Protein Expression and Purification 219 (2024) 106463

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Protein Expression and Purification

Review article

Escherichia coli as a versatile cell factory: Advances and challenges in

A R T I C L E I N F O A B S T R A C T

Keywords: E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of
E. coli recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a
Recombinant protein widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant
Optimized protein production
portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it
Solubility of protein
has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to
the production of misfolded, insoluble, or dysfunctional proteins.
This study focused on the challenges and engineering approaches for improved expression and solubility in
recombinant protein production in E. coli. In this context, solution strategies such as strain and vector selection,
codon usage, mRNA stability, expression conditions, translocation to the periplasmic region and addition of
fusion tags in E. coli were discussed.

1. Introduction and high efficiency of the transformation process are the important
factors that enable this progress [6]. In general, there are some diffi­
In the 1970s, a new area in the production of biopharmaceuticals culties in the production of recombinant proteins in various hosts, such
began with the discovery of recombinant DNA technology. Before re­ as high cost, complexity of the protein production process,
combinant insulin protein was produced, people used insulin derived post-translational modification processes, high protease activities of
from pig and bovine pancreas to treat diabetes. In 1978, the first re­ hosts, tendency to use unrelated codons, problems in the solubility of
combinant human insulin was expressed in E. coli K-12 and was intro­ recombinant proteins, failure to secrete recombinant proteins, and
duced for the treatment of diabetes in 1982 [1]. After this development, contamination with toxins and pathogens [7].
the idea that other therapeutic proteins could be produced quickly and Expression systems used in E. coli provide high protein yield, rapid
economically in E. coli gained importance. E. coli is a widely used and growth, low production costs (compared to eukaryotic systems), and
low-cost host for plasmid DNA and recombinant protein production. short production times. Despite its significant advantages, this system
Additionally, a significant portion of approved therapeutic proteins have also has disadvantages. One of these is the absence of eukaryotic post-
been produced in E. coli. Therefore, it is important for recombinant translational modifications that could result in the production of mis­
protein production in medical, food, chemical and other industries [2, folded, insoluble, or dysfunctional proteins. Therefore, complex proteins
3]. that require post-translational modifications cannot be produced
Escherichia coli (E. coli) is a rod-shaped gram-negative bacteria and a correctly in this system [8]. Some strategies have been developed over
mammalian intestinal pathogen that was first identified by Theodor the years to improve the production of recombinant proteins in the
Escherich [4]. E. coli genetics has received greater attention than those E. coli host system, and researchers continue to develop these strategies
of other bacteria. The use of E. coli bacteria for producing recombinant [9–11]. Additionally, new bioinformatic techniques have made it
proteins has gradually risen as a result of recent breakthroughs in possible to predict possible expression problems such as protein solu­
expression methods, protein folding mechanisms, and genetic tools [5]. bility during overexpression [12]. Furthermore, eliminating contami­
The ease of genetic manipulation in bacteria and the simple, low-cost nant endotoxins (such as lipopolysaccharides) formed in E. coli during

* Corresponding author.
E-mail addresses: ibrahimincir@[Link] (İ. İncir), [Link]@[Link] (Ö. Kaplan).

[Link]
Received 3 January 2024; Received in revised form 25 February 2024; Accepted 11 March 2024
Available online 12 March 2024
1046-5928/© 2024 Elsevier Inc. All rights reserved.
İ. İncir and Ö. Kaplan Protein Expression and Purification 219 (2024) 106463

