TABLE 1.

The various strategies to control and minimize the formation of recombinant protein inclusion bodies in E. coli.

Strategies	Specific approaches	Potential mechanism	Comments	References
Tailoring culture conditions	Lowering the culture temperature in induction phase	Reducing protein expression rate	Two-phase culture used. First-phase at 37°C for cell growth, second phase at 15–20°C for the induction of protein expression	Cabilly, 1989; Shirano and Shibata, 1990; Jung et al., 2013; Sina et al., 2015; Carere et al., 2018a; Wang et al., 2019
	Introducing a short time heat shock prior to expression induction	To induce chaperons’ production, meanwhile minimize IBs formation	E.g., 47°C for 20–30 min	Oganesyan et al., 2007
	Decreasing the concentration of inducer (e.g., IPTG)	Reducing protein expression rate	E.g., 0.01–0.05 mM instead of 0.5–1.0 mM	Jhamb and Sahoo, 2012; Sina et al., 2015
	Adding glucose in growth medium	Reducing protein expression rate through catabolic repression effect of glucose to the induction	The glucose concentration at 1–2% was often used	Grossman et al., 1998
	Adding chemical additives (e.g., D-sorbitol, glycerol, ethanol, NaCl et al)	Sorbitol, glycerol and NaCl will cause osmotic stress and further induce osmolytes synthesis or uptake. Ethanol will elicit heat shock response and induce the production of chaperones	Often used conditions: Sorbitol (0.5–1.0 M), NaCl (0.2–0.8 M), Betaine (1 mM), Ethanol [3% (v/v)]	Blackwell and Horgan, 1991; Diamant et al., 2001; Oganesyan et al., 2007
	Adding co-factors of target protein in growth medium	To assist proper protein folding	Many proteins require cofactors for their proper folding such as metalloenzymes	Bushmarina et al., 2006; Rosano and Ceccarelli, 2014
	Use buffer to control pH of growth medium	Controlling the pH fluctuation for the proper protonation states of proteins	No fluctuations to the protein, keeps it chemically stable	Castellanos-Mendoza et al., 2014
Expression host engineering	Engineered strains to catalyze di-sulfide bond formation—TrxB, gor mutants, CyDisCo system	Trxb– and gor– generate a more oxidizing environment. CyDisCo involves di-sulfide bonds catalyzed by a sulfhydryl oxidase Erv1p	Proteins requiring di-sulfide bonds can be successfully folded and functional. E.g., SHuffle and Origami strains, CyDisCo system	Xiong et al., 2005; Rasiah and Rehm, 2009; Nguyen et al., 2011; Lobstein et al., 2012; Hatahet and Ruddock, 2013
	Engineering strains to perform glycosylation	Addition of enzymes or pathways able to catalyze N- or O-linked glycosylation Knockouts of wecA and waaL to remove competing glycan pathways	Important implications for activity, structure, and stability E.g., CLM37 and CLM24 strains	Wacker et al., 2002; Feldman et al., 2005
	Co-expressing chaperone	Aid in the proper protein folding	E.g., GroEL, GroES, ClpB	Lee et al., 2004; de Marco et al., 2007; Jhamb and Sahoo, 2012
	Co-expressing foldase	Aid in the proper protein folding and disulfide bond formation	Include protein disulfide isomerases (PDI) and peptidyl prolyl isomerases (PPI)	Ngiam et al., 2000; Lee et al., 2004; Jung et al., 2013; Zhuo et al., 2014
	Strains engineered for membrane proteins or toxic proteins	Dampening of T7 RNA polymerase expression and/or activity	Aims to reduce expression levels to reduce toxicity and improve membrane protein expression E.g., E. coli strains C41 (DE3), C43 (DE3), Lemo21 (DE3), BL21 (DE3) pLysS, pAVEwayTM	Miroux and Walker, 1996; Wagner et al., 2008; Kwon et al., 2015; Kim et al., 2017
	Co-expressing multiple components of protein complex	The co-expression of protein components is beneficial for protein folding, stability and protect individual components from degradation	Using compatible duet vectors with different antibiotics resistance	Tolia and Joshua-Tor, 2006
	Engineered metal ion transport for metalloenzymes	Overexpress operons involved in uptake/transport of metal cofactors	Overexpressing cobalamin transport pathways and Suf pathways shown to produce proteins with full iron occupancy	Lanz et al., 2018; Corless et al., 2020
	Use weaker promotor	Reducing protein expression rate	Better balance between protein synthesis and folding, and lower metabolic burden to host cells	Kaur et al., 2018
	Linked to a soluble fusion tag or chaperone at either N- or C-terminus.	Improve protein expression yields, solubility and folding, facilitate protein purification.	E.g., maltose binding protein, glutathione-S-transferase, Spy	Vu et al., 2014; Ruan et al., 2020
Altering expression vector	Plasmid display technology, linking the target protein to a DBD	Target protein and DBD are attached to the plasmid itself, aids in stabilization	Ensure a soluble DBD partner E.g., Oct-1 DBD, GAL4 DBD	Xiong et al., 2005; Park et al., 2013, 2020
	Use a low copy number plasmid	Reducing protein expression rate	Better balance between protein synthesis and folding, and lower metabolic burden to host	Kaur et al., 2018
	Minimize the hydrophobic patch on the surface of protein	Site directed mutagenesis to change aggregation-promoting residues	Prediction using programs Ex. TANGO, PASTA 2.0, AMYLPRED 2.0, Protein-Sol, SoDoPE	Conchillo-Solé et al., 2007; Tsolis et al., 2013; Walsh et al., 2014; Hebditch et al., 2017; Bhandari et al., 2020
	Express partial protein (truncated and soluble domain)	Potential aggregation prone protein is expressed in a soluble state	Based on the purpose for the protein, as it may not be functional	Chen et al., 2003
Modifying the protein of interest	Add signal peptide to direct the expressed protein into periplasmic area	It is beneficial for folding with the more oxidized environment and foldases in the periplasmic space	Less proteolytic activity in periplasmic space	Dow et al., 2015; Malik, 2016