Biolabel Report Summary
A growth medium was prepared from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in 15N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.
Growth medium developed from Spirulina patensis autolysate showed to have good properties respect to traditional labeled media for mammalian cells. Among them, it is less expensive and allows a final protein yeld largely sufficient to observe the amide signals in the in-cell NMR spectra.
The expression levels of the proteins obtained in algal-derived medium relative to those obtained in the commercial medium were measured from the analysis of 1D 1H in-cell NMR spectra and from Coomassie-stained SDS-PAGE. SOD1 expression in algal-derived medium was about 60 % of the highest expression levels obtained in the commercial medium [150 ± 10 μM in 180 μL of extract from ~3 × 107 cells, (Banci et al. 2013a)], while Mia40 expression was about 40 % of the levels obtained in commercial medium [60 ± 10 μM in 180 μL of extract from ~3 × 107 cells. The isotopic incorporation in the algal-derived and in the commercial medium, qualitatively assessed from the 2D/1D signal intensity ratios, was comparable. In both cases, the protein yield of the algal-derived medium was lower than that of the commercial medium, but was still largely sufficient to observe the amide signals in the in-cell NMR spectra.
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