Abstract
Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.
Highlights
Antibodies in particular immunoglobulin G (IgGs) are some of the most important biopharmaceutical molecules with a highly relevant market volume
Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries
Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length
Summary
Antibodies in particular immunoglobulin G (IgGs) are some of the most important biopharmaceutical molecules with a highly relevant market volume. By introducing randomly selected nucleotides as a cost-efficient method during the construction of the synthetic library, stop codons can occur that significantly decrease its quality by lowering the number of clones expressing a full-length protein. A significant bottleneck of a phage display selection is the production of sufficient amounts of bioactive monoclonal binders since the low expression level of properly folded proteins from the periplasmic space can be challenging In this protocol, we describe a simple method for the construction of a synthetic vNAR library with codon-wise mutagenesis by using degenerated NNK codons (N means a 25% mix each of adenine, thymine, cytosine and guanine nucleotides; and K stands for a 50% mix each of thymine and guanine nucleotides). Using NNK primers decreases codon redundancy, i.e., arginine encoded with six codons is reduced
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