title: A simple high-throughput purification method for hit identification in protein screening.
abstract: Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.
authors.affiliation: Wyeth Research Ireland, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland. cummine4@wyeth.com
authors.forename: Emma
authors.initials: E
authors.lastname: Cummins
authors.forename: Deborah P
authors.initials: DP
authors.lastname: Luxenberg
authors.forename: Fionnuala
authors.initials: F
authors.lastname: McAleese
authors.forename: Angela
authors.initials: A
authors.lastname: Widom
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.forename: Alfredo
authors.initials: A
authors.lastname: Darmanin-Sheehan
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Whitters
authors.forename: Laird
authors.initials: L
authors.lastname: Bloom
authors.forename: Davinder
authors.initials: D
authors.lastname: Gill
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham