التفاصيل البيبلوغرافية
العنوان: |
Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4 |
المؤلفون: |
Miyauchi, Yuu, Kimura, Akane, Sawai, Madoka, Fujimoto, Keiko, Hirota, Yuko, Tanaka, Yoshitaka, Takechi, Shinji, Mackenzie, Peter I., Ishii, Yuji |
المساهمون: |
Japan Society for the Promotion of Science, Nakatomi Foundation |
المصدر: |
Frontiers in Pharmacology ; volume 13 ; ISSN 1663-9812 |
بيانات النشر: |
Frontiers Media SA |
سنة النشر: |
2022 |
المجموعة: |
Frontiers (Publisher - via CrossRef) |
مصطلحات موضوعية: |
Pharmacology (medical), Pharmacology |
الوصف: |
Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics ... |
نوع الوثيقة: |
article in journal/newspaper |
اللغة: |
unknown |
DOI: |
10.3389/fphar.2022.832931 |
DOI: |
10.3389/fphar.2022.832931/full |
الإتاحة: |
https://doi.org/10.3389/fphar.2022.832931Test |
حقوق: |
https://creativecommons.org/licenses/by/4.0Test/ |
رقم الانضمام: |
edsbas.9914B312 |
قاعدة البيانات: |
BASE |