Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set

Satoshi Akanuma, Takanori Kigawa, Shigeyuki Yokoyama*


研究成果: Article査読

82 被引用数 (Scopus)


We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.

ジャーナルProceedings of the National Academy of Sciences of the United States of America
出版ステータスPublished - 2002 10月 15

ASJC Scopus subject areas

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