heat shock. For the detection of clones that contain the insert, the plasmids were isolated using the GeneJET™ Plasmid Miniprep

Kit (Fermentas) and the DNA inserts were sequenced (GATC, Konstanz, Germany). Methyltransferase gene expression was performed in the E. coli expression strain BL21 (DE3) (New England Biolabs). The E. coli BL21 (DE3) cultures were grown in Luria–Bertani media containing 0.1 g L−1 Progesterone ampicillin and 0.1% glucose. The induction of MT Ivan C286A and MT Iver C277A was performed at 28 °C for 2 h with 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer. All the other mutants were induced at 18 °C for 16 h with 0.5 (MT Ivan D150A, MT Ivan D154A, MT Iver E88A, MT Iver C111A and C128A), 0.25 (MT Iver C210A) or 0.1 mM IPTG (all other mutants). After induction, the cells were harvested by centrifugation for 10 min at 10 000 g and 10 °C and stored at −20 °C. The cells were disrupted under aerobic conditions using a French pressure cell at 137 MPa and the cell debris was removed by centrifugation for 30 min at 10 000 g and 10 °C.