« Previous Abstract"Disulfide pairing and secondary structure of ASP1, an olfactory-binding protein from honeybee (Apis mellifera L)"    Next Abstract"Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)" »

Protein Expr Purif

Title:		Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris
Author(s):		Briand L; Lescop E; Bezirard V; Birlirakis N; Huet JC; Henry C; Guittet E; Pernollet JC;
Address:		"Unite de Recherches de Biochimie et Structure des Proteines, UR 477, INRA, Domaine de Vilvert, Jouy-en-Josas Cedex, F-78352, France. lbriand@jouy.inra.fr"
Journal Title:		Protein Expr Purif
Year:		2001
Volume:		23
Issue:		1
Page Number:		167 - 174
DOI:		10.1006/prep.2001.1478
ISSN/ISBN:		1046-5928 (Print) 1046-5928 (Linking)
Abstract:		"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"
Keywords:		"Animals Bees/*chemistry Carbon Isotopes/metabolism Carrier Proteins/biosynthesis/chemistry/isolation & purification Chromatography Cloning, Molecular/methods *Insect Proteins Nitrogen Isotopes/metabolism *Nuclear Magnetic Resonance, Biomolecular Pichia/*m;"
Notes:		"MedlineBriand, L Lescop, E Bezirard, V Birlirakis, N Huet, J C Henry, C Guittet, E Pernollet, J C eng 2001/09/26 Protein Expr Purif. 2001 Oct; 23(1):167-74. doi: 10.1006/prep.2001.1478"