Advances in Escherichia coli-Based Therapeutic Protein Expression: Mammalian Conversion, Continuous Manufacturing, and Cell-Free Production
Abstract
:1. Introduction
2. Background
3. Advantages
- It is the most well-understood expression system. The genome of Escherichia coli strain K-12 MG1655, which is the most studied and best-characterized strain, has been fully sequenced and annotated. It was first completely sequenced in 1997, and the annotation and analysis have been continually updated since then as our understanding of genomics and the biology of E. coli has advanced [9]. This knowledge base is critical in its utilization as a robust expression system.
- Numerous prokaryotic genes [10] are expressed in operons [11], where a solitary promoter leads to the synthesis of multiple proteins from a single mRNA molecule, which has a ribosome binding site (RBS) preceding the beginning AUG codon of each protein. This enables the simultaneous production [12] of subunits that assemble into complexes or the simultaneous expression of auxiliary components that may be necessary for the protein to attain its native shape.
- Simpler scale-up compared to eukaryotic systems, including mechanical cell disruption, which is less variable than eukaryotic cells, requires gentler lysis to preserve more fragile organelles and structures [16].
- Avoiding virus contamination risk. Proteins synthesized in mammalian cell lines, the host cells possess multiple copies of endogenous retrovirus-like sequences, which subsequently generate retrovirus-like particles (RVLPs) together with the target protein. While RVLPs are commonly regarded as dysfunctional, certain instances have demonstrated their ability to infect cell lines that are not of rodent origin. Exogenous viral contamination resulting from raw materials or persons is also possible; however, such concerns are not relevant in the context of E. coli-based expression systems [17].
- Low-cost growth medium, fast cellular proliferation, uncomplicated fermentation procedures, no viral contaminants in the final product, and high product yields [18].
4. Challenges
- Proteins overexpressed in E. coli may form insoluble aggregates known as inclusion bodies, requiring specific solubilization and refolding steps, adding complexity to the purification process compared to eukaryotic cells [19]; additional purification steps if inclusion bodies are formed. A frequently encountered challenge is the formation of inclusion bodies—insoluble aggregates of misfolded proteins. Several tactics have been developed to address this. Incorporating solubility-enhancing fusion tags, such as SUMO or maltose-binding protein (MBP), has proven to enhance the solubility of certain target proteins [20]. Additionally, co-expressing the protein of interest with molecular chaperones can help in its proper folding, making inclusion body formation less likely [21]. Like adjusting the temperature or the IPTG concentration, fine-tuning expression conditions can also modulate protein synthesis rates and improve solubility [22]. Even if inclusion bodies form, there is a workaround: the proteins can be solubilized with denaturants and then refolded, salvaging the protein for further use [23]. These adaptive strategies emphasize the versatility and adaptability of the E. coli expression system, showcasing the myriad tools researchers have at their disposal to optimize protein production.
- E. coli lacks the machinery for many eukaryotic PTMs, such as glycosylation, which may affect protein stability, folding, and activity [24].
- Unlike eukaryotic systems, E. coli produces endotoxin contamination from its lipopolysaccharide, which must be removed during purification [25].
- The toxicity of overexpressed proteins to E. coli often forces the expression of toxic protein fragments or domains retaining essential functions [26]. One strategy involves using signal sequences attached to the protein’s N-terminus, directing the protein’s export to the periplasm, and decreasing cytoplasmic accumulation, thereby reducing potential toxicity [27].
- Regulating the expression through weak promoters or controlled induction can temper any adverse impacts on the host cells. This requires codon optimization to enhance translation efficiency [28].
- Expressing monoclonal antibodies (mAbs) in Escherichia coli (E. coli) presents multiple challenges, stemming primarily from the intricacy of these proteins. One of the main hurdles is ensuring the proper folding of mAbs, especially since they possess multiple domains. E. coli often struggles to correctly fold such large eukaryotic proteins, especially when they have multiple disulfide bonds. Furthermore, bacteria lack the machinery for certain post-translational modifications like glycosylation, which are vital for the function of mAbs. This absence can compromise the mAb’s efficacy [29]. The reducing environment of the E. coli cytoplasm also makes disulfide bond formation problematic, while protein degradation can occur if the expressed proteins are unstable or perceived as foreign. Several strategies can be employed to counter these challenges. One approach is the expression of single-chain variable fragments (scFvs), which comprise the variable regions of the mAb’s heavy and light chains connected by a short peptide linker. Researchers can also leverage specialized E. coli strains designed for disulfide bond formation in the cytoplasm, such as SHuffle strains [30]. Directing mAbs or scFv expression to the periplasmic space of E. coli, which is more oxidizing than the cytoplasm, can also encourage proper disulfide bond formation. Adjustments in expression conditions, co-expression with molecular chaperones, and codon optimization for E. coli are additional strategies to improve yields [31]. The ability of bispecific antibodies (BsAbs) [32] to effectively target two entities concurrently enhances the practicality of antibody-based treatments. Genentech has successfully devised a periplasmic expression system in Escherichia coli, known as the BsAb expression system. This system utilizes either the Knobs-into-Holes (KiH) [33] technology or Fc domain HC heterodimerization [34]. Genentech has made significant advancements in the production process of bispecific antibodies (BsAbs), including two distinct heavy chains (HCs) and two distinct light chains (LCs). These improvements have been achieved by utilizing either a two-culture or a coculture strategy in Escherichia coli (E. coli) systems [35].
5. Bioinformatics Applications
- Exploiting the use of bioinformatics tools to determine the biophysical characteristics of the protein [41]. It is a complex process that involves various computational methods. These methods utilize algorithms and statistical models to analyze the protein’s primary sequence, infer its three-dimensional structure, and predict its interactions and functions.
