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 2530 nm in width and similar to 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 mu M 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.