Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

Reconstruction of variations of isotopic ratios of atmospheric methane: measurements of firn air /ice core and applications of firn air transport model

第6回極域科学シンポジウム分野横断セッション：[IA] 急変する北極気候システム及びその全球的な影響の総合的解明―GRENE北極気候変動研究事業研究成果報告2015―11月19日（木）　国立極地研究所１階交流アトリウ

Hysteretic Three-State Redox Interconversion among Zigzag Bisquinodimethanes with Non-fused Benzene Rings and Twisted Tetra-/Dications with [5]/[3]Acenes Exhibiting Near-Infrared Absorptions

Octaaryl-substituted bisquinodimethanes (BQDs) with a zigzag structure were designed as redox-switchable molecules that undergo four-electron oxidation to produce tetracationic pentacenes with a doubly twisted structure. In contrast to one-stage four-electron oxidation of BQDs, stepwise two-electron reduction of tetracationic pentacenes occurs to give dicationic anthracenes and then the original BQDs, step-by-step. Since both tetracations and dications exhibit near-infrared (NIR) absorptions (-1400 nm) based on an intramolecular charge-transfer interaction, changes in not only their structures but also their UV-vis-NIR spectra can be controlled by redox stimuli. In this Communication, we present an unprecedented one-step p-extension to pentacene from non-fused benzene rings by oxidation, and subsequent two-stage deannulation to benzene rings via anthracene upon reduction. All structures were determined by single-crystal X-ray analyses, and their properties were characterized by spectroscopic and theoretical studies

Geometrical and Electronic Structure of Cation Radical Species of Tetraarylanthraquinodimethane: An Intermediate for Unique Electrochromic Behavior

Two tetraarylanthraquinodimethane (Ar(4)AQD) derivatives having two different aryl groups (aminophenyl and methoxyphenyl) were prepared by sequential dibromomethylation and Suzuki-coupling reactions. X-ray analyses showed that they adopt a folded structure in the neutral state whereas the corresponding dications have a planar anthracene ring, to which diarylmethylium units are perpendicularly attached. Different from Ar(4)AQD having the same substituents that undergoes facile two-electron transfer during interconversion with the dicationic state, the intermediary cation radical becomes long-lived in the newly prepared unsymmetric derivatives. The geometric and electronic structures of the open-shell intermediates were elucidated through electrochemical and theoretical investigation, with revealing that the cation radicals adopt the twisted geometry like dications. Upon electrolyses of the dications, the twisted cation radicals were involved in the electrochromism whereas their steady-state concentration is negligible in the oxidation process, thus realizing unique tricolor electrochromic behavior with a hysteretic pattern of color change (colorless -> purple -> blue -> colorless)

Switching of Redox Properties Triggered by a Thermal Equilibrium between Closed-Shell Folded and Open-Shell Twisted Species

Thermally switchable redox properties have been reported to be due to a change in the spin state of newly designed overcrowded ethylenes, which can adopt closed-shell folded and open-shell twisted forms. In this study, tetrathienylanthraquinodimethane derivatives were designed to be in thermal equilibrium between a more stable folded form and less stable but more donating twisted diradical in solution, so that the oxidation potential can be controlled by heating/cooling. This is the first example of a switching of redox properties based on a thermally equilibrated twisted diradical, which can be more readily oxidized to the twisted dication

Pharmacokinetics of mycophenolic acid after haplo-hematopoietic stem cell transplantation in Japanese recipients

Purpose : Mycophenolate mofetil (MMF), a mycophenolic acid (MPA) prodrug, is used to prevent graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). Although previous studies have reported that enterohepatic circulation (EHC) of MPA, which is usually observed in MMF-treated patients, does not occur in HSCT patients, it is unclear what happens in haploidentical-HSCT (haplo-HSCT) patients, who are using post-transplant cyclophosphamide. This study was conducted to investigate MPA pharmacokinetics in haplo-HSCT patients. Methods : Seventeen haplo-HSCT patients, who received MMF for GVHD prophylaxis, were enrolled in this study. We collected blood samples on days 14 and 28, and plasma MPA concentrations were measured by high-performance liquid chromatography; pharmacokinetic parameters such as area under the curve (AUC), mean residence time (MRT), and apparent oral clearance (CL/F) were measured with moment analysis. We also evaluated EHC as AUC(6-12h)/AUC(0-12h.) Results : There was no significant difference in MPA pharmacokinetic parameters between days 14 and 28. There was also no difference between the pharmacokinetic parameter changes and diarrhea. Additionally, varying plasma MPA concentrations suggested that MPA EHC did not occur. Conclusion : In this study, we revealed the pharmacokinetics of MMF in Japanese haplo-HSCT recipients. Additionally, our study demonstrated that MPA EHC might not occur in Japanese haplo-HSCT recipients

9,10-Dihydrophenanthrene with Two Spiro(dibenzocycloheptatriene) Units : A Highly Strained Caged Hydrocarbon Exhibiting Reversible Electrochromic Behavior

The title dispiro hydrocarbon 1 was designed as a new electrochromic material. This multiply clamped hexaphenylethane-type electron donor was prepared from 2,2'-diiodobiphenyl via biphenyl-2,2'-diylbis(dibenzotropylium) 2(2+) salt. X-ray analysis of 1 revealed a highly strained structure as reflected by an elongated ethane bond [bond length: 1.6665(17) angstrom] and nearly eclipsed conformation. The weakened bond was cleaved upon two-electron oxidation to regenerate the deeply colored dication 2(2+). The reversible interconversion between 1 and 2(2+) is accompanied not only by a drastic color change but also by C-C bond formation/cleavage. Thus, the voltammogram showed a pair of well-separated redox waves, which is characteristic of "dynamic redox (dyrex)" behavior. The tetrahydro derivative of 1 with two units of spiro(dibenzocycloheptadiene), which suffers from more severe steric congestion, was also prepared. The crystallographically determined bond length for the central C-C bond [1.705(4) angstrom] is greatest among the values reported for 9,9,10,10-tetraaryl-9,10-dihydrophenanthrene derivatives

Albatross-borne loggers show feeding on deep-sea squids: implications for the study of squid distributions

How surface-feeding albatrosses feed on deep-sea squids has long been a mystery. We investigated foraging behavior during daylight hours of 20 Laysan albatrosses Phoebastria immutabilis breeding in Hawaii using GPS- and camera-loggers. The birds traveled to the North Pacific Transition Zone up to 600 km north of their breeding site. The camera images showed that Laysan albatrosses fed on large (～ 1 m body length), intact floating dead squids (6 events) and floating fragmented squids (10 events) over deep oceanic water (>2000 m) while they flew in a straight path without sinuous searching. Feeding events on squids were not observed during trips when fishing vessels were photographed and seemed to be distributed randomly and sparsely. Thus, this study suggests that Laysan albatrosses found large, presumably post-spawning, squids opportunistically while they were traveling during daylight hours. Although we did not find cetaceans in our surface pictures, we could not rule out the possibility that birds fed on squids, especially fragmented specimens, in the regurgitates of cetaceans at depth. This study demonstrates the usefulness of combining animal-borne GPS- and camera-loggers on wide-ranging top predators for studying the distribution of little known deep-sea squids and their importance in the diet of marine top predators