Fig. 2.

Schematic overview of one round of sorting. The Fc-wt gene was used as the template for an error prone PCR, resulting in an Fc-library with point mutations randomly distributed over the entire gene. This Fc-library, together with the linearized vector pYD1, was cloned into the yeast S. cerevisiae by homologous recombination. After induction of surface expression of the Fc-library the yeast suspension was incubated for 10 min at 79 °C, followed by cooling on ice and labeling with structurally specific Fc-ligands. Positive cells were selected by FACS and since the cells did not survive the heat shock applied before, plasmid DNA had to be isolated and amplified by PCR. With this Fc-library the next sorting round was performed, starting again with the yeast transformation step.