Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogaster

Protein Expression and Purification
Gilles RautureauFrançoise Schoentgen

Abstract

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 p...Continue Reading

Citations

Mar 25, 2009·Archives of Insect Biochemistry and Physiology·Gilles RautureauMartine Decoville
Jul 16, 2008·Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Médicas E Biológicas·P ZhangY Q Wei
Jun 26, 2009·Molecular and Cellular Biochemistry·Hualin SunXiaosong Gu
Feb 5, 2010·Proteins·Gilles J P RautureauLaurence Jouvensal

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