Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

1Inclusion bodies (IBs) are nuclear, cytoplasmic, or periplasmic aggregates of bio-macromolecules, mostly proteins. These proteins are generally expressed from foreign or mutated genes without proper post-translational modifications and/or folding. In humans, protein aggregation has been found associated with numerous protein misfolding diseases such as Huntington, Alzheimer, and Parkinson’s diseases. Besides humans, protein inclusion bodies have been also observed in almost all studied domains of life (e.g., animals, plants, fungi, and bacteria), and they are often related to stress and diseases as well.

2Recombinant proteins are becoming increasingly important as enzymes and non-catalytic proteins (e.g., antibodies, hormones, factors, vaccines) for industrial and agricultural applications. Escherichia coli is the most popular bacterial host for recombinant proteins production due to: (1) its fast growth rate with a generation time spanning 20 min under optimized conditions, (2) well-developed tools of molecular manipulations along with in-depth knowledge of its biology, and (3) the ability to achieve high cell density using inexpensive culture reagents. However, the heterogeneous expression of recombinant proteins in E. coli is often hampered by protein aggregation into IBs. This poses a serious challenge for producing soluble recombinant proteins with proper biological function at the laboratory and/or industrial scales. In this review, the mechanism and the factors that influence the formation of recombinant protein IBs will be discussed together with their unique features. In particular, strategies to minimize protein IB formation in E. coli will be comprehensively presented in the following sections with the new developments in the field.

3All living organisms have evolved a sophisticated mechanism to maintain their protein homeostasis. Protein homeostasis refers to the control of concentration, conformation, binding interactions, and localization of individual proteins making up the proteome by readapting the innate biology of the cell. The maintenance of protein homeostasis is critical for cell function and the overall health of the organism. It involves multiple pieces of the cellular machinery of transcription, translation, protein post-translational modification, folding, and degradation. Protein IBs formation in E. coli cells results from an unbalanced equilibrium among protein proper folding, aggregation, and degradation (Figure 1). It is associated with many factors including host cell metabolism, protein synthesis, and modification machinery, target protein properties, and environmental conditions.

4IBs formation can be triggered by a high rate of protein expression, which is the pinnacle of recombinant proteins production pipelines. This goal is often achieved by constructing expression vectors with strong promoters (e.g., T7 and Lac promotors), using plasmids with high copy numbers, optimizing codon usage, or engineering E. coli host-strains for their fast growth. However, when the rate of a recombinant protein expression exceeds the ability of the host cells to manage protein post-translational modifications and folding, the target protein will be increasingly misfolded, and then aggregated into IBs as the hydrophobic residues buried in the native protein are exposed on its surfaces. Furthermore, host cells cannot sustain protein expression at such high levels due to the cell stress and metabolic burden from the increased energy demand.

5The lack of proper host machinery for protein post-translational modifications (PTMs) is another factor leading to protein misfolding, particularly when expressing eukaryotic proteins (e.g., mammalian, plant, and fungal proteins) in E. coli. PTMs are important to achieve a native, biologically active conformation, and can significantly affect the characteristics of proteins including their charge, hydrophobicity, solvent accessibility, etc.. Among multiple PTMs, glycosylation particularly plays critical roles in protein sorting, folding, localization, and quality control in eukaryotic organisms. Yet another PTM, disulfide bond formation, is also crucial for producing stable proteins. E. coli lacks subcellular compartments such as the endoplasmic reticulum and Golgi apparatus to facilitate glycosylation and disulfide bridge formation. Secreting recombinant protein to the oxidative environment of E. coli periplasm has proven successful for disulfide bond formation in certain cases. However, secretion efficiency is often limited by the size and structure of the protein of interest as well as the secretory machinery of E. coli.

6Protein aggregation into inclusion bodies is also governed by physicochemical and structural features of the proteins themselves. These characteristics include the molecular weight, the number of contiguous hydrophobic residues, and low complexity regions. used a deep-mining approach to investigate the correlation between these factors and the possibility to produce soluble functional mammalian proteins in E. coli. Generally, single-domain proteins with low molecular weight tend to be produced in soluble form with retention of proteins functionality. This is partly due to the requirement for fewer folding intermediates in the protein folding pathway. Contrary to this, the folding of multi-domain proteins may be accompanied by folded or misfolded intermediates increasing the likelihood of aggregation. Other aggregation-prone proteins such as intrinsically disordered proteins and membrane proteins also pose significant challenges in protein expression studies. The presence of disordered, low complexity regions and stretches of hydrophobic regions predispose these proteins to IBs formation.

7Environmental conditions, such as culture temperature and pH, also affect IBs formation during recombinant proteins production in E. coli. The effect of heat stress at 45°C on inducing the aggregation of recombinant luciferases in E. coli has been reported earlier, as well as the ability of physiological pH conditions (pH = 7.5) to leverage a beneficial effect on the heterologous expression of Boophilus microplus sphingomyelinase-D in E. coli. Due the above reasoning, tailoring culture conditions is an approach that is often used as a solution to minimize IBs formation of recombinant proteins in E. coli.

