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KMID : 0364820070430030166
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Korean Journal of Microbiology
2007 Volume.43 No. 3 p.166 ~ p.172
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Cloning of tlrD, 23S rRNA Monomethyltransferase Gene, Overexpression in Escherichia and Its Activity
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Jin Hyung-Jong
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Abstract
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ERM proteins transfer the methyl group to A2058 in 23S rRNA, which reduces the affinity of MLS (macrolidelincosamide-
streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on microorganisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tlrD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at 37oC accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as 18oC did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.
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KEYWORD
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in vivo activity, minmal inhibitory concentraion (MIC), MLSB (macrolide-lincosamide-streptogramin B) antibiotic resistance factor protein, overexpression, TlrD
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