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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 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.

SUBMITTER: Zhao X

PROVIDER: S-EPMC5529518 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Zhao Xinrui X Hong Haofei H Cheng Xiaozhong X Liu Shaozhong S Deng Tao T Guo Zhongwu Z Wu Zhimeng Z

Scientific reports 20170726 1

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 manipula ...[more]

PMID: 28747746

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Project description:Immobilizing particles on beads, fibers, or filaments, when only one side is exposed to the reaction medium and therefore can be selectively functionalized, is a scalable and easy to control strategy for the fabrication of amphiphilic Janus particles. Here we describe a new, robust method for the fabrication of amphiphilic Janus particles based on immobilization of polymethylsilsesquioxane (PMSQ) particles on polycarbonate (PC), a high impact-resistance polymer with superior mechanical properties. The immobilization of the particles on the PC microspores is performed via inverse solvent displacement method. PMSQ particles are added to a PC solution in tetrahydrofuran (THF), a good solvent for PC. The solution is then precipitated by the introduction of aqueous surfactant solution (antisolvent for PC) under an ultrasonic field. It is important to note that THF and water are miscible and do not form emulsion. During precipitation, PMSQ particles are assembled onto the surface of the PC spherical precipitates/microspheres. The exposed hemispheres of the PMSQ particles are then selectively silanized by (3-Aminopropyl)triethoxysilane (APTES) to introduce amine groups on their surface. To increase the polarity of the functionalized hemispheres, the amine groups are further modified to introduce carboxyl groups. SEM characterization confirms the fine embedment of PMSQ particles onto the PC microspheres. Covalent attachment of silica nanoparticles (NPs) to the functionalized hemispheres of the resulting particles along with fluorescent confocal microscopy conclusively prove the successful fabrication of amphiphilic Janus particles. The immobilization of particles onto highly rigid polymeric microspheres such as PC may pave the way for the development of a robust fabrication procedure with high resistance to temperature fluctuations and harsh mixing conditions that can arise during preparation. This method can be implemented toward a large variety of other synthetic commercial polymers such as polyamide, polyether sulfones, Polyether, ether ketone, or similar.

Dataset Information

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

Publications

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

Similar Datasets

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