Selection of single domain binding proteins by covalent DNA display.
Selection technologies such as phage and ribosome display, which provide a physical linkage between genetic information and encoded polypeptide, are important tools for the engineering of proteins for diagnostic and therapeutic applications. We have recently described a selection strategy called covalent DNA display, in which individual proteins are covalently linked to the cognate encoding DNA template in separate droplets of a water-in-oil emulsion. We here report on the optimization of several experimental steps in covalent DNA display technology, such as the elution conditions and the PCR strategy used for the amplification of selected DNA templates. A PCR assembly strategy was developed, which allows the amplification of the DNA templates over repeated rounds of selection. In addition, we could demonstrate that approximately 50% of the DNA templates form a covalent adduct with the corresponding proteins in the compartments of a water-in-oil emulsion. In model selection experiments, differences in recovery efficiency <100 000 per round of selection could be observed when comparing a specific binding polypeptide with a binder of irrelevant specificity. Furthermore, the optimized protocol was successfully applied for the selection of single domain proteins, capable of specific binding to mouse serum albumin (MSA). A mutant derived from the SH3 domain of the Fyn kinase, with millimolar affinity to MSA, was affinity matured using covalent DNA display and yielded several MSA binding FynSH3 variants with dissociation constants in the 100 nM range.
Bertschinger
Julian
J
Institute of Pharmaceutical Sciences, ETH Hönggerberg, Wolfgang-Pauli-Strasse 10, HCI G394, CH-8093 Zurich, Switzerland. julian.bertschinger@pharma.ethz.ch
Grabulovski
Dragan
D
Neri
Dario
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004655:Emulsions; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D018909:src Homology Domains
D004268:DNA-Binding Proteins; D004655:Emulsions; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D004247:DNA; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn