Background

Biomolecules based on monoclonal antibodies are commonly utilized for disease detection and prevention (Borghardt et al., 2018; Parray et al., 2020; Kumaret al., 2022). The single-chain variable fragment (scFv) antibody is one of the most often exploited biomolecules because it is the smallest antibody unit and has low immunogenicity and low-cost production properties (Kumar et al., 2019b; Parray et al., 2020). The scFv is the most commonly employed combinatorial therapeutic entity, either alone or in combination with other medications (Frenzel et al., 2016; Kumar et al., 2019b). An antibody in the form of scFv has variable heavy (VH) and light-chain (VL) sections that are linked by an efficient linker that can be effectively produced in E. coli (Kumar et al., 2012; Kumaret al., 2019a). The phage display technique is the most popular and successful way of generating scFv antibody fragments among all in vitro display methods. The size and functional diversity of the library used for screening enhances the efficiency of isolating scFv molecules from phage display antibody libraries. The most common issue with the soluble expression of scFv clones from phage libraries is the higher frequency of amber codons within the scFv gene, resulting in the premature expression of scFv clones in non-suppressor E. coli strains. This is more common in the case of synthetic and semi-synthetic libraries because these libraries are constructed randomly at few residues—particularly at NNN, NNK, NWG, NWC, and NSG codons—which increase the biased inclusion of amber codons (Marcus et al., 2006). Due to the frequent presence of amber codons within antibody gene sequences, the isolation of functional soluble scFv molecules is the most prevalent problem encountered during the screening of synthetic and semi-synthetic libraries, resulting in premature expression of scFv clones in non-suppressor E. coli strains (Barderas et al., 2006; Perween et al., 2021a). However, the inclusion of an amber stop codon does not affect the display of scFvs on the phage surface in E. coli suppressor strains, but it reduces the overall yield in terms of the total number of functional soluble scFv protein-expressing clones.

Directing individual scFv genes to be resynthesized or using Kunkel mutagenesis are two popular ways to solve this problem. Both of these processes become expensive and time demanding, considering when the purpose is to screen a substantial proportion of clones, especially in viral targets where a large amount of screening is essential to generate a small number of neutralizing clones (Reader et al., 2019).

In this Bio-protocol, we describe a novel strategy for rapid screening of scFvs containing amber codons and turning them into usable soluble scFvs that can be applied to several phage antibody libraries. We discuss a fast and reliable screening strategy that can be used to screen a large number of phage antibody libraries with amber stop codons (TAG) in the encoding series.