production is a significant issue. Although new systems are being recombinant proteins. To overcome this issue, mutagenesis can change
actively researched, E. coli is still the most widely used bacterial strain the negative redox potential of the cytoplasm by changing the enzymes
[13] due to its adaptability to new constraints such as antibiotic-free of the trx and gor systems. Commercially available modified E. coli
selection and ease of engineering [14,15]. In this review, it was aimed strains, such as Origami B(DE3) (trxB mutant) and Rosetta-gami 2(DE3)
to examine the problems and solution suggestions encountered in the (double mutant with trxB and gor mutations), have been successfully
production of recombinant protein in E. coli. used in numerous studies for the high soluble production of proteins
requiring disulfide bonds [22–24]. E. coli Shuffle strain allows the
2. Challenges and engineering approaches in E. coli expression of a chromosomal copy of the disulfide isomerase DsbC, in
recombinant protein production for enhanced expression and addition to lacking trxB and gor functions. The utilization of the Shuffle
solubility strain has enabled high levels of soluble and correctly folded protein
production, particularly for proteins containing disulfide bonds [25].
Various factors affect the soluble synthesis of recombinant proteins Furthermore, the CyDisCo expression system in E. coli allows the soluble
in E. coli. Heterologous gene expression frequently results in the syn­ expression of proteins containing disulfide bonds in the cytoplasm. This
thesis of recombinant proteins with insoluble inclusion bodies. Several technique is based on the co-production of a protein, disulfide isom­
factors can contribute to the synthesis of insoluble proteins in E. coli. erase, and sulfhydryl oxidase [26].
These include the incapacity of E. coli to execute post-translational E. coli strains have also been modified to produce toxic proteins.
modifications, the rapid production facilitated by promoters, the insta­ Uncontrolled production of the desired protein in an insoluble form can
bility of mRNA, bias in codon usage, and the absence of distinct sub­ be caused by leaky or basal expression of T7 RNA polymerase. To
cellular compartments within the E. coli cytoplasm [16]. mRNA stability address this issue, tightly regulated expression of T7 RNA polymerase is
in E. coli directly affects the expression rate of recombinant proteins and preferred. For instance, the pLysS strain is used to inhibit the basal ac­
may result in rapid expression. Similarly, E. coli has restricted tivity of T7 polymerase. Through this strategy, strains like Rosetta-gami
post-translational modification processes, which can prevent some re­ pLysS, BL21 (DE3) pLysS, and Origami pLysS have been developed [27,
combinant proteins from folding properly. Furthermore, fast accumu­ 28]. Another strategy involves expressing the T7 RNA polymerase gene
lation of recombinant proteins inside the cytoplasmic compartment may under a stringent promoter. For example, the BL21-AI strain, controlled
interfere with correct folding [17,18]. by the araBAD promoter, regulates T7 polymerase activity in the
There are several techniques for increasing the soluble synthesis of absence of arabinose and presence of glucose. The BL21-AI strain has
recombinant proteins in E. coli. These consist of optimizing expression been utilized to successfully produce high amounts of toxic proteins in
conditions, employing modified E. coli expression strains, co-expressing soluble form [29].
with molecular chaperones, using low-copy plasmids with weak pro­ Most membrane proteins require entry into the membrane via a
moters, and attaching a solubility-enhancing tag to the target protein channel known as a translocon. This complex (SecYEG) is found in the
[19]. In this section, effective strategies to increase the solubility and inner membrane of E. coli, while the Sec61 complex (in eukaryotic cells)
expression efficiency of proteins in E. coli have been discussed. is found in the endoplasmic reticulum. Although this mechanism is
generally conserved, there are differences that cause difficulties during
2.1. Selection of E. coli host strains the synthesis of membrane proteins [30]. C41(DE3) and C43(DE3)
strains of E. coli were developed for the expression of the gene encoding
Various factors in E. coli influence the solubility and production of the membrane protein. These capacity of strains to overexpress mem­
recombinant proteins. Host strain selection is one of these factors, and brane proteins is owing to a mutation in the lacUV5 promoter, which
choosing the optimal host strain based on the characteristics of the gene leads in lower T7 RNA polymerase production [31]. In addition, C41
product is crucial. We can summarize host strains into six classes for (DE3) and C43(DE3) strains can help overcome plasmid instability
protein expression: 1. protease-free strains (e.g., BL21, BL21(DE3), and during toxic protein production [32]. Excessive production of mem­
BL21star (DE3)); 2. toxic protein-expressing strains (e.g., BL21-AI, BL21- brane proteins strains the cellular machinery, preventing overproduced
pLysS, and BL21-pLysE); 3. strains suitable for expressing proteins with recombinant proteins from entering the membrane and causing them to
disulfide bonds (e.g., Origami, and Shuffle); 4. strains designed to pro­ accumulate in the cytoplasm. To overcome this challenge, the tran­
duce proteins from genes with rare codons by overexpressing rare codon scripts of genes can be regulated by T7 RNA polymerase interacting with
t-RNAs (e.g., Rosetta and BL21-codonPlus); 5. strains capable of pro­ T7 lysozyme or through mutations in the lacUV promoter. The Lemo21
ducing membrane proteins and inducible protein expression systems (e. (DE3) strain exhibits the ability to properly overproduce both mem­
g., Lemo 21(DE3) and C41(DE3), C43(DE3)); 6. strains designed to ex­ brane and toxic proteins [33]. The ArcticExpress (DE3) strain is used to
press aggregation-prone proteins (e.g., ArcticExpress (DE3)). improve recombinant protein solubility by boosting low-temperature
E. coli BL21 and its derivatives are commonly preferred expression expression with active molecular chaperones and optimal folding
strains for recombinant protein production. This strains allows efficient under low-temperature settings [34].
production of soluble recombinant proteins due to the absence of ompT
and lon proteases [20]. The chromosomal copy of the T7 RNA poly­ 2.2. Codon usage
merase gene under the control of the lac promoter is carried by the E. coli
BL21 derivative strain DE3, allowing stimulated cells to synthesize The situation where the frequency of codons in foreign coding DNA
transcripts of the gene located just downstream of the T7 promoter. differs greatly from that of the host is known as codon bias [35].
Additionally, mRNA stability within the cell is another factor influ­ Depletion of low abundance tRNAs can occur during the synthesis of a
encing the soluble production of recombinant proteins, and an addi­ recombinant protein. This can result in protein misassemble or cleavage,
tional mutation in the RNaseE gene inhibits mRNA degradation. The rne affecting the expression and activity of the protein. Two strategies have
gene mutation in E. coli BL21Star (DE3) results in a considerable in­ been employed to address codon usage bias: codon optimization or
crease in the synthesis of soluble recombinant proteins in this mutant enhancing compatibility with the underrepresented tRNAs through host
strain [21]. modifications [36].
The reduced redox environment in the E. coli cytoplasm is one of the Codon optimization involves adapting the rare codons of a gene to be
primary factors contributing to recombinant protein misfolding and more compatible with the codon usage of host without altering the
aggregation. This reduced redox state is provided by the thioredoxin- amino acid sequence of the protein [37]. However, codon optimization
thioredoxin reductase (trxB) and glutaredoxin-glutaredoxin reductase can be laborious and costly, which limits its widespread use. On the
(gor) systems, which prevent the proper formation of disulfide bonds in other hand, rare codons function as genetic regulators to control the rate

