Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR–GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN–yhbJ–npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope

Strain engineering for improved expression of recombinant proteins in bacteria

Tomohiro Makino and George Georgiou are with the Department of Chemical Engineering, The University of Texas at Austin and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA -- George Gerorgiou is with the Department of Biomedical Engineering, The University of Texas at Austin and the Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712, USA -- Georgios Skretas is with the Insitute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece -- Tomohiro Makino is with the Asubio Pharma CO., LTD. 6-4-3, Minatojima-Minamimachi Chuo-ku, Kobe 650-0047, JapanProtein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.Chemical EngineeringInstitute for Cellular and Molecular BiologyBiomedical EngineeringMolecular [email protected]

Neural network-based virtual microphone estimation with virtual microphone and beamformer-level multi-task loss

Array processing performance depends on the number of microphones available. Virtual microphone estimation (VME) has been proposed to increase the number of microphone signals artificially. Neural network-based VME (NN-VME) trains an NN with a VM-level loss to predict a signal at a microphone location that is available during training but not at inference. However, this training objective may not be optimal for a specific array processing back-end, such as beamforming. An alternative approach is to use a training objective considering the array-processing back-end, such as a loss on the beamformer output. This approach may generate signals optimal for beamforming but not physically grounded. To combine the advantages of both approaches, this paper proposes a multi-task loss for NN-VME that combines both VM-level and beamformer-level losses. We evaluate the proposed multi-task NN-VME on multi-talker underdetermined conditions and show that it achieves a 33.1 % relative WER improvement compared to using only real microphones and 10.8 % compared to using a prior NN-VME approach.Comment: 5 pages, 2 figures, 1 tabl

Apparent stratospheric ozone loss rate over Eureka in 1994/95, 1995/96, and 1996/97 inferred from ECC ozonesonde observations

Many ECC-type ozonesondes were launched at the Canadian Arctic Eureka observatory(80°N , 86°W ), one of the most northern stations in the Arctic, during winters from 1993/94 to 2001/02, and the temporal evolutions of the vertical ozone profiles were obtained in detail. The lower stratospheric temperature over Eureka was very low inside the polar vortex and the largest ozone loss was observed in 1999/2000, as reported in a previous paper. Similarly, Eureka was often or persistently inside the vortex in the lower stratosphere(around the 470K isentropic surface level) in the winters of 1994/95, 1995/96, and 1996/97. Very low temperatures were observed inside the vortex in the lower stratosphere over Eureka, as indicated by detection of PSCs by Mie lidar. Observations of tracers(N_2O, total reactive nitrogen species(NOy), and others) inside the vortex during these winters using an ER-2 aircraft and balloons indicated that the effect of air parcel mixing across the vortex edge was minimal, based on the tracer-tracer relationship(e.g., Y. Kondo et al.; J. Geophys. Res., 104D, 8215, 1999). Therefore, significant decreases of the in-travortex ozone mixing ratio in the lower stratosphere were considered to be chemical ozone losses due to chlorine activation of PSCs following diabatic descent. The apparent ozone loss rate inside the vortex over Eureka was estimated for each year. The rates ranged from 0.01 to 0.03ppmv/day, less than that observed in 1999/2000(0.04ppmv/day). The observations were conducted at a single station; however, the apparent ozone loss rate over Eureka inside the vortex each year agrees with loss rates obtained in other studies

pH-dependent Formation of Membranous Cytoplasmic Body-like Structure of Ganglioside GM1/Bis(Monoacylglycero)Phosphate Mixed Membranes

Membrane structures of the mixtures of ganglioside GM1 and endosome specific lipid, bis(monoacylglycero)phosphate (BMP, also known as lysobisphosphatidic acid, LBPA) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering (SAXS). At pH 8.5 – 6.5, a GM1/BMP (1/1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and GM1 forms a micelle, the present results indicate that both GM1 and BMP are required to produce the closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies (MCB) in cells derived from patients suffering from GM1 gangliosidosis. Similar to GM1 gangliosidosis, cholesterol was trapped in BMP vesicles in GM1- and in a low pH-dependent manner. Studies employing different gangliosides and a GM1 analog suggest the importance of sugar chains and a sialic acid of GM1 in the pH-dependent structural change of GM1/BMP membranes

Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells

Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentration-dependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ER alpha. and ER beta, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ER alpha/beta and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells

A Case Report of Spindle Cell Carcinoma with Osteoid and Cartilage Formation in the Tongue

Spindle cell carcinoma (SCSCC) with osteoid and/or cartilage formation in the head and neck is rare; only one case was reported in the tongue. Herein, we report an SCSCC with osteoid and cartilage formation of the tongue developed in an 85-year-old man, and then review the report