In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments.
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 alpha 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.
Tsuji
Toru
T
Department of Biosciences and Informatics, Keio University, Yokohama 223-8522, Japan.
Onimaru
Michiko
M
Doi
Nobuhide
N
Miyamoto-Sato
Etsuko
E
Takashima
Hideaki
H
Yanagawa
Hiroshi
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques; D019020:Directed Molecular Evolution / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics*; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
D047628:Estrogen Receptor alpha; D008024:Ligands; D019204:GTP-Binding Proteins