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İ. İncir and Ö. Kaplan Protein Expression and Purification 219 (2024) 106463

of protein synthesis and influence secondary and tertiary structure for­ bacteriophage T7, enabling induction with lactose or its non-hydrolyzed
mation. These codons are often found in abundance at structural domain analogue, isopropyl β-D-1-thiogalactopyranoside (IPTG) [17]. Different
boundaries and β-sheet, providing sufficient time for the nascent protein combinations of promoters have been devised to enhance expression,
to acquire its correct structure [38]. It has also been shown that rare such as the powerful tac promoter and the T7 promoter system
codons can significantly affect protein folding. Codon adaptation has commonly used in pET vectors.
been successfully utilized to improve protein solubility by enabling Arabinose promoter-based systems (pBAD vectors) are also
slow-translating codons to occur at structural domain boundaries [39]. commonly used in protein expression. These vectors include the araC
Rare codons near the 5′ end of a gene can help to stabilize the ribosomal gene, which encodes the PBAD promoter from the araBAD (arabinose)
initiation complex and reduce the formation of mRNA secondary operon, as well as the promoter’s positive and negative regulators. AraC
structures. There is a correlation between decreased stability of RNA suppresses araBAD promoter expression in the absence of L-arabinose or
secondary structure and higher levels of protein expression [40,41]. In in the presence of glucose but stimulates transcription in the presence of
conclusion, rare codons are an important factor in recombinant protein both L-arabinose and glucose [49]. L-arabinose costs less than IPTG,
expression, and strategies such as codon optimization or codon adap­ making large-scale protein production more economical. High copy
tation can be employed to ensure proper expression and folding of such number pTTQ18 plasmids (containing the tac promoter, lacIQ repressor,
proteins. However, it is important to note that some studies have sug­ rrnB terminator and ampicillin resistance) have been successfully used
gested that codon adaptation might also affect ribosomal initiation in combination with strains of type BL21(DE3) to increase expression of
complex and translation speed [42]. Additionally, it has been reported various bacterial and archaeal membrane proteins [50].
that mRNA stability and protein yield may decrease in some cases where The pL promoter is associated with pH or temperature responsive
codon optimization is performed [43]. systems [51]. pQE vectors use the T5 promoter. E. coli RNA polymerase
Another strategy developed for codon bias is the use of a plasmid also transcribes the phage T5 promoter, however many of the pQE
encoding rare tRNAs, and pRARE was developed using this strategy. plasmids lack the lacIQ repressor gene, therefore a host with the
pRARE contains tRNA genes that recognize rare codons. E. coli Rosetta lacI-carrying compatible plasmid pRep4 or lacIQ is necessary. Eukary­
and BL21-CodonPlus strains were constructed using the pRARE plasmid. otic proteins produced in E. coli are mostly insoluble, however cooling
Later, E. coli versions containing rarer tRNA genes were also created. the culture during production can sometimes increases their solubility.
CodonPlus-RIL and CodonPlusRP strains are used to overcome the ten­ One proposed explanation for this improvement is because chaperones
dency of AT- and GC-rich genome, respectively [31]. activated by E. coli’s cold shock response assist foreign proteins in
Tegel et al. (2011) investigated the expression of a number of human folding correctly. In this context, plasmids from the pCold family with a
proteins in E. coli BL21(DE3) and Rosetta (DE3) cells and discovered that pUC118 backbone and a CspA promoter are employed [17]. Rhamex
the Rosetta (DE3) strain produced more soluble protein [44]. Arif et al. vectors contain the regulatory genes RhaR and RhaS and are capable of
(2016) used the E. coli BL21-CodonPlus (DE3)-RIL strain to express adjusting the expression level of target mRNA, which can increase
human interferon -2b and E. coli methionine amino peptidase genes and protein accumulation and protein solubility [52]. Finally, for recombi­
