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Recombinant protein is proteins expressed in host cells. The modified protein is obtained by gene coding of target proteins via recombinant DNA technology before being introduced into the host cells. Recombinant protein production consists of upstream expression and downstream purification. On account of the complexity of macro-biomolecules in expressed host cells, protein purification is going to be exceptionally challenging. As the chromatography technology develops and improves, chromatography resin is now playing an indispensable role in the bio-separation process.
Affinity resins usually function by adding tags(i.e. His,GST,MBP, Strep-tagⅡ,FLAG ) which can specifically bind with resin ligands on the recombinant protein terminus. For example, His tag has highly selective affinity with Ni2+ and other metal ions, enabling its specific bind with metal ion chelated resins(i.e Ni Bestarose FF and Ni Bestarose HP).Its elution can be performed by increasing imidazole concentration. This chromatography technology is referred as immobilized metal ion affinity chromatography(IMAC).In addition, MBP and GST are also very commonly used tags.However, due to their relatively large molecular weight, it is very often to introduce enzyme cutting sites, so as to remove tags in the follow-up processing.
Ion exchange(IEX) chromatography achieves purification purpose based on the variety in property of charges on the protein components. Due to the fact that interaction between protein surface charges and ligand charges is reversible, it is possible to elute proteins by enhancing ion strength or altering pH value. On account of advantages including high binding capacity, high resolution and easy scale-up, IEX resins are widely used in the purification of non-tagged recombinant proteins and the multiple step purification of tagged proteins.
Hydrophobic interaction chromatography is an effective purification method in IEX resins. It can function well in high salinity condition without the restriction of sample volume, making it suitable for subsequent steps of recombinant proteins after ammonium sulfide precipitation and high salinity elution. Since the proteins will gradually concentrate and be collected in concentration during bind process in addition to declined ion strength, it is easy to rapidly exchange buffer by dilution or de-salting column, connecting to the subsequent process step.
Size exclusion chromatography (SEC) resins function based on the size and shape of protein molecules. During chromatography process, proteins have no interaction with resin, providing advantages including mild operational condition, straightforward operation, negligible impact from buffer solution. However, due to limited sample volume can be loaded, SEC resins are more applicable after concentration step or polishing step in the purification process.
| Affinity chromatography | Ion exchange chromatography | Size-exclusion chromatography | Hydrophobic interaction chromatography | |
|---|---|---|---|---|
| Protein property | Biorecognition | Charge | Size | Hydrophobicity |
| Applications | Receptor and ligand, enzyme and substrate, antigen and antibody | Charged molecules | Large molecules, macromolecular complexes | Proteins and peptides with hydrophobic amino acid side chains on surfaces |
| Advantages |
• Able to isolate one specific protein at a time • High recovery yield • Rapid separation |
• High accuracy and precision • High matrix tolerance • High selectivity |
• High recovery yield • Well defined separation time • Narrow bands available |
• High selectivity • Mild, non-denaturing conditions |
| Disadvantages | Demand ligand with high selectivity | Inconsistency from column to column | Demand differences in MW | Too strong interactions |
In conclusion, the above mentioned purification strategies can work well in the purification process of soluble recombinant proteins. However, what is noteworthy is that a widely applicable purification method is almost nonexistent when it comes to challenging proteins including membrane proteins, toxic proteins and inclusion bodies. Thus, customization on the basis of the specific protein property is the right choice when facing purification tasks targeting on these proteins.
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