Introduction

Tremendous success in the fields of genetic engineering and molecular biology has enabled scientists to express any amino acid sequence and practically synthesize any protein. But, this success depends on one key factor; in order for the expressed proteins to be used commercially, it should be purified to a certain degree. Protein purification from a fermentation broth involves a number of combined techniques that resolve the proteins according to charge, binding ability, size, or hydrophobicity. Traditionally, these techniques have been optimized as individual process rather than being considered as a continuous integrated process.

Successful techniques should be able to address two primary objectives:
(1) protein selection (the preferred protein accumulated in one phase while the other proteins accumulate in another phase); and
(2) mechanical separation of the two phases (should allow high-flow rates and be able to process turbid solutions with minimal impurities).

Affinity Ligands

Most proteins have the intrinsic ability of binding to certain substances. For example, majority of the enzymes possess strong affinity toward their substrate or substrate molecules; protein inhibitors are capable of binding with proteins which they target allowing the antigen-antibody pairs to represent a perfect binding selectivity. Whenever the protein purification process faces a deficit in natural binding force of protein, the target protein should be genetically modified by fusing the many affinity tags or tails to facilitate the process of verification. The affinity ligands that are matrix coupled present an important tool that is capable of recognizing and biding the target protein thereby enriching the matrix when compared to crude extract in the aqueous phase.

Column Chromatography

Separating the enriched phase from the depleted crude extract was traditionally done using column chromatography. The matrix that bears the ligand is presented by chromatographic adsorbent and then packed into a column that has a porous bottom. Through the column, the starting mixture is pumped providing sufficient time for the target protein to take its place. High flow rates and high pressures are maintained to get the desired flow rate. The high-flow via the medium is completely blocked as a final result. In the affinity partitioning process two-phase systems, the liquid phase enriches the target protein while the other one is filtered using funnel for liquid-liquid separation.

Ultrafiltration

Molecules are separated based on their shape and size by the process of ultrafiltration. For maximum separation, the species that are to be separated should exhibit atleast 10-fold difference in their sizes. In order to make ultrafiltration process applicable for the protein purification process, the target protein should be included in a complex that is 10 times larger than the impurities (usually are of the size of the target protein) present. This can be achieved by either binding the target protein to a micro-particle-bearing affinity ligand or to a macro-ligand (soluble polymer); the macro-ligand should not be big. It should remain in a suspended or soluble state allowing the easy transfer of mass thereby avoiding aggregation. The high-MW micro-particles or soluble polymers are the primary choice for affinity ultrafiltration.

Affinity Ultrafiltration

In order to increase the processing potential and the specificity of membrane filtration, affinity ultrafiltration concept was introduced. Affinity ultrafiltration follows the principle that:
(1) when the target protein and impurities are free in a solution pass via the ultrafiltration membrane;
(2) whereas, when a macro-ligand is bound to the target protein, the resultant is a membrane-retained complex.

After the target protein binds to the macroligand, the impurities are washed away. Dissociation of the target protein from macroligand occurs when a buffer (elution) is introduced. It is then transported in its purified form through the flux-membrane. This process is operated either as a batch process or a semi-batch process; sometimes even as a continuous process.

The respective sizes of the macroligand and the target protein along with membrane cut-off will determine the efficacy of the purification process. The membrane should be able to prevent macroligand leakage and also should possess high permeability for free protein and impurities. In all the protein purification techniques, the affinity ultrafiltration protocol follows four major steps:

(1) binding (of the target protein to macroligand);
(2) washing (of complex macroligand- target protein using diafiltration mode);
(3) elution (achieved by buffer change); and
(4) regeneration (free macroligand is regenerated in the buffer).

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