Expression of Taiwan banded krait phospholipase A2 in Escherichia coli, a fully active enzyme generated by hydrolyzing with aminopeptidase.
A cDNA encoding Bungarus multicinctus (Taiwan banded krait) phospholipase A2 was expressed in Escherichia coli. The expressed PLA2 contained a Met residue fused to the N-terminus of PLA2. The expressed protein was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. Then the N-terminus Met residue of recombinant PLA2 was cleaved with aminopeptidase. The resulting recombinant PLA2 had the same amino acid composition and N-terminal sequence of native PLA2 and displayed the same CD spectra and pI value as native enzyme. Moreover, the recombinant PLA2 was indistinguishable from native PLA2 in enzymatic activity and antigenicity. These results suggest that a fully active PLA2 enzyme has been successfully expressed as its native form in snake venom.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan, Republic of China. lschang@mail.nsysu.edu.tu
Wu
P F
PF
Chang
C C
CC
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017838:Bungarus / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D000626:Aminopeptidases