Identification And Release Process Of Active Components In RhTRAIL Inclusion Bodies

Inclusion bodies(IBs)commonly exist in E.coli expressing exogenous gene.Classical IBs are considered as misfolded protein aggregations of less value.Instead,non-classical inclusion bodies(ncIBs)exhibit biological activity of potential value.Despite attention was drawn to the application of ncIBs,there were few studies for ncIBs to date.In this paper,we studied the preparation method and biological activity of recombinant human TNF-Related Apoptosis Inducing Ligand(rhTRAIL)IBs.The release behavior of active components from IBs in different environments was also studied.The main conclusions are introduced as follows:1)The preparation conditions of rhTRAIL IBs suitable for subsequent studies were preliminary established through the single element experiment.IBs with high protein purity were prepared and showed clear outline with no adhesion and residual bacteria under scanning electron microscopy.2)The in vitro cytotoxicity assay showed that rhTRAIL IBs could significantly induce apoptosis of NCI-H460 and SW1990 cell,while had no effect on FBHE cell.In comparison,Green Fluorescent Protein(GFP)IBs had no effect on all three cell lines.It indicated the activity of IBs was related to protein type.Furthermore,Flow cytometry showed that rhTRAIL IBs killed NCI-H460 cell by inducing apoptosis,indicating the active components in IBs were TRAIL proteins.A native-like structure was found in rhTRAIL IBs by Fourier transform infrared spectrum meanwhile.3)The release process of active components in TRAIL IBs was studied in different systems including serum-free medium,PBS buffer,and 2M urea.In serum-free medium,the amount of released active components increased over time and was constant after 18 h,which was similar to that in PBS buffer system.The residual IBs after 18 h remained active and native-like structure.In comparison,the release amount was greatly improved and achieved stable after 48 h in serum-free medium with 2M urea.The activity and secondary structure of residual IBs didn't change before 48 h.However,the residual IBs became biologically inert and showed alterant structure after 48 h.