Formation of microspheres

Controlled interactions and assembly of proteins with one another promise to be a powerful approach for generating novel supramolecular architectures. In this study, we report on the ability of oppositely charged globular proteins to self-assemble into well-defined micrometer-sized spherical particles under specific physicochemical conditions. We show that microspheres were spontaneously formed in all binary protein mixtures tested once the physicochemical conditions were optimized. The optimal pH value, initial protein concentrations needed to form microspheres, and protein stoichiometry in these microspheres varied and depended on the structural features of the mixed proteins. We show that charge compensation is required but not sufficient to guide optimal protein assembly and organization into microspheres. Size difference between protein couples (acidic and basic) is a key element that defines optimal pH value for microsphere formation and the protein molar ratio in the formed microspheres. Our findings give new elements that can help to predict the assembly behavior of various proteins in mixed systems.



Liquid-liquid phase transitions in binary and ternary mixtures of proteins.

Mixing two proteins of opposite net charge provokes a liquid-liquid phase transition. This is evidenced by an increase in turbidity and the formation of homogeneous and spherical “aggregates” (Nigen et al, 2007). This was first demonstrated for one couple a protein, alpha-lactalbumin and beta-lactoglobulin, and was proposed to be a specific phenomenon due to the metastable state of the molten globule of alpha-lactoablumin. We found that the transition could be observed for any kind of binary mixtures only at a pH where basic and acidic proteins bear an exact opposite net charge and if the size of the two partners is similar.

Table spheres_1

LYS: lysozyme, ALA: alpha-lactalbumin, OVA: ovalbumin, AVI: avidin, BSA: bovine serum albumin.



Avi Lys Ova


We were able to incorporate more than two proteins in the same sphere. Lysozyme and avidin are two basic proteins that cannot form spherical aggregates. We labelled them with either FITC or RITC. We prepared mixtures of Lysozyme-FITC, Avidin-RITC, and the acidic protein ovalbumin. We found that both basic proteins were detectable in the same microspheres.

The molar ratio of the three proteins is still to be determined.


The ability of incorporating more than two proteins at a time is encouraging if one considers that these structures may be used as vehicles to carry functional compounds.






Attemps to observe the fomation of microspheres in real time.

Ovalbumin and lyzozyme were mixed on ice to produce “amorphous aggregates”.

Agreg to spheres

The mixtures were placed on a microscope slide and covered with a coverslip. During the observation, the sample warmed up to room temperature, which allowed phase separation and sphere formation. With this setup, aggregates are not always spherical because  the samples were stuck between the slide and the coverslip.