Different proteins have different amino acid sequences and spatial structures, resulting in differences in their physical, chemical, and biological properties. The development of laboratory-scale purification schemes that produce pure protein (a single band on SDS-PAGE) should be relatively straightforward given the relatively high abundance of recombinant proteins in cell extracts.
The various steps in the downstream processing protocol separate the protein and nonprotein parts of the mixture and finally separate the desired protein from all other proteins while retaining the biological activity and chemical integrity of the polypeptide. Separation steps may exploit differences in chemical/structural/functional properties between the target protein and other proteins in the crude mixture. These properties include size, shape, charge, isoelectric point, charge distribution, hydrophobicity, solubility, density, ligand-binding affinity, metal binding, reversible association, posttranslational modifications, and specific sequences or structures.
Physicochemical Basis of Common Bioseparation Methods
|Separation||Basis of Separation||Resolution|
|Affinity precipitation||Molecular recognition, solubility||Low|
|Aqueous two-phase partition||Solubility/hydrophobicity||Low/medium|
|Three phase partitioning||Solubility/hydrophobicity||Low/medium|
|Ion exchange||Charge, charge distribution||High|
|Reverse-phase HPLC||Hydrophobicity, size||High|
|Affinity chromatography||Molecular recognition||High|
|Gel filtration/size exclusion||Size, shape||High|
For applications requiring the highest purity and relatively small Protein Purification amounts of protein, chromatography can be chosen to selectively purify the target protein. Chromatography is certainly the principal and commonly used operation in downstream processing. This can be explained by certain advantages of chromatography over other unit operations.
For example, chromatography displays high-resolution efficiencies which allow the resolution of complex crude mixtures with very similar molecular properties. (See Details...)
Labrou NE (2014) Protein purification: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 3-10.
Lacki KM, et al. (2011) High throughput screening techniques in protein purification. Methods Biochem Anal 54: 489-506.