Tailoring in vitro evolution for protein affinity or stability

We describe a rapid and general technology working entirely in vitro to evo lve either the affinity or the stability of ligand-binding proteins, depend ing on the chosen selection pressure. Tailored in vitro selection strategie s based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of s ingle-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (k(off) at 4 degrees C of 10(-4) s(-1)) was improved 30-fold by the use of off-rate selections o ver a period of several days. As a second example, a generic selection stra tegy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed pro tein occurs. We used decreasing redox potentials in the selection step to s elect for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to for m disulfides again, their stability had increased to 54 kJ/mol from an init ial value of 24 kJ/mol, Sequencing revealed that the evolved mutant protein s had used different strategies of residue changes to adapt to the selectio n pressure. Therefore, by a combination of randomization and appropriate se lection strategies, an in vitro evolution of protein properties in a predic table direction is possible.