Recombinant Single-chain Antibody Preparation-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

Recombinant Single-chain Antibody Preparation

Single-chain antibodies consist of antibody variable light (VL) and heavy (VH) chain fragments linked together and are therefore often also referred to as single-chain variable fragments (scFv). The molecular weight of scFv ranges from 27,000 to 30,000 Da and is essentially the smallest antibody fragment encoded by a single gene carrying the entire antigen-binding region for a given antibody. The carboxyl terminus of one variable fragment is linked to the amino group of another fragment via a polypeptide linker that is typically 10-25 amino acid residues in length. The choice of polypeptide linker depends on the suitability of the linker's conformation and length to link the two variable chains without imposing any major stereostructure, and may also vary depending on the intended application of the scFv.

Molecular design of scFv - Tekbiotech.png

Figure 1 Molecular design of scFv

1. Preparation of Recombinant Single-chain Antibodies

The selection of antibody fragments can be performed using display libraries or affinity binding. The selection of desired antibodies isolated from natural or synthetic sources through display libraries can be achieved using (1) phage display; (2) ribosome display; (3) cell surface display in bacterial or yeast systems.

1.1 Expression of scFv in E. coli

Bacterial systems offer the advantages of high protein expression (i.e., efficient yields of several grams per liter), simple culture procedures, and inexpensive fermentation costs, resulting in the production of functional and low-cost products. The lack of glycosylation capacity in bacterial systems does not negatively affect the expression of scFvs, as scFv molecules do not carry any constant heavy chain fragments and therefore do not require glycosylation for biological activity or solubility. As with other recombinant heterologous proteins, the efficiency of scFv expression depends on the efficiency of transcription, translation, and correct folding or refolding, which often depends on the intrinsic properties of each scFv and its interaction with the expression system.

Bacterial expression of scFv fragments can be obtained by cytoplasmic secretion (i.e., soluble expression in the cytoplasm), periplasmic secretion, or the formation of inclusion bodies in the cytoplasm. Periplasmic secretion increases the probability of correctly folded proteins, as the cellular environment is more oxidative, favoring correct disulfide bond formation. Extracytoplasmic expression of scFv proteins is advantageous for large-scale production due to the ease of subsequent product recovery. Extracytoplasmic pulling of periplasmic secreted products can be easily achieved by osmotic shock of the bacterial outer membrane. Subsequent purification of scFv proteins is also straightforward given the relatively small spectrum of contaminants present.

1.2 scFv in Mammalian and Insect Systems

When post-translational modification of the product is required, eukaryotic cells are fully capable of recognizing signals for eukaryotic protein synthesis, processing, and secretion, and the use of eukaryotic systems to produce recombinant scFv proteins is the optimal choice. Studies have shown that the efficiency of producing functional scFvs is comparable in both systems (i.e., between 1 and 10 mg/L). Complex proteins require a correctly folded conformation or post-translational modifications (such as glycosylation) to remain functional, and although the use of mammalian expression systems may be unavoidable, this is rarely the case for relatively simple scFvs. Microbial production of antibody fragments can prove to be more advantageous in terms of processing economy and production volume.

It is not uncommon to use other higher-order eukaryotic systems to produce scFv. The methods used are easily scaled up to prepare high-yield and high-purity scFv antibodies without any affinity chromatography or refolding steps. If the upstream economics can be optimized, the baculovirus system may be an effective alternative to the E. coli system for the co-expression of single-chain antibodies for large-scale production.

1.3 Expression of scFv in Yeast Systems

The production of scFv in yeast systems generally involves secretion directly into the cytoplasm in a correctly folded form, or directly into the culture medium outside the cell. Extracellular secretion is generally desirable because it allows for simultaneous product purification and recovery in a single step.

In yeast systems, high cell density fermentation is a well-established and advantageous feature, where scFv products have been reported to have grown to cell densities of 5 g/L. In different studies, the importance of adequate optimization of fermentation conditions (such as medium composition, aeration, pre-induction osmotic stress, induction components, and temperature) to improve scFv yield and quality has been shown to be critical. For secreted proteins, a common problem in large-scale production is their stability in solution. Optimizing the medium composition to improve the stability of scFv is an important first step to improve overall product yield. Although the production of scFv in yeast systems is generally reported to be high expression levels, downstream recovery strategies need to be developed to minimize product losses.

TekBiotech can provide one-stop recombinant antibody expression services from sequence analysis (including transmembrane analysis, hydrophilicity analysis, etc.), tag design (including but not limited to affinity tags, lytic tags, biotinylation tags, etc.), linker design, vector construction design, recombinant antibody expression to antibody purification. At the same time, TekBiotech can also provide downstream testing and verification experiments such as antibody labeling, affinity verification, ELISA verification, flow cytometry verification, immunological testing, drug antibody screening, etc.

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