Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogaster.
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFkappaB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly beta-sheets at pH 7. The good dispersion of the crosspeaks on the 1H-15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family.
Rautureau
Gilles
G
Centre de Biophysique Moléculaire, UPR 4301 CNRS affiliated to Orléans University and to INSERM, Rue Charles Sadron, 45071 Orléans cedex 2, France.
Jouvensal
Laurence
L
Decoville
Martine
M
Locker
Daniel
D
Vovelle
Françoise
F
Schoentgen
Françoise
F
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D029721:Drosophila Proteins; C515214:Pebp1 protein, Drosophila; D049488:Phosphatidylethanolamine Binding Protein; D011994:Recombinant Proteins