Endothelial differentiation-related factor1 (EDF-1) encodes a 16-kDa polypeptide, characterized by the presence of a conserved region of about 20 amino acids, the IQ motif, usually presents in CaM-binding domains1. EDF-1 serves two main functions in endothelial cells: it regulates CaM availability in the cytosol, and it acts in the nucleus as a transcriptional coactivator2. Modulators of calmodulin are potential therapeutic targets 3, so their structural and functional characterization is of great interest. With this work we present the protocol that we have optimized to obtain an NMR sample of EDF-1 and the preliminary screening of the protein folding. The gene coding for EDF-1 has been cloned into pETG20A (Invitrogen), that contains the DNA fragments coding for the fusion tag Trx-(his)6. E. coli host strain used for expression of EDF-1 was BL21 Codon plus. Protein expression has been induced with Isopropil β-thiogalactopiranoside (IPTG) and protein purification of EDF-1, produced in inclusion bodies, has been obtained with a denaturation-refolding step on the nickel chelate column. The correct folding of the final sample, without its fusion tag, has been verified by monodimensional and bidimensional HSQC NMR spectra. At present structural determination is under progress.
Federica Casadei, Elena Babini, Ivano Bertini, Francesco Capozzi, Emanuele Chirivino, Claudio Luchinat (2005). Expression and purification of the human Calmodulin target Endothelial Differentiation-related Factor 1.
Expression and purification of the human Calmodulin target Endothelial Differentiation-related Factor 1
CASADEI, FEDERICA;BABINI, ELENA;CAPOZZI, FRANCESCO;
2005
Abstract
Endothelial differentiation-related factor1 (EDF-1) encodes a 16-kDa polypeptide, characterized by the presence of a conserved region of about 20 amino acids, the IQ motif, usually presents in CaM-binding domains1. EDF-1 serves two main functions in endothelial cells: it regulates CaM availability in the cytosol, and it acts in the nucleus as a transcriptional coactivator2. Modulators of calmodulin are potential therapeutic targets 3, so their structural and functional characterization is of great interest. With this work we present the protocol that we have optimized to obtain an NMR sample of EDF-1 and the preliminary screening of the protein folding. The gene coding for EDF-1 has been cloned into pETG20A (Invitrogen), that contains the DNA fragments coding for the fusion tag Trx-(his)6. E. coli host strain used for expression of EDF-1 was BL21 Codon plus. Protein expression has been induced with Isopropil β-thiogalactopiranoside (IPTG) and protein purification of EDF-1, produced in inclusion bodies, has been obtained with a denaturation-refolding step on the nickel chelate column. The correct folding of the final sample, without its fusion tag, has been verified by monodimensional and bidimensional HSQC NMR spectra. At present structural determination is under progress.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
