Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
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Acknowledgments
The authors wish to acknowledge the National Natural Science Foundation of China (no. 81001477), National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (no. 2012ZX09506001-004), and Zhejiang Provincial Natural Science Foundation of China (no. R2110231).
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Pan, L.Q., **e, Z.M., Tang, X.J. et al. Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1. Appl Microbiol Biotechnol 97, 7253–7264 (2013). https://doi.org/10.1007/s00253-012-4604-0
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DOI: https://doi.org/10.1007/s00253-012-4604-0