Backbone and side chain resonance assignments of domain III of the tick-borne Langat flavivirus envelope protein.
Backbone and side chain resonance assignments of domain III of the tick-borne Langat flavivirus envelope protein.
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蜱传兰加特黄病毒包膜蛋白结构域 III 的主链和侧链共振分配。
DOI：
10.1023/b:jnmr.0000034345.94232.16
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发表时间：
2004
影响因子：
2.7
通讯作者：
Fox,RobertO
中科院分区：
文献类型：
--
作者：
Mukherjee,Munia;Dutta,Kaushik;Pascal,StevenM;Fox,RobertO
材料与方法Langat黄病毒E蛋白结构域Ⅲ（LGT-E-D3）在大肠杆菌中表达。大肠杆菌菌株BL 21（DE 3）Codon Plus（Stratagene）作为C-末端融合物与N-末端6个组氨酸-MBP（麦芽糖结合蛋白）标签，具有凝血酶切割位点，使用编码LGT-E-D3的残基300-401的质粒H-MBP-T。H-MBP-T载体的细节在别处报道（Alexandrov et al.，2001年）。细胞在37 ℃下在含有15 N标记的NH 4Cl（1 g/L）和均匀标记的13 C葡萄糖（3 gl-1）的M9基本培养基中生长，产生13 C/15 N标记的LGT-E-D3样品。通过超声处理实现细胞裂解，并将澄清的上清液（每升生长物）与5 ml Talon钴亲和树脂（Clonetech，Palo Alto，CA）孵育2小时。用10 mM Tris，10 mM NaCl，pH 7.5充分洗涤Talon树脂，然后过夜
Materials and methodsDomain III of Langat flavivirus E-protein (LGT-E-D3) was expressed in E. coli strain BL21 (DE3) Codon Plus (Stratagene) as a C-terminal fusion to an N-terminal six histidine-MBP (maltose binding protein) tag, with a thromibin cleavage site using plasmid H-MBP-T that encodes for residues 300-401 of LGT-E-D3. Details for the H-MBP-T vector are reported elsewhere (Alexandrov et al., 2001). Cells were grown at 37◦ C in M9 minimal media with 15N labeled NH4Cl (1 g/L) and uniformly labeled 13C glucose (3 gl− 1) producing 13C/15N labeled LGT-E-D3 sample. Cell lysis was achieved by sonication and the clear supernatent (per liter of growth) was incubated with 5ml Talon cobalt affinity resin (Clonetech, Palo Alto, CA) for 2 h. Extensive washing of the Talon resin by 10 mM Tris, 10 mM NaCl, pH 7.5 was followed by overnight