Optimisation of recombinant production of active human cardiac SERCA2a ATPase

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 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.

Figure 1. Small scale expression test for hSERCA2a-GFP-His₈ using different clones.

A. SDS-PAGE 4-12% Tris-Glycine in gel fluorescence analysis. M-fluorescent ladder, 1-6 different hSERCA2a-GFP-His₈ clones; B. Western Blotting using specific hSERCA2 antibodies, MM – prestained protein ladder.

Figure 2. Purification of hSERCA2a-Biotin fusion protein using Streptavidin-Sepharose resin.

A. Coomassie stained 12% Tris-Glycine SDS PAGE gel from purification, M- protein ladder, HS-high salt wash, LS-low salt wash, FT- flow-through after binding to Streptavidin resin, R-resin with bound hSERCA2a, FTC- flow-through concentrated, E – elution from Streptavidin resin, C- concentrated sample prior to gel filtration, GF- elution after gel filtration, rS1a- rabbit SERCA1a, 1µg. B. HPLC-SEC profile for hSERCA2a after Streptavidin affinity purification using 12L culture. DDM detergent was exchanged on gel filtration column with C₁₂E₈ detergent. C. Coomassie stained 4-12% Bis-Tris SDS PAGE gel. SEC fractions obtained for hSERCA2a purified with Streptavidin resin.

Figure 3. Purification of hSERCA2a-GFP-His₈ using Ni-NTA affinity chromatography.

Purification was done in the presence of DDM only throughout all steps, including SEC. Protein was obtained using rich media. A. Coomassie stained SDS-PAGE gel; B. In gel fluorescence 12% Tris-Glycine SDS-PAGE gel. MF- fluorescent protein ladder; MB- diluted membrane fraction; S- solubilised fraction; FT- flow-through after binding; W- wash fraction; E- elution; D- sample after cleavage with TEV protease and dialysis; R- sample after Ni-NTA rebinding after tag cleavage; C- sample concentrated using 50 kDa cut-off filter concentrator, before gel filtration; S1a- rabbit SERCA1a; GF- fraction containing human SERCA2a after gel filtration; M- prestained protein ladder. C. HPLC-SEC profile for hSERCA2a purified using Ni-NTA super-flow resin. D. Coomassie stained 4-12% Tris-Glycine SDS PAGE gel for SEC fractions obtained for purification of hSERCA2a using Ni-NTA super-flow resin.

Figure 4. Talon resin affinity purification of hSERCA2a-GFP-His₈.

Protein was obtained using minimal media. A. Coomassie stained SDS-PAGE gel; B. In gel fluorescence 12% Tris-Glycine SDS-PAGE gel, MF- fluorescent protein ladder; MB- membranes fraction; S- solubilised fraction; FT- flow-through after binding to Talon resin; W- wash fraction; E- elution; D- sample after cleavage with TEV protease and dialysis; R- sample after Ni-NTA reverse binding; C- sample concentrated, before gel filtration; rS1a- rabbit SERCA1a at; C- fraction containing human SERCA2a concentrated before gel filtration; W2- eluted material from Ni-NTA His trap after reverse Ni-NTA purification; M- protein ladder. C. HPLC-SEC profile for hSERCA2a purified using Talon resin. Protein was concentrated with 100 kDa cut-off filter concentrator and DDM detergent was exchanged on gel filtration column with C₁₂E₈ detergent. D. Coomassie stained 4-12% Tris-Glycine SDS PAGE gel for SEC fractions obtained for purification of hSERCA2a using Talon super-flow resin.

Figure 5. Typical activity assay profile for purified recombinant human Ca²⁺ ATPase isoform 2a (hSERCA2a).

The protein was purified using -GFP-His₈ tag and Talon resin affinity purification. Reaction buffer used was as in Materials and Methods; the reaction was triggered by adding 5 µg of purified protein. Addition of thapsigargin inhibits activity of purified protein. Calcium-dependent activity corresponds to the difference of slope before and after thapsigargin addition. Here, final calcium-dependent ATPase activity is about 3 µmol hydrolysed ATP/min/mg of hSERCA2a.

Figure 6. Normalised specific ATPase activity rate versus Ca²⁺ dependence for DDM solubilised hSERCA2a after HPLC-SEC purification.

100% specific ATPase activity corresponds to 3 µmol hydrolysed ATP/min/mg of hSERCA2a. The results are the means of seven measurements, using protein obtained from three independent membrane preparations; error bars represent ±S.D.