Immunoaffinity chromatography uses antibody ligands against given protein
antigens. Its major advantage is to be particularly specific, but antibodies are too
fragile ligands for repeated purification cycles. Polyclonal or monoclonal antibodies
are prepared first and then grafted onto a solid support for the separation of the target
protein (for review, see the following references [1, 20]).
Lectin affinity chromatography has been developed as an elegant way to separate
glycoproteins as a function of the glycan structure. The lectin ligand is a protein
mainly of plant origin. The capture of the protein to purify is accomplished under
physiological conditions, and the target glycoproteins are harvested by displacement
with competitive sugars. Here, also thousands of published examples are available; for
general information the reader should refer to the following general reviews [21, 22].
Another major advancement in affinity chromatography ligands, mentioned
above, is metal-chelate affinity chromatography, which uses transition metal ions
complexed on a solid chromatography material via a grafted chelating agent. This
method, developed in the 1970s, knew a superb success with recombinant protein
expressions where one extremity of the protein peptidic chain comprised an oligohistidine residue. With this specifically designed tag, the protein is captured by the
chelated transition metal and isolated from a crude extract, as pioneered by Porath
et al. [23]. The major application of this technology is focused toward the recombinant proteins expressed with His 6 tag, which is then enzymatically cleaved and
eliminated. Pertinent reviews in this domain are referenced here: [24–26].
When proteins to purify do not correspond to the above described categories, the
research of ligands was directed toward smaller groups of available molecules that
naturally interact with some proteins, such as enzyme cofactors or enzyme inhibitors. Examples of them are AMP [27], NAD [28], heparin for antithrombin III [5],
and benzamidine derivatives for serine proteases [29, 30].
While affinity chromatography was still at its early developments, scientists
realized that a major problem was the design of adapted affinity reversible ligands.
Thus, one of the selected research directions was to explore the properties of
collections of available molecules, such as textile reactive dyes [31, 32]. As such,
they could be grafted directly on agarose-based solid supports without extra chemical reactions. Although many papers and reviews have been published with this
principle, real successes were limited in numbers. Nevertheless, a few success stories
could be accounted for [33–35]. This technology though opened up a new domain,
which was the design of combinatorial structures (not necessarily colored) based on
the principle of the dye chemistry molecule collection with enhanced selectivity. The
concept of selecting affinity ligands from predesigned libraries of molecules was
thus initiated with promising potential developments [36, 37].
To reduce the library diversity and thus decrease the workload of ligand selection,
it has been suggested to combine the diversomer synthesis with informatics models
of chemical structure docking. This approach was successfully applied to chemicalbased ligands [38–40], as well as to peptide structures [9]. A recent general review
describes the most important approaches in both the library design and the selection
methods for ligands out of very large and complex scaffold structures [13].
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G. Perret and E. Boschetti
antigens. Its major advantage is to be particularly specific, but antibodies are too
fragile ligands for repeated purification cycles. Polyclonal or monoclonal antibodies
are prepared first and then grafted onto a solid support for the separation of the target
protein (for review, see the following references [1, 20]).
Lectin affinity chromatography has been developed as an elegant way to separate
glycoproteins as a function of the glycan structure. The lectin ligand is a protein
mainly of plant origin. The capture of the protein to purify is accomplished under
physiological conditions, and the target glycoproteins are harvested by displacement
with competitive sugars. Here, also thousands of published examples are available; for
general information the reader should refer to the following general reviews [21, 22].
Another major advancement in affinity chromatography ligands, mentioned
above, is metal-chelate affinity chromatography, which uses transition metal ions
complexed on a solid chromatography material via a grafted chelating agent. This
method, developed in the 1970s, knew a superb success with recombinant protein
expressions where one extremity of the protein peptidic chain comprised an oligohistidine residue. With this specifically designed tag, the protein is captured by the
chelated transition metal and isolated from a crude extract, as pioneered by Porath
et al. [23]. The major application of this technology is focused toward the recombinant proteins expressed with His 6 tag, which is then enzymatically cleaved and
eliminated. Pertinent reviews in this domain are referenced here: [24–26].
When proteins to purify do not correspond to the above described categories, the
research of ligands was directed toward smaller groups of available molecules that
naturally interact with some proteins, such as enzyme cofactors or enzyme inhibitors. Examples of them are AMP [27], NAD [28], heparin for antithrombin III [5],
and benzamidine derivatives for serine proteases [29, 30].
While affinity chromatography was still at its early developments, scientists
realized that a major problem was the design of adapted affinity reversible ligands.
Thus, one of the selected research directions was to explore the properties of
collections of available molecules, such as textile reactive dyes [31, 32]. As such,
they could be grafted directly on agarose-based solid supports without extra chemical reactions. Although many papers and reviews have been published with this
principle, real successes were limited in numbers. Nevertheless, a few success stories
could be accounted for [33–35]. This technology though opened up a new domain,
which was the design of combinatorial structures (not necessarily colored) based on
the principle of the dye chemistry molecule collection with enhanced selectivity. The
concept of selecting affinity ligands from predesigned libraries of molecules was
thus initiated with promising potential developments [36, 37].
To reduce the library diversity and thus decrease the workload of ligand selection,
it has been suggested to combine the diversomer synthesis with informatics models
of chemical structure docking. This approach was successfully applied to chemicalbased ligands [38–40], as well as to peptide structures [9]. A recent general review
describes the most important approaches in both the library design and the selection
methods for ligands out of very large and complex scaffold structures [13].
96
G. Perret and E. Boschetti