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

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Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes. / Kiiski, Iiro; Pihjala, Tea; Urvas, Lauri; Witos, Joanna; Wiedmer, Susanne K.; Jokinen, Ville; Sikanen, Tiina M.

julkaisussa: Advanced Biosystems, Vuosikerta 3, Nro 1, 1800245, 01.01.2019.

Tutkimustuotos: Lehtiartikkelivertaisarvioitu

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Kiiski, Iiro ; Pihjala, Tea ; Urvas, Lauri ; Witos, Joanna ; Wiedmer, Susanne K. ; Jokinen, Ville ; Sikanen, Tiina M. / Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes. Julkaisussa: Advanced Biosystems. 2019 ; Vuosikerta 3, Nro 1.

Bibtex - Lataa

@article{9614b50d183a43b88679006513d73e7d,
title = "Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes",
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.",
author = "Iiro Kiiski and Tea Pihjala and Lauri Urvas and Joanna Witos and Wiedmer, {Susanne K.} and Ville Jokinen and Sikanen, {Tiina M.}",
year = "2019",
month = "1",
day = "1",
doi = "10.1002/adbi.201800245",
language = "English",
volume = "3",
journal = "Advanced Biosystems",
issn = "2366-7478",
number = "1",

}

RIS - Lataa

TY - JOUR

T1 - Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes

AU - Kiiski, Iiro

AU - Pihjala, Tea

AU - Urvas, Lauri

AU - Witos, Joanna

AU - Wiedmer, Susanne K.

AU - Jokinen, Ville

AU - Sikanen, Tiina M.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

U2 - 10.1002/adbi.201800245

DO - 10.1002/adbi.201800245

M3 - Article

VL - 3

JO - Advanced Biosystems

JF - Advanced Biosystems

SN - 2366-7478

IS - 1

M1 - 1800245

ER -

ID: 30195091