SR3335 br Materials and methods br Results
Materials and methods
Discussion Schistosome cercarial elastase secreted from the acetabular glands of cercariae is recognized to play a critical role in mammalian host skin penetration upon infection. The present study extends the understanding of biochemical and immunological property of S. japonicum CE. Unlike S. mansoni, only one elastase was identified in S. japonicum, SjCE-2b, an ortholog of SmCE-2b. Immunofluorescence assay has confirmed the presence of SjCE in sporocyst and cercaria stages and in infected mice skin, implying its role in invasion of mammalian host (; Liu et al., 2015). However, little is known of its biochemical and immunogenic traits. In this study, the SjCE-2b gene was cloned and expressed in E. coli, which induced the formation of inclusion body in high level expression. Although optimization of SR3335 parameters including IPTG concentrations and expression temperatures was examined, the system could not produce soluble protein. In general, the IBs are resistant to proteolysis and contain a mass of comparatively pure proteins, their formation is often utilized for the production of proteins that are toxic, unstable or structurally reversible (Baneyx, 1999). It has been reported that a degradation product due to auto-proteolysis of natural S. mansoni cercarial elastase results in 16–17 kDa peptides (McKerrow et al., 1985; Salter et al., 2002). In our system, high level expression of the protein in IBs avoids self-degradation, as compared to soluble expression forms reported (Singh and Panda, 2005), thus having an apparent advantage to enable high yield of SjCE-2b protein. To recover bioactive protein, the two-step-denaturing and refolding method has proven to be highly efficient and applicable in many cases (Yang et al., 2011). The result of our study supports the applicability of this technique, achieving an 83% yield and 90% purity of refolded rSjCE-2b protein. In processing, the first strong guanidine hydrochloride denaturing step is used to completely unfold the misfolded protein in the IBs, while the subsequent dilution is used to obtain partially refolded intermediates in the precipitate. Resolubilization of the intermediates under a mild urea condition allows restoration of native structure. Notably, a suitable protein concentration is crucial during processing. In our case for SjCE-2b, 8 mg/ml in urea buffer was found optimum for avoiding possible re-aggregation in the process. The refolding pH is also an important parameter, as lower pH (<8.0) is prone to re-aggregation, whereas higher pH (>10.0) may cause protein instability. Thus an optimal pH at a range of 8.5–9.5 was used. Additionally, two purification steps ensure the quality of recovered bioactive form. The Ni-NTA affinity chromatography before refolding eliminated any potential influences of contaminated E. coli components, and the size exclusion chromatography after refolding removed possible auto-degraded molecules from the refolded rSjCE-2b product. Biochemical characterization of SjCE-2b has not been reported due to the insolubility and denatured conformation of the recombinant molecule in bacteria-expressed aggregates which failed to display its enzymic activity. Recovery of refolded bioactive protein possessing native conformation enabled us to perform biochemical assays. This study demonstrated the enzymatic activity of refolded rSjCE-2b with the substrate Suc-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin-like serine proteases, and determined a Km value 0.116 mM at an optimum pH of 9.0. Almost all previous investigations on schistosome proteinase activity were based on the assays with S. mansoni cercariae secretions or extracts. Earlier studies used a substrate succinyl-ala-ala-pro-phe p-nitroanilide, the same as we used, to assay the elastase activity in S. mansoni cercarial secretions at pH 9.0, and reported a Km value 0.029 mM (Salter et al., 2000, 2002; McKerrow et al., 1985). Another study to assess the elastase activity of S. mansoni in cercarial secretions employed a substrate Boc-Val-Leu-Gly-Arg-PNA, which reacts specifically with trypsin-like serine proteinases, and detected a Km value 0.095 mM at an optimum pH value around 10.5 (Bahgat and Ruppel, 2002). As the cercarial secretions may contain all isoforms of serine proteinases, the previous studies on S. mansoni used different substrate depending on the target form to be assayed. The SmCE is a biologically potent protease family capable of degrading insoluble elastin. Within the isoforms family, SmCE-1a and SmCE-1b are the most abundant in cercarial extracts, while SmCE-2b was at a detectable level, but purified bioactive protein was unavailable. We expect to further characterize the rSjCE-2b by examining the substrate specificity and inhibition kinetics, via scaling up the quantity of refolding production.