One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

Abstract Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of ex...

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Autores principales:	Xinrui Zhao, Haofei Hong, Xiaozhong Cheng, Shaozhong Liu, Tao Deng, Zhongwu Guo, Zhimeng Wu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4cba37056c2647fd9242f77dcb081564
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spelling	oai:doaj.org-article:4cba37056c2647fd9242f77dcb0815642021-12-02T11:40:33ZOne-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst10.1038/s41598-017-06856-y2045-2322https://doaj.org/article/4cba37056c2647fd9242f77dcb0815642017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06856-yhttps://doaj.org/toc/2045-2322Abstract Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.Xinrui ZhaoHaofei HongXiaozhong ChengShaozhong LiuTao DengZhongwu GuoZhimeng WuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xinrui Zhao Haofei Hong Xiaozhong Cheng Shaozhong Liu Tao Deng Zhongwu Guo Zhimeng Wu One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
description	Abstract Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.
format	article
author	Xinrui Zhao Haofei Hong Xiaozhong Cheng Shaozhong Liu Tao Deng Zhongwu Guo Zhimeng Wu
author_facet	Xinrui Zhao Haofei Hong Xiaozhong Cheng Shaozhong Liu Tao Deng Zhongwu Guo Zhimeng Wu
author_sort	Xinrui Zhao
title	One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
title_short	One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
title_full	One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
title_fullStr	One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
title_full_unstemmed	One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst
title_sort	one-step purification and immobilization of extracellularly expressed sortase a by magnetic particles to develop a robust and recyclable biocatalyst
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/4cba37056c2647fd9242f77dcb081564
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One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

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