27 In Vitro Selection and Evolution of Protein - . . Ligand Interactions by Ribosome Display

Ribosome display is a method for selecting and further evolving functional proteins for their properties of interaction and biophysical parameters. It is performed completely in vitro and does not involve living cells at any step (Hanes and Pliickthun 1997). The method has two main advantages over most other selection methods. First, the screening of very large libraries with up to 1014 members is possible. In contrast, other selection methods such as phage display (Smith 1985; Winter et al. 1994), the yeast two-hybrid system (Fields and Song 1989; Chien et al. 1991), or cell surface display methods (Georgiou et al. 1993; Boder and Wittrup 1997) all necessarily involve transformation steps in which the original library is introduced into cells. This step limits the size of the library to the transformation efficiency, which is usually about 109 to 1010 clones per microgram of DNA in Escherichia coli {Dower and Cwirla 1992) and -107 per microgram of DNA in yeast. Second, an evolution process occurs during the selection procedure. Low-fidelity DNA polymerases that introduce mutations are usually used during the many amplification steps that are part of the ribosome display protocol. In most cases, the mutations are detrimental to the interaction in question. However, in some cases, a mutation is INTRODUCTION, 517