Comparison of Fluorescence Labelling Techniques for the Selection of Affinity Ligands from Solid-Phase Combinatorial Libraries

Abstract

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

“…Synthesis of the combinatorial library of affinity ligands: Solid phase synthesis of the combinatorial library (64 affinity ligands) on agarose (solid support) was performed by using the multicomponent Ugi reaction as described elsewhere 17a. 28 The amines and carboxylic acids are listed in Tables 4 and 5.…”

Biomimetic Affinity Ligands for Protein Purification

“…This rapid qualitative screening methodology, which requires very low amounts of both reactants (conjugated protein and immobilized ligand) and facile instrumentation, was proved successful as a first approach to reduce the number of potential lead candidates of a combinatorial library designed to bind human IgG [38] and in the search for cutinase strong binders in a random evaluation of the same library [27]. The method tends to give false positives but not false negatives [38,39]. False positives may increase slightly the number of ligands selected to proceed for further assessment.…”

“…Recent studies have confirmed that the strength of binding between immobilized ligands and proteins can be indirectly affected by interactions with the support material [37], thus reinforcing the advantages of carrying out synthesis and screening on the same support. As so, on-bead fluorescence-based binding assessment [38,39] and affinity chromatography, alone or combined with an immunoassay, are the most common and effective strategies to screen large solid-phase combinatorial libraries of triazine-based synthetic affinity ligands [15,18,19,26,27].…”

Mild and cost-effective green fluorescent protein purification employing small synthetic ligands

“…Aldehyde-activated agarose beads were used as the starting solid support following previously reported methodologies [26,29]. The combinatorial library comprised a total of 64 affinity ligands and each ligand was obtained from the reaction between the aldehyde-functionalized agarose, an amine, a carboxylic acid and isopropyl isocyanide.…”

“…This property makes the formation of the chromophore easier in live organisms, tissues and cells. Other advantages of these proteins include the low-toxicity and high stability in a wide range of pH and solvents, the easy detection in a bulk cell suspension without cell disruption, and relatively small molecular weight (25)(26)(27)(28)(29)(30) which contributes to a low burden to host cells [5]. Due to the attractive properties of GFP, its use can be extended as an alternative protein tag for the purification of GFP fusion proteins through chromatography.…”