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- Sequence analysis involves comparing the amino acid sequence of a protein with known sequences in databases to identify conserved domains, motifs, or families [42];
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- Structure prediction includes methods like homology modeling, ab initio modeling, and threading to predict a protein’s three-dimensional (3D) structure based on its sequence [43];
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- Functional prediction identifies the biological role of a protein by assessing its structural and sequential features, often in conjunction with known protein–protein interactions and pathway analyses [44];
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- Molecular dynamics simulations and related techniques are used to study the movement and interactions of proteins, providing insight into their behavior in the cellular environment [45];
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- Specific bioinformatics tools are designed to predict sites in proteins likely to undergo post-translational modifications (PTMs) such as phosphorylation or glycosylation [46];
- ○
- Predicting how proteins interact with other proteins or ligands can be achieved through docking simulations and other modeling techniques [47].
- Accurate delineation [48]:
- ○
- Identifying the boundaries of protein domains is essential for understanding the function and evolution of proteins [49];
- ○
- Signal sequences are crucial for the targeting of proteins to specific cellular locations. Identifying these sequences helps in understanding the transportation and localization of proteins [50];
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- Transmembrane regions anchor proteins in membranes, playing essential roles in cellular communication, signaling, and transport. Accurate prediction of these regions aids in understanding membrane protein structure and function [51];
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- Identifying obligate oligomeric complexes is essential for understanding protein–protein interactions and the assembly of multi-protein complexes [52];
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- Identification of PTMs is vital for understanding protein regulation and signaling [53].
6. Gene Cloning and Design
6.1. Ribosomes
- The characteristics and location of the ribosome binding site (RBS) and the disparities in translation rates observed in prokaryotic and eukaryotic organisms [70]. The ribosome binding site (RBS) plays a crucial role in the translation initiation. The sequence and position of a gene relative to the initiation codon can influence the translation efficiency. Customizing the RBS to the host organism might enhance the efficiency of translating the desired protein [71];
- Correct use of the strain and media to optimize production, though with many limitations [72]. The optimization of production in E. coli strains through proper selection of the strain and media is a common strategy in biotechnology but comes with certain limitations;
- Optimization in E. coli can vary widely depending on the protein or other manufactured product. Selecting the right strain of E. coli, determining the optimal temperature, and choosing the appropriate culture media are crucial considerations for recombinant protein expression.
6.2. Promoter
- To create the promoter variation lac1G, the promoter lacUV5 and lac were joined again. (G was substituted for A at position +1) [82];
- The expression of T7 RNA polymerase (RNAP) is effectively regulated to prevent leakage by the presence of a mutant form of the Lac repressor protein (LacI), specifically the V192F variant. This mutant variant cannot bind to isopropyl β-D-1-thiogalactopyranoside (IPTG), hence preventing its activation. Consequently, the mutant LacI dynamically governs the levels of transcripts produced by T7 RNAP [83];
- Building a T7 RNAP RBS library quickly involves using the base editor and CRISPR/Cas9 to screen potential expression hosts [84];
- The ability of T7 RNA polymerase to bind to the PT7 promoter was impaired due to a specific amino acid substitution (A102D), resulting in an alteration in the rate of RNA production. The T7 RNA polymerase (T7 RNAP) was fragmented into two segments and co-expressed with a light-responsive dimerization domain, exhibiting functional behavior upon exposure to blue light [85].
6.3. Codons
6.4. Protein Folding
7. Enhanced Efficiency
7.1. Solubilization
7.2. Disulfide Bond
7.3. Post-Translational Modifications
7.4. Strain and Media
7.5. Fermentation Conditions
7.6. Purification
8. Cell-Free Protein Synthesis System (CFPS)
9. Continuous Manufacturing (CM)
10. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Listed Protein Class | Number |
---|---|
Monoclonal Antibody | 94 |
Hormone | 10 |
Enzyme | 8 |
Monoclonal Antibody Conjugate | 8 |
Cytokine | 4 |
Bispecific Antibody | 3 |
Coagulation Factor | 3 |
Growth Factor | 3 |
Peptide | 3 |
Carrier Protein | 1 |
Enzyme Inhibitor | 1 |
Fab | 1 |
Fusion Proteins | 1 |
Single-Domain Antibody | 1 |
Toxin | 1 |
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Niazi, S.K.; Magoola, M. Advances in Escherichia coli-Based Therapeutic Protein Expression: Mammalian Conversion, Continuous Manufacturing, and Cell-Free Production. Biologics 2023, 3, 380-401. https://doi.org/10.3390/biologics3040021
Niazi SK, Magoola M. Advances in Escherichia coli-Based Therapeutic Protein Expression: Mammalian Conversion, Continuous Manufacturing, and Cell-Free Production. Biologics. 2023; 3(4):380-401. https://doi.org/10.3390/biologics3040021
Chicago/Turabian StyleNiazi, Sarfaraz K., and Matthias Magoola. 2023. "Advances in Escherichia coli-Based Therapeutic Protein Expression: Mammalian Conversion, Continuous Manufacturing, and Cell-Free Production" Biologics 3, no. 4: 380-401. https://doi.org/10.3390/biologics3040021
APA StyleNiazi, S. K., & Magoola, M. (2023). Advances in Escherichia coli-Based Therapeutic Protein Expression: Mammalian Conversion, Continuous Manufacturing, and Cell-Free Production. Biologics, 3(4), 380-401. https://doi.org/10.3390/biologics3040021