Cell-free translation reconstituted with purified components

• Published in: Nature biotechnology 2001-08, Vol.19 (8), p.751-755
• Main Authors: Ueda, Takuya, Shimizu, Yoshihiro, Inoue, Akio, Tomari, Yukihide, Suzuki, Tsutomu, Yokogawa, Takashi, Nishikawa, Kazuya
• Format: Article
• Language: English
• Subjects: Agriculture; Amino acids; Amino Acids - chemistry; Bioinformatics; Biological and medical sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cell-Free System; Chromatography; Chromatography, Affinity; Diverse techniques; E coli; Electrophoresis, Polyacrylamide Gel; Enzymes; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Histidine - chemistry; Homogeneity; Kinases; Life Sciences; Molecular and cellular biology; Plasmids - metabolism; Productivity; Protein Biosynthesis; Protein synthesis; Proteins; Publishing; Recombinant Proteins - metabolism; Ribosomes - metabolism; RNA polymerase; RNA, Transfer - metabolism; RNA, Transfer, Amino Acyl - metabolism; Time Factors; Transfer RNA
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/90802

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).