PMC:5026484 / 47168-49316 JSONTXT

{"project":"2_test","denotations":[{"id":"27552051-20064470-26907328","span":{"begin":125,"end":129},"obj":"20064470"},{"id":"27552051-19116169-26907329","span":{"begin":173,"end":177},"obj":"19116169"},{"id":"27552051-7678431-26907330","span":{"begin":1588,"end":1592},"obj":"7678431"},{"id":"27552051-16506760-26907331","span":{"begin":1731,"end":1735},"obj":"16506760"},{"id":"27552051-11388804-26907332","span":{"begin":1753,"end":1757},"obj":"11388804"}],"text":"Protein expression and purification\nRab proteins were expressed and purified as described previously (Rab1 [Schoebel et al., 2009] and other Rab proteins [Bleimling et al., 2009]) and preparatively loaded with GppNHp (Guanosine-5’-[β-γ-Imido]-triphosphat) for interaction studies with their effector proteins. For this purpose, the proteins were incubated in the presence of 5 mM EDTA, 5–10% glycerol, three-fold molar excess of GppNHp over the Rab protein and 0.5 units alkaline phosphatase per mg Rab protein for 2 hr at 20°C or at 4°C overnight. Subsequently the proteins were purified via gel filtration in a buffer containing 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM DTE, 2 mM MgCl2 and 10 µM GppNHp. bMERB domains (amino acid boundaries and Uniprot accession IDs are shown in Figure 6—figure supplement 3) were cloned into a modified pET19 expression vector and proteins were expressed in E. coli BL21 DE3 RIL cells (growth at 37°C to OD600 nm = 0.8–1.0, stored at 4°C for 30 min, expression was induced by addition of 0.3–0.5 mM IPTG and cells were grown for 14–18 hr at 20°C). Subsequently the proteins were purified by Ni2+-affinity chromatography, cleavage of the His6-tag with TEV-protease and a second Ni2+-affinity purification. Final purification was achieved by gel filtration (Rabs: 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM DTE, 2 mM MgCl2, 10 µM GDP or GppNHp; Micals: 20 mM Hepes pH 7.5, 50 or 100 mM NaCl, 2 mM DTE). In order to express the selenomethionine labeled version of the coiled coil domain of Mical-3, the methionine biosynthesis inhibition method (Van Duyne et al., 1993) was used. The labeled protein was purified as described above. FTase and GGTase I were purified as described previously (Dursina et al., 2006; Kalinin et al., 2001).\nFor prenylation, EHBP11047-1231/EHBP1L11340-1523, substrate (farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP); Sigma) and prenyltransferase (FTase or GGTase I) were mixed in a 1:5:0.5 ratio in buffer containing 25 mM Hepes pH 7.2, 40 mM NaCl and 2 mM MgCl2 and incubated for 3 hr at room temperature. To check the extent of prenylation, samples were analyzed by ESI-MS."}

{"project":"MyTest","denotations":[{"id":"27552051-20064470-26907328","span":{"begin":125,"end":129},"obj":"20064470"},{"id":"27552051-19116169-26907329","span":{"begin":173,"end":177},"obj":"19116169"},{"id":"27552051-7678431-26907330","span":{"begin":1588,"end":1592},"obj":"7678431"},{"id":"27552051-16506760-26907331","span":{"begin":1731,"end":1735},"obj":"16506760"},{"id":"27552051-11388804-26907332","span":{"begin":1753,"end":1757},"obj":"11388804"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Protein expression and purification\nRab proteins were expressed and purified as described previously (Rab1 [Schoebel et al., 2009] and other Rab proteins [Bleimling et al., 2009]) and preparatively loaded with GppNHp (Guanosine-5’-[β-γ-Imido]-triphosphat) for interaction studies with their effector proteins. For this purpose, the proteins were incubated in the presence of 5 mM EDTA, 5–10% glycerol, three-fold molar excess of GppNHp over the Rab protein and 0.5 units alkaline phosphatase per mg Rab protein for 2 hr at 20°C or at 4°C overnight. Subsequently the proteins were purified via gel filtration in a buffer containing 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM DTE, 2 mM MgCl2 and 10 µM GppNHp. bMERB domains (amino acid boundaries and Uniprot accession IDs are shown in Figure 6—figure supplement 3) were cloned into a modified pET19 expression vector and proteins were expressed in E. coli BL21 DE3 RIL cells (growth at 37°C to OD600 nm = 0.8–1.0, stored at 4°C for 30 min, expression was induced by addition of 0.3–0.5 mM IPTG and cells were grown for 14–18 hr at 20°C). Subsequently the proteins were purified by Ni2+-affinity chromatography, cleavage of the His6-tag with TEV-protease and a second Ni2+-affinity purification. Final purification was achieved by gel filtration (Rabs: 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM DTE, 2 mM MgCl2, 10 µM GDP or GppNHp; Micals: 20 mM Hepes pH 7.5, 50 or 100 mM NaCl, 2 mM DTE). In order to express the selenomethionine labeled version of the coiled coil domain of Mical-3, the methionine biosynthesis inhibition method (Van Duyne et al., 1993) was used. The labeled protein was purified as described above. FTase and GGTase I were purified as described previously (Dursina et al., 2006; Kalinin et al., 2001).\nFor prenylation, EHBP11047-1231/EHBP1L11340-1523, substrate (farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP); Sigma) and prenyltransferase (FTase or GGTase I) were mixed in a 1:5:0.5 ratio in buffer containing 25 mM Hepes pH 7.2, 40 mM NaCl and 2 mM MgCl2 and incubated for 3 hr at room temperature. To check the extent of prenylation, samples were analyzed by ESI-MS."}