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The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.