A KD value of 100??9 nM was calculated by fitting the data to a hyperbolic equation using SigmaPlot? 9. a variety of chemical series and focused chemical libraries. was dramatically increased using an optimized buffer condition that was recognized through screening a series of purification buffers (Arai et al., 1981). In some cases, it is crucial to express the protein in the presence of a ligand (co-factor, ion, etc.) to promote proper conformational changes or folding and to thereby increase solubility. For example, the expression of the recombinant human 11b-hydroxysteroid dehydrogenase type 1 in was increased by more than one order of magnitude in the presence of an inhibitor (Elleby et al., 2004). In addition to screening for ligands, we adapted a combination of dynamic light scattering (DLS) and thermodenaturation-based screening at the SGC in order to find optimum buffer conditions that increase the solubility and stability of problematic proteins. DLS is very sensitive in detecting small particles in solution and can Ly6a differentiate between non-aggregated proteins and proteins that form soluble aggregates or oligomers in answer. On the other hand, differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) allow evaluation of the effect of buffer conditions on protein stability. Unlike DLS, Amsilarotene (TAC-101) DSLS is only sensitive to insoluble aggregates produced during protein precipitation and denaturation. The recombinant proteins may require several purification actions, which may take many hours. Furthermore, they Amsilarotene (TAC-101) may be stored for lengthy periods before being used and thus undergo a freezeCthaw process, and they are almost always subject to lengthy protocols such as crystallization screening and kinetic studies. It is therefore very helpful to select a buffer condition that makes the protein not only more soluble but also more stable. Conditions that make a protein more stable sometimes render improved solubility as well. We previously reported that by using thermodenaturation methods to screen for stability, a buffer condition was recognized in 50% of the cases that stabilized the protein by at least 4?C compared with the original buffer (HEPES buffer, pH 7.5, 150?mM NaCl) (Vedadi et al., 2006). Most proteins were also stabilized in this assay by the addition of higher concentrations of NaCl. Only 27% of proteins were more stable at lower NaCl concentrations. In some instances, the Amsilarotene (TAC-101) identification of a stabilizing solution increased the ability to purify, concentrate or crystallize the protein (Vedadi et al., 2006). Using buffers and additives recognized by DSF, Ericsson et al., reported a twofold increase in the number of crystallization prospects compared with testing in the absence of the additives (Ericsson et al., 2006). Buffer optimization to improve purification yield and protein quality has also been reported (Mezzasalma et al., 2007). In search for optimum buffer conditions, more than 100 malarial proteins from different species were screened for stability by DSLS at numerous pHs (6, 7, 8 and 9). This provided an opportunity to seek possible correlations between protein stability related to the pH of a buffer and physical properties of these proteins (Fig. 6). About 25% of the proteins showed no thermodenaturation transition up to 80?C in any buffer. Interestingly, a Amsilarotene (TAC-101) significant number of the remaining proteins ( 50%) appeared to be most stable in an alkaline condition, versus only about 20% that were more stable in an acidic buffer (pH 6). The rest of the proteins were either most stable at neutral pH or showed no preference for any specific buffer. Although no correlation was observed between the isoelectric points of the proteins and their stability, there may be a Amsilarotene (TAC-101) poor correlation between their stability and their molecular excess weight and percentage of charged residues. The proteins that showed no transition in all or some buffer conditions were often small proteins (Fig..
A KD value of 100??9 nM was calculated by fitting the data to a hyperbolic equation using SigmaPlot? 9
- by globalhealth