obtained higher recombinant protein yields by avoiding the codon bias nant proteins with fusion tags such as six histidines (6x His-tag), maltose
problem [45]. Valverde-Tercedor et al. (2015) investigated the best binding protein (MBP), and glutathione S-transferase (GST), different
expression conditions of MamC and MamCnts in four E. coli strains: E. coli expression vectors (pBAD, pQE, pUC, and pET) are extensively
TOP10, BL21 CodonPlus-RIL, BL21 star, and BL21 (DE3), and found that utilized [17].
the BL21 CodonPlus-RIL strain produced the highest quantity of soluble
protein [46]. The presumptive membrane protein of M. tuberculosis, 2.4. Secondary structure stability of mRNA
which has a high GC content, was efficiently produced in E. coli BL21
(DE3) CodonPlus-RP [47]. In E. coli, transcription and translation occur simultaneously, so
transcription affects translation and folding of proteins. To ensure
2.3. Selection of expression vectors effective translation initiation, the formation of stable mRNA secondary
structures is prevented at the 5′-end of the open reading frame. Various
Expression vectors have various combinations of promoters, multiple factors, such as GC content, influence mRNA secondary structures.
cloning sites, replicons, selection markers, and fusion tag. The choice of Therefore, in the 5′-end of the open reading frame, there is a preference
an appropriate vector for protein expression is a crucial factor in for 35–40 nucleotides rich in A/T nucleotides compared to stable G/C
achieving high levels of target protein production in E. coli. The copy nucleotides. This helps prevent the formation of mRNA secondary
number is a significant parameter to consider. High copy number vectors structures [53].
may result in higher recombinant protein synthesis within the cell. The Shine-Dalgarno (SD) sequence located upstream of the start
However, vectors with high copy numbers may impose a metabolic codon and the sequence complementary to SD in 16S rRNA affect the
burden and lead to decreased protein synthesis. The vector should have stability of the ribosome attached to the mRNA. Although the maximum
a tightly controlled strong promoter with low basal expression levels for length of this match is 12–13 nucleotides [54], it has been reported that
an optimum expression system. The lactose (lac) promoter has provided a 6-nucleotide match with the SD sequence (AGGAGG) gives the best
important insights into the development of prokaryotic expression results for translation [55]. Additionally, the distance between the start
vectors. However, the lac promoter and its derivatives are weak for re­ codon and the SD sequence is important for high-level translation. From
combinant protein production. Instead, synthetic combinations of experimental studies in E. coli, this optimal distance has been suggested
different promoters have been developed [48]. For example, the tac to be a length of 4–12 nucleotides [56].
promoter is formed by combining the − 35 region of the tryptophan (trp) A stable mRNA structure may influence the interaction with specific
promoter with the lac repressor recognition region in the − 10 region. It RNA regions (start codon and SD sequence) that can be recognized by
is approximately 10 times more potent than the standard lacUV5 pro­ the ribosome. Increased mRNA stability in the translation start region
moter. The tac promoter can be further enhanced by fusing it with lacIQ, has been demonstrated in experiments to potentially prevent effective
a mutated repressor of the lacI gene, allowing higher expression levels translation initiation, thus decreasing levels of gene expression [57]. For
(approximately 10-fold higher) than that achieved with lacI. The T7 example, the SD sequence in the mRNA of unexpressed genes may bind
promoter system, which is prevalent in pET vectors, is frequently to a very stable stem-loop structure, making it inaccessible to the ribo­
employed. In this system, the target gene is placed under transcriptional some. In contrast, SD sequence in expressed genes can form less hairpin
control of the bacterial genome’s lacUV5 promoter and cloned down­ structures, making it more accessible. Therefore, mutations on the
stream of a promoter recognized by the T7 RNA polymerase of the overall stability of the mRNA secondary structure may result in

