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

Iiro Matti Aleksi Kiiski, Tea Liisa Milena Pihlaja, Lauri Mikael Urvas, Susanne Kristina Wiedmer, Joanna Magdalena Witos, Ville P. Jokinen, Tiina Marjukka Sikanen

Research output: Contribution to journalArticleScientificpeer-review

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.
Original languageEnglish
Article number1800245
JournalAdvanced Biosystems
Volume3
Issue number1
Number of pages6
ISSN2366-7478
DOIs
Publication statusPublished - Jan 2019
MoE publication typeA1 Journal article-refereed

Fields of Science

  • 317 Pharmacy
  • 1182 Biochemistry, cell and molecular biology
  • 221 Nano-technology

Cite this

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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.",
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author = "Kiiski, {Iiro Matti Aleksi} and Pihlaja, {Tea Liisa Milena} and Urvas, {Lauri Mikael} and Wiedmer, {Susanne Kristina} and Witos, {Joanna Magdalena} and Jokinen, {Ville P.} and Sikanen, {Tiina Marjukka}",
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Overcoming the Pitfalls of Cytochrome P450 Immobilization Through the Use of Fusogenic Liposomes. / Kiiski, Iiro Matti Aleksi; Pihlaja, Tea Liisa Milena; Urvas, Lauri Mikael; Wiedmer, Susanne Kristina; Witos, Joanna Magdalena; Jokinen, Ville P.; Sikanen, Tiina Marjukka.

In: Advanced Biosystems, Vol. 3, No. 1, 1800245, 01.2019.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

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

AU - Kiiski, Iiro Matti Aleksi

AU - Pihlaja, Tea Liisa Milena

AU - Urvas, Lauri Mikael

AU - Wiedmer, Susanne Kristina

AU - Witos, Joanna Magdalena

AU - Jokinen, Ville P.

AU - Sikanen, Tiina Marjukka

PY - 2019/1

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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.

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