An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis.
A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.
Triccas
J A
JA
Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France. jtriccas@pasteur.fr
Parish
T
T
Britton
W J
WJ
Gicquel
B
B
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D020161:Physical Chromosome Mapping; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D000581:Amidohydrolases; C021445:acetamidase