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Protein Purification. Which method is for me?


Protein purification is vital for the characterisation of the function, structure, and interactions of proteins. Selecting the optimal approach for the separation of your protein of interest from all those around it is one of the most challenging aspects of protein purification. Chromatographic methods can purify your protein based on its different biochemical and biophysical properties such as net charge, shape, size and hydrophobicity.Purification strategy components such as which stationary phase to use and binding capacity of a specific column also need to be considered [1]. Here we describe some purification concepts and some of the differences between methods and strategies.


Ion Exchange Chromatography (IEX)


IEX separates proteins based on differences in electrostatic interactions in a protein’s net or local surface charges. This is achieved by a reversible interaction between the chromatography resin and the oppositely charged proteins. To provide the best chromatographic resolution, surface charges can be altered by adjusting the buffer pH, concentration of ion salts or ionic strength of the buffer solution [2]. Positively charged IEX matrices (anion-exchange), adsorb negatively charged proteins. While matrices bound with negatively charged groups (cation-exchange), adsorb positively charged proteins. IEX matrices of different ionic strength are available and can be used as part of a screen to help find the best format for the optimal resolution for your protein. IEX is an incredibly versatile method for protein purification, being able to be used at any stage of the process; isolate your protein, remove bulk impurities, and remove trace impurities.



Hydrophobic Interaction Chromatography (HIC)


HIC separates proteins based on differences in their surface hydrophobicity, through a reversible interaction between hydrophobic groups of the resin and hydrophobic patches on the surface of the protein. As salt concentration increases more water molecules interact with salt ions which decreases the solvation layer around the protein. This leads to increased hydrophobicity of the protein and alters the interaction of the protein with the resin. Protein-HIC matrix interactions are promoted by kosmotropic salts, e.g., ammonium sulphate, sodium citrate, potassium phosphate, and other salts. HIC is a great first-choice purification method that concentrates the target protein into a reduced volume, meaning that fractions containing your protein can be transferred directly to a polishing step.


Affinity Chromatography (AC)


AC is a technique used to purify enzymes, hormones, antibodies, nucleic acids and specific proteins [3]. A reversible complex forms between your protein and its ligand (conjugated to a support matrix) whilst non-specific proteins are washed away. The complexed protein is eluted by alterations in pH, addition of salt solution or competing compounds. AC includes non-biospecific interactions, such as Immobilised Metal Ion Chromatography (IMAC), as well as biospecific interactions, e.g. protein L chromatography. Target proteins may have intrinsic affinity for the ligand on the solid phase surface or they can carry an engineered tag that confers specificity. Most commonly chosen affinity tags include His6-tag, MBP-tag and GST-tag, to name a few. Polyclonal antibody purification from whole blood or sera and monoclonal antibody isolation from culture media can also be performed on several matrices, such as protein A, to achieve the best yield and purity.


Size Exclusion Chromatography (SEC)


SEC, or gel filtration, is the most intuitive chromatography technique as it separates proteins by differences in their size, or, more precisely, their hydrodynamic volume. SEC can be analytical as well as preparative as the proteins are in near-native conditions [4]. The physiological salt concentrations and circumneutral pHs generally limit unfavourable interactions between the protein and the matrix, meaning that buffer screening is rarely required for optimal resolution. Both analytical and preparative gel filtration solutions utilise state-of-the-art pre-packed columns with a wide fractionation range of 0.1 - 600 kDa on low to medium pressure chromatography systems, making this method amenable for researches in the R&D stage and those undertaking large scale clinical and industrial applications.


There are many ways to purify your protein of interest, with the above being some of the most common. There is usually more than one approach that may be applicable to your protein. Indeed, sequential chromatography steps are the norm for the purification of most proteins. Finding the right combination of techniques opens the door to a myriad of downstream analyses and application for your pure protein.


References

1. Coskun O. Separation techniques: Chromatography.North Clin Istanb. 2016;3(2):156–160. Published 2016 Nov 11. doi:10.14744/nci.2016.32757

2. Karlsson E, Ryden L, Brewer J Protein purification. Principles, High Resolution Methods, and Applications.Ion exchange chromatography.2nd ed. New York: Wiley; 1998.

3. Wilchek M, Chaiken I. An overview of affinity chromatography in affinity chromatography–Methods and protocols.Humana Press.2000:1–6.

4. Hong P, Koza S, Bouvier ES. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates.J Liq Chromatogr Relat Technol. 2012;35(20):2923–2950. doi:10.1080/10826076.2012.743724


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