GoxB is a marine bacterial flavoprotein that catalyzes the covalent modification from precursor to mature form of a glycine oxidase (GoxA). Grown and expressed in E. coli Rosetta cells, it elutes well off of a nickel column in the 30-150 mM imidazole range, showing a lot of yellow color as indication of flavin content. As I attempt to concentrate and exchange buffers, it begins to precipitate, clogging up the concentrator membranes. The elution is white, indicating the flavin is dissociating from the protein as a result of or causing the precipitation.
Anyone have experience stabilizing fragile flavoproteins?
Avoid concentration, run only diafiltration. What is the desired final buffer?
Usually we use 50 mM KPi pH 7.5 buffers for our proteins, with 5% glycerol when stored in -80 C. I tried using 50 mM Hepes 300 mM NaCl pH 8 10% glycerol instead but the protein was still precipitating.
You can avoid the use of concentration with a desalting column in order to change the buffer. But if you still need to concentrate try with a different membrane concentrator polyethersulfone, regenerated cellulose, PVDF, etc. In my hands with some precipitating protein at high concentrations I've observed better results with regenerated cellulose.
Article Asn336 is involved in the substrate affinity of glycine oxid...
Ashok, did you have trouble with the flavoprotein in that paper precipitating in solution?
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