To shorten the very long history of affinity chromatography ligands, combinatorial libraries of peptides and oligonucleotides must be mentioned. It is known that
proteins display the capability to recognize other structures in vivo. The latter are
frequently of proteinaceous nature; thus it is conceivable to have polypeptides usable
as ligands for specific protein capture. This molecular recognition involves regions
of a protein that can be of sequential or conformational nature. In the first case, short
peptides can be used as ligands; in the second case, more complex polypeptides are
necessary. Combinatorial peptide ligand libraries are obtained by well-defined
chemical synthesis, using natural or even nonnatural amino acids [41]. For instance,
combinatorial heptapeptides using 20 amino acids result in more than a billion of
possible sequential structures. It is among these complex mixtures that one or more
affinity ligands for a given protein can be selected and then used to prepare affinity
chromatography sorbents [11, 42, 43]. In a recent review, Fang et al. [44] made a
point on this technology that covers the domain of short linear and cyclic peptides. A
strategy approach is suggested for the design, the screening, and the coupling of the
peptide ligands onto solid supports.
Another combinatorial ligand strategy is based on polypeptides (small or medium
proteins). Scaffolds for this strategy are available from various sources as described
[45–47], and practical development is obtained mostly by phage display technology
[48]. Here, also the number of diverse polypeptide ligands can be very large, but its
production is rather of biological than of synthetic origin. Both technologies and
their developments were described in extenso in various published reviews during
the last two decades [13, 49, 50].
Combinatorial sources of ligands (small or large) necessitate a screening phase
to sort out the best selective structures, on the one hand, and subsequently their
production prior to grafting on a solid support, on the other hand. Both are technical
constraints that have been resolved nowadays, but they are still time- and resourceconsuming. Practical approaches for these processes are described in dedicated
reviews [48, 51, 52]. What mainly distinguishes the adoption of small peptides to
large polypeptides is the mode of preparation after the screening phase. In the first
instance, the peptide is generally a short chain of amino acids that can easily
be produced in quantity by well-established chemical synthesis procedures and
obtained under relatively pure state. Conversely, the production of a large polypeptide is made by recombinant technology necessitating a purification process to
remove many impurities of proteinaceous nature prior to grafting on the solid
support [48, 52]. A typical example of such an approach is represented by
recombinantly modified Protein A [16]. It represents one of the major success stories
of affinity chromatography, to the point that it has never been replaced despite of the
numerous attempts by either chemically made biomimetic ligands [53, 54] or by
peptides [55] and polypeptides [56].
At present, one novel promising milestone in affinity chromatography seems to
emerge with the use of aptamers. Affinity, specificity, elution possibilities, easiness
to sort them out, production by pure chemical synthesis, stability of constructs, and
finally easy grafting method onto solid supports are typical attributes for this type of
ligands.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
97
proteins display the capability to recognize other structures in vivo. The latter are
frequently of proteinaceous nature; thus it is conceivable to have polypeptides usable
as ligands for specific protein capture. This molecular recognition involves regions
of a protein that can be of sequential or conformational nature. In the first case, short
peptides can be used as ligands; in the second case, more complex polypeptides are
necessary. Combinatorial peptide ligand libraries are obtained by well-defined
chemical synthesis, using natural or even nonnatural amino acids [41]. For instance,
combinatorial heptapeptides using 20 amino acids result in more than a billion of
possible sequential structures. It is among these complex mixtures that one or more
affinity ligands for a given protein can be selected and then used to prepare affinity
chromatography sorbents [11, 42, 43]. In a recent review, Fang et al. [44] made a
point on this technology that covers the domain of short linear and cyclic peptides. A
strategy approach is suggested for the design, the screening, and the coupling of the
peptide ligands onto solid supports.
Another combinatorial ligand strategy is based on polypeptides (small or medium
proteins). Scaffolds for this strategy are available from various sources as described
[45–47], and practical development is obtained mostly by phage display technology
[48]. Here, also the number of diverse polypeptide ligands can be very large, but its
production is rather of biological than of synthetic origin. Both technologies and
their developments were described in extenso in various published reviews during
the last two decades [13, 49, 50].
Combinatorial sources of ligands (small or large) necessitate a screening phase
to sort out the best selective structures, on the one hand, and subsequently their
production prior to grafting on a solid support, on the other hand. Both are technical
constraints that have been resolved nowadays, but they are still time- and resourceconsuming. Practical approaches for these processes are described in dedicated
reviews [48, 51, 52]. What mainly distinguishes the adoption of small peptides to
large polypeptides is the mode of preparation after the screening phase. In the first
instance, the peptide is generally a short chain of amino acids that can easily
be produced in quantity by well-established chemical synthesis procedures and
obtained under relatively pure state. Conversely, the production of a large polypeptide is made by recombinant technology necessitating a purification process to
remove many impurities of proteinaceous nature prior to grafting on the solid
support [48, 52]. A typical example of such an approach is represented by
recombinantly modified Protein A [16]. It represents one of the major success stories
of affinity chromatography, to the point that it has never been replaced despite of the
numerous attempts by either chemically made biomimetic ligands [53, 54] or by
peptides [55] and polypeptides [56].
At present, one novel promising milestone in affinity chromatography seems to
emerge with the use of aptamers. Affinity, specificity, elution possibilities, easiness
to sort them out, production by pure chemical synthesis, stability of constructs, and
finally easy grafting method onto solid supports are typical attributes for this type of
ligands.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
97