Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli.
The secreted phospholipases A2 (sPLA2s) 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 A2 (hsPLA2-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 hsPLA2-IID in Escherichia coli, the DNA-coding sequence for hsPLA2-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 hsPLA2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A2. The refolded recombinant hsPLA2-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 hsPLA2-IID which will advance our understanding of the structure-function relationship and biological effects of the protein.
Department of Biochemistry and Immunology, FMRP-USP, Universidade de São Paulo, Brazil.
Raquel Gomes
RG
Fonseca
Tatiana Lopes
TL
Ferreira
Richard J
RJ
Ward
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000201:enzymology; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010743:Phospholipids; D011994:Recombinant Proteins; D054467:Phospholipases A2; D019791:Guanidine