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Pellis M Pardon E Zolghadr K Rothbauer U Vincke C Kinne J Dierynck I Hertogs K Leonhardt H Messens J Muyldermans S Conrath K 《Archives of biochemistry and biophysics》2012,526(2):114-123
Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings. 相似文献
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Paul D. Lampe Joerg Kistler Andreas Hefti Jacqui Bond Shirley Müller Ross G. Johnson Andreas Engel 《Journal of structural biology》1991,107(3)
Gap junction structures were assembled in vitro from octyl-β-
-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly. 相似文献
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Sabrina Stebe Katharina Schellig Florian Lesage Heinz Breer Joerg Fleischer 《Cellular and molecular neurobiology》2014,34(1):113-122
Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs. 相似文献
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Florian Wilfling Huajin Wang Joel T. Haas Natalie Krahmer Travis J. Gould Aki Uchida Ji-Xin Cheng Morven Graham Romain Christiano Florian Fröhlich Xinran Liu Kimberly K. Buhman Rosalind A. Coleman Joerg Bewersdorf Robert V. Farese Tobias C. Walther 《Developmental cell》2013,24(4):384-399
Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs 相似文献
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Christopher I Keeling Macaire MS Yuen Nancy Y Liao T Roderick Docking Simon K Chan Greg A Taylor Diana L Palmquist Shaun D Jackman Anh Nguyen Maria Li Hannah Henderson Jasmine K Janes Yongjun Zhao Pawan Pandoh Richard Moore Felix AH Sperling Dezene P W Huber Inanc Birol Steven JM Jones Joerg Bohlmann 《Genome biology》2013,14(3):R27