Amber codon-mediated expanded saturation mutagenesis of proteins using 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 of proteins, in which an amino acid at a specific site is substituted with each of the other 19 amino acids, is a powerful method for protein analysis and engineering. However, 19 mutated genes have to be prepared to express all possible amino acid-substituted proteins at one site. We previously reported a four-base codon-mediated saturation mutagenesis method for the expression of all 20 amino acid-substituted proteins from one four-base codon-containing gene using 20 types of chemically aminoacylated tRNAs corresponding to the four-base codon. In this study, an improved method for saturation mutagenesis using an amber codon was developed. By combining the use of Escherichia coli-derived amber suppressor tRNAs and chemically aminoacylated Mycoplasma-derived tRNAs, all 20 mutated proteins were successfully expressed from one amber mutant gene in a cell-free translation system. The use of E. coli-derived amber suppressor tRNAs simplified the preparation of the tRNA reagents required for saturation mutagenesis, and also improved the expression of some of the mutated proteins. The expressed mutant proteins were used to evaluate the effect of the amino acid substitutions on the ligand-binding activity. To further expand the possibilities of saturation mutagenesis, a series of nonnatural amino acids analogous to a naturally occurring amino acid was added to the amino acid repertoire. The expanded saturation mutagenesis was utilized to evaluate the effect of a series of atomic-level side chain substitutions on the protein activity.
著者
-
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
-
SHOZEN Naoki
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