Design, synthesis and characterisation of affinity ligands for glycoproteins

Abstract

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine-based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself.

Membrane adsorbers as purification tools for monoclonal antibody purification

“…The introduction of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. Among those protein A mimetics like A2P [25][26][27][28], D-PAM [29][30][31][32], hexamer peptide ligands [33] and CaptureSelect ® [34] have been the subject of intense investigation.…”

Ligands for mixed-mode protein chromatography: Principles, characteristics and design

“…A common strategy for establishing a small but promising library is to choose the functional groups that chemically and structurally complement the target protein (Palanisamy et al, 1999;Sproule et al, 2000;Platis et al, 2006). For instance, in order to develop affinity ligands for human insulin precursor, Sproule et al (2000) examined the structure of the protein and identified a possible binding region for ligands, which contains several aromatic residues.…”

“…This approach is especially applicable to libraries established by solid synthesis, of which the ligands are already immobilized. Lowe and co-workers (Palanisamy et al, 1999;Teng et al, 1999;Sproule et al, 2000;Roque et al, 2005;Platis et al, 2006) screened affinity ligands for various proteins by chromatographic methods. However, the scale of chromatographic evaluation is usually limited.…”

A comparison study between polymeric ligand and monomeric ligand for oligopeptide adsorption

“…To design a ligand with promising affinity to the target peptide, the first principle is to identify a target binding site or use a known biological interaction as a template which was suggested by Lowe and coworkers [31,44,45]. According to our previous data, two tryptophan residues (Trp) on the C-terminal of VW-8 were the main binding sites, and the ligands bearing both hydrophobic and electrostatic sites probably exhibited higher affinity to VW-8 [37,38].…”