Phage display is commonly understood as fusing the protein to be expressed with the phage coat protein so that the protein is expressed on the phage surface. Of course, in this process, it is necessary to ensure that the structure and function of the protein do not change. Because the protein maintains its own activity, the phage expressing active protein can be screened out through specific reaction identification. This technology directly links the phenotype and genotype of the protein, and can achieve high-throughput screening.

Phage display libraries can be widely used to select antibodies against a variety of different antigens. Phage display antibody (PDA) libraries can quickly isolate and identify highly specific monoclonal antibodies for treatment and diagnosis. Phage display antibody libraries can be divided into immune libraries, natural libraries, and random peptide libraries. In order to create the largest possible antibody library, random peptide libraries have received increasing attention, and many synthetic and semi-synthetic antibody libraries have been produced. Phage display antibody libraries are essentially a library construction and screening technology. Here we focus on the library construction step of inserting exogenous genes into phage coat genes and expressing them on the phage coat.

In order to display the antibody variable region on the surface of filamentous phage and subsequently recover the soluble antibody, an amber stop codon (TAG) is placed between the antibody coding region and the phage coat protein pill. When this amber stop codon TAG is expressed in an inhibitory strain of E. coli such as TG1 or XL1 blue, it is translated into glutamine about 20% of the time, and the other 80% of the time it is translated into a stop signal, so the number of clones that encode the amber stop codon as a stop signal is greater than the number of clones that encode the amber stop codon as glutamine, that is, most phages do not display the antibody on the phage surface. Since only phages that express foreign proteins and phage capsid proteins in fusion can be screened, and most translations are terminated, it is easier to screen antigen-specific phages in inhibitory strains. Once the antigen-specific phage is selected and cloned, it can be expressed as a soluble antibody by switching to a non-inhibitory strain of E. coli such as HB2151.

However, there is an inherent bias in the selection of positive binding clones from synthetic and semisynthetic libraries, namely that clones will contain randomly generated amber stop codons TAG, which slows down the scFv selection process and affects the isolation and production of scFv, complicating the identification of high affinity binding antibodies. Normally, the proportion of scFv containing stop codons fluctuates between 10-30%, but for certain specific antigen selection methods, this proportion may increase to 70-100%. Although more and more researchers are taking advantage of this technology, the frequent presence of amber stop codons in variable regions is a largely ignored issue, as stop codons are only considered when positive clones cannot be identified by soluble ELISA experiments. When stop codons are present in scFv, antigen-specific antibody selection must be followed by site-directed mutagenesis to remove the stop codon in each scFv, which is a rather tedious process because different specific primers need to be designed for each scFv, followed by sequencing. Therefore, a possible alternative is to express a soluble form of the antibody-pIII fusion protein, but this is also problematic - the antibody-pIII fusion protein is expressed at low levels in E. coli and is functionally heterogeneous due to spontaneous cleavage of the pIII protein. For these reasons, accurate quantification and affinity measurement of the antibody-pIII fusion protein is also difficult, and the key issue for selecting a functional antibody is to determine its solubility and affinity for binding to the antigen when expressed in E. coli.

Therefore, Marcus et al. mutated the amber stop codon to a glutamine codon (CAG), and Barderas et al. mutated the amber stop codon to an ochre stop codon (TAA), so that it only expressed the soluble target antibody without expressing the phage capsid protein pIII, which provided a solution. Currently, Perween et al. have applied the TAA mutation to SARS CoV 2 and successfully produced humanized recombinant scFv antibodies against SARS CoV 2 RBD.

Tek biotech phage antibody display technology platform service advantages:

-- Rapid preparation of soluble antigens: Combined with the company's existing recombinant protein expression system, high-purity, high-biologically active soluble antigen proteins can be prepared in a short time to support phage antibody immunization and screening.

--Mature and stable immune library construction technology: The library capacity of the primary phage immune library we have obtained can reach 10^8-10^9, and its sequence abundance is >99%, ensuring that the antibody affinity obtained by direct screening reaches nM or even pM level.

--Rapid and reliable candidate antibody screening and preliminary identification technology: According to different antibody drug functional requirements, a variety of screening systems have been developed, including direct screening, competition, negative screening, cell screening, etc.