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Abstract: Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl− and across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-β-d-maltoside. His-tagged PcAEP was purified as a single band on SDS–PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins. [Copyright &y& Elsevier]

We have previously described that oligonucleotides (ODN) containing phenylsulfoxide derivative of 2-amino-6-vinylpurine nucleoside analog (1) are activated within duplex to form cross-link toward cytidine selectively at the target site. In this paper, we wish to report the search for more stable precursor susceptible for activation within duplex. [ABSTRACT FROM AUTHOR]

Abstract: CC chemokine receptor 4 (CCR4) has been implicated as a preferential marker for T helper type 2 (Th2) cells, and is believed to be involved in the pathology of allergic diseases by controlling Th2 cell trafficking into inflamed tissues. The objective of the study was to characterize the pharmacological properties of E0001-163, a novel CCR4 antagonist. E0001-163 was tested in both in vitro chemotaxis assays as well as in vivo mouse models of CCR4 ligand-induced air pouch and antigen-induced airway inflammation by utilizing in vitro-polarized Th2 cells. In vitro, E0001-163 inhibited migratory response of human Th2-polarized cells to CCL22, a CCR4 ligand, with an IC50 value of 11.9nM. E0001-163 significantly suppressed CCL22-induced Th2 cell trafficking into mouse air pouch in a dose-dependent manner at doses of 3 and 10mg/kg, suggesting that E0001-163 has an inhibitory effect on CCR4-mediated T cell trafficking in vivo. In addition, E0001-163 partially decreased Th2 cell trafficking and the level of IL-4 in the lungs in Th2-tansferred and ovalbumin (OVA)-challenged mice. T cell trafficking involves multiple chemokine receptors both in acute and chronic phases, and our findings suggest that CCR4, together with other chemokine receptors, may be involved in Th2 cell trafficking under disease conditions. [Copyright &y& Elsevier]

Abstract: Structure–activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P1 agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P3 agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P1 agonistic activity with excellent selectivity over hS1P3 and in vivo PLL activity in mice. [Copyright &y& Elsevier]

Abstract: Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure–function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials. [Copyright &y& Elsevier]

Abstract: N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand–receptor binding assays. We found that five clones were expressed at greater than 10pmol/mg, 9 clones at 1–10pmol/mg and 11 clones at less than 1pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B max value of 51.2pmol/mg of membrane protein. Approximately 1.9mg of the human CHRM2 was produced from a 1-L culture. [Copyright &y& Elsevier]

Abstract: Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination. [Copyright &y& Elsevier]