2004
DOI: 10.1023/b:jsfg.0000029204.57846.7d
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Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

Abstract: As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this art… Show more

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Cited by 317 publications
(302 citation statements)
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“…Overall Structure and Comparison with Those of Bacteriorhodopsin, Halorhodopsins, and KR2-We synthesized the fulllength NM-R3 protein, using an E. coli cell-free protein production system (23,24). We obtained over 25 mg of NM-R3 from the 9-ml reaction mixture.…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…Overall Structure and Comparison with Those of Bacteriorhodopsin, Halorhodopsins, and KR2-We synthesized the fulllength NM-R3 protein, using an E. coli cell-free protein production system (23,24). We obtained over 25 mg of NM-R3 from the 9-ml reaction mixture.…”
Section: Resultsmentioning
confidence: 99%
“…Cell-free Protein Synthesis and Purification-Cell-free protein synthesis of NM-R3 was performed essentially according to the previously reported protocols used for Acetabularia rhodopsin I production (22)(23)(24). Briefly, the cell-free reaction mixture included 100 M all-trans-retinal, 0.4% digitonin, and 6.67 mg/ml egg yolk phosphatidylcholine.…”
Section: S1-08mentioning
confidence: 99%
“…An E. coli cell-free protein expression system, as described elsewhere (25), was used to synthesize the Eh-d, -F, and -DF polypeptides using plasmids harboring the corresponding genes or a mixture of plasmids for two genes. We used selenomethionine to synthesize the Eh-DF to facilitate X-ray crystallographic analysis.…”
Section: Methodsmentioning
confidence: 99%
“…We used selenomethionine to synthesize the Eh-DF to facilitate X-ray crystallographic analysis. More than 15 mg of the complex was synthesized with this system, using 27 mL of the reaction solution in the presence of 0.1 mg of plasmids (25). The expressed protein was purified as previously described (11).…”
Section: Methodsmentioning
confidence: 99%
“…The kinase domain of human Pim-1 was synthesized by the Escherichia coli cell-free system using the dialysis method (Kigawa et al, 2004Matsuda et al, 2007). Details of the purification and expression methods have been reported previously (Nakano et al, 2012).…”
Section: Pim-1 Expression and Purification For Crystallizationmentioning
confidence: 99%