8In essence, the aggregation process is driven by hydrophobic interactions that act as countermeasures shielding hydrophobic stretches of protein from the surrounding aqueous environment. Newly formed aggregates may then promote nucleation by acting as seeds for the aggregation of other highly similar proteins. This process is more likely to occur in prokaryotes such as E. coli, which do not possess the appropriate protein-modification machinery. High levels of expression coupled with a lack of modification machinery promote misfolding, resulting in the inadvertent exposure of hydrophobic residues on the protein surface.

9Relative to soluble proteins, IBs proteins in E. coli display unique characteristics in terms of structure, morphology, and composition. Morphologically, these aggregates are observed as dense refractile particles with smooth or irregular rough surfaces. These characteristics may vary from protein to protein and are dependent on the properties of the expressed proteins.

10Protein aggregates may consist of ordered structures termed amyloid fibrils or disordered, amorphous structures as observed for regular IBs proteins. Amorphous aggregation is a thermodynamically favorable process obviating the need for a high energy barrier necessary for nucleation processes such as amyloid fibrillation and crystallization. The catalytic activity were found for some amyloid aggregates which are characterized by cross β-sheet motifs. Conversely, E. coli protein IBs appear as amorphous aggregates lacking proper structure and function. Despite this, a growing body of evidence has suggested that certain IBs proteins may possess amyloid-like structures with an associated functionality. Amyloid-like properties were seen in β-galactosidase IBs expressed in E. coli, which were shown to be biologically active. Another study carried out by to characterize asparaginase IBs confirmed the high β-content as well as the catalytic activity of the amyloid-like aggregates. Furthermore, it was determined that temperature could modulate the levels of reported amyloid features. The parallel findings were reported in a similar study. The mechanism behind how temperature and time may influence IBs formation/structure is not well understood and speculations of protein-specific outcomes do exist.

11E. coli IBs while largely composed of self-aggregated protein, also contain traces of other bio-macromolecules including nucleic acids, and/or phospholipids. Within the biotechnological context, proteins in IBs predominantly consist of expressed foreign proteins. The nature of such highly specific protein self-aggregates was revealed through a pioneering study using two aggregation-prone proteins that were fluorescently labeled, namely the ΔF508 mutant of the cystic fibrosis transmembrane conductance regulator (F508) and the P23H rhodopsin mutant (P23H). Interestingly, the aggregation of P23H with other aggregation-prone proteins was not observed and P23H was seen aggregating predominantly with itself. This phenomenon was further validated by an experiment conducted thorough the co-expression of Aβ42 amyloid peptide with VP1 capsid protein from the foot-and-mouth disease virus. The Aβ42 amyloid peptide only co-aggregated with itself and was not observed aggregating with VP1. Given the highly homogenous protein composition, E. coli protein IBs provide a unique source of an almost pure target protein and might be further exploited for biotechnological applications (see “Potential Advantages of Protein Inclusion Bodies in Industrial Applications” section for further discussion).

12Bacterial IBs also show specific cellular distribution at the poles and/or septation sites of E. coli cells. The majority of IBs are formed near the poles, and 24% of formed IBs showed a migratory movement after formation. Similarly, a VP1 capsid protein tagged with green fluorescent protein aggregated specifically at E. coli cell poles. The results of the above studies suggest that IBs are exclusively assembled at the cellular pole(s) or are transported thereafter the formation event. Conceivably, this polar distribution of IBs will result in an asymmetrical pattern of inheritance during cell division with one daughter cell containing IBs while the other is free. This distribution pattern is beneficial for the later daughter cells as it places less burden with reduced or no inclusion bodies. Notably, daughter cells that inherited the new poles without inclusion bodies showed higher growth rates compared to the cells carrying the poles with IBs.

13A direct consequence of light scattering by protein IBs is that the E. coli lysate often appears as a milk-like broth when the recombinant protein forms IBs are released. This was often used as a primary indicator of IBs formation in the many protein expression trials of several research groups. Under light microscopy, protein IBs often appear as dense refractile particles with varying sizes inside cells. Additionally, SDS-PAGE analysis of soluble and insoluble fractions of bacterial cell lysates can also be applied to test the presence of IBs of the expressed protein(s). This can be visualized by a strongly stained protein band corresponding to the molecular size of the target protein in the insoluble fraction of the cell lysate. Amyloid-protein aggregates can also be identified by using dyes such as Congo Red. Congo red shows apple-green birefringence under the light microscope. Molecular motor dyes such as thioflavin-T and Proteostat may also be used for visualization. These types of dyes show fluorescence enhancement when rotary movement is constricted in microenvironments.

14Based on the above-discussed mechanisms governing proteins’ IBs formation, many strategies have been developed recently to minimize this phenomenon. In general, these strategies contribute to one or more of the following aspects (Table 1): (1) reducing protein synthesis rate, (2) inducing the production of endogenous chaperones, and/or the synthesis or the absorption of osmolytes in E. coli cells, (3) introducing additional cellular components (e.g., chaperones and foldases) to E. coli cells or adding chemical chaperones to the culture medium to assist protein folding and modification, and (4) modifying the protein of interest by removing structural elements contributing to protein IBs formation, and/or fusing the target protein to a soluble protein or peptide tags. The effectiveness of each approach may vary from one protein to another and it requires an empirical optimization. These strategies will be elaborated below with solid examples (Table 1).

