Da Yu Protein Sciences – Protein Stability
A protein’s stability is often measured through methods of accelerated degradation and one of the most common methods of assessing protein stability is through thermal stress. The thermal stability of a protein is indicated by its melting temperature, Tm, which is considered the temperature at which half the protein molecules in solution are unfolded. A protein’s Tm can be used to find environmental conditions that enhance protein stability. Variables such as pH, ionic strength and excipients that increase a protein’s Tm create an environment where protein stability is enhanced.
Da Yu Protein Sciences uses a number of techniques to examine the effect environmental conditions have on protein stability including:
Differential Scanning Fluorimetry - When a protein unfolds it reveals hydroophobic amino acid sequences to which an environmentally-sensitive fluorophore can bind. In aqueous environments the fluorophore has a low quantum yield and minimal fluorescence. However, the fluorophore’s quantum yield increases significantly in a hydrophobic environment. Thus, as more protein unfolds a greater number of fluorophores bind to the exposed hydrophobic regions and the fluorescence increases.
The Tm in a DSF profile is the inflection point on the leading side of the fluorescence peak. Determining the Tm by dropping a horizontal line to the temperature axis is difficult and not necessarily accurate. A better and more accurate approach to determine the Tm is to take the second derivative of the DSF profile. In the second derivative the Tm is simply the temperature where the curve crosses the abscissa.
DSF is performed in 96-well assay plates and therefore it is a high-throughput technique for screening conditions that increase protein stability. In the example shown, DSF was used to screen pH for a protein at three different concentrations. This particular protein was most stable at pH 4.5 with Tm = 73.1 – 73.7 oC. Noteworthy is the low amounts of protein needed for DSF. The pH profile produced for the lowest protein concentration required only 50 µg of total protein.
Subsequent to identifying the most stabilizing pH, DSF can then determine the most stabilizing buffer and its concentration. Using DSF and the protein at 0.07 mg/mL, 6 different buffers at 3 different concentrations were analyzed each on 3 different days. The results shown in the table are the Tm ± the standard deviation. These results suggest this protein's stability is independent of buffer concentration and is not significantly altered by buffer composition except for Buffer 1, which may modestly increase protein stability. The overall low standard deviations found for DSF attest to the technique’s high degree of intermediate precision.
|Buffer||25 mM||50 mM||100 mM|
|Buffer 1||75.0 ± 0.7||74.7 ± 0.1||74.4 ± 0.2|
|Buffer 2||73.1 ± 0.1||73.3 ± 0.1||73.4 ± 0.1|
|Buffer 3||74.1 ± 0.1||74.0 ± 0.3||74.2 ± 0.2|
|Buffer 4||74.1 ± 0.2||74.2 ± 0.5||73.6 ± 0.3|
|Buffer 5||74.1 ± 0.2||74.2 ± 0.3||73.5 ± 0.2|
|Buffer 6||74.0 ± 0.1||73.9 ± 0.2||74.1 ± 0.1|
In a similar manner, DSF can be used to examine the influence other excipients or mixtures of excipients have on a protein’s stability. With the low protein requirements of DSF and the fast experimental turnaround (A single multi-well plate can be completed in less than 24 hours!), DSF is an ideal high-throughput technique to examine a protein’s stability in a preformulation strategy.
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