To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor a ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results Of Our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution. (C) 2009 Elsevier Inc. All rights reserved.