Chapter 3: Expression and Purification of Bioactive Proteins/Peptides with Conventional Liquid Chromatography

Liquid chromatography has frequently been used to isolate particular proteins/peptides in their native state from extracts of various biological sources. These separations are based on the differences in physicochemical properties of proteins/peptides, such as molecular weight, net charge, hydrophobicity and affinity for ligand or substrate. Nowadays, a variety of resins in an open or packed column are commercially available, and researchers should establish an appropriate separation system, including a column and elution buffer, considering the physicochemical properties of the target proteins/peptides. Three types of liquid chromatography – ion exchange, gel filtration and hydrophobic interaction chromatography – have been most frequently used for the preparatory separation of native proteins/peptides. We describe here the expression and purification of a recombinant enzyme protein from tobacco plants, where the three types of column chromatography were successively exploited.