Recombinant DNA technology and recombinant protein expression provide a practical way to simplify the production of high-purity target proteins and the development of assay kits. Among them, antibodies and antibody fragments are being developed as key reagents for advanced diagnosis and therapy. Mammalian cells, insect cells, yeast, bacteria, and transgenic animals. have been used in the expression of recombinant antibodies.

Glycosylation modification is essential for full-length antibodies and must be produced by a mammalian cell expression system. However, it is not required for most antibody fragments and antibody-like molecules. Therefore, E. coli, with faster growth and higher expression levels, can be considered the host for recombinant antibody expression. According to research statistics, about 30% of recombinant proteins used in healthcare are expressed in E. coli.

E. coli Antibody Expression Host Characteristics

E. coli has become one of the most important tools for molecular biology since Escherich’s initial discovery. Compared with yeast and mammalian cells, E. coli has a clear and simple genetic background. As a recombinant protein expression system, it has the advantages of simple cultivation parameters, fast propagation, and high protein expression levels. The theoretical density limit of E. coli culture is about 200 g cells/L, which is equivalent to about 1×1013 viable bacteria/mL.

Most heterologous proteins expressed in the cytoplasm of E. coli, especially for proteins with disulfide bonds, are usually misfolded. In many cases, the optimal HC/LC ratios and protein folding rates are more important than high expression in the successful expression of antibody fragments. In addition, protein expression can be redirected to E. coli periplasm by using signal peptides at the N-terminal (SpA, PhoA, PelB, OmpA, OmpT, DsbA, TorT, and TolT), and the oxidation environment of the periplasm can be utilized to achieve stable disulfide bond formation. Several groups have also investigated the importance of Fab co-expression with molecular chaperones and increased soluble expression of Fab antibody fragments. Molecular chaperones are universal folding modulators, which play an important role in the conformational quality control of proteomics and facilitate the correct non-covalent assembly of non-natural peptides.

Common Antibody Fragments

Antibody fragments have several pharmacokinetic advantages over full-length antibodies, including better tissue penetration, shorter half-life period, and lower immunogenicity. Currently, single-domain antibodies (sdAb), antigen-binding fragments (Fab), and single-chain variable fragment (scFv) are the most widely used antibody fragments.

Single-Domain Antibodies (sdAb)

sdAbs are a type of natural heavy-chain antibody that is found in camels, lacking the light-chain and heavy-chain constant region of the antibody. Its’ small size, simple structure, high affinity, low immunogenicity, and low toxicity are all advantages. Some hydrophobic residues in the sequence are replaced by hydrophilic residues, which are water-soluble and make it difficult to form aggregates.

The drug form of sdAbs is very malleable and can be combined with other drugs to form multivalent and multi-specific molecules or as drug carriers with other compounds. Additionally, the excellent physical and chemical stability of sdAbs bring great prospects for drug development. Currently, most sdAb drugs developed are designed by polymolecular polymerization, coupling, or structural modification to prolong the half-life period.

Antigen-Binding Fragments (Fab)

Fabs are the region of the antibody structure that can bind to an antigen. Fabs have an antigen-binding region and a partially constant region. Not only do Fabs have a high antibody-antigen affinity and excellent tissue penetration, but they also have a more stable structure. Thus, Fabs play a large role in clinical diagnosis and treatment. Compared with full length IgGs, Fabs lack an Fc segment and won’t precipitate with the antigen. Due to its low immunogenicity, it can significantly reduce the probability of hypersensitivity reactions and improve product safety. Fab antibody fragments have the characteristics of high antibody affinity, high stability, low immunogenicity, short half-life period, and can be manipulated by genetic engineering.

Single-Chain Variable Fragments (scFv)

ScFvs are composed of the variable region of the heavy-chain and the variable region of the light-chain of an antibody connected by a short peptide of 15-20 amino acids, without an Fc fragment, and belong to genetically engineered, small-molecule antibodies. Compared with monoclonal antibodies, a single-chain antibody molecule size is small, with fast blood clearance and fewer negative reactions to the human immune system. Therefore, as a radionuclide carrier or tumor drug carrier, scFvs can produce larger tumor permeability and have broad application prospects in the fields of antiviral tumor therapy, targeted drug therapy, therapeutics, and diagnostics.

Characteristics of Expression Vectors Commonly Used in E. coli Protein Expression System

Plasmids combine different DNA elements to form vectors with different functions.
1. An effective vector must contain an origin of replication (ORI) and the position where the sequence begins to replicate, which can determine the purpose of the plasmid, the type/type of the host, and the number of plasmid copies.
2. Promoter selection determines the mechanism that regulates protein expression. An effective promoter should be able to achieve an accumulation of recombinant proteins higher than 10-30% of total cellular proteins. In addition, most expression vectors employ an inducible promoter system that, under normal conditions, exhibits minimal basal transcriptional activity, enabling simple and inexpensive induction under specific conditions. The induction point is critical for maximizing recombinant protein production and growth curve plots are usually performed to determine the optimal growth time before induction.
3. Antibiotic resistance genes allow cell selection of a target strain. The addition of this antibiotic to the culture inhibits the growth of cells that do not carry the desired plasmid.
4. Fusion tags commonly used in protein expression are generally divided into purified tags and dissolved tags. Affinity tags can be used to purify proteins quickly and effectively, while solubility tags can enhance the correct folding and solubility of proteins.

The pET vector system is a powerful and widely-used system for expressing recombinant proteins in E. coli using the Lac-controlled T7 promoter operon, a strong T7 promoter that regulates transcription and translation of target genes. Generally, the expression level of recombinant proteins can reach 50% of the total protein content, and the expression state of the target gene is very stable. Therefore, the pET vector system is suitable for most genes. The pBAD expression system modulates specific carbon sources (glucose, glycerol, arabinose) to achieve protein titer expression. pBAD is ideal for expression of E. coli’s toxic proteins and optimization of protein solubility.

About Synbio Technologies

Based on our extensive experience in gene synthesis, vector construction, and recombinant protein expression and purification, Synbio Technologies has developed an efficient E.coli protein expression and purification platform to provide our customers with high-quality protein products that save them time and money. Our codon optimization technology and mature expression system significantly improve sthe expression level of soluble proteins.

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References
[1] Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli expression system in producing antibody recombinant fragments. Int J Mol Sci. 2020; 21: 1–39.
[2] Hayat SMG, Farahani N, Golichenari B, et al. Recombinant protein expression in Escherichia coli (E. coli): what we need to know. Curr Pharm Des. 2018; 24(6): 718–725.
[3] Huleani S, Roberts Michael R, Beales Lucy et al. Escherichia coli as an antibody expression host for the production of diagnostic proteins: significance and expression . Crit Rev Biotechnol, 2022, 42: 756-773.