Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.
A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.
Gioffré
A
A
Instituto de Biotecnología, CICVyA, INTA, Castelar, Argentina.
Caimi
K
K
Zumárraga
M J
MJ
Meikle
V
V
Morsella
C
C
Bigi
F
F
Alito
A
A
Santángelo
M P
MP
Paolicchi
F
F
Romano
M I
MI
Cataldi
A
A
D000818:Animals; D001426:Bacterial Proteins / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D008074:Lipoproteins / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016927:Mycobacterium avium subsp. paratuberculosis / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000145:classification / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008074:Lipoproteins; D011994:Recombinant Proteins