Chemical definition, cloning, and expression of the major protein of the leprosy bacillus.
The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection. Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol. By committing 0.3 g of M. leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein. Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass. The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog. The work represents the first complete chemical definition of an M. leprae protein. PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein. Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein. The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.
Rivoire
B
B
Department of Microbiology, Colorado State University, Fort Collins 80523.
Pessolani
M C
MC
Bozic
C M
CM
Hunter
S W
SW
Hefta
S A
SA
Mehra
V
V
Brennan
P J
PJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D018835:Chaperonin 10; D018834:Chaperonin 60; D003001:Cloning, Molecular; D006360:Heat-Shock Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000737:chemistry*; D011994:Recombinant Proteins / Q000276:immunology
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins