column as reported were (1) milder elution operated at neutral pH versus acidic
conditions that are prone to form antibody aggregates, (2) usable for all IgG subclasses, and (3) stable under caustic conditions usually adopted for column cleaning.
Of particular interest is the protein elution under neutral conditions, which facilitates
obtaining functional proteins, as also underlined by Beloborodov et al. [62].
Aptamer selection for chromatography can also be performed at unconventional
pH. For instance, finding an aptamer with an affinity constant compatible with
protein purification at acidic pH may allow desorbing the captured target protein
in neutral conditions [145]. Such an approach may be very helpful for process
optimization, when the pH of the initial crude extract is slightly acidic. Thus, the
protein solution does not necessitate any preliminary conditioning. In addition,
eluting in neutral conditions may facilitate the following purification step (e.g.,
another chromatographic process) without further conditioning manipulations.
This approach has been adopted in our laboratories for the purification of human
plasma fibrinogen [146] and for immunoglobulins G [147]. In the first case, the antifibrinogen aptamer was selected in order to have an affinity interaction at pH 6.3
and the dissociation of such an affinity complex at pH 7.4 (Fig. 11). Fibrinogen
was eluted as almost homogeneous protein (SDS-PAGE, lane El) out of a very crude
biological material, as shown on lanes Start and FT. Concerning the human
immunoglobulins G, the SELEX operation was performed at pH 5.5 for the
B
200
116
97
66
55
36
31
21
14
6
Mr
Start FT
El
A
FT
El
Reg
0
5
10
15
20
25
25
20
15
10
5
0
mAU
KDa
mL
Fig. 11 Purification of human plasma fibrinogen by aptamer-based affinity chromatography
[146]. The primary amino-derived aptamer was grafted via NHS-agarose support. The load was
human plasma in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 6.3. Fibrinogen elution was performed
by 50 mM Tris-HCl buffer, pH 7.4. Regeneration of the column was performed with 2 M MgCl 2 ,
pH 7.4. (a) Chromatographic profile; (b) SDS-PAGE. Start human serum, FT flowthrough, El
elution of fibrinogen, Mr molecular ladder
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
119
conditions that are prone to form antibody aggregates, (2) usable for all IgG subclasses, and (3) stable under caustic conditions usually adopted for column cleaning.
Of particular interest is the protein elution under neutral conditions, which facilitates
obtaining functional proteins, as also underlined by Beloborodov et al. [62].
Aptamer selection for chromatography can also be performed at unconventional
pH. For instance, finding an aptamer with an affinity constant compatible with
protein purification at acidic pH may allow desorbing the captured target protein
in neutral conditions [145]. Such an approach may be very helpful for process
optimization, when the pH of the initial crude extract is slightly acidic. Thus, the
protein solution does not necessitate any preliminary conditioning. In addition,
eluting in neutral conditions may facilitate the following purification step (e.g.,
another chromatographic process) without further conditioning manipulations.
This approach has been adopted in our laboratories for the purification of human
plasma fibrinogen [146] and for immunoglobulins G [147]. In the first case, the antifibrinogen aptamer was selected in order to have an affinity interaction at pH 6.3
and the dissociation of such an affinity complex at pH 7.4 (Fig. 11). Fibrinogen
was eluted as almost homogeneous protein (SDS-PAGE, lane El) out of a very crude
biological material, as shown on lanes Start and FT. Concerning the human
immunoglobulins G, the SELEX operation was performed at pH 5.5 for the
B
200
116
97
66
55
36
31
21
14
6
Mr
Start FT
El
A
FT
El
Reg
0
5
10
15
20
25
25
20
15
10
5
0
mAU
KDa
mL
Fig. 11 Purification of human plasma fibrinogen by aptamer-based affinity chromatography
[146]. The primary amino-derived aptamer was grafted via NHS-agarose support. The load was
human plasma in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 6.3. Fibrinogen elution was performed
by 50 mM Tris-HCl buffer, pH 7.4. Regeneration of the column was performed with 2 M MgCl 2 ,
pH 7.4. (a) Chromatographic profile; (b) SDS-PAGE. Start human serum, FT flowthrough, El
elution of fibrinogen, Mr molecular ladder
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
119