Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli

Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System

“…However, it is also troublesome when the extra tags affect the protein conformation and stability, attenuating the protein function in the end [43]. To date, most of the recombinant TNFα proteins from various systems have been purified with affinity tags such as Polyhistidine and Glutathione S-transferase (GST) [8,14,44,45]. Based on our results, it might be better to remove the terminal tags to achieve the best performance of the cytotoxicity of TNFα.…”

“…It has been reported that recombinant human and murine TNFα has been successfully produced in other protein expression hosts with a milligram (mg) scale [8,10,12,13]. Compared with other protein expression systems such as the E. coli system, although most literature has claimed that up to mg protein per liter bacterial culture could be obtained, the expressed TNFα proteins formed inclusion bodies under which harsh denature reagents like 8 M urea before purification and protein refolding after purification are usually required to obtain active proteins [36,37].…”

“…Compared with other protein expression systems such as the E. coli system, although most literature has claimed that up to mg protein per liter bacterial culture could be obtained, the expressed TNFα proteins formed inclusion bodies under which harsh denature reagents like 8 M urea before purification and protein refolding after purification are usually required to obtain active proteins [36,37]. More recently, several attempts using the E. coli expression system under optimized culture and induction conditions claimed that soluble human rhTNFα proteins could also be obtained in a large amount at 7.2 mg/L~1.26 g/L of culture medium [8,9]. In contrast, the silkworm-BEVS produces soluble TNFα proteins so that no refolding and other unnecessary processes are needed.…”

“…The killing effects of the TNFR1 signaling pathway endows the mature type of TNFα a potential drug for the treatment of cancer in the clinical trial market. Because of its high demand, the productions of recombinant human and murine TNFα proteins with desirable functions have been investigated in various heterologous protein expression systems, including Escherichia coli, Streptomyces lividans, Spodoptera frugiperda Sf9 cells, or mouse embryonic 3T3 fibroblasts [8][9][10][11][12]. To achieve better productivity of TNFα, there have been constant efforts in optimizations in the genetic gene codon, expression promoters, culture, and induction conditions of the E. coli expression system targeting more soluble protein products over inclusion body in most cases [8,9,[13][14][15].…”

“…Because of its high demand, the productions of recombinant human and murine TNFα proteins with desirable functions have been investigated in various heterologous protein expression systems, including Escherichia coli, Streptomyces lividans, Spodoptera frugiperda Sf9 cells, or mouse embryonic 3T3 fibroblasts [8][9][10][11][12]. To achieve better productivity of TNFα, there have been constant efforts in optimizations in the genetic gene codon, expression promoters, culture, and induction conditions of the E. coli expression system targeting more soluble protein products over inclusion body in most cases [8,9,[13][14][15]. The eukaryotic protein expression systems such as mammalian cell expression systems and baculovirus expression vector system (BEVS) have advantages in achieving soluble targets without frustrating condition optimizations, which seems necessary in the E. coli system, although shortages also exist regarding the relatively higher cost due to cell culture and laboratory maintenance [16][17][18].…”