# E. coli Expression System: Principles and Applications

## Introduction to the E. coli Expression System

The *E. coli* expression system is a molecular biology platform that uses the bacterium *Escherichia coli* as a host to produce recombinant proteins. The system's purpose is straightforward: to introduce a gene of interest into *E. coli* cells, induce [transcription and translation](/knowledge/molecular-biology/transcription-translation) of that gene, and harvest the resulting protein for research, therapeutic, or industrial applications. Since the advent of recombinant DNA technology in the 1970s, *E. coli* has become the most widely used prokaryotic host for heterologous protein production, and it remains the first choice for many applications despite the availability of yeast, insect, and mammalian systems.

The importance of the *E. coli* expression system in biotechnology cannot be overstated. It is the production platform for numerous FDA-approved therapeutic proteins, including insulin (Humulin), human growth hormone (somatropin), and granulocyte colony-stimulating factor (filgrastim). In research laboratories, it is the standard tool for producing enzymes, antigens for antibody generation, structural biology targets for [X-ray crystallography](/knowledge/molecular-biology/x-ray-crystallography) and cryo-electron microscopy, and reporter proteins such as green fluorescent protein (GFP) and luciferase. The system's dominance stems from several practical advantages: *E. coli* grows rapidly to high cell densities in inexpensive media, its genetics are extensively characterized, a vast array of [expression vectors](/knowledge/molecular-biology/expression-vector) and host strains are commercially available, and transformation with plasmid DNA is efficient and straightforward. For a detailed overview of the underlying biology, see the entry on [E. coli Protein Expression](/knowledge/molecular-biology/e-coli-protein-expression).

However, the system has limitations. *E. coli* is a prokaryote, so it cannot perform many eukaryotic post-translational modifications, including N-linked glycosylation and complex phosphorylation. Proteins requiring such modifications must be expressed in eukaryotic systems. Additionally, many eukaryotic proteins misfold when overexpressed in bacteria, forming insoluble aggregates called inclusion bodies. Understanding the principles that govern successful expression—vector design, host strain selection, induction strategy, and protein solubility optimization—is essential for anyone working with this system.

## Key Components of an E. coli Expression System

A functional *E. coli* expression system requires four essential elements: an expression vector carrying the gene of interest, a promoter and operator that control transcription, a ribosome binding site (RBS) for translation initiation, and a selection marker to maintain the plasmid in the bacterial population. The host strain itself is the fifth component, providing the cellular machinery for transcription, translation, and protein folding.

### Expression Vectors and Plasmids

The expression vector is typically a plasmid—a small, circular, double-stranded DNA molecule that replicates independently of the bacterial chromosome. Plasmids used for protein expression share several common features. The origin of replication (ori) controls plasmid copy number. High-copy-number origins, such as pUC or pMB1 derivatives, maintain 500–700 copies per cell, which generally leads to higher protein yields because more gene copies mean more mRNA transcripts. Lower-copy origins, such as p15A or pSC101, maintain 10–20 copies per cell and are useful when high expression levels are toxic to the host.

The [multiple cloning site](/knowledge/diagnostics/molecular/multiple-cloning-site-plasmids-structure-function) (MCS) is a short DNA sequence containing several unique restriction enzyme recognition sites, allowing the gene of interest to be inserted in a defined orientation. Most commercial vectors position the MCS downstream of the promoter and RBS, with optional sequences for fusion tags. The selection marker, usually an antibiotic resistance gene such as *bla* (ampicillin resistance, encoding β-lactamase) or *kan* (kanamycin resistance, encoding aminoglycoside phosphotransferase), ensures that only plasmid-bearing cells survive when the corresponding antibiotic is present in the growth medium. Ampicillin is common but problematic because β-lactamase is secreted into the medium and degrades the antibiotic, allowing plasmid-free cells to grow during extended culture. Kanamycin is often preferred for long cultures because resistance is mediated by an intracellular enzyme that does not inactivate the antibiotic in the medium. For a deeper discussion of plasmid design, see the entry on [Expression Vector](/knowledge/molecular-biology/expression-vector).

### Promoters and Induction Systems

The promoter is the DNA sequence where RNA polymerase binds to initiate transcription. For protein expression, the promoter must be strong (producing high levels of mRNA) and tightly regulated (silent before induction, active after induction). The most widely used system is the *lac* promoter and its derivatives, which are based on the native *E. coli* lactose operon.