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İ. İncir and Ö. Kaplan Protein Expression and Purification 219 (2024) 106463

increased overall free energy and allow exposure of the SD sequence folding [67]. The culturing temperature influences not only the pro­
[58]. This may lead to overexpression of the protein. In a study on the duction of inclusion bodies, but also the activity of the recombinant
expression of hepatitis B virus surface antigens and hepatitis E virus ORF protein. It should be emphasized, however, that expression at low
2 protein similar mRNA secondary structures were observed in temperatures might result in a decrease in recombinant protein pro­
non-expressed genes, preventing access to the SD sequence and AUG duction [16].
codon. Then, as a result of modifications made at the 5′-end of these A few methods have been developed to increase recombinant protein
genes, their expression was successfully achieved in E. coli [59]. Zhang synthesis at low temperatures. One of them is gene expression induction
et al. found that mutating the 5′ end of human IL-10 and IFN-α mRNA using cold shock promoters like CspA (Cold-shock protein A), which
enhanced the free energy of secondary structures and revealed the AUG work best at temperatures below 30 ◦ C. Low-temperature CspA pro­
start codon. As a result, the expression efficiency of both genes increased moter use may benefit membrane-spreading domains or other labile
significantly [60]. gene products in E. coli. E. coli cells can grow rapidly even at 4 ◦ C when
two cold-inducible chaperones, Cpn10 and Cpn60, are induced. Heat
2.5. Expression conditions shock protein stimulation in E. coli may also be advantageous for
increasing recombinant protein solubility. Pre-induction of culture at
Expression conditions play a critical role in the production of re­ 42 ◦ C enhances protein solubility and inhibits the development of in­
combinant proteins and should be optimized to increase soluble product clusion bodies [68]. The co-expression of aminoacylase and the chap­
yield. Several methods for preventing the development of inclusion erone GroEL/S in E. coli was found to increase the enzyme’s activity
bodies have been developed. Some of these methods are optimization of (1.8-fold) in research conducted by Haeger et al. It was also reported
inducing agent concentration and induction temperature, adjustment of that the aminoacylase produced in E. coli ArcticExpress (DE3) that
culture density, addition of chemical and biological chaperones [3]. co-expresses with cold-inducible chaperonins Cpn60/10 at 12 ◦ C had
2-fold higher activity than the aminoacylase produced in E. coli BL21
2.5.1. Optical density at induction (DE3) with GroEL/S co-expression at 20 ◦ C [69].
The optical density of the culture during induction is critical in
influencing recombinant protein synthesis and solubility. In most cases, 2.5.4. Addition of chemical chaperones
protein expression during the early mid-log phase or early log phase or is In response to different stresses, organisms use various methods to
linked with higher protein solubility. This also helps with purification maintain cellular integrity. Under osmotic stress, bacterial defense
since the E. coli protein to recombinant protein ratio is low at these mechanisms prevent denaturing of proteins by accumulating high con­
stages of growth. As a result, for best output, induction is normally centrations of osmo-protectors [70]. The osmo-protectants also act as
undertaken in the early mid-log phase, but induction outside of the mid- chemical chaperones. By adding these chemical chaperones to the
log phase might result in a considerable decrease in recombinant protein growth medium, such as polyols, sugars, glycerol, dimethyl sulfoxide,
synthesis and activity. Nutrient limitation minimizes cellular activity at ethanol, low molecular weight thiols, amino acids, and methylamines,
larger cell densities due to metabolic load, limited dissolved oxygen the solubility of recombinant proteins can be increased [71]. For
availability, acetate synthesis, and high levels of carbon dioxide. As a instance, the addition of polyols and sugars indirectly affects the solu­
result, the expression of recombinant genes is lowered. Induction in the bility of recombinant proteins by increasing the levels of other
late log phase of culture, on the other hand, may be helpful in some osmo-protectants, thus enhancing the protein’s stability [72]. On the
circumstances, notably for hazardous proteins and proteases, because other hand, when glycerol and dimethyl sulfoxide are added, it has a
there are enough cells to generate these negatively impacting proteins direct effect on protein solubility. Ethanol addition increases the stress
[61]. response of cells and improves the solubility of recombinant proteins by
increasing the production of molecular chaperones [73]. The use of low
2.5.2. Induction concentration molecular weight thiols leads to an increase in the solubility of
For strong promoters, the amount of inducer employed is frequently disulfide-bonded proteins. This increases the solubility by affecting the
linked with the level of expression. Induction levels that are too low redox state of thiols [74]. The addition of amino acids and methylamines
result in low expression, whereas induction levels that are too high has also been effective in suppressing protein aggregation and
might be harmful to the cells. Inclusion bodies can occur when the increasing the solubility of recombinant proteins [75]. Additionally, the
translation rate exceeds the folding capability of the chaperone ma­ addition of certain cofactors or prosthetic groups can prevent the for­
chinery in the presence of a high inducer concentration [62]. As a result, mation of insoluble protein aggregates, thereby enhancing the solubility
the inducer concentration should be carefully determined. To obtain of recombinant proteins. These chemical chaperones can also be added
best yield, the concentration of the inducer, iso­ during cell lysis to aid in obtaining soluble fractions of otherwise
propyl-D-1-thiogalactopyranoside (IPTG), in E. coli is typically between insoluble proteins. In some cases, these chemical chaperones have been
0.1 and 1 mM [63]. observed to increase activity by improving the functional conformation
of soluble proteins [63].
2.5.3. Cultivation temperature
Energy consumption during heterologous gene expression raises the 2.5.5. Co-expression with chaperone proteins
metabolic burden and consumes the host’s energy necessary for appro­ Chaperone proteins help in the correct folding of proteins and the
priate protein folding. This frequently results in the creation of inclusion avoidance of aggregation. Recombinant proteins are produced quickly
bodies [64,65]. Because of the very hydrophobic interactions of the with high numbers in E. coli. While small heterologous proteins tend to
newly synthesized peptides, the creation of inclusion bodies is strongly fold faster, the risk of misfolding increases as the size of the protein
temperature sensitive. Lowering the temperature of the bacterial culture increases. Chaperone proteins can play a crucial role for heterologous
is one technique to minimize inclusion body development [66]. This is a proteins prone to aggregation [76]. E. coli has many chaperone protein
well-known method for minimizing inclusion body development in systems, including GroES/EL and DnaK-DnaJ-GrpE, each with a unique
E. coli. Lower temperatures aid in the proper folding of newly formed function. The GroES/EL system aids in the repair of misfolded proteins.
proteins due to several variables such as a slower translation rate, The DnaK-DnaJ-GrpE system, on the other hand, not only assists in the
reduced protein self-assembly, and changes in the folding kinetics of the appropriate folding of freshly translated peptides, but also operates
polypeptide chain. Furthermore, at lower temperatures, the activity and during co-translation and post-translational modification. Over­
synthesis of several E. coli chaperones increases. These elements expression of molecular chaperones can improve folding efficiency;
contribute to improved stability at lower temperatures and appropriate typically, three combinations are used: DnaK-DnaJ-GrpE, GroES/GroEL,

