The DOI matches its Crossref record; 23 of the 23 references it cites were found in a registry or an index.
A cell-free protein synthesis system for high-throughput proteomics
Open accessAbstract
We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.
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The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology
Garamella, J., Marshall, R., Rustad, M., & Noireaux, V. (2016) · ACS Synthetic Biology · doi:10.1021/acssynbio.5b00296 · Cited by 419
EmrE, a model for studying evolution and mechanism of ion-coupled transporters
Schuldiner, S. (2009) · Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics · doi:10.1016/j.bbapap.2008.12.018 · Cited by 164
Robotic cloning and protein production platform of the northeast structural genomics consortium
Acton, T. B., Gunsalus, K. C., Xiao, R., Ma, L. C., Aramini, J., Baran, M. C., Chiang, Y.-W., Climent, T., Cooper, B., Denissova, N. G., et al. (2005) · Methods in Enzymology · doi:10.1016/s0076-6879(05)94008-1 · Cited by 129
Structural bases of IMiD selectivity that emerges by 5-hydroxythalidomide
Furihata, H., Yamanaka, S., Honda, T., Miyauchi, Y., Asano, A., Shibata, N., Tanokura, M., Sawasaki, T., & Miyakawa, T. (2020) · Nature Communications · doi:10.1038/s41467-020-18488-4 · Cited by 89
Self-citationYamanaka, S., Murai, H., Saito, D., Abe, G., Tokunaga, E., Iwasaki, T., Takahashi, H., Takeda, H., Suzuki, T., Shibata, N., et al. (2021) · The EMBO Journal · doi:10.15252/embj.2020105375 · Cited by 89
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Metadata
- Authors
- Sawasaki, T., Ogasawara, T., Morishita, R., & Endo, Y.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 2002-10-30
- Volume
- 99
- Issue
- 23
- Pages
- 14652-14657
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2121553505
- Cited by
- 517
- References
- 23
- FWCI
- 7.34
- Type
- journal article
- Language
- en
- Open access
- green
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