A modified two-step phage display selection for isolation of polycystin-1 ligands.
The identification of proteins that interact with polycystin-1, the product of the autosomal dominant polycystic kidney disease gene, is an important step towards understanding the molecular pathogenesis of the disease. We have developed a two-step approach for the efficient identification of potential polycystin-1 ligands using the T7 phage display system. The first enrichment step of 4-5 rounds of biopanning is followed by a second step of reverse protein overlay assay. Thus, the sequencing efforts are minimized to the analysis of only positive rather than randomly chosen clones from the enriched population as in the standard phage display approach. Most importantly, the modified approach immediately provides the confirmation of the specificity of interaction and discriminates between strong and weak interactions. Here we present several potential interactors with distinct regions of polycystin-1, representing high-affinity binding partners.
Bukanov
N O
NO
Genzyme Corporation, 1 Mountain Road, Framingham, MA 01701-9322, USA.
Meek
A L
AL
Klinger
K W
KW
Landes
G M
GM
Ibraghimov-Beskrovnaya
O
O
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050396:TRPP Cation Channels
D005982:Glutathione Transferase; D008024:Ligands; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels