Studies on gene ARR expression
Date
2008-05-19T07:58:31Z
Authors
Gianniosis, Mary
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Abstract
ABSTRACT
Rifampicin is a major chemotherapeutic agent used against mycobacterial and
nocardial infections. High level resistance is primarily due to mutational
alterations in the rpoB gene encoding the β subunit of RNA polymerase. When
challenged, these bacteria may inactivate rifampicin by one of four mechanisms:
decomposition, ADP-ribosylation, glucosylation and phosphorylation. ADPribosylation
occurs in many mycobacterial pathogens but nothing is known about
the properties of the enzyme responsible. Consequently mutational analysis may
be used to explore structure-function relationships in this protein.
Three mutants with changes in the open reading frame were selected and studied.
The altered arr gene in pMG1 was obtained by in vivo selection whilst in pMG2
and pMG4 by in vitro mutagenesis. The mutated arr gene in pMG1 and pMG2
conferred resistance to 50 μg/ml of rifampicin while in pMG4 to 200 μg/ml. This
suggested that alterations near the N-terminus resulted in lowered activity because
of closer proximity to the active site. This is the first successful report of induced
arr gene expression. This over-expression of the Arr ADP-ribosyltransferase and
its mutants assisted in their later purification by metal affinity chromatography.
Description
Keywords
Over-expression and purification of ADP-ribosyltransferase that inactivates Rifampicin by ribosylation