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İ. İncir and Ö. Kaplan Protein Expression and Purification 219 (2024) 106463

or co-expression [77]. particular the GroEL-GroES complex, are higher than when expressed
In the GroEL/ES complex, GroEL consists of two ring-shaped hep­ without chaperones. In addition, an increase in enzyme activities was
tamers, and when a protein enters the cavity of GorEL, the lid-like observed not only in secretory enzymes but also in the tested cytosolic
structure GroES seals it. This structure exhibits its protein folding ac­ Benzoylformate decarboxylase, Benzaldehyde dehydrogenase and
tivity through ATP hydrolysis [78]. Co-expression of GroES and GroEL D-phenylglycine aminotransferase enzymes, especially as a result of
has been shown to enable approximately 65% of the single-chain anti­ co-expression with GroEL-GroES [96].
bodies (scFv) to be produced in soluble form [79]. Goyal and Chaudhuri
found that co-expression of recombinant yeast mitochondrial aconitase 2.5.6. Medium composition
and maltodextrin glucosidase with GroEL/ES increased the yield, solu­ When the volumetric yield of a recombinant protein is low and
bility, and enzyme activity [80]. Additionally, Liu et al. reported that the cannot be improved by known techniques, the volumetric yield of the
solubility of the ovine growth hormone was improved through protein can be enhanced by growing the culture at greater densities. LB
co-expression with GroEL/ES [81]. The chaperone function of GroEL/ES medium is a typical E. coli culture medium and is easily prepared, of­
has been shown to be more effective for small proteins that can fit into fering a rich nutrient content. However, it may not be the ideal choice
the gap. These results indicate that while selecting GroEL/ES, the for low cell density cultures. Due to its limited amount of carbohydrates
properties and size of the protein should be taken into account [82]. and the presence of divalent cations, LB medium restricts cell growth at
However, a recent study by Yurkova and Fedorov suggested that GroEL relatively low densities. It has been hypothesized that increasing the
may be able to assist in folding some proteins even without GroES and quantity of peptone or yeast extract in the LB medium will result in
may have the ability to fold substrates outside of the cavity [83]. larger cell densities [97]. An important advancement in medium
The chaperone protein DnaK operates in an ATP-dependent manner composition has been made through the concept of auto-induction.
and collaborates with the chaperone proteins DnaJ and GrpE to facilitate Auto-induction medium use a carefully optimized mixture of lactose,
protein folding under both normal and stressful growth conditions [84]. glucose, and glycerol. Glucose is the primary carbon source and is
The DnaK-DnaJ and GrpE system plays a crucial role in various cellular largely digested throughout development, inhibiting lactose absorption
processes including polypeptide folding, protein translocation across until glucose is exhausted and triggering expression in the mid to late log
membranes, assembly of multi-subunit protein structures, DNA repli­ phase. As cell density increases, the availability of oxygen becomes
cation, cell division, solubility of protein aggregation [85]. Hu et al. critical for growth [98]. Increasing the shaking speed in shaking flasks
demonstrated that the soluble production of scFv protein increased by enhances oxygen levels, crucial for proper aeration. Furthermore, the
up to 100-fold through expression with DnaK-DnaJ-GrpE (DnaKJE) culture volume to flask capacity ratio effects adequate aeration [99].
[86]. Ying et al. noticed that the expression system involving DnaK-DnaJ The initial culture’s preparation and the timing of induction are other
and GrpE significantly improved the solubility of the anti-BSA single-­ critical aspects. A saturated overnight pre-culture may lead to unstable
chain antibody fragment [87]. Niwa et al. found that the DnaK/J/GrpE protein expression and low yield. An appropriate pre-culture can be
system was the most successful after evaluating over 800 cytosolic obtained from actively and evenly growing cells [100].
proteins with a cell-free translation system. It enhanced the solubility of
409 proteins by more than 50% as compared to GroEL/ES’s 287 pro­