The *lac* operon promoter (P*lac*) is regulated by the LacI repressor protein. In the absence of lactose (or an inducer), LacI binds to the operator sequence (O1) located just downstream of the promoter, physically blocking RNA polymerase and preventing transcription. When the inducer is present, it binds LacI, causing a conformational change that releases the repressor from the operator, allowing transcription to proceed. The most common inducer is isopropyl β-D-1-thiogalactopyranoside (IPTG), a synthetic analog of lactose that is not metabolized by *E. coli*, so its concentration remains constant throughout the induction period.

The T7 promoter system is another powerful approach. In this system, the gene of interest is placed under the control of a promoter recognized by T7 RNA polymerase, a highly processive enzyme from bacteriophage T7. The host strain carries a chromosomal copy of the T7 RNA polymerase gene under the control of the *lac* promoter (in strains such as BL21(DE3)). Addition of IPTG induces T7 RNA polymerase expression, which then transcribes the target gene at very high rates. The T7 system produces extremely high protein levels, often 20–50% of total cellular protein, but the system is "leaky"—some T7 RNA polymerase is produced even without IPTG, which can be toxic if the target protein is harmful to the cell. The pLysS and pLysE plasmids, which encode T7 lysozyme (a natural inhibitor of T7 RNA polymerase), are used to suppress basal expression.

## Choosing the Right E. coli Host Strain

The choice of host strain is as important as the vector design. Different strains have been engineered to address specific challenges in recombinant protein production, including protease degradation, codon usage, and disulfide bond formation. The two most common lineage families are K-12 and B strains. For background on the organism itself, see [E. coli Bacteria](/knowledge/molecular-biology/e-coli-bacteria).

### Protease-Deficient Strains

The B strain BL21 is the most popular host for protein expression. Unlike K-12 strains, BL21 is naturally deficient in the Lon protease, an ATP-dependent protease that degrades abnormal or misfolded proteins, and it lacks the OmpT outer membrane protease. These deficiencies reduce degradation of recombinant proteins during expression and purification. BL21 also grows faster than K-12 strains and reaches higher cell densities. The most common derivative, BL21(DE3), carries the λDE3 prophage containing the T7 RNA polymerase gene under *lac* control, making it compatible with T7 promoter-based vectors.

K-12 strains, such as DH5α and JM109, are primarily used for plasmid construction and propagation rather than protein expression. They have intact protease systems and are not optimized for high-level production. However, K-12 derivatives such as Rosetta and Origami are engineered for specific applications. Rosetta strains carry a plasmid encoding tRNAs for rare *E. coli* codons (AGA, AGG, AUA, CUA, GGA, CCC, and CGG), which improves expression of genes from organisms with different codon usage. Origami strains carry mutations in the thioredoxin reductase (*trxB*) and glutathione reductase (*gor*) genes, creating a more oxidizing cytoplasm that permits disulfide bond formation.

### Specialized Strains for Disulfide Bonds

Disulfide bond formation is a significant challenge in *E. coli* because the cytoplasm is a reducing environment, maintained by the thioredoxin and glutaredoxin systems, which keep cysteine residues in their reduced form. Proteins requiring disulfide bonds for proper folding often misfold in the cytoplasm. The standard solution is to target the protein to the periplasm, the oxidizing compartment between the inner and outer membranes, using a signal sequence such as PelB or OmpA. The periplasm contains the DsbA and DsbC enzymes, which catalyze disulfide bond formation and isomerization, respectively.

For cytoplasmic expression of disulfide-bonded proteins, strains with mutations in the reducing pathways are available. SHuffle strains (based on BL21) have both *trxB* and *gor* deleted and additionally express a cytoplasmic version of DsbC, providing both an oxidizing environment and an active isomerase to correct mispaired disulfides. These strains are particularly useful for expressing antibody fragments and other cysteine-rich proteins.

## Induction Strategies for Protein Expression

The timing and method of induction critically affect protein yield and quality. The goal is to separate cell growth from protein production: cells are first grown to an optimal density, then induced to express the target protein for a defined period before harvest.

