Furthermore, posttranslationally modified factor VII in its des-Gla form was successfully separated from the native form. The DNA aptamer ligands used in these
applications were stable throughout several cycles of 1 M sodium hydroxide
cleaning. Binding capacity was also determined and appeared close to 6 mg of
proteins per mL of affinity sorbent.
5.3.2 DNA-Related Proteins
Very early, the immobilization of DNA on a solid support was developed for affinity
chromatography separations [151]. At that time, the protein capture was not specific
but could be qualified as group specific for all proteins able to interact with nucleic
acids. Then, more specific interactions were targeted, such as anti-DNA antibodies
[152] and transcription factors [132]. It is thus not surprising to see the application of
aptamers as specific ligands for the separation of proteins related to DNA. In 2007, a
DNA aptamer recognizing specifically a Taq polymerase was attached chemically to
magnetic beads previously activated with glutaraldehyde and used for protein
purification [129]. The first interest of this work was the possibility to produce
small quantities of this recombinant protein for characterization purposes. Simplicity
and rapidity of results were the most important motivations for this development. It
was observed that the best conditions of aptamer grafting were obtained when the pH
was between 6 and 8. With this affinity sorbent, a crude physiological bacterial
extract containing the recombinant enzyme could be loaded in a mini batch mode.
After washing, the Taq polymerase was desorbed by decreasing the pH to 4. Compared to conventional purification techniques, the process was considered more
rapid, without the need of various chemicals and very reliable for single-step
purification.
In another more recent paper, Beloborodov et al. [62] described the facilitated
aspect of functional protein purification of two proteins with the use of DNA
aptamers as ligands. They compared the purification of proteins to immunoaffinity
chromatography and IMAC chromatography. Outside the purity aspect, immunoaffinity chromatography necessitates acidic condition for elution that can be detrimental to the biological activity. In a similar way, the presence of metal ions in
IMAC may have other deleterious effects. On the contrary, with the use of aptamer
ligands, the elution could be performed at neutral pH with the assistance of Mg
++
cations. The purity and the biological activity of AlkB, a protein involved in the
DNA alkylation damage, were above 85%. MutS, a protein involved in DNA
replication and repair, was very tightly associated to the aptamer and only desorbed
by 2 M MgCl 2 ; moreover, it retained its binding activity.
5.3.3 Group-Specific Proteins
In affinity chromatography, certain ligands are used for the separation of groups of
proteins. Transition metal ions chelated by immobilized complexing agents interact
122
G. Perret and E. Boschetti
applications were stable throughout several cycles of 1 M sodium hydroxide
cleaning. Binding capacity was also determined and appeared close to 6 mg of
proteins per mL of affinity sorbent.
5.3.2 DNA-Related Proteins
Very early, the immobilization of DNA on a solid support was developed for affinity
chromatography separations [151]. At that time, the protein capture was not specific
but could be qualified as group specific for all proteins able to interact with nucleic
acids. Then, more specific interactions were targeted, such as anti-DNA antibodies
[152] and transcription factors [132]. It is thus not surprising to see the application of
aptamers as specific ligands for the separation of proteins related to DNA. In 2007, a
DNA aptamer recognizing specifically a Taq polymerase was attached chemically to
magnetic beads previously activated with glutaraldehyde and used for protein
purification [129]. The first interest of this work was the possibility to produce
small quantities of this recombinant protein for characterization purposes. Simplicity
and rapidity of results were the most important motivations for this development. It
was observed that the best conditions of aptamer grafting were obtained when the pH
was between 6 and 8. With this affinity sorbent, a crude physiological bacterial
extract containing the recombinant enzyme could be loaded in a mini batch mode.
After washing, the Taq polymerase was desorbed by decreasing the pH to 4. Compared to conventional purification techniques, the process was considered more
rapid, without the need of various chemicals and very reliable for single-step
purification.
In another more recent paper, Beloborodov et al. [62] described the facilitated
aspect of functional protein purification of two proteins with the use of DNA
aptamers as ligands. They compared the purification of proteins to immunoaffinity
chromatography and IMAC chromatography. Outside the purity aspect, immunoaffinity chromatography necessitates acidic condition for elution that can be detrimental to the biological activity. In a similar way, the presence of metal ions in
IMAC may have other deleterious effects. On the contrary, with the use of aptamer
ligands, the elution could be performed at neutral pH with the assistance of Mg
++
cations. The purity and the biological activity of AlkB, a protein involved in the
DNA alkylation damage, were above 85%. MutS, a protein involved in DNA
replication and repair, was very tightly associated to the aptamer and only desorbed
by 2 M MgCl 2 ; moreover, it retained its binding activity.
5.3.3 Group-Specific Proteins
In affinity chromatography, certain ligands are used for the separation of groups of
proteins. Transition metal ions chelated by immobilized complexing agents interact
122
G. Perret and E. Boschetti