Simplified Synthetic Antibody Libraries

Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
Methods in enzymology (Impact Factor: 2.09). 01/2012; 502:3-23. DOI: 10.1016/B978-0-12-416039-2.00001-X
Source: PubMed


Synthetic antibody libraries are constructed from scratch using designed synthetic DNA. Precise control over design enables the use of highly optimized human frameworks and the introduction of defined chemical diversity at positions that are most likely to contribute to antigen recognition. We describe complete methods for the design, construction, and application of simplified synthetic antibody libraries built on a single human framework with diversity restricted to four complementarity-determining regions and two amino acids (tyrosine and serine). Despite the extreme simplicity of design, these libraries are capable of generating specific antibodies against diverse protein antigens. Moreover, the same methods can be used to build more complex libraries that can produce synthetic antibodies with affinities and specificities beyond the scope of natural antibodies. Most importantly, these simplified methods rely on standard supplies, equipment, and methods that are accessible to any molecular biology laboratory.

1 Follower
10 Reads
  • Source
    • "For a typical library construction, an oligonucleotide library is designed complementary to the ssDNA with flanking regions corresponding to the phagemid vector. The oligonucleotides are then annealed to the vector and the complementary strand is synthesized and ligated together to form a circular, double stranded DNA vector, which is then electroporated into E. coli [34]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.
    BioMed Research International 09/2014; 2014:176172. DOI:10.1155/2014/176172 · 2.71 Impact Factor
  • Source
    • "Antibodies have been extensively studied and many experimental methods are available for their construction, including hybridoma technology [7], phage display [8], yeast surface display [9], and synthetic libraries [10] (see [11] for a review). Immunoinformatics tools have been developed to identify the genes used to create antibodies from nucleotide sequences [12-17], amino acid sequences [17-22], and three-dimensional structures [19,23]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The de novo design of a novel protein with a particular function remains a formidable challenge with only isolated and hard-to-repeat successes to date. Due to their many structurally conserved features, antibodies are a family of proteins amenable to predictable rational design. Design algorithms must consider the structural diversity of possible naturally occurring antibodies. The human immune system samples this design space (2 1012) by randomly combining variable, diversity, and joining genes in a process known as V-(D)-J recombination. Description By analyzing structural features found in affinity matured antibodies, a database of Modular Antibody Parts (MAPs) analogous to the variable, diversity, and joining genes has been constructed for the prediction of antibody tertiary structures. The database contains 929 parts constructed from an analysis of 1168 human, humanized, chimeric, and mouse antibody structures and encompasses all currently observed structural diversity of antibodies. Conclusions The generation of 260 antibody structures shows that the MAPs database can be used to reliably predict antibody tertiary structures with an average all-atom RMSD of 1.9 Å. Using the broadly neutralizing anti-influenza antibody CH65 and anti-HIV antibody 4E10 as examples, promising starting antibodies for affinity maturation are identified and amino acid changes are traced as antibody affinity maturation occurs.
    BMC Bioinformatics 05/2013; 14(1):168. DOI:10.1186/1471-2105-14-168 · 2.58 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.
    Proceedings of the National Academy of Sciences 02/2014; 111(7):2542-7. DOI:10.1073/pnas.1312296111 · 9.67 Impact Factor
Show more