DARTs: A DNA-based in vitro polypeptide display technology.
Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.
de Figueiredo
Paul
P
Department of Microbiology, University of Washington, Seattle, WA 98195-7242, USA.
Roberts
Radclyffe L
RL
Nester
Eugene W
EW
D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019020:Directed Molecular Evolution; D005798:Genes, Bacterial; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012231:Rhizobium / Q000235:genetics
D018076:DNA, Complementary; D009841:Oligonucleotides; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D004247:DNA