15A reduction in the level of IBs formation can be achieved by modifying culture conditions, including growth temperature, inducer concentrations, and culture additives. These factors often influence the rate of protein expression, and aid in the protein folding process in E. coli cells (Table 1).

16Lowering culture temperatures below the optimal 37°C can decrease the rate of E. coli cells growth and enhance their protein expression in a soluble form. This approach is usually carried out in two phases, where the first-phase culture at 37°C is optimal for the growth of E. coli to reach a high cell density. Second-phase temperatures are set to low ones (e.g., 15–20°C) for the protein expression induction with a low rate. Such an approach has been very effective in our efforts to produce multiple soluble and active enzymes of agricultural importance, including Tri101 acetyltransferase, DepA, and DepB for mycotoxins detoxification in addition to several carbohydrate-active enzymes (CAZymes) for agricultural biomass valorization. In contrast, culture temperatures greater than 37°C have been also scrutinized in the past for their effect on protein expression, as the heat shock could presumptively induce the production of molecular chaperones. However, the use of high cultivation temperatures (>37°C) generally resulted in elevated inclusion bodies formation, likely due to the increase in hydrophobic interactions, as well as cellular stress poised by protein denaturation at temperatures greater than 37°C. To take the advantage of the production of the molecular chaperones, induced by high temperatures, while minimizing the formation of IBs, a short heat shock (47°C, 30 min) was used to treat E. coli cells before inducing protein expression under 20°C. This culture program improved the expression of several recombinant proteins into their soluble form.

17Protein expression rates can also be reduced by lowering inducer concentrations or adding glucose into the culture medium (Table 1). In E. coli protein expression systems, the lac operon has been extensively tailored for the induction of heterogenous proteins expression, where lactose is an activator of the lac operon. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a structural analog of allolactose, a lactose metabolite, and therefore efficiently activates the lac operon through binding to lac repressor protein. However, since IPTG is not easily metabolized, its concentration remains consistent compared to lactose in bacterial cultures. Therefore, IPTG has been a widely used chemical inducer for recombinant proteins expression. Reducing IPTG concentrations in the culture medium can decrease protein expression rates to manageable levels without placing a metabolic burden on E. coli cells, which is ultimately favorable for the proper folding of recombinant proteins. Alternatively, adding glucose to the medium can also decrease the rate of protein expression through competitive catabolic repression. Glucose inhibits adenylate cyclase (AC) activity on synthesizing cAMP from ATP. The cAMP is able to regulate the lac operon by binding to the catabolic activator protein (CAP). The latter then binds to the promoter CAP site and further stimulates the binding of RNA polymerase to the promoter region to initiate transcription. In the presence of glucose, low levels of cAMP lead to a low level of CAP activation, resulting in a low transcription level for the target gene.

18Some chemical additives in culture media display beneficial effects on the expression of soluble recombinant proteins. These additives generally include osmotic stress-triggering chemicals, heat shock-inducing chemicals, and protein cofactors. For example, the presence of salt or sorbitol in the culture medium facilitated the expression of target proteins in their soluble form by triggering osmotic stress and stimulating osmolyte (e.g., betaine, trehalose) synthesis/uptake in E. coli cells. These osmolytes can act as “chemical chaperones” by increasing the stability of native proteins and assisting in the refolding of unfolded polypeptides. This group of osmotic stress-triggering chemicals could also include glycerol, mannitol, and other polyols. Within them, D-sorbitol (along with betaine) is the most widely used additive in minimizing recombinant proteins IBs formation. D-sorbitol displayed beneficial effects in our earlier efforts to produce several CAZymes for their functional and structural characterization, and their application in agricultural by-products valorization. The inclusion of ethanol in culture media (e.g., 3%, v/v) has been reported to enhance the solubilities of some recombinant proteins. Ethanol here elicits a heat shock response and induces the production of chaperones. Additionally, adding co-factors to the growth media could be another consideration to improve protein expression in soluble form, as many proteins require cofactors for their proper folding and function.

19The pH of bacterial cultures often affects the charges of present proteins and can further impact their properties. For instance, pH has been reported to affect the tendency of β peptides to form amyloid-like structures in vitro, suggesting that pH might be an important factor that can significantly influence the levels of IBs formation. Mendoza-Castellanos’s group compared the effects of controlled and uncontrolled pH conditions on the formation of sphingomyelinase-D IBs when grown in a super broth medium. The results demonstrated that protein’s aggregation occurred at higher levels under uncontrolled pH condition (as pH fluctuated with cellular metabolism) than controlled ones, where the controlled pH environment (pH = 7.5) was achieved by coupling with an automatic supplementation of 1.0 M NaOH (when needed). Therefore, keeping culture pH stable at a certain level could be beneficial in minimizing the formation of recombinant protein IBs in E. coli.

20Taken together, it is worth noting that altering culture conditions usually present the simplest solution to reduce IBs formation in E. coli. However, culture conditions favorable for soluble protein production may vary depending on the involved proteins of interest and the used host strains of E. coli, and thus require experimental optimization.

21Recent advancements in biotechnology and engineering have led to the development of many efficient and flexible systems for recombinant proteins expression/production in E. coli. In this section, we address a few engineered E. coli strains to aid disulfide bond formation, glycoengineered strains for glycosylation, strains which offer fine tuning over protein expression, expression of chaperones and finally we briefly mention host engineering for certain metallo-enzymes (Table 1).

