4.

1 APPLICATIONS OF AFFINITY CHROMATOGRAPHY

Affinity chromatography is basically separation based on biological affinity. Affinity

chromatography can be known by the following characteristics:
 Its selectivity is high;
 The capacity can be very high depending on the ligand;
 High speed separation

In the development of successful procedure for affinity chromatography purification,

different classes of affinity targets, as well as different purification goals, which require
consideration of different priorities that is either high purity or high yield, technical limitations
and buffer conditions.
Affinity chromatography is used from small-scale research to large scale
purification. However, it is not used extensively in the large scale manufacture due to cost
because the materials are expensive. Thus, it is necessary for choosing the right matrices
for substance to be purified.

4.2 APPLICATIONS FOR PURIFICATION

Affinity chromatography is basically used in biotechnology, genetic engineering, biomedical

and basic metabolic research at which can be used to purify these bio-molecules
substances1:
(i.) Antibodies and antigens
Antigens are usually a protein substance foreign to the body and because
being so, it stimulates the body’s immune system to produce the unique
antibodies that can fight this antigen.
(ii.) Enzymes and inhibitors
(iii.) Regulatory enzymes
(iv.) Recombinant proteins including hormone, enzyme and other binding proteins
(v.) Receptors
(vi.) RNA and DNA (genes)
(vii.) Bacteria
(viii.) Viruses and phages
(ix.) Cells
(x.) Others

1
B.Pascal, K.E. George, F. Wen Jian, B. Wolfgang, Affinity Chromatography: Methods and Protocols.
 Affinity chromatography effectively binds specific solutes by taking advantages on
the solutes biological interactions. Antibodies, antigens, or dyes are conjugated to polymer
resins for the purpose of binding specific solutes from a mixture. For example, an antibody
could be raised against a target protein. The antibody then captures the solute out of the
mixture, and the impurities flow through the column. The solute can be recovered by
changing the pH, increasing the salt concentration, or adding displacer, that is, a molecule
that also has some affinity for the antibody, or other binding agent (affinity ligand) on the
stationary phase.
Other examples such as isolation of proteins, it is made possible by using the
special interactions of enzyme-ligand, enzyme-cofactor, receptor-agonist (antagonist). In
each case, one member of any of these pairs may be immobilized to a resin to isolate the
desired partner. Affinity chromatography is commonly coupled with cloning techniques to
synthesize the target molecule with an “epitope” or recognition sequence that can be
captured by the affinity ligand.
Below are a few descriptions on application of affinity chromatography:

4.1.1 Purification of Recombinant Proteins

Recombinant protein is a protein that carries a code by a recombinant DNA.

Recombinant DNA means that there are two segments of DNA in a plasmid. Plasmids are
usually occurring in bacteria. When a recombinant DNA is inserted into bacteria, the bacteria
will produce protein based on this recombinant DNA. This protein is called recombinant
protein. The use of bacteria in order to produce recombinant protein has grown in the recent
years. Using recombinant DNA and inserting it to a plasmid of rapidly reproducing bacteria
enables the manufacture of recombinant protein. These recombinant proteins can be variety
of types; they can be antibodies, antigens, hormones and enzymes.
Recombinant proteins are commonly purified using this method. Proteins with a
known affinity are protein tagged in order to aid their purification. The protein may have been
genetically modified so as to allow it to be selected for affinity binding; this is known as a
fusion protein. Tags include glutathione-S-transferase, hexahistidine (his), and maltose
binding protein (MBP). His tags have an affinity for nickel or cobalt ions which are coordinate
covalent bond with a chelator for the purposes of solid medium entrapment. For elution, an
excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used.
GST has an affinity for glutathione which is commercially available immobilized as
 glutathione agarose. During elution, excess glutathione is used to displace the tagged
protein.

4.1.2 Purification of Immunoglobulin

Immunoglobulin or antibody is a protein used by the immune system for the purpose
of defence against foreign substances such as bacteria and or viruses. The antibody
recognizes the antigen. The antibody contains a paratope that is specific for one antigen,
thus, aids in high attraction between the antibody and antigen. So, the antibody can tag a
microbes or infected cell that may attack immune system.
Due to the variety of antibody-antigen interactions has created many uses for
antibodies and antibody fragments. They are used for therapeutic and diagnostic
applications as well as for immunochemical techniques within general research. The use of
recombinant technology has greatly expanded our ability to manipulate the characteristics of
these molecules to our advantage. The potential exists to create an infinite number of
combinations between immunoglobulin and immunoglobulin fragments with tags and other
selected proteins. A significant advantage for the purification of antibodies and their
fragments is that a great deal of information is available about the properties of the target
molecule and the major contaminants, no matter whether the molecule is in its a native state
or has been genetically engineered and no matter what the source material.