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  • Title: Amplification of six putative RD1 genes of Mycobacterium tuberculosis for cloning and expression in Escherichia coli and purification of expressed proteins.
    Author: Amoudy HA, Mustafa AS.
    Journal: Med Princ Pract; 2008; 17(5):378-84. PubMed ID: 18685277.
    Abstract:
    OBJECTIVES: To amplify, clone and express in Escherichia coli six open reading frames (ORFs) predicted in the RD1 DNA segment of Mycobacterium tuberculosis and purify the expressed proteins to homogeneity. MATERIALS AND METHODS: DNA corresponding to the coding regions of six RD1 ORFs, i.e. ORF10 to ORF15, was amplified from genomic DNA of M. tuberculosis, cloned in the plasmid vector pPCR-Script and subcloned in expression plasmid vectors pET29a and/or pGEX-4T for expression in E. coli as fusion proteins. The recombinant fusion proteins were identified by sodium dodecyl polyacrylamide gel electrophoresis and Western immunoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. RESULTS: DNA corresponding to all six targeted RD1 ORFs was amplified from the genomic DNA of M. tuberculosis and five of the six ORFs, with the exception of ORF13, were cloned in the plasmid vectors and expressed in E. coli. Because of extensive degradation of ORF10 and ORF12 fusion proteins or nonbinding to the affinity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homogeneity. CONCLUSION: All of the six targeted RD1 genes were amplified and five expressed using E. coli hosts, but only two of the expressed proteins were purified to homogeneity. Alternative expression systems are required to obtain all RD1 proteins for functional characterization.
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