Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris.
The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.
Boué
O
O
Division of Mammalian Cell Genetics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, 10 600 Havana, Cuba. oscar.boue@cigb.edu.cu
Farnós
O
O
González
A
A
Fernández
R
R
Acosta
J A
JA
Valdés
R
R
González
L J
LJ
Guanche
Y
Y
Izquierdo
G
G
Suárez
M
M
Domínguez
I
I
Machado
H
H
Rodríguez
M
M
Lleonart
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D026863:Ixodidae / Q000235:genetics / Q000276:immunology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010316:Particle Size; D010843:Pichia / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D000941:Antigens; D011994:Recombinant Proteins