Expression and purification of functional recombinant CUL2•RBX1 from E. coli

Abstract Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable sub...

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Autores principales:	Stephanie Diaz, Lihong Li, Kankan Wang, Xing Liu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b1c3ddfa0b8b499594be9428b726f52b
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spelling	oai:doaj.org-article:b1c3ddfa0b8b499594be9428b726f52b2021-12-02T15:00:55ZExpression and purification of functional recombinant CUL2•RBX1 from E. coli10.1038/s41598-021-90770-x2045-2322https://doaj.org/article/b1c3ddfa0b8b499594be9428b726f52b2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-90770-xhttps://doaj.org/toc/2045-2322Abstract Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.Stephanie DiazLihong LiKankan WangXing LiuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stephanie Diaz Lihong Li Kankan Wang Xing Liu Expression and purification of functional recombinant CUL2•RBX1 from E. coli
description	Abstract Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.
format	article
author	Stephanie Diaz Lihong Li Kankan Wang Xing Liu
author_facet	Stephanie Diaz Lihong Li Kankan Wang Xing Liu
author_sort	Stephanie Diaz
title	Expression and purification of functional recombinant CUL2•RBX1 from E. coli
title_short	Expression and purification of functional recombinant CUL2•RBX1 from E. coli
title_full	Expression and purification of functional recombinant CUL2•RBX1 from E. coli
title_fullStr	Expression and purification of functional recombinant CUL2•RBX1 from E. coli
title_full_unstemmed	Expression and purification of functional recombinant CUL2•RBX1 from E. coli
title_sort	expression and purification of functional recombinant cul2•rbx1 from e. coli
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b1c3ddfa0b8b499594be9428b726f52b
work_keys_str_mv	AT stephaniediaz expressionandpurificationoffunctionalrecombinantcul2rbx1fromecoli AT lihongli expressionandpurificationoffunctionalrecombinantcul2rbx1fromecoli AT kankanwang expressionandpurificationoffunctionalrecombinantcul2rbx1fromecoli AT xingliu expressionandpurificationoffunctionalrecombinantcul2rbx1fromecoli
_version_	1718389159303839744

Expression and purification of functional recombinant CUL2•RBX1 from E. coli

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