Cell culture condition
Changing the culture condition of engineered E. coli , including temperature, isopropyl-β-D-thiogalactoside (IPTG) concentration, time of induction, buffers, pH, ionic strength, etc. can further enhance the expression level and solubility of recombinant proteins (Hamada, Arakawa, & Shiraki, 2009). For more information on hosts, promoters, concentration of the additives and other factors in detail see this article (Lebendiker & Danieli, 2014). The addition of charged amino acids L-Glu and L-Arg at 50 mM to the buffer can increase the maximum concentration of soluble protein (up to 8.7 times) (Golovanov, Hautbergue, Wilson, & Lian, 2004). The anaerobic effects and pH additives could increase the β-galactosidase  expression level 200 folds, where the pH value of cell culture was lowered from 5.5 to 7 (Tolentino, Meng, Bennett, & San, 1992). Various additives, including natural ligands, detergents, salts, buffers, and chemicals were used to increase the stability and solubility of recombinant proteins expressed in E. coli (Leibly et al., 2012). Evidently, the solubility of heterologous proteins increases following prolonged induction with low amounts of IPTG at decreased temperatures (Hesaraki et al., 2013; Saadati et al., 2010; Soulari, Basafa, Rajabibazl, & Hashemi, 2020). The solubility of granulocyte-macrophage colony-stimulating factor (GM-CSF) was improved by adding chemical chaperones and osmolytes such as sucrose (0.5 M), NaCl (0.5 M), sorbitol (0.5 M) and MgCl2 (1 mM) to the growth media (Malekian, Sima, Jahanian-Najafabadi, Moazen, & Akbari, 2019). Generally, the aggregation of expressed recombinant proteins in bacteria occurs at higher temperatures due to the hydrophobic interactions among overexpressed polypeptides [9]. The three factors of post-induction temperature, post-induction time and IPTG concentration were routinely optimized for improved expression conditions toward higher protein solubility (Gutiérrez-González et al., 2019). Some of the heat shock proteases expressed under overexpression conditions are eliminated as a result of temperature reduction [10]. Furthermore, the expression and activity of some E. coli chaperones are raised at temperatures around 30 °C [11,12]. Some studies reported soluble expression of the target protein at 4 °C. It should be noted that a sudden decrease in cultivation temperature triggers inhibition of replication, transcription and translation. Some chemical additives in the culture medium such as ethanol, benzyl alcohol and osmolytes along with ionic strength of the buffer may increase the expression level of recombinant proteins (Papaneophytou & Kontopidis, 2014). The formation of inclusion bodies is detectable even at low levels in fed-batch cultivations insisted of batch cultivations, by flow cytometry technology.