Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.
The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.
Witmer
M R
MR
Macromolecular Structure Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Micanovic
R
R
Tredup
J
J
Lin
W
W
Hail
M
M
Villafranca
J J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli; D015870:Gene Expression; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D002414:Cations, Monovalent; D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004492:Edetic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D005990:Glycerol; D002118:Calcium