sequences and to remove the formed complex from the mixture without denaturation.
This is the genuine principle of affinity chromatography of proteins. Even if variants to
this principle exist, the main dilemma is how to design a perfect specific ligand to fish
the target protein out of a complex crude extract. Many attempts have been experimentally performed, based on molecular recognition inspired from known interactions
in nature. Antibody-antigen protein interaction is one of the most important [1], but not
the only one, since proteins have various roles such as the transportation of small
molecules as lipids [2] or hormones [3]. They are capable of very specific enzymatic
modifications of substrates that can be exploitable in affinity chromatography, where
the interaction partner (or its analog) is chemically attached to a chromatographic
support [4]. In spite of these logical approaches, only a very limited number of proteins
can be purified because of the rare availability of specific affinity ligands.
Outside the natural antibody-ligand affinity strategy and other specific interactions, such as heparin with heparin-binding proteins [5] and biotin with avidin or
streptavidin [6], the creativity of researchers was developed toward two different
directions: the first was the rational design of affinity ligands for a given protein [7–
10], and the second was the development of large combinatorial libraries of ligands
[11, 12] from where a specific partner could be identified. The former is illustrated
by the clever synergistic combination of informatics and chemistry for the de novo
design of ligands using predefined scaffolds. The latter is illustrated by the preparation of millions of molecules (the libraries) made using organic chemistry or even
biology such as peptides, polypeptides, and nucleic acids (for a complete information, see ref. [13]). From these sources it is then possible to enucleate the most
appropriate ligand by various methodologies.
It is within this context that very effective affinity ligands have been found in the
last two decades.
In this review we will first present a short historical recall of the ligand evolution
in affinity chromatography and then focus the attention of the reader on effective
ways of selecting single-chain oligonucleotide affinity ligand molecules in view of
protein purification.
2 Major Milestones in Affinity Chromatography
After about half a century quest for affinity ligands, four main avenues have
emerged; they still play a major role today. Protein A affinity chromatography,
immunoaffinity chromatography, lectin chromatography, and metal-chelating affinity chromatography are the subjects of thousands of published reports. Although
they represent group-specific ligands for “families” of proteins, they are widely used.
Protein A, a polypeptide docking selectively on Fc region [14], could be used for the
purification of numerous immunoglobulins G from various mammals, as well as for
monoclonal antibodies from cell culture supernatants. For general reviews, see the
following references [15, 16]. Protein A belongs to a class of affinity ligands
deriving from bacterial surface proteins [17]. This class of ligands includes also
protein G [18] and protein L [19], both mainly used in laboratory-scale applications.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
95
This is the genuine principle of affinity chromatography of proteins. Even if variants to
this principle exist, the main dilemma is how to design a perfect specific ligand to fish
the target protein out of a complex crude extract. Many attempts have been experimentally performed, based on molecular recognition inspired from known interactions
in nature. Antibody-antigen protein interaction is one of the most important [1], but not
the only one, since proteins have various roles such as the transportation of small
molecules as lipids [2] or hormones [3]. They are capable of very specific enzymatic
modifications of substrates that can be exploitable in affinity chromatography, where
the interaction partner (or its analog) is chemically attached to a chromatographic
support [4]. In spite of these logical approaches, only a very limited number of proteins
can be purified because of the rare availability of specific affinity ligands.
Outside the natural antibody-ligand affinity strategy and other specific interactions, such as heparin with heparin-binding proteins [5] and biotin with avidin or
streptavidin [6], the creativity of researchers was developed toward two different
directions: the first was the rational design of affinity ligands for a given protein [7–
10], and the second was the development of large combinatorial libraries of ligands
[11, 12] from where a specific partner could be identified. The former is illustrated
by the clever synergistic combination of informatics and chemistry for the de novo
design of ligands using predefined scaffolds. The latter is illustrated by the preparation of millions of molecules (the libraries) made using organic chemistry or even
biology such as peptides, polypeptides, and nucleic acids (for a complete information, see ref. [13]). From these sources it is then possible to enucleate the most
appropriate ligand by various methodologies.
It is within this context that very effective affinity ligands have been found in the
last two decades.
In this review we will first present a short historical recall of the ligand evolution
in affinity chromatography and then focus the attention of the reader on effective
ways of selecting single-chain oligonucleotide affinity ligand molecules in view of
protein purification.
2 Major Milestones in Affinity Chromatography
After about half a century quest for affinity ligands, four main avenues have
emerged; they still play a major role today. Protein A affinity chromatography,
immunoaffinity chromatography, lectin chromatography, and metal-chelating affinity chromatography are the subjects of thousands of published reports. Although
they represent group-specific ligands for “families” of proteins, they are widely used.
Protein A, a polypeptide docking selectively on Fc region [14], could be used for the
purification of numerous immunoglobulins G from various mammals, as well as for
monoclonal antibodies from cell culture supernatants. For general reviews, see the
following references [15, 16]. Protein A belongs to a class of affinity ligands
deriving from bacterial surface proteins [17]. This class of ligands includes also
protein G [18] and protein L [19], both mainly used in laboratory-scale applications.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
95