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

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

Abstrakti

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.
Alkuperäiskielienglanti
Artikkeli1800245
LehtiAdvanced Biosystems
Vuosikerta3
Numero1
Sivumäärä6
ISSN2366-7478
DOI - pysyväislinkit
TilaJulkaistu - tammikuuta 2019
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä, vertaisarvioitu

Tieteenalat

  • 317 Farmasia
  • 1182 Biokemia, solu- ja molekyylibiologia
  • 221 Nanoteknologia

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