Bacterial Display Peptides for Use in Biosensing Applications

Abstract

Peptide affinity reagent technology including bacterial display encapsulates an in vitro analog of classical Darwinian evolution through a physical linkage between polypeptide sequence and the encoding of genetic information. Similar to the inate human imune system a unique binder is isolated, however, in this directed evolution method a large library displaying billions of diverse peptide sequences is created and the sythetic binder to the target of interest is determined. Subsequently the synthetic reagent is mass-produced for use in the specific biosensing application (Park & Cochran, 2010; Stratis-Cullum & Sumner 2008). Currently, a number of systems, including messenger ribonucleic acid (mRNA) and ribosome display (Wilson, Keefe, & Szostak, 2001 ), eukaryotic virus display (Bupp and Roth, 2002; Muller, 2003), and bacterial and yeast surface display (Georgiou et al., 1997, Boder & Wittrup, 1997), are used to rapidly generate affinity reagents that can be used for diagnostics, proteomics, and therapeutic applications (Kodadek, 2001; Nixon, 2002). Bacterial cell surface display is advantageous because the use of bacterial cells simplifies the polypeptide selection method and enables fast screening of potential recognition elements typically using fluorescence activated cell-sorting (FACS) and/or magnetic activated cellsorting (MACS). Polypeptide affinity reagents can offer more stable alternatives to antibody technology, enabling more rugged application in the field (e.g., enhanced thermal stability). A key advantage of bacterial display technology over traditional antibody creation as well as other peptide display alternatives is that it offers an strategy for generating tailor-made affinity ligands in a very short time period of several days (Georgiou et al., 1997; Bessette, Rice and Daugherty 2004), see Figure 1. The development of a bacterial display system suitable for robust reagent discovery has proven challenging (Lee, Choi & Xu, 2003). There are three main challenges in creating a high speed affinity ligand isolation technology against unknown/un-catalogued pathogens: (1) creation of a large (high diversity) and robust cell based library, (2) creation of an ultrahigh throughput, disposable screening system, and (3) gaining a fundamental understanding of the factors which influence binder performance along with this understanding the development of methodologies to enable universal isolation and optimization of ligand binder performance.