Production of Soluble Murine TRAILs in Escherichia coli with Zn²⁺ Supplementation

Title:Production of Soluble Murine TRAILs in Escherichia coli with Zn²⁺ Supplementation

Volume: 29 Issue: 12

Author(s): Xupu Wang, Lizheng Wang, Wenmo Liu, Xinyao Feng, Hui Wu, Haihong Zhang, Jiaxin Wu, Wei Kong, Xianghui Yu and Bin Yu*

Affiliation:

• National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China

Keywords: murine TRAIL, soluble expression, anti-tumor activity, prokaryotic expression, apoptosis, homotrimer

Abstract:

Background: Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported.

Objective: In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein.

Methods: Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo.

Results: Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn²⁺ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models.

Conclusion: A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Cite this article as:

Wang Xupu, Wang Lizheng, Liu Wenmo, Feng Xinyao, Wu Hui, Zhang Haihong, Wu Jiaxin, Kong Wei, Yu Xianghui and Yu Bin*, Production of Soluble Murine TRAILs in Escherichia coli with Zn²⁺ Supplementation, Protein & Peptide Letters 2022; 29 (12) . https://dx.doi.org/10.2174/0929866529666220912112328

DOI https://dx.doi.org/10.2174/0929866529666220912112328	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305