Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.
A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.
Lakey
D L
DL
Divisions of Infectious Diseases, Departments of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. dlakey@uthct.edu
Voladri
R K
RK
Edwards
K M
KM
Hager
C
C
Samten
B
B
Wallis
R S
RS
Barnes
P F
PF
Kernodle
D S
DS
D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013482:Superoxide Dismutase / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D003062:Codon; D017931:DNA Primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013482:Superoxide Dismutase