Now that there are few contaminants and similar sizemolecules present, a more selective procedure is required to purify the desiredCSP protein. Chromatography refers to a group of separation techniquesthat involves a retardation of molecules with respect to the solvent front thatprogresses through the material. Column chromatography is the most commonphysical configuration, in which the stationary phase is packed into a columnthrough which the mobile phase (the eluent), is pumped.
The degree to which themolecule adsorbs or interacts with the stationary phase will determine how fastit will be carried by the mobile phase. Chromatographic separation of proteinmixtures is a very selective way of purifying proteins.Ion exchange chromatography (IEC) is based on ionicinteractions as the basis for purification.
The separation is due tocompetition between proteins with different surface charges for oppositelycharged groups on an ion exchanger adsorbent. Proteins are complex ampholytesthat have both positive and negative charges. The isoelectric point of a proteinis the pH at which the net charge is zero. This depends on the proportions ofionisable amino acid residues in its structure. This makes it possible toseparate proteins using either fixed positive charges on the stationary phase (anionexchanger) or fixed negative charges (cation exchanger). Hydrophobic interactions are responsible for theself-association of phospholipids and other lipids to form the biologicalmembrane bilayer and the binding of integral membrane proteins. HydrophobicInteraction Chromatography (HIC) is based on the reversible interaction betweena protein surface and chromatographic sorbents of hydrophobic nature. Theproteins are separated according to differences in the amount of exposedhydrophobic amino acids.
To enable hydrophobic interactions, the proteinmixture is loaded on the column in a buffer with a high concentration of salt.The biological function of proteins often involves specificinteractions with other molecules, called ligands. An interacting protein hasbinding sites with complementary surfaces to its ligand. The binding caninvolve a combination of electrostatic or hydrophobic interactions as well asshort-range molecular interactions such as van der Waals forces and hydrogenbonds.
Affinity chromatography owes its name to the exploitation of thesevarious biological affinities for laboratory purification of proteins. Aspecific ligand is then covalently attached to an inert chromatographic matrix.Since only the intended protein is adsorbed from the extract passing throughthe column, other substances will be washed away.
Immobilised metal affinity chromatography (IMAC) relies onthe formation of weak coordinate bonds between immobilised metal ions and someamino acids on proteins (mainly containing histidine). The interaction used inIMAC depends on the formation of coordinated complexes between metal ions andelectron donor groups on the protein surface. Some amino acids are especiallysuitable for binding and histidine is the one that exhibits the strongestinteraction. This is because electron donor groups on the imidazole ring inhistidine readily forms coordination bonds with the immobilised transitionmetal.
Immobilised metal affinity chromatography on Ni–NTAsuperflow resinThe first chromatographic step chosen is IMAC, this isbecause the CSP protein has an affinity towards a Ni-NTA superflow resin. Afterloading, non-specific and unbound proteins were removed by washing the resinwith buffer N3. This was followed by elution of a major protein peak withbuffer N5. Collection of the protein started when the OD280 wentabove the initial baseline of 0.01 and finished when a new baseline wasreached, higher than the starting baseline due to the presence of the imidazolein the elution buffer. The protein sample eluted from the Ni–NTA column couldbe stored at 4°C after adding EDTA to a final 1 mM concentration.
The next step will involve the use of an ion exchangecolumn. Anion exchange resins will bind to negatively charged molecules. Theresin that is used is a Q sepharose fast flow resin. DNA and RNA are highly negativelycharged molecules. This means that they will bind to the resin, leaving the CSPproduct to pass through in a highly purified state.Polishing stepDiafiltration is a technique that uses ultrafiltrationmembranes to completely remove, replace, or lower the concentration of salts orsolvents from solutions containing proteins, peptides, nucleic acids, and otherbiomolecules. The process selectively utilizes permeable (porous) membranefilters to separate the components of solutions and suspensions based on theirmolecular size.
With diafiltration, salt or solvent removal as well as bufferexchange can be performed quickly and conveniently. Another big advantage ofdiafiltration is that the sample is concentrated on the same system, minimisingthe risk of sample loss or contamination. It will be used in this procedure toensure that there are no final contaminants present and will ultimately allowfor safe storage of the CSP protein due to the presence of a buffer.