Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

Autor(es)

Fonseca, Raquel Gomes ^[1] ; Ferreira, Tatiana Lopes ^[2] ; Ward, Richard J. ^[3]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Biochem & Immunol, FMRP USP, BR-05508 Sao Paulo - Brazil

^[2] Verdartis Desenvolvimento Biotecnol SS Ltda, Ribeirao Preto - Brazil

^[3] Univ Sao Paulo, FFCLRP USP, Dept Chem, BR-05508 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 67, n. 2, p. 82-87, OCT 2009.
• Citações Web of Science: 5

Resumo

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 02/12746-2 - Investigação de mudanças conformacionais de bothropstoxina I (PLA2-Lys49) do veneno de Bothrops jararacussu em associação com membranas artificiais
• Beneficiário: Tatiana Lopes Ferreira
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 06/54285-2 - Atividade da fosfolipase A2 secretada humana do grupo IID (hsPLA2-IID) contra membranas artificiais e biológicas
• Beneficiário: Raquel Fonseca Maldonado
• Modalidade de apoio: Bolsas no Brasil - Mestrado