title: Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.
abstract: In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies.
authors.affiliation: Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.affiliation: Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.affiliation: Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Norikazu
authors.initials: N
authors.lastname: Ichihashi
authors.affiliation: Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: yomo@ist.osaka-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D003956:Dialysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D066298:In Vitro Techniques / Q000379:methods*; D014176:Protein Biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase; D001616:beta-Galactosidase
keywords: Dialysis; Green fluorescent protein; In vitro protein synthesis; PURE system; Synthetic biology