Hadipour, Sara; (1996) Purification, characterisation and mutagenesis of aminoglycoside (3') (9) nucleotidyltransferase, ant(3")-I. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The antibiotic resistance gene, aminoglycoside(3")nucleotidyltransferase (ant(3")-I), which confers resistance to spectinomycin and streptomycin was isolated on a 1.95Kb HindIII restriction fragment from plasmid pIJ4642 (Davey, Ph.D. thesis 1992) and cloned into pBluescriptKS- vector in both orientations to form pQR601 and pQR602. Plasmid pQR601 was found to contain the gene in the same orientation as the lac promoter, whereas pQR602 was in the opposite orientation to the lac promoter of pBluscriptKS-. To enhance expression of ant(3")-Ia, the gene was further cloned into other expression vectors namely, pTTQ19 and pUC19. This gave rise to pQR603 and pQR604 respectively upon transformation into E.coli JM107. To further increase expression levels, PCR was used to remove the large non-coding sequences flanking the gene and introduce two restriction sites at either end of the gene. The PCR product was further cloned into two other expression vectors, pMTL2023 containing the trp promoter and pMTL1005 containing the mdh (malate dehydrogenase) promoter, this gave rise to recombinant plasmids pQR606 and pQR609 respectively. Expression studies indicated a two fold increase in expression from plasmid pQR606 and a 22 fold increase in expression from plasmid pQR609 with respect to pQR601. The enzyme has subsequently been purified to homogeneity by a combination of ammonium sulphate precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gel filtration steps. Kinetic and molecular characteristics of the enzyme have been determined. Comparative amino acid sequence homology studies have been performed with other members of ANT(3")-I enzymes and nucleotidyltransferases. This has revealed extensive homology throughout, although certain regions of the gene appear to be more highly conserved than others. In order to test the biological importance of these regions, site-directed mutagenesis has been carried out to alter some highly conserved aspartic acid residues. The ability of mutant enzymes to confer resistance to antibiotic subtrates was evaluted.

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