Optimisation of recombinant production of active human cardiac SERCA2a ATPase.
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- 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.
School of Biological Sciences, Whiteknights Campus, University of Reading, Reading, Berkshire, United Kingdom.
Ana V
AV
Antaloae
Cédric
C
Montigny
Marc
M
le Maire
Kimberly A
KA
Watson
Thomas L-M
TL
Sørensen
D002846:Chromatography, Affinity / Q000379:methods; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D009206:Myocardium / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases