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.