Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Abstract

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phagedisplayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10 4 -fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries. (Journal of Biomolecular Screening 2005:108-117)

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

“…To evaluate the chromato-panning procedure it was compared with the traditional biopanning procedure (Table 2 ) as well with other protocols (Table 3 ) described in the literature [ 28 - 30 ]. Special attention was paid to the total time required for the procedure, the efficiency of the elution media, and the efficiency of the procedure in terms of the number of rounds necessary to obtain high-affinity binders.…”

Class III peroxidases are activated in proanthocyanidin-deficient Arabidopsis thaliana seeds

“…Following staining with 200 mL TMB solution, the supernatant was collected after being centrifuged at 10 000 g for 1 min. The reaction was stopped after 10 min with 50 mL H 3 PO 4 and the absorbance at 450 nm was measured (Vanhercke et al, 2005).…”

Progress in phage display: evolution of the technique and its applications

“…Vanhercke et al [103] developed a procedure that they termed RISE (rescue and in situ selection and evaluation). Their method eliminates the need to purify the amplified phage pool before each panning round.…”