Optimization of crystals from nanodrops: crystallization and preliminary crystallographic study of a pheromone-binding protein from the honeybee Apis mellifera L.
Pheromone-binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in various sensory organs from moths and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. Here, crystals of a PBP (Amel-ASP1) originating from honeybee (Apis mellifera L.) antennae and expressed as recombinant protein using the yeast Pichia pastoris are reported. Crystals of Amel-ASP1 have been obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot under the following conditions: 200 nl of 40 mg ml(-1) protein solution in 10 mM Tris, 25 mM NaCl pH 8.0 was mixed with 100 nl of well solution containing 0.15 M sodium citrate, 1.5 M ammonium sulfate pH 5.5. The protein crystallizes in space group C222(1), with unit-cell parameters a = 74.8, b = 85.8, c = 50.2 A. With one molecule in the asymmetric unit, V(M) is 3.05 A(3) Da(-1) and the solvent content is 60%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). The nanodrop crystallization technique used with a novel optimization procedure made it possible to consume small amounts of protein and to obtain a unique crystal per nanodrop, suitable directly for data collection in-house or at a synchrotron-radiation source.
Lartigue
Audrey
A
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités Aix-Marseille I and 2, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20, France.
Gruez
Arnaud
A
Briand
Loïc
L
Pernollet
Jean-Claude
JC
Spinelli
Silvia
S
Tegoni
Mariella
M
Cambillau
Christian
C
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001516:Bees / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; C403713:pheromone binding protein, insect; D000645:Ammonium Sulfate