22Many recombinant proteins require correct disulfide bonding to attain their biological functionality. In E. coli, the disulfide bond formation often occurs in the periplasm through the oxidation of a pair of cysteine residues. In contrast, the S-S bond formation in the cytoplasm is disfavored due to the existence of a reducing environment maintained by two separate thioredoxin reductase (trxB) and glutaredoxin reductase (gor) systems. To attain the correct disulfide bonding of recombinant proteins expressed in the cytoplasm of E. coli, specific strains with a unique trio of modifications have been engineered to a more oxidative environment by eliminating glutathione reductase (gor gene) and thioredoxin reductase (trxB gene) along with a suppressor mutation in the ahpC gene. These strains include commercially available Origami and SHuffle strains. The SHuffle strain has both mutations of the Origami strain but also constitutively expresses a disulfide bond isomerase which promotes the correction of mis-oxidized proteins (; Figure 2A). Another alternative to these strains is the CyDisCo system, which enables the expression of disulfide bond containing proteins in the cytoplasm of E. coli without modifications to endogenous reducing pathways. Here, de novo disulfide bond formation is catalyzed by Erv1p, a eukaryotic sulfhydryl oxidase while a protein disulfide isomerase (PDI) rectifies errors in disulfide bond formation. Although a CyDisCo system could be constructed using periplasmic E. coli DsbB or vitamin K epoxide reductase, these transmembrane proteins require extensive engineering to invert their membrane topology to function stably in the cytoplasm. Recently, CyDisCo was benchmarked by expressing domain constructs of mammalian extracellular matrix proteins including mucin-2 (MUC2), alpha tectorin (TECTA) which possess von Willebrand Factor D domains implicated in several human diseases. Both proteins were successfully purified in a soluble state; however, the only limitation to this system appears to be the number of disulfide rich regions in the expressed protein.

23Protein glycosylation is a post-translational modification that involves the addition of glycans to proteins. Glycosylation has important implications for the folding, activity, structure, solubility, and stability of the protein. Two forms of glycosylation exist: O-linked glycosylation, found in bacteria and N-linked glycosylation which is a rare occurrence for bacteria and more exclusive to the eukaryotic and archaea domain. Prokaryotes like E. coli have difficulty in processing N-linked glycosylation, and mistakes often lead to the formation of aggregation-prone proteins. To accommodate the lack of N-glycosylation machinery, engineering E. coli strains with specific enzymes able to catalyze glycosylation presents a promising solution (Figure 2B). PglB, an oligosaccharyl transferase, is a key enzyme for the N-glycosylation pathway in Campylobacter jejuni where its function is to transfer an O antigen or O-polysaccharide to an L-asparagine residue on the acceptor protein. As proof of concept, PglB was co-expressed in a host plasmid, enabling the transfer of an O polysaccharide from a lipid carrier to the model protein, AcrA. This alternative allows for proper glycosylation to take place as well as ensuring that expressed recombinant proteins properly fold. Optimized E. coli strains for glycoprotein production include E. coli CLM37 and E. coli CLM24, which possess knockouts of wecA and waaL genes to remove competing glycan pathways present and reduce the metabolic load due to expression of plasmids containing the PglB gene. Several research groups have also employed “leaky” glycoengineered E. coli strains. In this context, the term “leaky” refers to strains with increased permeability of the outer membrane. These strains possess mutations or knockouts of the lpp gene which encodes a key lipoprotein (lpp), often referred to as Braun’s lipoprotein. In E. coli, lpp maintains connectivity between the outer membrane and the peptidoglycan layer through covalent interactions. Prior studies revealed that E. coli lpp mutants (E. coli E609YΔlpp, E. coli JM109 (DE3)Δlpp) have increased permeability of the outer membrane which is advantageous for recombinant production of glycoproteins including therapeutic proteins including glycosylated antibody fragments. One caveat that limits the use of such “leaky” host systems is the size of the protein as well as the dynamics of the secretion system in question. introduced the lpp mutation into a glycoprotein engineered E. coli strain of CLM37 to produce N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2). Levels of the secreted protein were at least 11–15 times higher compared to the parent E. coli CLM37 strain (no lpp deletion) with yields of 70 ± 3.4 mg/L.

24Another proprietary “leaky” strain, E. coli enGenes-X-pressTM possesses an expression system which incorporates the expression system described by and enables delivery of the recombinant protein to the culture medium which streamlines downstream processing steps.

25Foldases are a group of proteins whose function is to assist in the proper folding of proteins. Important foldases include thiol-disulfide oxidoreductase and protein disulfide isomerases (PDI) responsible for disulfide bonds formation, and peptidyl-prolyl isomerases (PPI) catalyzing the cis-trans isomerization of peptide bonds N−terminal to proline (Pro) residues within polypeptide chains. The purpose of co-expressing PDIs and PPIs in E. coli is to accelerate the rate-limiting step that leads to errors in protein folding. PDIs are found in eukaryotes as well as the periplasmic space of bacteria. Examples of bacterial PDIs include DsbA, DsbC, and DsbG. For instance, the co-expression of DsbA, DsbC, was shown to greatly enhance target protein solubility. PDIs (e.g., DsbG) also display chaperone-like activities in aiding protein folding. Moreover, PPIs were reported to help directly in addressing the above issue by catalyzing isomerization.

