Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

Kigawa, T.; Yabuki, Takashi; Matsuda, Natsuko; Matsuda, Takayoshi; Nakajima, Rie; Tanaka, Akiko; Yokoyama, Shigeyuki

doi:10.1023/b:jsfg.0000029204.57846.7d

Cited by 316 publications

(302 citation statements)

References 24 publications

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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%

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Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

Hosaka

Yoshizawa

Nakajima

et al. 2016

Journal of Biological Chemistry

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The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

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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%

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

Hosaka

Yoshizawa

Nakajima

et al. 2016

Journal of Biological Chemistry

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“…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%

Crystal structure of the central axis DF complex of the prokaryotic V-ATPase

Saijo

Arai

Hossain

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long lefthanded coiled coil with a unique short β-hairpin region that is effective in stimulating the ATPase activity of V 1 -ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A 3 B 3 complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.I on-transporting rotary ATPases are divided into three types based on their function and taxonomic origin: F-, V-, and A-type ATPases. F-ATPases function as ATP synthases in mitochondria, chloroplasts, and oxidative bacteria (1). V-ATPases function as proton pumps in the membranes of acidic organelles and plasma membranes of eukaryotic cells (2). A-ATPases in archaea function as ATP synthases similar to the F-ATPases (the "A" designation in A-type refers to archaea), although the structure and subunit composition of A-ATPases are more similar to those of V-ATPases (3). These ATPases possess an overall similar structure that is composed of a hydrophilic catalytic portion (F 1 -, V 1 -, or A 1 -ATPase) and a membrane-embedded ion-transporting portion (F o -, V o -, or A o -ATPase), and they have a similar reaction mechanism that occurs through rotation (2).V-ATPases are found in bacteria, such as Thermus thermophilus and Enterococcus hirae. In T. thermophilus, V-ATPase physiologically functions as an ATP synthase (4). Therefore, it has sometimes been called an A-ATPase, although T. thermophilus is a eubacterium that does not belong to archaea (5). However, the E. hirae V-ATPase acts as a primary ion pump (6), which transports Na þ or Li þ instead of H þ (7). The enzyme is composed of nine subunits (Eh-A, -B, -d, -D, -E, -F, -G, -a, -c; previously designated as NtpA, -B, -C, -D, -E, -G, -F, -I, -K) having amino acid sequences that are homologous to those of the corresponding subunits (A, B, d, D, E, F, G, a, c) of eukaryotic V-ATPases (7) (see Fig. 1). The core of the V 1 domain of this ATPase is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis (8). The V o domain, in which rotational energy is converted to drive Na þ translocation, is composed of oligomers of the 16-kDa c subunits and an a subunit (9, 10). The V 1 and V o domains are connected by a central stalk, which is composed of D, F, and d subunits, and two peripheral stalks, which a...

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“…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%

Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor

et al. 2012

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PDB References: Pim-1, complex with compound 1, 3umx; complex with compound 2, 4eny; complex with compound 3, 4enx.The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the 3 strand located above the binding site, is not usually observed in Pim-1 structures.

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Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

Cited by 316 publications

References 24 publications

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

Crystal structure of the central axis DF complex of the prokaryotic V-ATPase

Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor

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