Phage Display-Special Topic on Phage Display Technology-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Phage Display

The key to treating cancer is finding proteins, specifically antibodies, that can specifically recognize markers on the surface of cancer cells. However, traditional antibody production techniques are time-consuming and difficult to customize for arbitrary targets. In 1985, Smith discovered a new technology, phage display, which enables the rapid and efficient screening of high-affinity, high-specificity molecules from vast pools of candidates.

Principle

The M13 filamentous phage expresses the major coat protein pVIII and the minor coat protein pIII, along with other minor coat proteins pVI, pVII, and pIX. However, these have very low copy numbers, making them suitable for monovalent or low-copy-number display. They can sometimes be used in combination with pVIII or pIII for specialized displays, where a single viral particle displays two different molecules.

Phageprotech's core principle involves fusing the gene of an exogenous peptide or protein with a coat protein gene. As the phage assembles, the exogenous molecule is displayed on the phage surface, while its corresponding gene resides inside the phage. This creates a precise link between phenotype and genotype, providing the basis for downstream engineering.

pVIII Protein: Smaller in size. Although it is high-copy, pVIII is a critical protein for phage assembly; therefore, the N-terminal region can only accommodate very short peptide sequences. It is suitable for displaying short peptide epitopes and is often used for epitope mapping, antigen mimicry, or screening short peptides with specific functions.

pIII Protein: Low copy number, larger in size. The C-terminus of pIII is essential for infection, so it should be preserved during engineering. Its N-terminus can accommodate larger proteins (e.g., antibody fragments scFv, Fab), making it commonly used for high-affinity ligands.

Figure 1: M13 filamentous phage displaying OVA on pVIII ^[1]

Technical Workflow for Library Construction (using scFv library as an example):

1.Obtain cDNA: First, isolate B cells. Extract total mRNA using a kit or the TRIzol/phenol-chloroform method, then reverse transcribe it into total cDNA.

2.In Vitro Synthesis of VH and VL Genes: Use degenerate primers (a mixed pool of primers whose sequences contain a mixture of bases at certain positions) to amplify the variable regions of all B cells in vitro. Because the sequences at the two ends of antibody variable regions are relatively conserved while the middle regions are highly variable, degenerate primers can amplify VH and VL genes from different families, ensuring library diversity.

3.In Vitro Ligation to Form scFv Molecules: Design a flexible peptide linker. Assemble the VH, linker, and VL via overlapping sequences. The VH and VL may originate from different B cells; they are randomly assembled into complete scFv genes.

4.In Vitro Construction of Complete Phagemid: In a test tube, mix a defined ratio of the diverse scFv gene fragment pool with linearized vector and ligase. Individual scFv gene fragments randomly ligate with individual vector molecules to form a complete phagemid.

5.Electroporation into E. coli: Electroporate the recombinant phagemid into E. coli. Theoretically, each E. coli cell carries only one type of phagemid. The C-terminus of the M13 pIII protein recognizes and binds to the tip of the F pilus, which is then depolymerized [2]. However, this depolymerization is transient, so the infection titer of the recombinant phagemid must be controlled.

6.Library Amplification: Collect and culture the bacterial colonies from the previous step. Infect the mixed bacterial colonies with helper phage. The helper phage contains the essential elements for complete phage amplification. This allows the recombinant phagemid to replicate and assemble within E. coli, with each bacterium producing only one type of phage displaying the exogenous gene. Collecting the plaques yields a diverse scFv phage library, which can be stored in glycerol at -80°C.

Once the phage library is obtained, the next step is library screening.

TekBiotech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform based on phage display technology. We provide high-quality VHH neutralizing monoclonal antibody development services to scientists worldwide, including but not limited to the early-stage development and validation of therapeutic monoclonal antibodies targeting GPCRs, oncology targets, and various disease targets. We offer one-stop technical services from project design to developability assessment, meeting the diverse needs of clients for therapeutic monoclonal antibody development.

References

[1] Hess KL, Jewell CM. Phage display as a tool for vaccine and immunotherapy development. Bioeng Transl Med. 2019 Sep 18;5(1):e10142.

[2] Schmitz U, Versmold A, Kaufmann P, Frank HG. Phage display: a molecular tool for the generation of antibodies--a review. Placenta. 2000;21 Suppl A:S106-S112.

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