Letter to the Editor: 1H, 13C, and 15N Resonance Assignments of Human Microtubule-associated Protein Light Chain-3
Letter to the Editor: 1H, 13C, and 15N Resonance Assignments of Human Microtubule-associated Protein Light Chain-3
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致编辑的信：人类微管相关蛋白轻链 3 的 1H、13C 和 15N 共振分配
DOI：
10.1023/b:jnmr.0000032505.99071.ea
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发表时间：
2004
影响因子：
2.7
通讯作者：
K. Kawano
中科院分区：
文献类型：
--
作者：
T. Kouno;M. Mizuguchi;I. Tanida;T. Ueno;E. Kominami;K. Kawano
方法与结果利用pGEX载体在大肠杆菌中表达了MAP-LC 3-I与谷胱甘肽S-转移酶（GST）的融合蛋白。coli BL21。将细菌提取物应用于谷胱甘肽固定化柱以纯化GST融合蛋白。GSTMAP-LC 3-I融合蛋白经蛋白酶消化后得到GST和MAP-LC 3-I。消化后，通过阳离子交换色谱进一步纯化MAP-LC 3-I。通过在分别含有15 NH 4Cl和13 C-葡萄糖/15 NH 4Cl的基本培养基中培养细菌来制备15 N-标记和13 C-/15 N-标记的蛋白。所有NMR样品均含有0.8-1.0 mM MAP-LC 3-I、25 mM磷酸钠（pH 7.0）、100 mM NaCl、0.02 mM NaN 3和10%或100% D2 O。NMR实验在25 ° C下在Bruker DMX-500光谱仪上进行。以下光谱用于1H、15 N、13 C α、13 C β和13 C共振归属：1H-15 N HSQC、HNCA、CBCANH、CBCA（CO）NH、HNCO、HN（CA）CO、H（CCO）NH、C（CO）NH、HBHA（CBCA）NH、HBHA（CBCACO）NH、HCCH-COSY、HCCH-TOCSY（18.3 ms混合时间）、15 N编辑的TOCSY（75.9 ms）和15 N编辑的NOESY（85 ms）。
Methods and resultsThe recombinant MAP-LC3-I was expressed as glutathione S-transferase (GST)-fusion proteins using pGEX vector in E. coli strain BL21. Bacterial extract was applied to a glutathione-immobilized column in order to purify the GST-fusion protein. The GSTMAP-LC3-I fusion protein was digested by protease into GST and MAP-LC3-I. After the digestion, MAP-LC3-I was additionally purified by cationexchange chromatography. The 15N-labeled and 13C-/15N-labeled proteins were prepared by growing the bacteria in minimal medium containing 15NH4Cl and 13C-glucose/15NH4Cl, respectively. All of the NMR samples contained 0.8-1.0 mM MAP-LC3-I, 25 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.02 mM NaN3, and 10% or 100% D2O. NMR experiments were performed at 25◦ C on a Bruker DMX-500 spectrometer. The following spectra were used for the 1H, 15N, 13C α, 13C β, and 13C resonance assignments: 1H-15N HSQC, HNCA, CBCANH, CBCA (CO) NH, HNCO, HN (CA) CO, H (CCO) NH, C (CO) NH, HBHA (CBCA) NH, HBHA (CBCACO) NH, HCCH-COSY, HCCH-TOCSY (18.3 ms mixing time), 15N-edited TOCSY (75.9 ms), and 15N-edited NOESY (85 ms).
影响因子：
64.8
作者：
Ichimura, Y;Kirisako, T;Ohsumi, Y
通讯作者：
Ohsumi, Y
影响因子：
11.4
作者：
Kabeya, Y;Mizushima, N;Yoshimori, T
通讯作者：
Yoshimori, T