Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Original Paper Med Princ Pract 2008;17:378–384 DOI: 10.1159/000141501 Amplification of Six Putative RD1 Genes of Mycobacterium tuberculosis for Cloning and Expression in Escherichia coli and Purification of Expressed Proteins Hanady A. Amoudy Abu S. Mustafa Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait ity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homo- geneity. 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. Al- ternative expression systems are required to obtain all RD1 proteins for functional characterization. Copyright © 2008 S. Karger AG, Basel Introduction Tuberculosis (TB) is a major infectious disease prob- lem around the world with an estimated incidence of 8.9 million cases and 1.7 million deaths annually [1]. The global problem of TB is worsening mainly due to the in- crease in multidrug-resistant TB and coinfection with HIV [1]. The effective control of TB requires identifica- tion and availability of Mycobacterium tuberculosis-spe- cific proteins as immunological reagents for diagnosis of TB. Although a large number of proteins of M. tubercu- losis have been identified in the past, most of them are shared with the vaccine strains of Mycobacterium bovis BCG [2–5] , and thus differentiation between individuals infected with M. tuberculosis and vaccinated with M. bo- Key Words Mycobacterium tuberculosis  RD1 genes  Cloning and expression  Protein purification 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 ex- pressed 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 re- combinant fusion proteins were identified by sodium do- decyl polyacrylamide gel electrophoresis and Western im- munoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. Results: DNA correspond- ing to all six targeted RD1 ORFs was amplified from the ge- nomic 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 affin- Received: July 31, 2007 Revised: January 8, 2008 Dr. Hanady A. Amoudy Department of Microbiology, Faculty of Medicine, Kuwait University PO Box 24923 13110 Safat (Kuwait) Tel. +965 498 6506, Fax +965 533 2719, E-Mail amoudy@hsc.edu.kw © 2008 S. Karger AG, Basel 1011–7571/08/0175–0378$24.50/0 Accessible online at: www.karger.com/mpp