cDNA cloning and bacterial expression of phospholipase A(2) inhibitor PLIalpha from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus(1).
The cDNA encoding of a phospholipase A(2) inhibitor (PLIalpha) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIalpha. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIalpha. The PLIalpha cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIalpha expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIalpha fusion protein underwent folding to form a trimeric structure like the intact PLIalpha, and showed inhibitory activity against the group II acidic PLA(2) from A. blomhoffii siniticus venom; although its binding constant (1/K(i)) value was 30-fold lower than that of the natural PLIalpha. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIalpha against the acidic PLA(2). Furthermore, the carbohydrate chains of the natural PLIalpha were found to play an important role in the inhibitory activity against the basic PLA(2).
Okumura
K
K
Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka, Japan.
Inoue
S
S
Ikeda
K
K
Hayashi
K
K
D017836:Agkistrodon / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015723:Gene Library; D008969:Molecular Sequence Data; D009154:Mutation; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001798:Blood Proteins; D018076:DNA, Complementary; C400481:PLIalpha; D010726:Phosphodiesterase Inhibitors; D010741:Phospholipases A