A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha

Abstract

Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

A novel therapeutic vaccine targeting the soluble TNFα receptor II to limit the progression of cardiovascular disease: AtheroVax™

“…The inhibition of PBMCs by rhTNF-α, IC 50 , is 1,728 ± 11 ng/ml and the effective concentration, EC 50 , is 250 ± 1 ng/ml. The IC 50 in Caco-2 cells was 297 ng/ml, HepG-2 cells was 197 ng/ml, and MCF-7 cells was 374 ng/ml ( 11 ).…”

Challenging Post-Translational Modification in the Cell-Free Protein Synthesis System

“…Tumor Necrosis Factor-alpha (TNF-α) plays a crucial role in inflammatory responses, immune cell signaling, and apoptosis of cancer cells [23]. In 2022, a team utilized CFPS to produce human TNF-α, which carries a single disulfide bond between cysteine residues 69 and 101 [23].…”

“…Tumor Necrosis Factor-alpha (TNF-α) plays a crucial role in inflammatory responses, immune cell signaling, and apoptosis of cancer cells [23]. In 2022, a team utilized CFPS to produce human TNF-α, which carries a single disulfide bond between cysteine residues 69 and 101 [23]. The team optimized the yield of soluble TNF-α protein using response surface methodology and then tested the cytotoxicity of the recombinant TNF-α against three different human cancer cell lines, Caco-2, HepG-2, and MCF-7, as well as normal human cells.…”