A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha
Nawal Abd El-Baky,
Esmail M. EL-Fakharany,
Soraya A. Sabry,
Ehab R. El-Helow,
Elrashdy Mustafa Redwan,
Amira Sabry
Affiliations
Nawal Abd El-Baky
Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, Alexandria P.O. Box 21934, Egypt
Esmail M. EL-Fakharany
Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, Alexandria P.O. Box 21934, Egypt
Soraya A. Sabry
Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria P.O. Box 21568, Egypt
Ehab R. El-Helow
Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria P.O. Box 21568, Egypt
Elrashdy Mustafa Redwan
Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah P.O. Box 80203, Saudi Arabia
Amira Sabry
Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, Alexandria P.O. Box 21934, Egypt
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.
