Algal autolysate medium to label proteins for NMR in mammalian cells.
In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium 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 (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.
Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.
Carmelo
C
Fuccio
Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.|Department of Biomedical, Clinical and Experimental Sciences, University of Florence, Viale Morgagni 50, 50134, Florence, Italy.
Enrico
E
Luchinat
Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.|Giotto Biotech S.R.L., Via Madonna del Piano 6, 50019, Sesto Fiorentino, Florence, Italy.
Letizia
L
Barbieri
Giotto Biotech S.R.L., Via Madonna del Piano 6, 50019, Sesto Fiorentino, Florence, Italy.
Sara
S
Neri
Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy. fragai@cerm.unifi.it.|Department of Chemistry "Ugo Schiff", University of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Florence, Italy. fragai@cerm.unifi.it.
Marco
M
Fragai
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D000458:Cyanobacteria / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling
D011506:Proteins
Cell culture; In-cell NMR; Isotopic labeling; Mammalian cells; Protein expression; Spirulina platensis