teins. The simultaneous inclusion of two chaperone systems was 2.6. Translocation to periplasmic region
particularly successful in producing the group of resistant proteins in
soluble forms, suggesting that the DnaK/J/GrpE and GroEL/ES systems In E. coli, the periplasm is more oxidized than the cytoplasm, which
had a synergistic impact in recombinant protein folding [88]. has a negative redox potential. Cysteine residues carry out crucial redox
The trigger factor (TF) binds near the peptide exit site of ribosomes events in the periplasm in an oxidative environment, boosting disulfide
and interacts with short chains [89]. It has been demonstrated that TF bond formation and maintaining the bonds oxidized. An N-terminal
interacts with GroEL and enhances GroEL-substrate binding to facilitate signal sequence is added to direct recombinant proteins in E. coli to the
protein folding [90]. TF and heat shock proteins (GroEL/ES and periplasm. ompT and pelB signal sequences are routinely utilized for
DnaK/J/GrpE) were coexpressed alone and in combination to produce periplasmic localization [101]. Protein secretion into the periplasmic
three recombinant proteins (human lysozyme, human oxygen-regulated localization with these signal sequences provides a good environment
protein, mouse endostatin) in soluble form [91]. In 2020, Maksum et al. for the folding of the recombinant protein. Disulfide bond system (Dsb)
reported that co-expression of prethrombin-2, GroEL/ES, and proteins are involved in the formation as well as regulation of disulfide
DnaK-DnaJ-GrpE in E. coli ER2566 generated more soluble protein than bonds in E. coli. DsbA-DsbB promotes the formation of disulfide bonds in
co-expression with GroEL/ES [92]. TF, GroEL/ES, and DnaK/J/GrpE proteins that transport electrons to the respiratory chain in the peri­
were used to increase the solubility of the recombinant spike glyco­ plasm. Furthermore, the DsbC-DsbD pair isomerizes the mismatched
protein. Among the three chaperone systems, TF was found to be the disulfide bonds by using electrons provided from the inner membrane
most effective in increasing solubility [91]. A recent study reported re­ [102].
combinant production of chondroitin 4-O-sulfotransferase-1(C4ST-1). Periplasmic expression can be achieved by the post-translational Sec-
They developed a TF-C4ST-1 fusion system that provided 60% of the dependent mechanism. For recombinant proteins, this approach permits
catalytic efficiency of the recombinant enzyme compared to production extra-cytoplasmic targeting and is paired with an appropriate leader
without TF [93]. Kudhair and Green reported that co-expression of the peptide. Alternatively, the SRP (signal recognition particle) route, a
WhiB3 protein of Mycobacterium tuberculosis with the trigger factor frequent translation translocation mechanism, can be employed. With a
increased the solubility of the WhiB3 protein [94]. hydrophobic signal sequence at its N-terminus, SRP detects substrates,
The plasmids encompass various combinations of chaperone proteins and the resultant chain-ribosome complex is transported to the SecYEG
in five different types: pG-KJE8 (DnaK-DnaJ-GrpE/GroES-GroEL), translocon, which interacts with the membrane receptor FtsY. The di­
pGro7 (GroES-GroEL), pKJE7 (DnaK-DnaJ-GrpE), pG-Tf2 (GroES- sulfide isomerase I (DsbA) signal sequence was also exploited in the SRP
GroEL/TF), pTf16 (TF). These plasmids are commonly utilized for co- pathway to drive recombinant proteins to the periplasm [103]. These
expression with chaperone proteins [79,95]. In a study by Jomrit processes meant that essential recombinant proteins like thioredoxin
et al., it was reported that the yield was increased when secretory en­ and human growth hormone were successfully expressed in the peri­
zymes xylanase, glucanase and mannanase were produced in E. coli BL21 plasm. It has been reported that YebF protein, containing MdoD and
(DE3) without the sequence corresponding to the signal peptides. It has AmiC signal peptides produced using the CyDisCo system, is sent to the
been reported that enzyme activities obtained from co-expression of periplasm in high amounts via the Tat (twin-arginine translocation)
these enzymes without signal peptide sequences with chaperones, in pathway [104].

