*Review* **Heterogeneous Nucleation in Protein Crystallization**

**Hao Liu <sup>1</sup> , Yue Zhao <sup>1</sup> and Jing Sun 2,\***


**Abstract:** Protein crystallization was first discovered in the nineteenth century and has been studied for nearly 200 years. Protein crystallization technology has recently been widely used in many fields, such as drug purification and protein structure analysis. The key to successful crystallization of proteins is the nucleation in the protein solution, which can be influenced by many factors, such as the precipitating agent, temperature, solution concentration, pH, etc., among which the role of the precipitating agent is extremely important. In this regard, we summarize the nucleation theory of protein crystallization, including classical nucleation theory, two-step nucleation theory, and heterogeneous nucleation theory. We focus on a variety of efficient heterogeneous nucleating agents and crystallization methods as well. The application of protein crystals in crystallography and biopharmaceutical fields is further discussed. Finally, the bottleneck of protein crystallization and the prospect of future technology development are reviewed.

**Keywords:** protein crystallization; crystallography; heterogeneous nucleation; crystallization strategy
