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標題: Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules
作者: Chang, Gary Ro-Lin
Wang, Min-Ying
Liao, Jiahn-Haur
Hsiao, Yu-Ping
Lai, Su-Yuan
關鍵字: endopeptidase activity
immobilized metal-ion affinity chromatography (IMAC)
infectious bursal disease virus
nickel ions
Lys dyad
tubule-like particles
摘要: Viral protein 4 (VP4) is a serine protease that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 of infectious bursal disease virus. In this report, the recombinant VP4 with a His-tag and three mutants (VP4-S652A, VP4-K692A and VP4-S652A.K692A) were expressed in Escherichia coli. Soluble VP4 was purified using immobilized metal-ion affinity chromatography or sucrose density gradient following with gel-filtration chromatography. The purified VP4 has a tubular structure with 25–30 nm in width and ∼300 nm in length, as observed by transmission electron microscope. A similar tubular structure was also found for these three mutants. The endopeptidase activity of these VP4 tubules was characterized by fluorescence resonance energy transfer using a synthetic fluorogenic oligopeptide as a substrate. The results show that the tubule-like VP4 is a functional enzyme with Km of 43+ 2 mM and kcat of 0.04+0.01 min21; however, kcat of three mutants were significantly reduced. This is the first report to demonstrate that VP4 protein expressed in E. coli can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni12 ions.
Appears in Collections:生物科技學研究所



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