In this study, we have three recombinant proteins with different culture conditions. The changes in some parameters in culture conditions like rise in temperature after induction, optical density, and concentration of inducer, showed effects in soluble expression levels.
Inclusion bodies formation can be decreased by various methods. One of it is, decreased culture temperature after induction effect in the production of soluble protein (Schein 1989). The effect of lowering temperature is hard to pinpoint, but can be useful by permitting slower production. In some cases, this had a positive effects on solubility, especially when using the proper promoters/inducers, such as the T7 promoter (Weickert et al. 1996; Baneyx and Mujacic 2004). Many recombinant proteins from different sources are more soluble, when expressed in E. coli at lower growth temperature (25-30 C) (Shaw and Ingraham 1967; Chesshyre and Hipkiss 1989), because the cells will grow quite slowly after induction (Schein 1990).
Generally, at higher temperatures inclusion body formation is favored in E. coli
because the high temperature increases the hydrophobic interactions, leading to aggregation (Kiefhaber et al. 1991). Lowering the cultivation temperature reduces the expression of heat shock proteases, which in turn increases the recombinant protein stability and solubility (Chesshyre and Hipkiss 1989).
In this study, reducing the inducer concentration also plays a major role in producing soluble form in recombinant protein. Several studies, refers that, high yield of soluble protein can be attained, if IPTG inducer concentration is reduced to less than 1 mM. Rabhi-Essafi et al. (2007) and Soleymani and Mostafaie (2019) demonstrated that reducing IPTG concentration with low post-induction temperature increase the production of soluble recombinant IFNα 2B protein and recombinant bovine SRY protein in E. coli BL21 cells, respectively.
IPTG induces T7 RNA polymerase and protein production in E. coli. In the pRSET system with the strong T7 promoter and high inducer concentrations, product yield high proteins, this leads to an increase in misfolding and inclusion body formation (Baneyx and Mujacic 2004). Low IPTG concentration probably the strong affinity of the T7 promoter and lac repressor occurred in a suitable form; Therefore, the production during cultivation also occurred slowly, which increases the solubility form of protein. One possible reason for this increased solubility at low IPTG concentration is the slow production which eventually results increase in solubility.
The type of host strains used to produce recombinant proteins also plays an important role in protein expression yield, and solubility. Several E. coli strains that strongly improve membrane protein production have been engineered. E. coli BL21 and its derivatives are widely used for the production of recombinant protein in E. coli. These strains are deficient in the proteases Lon and OmpT, which can increase the stability of the protein. The strain BL21(DE3) contains a chromosomal copy of the T7 RNA polymerase gene for the simple and efficient expression of genes under the control of the T7 promoter ( Long et al. 2014; Joseph et al. 2015). E. coli GJ1158 strain is a simple, effective, and generally applicable method for the production of recombinant proteins in E. coli that makes use of NaCl as an inducer replaces IPTG (Bhandari and Gowrishankar 1997).
The media composition is affected in solubility for expressed protein. LB medium is most commonly used for culturing expression hosts. E. coli grows in low density because its low amounts of carbohydrates contain and divalent cations (Sezonov et al. 2007). Compatible co-solvents are add during culturing, to maintain protein solubility (Schein 1999; Saluta and Bell 1999).
However, these protocols for the production of soluble forms of recombinant protein, have not yielded successful results for all recombinant streptokinases. Hence, as there is no universal strategy, for production of soluble form for all proteins, therefore, a trial-and-error method is pursued.