26Chaperones are a group of proteins whose function is to stabilize unfolded proteins, unfold them for translocation across membranes or degradation, and/or to assist in their proper folding and assembly. In E. coli, the co-expression of chaperone systems has been shown to improve protein solubility and enhance proteins correct folding, hence leading to reduced IBs accumulation. Specifically, plasmids harboring genes of the following chaperones: GroEL/GroES, DnaK/DnaJ/GrepE (KJE), ClpB, and the small heat shock chaperones IbpA and IbpB, enhanced the level of soluble and functional recombinant proteins in E. coli. Using a similar approach, soluble cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was successfully expressed in E. coli. CHMO is quite prone to IB formation however, by expressing the protein in concert with either GroEL/GroES or DnaK/DnaJ/GrpE gave a 38-fold improvement. Additionally, expression of foldases DsbA, DsbC, hPPIase with the CHMO gene also improved soluble protein expression, though not as high as with molecular chaperones.

27Reducing culture temperature represents one strategy for increasing the production of soluble proteins as discussed in a previous section. The slight reduction in temperature has been identified to improve the soluble protein expression slightly. However, lowering the temperature significantly compromises the ability of chaperones of assisting protein folding in the regular mesophilic E. coli cells. To overcome this obstacle, specific strains of E. coli that possess cold-adapted chaperone systems have been developed. These strains show improved protein processing abilities at low temperatures. A prime example is the ArcticExpress strain, which contains cold-adapted chaperonins of Cpn60 and co-Cpn10 from the psychrophilic bacterium, Oleispira antarctica.

28Membrane proteins overexpression is often met with significant difficulties due to host toxicity and IBs formation. In such cases, the re-solubilization of IBs proteins involves harsh treatments with either detergents, organic solvents, or chaotropes with limited improvement to the overall protein yield. Engineered E. coli strains such as the Walker strains C41 (DE3), C43 (DE3), Lemo21, and BL21 (DE3) pLysS are commonly used to produce soluble membrane proteins as well as toxic or particularly challenging proteins. The success of these strains is attributed to their reduced transcriptional activity owing to a variety of beneficial mutations addressed in this section (Figure 2C).

29The E. coli C41 (DE3) and C43 (DE3) strains both possess mutations in the lacUV5 promoter and additionally, the C43 (DE3) strain also possesses a mutation in the lac repressor gene (LacI). These dampen T7 RNA polymerase expression compared with the wild type lac promoter, thus enabling these strains to withstand toxic effects associated with the overexpression of certain membrane proteins. By drawing on the same idea, a novel expression system which lowers T7 RNA polymerase expression via repression by a mutant LacI repressor protein (mLacI) was designed. mLacI possesses the same mutations found in the LacI gene in the C43 (DE3) strain which limits its ability to bind inducer molecules such as IPTG. In the mLacI system, expression of this mutant repressor is governed by an L-rhamnose inducible promoter (prhaBAD) and fine tuning over transcription can be implemented in a wide range of lacO expression systems. The strain E. coli BL21 (DE3) pLysS lowers basal level suppression of T7 RNA polymerase with a constitutively expressed T7 lysozyme (carried on the pLysS plasmid). Induction with IPTG then enables the T7 RNA polymerase to overcome this inhibition and carry out transcription of the target protein. Lemo21 (DE3) functions in a similar way, but allows tunable expression of the T7 lysozyme by placing it under the control of the prhaBAD promoter.

30While these strains enable control over orthogonal T7 RNA polymerases, another means to control protein expression entails the decoupling of recombinant protein expression from host cell growth. The design of this strain, E. coli BL21 (DE3):TN7 was inspired by bacteriophage-mediated hijacking of host RNA polymerases to reduce host cell growth, and instead, re-route resources toward the production of viral proteins. The system employs a T7 phage RNA polymerase inhibitor, Gp2, to inhibit endogenous E. coli RNA polymerases. Expression of Gp2 is under the control of the pBAD promoter and when induced with arabinose, binds to the ß’ jaw domain of E. coli RNA polymerase. This results in inhibition of host cell protein production making translation machinery more available for recombinant protein production, thus mitigating inclusion body formation.

31Other difficult proteins may also be expressed using E. coli SoluB21TM from Genlantis which facilitates soluble expression of particularly difficult mammalian proteins. As a proprietary strain, the mechanism behind soluble protein expression is not well understood. However, certain research groups have had reasonable success with the production of soluble proteins when this strain was used. purified over 2 mg/L of recombinant human μ-calpain compared with E. coli BL21 (DE3) where yields of purified protein was <0.2 mg/L.

32Finally, for industrially relevant biologics, robust E. coli expression systems have been designed for scale-up purposes including the E. coli pAVEwayTM system and more recently, the E. coli SoluProTM systems.

33For the soluble expression of metalloenzymes, the addition of metal cofactors is a key factor for proper folding and function. While supplementation of metal ions may mitigate inclusion body formation, in certain cases, the overexpression of operons involved in the uptake and transport of specific metal cofactors is required.