### IPTG-Inducible Systems

The standard induction protocol for *lac*-based systems is as follows:

1. Inoculate a single colony into 5–10 mL of LB medium containing the appropriate antibiotic and grow overnight at 37°C with shaking (200–250 rpm).
2. Dilute the overnight culture 1:100 into fresh medium (typically 50–500 mL) and grow at 37°C until the optical density at 600 nm (OD₆₀₀) reaches 0.4–0.8, which corresponds to mid-log phase (approximately 2–3 hours).
3. Add IPTG to a final concentration of 0.1–1.0 mM. Lower concentrations (0.1–0.3 mM) are often sufficient and reduce the formation of inclusion bodies.
4. Continue incubation for 2–4 hours at 37°C, or overnight at lower temperatures (16–25°C) for proteins that are prone to aggregation.
5. Harvest cells by centrifugation (4,000–6,000 × g for 15 minutes at 4°C) and store the cell pellet at −80°C until lysis.

The IPTG concentration and induction temperature are the two most important variables. High IPTG concentrations (1 mM) drive maximal transcription but can overwhelm the protein folding machinery, leading to inclusion body formation. Lower temperatures slow protein synthesis, giving the cell more time to fold the protein correctly, which often improves solubility.

### Auto-Induction and Alternative Inducers

Auto-induction is a method that eliminates the need to monitor cell density and add IPTG manually. The medium contains glucose, lactose, and glycerol. During early growth, *E. coli* preferentially consumes glucose, and the *lac* promoter remains repressed. When glucose is depleted, the cells switch to lactose, which is converted to allolactose—the natural inducer of the *lac* operon—by β-galactosidase. This triggers protein expression automatically at high cell density. Auto-induction is convenient for high-throughput applications and often produces higher yields than IPTG induction because expression begins when the culture is already dense.

Alternative inducers include anhydrotetracycline (aTc) for Tet promoter systems and arabinose for the araBAD promoter (PBAD). The arabinose system has a particularly tight off state, making it useful for expressing toxic proteins. Arabinose concentrations of 0.01–0.2% (w/v) are typical, and expression levels can be tuned by varying the arabinose concentration. The rhamnose promoter (PrhaBAD) offers similar tight regulation.

## Optimizing Protein Solubility and Yield

Even with a well-designed vector and appropriate host strain, many recombinant proteins fail to express in soluble form. The protein may aggregate into inclusion bodies, be degraded by proteases, or simply express at low levels. Several strategies can be employed to improve solubility and yield.

### Fusion Tags (GST, His-tag)

Fusion tags are short peptide sequences or entire proteins attached to the N- or C-terminus of the target protein. They serve two primary purposes: facilitating purification and improving solubility.

The polyhistidine tag (His-tag) consists of 6–10 consecutive histidine residues. Histidine has an imidazole side chain that coordinates with divalent metal ions (Ni²⁺, Co²⁺) immobilized on a chromatography resin. His-tagged proteins bind to the resin and can be eluted with imidazole (typically 250–500 mM) or low pH. The His-tag is small (approximately 1 kDa), rarely affects [protein structure](/knowledge/bioinformatics/protein-structure-biophysical-levels-folding) or function, and can be used under denaturing conditions, making it the most popular purification tag.

Glutathione S-transferase (GST) is a 26 kDa protein from *Schistosoma japonicum* that is fused to the N-terminus of the target protein. GST tags dramatically improve solubility, often rescuing proteins that would otherwise aggregate. Purification uses glutathione-agarose resin, and elution is achieved with reduced glutathione (10–20 mM). The large size of GST can be a drawback, as it may interfere with the structure or function of small proteins, and it must often be removed by proteolytic cleavage.

Maltose-binding protein (MBP) is another solubility-enhancing tag. MBP is a 40 kDa *E. coli* periplasmic protein that binds maltose and maltodextrins. It is one of the most effective solubility enhancers known, and fusion proteins are purified on amylose resin, eluting with 10 mM maltose. Other tags include the small ubiquitin-like modifier (SUMO), which not only improves solubility but can be cleaved by SUMO protease to yield a native N-terminus, and the NusA tag, a 55 kDa *E. coli* protein with strong chaperone-like properties.

