Expression, purification, and crystallization of the extracellular domain of a mammalian ZIP4

Zinc transporters are of vital importance in maintaining zinc homeostasis in all living organisms. In humans, ZIP4 is exclusive for dietary zinc uptake. Obtaining enough purified protein by heterologous expression is necessary for structural characterization to understand working mechanisms at the atomic level. However, due to the major obstacle in membrane protein expression, there is no structural information of the full-length human ZIP4 till now. A "divide and conquer" strategy has been applied to ZIP4 to study the extracellular domain (ECD) and the transmembrane domain separately, which has led to the first ECD structure in the entire ZIP family. In this chapter, we provide detailed protocols for the expression, purification, and crystallization of ZIP4-ECD from a mammalian species.