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171.
Hyuk Lee Si Jae Park Mee-Jung Han Gyeong Tae Eom Min-Jung Choi Seong Ho Kim Young Hoon Oh Bong Keun Song Seung Hwan Lee 《Biotechnology letters》2013,35(10):1677-1683
Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis. 相似文献
172.
Yudai Nagata Jae Man Lee Hiroaki Mon Shigeo Imanishi Sun Mee Hong Shoji Komatsu Yuji Oshima Takahiro Kusakabe 《Biotechnology letters》2013,35(7):1009-1016
Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans. 相似文献
173.
Joong-Youn Shim Kwang H. Ahn Debra A. Kendall 《The Journal of biological chemistry》2013,288(45):32449-32465
The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of β2-adrenergic receptor (β2AR) in complex with Gs (β2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-2183.54, Tyr-224IC2, Asp-3386.30, Arg-3406.32, Leu-3416.33, and Thr-3446.36, as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation. 相似文献
174.
Jooyeon Woo Seok-Kyu Kwon Jungyong Nam Seungwon Choi Hideto Takahashi Dilja Krueger Joohyun Park Yeunkum Lee Jin Young Bae Dongmin Lee Jaewon Ko Hyun Kim Myoung-Hwan Kim Yong Chul Bae Sunghoe Chang Ann Marie Craig Eunjoon Kim 《The Journal of cell biology》2013,201(6):929-944
Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABAA receptor– and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABAA receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2. 相似文献
175.
176.
One of the most widely accepted ideas related to the evolutionary rates of proteins is that functionally important residues or regions evolve slower than other regions, a reasonable outcome of which should be a slower evolutionary rate of the proteins with a higher density of functionally important sites. Oddly, the role of functional importance, mainly measured by essentiality, in determining evolutionary rate has been challenged in recent studies. Several variables other than protein essentiality, such as expression level, gene compactness, protein–protein interactions, etc., have been suggested to affect protein evolutionary rate. In the present review, we try to refine the concept of functional importance of a gene, and consider three factors—functional importance, expression level, and gene compactness, as independent determinants of evolutionary rate of a protein, based not only on their known correlation with evolutionary rate but also on a reasonable mechanistic model. We suggest a framework based on these mechanistic models to correctly interpret the correlations between evolutionary rates and the various variables as well as the interrelationships among the variables. 相似文献
177.
Seung Kyoon Woo Min Seong Kwon Alexander Ivanov Volodymyr Gerzanich J. Marc Simard 《The Journal of biological chemistry》2013,288(5):3655-3667
The sulfonylurea receptor 1 (Sur1)-NCCa-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NCCa-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and KATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury. 相似文献
178.
Moonil Son Kyung-Mi Lee Jisuk Yu Minji Kang Jin Man Park Sun-Jung Kwon Kook-Hyung Kim 《Journal of virology》2013,87(18):10356-10367
The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus. 相似文献
179.
Brooke M. Emerling Jonathan B. Hurov George Poulogiannis Kazumi S. Tsukazawa Rayman Choo-Wing Gerburg M. Wulf Eric L. Bell Hye-Seok Shim Katja A. Lamia Lucia E. Rameh Gary Bellinger Atsuo T. Sasaki John M. Asara Xin Yuan Andrea Bullock Gina M. DeNicola Jiaxi Song Victoria Brown Sabina Signoretti Lewis C. Cantley 《Cell》2013
180.
Ramesh Prasad Pandey Sailesh Malla Dinesh Simkhada Byung-Gee Kim Jae Kyung Sohng 《Applied microbiology and biotechnology》2013,97(5):1889-1901
Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h. 相似文献