Abstract
Simple SummaryBaculovirus expression vector system (BEVS) is widely employed to produce eukaryotic recombinant proteins with desired post-translational modifications. The tumor necrosis factor α (TNFα) is a promising reagent in treating autoimmunity and cancer diseases. In the current study, we designed to express and purify human and murine TNFα proteins in a silkworm larva-based baculovirus expression vector system (silkworm-BEVS). The results demonstrated that the desirable productivity of proteins with similar biological activity was experimentally confirmed. It was revealed that the C-terminal fusion tags negatively impacted their biological activity, as confirmed in the cytotoxicity assay. Taken together, silkworm-BEVS is an alternative platform for supplying high-quality TNFα products for various purposes.The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.
Highlights
The tumor necrosis factor α (TNFα) is a pleiotropic cytokine produced from activated macrophages and lymphocytes that regulates both pro-inflammatory responses and cellular communications [1,2]
The biological activity of the purified rh/rmTNFα proteins was assayed in culture murine L929 cells (RCB2619, supplied by Riken Cell Bank, Tsukuba, Japan) [30], which have been routinely employed for the cytotoxicity assay of TNFα
Since both mature type TNFα cytokines execute biological functions as an extracellular form, we employed a signal peptide from the silkworm 30K protein 6G (30K6G) to support the expressed protein to be secreted into silkworm hemolymph [23,27]
Summary
The tumor necrosis factor α (TNFα) is a pleiotropic cytokine produced from activated macrophages and lymphocytes that regulates both pro-inflammatory responses and cellular communications [1,2]. High productivity of protein with reasonable post-translational modifications and great flexibility for large protein complexes are attractive hallmarks of BEVS using either lepidopteran cells or insects such as the domestic silkworm, Bombyx mori, one of the critical economic insects in many Asian countries [18,19,20,21]. It is the primary source of the silk industry and is recognized as a model organism for fundamental research. Our current attempt demonstrates an alternative platform for supplying high-quality TNFα products for further cytokine-related academic research and pharmaceutical clinical trials
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