Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase
Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase
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LRSAM1（一种 LRR 和 RING 结构域 E3 泛素连接酶）在大肠杆菌中的表达、纯化和表征
DOI：
10.1016/j.pep.2016.05.002
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发表时间：
2017-01-01
影响因子：
1.6
通讯作者：
Li, Hongtao
中科院分区：
文献类型：
--
作者：
Guo, Yanmin;Bian, Weixiang;Li, Hongtao
LRSAM 1是一种典型的环指E3泛素连接酶，在许多生物过程中发挥重要作用。LRSAM 1在大肠杆菌中的表达和纯化尚未见报道。本研究旨在探讨在大肠杆菌中克隆、表达和纯化重组LRSAM 1的策略。coli细胞。LRSAM 1以包涵体的形式高产量表达，并通过随后的变性和复性步骤成功地以可溶性形式回收。复性后的LRSAM 1经两步硫酸铵沉淀直接纯化，纯度可达95%，产率约为6 mg/L。纯化的重组LRSAM 1表现出pH依赖性的E3连接酶活性。其连接酶活性依赖于RING指结构域，其泛素化有利于K6-，K27-，K29-和K48-键与UbcH 5型E2酶的合作。(C)2016 Elsevier Inc. All rights reserved.
LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichia coli had not yet been reported. Here, strategies to clone, express and purify recombinant LRSAM1 in E. coli cells were developed. LRSAM1 was expressed with high yield as inclusion bodies and successfully recovered in soluble form by subsequent denaturation and renaturation steps. Refolded LRSAM1 was directly purified through two steps of ammonium sulfate precipitation, resulting in a purity of up to 95% and a yield of about 6 mg/L bacterial culture. Purified recombinant LRSAM1 exhibited a pH-dependent E3 ligase activity. Its ligase activity was RING finger domain-dependent, and its ubiquitination favors K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes. (C) 2016 Elsevier Inc. All rights reserved.