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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 Ca 2+  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- His 8  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-His 8  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-His 8  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.

Optimisation of Recombinant Production of Active Human Cardiac SERCA2a ATPase