HIGH-LEVEL HETEROLOGOUS EXPRESSION AND SECRETION IN RAPIDLY GROWING NONPATHOGENIC MYCOBACTERIA OF 4 MAJOR MYCOBACTERIUM-TUBERCULOSIS EXTRACELLULAR PROTEINS CONSIDERED TO BE LEADING VACCINE CANDIDATES AND DRUG TARGETS

Citation
G. Harth et al., HIGH-LEVEL HETEROLOGOUS EXPRESSION AND SECRETION IN RAPIDLY GROWING NONPATHOGENIC MYCOBACTERIA OF 4 MAJOR MYCOBACTERIUM-TUBERCULOSIS EXTRACELLULAR PROTEINS CONSIDERED TO BE LEADING VACCINE CANDIDATES AND DRUG TARGETS, Infection and immunity, 65(6), 1997, pp. 2321-2328
Citations number
26
Categorie Soggetti
Immunology,"Infectious Diseases
Journal title
ISSN journal
00199567
Volume
65
Issue
6
Year of publication
1997
Pages
2321 - 2328
Database
ISI
SICI code
0019-9567(1997)65:6<2321:HHEASI>2.0.ZU;2-1
Abstract
Mycobacterium tuberculosis, the primary etiologic agent of tuberculosi s, is the world's leading cause of death from a single infectious agen t, and new vaccines and drugs to combat it are urgently needed, The ma jor extracellular proteins of M. tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading c andidates for a vaccine and prime targets for new drugs, However, the development of these biologicals has been hampered by the unavailabili ty of large quantities of recombinant extracellular proteins identical to their native counterparts, In this report, we describe the heterol ogous expression and secretion of four major M. tuberculosis extracell ular proteins (the 30-, 32, 16-, and 23.5-kDa proteins-the first, seco nd, third, and eighth most abundant, respectively) in rapidly growing, nonpathogenic mycobacterial species, Multiple attempts to obtain secr etion of the proteins by using Escherichia coli- and Bacillus subtilis -based expression systems were unsuccessful, suggesting that high-leve l expression and secretion of these Mycobacterium-specific proteins re quire a mycobacterial host, All four recombinant proteins were stably expressed from the cloned genes' own promoters at yields that were 5- to 10-fold higher than those observed for the native proteins, The fou r proteins were purified to apparent homogeneity from culture filtrate s by ammonium sulfate precipitation and ion-exchange and molecular sie ve chromatography. The recombinant proteins were indistinguishable fro m their native counterparts by multiple criteria, First, N-terminal am ino acid sequence determination demonstrated that processing of the le ader peptides was highly accurate, Second, sodium dodecyl sulfate-poly acrylamide gel electrophoresis analysis revealed identical migration p atterns, Third, mass spectrometry analysis confirmed that differences in mass were less than or equal to 5 Ha, A homolog of the M. tuberculo sis 30-kDa protein was identified in M. smegmatis by means of DNA anal yses and immunoscreening. This is the first time that secretion of rec ombinant M. tuberculosis extracellular proteins in their native form h as been achieved, This study opens the door to mass production of corr ectly processed and secreted extracellular proteins of M. tuberculosis in a heterologous host and allows ready evaluation of their biologic and immunologic function.