Four-Base Codon-Mediated Saturation Mutagenesis in a Cell-Free Translation System(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
スポンサーリンク
概要
- 論文の詳細を見る
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 amino-acyl-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.
- 社団法人日本生物工学会の論文
- 2008-03-25
著者
-
Hohsaka Takahiro
School of Materials Science, Japan Advanced Institute of Science and Technology
-
Takagi Hiroaki
Proteinexpress Co. Ltd
-
Hohsaka Takahiro
School Of Materials Science Japan Advanced Institute Of Science And Technology
-
MURANAKA Norihito
School of Materials Science, Japan Advanced Institute of Science and Technology
-
Hohsaka Takahiro
School Of Materials Sci. Japan Advanced Inst. Of Sci. And Technol.
-
Watanabe Takayoshi
School Of Materials Science Japan Advanced Institute Of Science And Technology
-
Muranaka Norihito
School Of Materials Science Japan Advanced Institute Of Science And Technology
関連論文
- Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- 2P081 二重標識したマルトース結合タンパク質の基質結合の一分子観察(蛋白質-機能(反応機構,生物活性など),第48回日本生物物理学会年会)
- 1P-064 二重標識したマルトース結合蛋白質の基質結合と折りたたみの一分子観測(蛋白質-機能(反応機構,生物活性など),第47回日本生物物理学会年会)
- Four-Base Codon-Mediated Incorporation of Nonnatural Amino Acids into Proteins in a Eukaryotic Cell-Free Translation System(Methods)
- Incorporation of Non-Natural Amino Acids with Two Labeling Groups into the N-Terminus of Proteins
- Incorporation of Fluorescent-Labeled Non-α-Amino Carboxylic Acids into the N-Terminus of Proteins in Response to Amber Initiation Codon
- 3P-062 非天然アミノ酸の二重導入によるタンパク質構造変化のFRET分析(蛋白質・計測,解析の方法論,第46回日本生物物理学会年会)
- Incorporation of Nonnatural Amino Acids into Proteins through Extension of the Genetic Code
- Four-Base Codon-Mediated Saturation Mutagenesis in a Cell-Free Translation System(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Amber codon-mediated expanded saturation mutagenesis of proteins using a cell-free translation system(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Synthesis of Novel BRET/FRET Protein Probes Containing Light-Emitting Proteins and Fluorescent Nonnatural Amino Acids
- Amber codon-mediated expanded saturation mutagenesis of proteins using a cell-free translation system