### Co-expression with Chaperones

Molecular chaperones assist in protein folding by binding to exposed hydrophobic surfaces of partially folded or misfolded proteins, preventing aggregation and promoting correct folding. Co-expressing chaperones with the target protein is a powerful strategy for improving solubility.

The most commonly used chaperone systems are:

- **DnaK-DnaJ-GrpE (Hsp70 system):** Binds to nascent polypeptide chains and prevents aggregation. DnaK is ATPase that cycles between ATP-bound (low affinity) and ADP-bound (high affinity) states, with DnaJ delivering substrates and GrpE promoting nucleotide exchange.
- **GroEL-GroES (Hsp60 system):** A large barrel-shaped complex that encapsulates partially folded proteins, providing an isolated environment for folding. GroEL is a tetradecamer arranged in two stacked heptameric rings, and GroES is a heptameric cap that binds to GroEL in the presence of ATP.
- **Trigger factor (Tig):** A ribosome-associated chaperone that binds to nascent chains as they emerge from the ribosome.

Co-expression is typically achieved by cloning the chaperone genes on a separate plasmid with a compatible origin of replication and a different antibiotic resistance marker. For example, the pKJE7 plasmid encodes DnaK-DnaJ-GrpE under an arabinose-inducible promoter, while pGro7 encodes GroEL-GroES. The chaperones are induced with arabinose (0.5–2 mg/mL) or tetracycline (for pTf16, encoding trigger factor) 30 minutes before IPTG induction of the target protein. Growth at reduced temperature (20–25°C) further enhances chaperone function.

## Common Methods to Analyze Expressed Proteins

After induction and cell lysis, the expressed protein must be detected and characterized. Several standard methods are used, often in combination.

**SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)** is the first-line method for detecting recombinant proteins. Cells are lysed, and the total protein is separated by size on a polyacrylamide gel. Proteins are denatured by SDS, which binds to hydrophobic regions and imparts a uniform negative charge, so proteins migrate according to molecular weight alone. The gel is stained with Coomassie Brilliant Blue, which binds to basic and aromatic amino acid residues, allowing visualization of protein bands. A highly expressed recombinant protein typically appears as a prominent band at its expected molecular weight, often comprising 10–40% of total protein. To distinguish soluble protein from inclusion bodies, the lysate is centrifuged (12,000–15,000 × g for 10–15 minutes at 4°C), and the supernatant (soluble fraction) and pellet (insoluble fraction) are analyzed separately.

**Western blotting** provides specific detection using antibodies. Proteins separated by SDS-PAGE are transferred electrophoretically to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane is blocked with a protein solution (e.g., 5% bovine serum albumin or non-fat dry milk) to prevent non-specific antibody binding, then incubated with a primary antibody specific to the target protein or its tag (e.g., anti-His or anti-GST). After washing, a secondary antibody conjugated to horseradish peroxidase (HRP) or [alkaline phosphatase](/knowledge/molecular-biology/alkaline-phosphatase) is added. The blot is developed with a chemiluminescent substrate (e.g., enhanced chemiluminescence, ECL) and exposed to X-ray film or imaged with a digital system.

**Activity assays** confirm that the expressed protein is functional. The specific assay depends on the protein. For enzymes, a substrate that produces a measurable product is used. For example, β-galactosidase activity is measured using the chromogenic substrate ONPG (o-nitrophenyl-β-D-galactopyranoside), which produces yellow o-nitrophenol with absorbance at 420 nm. For kinases, radioactive or fluorescent peptide substrates are used. For DNA-binding proteins, electrophoretic mobility shift assays (EMSAs) are common. Activity assays are essential because a protein can be present at high levels (as judged by SDS-PAGE) yet be completely non-functional due to misfolding.

## Troubleshooting and Common Pitfalls

Despite careful planning, protein expression frequently fails. Understanding the most common failure modes is essential for efficient troubleshooting.

### Inclusion Bodies and Refolding

Inclusion bodies are dense, insoluble aggregates of misfolded protein that form in the cytoplasm when the rate of protein synthesis exceeds the capacity of the folding machinery. They are visible under phase-contrast microscopy as refractile bodies and can be isolated by centrifugation after cell lysis. Inclusion bodies are not entirely useless—they protect the protein from proteolysis, and the protein within them is often correctly folded in terms of primary structure, requiring only solubilization and refolding.

