immobilized sugar sequences, the risk of co-purifying other proteins having an
affinity for the sugar ligands is eliminated.
Affinity chromatography with aptamer ligands can be applied to many proteins
from various biological samples even when the concentration of the target protein is
low. This situation has been reported by Javaherian et al. [158] with the purification
of recombinant of T. aquaticus protein MutS expressed in E. coli. After the SELEX
process, the aptamer was biotinylated and immobilized on a streptavidin solid
support. The affinity sorbent was then used for the capture of the expressed protein
by direct loading of the bacterial crude extract. In parallel, a similar chromatography
was performed using a cell lysate from native E. coli as sample. SDS-PAGE analysis
demonstrated that the purity of MutS proteins was particularly high in spite of the
poor expression in recombinant cells. Only few thin bands were found as trace
protein contaminants.
Outside protein purification, but still within the domain of selective protein
extraction, immobilized aptamers were employed for apheresis which consists of
removing specifically undesired proteins from blood and reinjecting the depleted
blood into circulation. Several examples have been published throughout the past
few years [159, 160]. One of the major applications of this extracorporeal therapy is
the elimination of pathogenic autoantibodies. Within this field, the first apheresisbased aptamer application has been reported by Wallukat et al. [161], targeting β-1
receptor autoantibodies (β1-AABs). This affinity solid support is put in a cartridge
where the blood or the plasma circulates. In this study, a 12-mer aptamer was
derivatized with an aminohexyl spacer and was grafted on NHS-activated agarose
beads. Reported experimental data from rats demonstrated a strong reduction of β1AABs. The process did not affect the concentration of other immunoglobulins, thus
demonstrating the specific property of the affinity adsorbent. Further developments
of this pioneering work have been recently reported [162].
Although affinity chromatographic separations, as described above, apply to
proteins, a number of applications are devoted to the separation of other molecular
entities or even biological particles. Typical examples are antibiotics [163], toxins
[164], and adenosine analogs [118]. Additionally the resolution of enantiomers was
also resolved elegantly with immobilized specific aptamers [165, 166]. Other examples extend to living biological particle separation, such as the specific capture of
circulating tumor cells via a grafted aptamer ligand selected for its specificity for a
cell surface antigen [167]. On a similar topic, Zamay et al. [168] reported the
purification of live cells with the use of aptamer ligands. Virus isolation has also
been published, for instance, hepatitis C virus, with great success [169].
5.4 Questions Around Aptamer Release, Hydrolysis,
and Sanitization
Preparative classical affinity chromatography technology, involving any type of
ligand, is threatened by undesired effects related to the release of ligands during
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
125
affinity for the sugar ligands is eliminated.
Affinity chromatography with aptamer ligands can be applied to many proteins
from various biological samples even when the concentration of the target protein is
low. This situation has been reported by Javaherian et al. [158] with the purification
of recombinant of T. aquaticus protein MutS expressed in E. coli. After the SELEX
process, the aptamer was biotinylated and immobilized on a streptavidin solid
support. The affinity sorbent was then used for the capture of the expressed protein
by direct loading of the bacterial crude extract. In parallel, a similar chromatography
was performed using a cell lysate from native E. coli as sample. SDS-PAGE analysis
demonstrated that the purity of MutS proteins was particularly high in spite of the
poor expression in recombinant cells. Only few thin bands were found as trace
protein contaminants.
Outside protein purification, but still within the domain of selective protein
extraction, immobilized aptamers were employed for apheresis which consists of
removing specifically undesired proteins from blood and reinjecting the depleted
blood into circulation. Several examples have been published throughout the past
few years [159, 160]. One of the major applications of this extracorporeal therapy is
the elimination of pathogenic autoantibodies. Within this field, the first apheresisbased aptamer application has been reported by Wallukat et al. [161], targeting β-1
receptor autoantibodies (β1-AABs). This affinity solid support is put in a cartridge
where the blood or the plasma circulates. In this study, a 12-mer aptamer was
derivatized with an aminohexyl spacer and was grafted on NHS-activated agarose
beads. Reported experimental data from rats demonstrated a strong reduction of β1AABs. The process did not affect the concentration of other immunoglobulins, thus
demonstrating the specific property of the affinity adsorbent. Further developments
of this pioneering work have been recently reported [162].
Although affinity chromatographic separations, as described above, apply to
proteins, a number of applications are devoted to the separation of other molecular
entities or even biological particles. Typical examples are antibiotics [163], toxins
[164], and adenosine analogs [118]. Additionally the resolution of enantiomers was
also resolved elegantly with immobilized specific aptamers [165, 166]. Other examples extend to living biological particle separation, such as the specific capture of
circulating tumor cells via a grafted aptamer ligand selected for its specificity for a
cell surface antigen [167]. On a similar topic, Zamay et al. [168] reported the
purification of live cells with the use of aptamer ligands. Virus isolation has also
been published, for instance, hepatitis C virus, with great success [169].
5.4 Questions Around Aptamer Release, Hydrolysis,
and Sanitization
Preparative classical affinity chromatography technology, involving any type of
ligand, is threatened by undesired effects related to the release of ligands during
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
125