Sortase-Mediated Quantifiable Enzyme Immobilization on Magnetic Nanoparticles

Fauser, Joel; Savitskiy, Sergey; Fottner, Maximilian; Trauschke, Vanessa und Gulen, Burak (2020): Sortase-Mediated Quantifiable Enzyme Immobilization on Magnetic Nanoparticles. In: Bioconjugate Chemistry, Vol. 31, No. 8: pp. 1883-1892

Abstract

Protein immobilization has gained high interest in recent years for its valuable applications in life sciences involving drug delivery and protein arrays. Herein, we combine sortase-mediated protein immobilization with the versatility of magnetic nanoparticles and a sensitive GFP-based quantification system. Using this method, we successfully immobilized and quantified the amount of coupled enzymes by fluorescence spectroscopy and assessed their activity by kinetic measurements. We show that sortase-mediated coupling of enzymes enables preparation of biological samples with a high demand of purity as demonstrated by single-molecule FRET. Here, we report that sortase-mediated protein ligation allows both N- and C-terminal site-specific protein immobilization. Additionally, we demonstrate that sortase-mediated protein immobilization is suitable for direct protein immobilization from complex lysates. Direct immobilization from lysate allows study of enzyme functionality without the need of time-consuming enzyme purification, while magnetic nanoparticles permit easy addition and removal of coupled enzymes to and from a reaction mixture.

• Item Type: Journal article
• Faculties: Chemistry and Pharmacy > Department of Chemistry
• Research Centers: Center for NanoScience (CENS)
• Subjects: 500 Science > 540 Chemistry; 500 Science > 500 Science
• ISSN: 1043-1802
• Language: English