To recover protein from inclusion bodies, the following protocol is typical:

1. Lyse cells and centrifuge to collect the inclusion body pellet.
2. Wash the pellet with buffer containing 0.5–2% Triton X-100 and 1–2 M urea to remove membrane debris and associated proteins.
3. Solubilize the inclusion bodies in a denaturing buffer containing 6–8 M urea or 6 M guanidine hydrochloride, plus a reducing agent such as 10 mM dithiothreitol (DTT) or β-mercaptoethanol to reduce disulfide bonds.
4. Refold the protein by removing the denaturant, either by dialysis against a refolding buffer (e.g., 50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.0) or by rapid dilution into a large volume of refolding buffer.
5. Add redox reagents such as oxidized and reduced glutathione (1 mM GSSG, 2 mM GSH) to promote correct disulfide bond formation.

Refolding yields are often low (5–30%), and the process must be optimized for each protein. Prevention is preferable: reducing induction temperature, lowering IPTG concentration, using a weaker promoter, or co-expressing chaperones can often prevent inclusion body formation altogether.

### Codon Bias and Rare tRNAs

Codon bias refers to the fact that different organisms prefer different synonymous codons for the same amino acid. *E. coli* has a particular codon usage pattern, and genes from organisms with very different patterns (e.g., humans, plants, or GC-rich organisms such as *Streptomyces*) may contain codons that are rare in *E. coli*. When the ribosome encounters a rare codon, translation pauses, which can lead to premature termination, frameshifting, or [protein misfolding](/knowledge/molecular-biology/protein-misfolding).

The most problematic rare codons in *E. coli* are AGA and AGG (arginine), AUA (isoleucine), CUA (leucine), and GGA (glycine). The AGA codon, for example, is recognized by the tRNA encoded by *argU*, which is present at very low levels. Solutions include:

- **Codon optimization:** Synthesizing the gene with codons optimized for *E. coli* is the most reliable solution. Commercial gene synthesis services routinely provide codon-optimized genes.
- **Using specialized strains:** Rosetta strains carry a plasmid (pRARE) encoding tRNAs for rare codons, supplementing the endogenous tRNA pool.
- **Reducing expression level:** Lowering the induction temperature or IPTG concentration gives the ribosome more time to navigate rare codons.

## Practical Summary: Key Takeaways for Students

The *E. coli* expression system is a powerful, versatile platform for recombinant protein production. Success depends on understanding and controlling each component of the system. The key principles are summarized below.

## Frequently Asked Questions

### What is an E. coli expression system?

An *E. coli* expression system is a molecular biology platform that uses *Escherichia coli* bacteria as living factories to produce recombinant proteins. A gene encoding the desired protein is cloned into an expression plasmid, introduced into *E. coli* cells by transformation, and the cells are induced to transcribe and translate the gene at high levels. The system is widely used because *E. coli* grows rapidly, is inexpensive to culture, and its genetics are well understood. For more background, see [E. coli Definition](/knowledge/molecular-biology/e-coli-definition).

### How does IPTG induction work?

IPTG (isopropyl β-D-1-thiogalactopyranoside) is a synthetic molecule that mimics allolactose, the natural inducer of the *lac* operon. In *lac*-based expression systems, the LacI repressor protein binds to the operator sequence and blocks transcription. IPTG binds to LacI, causing a conformational change that releases the repressor from the operator. This allows RNA polymerase to transcribe the target gene. IPTG is not metabolized by *E. coli*, so its concentration remains constant, providing sustained induction.

### Why do some proteins form inclusion bodies in E. coli?

Inclusion bodies form when the rate of protein synthesis exceeds the capacity of the cellular folding machinery. Overexpressed proteins accumulate as partially folded intermediates that expose hydrophobic surfaces, which drive aggregation. High induction temperatures, high IPTG concentrations, and proteins with complex folding requirements (multiple domains, disulfide bonds, or membrane-spanning regions) all increase the likelihood of inclusion body formation.

### What is the difference between BL21 and K-12 strains?

BL21 is a B strain that is naturally deficient in the Lon and OmpT proteases, making it the preferred host for high-level protein expression because recombinant proteins are less likely to be degraded. BL21 also grows faster than K-12 strains. K-12 strains (DH5α, JM109, TOP10) have intact protease systems and are primarily used for plasmid construction and cloning. K-12 derivatives like Rosetta and Origami are engineered for specific applications such as rare codon supplementation and disulfide bond formation.

