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Cloning the embC gene of Mycobacterium tuberculosis H37Rv
Author(s) -
Ongaro James,
Rawat Mamta,
Meyer Christopher R.,
Kantardjieff Katherine A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb54
Subject(s) - mycobacterium smegmatis , genomic dna , mycobacterium tuberculosis , cloning (programming) , ethambutol , gene , antimycobacterial , biology , plasmid , cloning vector , computational biology , recombinant dna , genetics , chemistry , microbiology and biotechnology , tuberculosis , vector (molecular biology) , medicine , pathology , computer science , programming language , streptomycin , antibiotics
Integral membrane arabinosylindolylacetylinositol synthases (embCAB) catalyze key regulatory steps in the cell wall biosynthesis of arabinogalactan and lipoarabinomanan in Mycobacterium spp. Furthermore, mutations to residues in several predicted loop regions facilitate ethambutol drug resistance—a first‐line antimycobacterial. To date the mechanisms of regulation and catalysis of embCABs are not understood and, as there are no high‐resolution X‐ray crystallographic structures of these enzymes, no structure‐function relationships have been deduced. We have successfully amplified the embC gene of Mycobacterium tuberculosis from genomic DNA using genomic sequence data from TubercuList and a two‐step nested PCR. The embC amplicon was subsequently ligated to the pALACE expression vector, which encodes for a 6x N‐terminal His‐tag, to generate pOKMC. Future studies involve the electroporation of pOKMC into M. smegmatis mc2155, purification via immobilized metal affinity chromatography, characterization and crystallization.