High-Level Expression Of The Staphylococcal Efflux Pump Gene Qaca In Escherichia Coli By Codon Optimization

Objective:Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels. This study was to enhance the expression level of the active effux pump gene qacA from Staphylococcus aureus in Escherichia coli (E.coli) using codon-optimized genes.Methods:Using the previous recombinant plasmid pBluescript II SK (+)/qacA as template, qacA gene with AatⅡ and Nhel sites was amplified using PCR method. After digested with Aatll and NheI enzymes on the qacA gene and the expression vector pET-cocol simultanously, the purified target gene was inserted into the compatible sites of the vector pET-cocol using T4DNA ligase; Based on the known codon bias of E. coli, codon-optimized qacA was designed and synthesized, followed by inserted into the pET-cocol vector. The recombinant expression plasmids of pET-cocol/qacA and codon-optimized pET-cocol/qacA were transformed into E. coli BL21(DE3), respectively. And cell cultures were induced by the addition of IPTG. The expression product was analyzed by SDS-PAGE and identified using Western blotting test. Minimum inhibitory concentration assay was used to analyze the resistance against disinfectants of the strains carrying codon-optimized pET-cocol/qacA.Results:(1).Restriction enzyme (RE) digestions confirmed that both qacA and codon-optimized qacA were inserted into prokaryotic expression vector pET-cocol, indicating successful construction of the prokaryotic expression plasmid pET-coco1/qacA and codon-optimized pET-cocol/qacA.(2).The recombinant expression plasmid pET-cocol/qacA successfully expressed QacA protein in E. coli BL21(DE3) after induction of IPTG, with the size of55kD. The codon-optimized pET-cocol/qacA was highly expressed in E.coli BL21(DE3).(3).Western blotting test demonstrated that the QacA protein showed specific immunological reaction with a QacA antisera raised against a peptide comprising of the19C-terminated QacA amino acids.(4) MIC assay showed that there was no significant difference on the resistance against disinfectants tested between the strain bearing the plasmid pET-cocol/oqacA and that bearing pET-cocol/qacA, indicating the correctly functional expression of oqacA.Conclusion: Codon-optimized qacA was highly expressed in E.coli BL21(DE3), which providing the basis for further studies on the structure and function of qacA gene and the designing of effective efflux pump inhibitor.