Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes

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Kiiski , I M A , Pihlaja , T L M , Urvas , L M , Wiedmer , S K , Witos , J M , Jokinen , V P & Sikanen , T M 2019 , ' Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes ' , Advanced Biosystems , vol. 3 , no. 1 , 1800245 . https://doi.org/10.1002/adbi.201800245

Title: Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes
Author: Kiiski, Iiro Matti Aleksi; Pihlaja, Tea Liisa Milena; Urvas, Lauri Mikael; Wiedmer, Susanne Kristina; Witos, Joanna Magdalena; Jokinen, Ville P.; Sikanen, Tiina Marjukka
Contributor: University of Helsinki, Preclinical Drug Formulation and Analysis group
University of Helsinki, Division of Pharmaceutical Chemistry and Technology
University of Helsinki, Division of Pharmaceutical Chemistry and Technology
University of Helsinki, Laboratory for Instruction in Swedish (-2016)
University of Helsinki, Aalto University
University of Helsinki, Drug Research Program
Date: 2019-01
Language: eng
Number of pages: 6
Belongs to series: Advanced Biosystems
ISSN: 2366-7478
URI: http://hdl.handle.net/10138/298854
Abstract: This work describes a new nanotechnology-based immobilization strategy for cytochrome P450s (CYPs), the major class of drug metabolizing enzymes. Immobilization of CYPs on solid supports provides a significant leap forward compared with soluble enzyme assays by enabling the implementation of through-flow microreactors for, for example, determination of time-dependent inhibition. Immobilization of the complex CYP membrane-protein system is however particularly challenging as the preservation of the authentic enzyme kinetic parameters requires the full complexity of the lipid environment. The developed strategy is based on the spontaneous fusion of biotinylated fusogenic liposomes with lipid bilayers to facilitate the gentle biotinylation of human liver microsomes that incorporate all main natural CYP isoforms. The same process is also feasible for the biotinylation of recombinant CYPs expressed in insect cells, same as any membrane-bound enzymes in principle. As a result, CYPs could be immobilized on streptavidin-functionalized surfaces, both those of commercial magnetic beads and customized microfluidic arrays, so that the enzyme kinetic parameters remain unchanged, unlike in previously reported immobilization approaches that often suffer from restricted substrate diffusion to the enzyme's active site and steric hindrances. The specificity and robustness of the functionalization method of customized microfluidic CYP assays are also carefully examined.
Subject: 317 Pharmacy
1182 Biochemistry, cell and molecular biology
221 Nano-technology
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