specific aptamers for sugars or glycan structures that are supported by glycoproteins
could be prepared, as this is reported for sialic acid [155] or sugar sequences
[156]. These studies could be advantageously applied to the preparation of lectinlike aptamers and used for the separation of groups of glycoproteins.
5.3.4 Other Proteins Purified by Aptamer Affinity Chromatography
In 2003, Murphy et al. [119] reported one of the earliest aptamer applications to
protein purification. A DNA aptamer against thyroid transcription factor 1 (TTF1)
was selected following 15 rounds of SELEX process. Then, the biotinylated form of
the aptamer was immobilized on streptavidin magnetic beads and used for the
capture of TTF1 directly from a soluble fraction of a bacterial lysate. Elution
conditions were not optimized during the SELEX process; thus it was not obvious
to decide how to recover the captured TTF1. Since an increase of concentration of
sodium chloride was ineffective to desorb the protein, a SDS elution was performed
and the collected solution directly analyzed by SDS-PAGE. Obtained results showed
that the purification of the protein was particularly specific with a single protein
band. However, the eluted protein was denatured by the action of SDS. It was then
decided to use a recombinant DNase, capable to hydrolyze the aptamer, and thus
release the captured protein. The purity of the TTF1 was very high, but the recovery
depended on the contact time with the DNase.
In a completely different domain, a DNA aptamer was selected and used for the
purification of egg white lysozyme [130]. After aptamer coupling at a density of
290 pmol of aptamer ligand per μL beads, the affinity sorbent was loaded with a
crude sample of egg white in 10 mM Tris-HCl buffer pH 7.5 containing 5 mM
MgCl 2 . After washing, the adsorbed lysozyme was harvested using an elution
solution of 1 M NaClO 4 , pH 6.4. Analytical methods demonstrated the good purity
and specificity of the affinity column. The stability of the affinity column was
satisfactory over 20 separation cycles.
A single-step purification of concanavalin A recently contributed to enlarge the
applicability of DNA aptamer ligands for affinity chromatography [138]. Concanavalin A is a glycoprotein belonging to a large group of lectins [157]. In this study, a
41-nucleotide aptamer, conjugated with a 6-spacer arm ending with a primary amine,
was grafted on a NHS-activated solid support under physiological conditions,
reaching an immobilization rate of about 5 mM. After active group quenching
with a solution of 1 M Tris-HCl buffer pH 9, the affinity sorbent was loaded with
a crude extract of jack beans. All foreign proteins were eliminated by washing with
0.1 M phosphate buffer pH 7.4 containing 0.15 M sodium chloride. Concanavalin A
elution was then performed by addition of 1.5 M sodium chloride in a 10 mM TrisHCl buffer, pH 7. The purity of concanavalin A attested by SDS-PAGE was in
excess of 90% with an enrichment factor of about 366 and an overall yield of 66%.
The main critical point was the binding capacity that was estimated to be less
than 1 mg per mL of sorbent. Confronted with current lectin separation using
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G. Perret and E. Boschetti
could be prepared, as this is reported for sialic acid [155] or sugar sequences
[156]. These studies could be advantageously applied to the preparation of lectinlike aptamers and used for the separation of groups of glycoproteins.
5.3.4 Other Proteins Purified by Aptamer Affinity Chromatography
In 2003, Murphy et al. [119] reported one of the earliest aptamer applications to
protein purification. A DNA aptamer against thyroid transcription factor 1 (TTF1)
was selected following 15 rounds of SELEX process. Then, the biotinylated form of
the aptamer was immobilized on streptavidin magnetic beads and used for the
capture of TTF1 directly from a soluble fraction of a bacterial lysate. Elution
conditions were not optimized during the SELEX process; thus it was not obvious
to decide how to recover the captured TTF1. Since an increase of concentration of
sodium chloride was ineffective to desorb the protein, a SDS elution was performed
and the collected solution directly analyzed by SDS-PAGE. Obtained results showed
that the purification of the protein was particularly specific with a single protein
band. However, the eluted protein was denatured by the action of SDS. It was then
decided to use a recombinant DNase, capable to hydrolyze the aptamer, and thus
release the captured protein. The purity of the TTF1 was very high, but the recovery
depended on the contact time with the DNase.
In a completely different domain, a DNA aptamer was selected and used for the
purification of egg white lysozyme [130]. After aptamer coupling at a density of
290 pmol of aptamer ligand per μL beads, the affinity sorbent was loaded with a
crude sample of egg white in 10 mM Tris-HCl buffer pH 7.5 containing 5 mM
MgCl 2 . After washing, the adsorbed lysozyme was harvested using an elution
solution of 1 M NaClO 4 , pH 6.4. Analytical methods demonstrated the good purity
and specificity of the affinity column. The stability of the affinity column was
satisfactory over 20 separation cycles.
A single-step purification of concanavalin A recently contributed to enlarge the
applicability of DNA aptamer ligands for affinity chromatography [138]. Concanavalin A is a glycoprotein belonging to a large group of lectins [157]. In this study, a
41-nucleotide aptamer, conjugated with a 6-spacer arm ending with a primary amine,
was grafted on a NHS-activated solid support under physiological conditions,
reaching an immobilization rate of about 5 mM. After active group quenching
with a solution of 1 M Tris-HCl buffer pH 9, the affinity sorbent was loaded with
a crude extract of jack beans. All foreign proteins were eliminated by washing with
0.1 M phosphate buffer pH 7.4 containing 0.15 M sodium chloride. Concanavalin A
elution was then performed by addition of 1.5 M sodium chloride in a 10 mM TrisHCl buffer, pH 7. The purity of concanavalin A attested by SDS-PAGE was in
excess of 90% with an enrichment factor of about 366 and an overall yield of 66%.
The main critical point was the binding capacity that was estimated to be less
than 1 mg per mL of sorbent. Confronted with current lectin separation using
124
G. Perret and E. Boschetti