LAUSR

Simple Summary As a result of increasing demand for the pleiotropic cytokine TNF-α, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous in vivo expression systems. While in vivo expression of this cytokine is laborious and lengthy, cell-free or in vitro expression system has the benefits of speed, simplicity, flexibility, focus of all the system energy on target protein synthesis alone, besides high soluble and functional protein yield. Therefore, we employed and optimized an E. coli-based cell-free system for the first time to express recombinant human TNF-α. Our findings revealed that cell-free expression system can be an alternative platform for producing soluble and functionally active recombinant TNF-α with a yield of 390 µg/mL in only 2 h at a temperature of 40 °C for further research and clinical trials. 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.