Use of EF-Tu mutants for determining and improving aminoacylation efficiency and for purifying aminoacyl tRNAs with non-natural amino acids.
We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.
Ohtsuki
Takashi
T
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. ohtsuk@cc.okayama-u.ac.jp
Yamamoto
Hiromichi
H
Doi
Yoshio
Y
Sisido
Masahiko
M
D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification*; D046249:Transfer RNA Aminoacylation / Q000235:genetics*
D012346:RNA, Transfer, Amino Acyl; D010444:Peptide Elongation Factor Tu