Now ion exchanger adsorbent. Proteins are complex ampholytes that

Now that there are few contaminants and similar size
molecules present, a more selective procedure is required to purify the desired
CSP protein.

Chromatography refers to a group of separation techniques
that involves a retardation of molecules with respect to the solvent front that
progresses through the material. Column chromatography is the most common
physical configuration, in which the stationary phase is packed into a column
through which the mobile phase (the eluent), is pumped. The degree to which the
molecule adsorbs or interacts with the stationary phase will determine how fast
it will be carried by the mobile phase. Chromatographic separation of protein
mixtures is a very selective way of purifying proteins.

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Ion exchange chromatography (IEC) is based on ionic
interactions as the basis for purification. The separation is due to
competition between proteins with different surface charges for oppositely
charged groups on an ion exchanger adsorbent. Proteins are complex ampholytes
that have both positive and negative charges. The isoelectric point of a protein
is the pH at which the net charge is zero. This depends on the proportions of
ionisable amino acid residues in its structure. This makes it possible to
separate proteins using either fixed positive charges on the stationary phase (anion
exchanger) or fixed negative charges (cation exchanger).

 

Hydrophobic interactions are responsible for the
self-association of phospholipids and other lipids to form the biological
membrane bilayer and the binding of integral membrane proteins. Hydrophobic
Interaction Chromatography (HIC) is based on the reversible interaction between
a protein surface and chromatographic sorbents of hydrophobic nature. The
proteins are separated according to differences in the amount of exposed
hydrophobic amino acids. To enable hydrophobic interactions, the protein
mixture is loaded on the column in a buffer with a high concentration of salt.

The biological function of proteins often involves specific
interactions with other molecules, called ligands. An interacting protein has
binding sites with complementary surfaces to its ligand. The binding can
involve a combination of electrostatic or hydrophobic interactions as well as
short-range molecular interactions such as van der Waals forces and hydrogen
bonds. Affinity chromatography owes its name to the exploitation of these
various biological affinities for laboratory purification of proteins. A
specific ligand is then covalently attached to an inert chromatographic matrix.
Since only the intended protein is adsorbed from the extract passing through
the column, other substances will be washed away.

Immobilised metal affinity chromatography (IMAC) relies on
the formation of weak coordinate bonds between immobilised metal ions and some
amino acids on proteins (mainly containing histidine). The interaction used in
IMAC depends on the formation of coordinated complexes between metal ions and
electron donor groups on the protein surface. Some amino acids are especially
suitable for binding and histidine is the one that exhibits the strongest
interaction. This is because electron donor groups on the imidazole ring in
histidine readily forms coordination bonds with the immobilised transition
metal.

 

Immobilised metal affinity chromatography on Ni–NTA
superflow resin

The first chromatographic step chosen is IMAC, this is
because the CSP protein has an affinity towards a Ni-NTA superflow resin. After
loading, non-specific and unbound proteins were removed by washing the resin
with buffer N3. This was followed by elution of a major protein peak with
buffer N5. Collection of the protein started when the OD280 went
above the initial baseline of 0.01 and finished when a new baseline was
reached, higher than the starting baseline due to the presence of the imidazole
in the elution buffer. The protein sample eluted from the Ni–NTA column could
be stored at 4°C after adding EDTA to a final 1 mM concentration.

The next step will involve the use of an ion exchange
column. Anion exchange resins will bind to negatively charged molecules. The
resin that is used is a Q sepharose fast flow resin. DNA and RNA are highly negatively
charged molecules. This means that they will bind to the resin, leaving the CSP
product to pass through in a highly purified state.

Polishing step

Diafiltration is a technique that uses ultrafiltration
membranes to completely remove, replace, or lower the concentration of salts or
solvents from solutions containing proteins, peptides, nucleic acids, and other
biomolecules. The process selectively utilizes permeable (porous) membrane
filters to separate the components of solutions and suspensions based on their
molecular size. With diafiltration, salt or solvent removal as well as buffer
exchange can be performed quickly and conveniently. Another big advantage of
diafiltration is that the sample is concentrated on the same system, minimising
the risk of sample loss or contamination. It will be used in this procedure to
ensure that there are no final contaminants present and will ultimately allow
for safe storage of the CSP protein due to the presence of a buffer.