Bacterial Display Screening Systems

The bacterial surface display of proteins has been successfully employed in the affinity-based screening of cell surface display libraries by means of immobilised beads. Typical combined selection and screening strategies for large libraries use biotinylated target proteins for sequential magnetic separation (MACS) with streptavidin-functionalized magnetic particles followed by fluorescence-activated cell sorting (FACS) of the enriched population or fine affinity resolution (1,2,3). The project aims to develop novel screening systems for protein based therapeutics and diagnostics using bacterial display technology to select for binding affinity and specificity. The system we are proposing to develop features a number of improvements to those which are currently used in research. First, we propose dispensing with magnetic beads and expressing both the target protein and the potential binding candidate proteins on the outside of bacterial cells - the former being magnetized cells, the latter being 'normal' non-magnetized cells; when the two sets of cells are mixed and binding takes place, the resultant conjugates being cells attached to cells rather than cells attached to beads. Second, we propose using an expression system for the candidate binder proteins that can express 'unnatural' proteins - i.e. those comprising fluorinated or brominated amino acids having greater functionalities than their natural, wild-type counterparts. Third, we propose being able to express the fluorescent proteins used in the FACS part of system both extracellularly and intracellularly as required. 1. Samuelson P, Gunneriusson E, Nygren PA, & Ståhl S. Display of proteins on bacteria. J Biotechnol. 2002 Jun 26;96(2):129-54. Review. 2. Ståhl S & Uhlén, M. Bacterial surface display: trends and progress 15, 5, 1997, 185-192 3. Dane KY, Chan LA, Rice JJ, Daugherty PS. Isolation of cell specific peptide ligands using fluorescent bacterial display libraries. J Immunol Methods. 2006 Feb 20;309(1-2):120-9. Epub 2006 Jan 11.