Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris.
Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.
Briand
L
L
Unité de Recherches de Biochimie et Structure des Protéines, UR 477, INRA, Domaine de Vilvert, Jouy-en-Josas Cedex, F-78352, France. lbriand@jouy.inra.fr
Lescop
E
E
Bézirard
V
V
Birlirakis
N
N
Huet
J C
JC
Henry
C
C
Guittet
E
E
Pernollet
J C
JC
D000818:Animals; D001516:Bees / Q000737:chemistry*; D002247:Carbon Isotopes / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D002845:Chromatography; D003001:Cloning, Molecular / Q000379:methods; D019476:Insect Proteins; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000378:metabolism*; D018035:Receptors, Odorant / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
C108516:ASP2 protein, Apis mellifera; D002247:Carbon Isotopes; D002352:Carrier Proteins; D019476:Insect Proteins; D009587:Nitrogen Isotopes; D018035:Receptors, Odorant; C121271:antennal-specific protein 1, Apis mellifera; C045648:odorant-binding protein