Fundamental aspects of protein isolation and purification

Proteins come from plants, tissues or, microorganisms, and recombinant organisms. Native protein purification involves several steps based on their differences in size, charge, hydrophobicity, and affinity binding to specific ligands. Extracellular proteins require minimal sample preparation whereas cell lysis by mechanical and non-mechanical procedures is required for intracellular proteins. A precipitation step using salts or chemicals is then conducted to obtain a target protein in complex with other biomolecules. Ion exchange, hydrophobic, and/or affinity chromatography is the final step, which might be used in sequence to purify the protein. As proteases are also released during cell lysis, proteolysis must be prevented by using a protease inhibitor or the purification must be performed at low temperature. Emerging novel fusion tags will be routinely introduced into the host cells to enhance protein solubility and enable tandem affinity purification. This powerful approach can make any desired protein from a recombinant host with all desired properties.