Tagged recombinant proteins are usually straightforward to purify. Use an affinity resin that corresponds to the tag system used and the natural conditions for the protein to avoid precipitation and degradation.
If the requirement for purity is high, add an additional intermediate step of ion exchange or hydrophobic interaction chromatography. However, try to use as few steps as possible as adding steps will decrease overall protein yield. Gravity columns are common to use in affinity steps and sometimes peristatic pumps in the other chromatography steps, however a protein purification system will deliver significantly more control, obtain more detailed information on target protein and impurities, and gives much better protection of columns.
The isolation methods used to isolate soluble recombinant or non-recombinant proteins depend on the intrinsic physiochemical properties of the proteins unless they are tagged. A typical purification protocol is shown as below (with Ion-Exchange Chromatography).
centrifuge (90 min at >60000 ×g) filter or salt fractionate and exchange buffer
Perform affinity methods
(2) conduct ion exchange
(3) perform other chromatography methods
|Perform gel filtration|
1 Determining the Isoelectric Point
2 Breaking Cells
3 Clarifying Cell Extract by Centrifugation or Selective Precipitation
4 Applying Clarified Extract to a Weak Anion Exchanger
5 Preparing for Repeat Ion-Exchange Step
6 Repeating Ion-Exchange Chromatography
7 Performing Gel Filtration
Recombinant proteins expressed in E. coli that are located in the low-speed pellet fraction following cell lysis are highly aggregated. Inclusion bodies are normally derived from protein aggregation in the cytoplasm, or in the periplasm if a secretion vector was used. As mentioned above, protein can also be located in either the low-or high-speed pellet fractions because of interaction with bacterial nucleic acids.
The protein is extracted with protein denaturants such as guanidine·HCl (Gu·HCl), urea, or an organic acid. The reductant dithiothreitol (DTT) is included to prevent artificial disulfide bond formation (especially intermolecular bonds). The denatured protein can be purified by various methods and then folded, or it can be directly folded. Typically, some purification (e.g., gel filtration in Gu·HCl) prior to folding is recommended as it often results in higher folding yields.
Ferrer-Miralles N, et al. (2015) General introduction: Recombinant protein production and purification of insoluble proteins. Methods in molecular biology (Clifton, N.J.) 1258: 1-24.
Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 147-168.