Phage Display Library Construction Services

Background

Phage display is a revolutionary technique in molecular biology that allows peptides, proteins, or antibodies to be presented on the surface of bacteriophages. Since its inception, it has become an indispensable tool for identifying molecules with high affinity and specificity, playing a crucial role in drug discovery, diagnostics, and understanding protein interactions.

The process involves creating large, diverse libraries of genetic variants displayed on phage surfaces, enabling rapid screening for desired binding properties. Advances in cloning, amplification, and phage packaging technologies have streamlined the construction of these libraries, improving their diversity and functional relevance.

Figure 1. Phage display library construction methodology. (Adapted from Almagro et al., 2019)

Innovative techniques like error-prone PCR and DNA shuffling further enhance library complexity, expanding the potential for discovering novel biomolecules. Phage display libraries continue to drive breakthroughs across biotechnology and pharmaceutical research, offering unmatched precision and scalability.

Applications

• Drug Discovery: Identifying peptides or proteins with high binding affinity for target molecules.
• Antibody Engineering: Developing therapeutic antibodies by displaying antibody fragments (scFv, Fab, sdAb) on phage surfaces.
• Epitope Mapping: Identifying specific regions (epitopes) of an antigen that are recognized by antibodies.
• Protein-Protein Interaction Studies: Exploring interactions between proteins to understand cellular processes.
• Enzyme Engineering: Screening enzyme variants to discover improved catalytic properties.

At Creative BioMart Microbe, we provide expert phage display library construction services tailored to meet the unique needs of therapeutic, diagnostic, and research applications. Contact us today to discuss your phage display project!

Service Procedure

Service Details

• Vector Preparation: Selection and optimization of suitable phagemid vectors, typically M13-based, followed by linearization and purification to enable efficient insert cloning.
• Insert Amplification: High-fidelity PCR amplification of target DNA sequences with custom primer design to maintain reading frame and expression fidelity, coupled with rigorous purification and quality verification.
• Ligation and Transformation: Precise ligation of inserts into linearized vectors using optimized protocols, followed by transformation into competent E. coli strains for robust library propagation.
• Phage Packaging and Amplification: Infection with helper phages to produce functional phage particles displaying the target peptides or proteins on their surfaces, ready for downstream screening.
• Library Diversity Enhancement: Optional incorporation of techniques such as error-prone PCR, DNA shuffling, and codon optimization to maximize sequence variability and expression efficiency.
• Quality Control: Verification of library size, diversity, and functionality through sequencing, titer measurement, and phenotypic assays to ensure library integrity and performance.

Our Advantages

• Tailored Designs: Custom library formats, CDR architectures, and mutagenesis schemes aligned with your research goals.
• High Diversity and Stability: Libraries designed with structural considerations to ensure optimal folding, expression, and diversity coverage.
• Advanced Bioinformatics Support: Proprietary algorithms and structural modeling tools guide framework selection and residue-level design.
• Flexible Scaffold Options: Extensive experience with scFv, Fab, VHH (nanobody), and peptide libraries in linear and cyclic formats.
• Comprehensive QC: NGS-based analysis, sequence entropy checks, and phage viability assessments ensure high library quality.

Case Study

Case Study 1: Phage display is the foundational technology for in vitro antibody selection.

First introduced by George P. Smith in 1985, phage display remains the most widely used method for in vitro antibody selection. By fusing antibody fragments, such as single-chain variable fragments (scFvs) or fragments of antigen-binding sites (Fabs), to the pIII coat protein of bacteriophage M13, researchers can construct vast libraries of up to 10^{11 variants for high-throughput screening of antigen-specific antibodies. These libraries have since become essential for generating potent, fully human monoclonal antibodies. This approach's strength lies in its direct linkage between antibody genotype and phenotype, which enables the efficient selection and development of therapeutic antibodies.}

Figure 2. Construction and affinity selection with phage-display antibody library. A. Outline of the procedure for constructing a phage-displayed antibody (Fab or scFv) library. B. Structure of IgG molecule. C. Biopanning with a phage-displayed library. (Lu et al., 2020)

Case Study 2: Protocol for design, construction, and selection of genome phage (gPhage) display libraries.

This protocol outlines the genomic phage (gPhage) display platform, a high-throughput method for comprehensive antigen and epitope mapping. In this study, researchers developed a gPhage display peptide library derived from the eukaryotic pathogen Trypanosoma cruzi, the causative agent of Chagas disease, to investigate the host antibody response to the parasite. While this approach was applied to an organism with a relatively large, intronless genome, it is also adaptable for use with other widespread or (re)emerging infectious agents that possess genomes with few introns.

Figure 3. Building and quality control of a Trypanosoma cruzi genomic DNA library. (A) T. cruzi genomic DNA can be fragmented with the COVARIS S2 ultrasonicator. The image shows an agarose gel electrophoresis comparing T. cruzi DNA before and after fragmentation. The obtained fragments were mainly distributed between 100-500 bp. (Rodriguez Carnero et al., 2021)

FAQs

References:

1. Almagro JC, Pedraza-Escalona M, Arrieta HI, Pérez-Tapia SM. Phage display libraries for antibody therapeutic discovery and development. Antibodies. 2019;8(3):44. doi:10.3390/antib8030044
2. Lu RM, Hwang YC, Liu IJ, et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci. 2020;27(1):1. doi:10.1186/s12929-019-0592-z
3. Rodriguez Carnero LA, Reis Teixeira AA, Fen Tang FH, et al. Protocol for design, construction, and selection of genome phage (gPhage) display libraries. STAR Protocols. 2021;2(4):100936. doi:10.1016/j.xpro.2021.100936