Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

Abstract

Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity-like interactions with a biomimetic triazine-scaffolded ligand. A synthetic lead compound (ligand 3'/11, K(a) ≈ 10(4) M(-1)) was selected from a previously screened solid-phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well-established solid-phase synthesis method, was able to strongly bind cutinase and led to impressive half-lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25- and 57-fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability.

Biomimetic Affinity Ligands for Protein Purification

“…When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free protein [27]. Thermostability assessment with cutinase adsorbed to a lead compound at elevated temperatures highlighted the potential of biomimetic affinity ligands as a novel protein-stabilizing tool [28]. As a general strategy, designing and screening selective biomimetic affinity ligands that simultaneously have a stabilizing effect may be useful either for the purification of proteins or the utilization of the macromolecular ligand-protein supports (e.g., as biocatalysts) under extreme, unfavorable conditions.…”

“…This was found to be a suitable strategy to find a number of triazine-scaffolded synthetic compounds binding cutinase with high affinity and selectivity while stabilizing its biological activity [27,28]. Figure 5 shows the structure of a lead stabilizing ligand and the analysis by SDS-PAGE of the purification of a recombinant E. coli extract of cutinase from Fusarium solani pisi with a ligand-derivatized adsorbent.…”

“…Activity assessment after ligand binding. Affinity synthetic ligands were shown to be potentially useful for enzyme immobilization and stabilization purposes [27,28]. When that is the aim of a ligand screening, it is necessary to verify whether enzymatic activity is retained upon binding.…”

“…Although often neglected in the screening process, a high activity recovery of the target protein should be obtained after the elution step in order to validate large-scale applications. Recently, triazine-scaffolded biomimetic ligands were assessed for their ability to bind a protein while, simultaneously, preserving its biological activity and enhancing its stability [27,28]. In this study, a small lipolytic enzyme (cutinase) was used as a model protein.…”

“…We present the materials used for the biological activity screening of ligands targeted at an enzyme, cutinase, with the aim of binding the protein while preserving its enzymatic activity [27,28]. Such screening includes standard adsorption and activity assays performed with the enzyme bound to the ligand support.…”