E. coli Expression Systems: Host Selection, Vectors, and Industrial Titers

Posted on Oct 7, 2025 in Biotechnology

Escherichia coli Expression Systems

Advantages of Microbial Expression Systems

Bacterial and yeast systems share several advantages over mammalian expression systems:

• Higher growth rate and cell densities.
• Robustness at the cellular level.
• High expression levels.
• Simple cultivation and are easy to scale-up.

The selection of the appropriate expression system is determined by the characteristics of the target protein or product.

Why E. coli Dominates Microbial Expression

E. coli has dominated microbial expression for over 30 years. Its popularity stems from several key factors:

• Historical Significance: E. coli and its phages were early objectives for studying molecular biology (gene functions and regulation), leading to more than 10 Nobel prizes.
• Pioneering Biopharma: The launch of the first (1^st) biopharma product (Humulin, 1982) was E. coli based.
• Genetic Simplicity: Low genome complexity and the presence of extra-chromosomal genetic elements (plasmids) allow for ease of genetic manipulation and gene insertion.
• Regulatory Acceptance: Low safety concerns, high regulatory acceptance, and ease of use.
• Continuous Improvement: Ongoing system improvements lead to:
  1. High control over product quality.
  2. High volumetric productivity (Q_P).

Core Elements of Bacterial Expression Systems

The bacterial expression system relies on two main components: the host organism and the expression vector.

The Expression Vector Components

The vector contains essential genetic components:

• Product coding DNA.
• Selection markers.
• Regulatory elements (promoters, signal sequence, ribosome binding site, transcription terminators, vector replication, and integration region).

Host Organism Characteristics

The host organism provides specific characteristics crucial to the expression system:

• Specific growth rate.
• Maximum achievable cell densities.
• Nutritional needs.
• Robustness at cellular and genetic levels.
• Control of product degradation.
• Secretion capacity (preferably into the medium).
• Amount of endotoxins produced.
• Post-translational modifications.

High cell densities are desirable because the product production rate (r_p) correlates positively with the amount of biomass (X): r_p = q_p * X.

Vector Features I: Target Gene and Selection Markers

• Product Coding DNA: Optimized codon usage is essential for high expression.
• Selection Markers: Used during the cloning process and to ensure plasmid stability.

Types of Selection Markers

1. Antibiotic Resistance Genes: (Constitutive expression)
   • Prevalence of β-lactam allergies is a concern.
   • High metabolic burden on the host cell.
2. Antidote/Poison Gene Systems: The antidote gene is on the plasmid, and the poison gene is on the chromosome (both constitutively expressed).
3. Auxotrophic Complementation: A marker gene on the plasmid complementing an auxotrophic mutation on the chromosome.

Vector Features II: Regulatory Elements (Promoters)

Promoters control the expression of mRNA, determining when and how much protein is synthesized (modulation of the rate of protein synthesis).

• Dominant Promoters: T7, Lactose, or IPTG induced.
• Classical Promoters: T5, araB, phoA, lambda (λ).
• Novel High-Performing Promoters: Disaccharide induced and stationary phase inducible promoters (e.g., Lonza XS Technologies).
• Less Common Promoters: lac, trp, P_L, P_R, tetA, trc/tac.

The nature of the induction signal can be chemical or physical.

Promoter Selection Considerations

The choice of promoter depends on the target protein characteristics and potential interactions:

• For proteins of low complexity (e.g., small protein).
• For proteins with disulfide bonds.
• Is there an interaction between the promoter system and the recombinant target protein?

Signal Sequences for Protein Secretion

Why Use Signal Sequences?

Signal sequences drive the secretion of the recombinant protein.

Which Proteins Require Signal Sequences?

Proteins intended for secretion should be expressed with an attached signal sequence.

• Typical E. coli Signal Sequences: MalE, OmpA, PelB.
• Secretion Mechanism:
  • With yeast and Gram-positive microorganisms: Secretion into the medium.
  • With Gram-negative microorganisms (like E. coli): Secretion into the periplasm.

Often, partitioning of the product between the periplasm and the medium is observed. This distribution can partly be controlled by fermentation conditions.

Industrial E. coli Expression Modes and Titers

Industrial E. coli systems allow for high intracellular expression levels. Recombinant protein can be localized in four effective expression modes:

1. Cytoplasm (insoluble as inclusion bodies).
2. Cytoplasm (soluble form).
3. Cell-free medium (soluble form).
4. Periplasm (soluble form).

1. & 2. Cytoplasmic Expression

Cytoplasmic expression is used for highly soluble recombinant proteins as well as proteins prone to high aggregation propensity. Product classes include recombinant vaccines, novel non-antibody based binders, therapeutic and non-therapeutic enzymes, virus-like particles (VLPs), peptides (hormones and cardiovasculars), monomers of biopolymers, and affinity ligands. These proteins are mostly monomeric, ranging in size from 2 to 80 kDa.

Soluble Cytoplasmic Expression (Mode 2)

Highest expression titers are obtained in the case of cytoplasmic soluble expression, with a median titer of 11 g/L culture broth (range: 3 – 20 g/L).

Insoluble Cytoplasmic Expression (Inclusion Bodies, Mode 1)

Intentional intracellular expression of recombinant protein in an insoluble state as inclusion bodies is often used for hydrophobic proteins or those exhibiting low solubility. This resulted in a median titer of about 9 g/L (range: 3 – 15 g/L), dependent on the target protein.

3. & 4. Extracellular Expression (Periplasmic and Medium)

Products expressed with a signal sequence include fragment antibodies (Fab), Fab fusion proteins, single-chain antibodies (scFv), growth factors, enzymes, and various formats of amphipathic proteins (apolipoprotein). The size of these products varies between 20 and 220 kDa, encompassing both highly soluble and fairly soluble monomers and multimers (some Fab formats).

Cell-Free Medium Expression (Mode 3)

Extracellular product reached concentrations in the range of 0.5 to 8.5 g/L in the cell-free medium, with a median of 1.5 g/L.

Periplasmic Expression (Mode 4)

Proteins which accumulated in the periplasm reached titers of functional product between 0.5 and 10 g/L, with a median titer of 2.0 g/L.

Note on Periplasmic Precipitation: Dependent on the product-specific aggregation propensity, significant amounts of precipitated recombinant protein were sometimes observed in the periplasm. This insoluble fraction has been ignored, since it does not contribute to functional product. The extent of product precipitation can be influenced by the choice of the promoter system, the related induction mode, and fermentation conditions. Similarly, the distribution of product between the periplasm and the cell-free medium can be partly controlled by changes in physical and chemical environmental conditions. However, ideal conditions must be identified empirically.