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Enzyme activity of Schistosoma japonicum cercarial elastase SjCE-2b ascertained by in vitro refolded recombinant protein

Author:
Zhang, Ting, Mo, Xiao-Jin, Xu, Bin, Yang, Zhong, Gobert, Geoffrey N., Yan, Shuai, Feng, Zheng, Hu, Wei
Source:
Acta tropica 2018
ISSN:
0001-706X
Subject:
Schistosoma japonicum, bacteria, cercariae, elastase, enzyme activity, inclusion bodies, mice, proteolysis, recombinant proteins, variance
Abstract:
Cercarial elastase (CE) secreted from cercariae is evinced to play a pivotal role in initial skin penetration of mammalian host. SjCE-2b, a Schistosoma japonicum CE orthologous to SmCE-2b in S. mansoni, was previously found present in cercarial stage to aid skin invasion, but its enzyme activity has not been validated due to the insolubility and altered conformation when expressed recombinantly in bacteria as inclusion bodies. We report here for the first time a bioactive and soluble recombinant SjCE-2b recovered successfully from inclusion bodies by refolding approaches, enabling our biochemical and immunological investigation of this enzyme. Using a “two-step-denaturing and refolding” method, we recovered an 83% yield with 90% purity of refolded protein. Proteolytic activity of rSjCE-2b was demonstrated and characterized by enzymatic assay, showing a Km of 0.116 mM and a specific activity of 1900 nmol p-nitroaniline/min/mg protein. A significant immunoprotective response was evidenced in mice immunized with refolded rSjCE-2b. The result of immunoprotection test is at apparent variance with previously reported findings using S. mansoni CE preparation, which was poorly immunogenic in immunized animals. This work extends the knowledge of schistosome cercarial protease, and presents a bioactive form of S. japonicum recombinant CE with high yield and good quality. This will allow further biochemical and biological investigations to explore schistosome CE activity and better understand the molecular mechanisms associated with cercarial skin invasion of the mammalian host.
Agid:
6092984