Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification.
The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.
Prestidge
R L
RL
Genesis Research and Development Corporation Ltd, Auckland, New Zealand.
Grandison
P M
PM
Chuk
D W
DW
Booth
R J
RJ
Watson
J D
JD
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002451:Cell Compartmentation; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility / Q000187:drug effects
C087864:19 kDa antigen, Mycobacterium; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003902:Detergents; D021382:Protein Sorting Signals; D011994:Recombinant Proteins