Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules.
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 K(m) of 43 ± 2 μM and k(cat) of 0.04 ± 0.01 min⁻¹; however, k(cat) 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 Ni⁺² ions.
Graduate Institute of Biotechnology,  National Chung Hsing University, Taichung,  Taiwan.
Gary Ro-Lin
GR
Chang
Min-Ying
MY
Wang
Jiahn-Haur
JH
Liao
Yu-Ping
YP
Hsiao
Su-Yuan
SY
Lai
D018175:Birnaviridae Infections / Q000821:virology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D007243:Infectious bursal disease virus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009532:Nickel / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D015678:Viral Structural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure
D011994:Recombinant Proteins; C478245:VP4 protein, infectious bursal disease virus; D015678:Viral Structural Proteins; D009532:Nickel; D010450:Endopeptidases