Open AccessUse of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase
Author(s) -
Usha Veeraraghavan,
Dover Lynn G.,
Roper David L.,
Lloyd Adrian J.,
Besra Gurdyal S.
Publication year - 2006
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2006.00356.x
Subject(s) - mycobacterium tuberculosis , escherichia coli , biology , antimycobacterial , cloning (programming) , translation (biology) , microbiology and biotechnology , gene , enzyme , recombinant dna , diaminopimelic acid , tuberculosis , mutation , genetics , biochemistry , messenger rna , peptidoglycan , medicine , pathology , computer science , programming language
Previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli resulted in undetectable enzyme yields. We used silent mutation of the first 10 codons of the recombinant ORF in an attempt to reduce the formation of secondary structures that might occur near the 5′ end of the mRNA and inhibit translation. This significantly increased the yield of the enzyme, which was purified and characterized biochemically. This strategy could be generally applied to other mycobacterial genes that are difficult to express hetero‐specifically and here provided pure M. tuberculosis DapF, a good foundation for future research in antimycobacterial agents.