Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons

Abstract

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 fourto sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.Despite widespread administration of bacillus CalmetteGuérin vaccine throughout the world, tuberculosis remains the leading cause of death from a single pathogen (3). Development of subunit tuberculosis vaccines has been spurred by findings that partial immunity is conferred by vaccination of animals with culture filtrate proteins (1, 18), purified antigen 85B (12), and naked DNA encoding mycobacterial antigens (13, 23). A major problem with developing a subunit vaccine is that Escherichia coli overexpression systems do not provide good yields of some Mycobacterium tuberculosis proteins, even when mycobacterial genes are placed behind strong E. coli promoters (15). Because the GC content of E. coli genes is only 50%, E. coli may lack the transcriptional and translational machinery needed to efficiently produce proteins from mycobacterial genes, which have a GC content of 65 to 70% (7). Difficulties in overexpressing mycobacterial genes in E. coli have led investigators to produce mycobacterial proteins in baculovirus expression systems (2) or to use bacteria that are phylogenetically closer to mycobacteria, such as Streptomyces lividans, Corynebacterium spp., and Mycobacterium smegmatis (7,10,16,26).When the gene encoding antigen 85B was first sequenced and cloned behind a trc promoter in E. coli, less than 0.5 mg of recombinant antigen 85B per liter was produced (15). Whereas this yield can be improved to 10 mg/liter by using a stronger promoter such as T7 (9), problems with achieving higher-level expression and solubility of antigen 85B have remained. Therefore, investigators studying antigen 85B and many other secreted M. tuberculosis antigens have generally purified them from M. tuber...

Construction and expression of an ethanol production operon in Gram-positive bacteria

“…Other studies have shown that changing rare codons to codons optimal for the recombinant host can increase protein levels. For example, expression of cyt2Aa1 of Bacillus thuringiensis in Pichia pastoris is improved (Gurkan & Ellar, 2003) and production of antigen 85A from Mycobacterium tuberculosis in E. coli is increased 54-fold (Lakey et al, 2000).…”

TheMycobacterium tuberculosis19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Regulated HLA-DR and FcγR1 on Human Macrophages through Toll-Like Receptor 2

“…The 2392 anti-TLR-2 blocking Ab was graciously provided by Paul Godowski, Genentech Inc. (San Francisco, Calif.). Recombinant Ag85B of M. tuberculosis was produced as described previously (19).…”

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

“…No adipic acid was seen in the control culture lacking the cosmids (data not shown). The low levels of conversion most likely result from inefficient expression of high GϩC gram-positive genes and operons in E. coli (8,35). Previous work with the cyclohexanone degradation genes of Brevibacterium had shown that the flavin cyclohexanone monooxygenases can be easily expressed in E. coli in an active form, unlike the other genes of the pathway (8,9).…”