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Expression and Purification of AAC(3)-II， a Aminoglycoside Acetyltransferase， and Its Activity
Zhen Wang1,2, Yun-Bo Chen1,2, Chang-Sen Zha1,2, Li-Lin Zou1,2, Ying Peng1,2, Jing-Zhang Ji1,2, Xiu-Shu Chen3, Jian-Xin Lü1,2*
1Zhejiang Provincal Key Laboratory of Medical Genetics, 2Institute of Cellular and Molecular Medicine,3Institute of Microecology, Wenzhou Medical College, Wenzhou 325035, China
Abstract: The aac(3)-II was amplied by PCR and cloned into pET28a expression plasmid， then the recombinant plasmid was transformed into E.coli BL21(DE3)， lastly AAC(3)-II was purified from the recombinant bacteria. The activity of AAC(3)-II was analyzed by comparing minimum inhibitory concentration (MIC) between the recombinant bacteria owning aac(3)-II and not. Bacteria were disrupted by ultrasonic treatment. Bacteria debris was removed by centrifugation， and the supernatant and AAC(3)-II purified from the supernatant were analyzed by SDS-PAGE and Western Blot electrophoresis. Results demonstrated that the bacteria owning aac(3)-II were 256-fold higher in gentamicin (GEN) MIC， 16-fold higher in tobramycin (TOB) and netilmicin (NTL) MIC than the bacteria not owing aac(3)-II. SDS-PAGE and Western blot electrophoresis proved purified protein was AAC(3)-II that its molecular weight was 32.0 kDa， purity of that was more than 95 percentage. The purified 10 μg AAC(3)-II transfered acetyl to GEN in 10 s， to TOB and NTL in 30 s， so the antibiotics were invalidated. The research provided fundamental conditions for exploiting the drug eliminating aminoglycosides.
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