### How can I improve solubility of my recombinant protein?

Several strategies can improve solubility: (1) reduce the induction temperature to 16–25°C, which slows protein synthesis and gives the folding machinery more time; (2) lower the IPTG concentration to 0.1–0.3 mM; (3) fuse the target protein to a solubility-enhancing tag such as MBP, GST, or SUMO; (4) co-express molecular chaperones such as DnaK-DnaJ-GrpE or GroEL-GroES; (5) change the host strain to one with an oxidizing cytoplasm (SHuffle) if disulfide bonds are required.

### What is codon bias and why does it matter?

Codon bias refers to the non-random usage of synonymous codons in different organisms. Each organism has a preferred set of codons for each amino acid, reflecting the abundance of corresponding tRNAs. When a gene from one organism is expressed in another, codons that are rare in the host can slow translation, cause ribosome stalling, and lead to truncated or misfolded proteins. Codon optimization—resynthesizing the gene with host-preferred codons—or using strains that supply rare tRNAs (e.g., Rosetta) can overcome this problem.

### What are common fusion tags used in E. coli expression?

Common fusion tags include: polyhistidine (His-tag, 6–10 histidines) for purification by immobilized metal affinity chromatography; glutathione S-transferase (GST, 26 kDa) for purification on glutathione resin and improved solubility; maltose-binding protein (MBP, 40 kDa) for strong solubility enhancement and purification on amylose resin; SUMO for solubility and precise cleavage to yield native N-termini; and small epitope tags such as FLAG, HA, and c-Myc for detection by Western blotting.

## Key Takeaways

- The *E. coli* expression system uses plasmids with strong, inducible promoters (e.g., *lac*/IPTG or T7) to produce recombinant proteins in a fast-growing, inexpensive bacterial host.
- Host strain selection matters: BL21 is protease-deficient and ideal for expression; K-12 strains are better for cloning; specialized strains address rare codons (Rosetta) and disulfide bonds (SHuffle, Origami).
- Induction is controlled by IPTG concentration, temperature, and timing; lower temperatures and lower inducer concentrations often improve protein solubility.
- Fusion tags (His-tag, GST, MBP, SUMO) serve dual purposes: purification and solubility enhancement; chaperone co-expression is a powerful tool for difficult proteins.
- Inclusion bodies are a common failure mode caused by overexpression exceeding folding capacity; they can sometimes be refolded, but prevention through optimization is preferable.
- Codon bias can severely reduce expression of genes from organisms with different codon usage; codon optimization or rare-tRNA-supplementing strains are the solutions.
- Analysis of expressed proteins requires SDS-PAGE for size and quantity, Western blot for specific detection, and activity assays to confirm functionality.

## Further Reading

- Jia L et al. *Expression and purification of amyloid β-protein, tau, and α-synuclein in Escherichia coli: a review*. Critical reviews in biotechnology. 2020. [PubMed 32202164](https://doi.org/10.1080/07388551.2020.1742646)
- Costa S et al. *Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system*. Frontiers in microbiology. 2014. [PubMed 24600443](https://doi.org/10.3389/fmicb.2014.00063)
- Piselli C. *How to isolate channel-forming membrane proteins using the E. coli expression system*. Nature protocols. 2025. [PubMed 39367089](https://doi.org/10.1038/s41596-024-01055-2)
- Li H et al. *High level expression, purification and characterization of active fusion human C1q and tumor necrosis factor related protein 2 (hCTRP2) in Escherichia coli*. [Protein expression and purification](/knowledge/molecular-biology/protein-expression-and-purification). 2011. [PubMed 21453774](https://doi.org/10.1016/j.pep.2011.03.013)
- Plückthun A, Stadlmüller J. *Catalytic antibodies: contributions from engineering and expression in Escherichia coli*. Ciba Foundation symposium. 1991. [PubMed 1959443](https://doi.org/10.1002/9780470514108.ch8)
- Murakami M, Murakami AM, Itagaki S. *A dual prokaryotic (E. coli) expression system (pdMAX)*. PloS one. 2021. [PubMed 34673793](https://doi.org/10.1371/journal.pone.0258553)

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