Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties

Abstract

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

Keywords: Calcium; Ischemia/reperfusion; Mapacalcine; Pichia pastoris; Protein production.

Fig. 1

Synthetic gene construction of mapacalcine from its aminoacid sequence. A: Mapacalcine monomer (89 aminoacids) and its deduced synthetic gene sequence (270 nucleotides). B: Map of the expressed 9HIS-mapacalcine cassette inserted in Pichia pastoris genome.

Fig. 2

Purification steps of 9HIS-mapacalcine. A: Elution profile (280 nm) obtained during the size exclusion chromatography step. Vertical lines delimitate the different pools. B: SDS PAGE (Coomassie staining) and WB analyses of pools P1–P4. C: Chromatogram (280 nm) obtained after injection of pool P1 on the C8 reverse phase column. Vertical lines indicate the recombinant mapacalcine.

Fig. 3

ESI-MS analysis of 9HIS-mapacalcine. Deconvoluted spectra and zooms on regions of interest. 9HIS-mapacalcine was analyzed after different treatments: A: Untreated. B: treated with DTT. C: treated with iodoacetamide.

Fig. 4

Effects of 9HIS-mapacalcine and native mapacalcine on cortical neurons during OGD. A: Results were expressed as the mean value of cells per mm² ± SEM. Incubations were performed in the absence (Control) or in the presence of 1µM of 9HIS-mapacalcine (9-His mapa), or 1 µm of native mapacalcine (Nat mapa), ***p<0.005 (Student t-test). B: Typical pictures of one counted field in control, 9HIS-mapacalcine and native mapacalcine conditions. C: Comparison of effects of 1 µM of 9HIS-mapacalcine or native mapacalcine on electrophysiological recordings in cortical neurons. The holding potential was −80 mV. Calcium currents were recorded from −70 to +50 mV in control condition (●), in the presence of 9HIS-mapacalcine () or native mapacalcine (). In each condition, current densities (pA/pF) are shown as a function of membrane potential (mV). Bars represent the SEM values, n=10 for each condition, p<0.05. D: Basal calcium levels. The fluorescence intensity of a calcium indicator Fluo-2/AM was measured in the absence (Control) or in the presence of 1 µM of 9HIS-mapacalcine (9-His mapa) or native mapacalcine (Nat mapa). Results are means ± SEM of 6 independent dishes, ***p< 0.005 (Student t-test).