Abstract
Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications.
Highlights
Thirty years ago George Smith introduced ‘phage display’ as a means to express vast collections of recombinant proteins and peptides to be screened by affinity selection [1](a process coined ‘biopanning’ [2])
While the percentage of UAG was similar after 1 h a substantial difference of over 40% was observed after 19 h (Figure 2). These results demonstrate that production of libraries in bacteria that are devoid of suppression of amber stop codon leads to over representation of phages containing UAG bearing inserts
The presumption in all these applications is that the selected peptides are derived from biopanning of bone fide unbiased random peptide libraries
Summary
Thirty years ago George Smith introduced ‘phage display’ as a means to express vast collections of recombinant proteins and peptides to be screened by affinity selection [1](a process coined ‘biopanning’ [2]). Two basic systems evolved: (i) phage display of random peptide libraries and (ii) the production of phage displayed antibodies. The latter has been used extensively to generate human monoclonal antibodies [3,4,5,6], replacing to some extent the production of murine monoclonal antibodies via classical hybridoma technology. The first system, random peptide libraries, was initially applied to the epitope analysis of a specific monoclonal antibody with the ultimate intent of affinity selection of a peptide-mimetic assumed to simulate and represent the cognate epitope of the antibody being studied [7,8]. In many cases the application of phage display libraries was performed to clone out a specific peptide or antibody for further development and study. Phage display has served as the basis for computational prediction of epitope-structures [15,16,17,18,19,20,21], as reviewed by Sun et al [22] and for the critical profiling of the repertoire of antibody specificities of polyclonal sera, i.e. profiling the IgOme [23,24], see [25]
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