Synthesis and Application of Dye-Ligand Affinity Adsorbents

Abstract

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity towards a large number of proteins. They are commercially available, are inexpensive, and can easily be immobilized. Important factors that contribute to the successful operation of a dye-ligand chromatography include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions used in the adsorption and elution stages, and contacting parameters like flow rate and column geometry. In general, with dye-ligand affinity chromatography, the specificity is provided by the adsorption and elution conditions employed in a particular purification, and these must often be worked out by trial and error. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris crude extract on Cibacron Blue 3GA-Sepharose is given as an example.

Rational design of affinity ligands for bioseparation

“…By using multicomponent synthetic reactions as templates and changing the substituents (e.g. R1,R2 in figure 3) used in the synthesis, it was possible to construct and screen multi-dimensional libraries (usually up to 100 variants) in a fast and simple way [10,13,[53][54][55]. The rational for selecting the substituents and library design was inspired in structural information on the critical residues known to play a role in target binding.…”

Reduce, Reuse and Recycle in Protein Chromatography: Development of an Affinity Adsorbent from Waste Paper and Its Application for the Purification of Proteases from Fish By-Products

“…The immobilization of CB3GA on cellulose microfibers was accomplished as described previously [15,16], with minor modifications. The nucleophilic substitution reaction was carried out at 60 • C under stirring for 7.5 h, using 28 mg CB3GA per g of cellulose microfibers.…”

“…The immobilized ligand is the key factor that affects the effectiveness of any affinity chromatographic method, since it provides the selectivity with the target protein [1]. However, the matrix itself is an additional parameter that determines the performance of the affinity chromatography as it affects the specificity of interaction, the capacity for the target protein as well as the stability of the adsorbent [15,16]. Examples of frequently used support matrices are agarose, cellulose, dextran, silica, glass and polyacrylamide derivatives.…”

“…Triazine dyes display several advantages compared to specific biological ligands due to their low cost, resistance to biological and chemical degradation and high protein binding capacity [10][11][12][13][18][19][20][21][22]. In addition, the presence of the triazine ring allows their direct immobilization to the matrix (e.g., Sepharose or cellulose) through a nucleophilic substitution reaction [15,16].…”