title: Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase.
abstract: LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichiacoli 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.
authors.affiliation: The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China.
authors.forename: Yanmin
authors.initials: Y
authors.lastname: Guo
authors.affiliation: The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China.
authors.forename: Weixiang
authors.initials: W
authors.lastname: Bian
authors.affiliation: The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China. Electronic address: htli@swu.edu.cn.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Li
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D054875:Ubiquitination
chemical_list: D011994:Recombinant Proteins; C489657:LRSAM1 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Activity; LRSAM1; Purification; RING-finger; Ubiquitin ligase