Academic Journal
Optimisation of recombinant production of active human cardiac SERCA2a ATPase
| العنوان: | Optimisation of recombinant production of active human cardiac SERCA2a ATPase |
|---|---|
| المؤلفون: | Antaloae, Ana V., Montigny, Cedric, le Maire, Marc, Watson, Kimberly A., Sorensen, Thomas L.-M. |
| بيانات النشر: | Public Library of Science |
| سنة النشر: | 2013 |
| المجموعة: | CentAUR: Central Archive at the University of Reading |
| الوصف: | Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins. |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | text |
| اللغة: | English |
| Relation: | https://centaur.reading.ac.uk/33947/2/journal.pone.0071842.pdf; Antaloae, A. V., Montigny, C., le Maire, M., Watson, K. A. orcid:0000-0002-9987-8539 and Sorensen, T. L.-M. (2013) Optimisation of recombinant production of active human cardiac SERCA2a ATPase. PLoS ONE, 8 (8). e71842. ISSN 1932-6203 doi: https://doi.org/10.1371/journal.pone.0071842 |
| الاتاحة: | https://centaur.reading.ac.uk/33947/ https://centaur.reading.ac.uk/33947/2/journal.pone.0071842.pdf |
| Rights: | cc_by |
| رقم الانضمام: | edsbas.398A6A1C |
| قاعدة البيانات: | BASE |
| الوصف غير متاح. |