Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli

Abstract

Different nutrient-feeding cultures were carried out in producing recombinant protein of truncated tumor necrosis factor related apoptosis-inducing ligand (TRAIL) (114-281 amino acids of TRAIL) in Escherichia coli strain C600/pBV-TRAIL. The effects of preinduction specific growth rate, postinduction carbon source (glucose and glycerol), and feeding strategies were investigated. The higher preinduction specific growth rate (mu = 0.22 h(-1)) contributed to the increase in the TRAIL production, at which TRAIL was accumulated in bacterial cells as 7.2% of total cellular protein, corresponding to 1.99 g l(-1) in contrast with 5.1% (1.29 g l(-1)) at preinduction specific growth rate (mu = 0.1 h(-1)) during high-cell-density culture. Glycerol was superior to glucose as the postinduction carbon source for TRAIL production. Under similar culture conditions, the final concentration of TRAIL was produced 1.59-fold more when glycerol was used as postinduction carbon source than when glucose was used. At the same time, the results showed that it is efficient to adopt the pH-stat feeding strategy at postinduction for the overproduction of TRAIL. The TRAIL production was increased up to 4.51 g l(-1), approximately 16.1% of total cellular protein. The mechanisms behind the preinduction specific growth rate effect on the expression level may be ascribed to the leakage secretion of acetate.

Application of a Fed-Batch Bioprocess for the Heterologous Production of hSCOMT in Escherichia coli

“…The media under study had the following carbon range composition: glycerol (10,20,30,40, and 60 g/l) and glucose (15,20, and 30 g/l). In addition, the pH was close to 7.2 without adjustment before sterilization, and all the components were mixed and dissolved in deionized water and autoclaved for 15 min at 120 o C. After autoclaving, a specific volume of a standard glucose solution was added under aseptic environment conditions to the medium in order to obtain the desired substrate concentration.…”

“…Recently, several studies have demonstrated that glycerol is superior to glucose in reducing acetate formation during the growth phase, and interestingly, increased recombinant protein formation when supplied post induction [20]. In flask experiments, it was observed that an increase in the glycerol concentration did not affect considerably hSCOMT production and specific activity levels.…”

“…Nevertheless, this main drawback in E. coli growth can be avoided by keeping low specific growth rates (e.g., fedbatch control algorithms on dissolved oxygen tension and pH), by genetically altering the pathways involved in the formation of acetate [7,28] or merely by selecting strains that have a particular genotype [27] and properly selecting the fermentation medium components [9]. For instance, recent results have demonstrated that glycerol is superior to glucose for reduced acetate and increased recombinant protein production when supplied post induction [20,26].…”

Sensitization of rhTRAIL-resistant Triple-negative Breast Carcinoma Through Silibinin Co-Treatment

“…rhTRAIL was produced according to well defined and previously detailed protocols (3)(4)(5). Silibinin (molecular weight of 482.44 g/mol, Sigma, Milwaukee WI, USA) was dissolved in Polyethylene Glycol (PEG) molecular weight 400 (Fisher Scientific, Pittsburgh PA, USA) at 10 mg/mL.…”

“…rhTRAIL consists of the extracellular C-terminus of TRAIL amino acids 114-281 lacking exogenous sequence tags (2-6). The appealing aspect of rhTRAIL is that it possesses the capacity to induce apoptosis in cancer cells without producing a toxic impact on normal, non-transformed cells (2)(3)(4)(5)(6). The extrinsic pathway of apoptosis is induced when rhTRAIL interacts with the extracellular death receptors (DRs) 4 and 5 stimulating the trimerization of the receptors, and consequently leading to activation of caspase 8, activation of the executioner caspases 3, 6, and 7, and the ultimate cleavage of poly (ADP-ribose) polymerase or PARP (hallmark of apoptosis) (7).…”