Optimisation of recombinant production of active human cardiac SERCA2a ATPase

Antaloae, A. V., Montigny, C., le Maire, M., Watson, K. A. ORCID: https://orcid.org/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: 10.1371/journal.pone.0071842

Abstract/Summary

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.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/33947
Identification Number/DOI	10.1371/journal.pone.0071842
Refereed	Yes
Divisions	Interdisciplinary centres and themes > Institute for Cardiovascular and Metabolic Research (ICMR) Life Sciences > School of Biological Sciences > Biomedical Sciences
Publisher	Public Library of Science
Download/View statistics	View download statistics for this item