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Applied and Environmental Microbiology, February 2004, p. 679-685, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.679-685.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

Martha Lara,1 Luis Servín-González,2 Mahavir Singh,3 Carlos Moreno,4 Ingrid Cohen,1 Manfred Nimtz,3 and Clara Espitia1*

Departamento de Inmunología,1 Departamento de Biología Molecular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F., México,2 GBF, German National Research Center for Biotechnology, Braunschweig, 38124 Braunschweig, Germany,3 Department of Bacteriology, Royal Free and University College Medical School, Windeyer Institute, London W1P 6DB, United Kingdom4

Received 22 May 2003/ Accepted 30 October 2003

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean {alpha}-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

* Corresponding author. Mailing address: Instituto de Investigaciones Biomédicas, Departamento de Inmunología, Apartado Postal 70-228, 04510 México, D.F., México. Phone (525) 6223818. Fax: (525) 6223369. E-mail: espitia{at}servidor.unam.mx.

Applied and Environmental Microbiology, February 2004, p. 679-685, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.679-685.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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