Movements in proteins reveal information about antibioticresistance spreading
1 construct was used for biochemical studies, for solving the Rep-apo structure and as template to generate the mutant variants. Site-directed mutagenesis was employed to generate the Rep1 Triple Mutant and PriRep5 K591A variants from the aforementionedgenes cloned into pET28a using primer pairs PriRep1_248A_251A_253A_F/PriRep1_248A_251A_253A_R and Rep5_K591A-F/Rep5_K591A-R, respectively. All of the resulting proteins had a His-tag moiety at their N-terminus.
Positive fractions were dialyzed, concentrated and stored in the same way as previous constructs. Rep1 Triple Mutant was purified in a similar manner to PriRep1 and PriRep5-ΔCTD, except the dialysis prior to injection into the HiTrap™ Heparin HP column was done in 20 mM Tris pH 8.0, 0.2M NaCl, 5% glycerol, 0.5 mM EDTA, 1 mM DTT . Purification of PriRep5 K591A was done in a similar manner to Rep1 Triple Mutant, except all buffers were adjusted to pH 8.
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