Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase

被引:9
作者
Guo, Yanmin [1 ]
Bian, Weixiang [1 ]
Zhang, Yuan [1 ]
Li, Hongtao [1 ]
机构
[1] Southwest Univ, State Key Lab Breeding Base Bioresources & Ecoenv, Key Lab Freshwater Fish Reprod & Dev, Minist Educ,Lab Mol Dev Biol,Sch Life Sci, Chongqing 400715, Peoples R China
关键词
LRSAM1; Ubiquitin ligase; RING-finger; Purification; Activity; POLYUBIQUITIN CHAINS; AUTOPHAGY; BACTERIA;
D O I
10.1016/j.pep.2016.05.002
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:158 / 161
页数:4
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