Functional experiments of mouse (Mus musculus), dolphin (Tursiops truncatus), and microbat (M. lucifugus) calsequestrin 1
This protocol is extracted from research article:
Molecular parallelism in fast-twitch muscle proteins in echolocating mammals
Sci Adv, Sep 26, 2018; DOI: 10.1126/sciadv.aat9660

Protein expression and purification. Rosetta(DE3)pLysS E. coli cells containing the Casq1 vectors were grown in LB media at 37°C until an OD600 (optical density at 600 nm) of 0.6 was reached. Next, the incubation temperature was reduced to 25°C, and the cells were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside and left to grow overnight. Following induction, the cells were harvested by centrifugation at 4000g for 30 min and then frozen at −20°C for later use.

For protein purification, cell pellets were suspended in 20 mM tris and NaN3 (0.5 g/liter) (pH 7.5) and sonicated at 8000 rpm using 450 Sonifier (Branson Ultrasonics) until they reached apparent homogeneity. The resulting lysate was clarified by centrifugation at 20,000g and loaded onto a GE Healthcare AKTA pure fast protein liquid chromatography (FPLC) with an attached Toyopearl DEAE-650M (Tosoh Biosciences) column equilibrated with DEAE loading buffer [20 mM tris, NaN3 (0.5 g/liter) (pH 7.5)]. Casq1 was eluted from the DEAE column between 12.5 and 25% buffer B by running a linear gradient of buffer A [20 mM tris, NaN3 (0.5 g/liter) (pH 7.5)] to buffer B [20 mM tris, NaN3 (0.5 g/liter), 2 M NaCl (pH 7.5)]. The fractions containing Casq1 were buffer-exchanged to buffer C [5 mM sodium phosphate, NaN3 (0.5 g/liter) (pH 6.8)] and further purified with FPLC using a ceramic hydroxyapatite (HA) column (Bio-Rad Laboratories). Casq1 protein was loaded onto an HA column preequilibrated with buffer C. Casq1 was eluted from the HA column between 50 and 100% buffer D by running a linear gradient of buffer C to buffer D [0.5 M sodium phosphate, NaN3 (0.5 g/liter) (pH 6.8)]. For the final purification step, the HA Casq1 elution was buffer-exchanged to buffer E [20 mM tris, NaN3 (0.5 g/liter) (pH 8.5)] and loaded onto a preequilibrated Mono Q column (GE Healthcare). Casq1 was eluted form the Mono Q column by running a linear gradient from buffer E to buffer F [20 mM tris, NaN3 (0.5 g/liter), 2 M NaCl (pH 8.5)]. Fractions containing Casq1 were then buffer-exchanged into Casq1 assay buffer (20 mM Mops, 0.3 M KCl (0.5 g/liter) (pH 7.2)]. Throughout the purification process, fractions containing Casq1 were identified with SDS–polyacrylamide gel electrophoresis. Protein concentrations were determined using the bicinchoninic acid assay (Thermo Fisher Scientific).

Molecular mass determination by multiangle light scattering. We injected 100 μl of each protein at a concentration of 1 mg/ml, which was preequilibrated with chromatography running buffer [20 mM tris-HCl (pH 7.5), 300 mM KCl, and either with or without 1 mM CaCl2], onto a Bio-Sep S-2000 column (Phenomenex). The chromatography was carried out at a flow rate of 0.5 ml/min using an Acuflow series IV pump (Analytical Scientific Instruments). The eluate was passed in tandem through an ultraviolet detector (Gilson), a refractometer (Optilab DSP, Wyatt Technology), and a multiangle laser light scattering detector (Dawn EOS, Wyatt Technology). All the chromatography experiments were performed at 25°C. Scattering data were analyzed using ASTRA software (Wyatt Technology) supplied with the instrument. Relative weight-averaged molecular masses were determined from the scattering data collected for a given condition using the Zimm fitting method, in which K*c/R(Q) is plotted against sin2(Q/2), where Q is the scattering angle, R(Q) is the excess intensity (I) of scattered light at the angle Q, c is the concentration of the sample, and K* is a constant equal to 4π2n2(dn/dc)204NA (where n is the solvent refractive index, dn/dc is the refractive index increment of scattering sample, λ0 is the wavelength of scattered light, and NA is Avogadro’s number). Extrapolation of a Zimm plot to zero angle was used to estimate the weight-averaged molecular mass.

Turbidity assays. The turbidity of Casq1 solutions (that is, absorbance at 350 nm) as a function of Ca2+ concentration was monitored using a Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific). Assays were performed using 1.3 ml of 15 μM mouse, bat, and dolphin Casq1 in the assay buffer. Concentrated Ca2+ solutions (0.10, 0.25, 0.50, and 1.0 M) were added in 1.0 to 2.0 μl aliquots to the 1.3 ml of Casq1 solutions in a quartz cuvette to achieve the proper calcium concentration. Upon addition of each concentrated Ca2+ aliquot, the samples were mixed by stirring with a small stir bar and allowed to equilibrate (dA350/dt = 0) before addition of the next aliquot. Dilutions from adding Ca2+ aliquots were included in the data analysis.

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