Once the protein is expressed cells will be pelleted and the pellet will be resuspended in a suitable buffer and lysed. If present in the soluble fraction, the recombinant protein will be purified using appropriate affinity chromatographic columns.
If the protein is expressed in inclusion bodies the protein needs to be solubilized and refolded before column chromatography can be conducted (refolding).
1) Purification by affinity chromatography (column or batch)
|Immobilized||Tag in fusion protein|
|Metal (mostly Ni2+)||Hexa-His|
|Chitin||Chitin binding sites in intein|
2) Purification of tag-free proteins by
- Cation- or anion-exchange chromatography (MonoS and MonoQ)
- Hydrophobic interaction chromatography (Phenyl-sepharose)
- Size exclusion chromatography
|Example of step gradient Ni column purification|
|SDS-PAGE of individual fractions.|
Specialty Protein Services Provided