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Infection and Immunity, January 2000, p. 233-238, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

David L. Lakey,1,2,3,4,5,* Rama K. R. Voladri,2 Kathryn M. Edwards,1 Cynthia Hager,1 Buka Samten,4 Robert S. Wallis,6 Peter F. Barnes,4,5 and Douglas S. Kernodle2,3

Divisions of Infectious Diseases, Departments of Pediatrics1 and Medicine,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232; Department of Veterans Affairs Medical Center, Nashville, Tennessee 372123; Center for Pulmonary and Infectious Disease Control4 and Departments of Medicine and Cell Biology,5 The University of Texas Health Center at Tyler, Tyler, Texas 75708; and Department of Medicine, Case Western Reserve University, Cleveland, Ohio 441066

Received 22 February 1999/Returned for modification 14 September 1999/Accepted 4 October 1999

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.

* Corresponding author. Mailing address: Center for Pulmonary and Infectious Disease Control, The University of Texas Health Center at Tyler, 11937 U.S. Highway 271, Tyler, TX 75708-3154. Phone: (903) 877-5957. Fax: (903) 877-7989. E-mail: dlakey{at}uthct.edu.

Infection and Immunity, January 2000, p. 233-238, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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