Abstract:
Background: Today, despite the increased availibility of antibiotics, mortality of patients with infectious diseases and cancer are going to increase due to antibiotic resistance. On the other hand, resistance to antibiotics will be a major blow to the global economy in the future. Integrons play an important role in multidrug resistance (MDR). Overall, integrons consist of three key component including integrase (IntI) gene coding a recombinase that is required for site-specific recombination, an simple recombination site involved in gene cassette integration and a strength promoter for the gene casset array expression. Cassette integration mainly occur trough recombination between attI and attC sites. Integrase enzyme is a member of tyrosin recombinase family which catalyzes integration and class 1 integron cassete excision by site specific recombinetain mechanism. The recombination crossover, which catalyzes by integrase, occurs between the G and T nucleotides in nonpalindromic attI and palindromic attC sites.These sites are critical point for recombination process. The aim of this study was cloning, expression and purification of integrase and also representation of an efficient method for its in vitro assay. Purification of integrase and meauserment of its activity could serve as the base study for selection of inhibitory molecule and further assessment of biochemical charectristics.
Method: To this end, the IntI gene was cloned into the pET-22b plasmid. The resulting recombinant plasmid was transformed into the bacterial strain TOP10 F' and then transformed to Origami after approvement. Expression of the recombinant protein was investigated by SDS-PAGE and western blot assays. The recombinant IntI protein containing His-Tag in its C- terminal was purified by Ni–NTA affinity chromatography, and its activity was measured by a newly-introduced assay. This assay was based on the incubation of enzyme with its substrate and subsequently product evaluation by PCR.
Results: SDS-PAGE and western-blotting confirmed the presence of a recombinant protein with the correct size of 38 kDa. The colony PCR assessment was indicative of cloning of integrase. The presence of a recombinant protein with correct size of 38 kDa was confirmed by SDS-PAGE and western-blotting. Recombinant activity of enzyme was assessed by PCR method and specific primers.
Conclusion: Results of this research could be used for production of recombinant integrase. In this research, newly-introduced assay was used to assay integrase activity based on PCR. So, this method could be used instead of radioactive base expensive method
Keywords : Antibiotic Resistance, Integrase, Site-Specific Recombination, attI site, attC site