Journal
BIOCONJUGATE CHEMISTRY
Volume 31, Issue 8, Pages 1883-1892Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.bioconjchem.0c00322
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Funding
- German Research Foundation [SFB1035, GRK1721]
- Alexander von Humboldt Postdoctoral Fellowship (AvH, 2017-2019)
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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.
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