34Class B Radical S-Adenosylmethionine Methylases (SAM) belong to the family of Radical SAM (RS) enzymes which methylate inactivated carbon and phosphorus centers. This class of RS methylases are cobalamin (Vitamin B12) dependent and possess an iron-sulfur cluster. Attempts to express this protein in E. coli have resulted in inclusion bodies, however by overexpressing the cobalamin transport system of E. coli (btuCEDFB operon) under the control of the pBAD promoter in E. coli BL21 (DE3) yielded a significant improvement. In this way, Fom3 from S. wedmorensis which produces IBs in E. coli achieved 95% soluble expression at a yield of 3 mg/L.

35Alternatively, strains such as E. coli SufFeScient have been engineered to overexpress biogenic pathways (Suf pathway) to produce iron-sulfur cluster containing proteins with full iron occupancy. Generally, iron-sulfur proteins are re-folded from IBs or expressed as fusion proteins for soluble expression. Utilizing this strain in conjunction with soluble fusion tags may provide an improvement in soluble protein yield, but further research is required to examine the potential benefits of using such a strain to minimize IBs.

36The numerous strains described here follow a similar theme of modulating the initial burst of transcription associated with strong T7 promoters. However, if the usage of engineered E. coli, specifically designed to reduce IBs, is deemed unsuccessful; an alternative host organism may be utilized. Alternative eukaryotic hosts may have the correct PTM machinery, as well as an internal environment that is better suited to express and fold target proteins. Yeast is a popular unicellular host organism that can perform sufficient PTMs with their cell machinery. Many yeast species have been developed for heterogeneous proteins expression including Pichia pastoris, Saccharomyces cerevisiae, and Kluyveromyces lactis. In addition, insect and mammalian cell lines, transgenic plants, and animals are among the other host systems for recombinant proteins expression systems. Each one of these systems comes with their advantages and disadvantages.

37Introducing certain features to the backbone of used expression vectors may reduce IBs formation and improve the solubility of recombinant proteins. Among these changes are adding a soluble fusion tag to the protein of interest, using weaker promoters to drive protein synthesis, constructing expression vector with low copy number plasmids, and the co-expression of multiple protein components using compatible dual plasmids.

38Fusing the target protein to a soluble protein or peptide tags can enhance the solubility and reduce IBs levels. There are many fusion tags available for the attachment to either the N or C termini of target proteins. In such cases, the soluble fusion tag helps the expressed fusion protein (fusion tag linked with a target protein) to achieve a better overall solubility. While the mechanism is not well understood, it is thought that fusion to a stable partner assists in stabilizing and promoting proper folding of the insoluble protein. Notably, these fusion tags can be removed from target proteins using the cleavage power of specific proteases working at sites between fusion tags and the target proteins. The widely used fusion partners include glutathione-S-transferase (GST) and maltose-binding protein (MBP). These tags have displayed beneficial effects in improving the solubility of some heterologous proteins, which mainly formed IBs when expressed alone in E. coli. Examples of other popular tags used in enhancing recombinant protein solubility include the small ubiquitin-like modifier (SUMO), thioredoxin (Trx), and N-utilization substance A (NusA) tags. An alternative to protein fusion tags are small peptide tags such as the commonly employed His tag. While peptide tags employ much of the similar benefits to protein fusions, peptide tags are smaller, generally up to 15 amino acids. The benefit of a smaller tag is that they are less likely to interfere with the structure and potentially function of the target protein. Not all fusion tags will work efficiently with any protein, therefore, the fusion must be chosen to be compatible with the protein of interest (Figure 3).

39Fusion of the target protein directly to molecular chaperones themselves presents an interesting alternative of utilizing the ability of chaperones through a fusion system. This interaction allows for a more facilitated interaction between the protein and chaperone leading to the mediated folding via the chaperone and therefore preventing formation of the inclusion bodies. Fusions utilizing chaperones have been validated in the past. This methodology was brought back to attention recently using spheroplast protein Y (Spy). Spy is a periplasmic chaperone found in bacteria that inhibits aggregation independent of ATP and other chaperones. Spy was fused to the N terminus of different mammalian proteins in the experiment and was able to retain its chaperone activity. Furthermore, a clear improvement to the solubility of the proteins was observed vs. the non-fused proteins. These recent advancement in utilizing the Spy chaperone highlighted the potential of this method and will likely propel this research and application forward.

40Plasmid display system are another approach on fusions proteins wherein the proteins expressed are directly attached to the plasmid. The target protein is expressed in vivo and binds to a specific DNA sequence on the encoding plasmid via the DNA binding domain (DBD) that is fused to the target protein. Selection of the DNA-binding protein is critical to the success of the display system. The DNA-binding protein must be soluble when expressed in E. coli but must also possess a high binding affinity (Kd) with the recognition sequence on the plasmid to be effective. Although numerous display systems exist such as phage display, cell display, ribosome display and mRNA display, the stability of DNA allows for more variable conditions during downstream processes. The use of plasmid display technology has been validated in the past and is picking up more traction in recent times (;; Figure 3). Park et al. utilized the Oct-1’s DBD fused to target proteins for the purposes of screening engineered proteins. One of the protein, an antibody fragment (M18 scFv) requires disulfide bonds for functional activity, despite this, efficient expression of M18 scFv was observed utilizing this technology within the E. coli SHuffle T7 express lysY strain. Heterologous enzymes expressed within E. coli may aggregate or be unstable, however, being bound to a stable molecule such as DNA may improve their stability and reduce levels of aggregation. More recently, Park et al. was involved in the stable expression of immobilized enzymes within E. coli using the plasmid display method. Once again the Oct-1 DBD was fused to two fucosyltransferases, and soluble expression and functional activity was improved in the fused constructs. Further research into this technology tackling the issue of inclusion bodies is needed to optimize and provide further proof of concept. However, the technology presents potential as a novel and alternative application in preventing IB formation and may be of further interest in the field.

