Serial seeding in bulk protein precipitation

Abstract

Protein precipitation relies on a supersaturation driving force generated by a change in the system, which promotes protein aggregation and sedimentation. It is a unit operation often used at the beginning of protein recovery and purification processes. Crystallization (formation of a solid phase with a well-defined tridimensional shape) also used on bioproduct downstream processing, relies on the same principle, but is designed to work on a different region of a proteins phase diagram where supersaturation is lower. These solids are usually larger and more pure than those generated by precipitation. The transition between crystal formation (nucleation) and crystal growth steps is not easily achieved. A common practice in crystallization is to add previously formed crystals to it in order to avoid the nucleation phase, and better control crystal shape and size distribution. Serial seeding can be used to increase crystal size and reduce its formation time. However, studies of the use of seeds in protein precipitation are scarce. A recent study found that it can increase enzyme recovery in protein precipitation, with similar results found for seeding in mineral and drug precipitation. This project intends to analyze the effect of serial seeding on yield, purity, precipitate morphology, and size distribution when applied to a recombinant ²-glicosidase (used as protein model) precipitation. This enzyme carries a histidine tail, so it can be purified by immobilized metal affinity chromatography (IMAC) after being produced by fermentation. After drying, this purified protein will be used as first seed to precipitate the enzymes present in the fermentation broth, together with a to be selected precipitating agent. Then, serial seeding will be done until a precipitate with stationary characteristics is obtained. The results will contribute to the better use of this technique in protein recovery. (AU)