A new method for the screening of solid‐phase combinatorial libraries for affinity chromatography

Abstract

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.

Synthetic affinity ligands as a novel tool to improve protein stability

“…It was formerly reported that the FITC-based screening methodology utilized as a first rapid assessment of the 136-membered random library, tends to give false positives but not false negatives (Roque et al, 2004). Therefore, most of the ligands showing negative results by this method were not included in a following screening step by affinity chromatography ( Second-generation ligand library: dual screening for cutinase binding and biological activity retention…”

“…The labelled protein was purified from the unconjugated fluorescein by gel filtration using a Sephadex G-25M column. The screening of the random combinatorial library was performed using the method described by Roque and co-workers (Roque et al, 2004) with the conjugate FITC-cutinase in equilibration buffer (20 mM Tris-HCl, pH 8.0). Imaging of the agarose beads was performed under a fluorescence microscope.…”

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

“…These are: (a) reduced production costs (protein A chromatography alone can account for more than 35% of the downstream processing costs), (b) resistance to chemical and biological degradation, (c) resistance to harsh sanitation or sterilization conditions imposed by GMP protocols, and (d) they are readily amenable to scale up procedures. To address some of these issues, several researchers over the years have attempted to develop biomimetic ligands that have specific binding to immunoglobulins and can potentially replace protein A (Li et al, 1998;Teng et al, 1999;Labrou, 2003;Roque et al, 2004b;Ghose et al, 2006;.…”

Phosvitin Plays a Critical Role in the Immunity of Zebrafish Embryos via Acting as a Pattern Recognition Receptor and an Antimicrobial Effector

“…The purity of the conjugate, FITC-labeled rPv, was confirmed by SDS-PAGE and Coomassie Blue staining, and its F/P ratio (F/P ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate) was calculated by the equation F/P ϭ (2.77 ϫ A 495 )/(A 280 Ϫ(0.35 ϫ A 495 )) from the absorbance readings of the conjugated samples (42). To assess if rPv activity is affected by labeling with FITC, colony-forming unit assay was conducted to compare the growth inhibition rates of E. coli by FITC-labeled rPv and nonlabeled rPv.…”