{"id":200,"date":"2018-12-27T08:51:45","date_gmt":"2018-12-27T07:51:45","guid":{"rendered":"http:\/\/bioactivecompoundlibrary.com\/?p=200"},"modified":"2022-01-11T17:10:37","modified_gmt":"2022-01-11T09:40:37","slug":"constructed-set-plasmids-encoding-fusion-proteins-fused","status":"publish","type":"post","link":"http:\/\/bioactivecompoundlibrary.com\/index.php\/2018\/12\/27\/constructed-set-plasmids-encoding-fusion-proteins-fused\/","title":{"rendered":"We constructed a set of plasmids encoding fusion proteins was fused"},"content":{"rendered":"<p>Notably, EBC5-16 induced a statistically-significant 50% increase in CAT activity compared to TC2-3, indicating that the transmembrane domain of EBC5-16 forms a stronger oligomer than TC2-3. This finding corroborates the biochemical results that a higher fraction of EBC5-16 is present as a dimer in murine cells. The results presented above <a href=\"www.abmole.com\/products\/echinatin.html\">Echinatin<\/a> demonstrated that EBC5-16 displays increased dimerization compared to TC2-3. To assess the importance of dimerization in EBC5-16 activity, we mutated both cysteines in the C-terminus of EBC5-16 to serine. This mutant was expressed in BaF3\/HAhEPOR cells, and growth factor independence was assessed. As shown in Figure 5C, EBC5-16-CCSS did not confer growth factor independence, demonstrating that the cysteines, and presumably dimerization, are necessary for EBC5-16 activity. Taken together, these results raised the possibility that the increased activity of EBC5-16 is due to increased dimerization. To determine which amino acids constitute the homodimer interface of EBC5-16, we used an approach we developed to identify the dimer interface of the BPV E5 oncoprotein, which was subsequently confirmed by biophysical studies. We constructed a set of plasmids encoding fusion proteins in which EBC5-16 was fused at seven consecutive residues to the dimerization domain of the yeast transcription factor, Put3, <a href=\"www.abmole.com\/products\/isoliquiritigenin.html\">Isoliquiritigenin<\/a> containing an N-terminal AU1 epitope tag. This segment of Put3 contains a leucine zipper motif that forms a left-handed coiled-coil homodimer, which will in essence force the fused protein of interest into a left-handed coiled-coil, whose interface residues can be predicted from the known structure of the Put3 dimer and the point of fusion. By fusing the Put3 segment at sequential residues of EBC5-16, each of the seven possible lefthanded coiled-coil helical registers of the dimeric EBC5-16 segment is generated. The residues that constitute the homodimer interface of native EBC5-16 can be inferred from the fusion protein that displays the highest biological activity. Each of the Put3\/EBC5-16 chimeras was cloned into the pRVY-puro vector and used to infect BaF3\/HA-hEPOR cells. <\/p>\n","protected":false},"excerpt":{"rendered":"<p>Notably, EBC5-16 induced a statistically-significant 50% increase in CAT activity compared to TC2-3, indicating that the transmembrane domain of EBC5-16 forms a stronger oligomer than TC2-3. This finding corroborates the biochemical results that a higher fraction of EBC5-16 is present as a dimer in murine cells. The results presented above Echinatin demonstrated that EBC5-16 displays&hellip; <a class=\"more-link\" href=\"http:\/\/bioactivecompoundlibrary.com\/index.php\/2018\/12\/27\/constructed-set-plasmids-encoding-fusion-proteins-fused\/\">Continue reading <span class=\"screen-reader-text\">We constructed a set of plasmids encoding fusion proteins was fused<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[],"_links":{"self":[{"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/200"}],"collection":[{"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/comments?post=200"}],"version-history":[{"count":1,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/200\/revisions"}],"predecessor-version":[{"id":201,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/200\/revisions\/201"}],"wp:attachment":[{"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/media?parent=200"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/categories?post=200"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/bioactivecompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/tags?post=200"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}