41Besides reducing protein synthesis rates (“Host Engineering for Recombinant Protein Expression in Soluble Form” section), the use of weak promoters and/or a low copy number plasmid is considered another distinctive approach to achieve the above goal. T7 is a popular and strong promoter for protein expression in E. coli. Alternative moderately strong or weak promoters may be beneficial to express recombinant proteins that are prone to inclusion bodies formation. Examples of such promoters for this purpose include the tac, araC, and synthetic trc promoters.

42Similarly and while high-copy number plasmids are capable of providing the host with many functions that are important for recombinant proteins expression/production including the screening/cloning of genes of interest through selection markers utilization (drug or antibiotic resistance), such plasmids can increase the metabolic burden of the host. To minimize such effects in E. coli cells, plasmids are engineered to control their replication with a defined copy number. A high copy number generally corresponds to 100 copies/cell, while a low copy number is anywhere from 0 to 50 copies/cell. High copy number expression plasmids can lead to inclusion bodies formation due to the high rate of heterogeneous protein expression, thus a low copy number plasmid is more beneficial to yield soluble proteins.

43The co-expression of proteins using dual vectors has the potential to achieve soluble, and active protein complexes while protecting individual subunits from degradation. This is true as the expression of separate components of protein complexes individually often results in IBs formation. For example, the co-expression of bphI and bphJ in E. coli using two compatible plasmids (e.g., pBTL4 and pET28a) yielded a soluble and functional BphI-BphJ complex. Dual vectors, driven by T7 promoters, are designed to co-express two (up to eight) target proteins in E. coli, which allow host strains to simultaneously express any targeted proteins/chaperone combinations.

44Given the tight correlation between certain properties of proteins and their propensity to form IBs as discussed in “The Mechanisms and the Influencing Factors of Protein Inclusion Bodies Formation in E. coli” section, several bioinformatics tools have been developed to predict protein solubility such as Protein-Sol and SoDoPE or identify sequence signatures that lead to protein aggregation, including PASTA 2.0, AMYLPRED 2, Aggrescan, and more others. These tools can be used to minimize protein IBs formation through identifying and then modifying such sequence signatures through site-specific mutagenesis. Such an approach was exemplified by a native Candida Antarctica lipase B (CAL-B) that is prone to form IBs in E. coli. After replacing five hydrophobic residues (Leu147, Leu199, Leu219, Leu261, and Ileu255) with aspartate on the surface of CAL-B, the mutated CAL-B displayed a substantial increased activity and yield in comparison with the wild type enzyme. Furthermore, the expression of soluble and truncated domains of the desired recombinant protein can aid in the production of soluble yet functional proteins. However, these two approaches may not be a viable solution in case modifying targeted protein sequences impairs their functions.

45While the inability to form disulfide bonds within the cytoplasmic space of E. coli might be addressed by engineering trxB– and gor– mutants as mentioned earlier, an alternative solution is through expressing proteins within the periplasmic space that supports the proper disulfide bonding through its oxidative environment and the presence of foldases (including DsbA and DsbC). Linking specific signal peptide to a target protein will localize the target protein to the periplasm via the secretory dependent pathway (Sec), signal recognition particle pathway, or twin-arginine translocation pathway (Tat). The prokaryotic signal peptide sequences OmpA and PhoA are often used for this purpose. The pelB signal sequence from Pectobacterium carotovorum was successfully used to secrete mouse scFv 13R4 antibody fragment into the periplasmic space. Among the additional advantage of expressing proteins in the periplasmic space is their protection against proteolytic cleavages, which can also be obtained by secretory expression outside the outer membrane as mentioned earlier (“Optimized Glycoengineered E. coli Strains for N-Linked Glycosylation” section). This is often desirable to obtain higher yields of the target protein/enzymes as they are not subject to proteases.

46IBs have unique characteristics that may be exploited for medical research and biotechnological applications. First, they are mechanically and chemically stable which constitutes the foundation for their emerging applications as a biomaterial in biomedicine. Second, the propensity of IBs to aggregate homogenously with a high density presents an opportunity for quick isolation of expressed proteins. Moreover and in certain cases, research has demonstrated that inclusion bodies formed by certain proteins might contain bio-functional preparations despite the earlier common notion of mostly un-functional and inactive protein complexes within purified IBs. Such promising observations can be further investigated for the factors that affect such functionalities and decipher the mechanism(s) behind the observed differences among individual proteins.

