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Prokaryotic expression and characterization of prophenoloxidase from the Asian corn borer,Ostrinia furnacalis( Lepidoptera: Pyralidae)
Author(s): 
Pages: 798-805
Year: Issue:  7
Journal: Acta Entomologica Sinica

Keyword:  Ostrinia furnacalisprophenoloxidase(PPO)prokaryotic expressionprotein purificationphenoloxidase(PO)enzymatic characteristics;
Abstract: 【Aim】Prophenoloxidase( PPO) is an important immune protein in insects,which is involved in insect humoral immunity and cellular immunity. In our study,the prokaryotic expression system was applied to express soluble recombinant PPO with activity on a large scale. The recombinant PPO was used to screen various phenoloxidase( PO) inhibitors so as to create novel insecticides targeted on the insect immune system. 【Methods】Using the PPO gene cloned from the 5th instar larvae of the Asian corn borer,Ostrinia furnacalis( Guenée),the prokaryotic expression vector,pET-28b-PPO,was constructed,and the recombinant PPO protein was expressed in Escherichia coli. The fusion protein was purified from the supernatant of the lysis of E. coli cells with Ni-NAT affinity chromatography column,and identified with Western blotting. Enzymatic characteristics of phenoloxidase( PO) activated from the recombinant PPO with cetylpyridinium chloride( CPC) and the effects of metal ions,such as Mg2 +,Cu2 +and Fe2 +on the secondary structure of the recombinant PPO were also assayed and analyzed. 【Results】The recombinant PPO protein was expressed and purified. The suitable temperature for PO obtained throughthe activation of the recombinant PPO was 30℃,and the suitable pH was 7. 2. The kinetic parameters calculated for substrate oxidation were Vmax= 140. 8 U /mg·min and Km = 0. 92 mmol /L for L-DOPA.The content of β-sheet in the recombinant PPO increased dramatically to 53. 7% ± 4. 6% when Fe2 +ions existed. However,the content of α-helix decreased significantly to 2. 6% ± 1. 2%( P < 0. 05). The content of β-sheet in the recombinant PPO reduced clearly and the content of α-helix increased slowly when Mg2 +ions existed. The content of β-sheet in the recombinant PPO declined remarkably to 10. 0% ±1. 6% when Cu2 +ions were present,but the content of α-helix increased significantly to 35. 3% ±6. 9%. 【Conclusion】The results suggest that the secondary structure of the recombinant PPO is affected notably by different metal ions.
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