The liquid protein phase in crystallization: a case study—intact immunoglobulins

Abstract

A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in ''salting out'' or ''salting in' procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The ''liquid protein'' phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO 4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase. #

Two-step mechanism for the nucleation of crystals from solution

“…There is abundant evidence in literature that shows that the long-lifetime macroscopic dense liquid phase does not promote the nucleation of crystals [40]. In many cases, similar to the example discussed above, crystals and dense liquid droplets were found to coexist for extended periods of time without the droplets generating additional crystal nuclei.…”

Crystalline, Porous Microspheres Made from Amino Acids by Using Polymer‐Induced Liquid Precursor Phases

“…[27] During protein crystallization similar effects can be observed when "liquid protein" phases are built. [28] While this effect is undesirable during protein crystallization, application of such precursors for crystal morphogenesis is very promising. Since the crystallization proceeds by the Ostwald rule of stages, including a liquid precursor step, the drowning-out process now becomes more attractive for crystal engineering.…”

Polymer‐Controlled Biomimetic Mineralization of Novel Inorganic Materials

“…A liquid/liquid phase separation can be observed as an undesired effect during protein crystallization from solution [73] and has already been known for more than a century for CaCO 3 [74][75][76]. If such a phase is used as a precursor phase for a subsequent crystallization process, crystal morphologies with complex shape, molten appearance, and crystalline coatings can be realized.…”