Academic Journal
Recovery of the histamine H3 receptor activity lost in yeast cells through error-prone PCR and in vivo selection
| العنوان: | Recovery of the histamine H3 receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
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| المؤلفون: | Watanabe, Ayami, Nakajima, Ami, Shiroishi, Mitsunori |
| المساهمون: | Japan Society for the Promotion of Science, Ministry of Education, Culture, Sports, Science and Technology, Japan Agency for Medical Research and Development, Kato Memorial Bioscience Foundation |
| المصدر: | Scientific Reports ; volume 13, issue 1 ; ISSN 2045-2322 |
| بيانات النشر: | Springer Science and Business Media LLC |
| سنة النشر: | 2023 |
| الوصف: | G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae , is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and then an in vivo screening to obtain mutants that recovered the activity of the human histamine H 3 receptor (H 3 R), which loses its signaling function when expressed in yeast. Four mutations with recovered activity were identified after screening. Three of the mutations were identified near the DRY and NPxxY motifs of H 3 R, which are important for activation and are commonly found in class A GPCRs. The mutants responded exclusively to the yeast YB1 strain harboring G i -chimera proteins, showing retention of G protein specificity. Analysis of one of the mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The strategy used in this study may enable the recovery of the activity of other GPCRs that do not function in S. cerevisiae and may be useful in creating GPCRs mutants stabilized in their active conformations. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| DOI: | 10.1038/s41598-023-43389-z |
| الاتاحة: | http://dx.doi.org/10.1038/s41598-023-43389-z https://www.nature.com/articles/s41598-023-43389-z.pdf https://www.nature.com/articles/s41598-023-43389-z |
| Rights: | https://creativecommons.org/licenses/by/4.0 ; https://creativecommons.org/licenses/by/4.0 |
| رقم الانضمام: | edsbas.2F844F79 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1038/s41598-023-43389-z |
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