Stephen Poon, Robert C. Rivers, Department of Chemistry, University of Cambridge, Lensfield road, Cambridge, CB21EW, UK., 12/2005
The importance of studying protein aggregation becomes apparent when we realise that not only is it the underlying cause of many debilitating and often incurable human diseases (1) but that it is also one of the most significant obstacles to the development of protein-based therapeutics; aggregation during drug formulation invariably leads to reduced efficacy, low yield, poor storage capacity, and increased production costs.
The ability to understand why and how proteins aggregate will undoubtedly help improve upon current strategies aimed at eliminating the causes and effects of this phenomenon. This may sound easy but the reality is that, due to the unique structural and physicochemical properties (e.g. thermodynamic stability, hydrophobicity, and net protein charge) of the vast numbers of proteins described to-date, the conditions required to induce protein aggregation are often highly protein-specific and are as varied as the proteins themselves.
Compounding this problem is the existence of the many biophysical factors (e.g. temperature, pH, molecular crowding, sequence mutations, proteolysis, and molecular chaperones) that also have profound influences on protein aggregation. With such an array of aggregation determinants at hand, the task of assessing what effect one or more of these factors have on the aggregation of a given protein would be almost impossible if not for the availability of firmly established screening techniques such as those that make use of multi-well microplate readers.
In this report, we explore some of the features of the BMG LABTECH FLUOstar OPTIMA microplate reader by highlighting examples of their potential uses in the study of protein aggregation, with particular emphasis on key areas of:
The results presented here were obtained from recent aggregation studies of a protein which, due to its inherent propensity to self-aggregate to form fibrils, has severely diminished therapeutic potentials, and as such has been the prime focus of our research. Due to the sensitive nature of this study, the identity of the protein concerned will not been disclosed; it shall be referred in this report simply as “Protein X”.
1. Assessment of protein aggregation by absorbance and fluorescence
Since the wild-type form of “Protein X” was inherently susceptible to aggregation, we designed a number of variants containing minimal residue substitution. By adopted a mass-screening approach using the FLUOstar OPTIMA, we proceeded to test their ability to aggregate in a hope of finding a suitable substitute for the therapeutically valued WT sequence. The time-dependent aggregation by “Protein X” and variants were followed by measuring the turbidity associated with the formation of precipitated protein at A340. The end-point formation of fibrous aggregates was assessed by measuring the fluorescence associated with the specific binding of the dye, Thioflavin T (Th-T), to the ß-sheet-rich fibrils.
i) Prepare “Protein X” by dissolving in a suitable buffer at a protein concentration of 1 mg/mL
ii) Add 100 mL aliquots to separate wells of a 96- or 384-well microplate, depending on how many tests are required.
iii) Insert the plate into the FLUOstar OPTIMA microplate reader and adjust the instrument settings as shown:
iv) Start program
* Shaking the samples will help promote protein aggregation and lessen the time for this
event to occur.
2. Assessment of the dose-dependent inhibition of temperature-induced protein aggregation by molecular chaperones
Molecular chaperones are proteins that possess the unique ability to stabilise and prevent the aggregation of partially unfolded proteins. Due to this reason, they are seen as a possible solution to the aggregation problem. Here, we illustrate how the FLUOstar OPTIMA can be used to assess the suppressive effects of molecular chaperones on the aggregation of “Protein X”. As “Protein X” is heat labile, we have chosen to take advantage of the heating capabilities of the FLUOstar OPTIMA (which, unlike many other plate readers on the market, can heat beyond 45°C) by subjecting the protein to the maximum 60°C allowed by the plate reader, in the presence or absence of the chaperone of our choice.
i) Prepare solutions of “Protein X” alone, chaperone alone, or “Protein X” with different concentrations of chaperone and then add them into separate wells of a microplate.
ii) Insert the plate into the pre-heated (60°C) FLUOstar OPTIMA microplate reader and adjust the instrument settings as shown:
iii) Run time course
Figure 1 shows the plot of protein aggregation by wild-type “Protein X” and its variants. Out of the 6 proteins tested 3 did not exhibit any detectable tendencies to self-aggregate. Electron microscopy imaging of these samples confirmed this.
Fig. 1: Time course of protein aggregation by wild-type “Protein X” and its variants.
Thioflavin T binding studies showed high fluorescence associated with the presence of fibrous aggregates in 3 of the 6 samples; these were the same 3 samples that showed extensive aggregation by absorbance. The results of the fluorescence measurements are shown in figure 2.
Fig. 2: End point fluorescence measurements associated with binding of fibril-specific Thioflavin-T.
By implementing the plate format for the mass screening of protein aggregation, we were able to incorporate this and other data obtained from activity tests to eventually show that variants 2 and 4 were good candidates for further therapy-related studies.
The time course of heat-induced “Protein X” aggregation in the presence or absence of molecular chaperones is shown in figure 3. Heating “Protein X” at 60°C resulted in extensive aggregation which reaches maximum by 20 min. In the presence of increasing amounts of the chaperone, the rate and extent of aggregation is significantly and proportionally reduced. These results clearly show that the molecular chaperone can inhibit the aggregation of “Protein X” and as such, warrants further investigation into their potentials for solving the greater aggregation problem.
Fig. 3: Dose-dependent effects of the molecular chaperone (MC) on the heat-induced aggregation of “Protein X” (Px). Results shown are representative of 3 independent experiments.
Showing that the molecular chaperone can inhibit the aggregation of “Protein X” highlights their possible use during drug formulation and storage. Certainly, it would be better to produce therapeutics using wild-type proteins as this minimises the problems of immunogenicity associated with the non-natural forms.
In this report, we have demonstrated that the FLUOstar OPTIMA is well-suited in medium to high throughput labs where screening of large sets of experimental conditions, required for understanding of the protein aggregation phenomenon, is required. We have demonstrated, for example, that the FLUOstar OPTIMA can be used efficiently to screen for sequences that, due to their reduced aggregation propensity, may be better therapeutic alternatives than the aggregation-prone protein from which they are derived. The availability of mass screening techniques, made possible by equipments such as the FLUOstar OPTIMA plate reader, will undoubtedly improve upon the efficiency of current methods aimed at addressing the protein aggregation issue.