High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides.
Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.
Rahim
Ahad
A
Gene Therapy Research Group, Biomedical Sciences, Imperial College, Faculty of Medicine, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London, SW7 2AZ, UK. ahad.rahim@imperial.ac.uk
Coutelle
Charles
C
Harbottle
Richard
R
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods; D013997:Time Factors
D016895:Culture Media, Serum-Free; D008024:Ligands; D019151:Peptide Library; D010455:Peptides