The three phase purification strategy has been used in simple laboratory purification to large or industrial scale production. This three phase purification system is also used to purify large scale production of pharmaceutical compounds or biomolecules such as monoclonal antibodies or any recombinant enzymes. Protein purification system uses a standard protocol such as sample extraction and clarification, capture, then intermediate purification and polishing. There are some instances where strategies for protein purification were designed with fewer steps, by selecting the most appropriate technique and also by selecting the appropriate media that fulfil all the required purification objectives.

Three Step Purification of a Protein (Recombinant Enzyme):

This three step purification strategy is most commonly used to purify the proteins. Ion exchange chromatography is used for capturing the protein, hydrophobic interaction chromatography for intermediate purification and gel filtration (GF) for the protein polishing step. The objective of using this three step purification method is to get highly purified form of protein that can be used for crystallization and structural determination.

Target molecule or protein is an oxygen-sensitive enzyme known as Deacetoxycephalosporin C synthase (DAOCS). Source material is this recombinant protein over expressed in the cytoplasm of E.coli bacterial cell.

Sample Extraction and Clarification:

1. Transformed and over expressing E.coli bacterial cells were suspended in a lysis buffer. This lysis buffer is formed by using chemical compounds such as 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMSF, pH 7.5
2. These E.coli bacterial cells are then lyzed using ultrasonication technique.
3. Streptomycin sulphate (1%) and polyethyleneimine (0.1%) were added to this lysed cell mixture to precipitate DNA. The extraction was clarified using centrifugation technique.
4. Chemical compounds EDTA, DTT, Benzamidine-HCl and PMSF were used in the lysis buffer to inhibit the activity of proteases and to minimise damage to the oxygen-sensitive enzyme Deacetoxycephalosporin C synthase. Also keeping the extracted sample in ice also reduces the activity of proteases.


The capture step used to remove the most harmful contaminants from the relatively unstable oxygen-sensitive protein molecule. This, along with the calculated isoelectric point of enzyme Deacetoxycephalosporin C synthase, led to the selection of anion exchange purification system.

An array of anion exchange columns are screened first to aid in the selection of the optimum medium before selecting and using a larger column for the optimization of the capture step. For the capturing of oxygen-sensitive enzyme Deacetoxycephalosporin C synthase Sepharose XL, is most commonly used because it is well suited for capturing. Step elusion is used at high flow rate to speed up the purification of recombinant enzyme Deacetoxycephalosporin C synthase. This step can be used for all the capturing of potentially unstable compound.

Intermediate Purification:

Hydrophobic interaction chromatography (HIC) was selected as the separation principle is complementary to that of ion exchange chromatography and also in this method minimum quantity of sample was required. As hydroscopic property is very difficult to predict therefore always recommended to screen different media. The intermediate purification strategy was developed by using and screening pre-packed hydrophobic interaction media. This helps in selecting optimum media for the separation. In this intermediate purification step maximum possible speed can be used for separation of enzyme or proteins were avoided in order to achieve resolution and also this reduces significantly impurities present in the mixture.


The main aim of this polishing step is to remove minor contaminants and also aggregates present in the mixture. This step is also used to transfer the purified sample into suitable buffer, which can be used for further structural studies. For example Superdex 75 prep grade, a gel filtration medium can be used as it gives relatively high resolution at relatively short separation time. Enzyme Deacetoxycephalosporin C synthase can be separated using this media as molecular weight of the enzyme is within the optimal separation range of this medium.

Uses of Three Phase Purification Strategy:

1. Three phase purification strategy can also be used to extract and purify recombinant Antigen binding fragment protein.
2. Three phase purification strategy can also be used to extract and purify monoclonal antibodies.
3. Three phase purification strategy can also be used to extract and purify Integral membrane proteins.

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