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İ. İncir and Ö. Kaplan Protein Expression and Purification 219 (2024) 106463

2.7. Attachment of fusion tag 3. Conclusions

The addition of fusion tags is a frequently used approach for In this review, E. coli was evaluated as a versatile cell factory for
improving recombinant protein solubility, stability, and purification recombinant protein production. The industrial importance and ad­
[105]. Furthermore, fusion tags can shield the recombinant protein from vantages of recombinant protein production in E. coli were emphasized.
proteolysis and minimize its antigenicity. Fusion tags can be introduced Despite these advantages, E. coli-based recombinant protein production
at the protein’s N-terminus, C-terminus, or inside the coding sequence. has challenges such as inclusion bodies, protein misfolding, proteolytic
His, CBP, and FLAG tags with low molecular weight do not need to be degradation, codon bias, and host cell toxicity. We discussed in detail
removed following protein purification. Some fusion tags, on the other the engineering approaches developed to overcome the mentioned
hand, are often bigger in size and may interfere with protein folding. challenges and improve protein expression and solubility. These ap­
Tags such as MBP and GST, for example, can cause misfolding, which proaches include the use of fusion tags, co-expression with chaperone
can result in changes in biological function, decreased yield, toxicity, proteins, host selection, inducer concentration, and other strategic
and structural flexibility. The tag can be removal after purification to techniques.
prevent misfolding. Tag removal can be accomplished via a variety of In conclusion, E. coli is a valuable and versatile cell factory to pro­
protease systems, including TEV, thrombin, factor Xa, and enterokinase duce recombinant proteins. Engineering research and development has
[106]. increased the importance of E. coli in recombinant protein production.
The His-tag, which comprises six or more consecutive histidine res­ Future research and development could increase the value and efficacy
idues, is often utilized for successful affinity purification of fused pro­ of E. coli in recombinant protein synthesis, allowing more extensive and
teins. Following expression, the fusion protein is purified using a Ni-NTA effective applications in the biotechnology and pharmaceutical sectors.
affinity purification method, and the His-tag is later removed using
proteases. Funding
Various protein tags, such as MBP, GST, NusA, Trx, and SUMO, are
commonly utilized as fusion tags in E. coli to create soluble, active, or This work did not receive any funding.
partly active proteins. MBP is a common fusion tag for increasing the
solubility of proteins. MBP performs as a molecular chaperone, facili­
CRediT authorship contribution statement
tating the proper folding of the fusion protein. To avoid aggregation or
proteolysis, it is thought to interact with unfolded proteins’ hydrophobic
İbrahim İncir: Writing – review & editing, Writing – original draft,
amino acid residues [107]. In a study conducted with the MBP fusion
Conceptualization. Özlem Kaplan: Writing – review & editing, Writing
tag, it was shown that the amount of soluble protein increased approx­
– original draft, Conceptualization.
imately 10-fold compared to the expression of Tth DNA polymerase
produced with the MBP fusion tag in E. coli BL21 (DE3) without the MBP
fusion tag [108].
Declarations of completing interest
Glutathione S-Transferase (GST) is a protein with a molecular weight
of 26 kDa. When compared to other commonly used fusion tags, GST’s
The authors declare no potential conflicts of interest.
ability to enhance solubility is rather limited [109]. NusA is a 55 kDa
protein that delays translation in transcriptional terminations, giving
Data availability
additional time for protein folding and also stabilizing the passenger
protein throughout translation [106]. According to the findings, NusA
No data was used for the research described in the article.
interacts with chaperones such as GroEL [110]. In E. coli, Trx (Thio­
redoxin) is a 12-kDa intracellular thermostable protein. It is widely used
as a fusion tag to improve target protein solubility by using its native References
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