engleski

Stability of proteins and viruses determined by differential scanning fluorimetry

Differential scanning fluorimetry (DSF) is readily employed high-throughput method for probing protein stability. Typically, DSF monitors thermal unfolding of proteins using a fluorescent reporter dye (e.g. Sypro Orange) and a real-time PCR instrument[1]. Recently, a new method, nanoDSF, was developed which enables measuring of intrinsic tryptophan and tyrosine fluorescence[2, 3], its main advantage being that no fluorescent dye is needed, thus avoiding any detrimental dye-protein interactions. Both in DSF and nanoDSF, the fluorescence intensity increases with the exposure of hydrophobic protein parts. When plotted as a function of temperature, it gives a value of Tm at which the concentrations of folded and unfolded proteins are equal. Effect of different compounds on protein stability is seen as a change in Tm. Stability of human immunoglobulin G (hIgG) and human serum albumin (HSA) is very interesting because of their therapeutical use. DSF and nanoDSF were used to examine the stability of hIgG, HSA and ovalbumin (OVA) in different formulations (e.g. pH, salts, amino acids, sugars) and the methods showed excellent correlation. Formulations containing ammonium sulphate and sucrose proved as best stabilizers, while pronounced destabilizing effects were observed with imidazole, arginine and at low pH (pH 4). Thermal stability of measles and mumps virus was also probed, but several problems occurred – acquiring freshly concentrated viruses, damage to the viruses during concentration and lack of clear interpretation of the obtained Tm. Protein stability was also monitored by measuring protein aggregation by SEC-HPLC and virus stability by CCID50. Obtained results correlate well with DSF results. [1] Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221. [2] http://www.nanotemper- technologies.com/technologies/nanodsf/ (accessed on 21-06-2016). [3] Temel DB, Landsman P, Brader ML (2016) Orthogonal methods for characterizing the unfolding of therapeutic monoclonal antibodies: differential scanning calorimetry, isothermal chemical denaturation, and intrinsic fluorescence with concomitant static light scattering. Methods Enzymol 567:359–389.

protein ; virus ; differential scanning fluorimetry ; SEC-HPLC

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