Figure 4
Analysis of the induction pattern of recombinant TsoI REase. (A) Enzymatic activity in crude E. coli ER2566/pET21NS-TsoIRM extracts. Two selected clones of the confirmed tsoIRM gene nucleotide sequence after the 3rd round of biochemical selection were subjected to expression experiments, giving similar TsoI induction patterns. For clarity, the expression experiment of only one selected clone was shown. Lanes M, DNA size standards (Gene RulerTM Ladder Mix (Thermo Fisher Scientific (Fermentas); selected bands marked); lane 1, control (untreated) λ DNA; lane 2, λ DNA cleaved with native (wt) TsoI; lanes 3, 4, 5, various amounts (1, 0.1, 0.02 μl, respectively) of cell extracts prior to induction incubated with λ DNA in TsoI digestion buffer; lanes 6, 7, 8, various amounts (1, 0.1, 0.02 μl, respectively) of induced cell extracts incubated with λ DNA in TsoI digestion buffer. Samples were resolved on 1.2% agarose gel in TBE buffer, stained with EtBr. (B) SDS/PAGE evaluation of TsoI induction in E. coli ER2566/pET21NS-TsoIRM. Two selected clones as in A were analysed for the presence of TsoI protein bands on 8% SDS/PAGE gels. Electrophoresis was subjected to an extended run in order to clearly visualize protein bands in the high molecular weight range. Lanes M, protein standards (Gene RulerTM Prestained Protein Ladder Plus (Thermo Fisher Scientific (Fermentas); lane TsoIWT, native (wt) TsoI (1 unit); lanes 1 and 3, samples prepared from two colonies of E. coli ER2566/pET21NS-TsoIRM clones as in A, before induction; lanes 2 and 4, as in lanes 1 and 3, after IPTG induction.