High-Throughput Screening of Dye-Ligands for Chromatography

Abstract

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

An alternative, arginase‐independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

“…While guanidinobutyrase activity had previously been measured in Aspergillus niger (Kumar and Punekar, ), no gene encoding this activity has been identified in eukaryotes, a phylogenetic analysis was performed by searching for KlCar1 (arginase) and KlGbu1 (guanidinobutyrase) orthologues among the predicted protein sequences encoded in the genomes of yeasts belonging to the Saccharomycotina group, a subphylum of the Ascomycota (Kurtzman, ; Dujon, ). Using a phylogenetic tree topology proposed by Dujon () for the Saccharomycotina group, orthologues of KlCar1 were found in all interrogated yeast proteomes.…”

Chemokine-Releasing Nanoparticles for Manipulation of the Lymph Node Microenvironment

“…The chemical structures of the dyes containing multiple aromatic, condensed, and heterocyclic rings, as well as 2–4 negatively-charged sulfate groups per molecule, are shown in Figure A1 . Overall, the dyes are capable of electrostatic, hydrophobic, and hydrogen-bonding interactions with the proteins [ 32 , 33 , 34 ].…”

“…A variety of inner baits of different chemical nature can be quickly incorporated into the NPs to capture from the environment in one step a wide range of molecules including the CKs [ 19 ]. Among such baits the textile dyes of triazine, acidic, basic and disperse types attracted attention due to their affinity interactions with broad classes of protein ligands on the basis of specific molecular recognition processes [ 32 , 33 ]. The dye-ligand matrices used in affinity chromatography are thought to mimic the structural features of the corresponding natural substrates, cofactors, etc.…”