Controlled co-immobilisation of proteins via 4'-phosphopantetheine-mediated site-selective covalent linkage.

Autor: Zheng Y; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Luo W; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Yang J; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Wang H; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Hu Q; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Zeng Z; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Li X; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China., Wang S; Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 541642, PR China; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China. Electronic address: swwl@scau.edu.cn.
Jazyk: angličtina
Zdroj: New biotechnology [N Biotechnol] 2022 Dec 25; Vol. 72, pp. 114-121. Date of Electronic Publication: 2022 Oct 25.
DOI: 10.1016/j.nbt.2022.10.004
Abstrakt: In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (k cat /Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.
Competing Interests: Declaration of Interest Statement The authors declare that they have no any competing financial interests to influence the work reported in this paper.
(Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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