Abstract
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.
Highlights
Cellular calcium homeostasis is regulated by a large number of proteins, with the Sarco/Endoplasmic Reticulum Ca2+ATPase (SERCA) pumps as key players in this process
An estimated expression level of up to 5 mg of solubilised hSERCA2a-GFP-His8 per litre of culture was obtained, which is comparable to that reported for heterologous expression of rSERCA1a isoform using various constructs in different cell lines, including mammalian and S. cerevisiae [16,17,18,41]
Given the comparable yields for the two constructs, the hSERCA2a-GFP-His8 construct offers two main advantages: a) the purification is more cost effective as the resin for affinity chromatography can be re-used and b) the GFP-fusion protein can be monitored throughout the expression and purification steps by in-gel fluorescence, with the GFP detection being as sensitive as or better than Western blot detection using a hSERCA2 specific antibody
Summary
Cellular calcium homeostasis is regulated by a large number of proteins, with the Sarco/Endoplasmic Reticulum Ca2+ATPase (SERCA) pumps as key players in this process. SERCA pumps are integral membrane proteins responsible for Ca2+ uptake from the cytosol into the sarcoplasmic/ endoplasmic reticulum (SR/ER), using the energy derived from ATP hydrolysis to fuel the ion translocation. This process is vital for preserving low intracellular calcium levels in a resting cell, a prerequisite for the use of calcium as a secondary messenger to control essential cellular processes such as muscle relaxation/contraction, cell signalling and apoptosis. The cytoplasmic domain contains three distinct subdomains; the nucleotide binding domain (N), the phosphorylation domain (P) and the actuator domain (A) These subdomains are jointly responsible for ATP binding and hydrolysis, and serve as the motor driving ion translocation through long-range intra-molecular interactions with the integral membrane domain. The integral membrane domain consists of ten, or for SERCA2b eleven, transmembrane (TM) helices and is responsible for calcium binding and translocation [1,2]
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