DNA gyrase is an enzyme belonging to the group IIA topoisomerases, known to be a common target of antimicrobial agents. The enzyme consists of two subunits; gyrase A and gyrase B. The B subunit contains the ATPase domain, which is responsible for the hydrolysis of ATP and for the normal functioning of the enzyme. Quinolones are a class of antimicrobial agents that work by binding to the A subunit of DNA gyrase and stabilising the complex between the enzyme and the DNA molecule. Bacteria have adapted to the effects of antibiotics by spontaneous mutations through which they have acquired resistance to these agents. One clinically relevant mutation is the mutation of arginine 136 to cysteine (R136C) on the ATPase domain of gyrase B.
To help researchers with the design and evaluation of DNA gyrase inhibitors, we prepared a mutant of the ATPase domain of gyrase B, R136C, with site-specific mutagenesis by overlap extension method. A site-directed mutation was introduced into the gyrase B sequence with three consecutive polymerase chain reactions with the corresponding mutation-carrying primers. At position 136, a single base was substituted in place of the CGC triplet encoding the amino acid arginine to give the TGC triplet encoding the amino acid cysteine. The resulting polymerase chain reaction product was purified, cut and ligated into the corresponding cut plasmid pET44. The vector prepared was used to transform the E. coli TOP 10 cloning strain and subsequently transferred into the E. coli NiCo21 expression strain, in which the gyrase mutant was expressed. The mutagenesis was verified by sequencing. The mutant was purified by IMAC (Immobilized metal affinity chromatography) and gel filtration (desalting column) on an ÄKTA system. The presence of the mutant in the resulting fractions was confirmed with SDS-PAGE and the western blot technique. We isolated 3,74 mg of the ATPase domain of gyrase B R136C mutant.
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