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51.
Gal-Tanamy M Zemel R Berdichevsky Y Bachmatov L Tur-Kaspa R Benhar I 《Journal of molecular biology》2005,347(5):991-1003
Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources. 相似文献
52.
Podbilewicz B Leikina E Sapir A Valansi C Suissa M Shemer G Chernomordik LV 《Developmental cell》2006,11(4):471-481
During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery. 相似文献