47Industrial applications of protein IBs have gained momentum with the current technological advancements and research efforts invested in recent years. Lately, IBs have been used in establishing protein scaffolds through tissue engineering to stimulate cell proliferation and enhance cell attachment through adhesion, immobilizing enzymes and the use of cascade enzymatic reactions for enhanced formation of products, and serving as drug-delivery systems. Additionally, IBs formed from therapeutic proteins (e.g., Hsp70, catalase, dihydrofolate reductase, and leukemia inhibitory factor) have been shown to increase the viability of target cells placed under certain stress conditions when such IBs are added to the culture media. Moreover, the use of IBs, as a controlled protein packaging and delivery system, was demonstrated to contribute to the partial reconstruction of cytoskeleton through the utilization of keratin inclusion bodies. Catalytically active IBs have also been used to synthesize key precursors of important pharmaceutical drugs. For example, sialic acid aldolase (SAA) is used in industrial settings to produce neuraminic acid, which is the precursor of the antivirotic drug Relenza®. In this case, an N-terminal cellulose-binding domain from Clostridium cellulovorans with self-aggregation was fused to the SAA gene and expressed in E. coli BL21 (DE3) cells forming catalytically active IBs. These IBs showed the same activity as the soluble SAA enzyme while the lyophilized catalytic IBs showed 93% of the original activity. Significant strides have also been made toward boosting E. coli IBs formation of other pharmaceutically relevant drugs using continuous fed-batch cultivation. For example, catalytically active inclusion bodies were engineered most recently to produce 1,5-diaminopentane (DAP), a natural polyamine with broad prospects for various applications/bio-based polyamides/bioactivities. By using catalytically active IBs of the constitutive L-lysine decarboxylase (expressed in E. coli) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum, high conversions to DAP (87–100%) were obtained in 30–60 mL batch reactions.

48Similarly, an industrial lysozyme of SLLyz from the insect Spodoptera litura was produced as IBs containing a 121 amino acids polypeptide fused at the C-terminal to GST. After purification, this lysozyme demonstrated strong antibacterial activity against Bacillus megaterium, providing a dependable approach for maximizing production and purification of such important recombinant polypeptides. Moreover, IBs proved to be a pivotal tool for the expression and purification of numerous antimicrobial peptides that are of commercial importance. The antibacterial, antiviral, antifungal, and antiparasitic properties of such short (10–100 amino acids) positively charged polypeptides makes them a very interesting target for commercial implementations, especially in light of the current unprecedented spread of multi-drug resistant microbes. The decreased solubility, small-size, susceptibility to degradation, and elevated host-toxicity of such peptides makes the use of IBs heterologous expression the most efficient approach to manage production costs, as well as masking host toxicity issues and protecting the expressed peptides from proteolytic cleavage.

49It should be noted however that E. coli IBs of therapeutic proteins can trigger in some cases endotoxic immune responses in humans due to the presence of lipopolysaccharides. Cases such as these have revived interest in the use of gram-positive strains such as food-grade Lactococcus lactis for the production of functional IBs. Notably, there are commercially available strains of E. coli such as ClearColiTM that do not produce LPS.

50Finally, a good example for IBs potential industrial usages which was recently explored is the use of IBs in the production of 1-Butanol. This solvent is commonly utilized in many industries including but not limited to flavorings, cosmetics, and brake fluids. In addition to its use in repellants production, it is also considered an essential factor in the manufacturing of dietary vitamins and vegetable oils as well as antibiotics and hormones. The natural presence of 1-Butanol in alcoholic beverages, chesses, fruits, and a variety of other foods (as a by-product of carbohydrate fermentation), negates any concerns to its safety within the reported concentrations. In the above explored application, IBs were designed to harbor heterologous enzymes that are involved in 1-Butanol production alongside a carbon binding domain (CBD). The enzymes and the CBD interacted through a leucine zipper motive mimicking a prey-bait system to achieve active IBs for 1-Butanol production, leading to a 1.5-fold increase in 1-Butanol yields compared to the control.

51E. coli is a popular microbial host for protein expression, where recombinant proteins applications often encounter issues with IBs formation. Many strategies have been developed to reduce the formation of IBs in E. coli, involving the fine-tuning of protein expression rates, engineering host strains to enable key post-translational modifications, tuning the expression vector appropriately, and using bioinformatics tools to predict the tendency of proteins to aggregate. These strategies were discussed extensively for the aims to reduce and prevent IBs formation. Furthermore, the review discussed specific methods and examples to achieve the desired goals of many industrial applications while addressing any shortcomings. The future better understanding in the regulation mechanism of protein homeostasis network will certainly facilitate the development of strategies to minimize protein IBs formation in E. coli. In addition, the recently changing views on classical IBs may be attributed to the discovery of functionally active IBs. These changing views have enabled the breakthrough of IBs into industrial areas for the production of compounds, and future use in medicines. While the field agreeably is at its infancy with a limited number of reports/experiments, research endeavors focusing on IBs within the large industrial scale are encouraged in order to refine future applications, better understand the various aspects of E. coli IBs, and identify mitigation strategies.

52AB and WW were involved in the development of the topic and initial drafts. YH and NA contributed by including and writing further relevant ideas of discussion to the topic. X-ZL and all other listed authors assisted in editing and peer-reviewing the manuscript. TZ conceived and coordinated the study. All authors contributed to the article and approved the submitted version.

53The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.