Use of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase.
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.
Usha
Veeraraghavan
V
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Dover
Lynn G
LG
Roper
David L
DL
Lloyd
Adrian J
AJ
Besra
Gurdyal S
GS
D000591:Amino Acid Isomerases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D003062:Codon; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase