5.3 A Focus on Major Published Applications
To date, the number of aptamer-based affinity chromatography examples is relatively limited; however, it is fully representative of the large potential of this
technology. They cover various aspects such as the diversity of grafting methods
on solid supports, the mechanisms of docking, the desorption, the type, and the mass
of purified proteins. At the beginning, protein separation was essentially applied at
small scale for research purposes. Progressively, the method moved toward more
preparative examples and the repeatability of separation cycles, thus paving the way
to future large-scale applications. To illustrate the state of the art, the examples given
below are presented by groups.
5.3.1 Blood Proteins
The first human blood protein purified by affinity chromatography using a DNA
aptamer as ligand was thrombin [121]. Here, the biotinylated oligonucleotide
was immobilized on a streptavidin solid phase and captured proteins eluted by an
increase of ionic strength. This system was described as useful for the detection and
the separation of the target protein.
Among the numerous human plasma proteins, immunoglobulins G have been
targeted for their separation using RNA aptamer ligands [137]. A calcium-dependent
aptamer against the Fc fragment of IgG was first selected from a large library and
then attached to a chromatographic support. The grafting process involved the use of
a C18 amino spacer attached on either 3
0 or 5
0 oligonucleotide end and immobilized
on tresyl- or NHS-activated sorbent. The resulting affinity chromatography columns
were loaded with human serum and then washed repeatedly with a 20 mM Tris-HCl
pH 7.6 containing 145 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM
magnesium chloride, and 1.8 mM calcium chloride. The IgG elution was performed
with 10 mM Tris-HCl buffer pH 7.6 containing 200 mM potassium chloride and
10 mM EDTA. The SDS-PAGE analysis of the eluate demonstrated a purity level
similar to the one obtained using current affinity chromatography with protein A or
protein G ligands. According to the data reported by the authors, this purification
process could be used for preparative applications with repeated separation cycles
and a binding capacity close to 30 mg per mL of affinity resin. Ten years later, the
same research team published a second report focused on an alkaline-tolerant
aptamer ligand for the purification of acid-sensitive antibodies [110]. In this study,
several nucleotides were modified in the 2
0 position of the ribose with the introduction of 2’-OMe groups. With this modification, the aptamer ligand could withstand
treatments with 1 M sodium hydroxide. The 3
0 -aminogroup-modified aptamer was
grafted on NHS-activated Sepharose. The IgG separation process from crude human
serum was performed using the same buffer as described in the first article; the
IgG elution was also performed similarly by using EDTA as complexation agent
for calcium ions. Beyond satisfactory purity, further investigations showed that
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
117
To date, the number of aptamer-based affinity chromatography examples is relatively limited; however, it is fully representative of the large potential of this
technology. They cover various aspects such as the diversity of grafting methods
on solid supports, the mechanisms of docking, the desorption, the type, and the mass
of purified proteins. At the beginning, protein separation was essentially applied at
small scale for research purposes. Progressively, the method moved toward more
preparative examples and the repeatability of separation cycles, thus paving the way
to future large-scale applications. To illustrate the state of the art, the examples given
below are presented by groups.
5.3.1 Blood Proteins
The first human blood protein purified by affinity chromatography using a DNA
aptamer as ligand was thrombin [121]. Here, the biotinylated oligonucleotide
was immobilized on a streptavidin solid phase and captured proteins eluted by an
increase of ionic strength. This system was described as useful for the detection and
the separation of the target protein.
Among the numerous human plasma proteins, immunoglobulins G have been
targeted for their separation using RNA aptamer ligands [137]. A calcium-dependent
aptamer against the Fc fragment of IgG was first selected from a large library and
then attached to a chromatographic support. The grafting process involved the use of
a C18 amino spacer attached on either 3
0 or 5
0 oligonucleotide end and immobilized
on tresyl- or NHS-activated sorbent. The resulting affinity chromatography columns
were loaded with human serum and then washed repeatedly with a 20 mM Tris-HCl
pH 7.6 containing 145 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM
magnesium chloride, and 1.8 mM calcium chloride. The IgG elution was performed
with 10 mM Tris-HCl buffer pH 7.6 containing 200 mM potassium chloride and
10 mM EDTA. The SDS-PAGE analysis of the eluate demonstrated a purity level
similar to the one obtained using current affinity chromatography with protein A or
protein G ligands. According to the data reported by the authors, this purification
process could be used for preparative applications with repeated separation cycles
and a binding capacity close to 30 mg per mL of affinity resin. Ten years later, the
same research team published a second report focused on an alkaline-tolerant
aptamer ligand for the purification of acid-sensitive antibodies [110]. In this study,
several nucleotides were modified in the 2
0 position of the ribose with the introduction of 2’-OMe groups. With this modification, the aptamer ligand could withstand
treatments with 1 M sodium hydroxide. The 3
0 -aminogroup-modified aptamer was
grafted on NHS-activated Sepharose. The IgG separation process from crude human
serum was performed using the same buffer as described in the first article; the
IgG elution was also performed similarly by using EDTA as complexation agent
for calcium ions. Beyond satisfactory purity, further investigations showed that
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
117