Bacterial expression and characterization of human pancreatic phospholipase A2.
Mammalian pancreatic phospholipases A2 (PLA2) have recently been implicated in cell surface receptor-mediated inflammation. As a first step toward understanding how human pancreatic PLA2 (hp-PLA2) interacts with membranes and other biological targets including cell-surface receptors, we constructed its bacterial expression vector which can be used for the mutagenesis and protein over-expression. The expression vector (pSH-hp) was constructed using a synthetic hp-PLA2 gene whose transcription is controlled by T7 promoter. hp-PLA2 was expressed as a mature protein in high concentration in Escherichia coli cells and formed inclusion body. The solubilization of inclusion body protein followed by the refolding and purification produced ca. 5 mg of pure protein from one liter of growth medium. Kinetic studies of recombinant human, bovine and porcine pancreatic PLA2s using polymerized mixed liposomes and micelles as substrates showed that despite their highly homologous structures these mammalian pancreatic PLA2s have distinct phospholipid head group specificity and different activity toward various lipid substrates.
Han
S K
SK
Department of Chemistry, University of Illinois at Chicago, USA.
Lee
B I
BI
Cho
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054467:Phospholipases A2; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2