Thomas Shamberia, Dougay Joel A, Annunziata Onofrio
Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76109, USA.
Molecules. 2025 May 29;30(11):2371. doi: 10.3390/molecules30112371.
Preparative protein crystallization is regarded as an economically sustainable protein purification alternative to chromatography in biotechnological downstream processing. However, protein crystallization is a not-well-understood process that is usually slow and poorly reproducible. A promising strategy for enhancing protein crystallization is exploiting the metastable liquid-liquid phase separation (LLPS) of protein solutions. Here, we report an enhancement of lysozyme-crystallization yield by using a combination of two additives under LLPS conditions. The first additive, NaCl (0.15 M), is necessary to introduce protein-protein attractive interactions and induce LLPS by lowering temperature. The second additive, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.10 M, pH 7.4), accumulates in the metastable protein-rich liquid phase and thermodynamically stabilizes lysozyme crystals. We found that this combination of additives leads to crystallization yields of higher than 90% under LLPS conditions at a lysozyme concentration of 5% by weight and a fairly low ionic strength (0.2 M) within an operational time of the order of one hour. This crystallization yield is more than three-fold larger than that obtained from samples containing NaCl without HEPES at the same pH and ionic strength. Moreover, we determined crystallization yield as a function of incubation time, and temperature below and above the LLPS boundary. As crystallization temperature intersects with LLPS temperature, a significant increase in crystallization yield is observed. This is consistent with LLPS boosting protein crystallization. Our work suggests a possible strategy for increasing the crystallization success of other proteins, with applications in protein purification.
在生物技术下游加工中，制备性蛋白质结晶被视为一种经济上可持续的蛋白质纯化方法，可替代色谱法。然而，蛋白质结晶是一个尚未被充分理解的过程，通常速度缓慢且重现性差。一种有前景的增强蛋白质结晶的策略是利用蛋白质溶液的亚稳态液-液相分离（LLPS）。在此，我们报告了在LLPS条件下使用两种添加剂的组合提高溶菌酶结晶产率的方法。第一种添加剂NaCl（0.15 M）是引入蛋白质-蛋白质吸引相互作用并通过降低温度诱导LLPS所必需的。第二种添加剂4-(2-羟乙基)-1-哌嗪乙磺酸（HEPES，0.10 M，pH 7.4）在富含蛋白质的亚稳态液相中积累，并在热力学上稳定溶菌酶晶体。我们发现，在LLPS条件下，当溶菌酶浓度为5%（重量）且离子强度相当低（0.2 M）时，这种添加剂组合在大约一小时的操作时间内可使结晶产率高于90%。该结晶产率比在相同pH和离子强度下不含HEPES的NaCl样品所获得的产率高出三倍多。此外，我们确定了结晶产率与孵育时间以及低于和高于LLPS边界的温度的函数关系。当结晶温度与LLPS温度相交时，观察到结晶产率显著增加。这与LLPS促进蛋白质结晶一致。我们的工作提出了一种提高其他蛋白质结晶成功率的可能策略，可应用于蛋白质纯化。
