Cell-free translation reconstituted with purified components

Y Shimizu; A Inoue; Y Tomari; T Suzuki; T Yokogawa; K Nishikawa; T Ueda

doi:10.1038/90802

Cell-free translation reconstituted with purified components

Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802.

Authors

Y Shimizu¹, A Inoue, Y Tomari, T Suzuki, T Yokogawa, K Nishikawa, T Ueda

Affiliation

¹ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bldg FSB401, 5-1-5, Kasiwanoha, Kashiwa, Chiba prefecture 277-8562, Japan.

PMID: 11479568
DOI: 10.1038/90802

Abstract

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 microg/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).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acids / chemistry
Cell-Free System*
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
Escherichia coli / metabolism
Histidine / chemistry
Plasmids / metabolism
Protein Biosynthesis
RNA, Transfer / metabolism
RNA, Transfer, Amino Acyl / metabolism
Recombinant Proteins / metabolism*
Ribosomes / metabolism
Time Factors

Substances

Amino Acids
RNA, Transfer, Amino Acyl
Recombinant Proteins
Histidine
RNA, Transfer